Publications

Doppler Detection of Pathogenic Activity in Living Tissue by Biodynamic Imaging

 

 

Acetic Acid Removal from Corn Stover Hydrolysate Using Ethyl Acetate and the Impact on Saccharomyces cerevisiae Bioethanol Fermentation

2016

Authors: M. Aghazadeh, M. R. Ladisch, A. S. Engelberth
Journal: Biotechnology Progress, 32(4), 929-937
Book Chapter: 

Abstract: Acetic acid is introduced into cellulose conversion processes as a consequence of composition of lignocellulose feedstocks, causing significant inhibition of adapted, genetically modified and wild-type S. cerevisiae in bioethanol fermentation. While adaptation or modification of yeast may reduce inhibition, the most effective approach is to remove the acetic acid prior to fermentation. This work addresses liquid-liquid extraction of acetic acid from biomass hydrolysate through a pathway that mitigates acetic acid inhibition while avoiding the negative effects of the extractant, which itself may exhibit inhibition. Candidate solvents were selected using simulation results from Aspen Plus, based on their ability to extract acetic acid which was confirmed by experimentation. All solvents showed varying degrees of toxicity toward yeast, but the relative volatility of ethyl acetate enabled its use as simple vacuum evaporation could reduce small concentrations of aqueous ethyl acetate to minimally inhibitory levels. The toxicity threshold of ethyl acetate, in the presence of acetic acid, was found to be 10 g L-1. The fermentation was enhanced by extracting 90% of the acetic acid using ethyl acetate, followed by vacuum evaporation to remove 88% removal of residual ethyl acetate along with 10% of the broth. NRRL Y-1546 yeast was used to demonstrate a 13% increase in concentration, 14% in ethanol specific production rate, and 11% ethanol yield. This study demonstrated that extraction of acetic acid with ethyl acetate followed by evaporative removal of ethyl acetate from the raffinate phase has potential to significantly enhance ethanol fermentation in a corn stover bioethanol facility.

Research Area: Bioseparations Bioenergy   

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Fiber-Based Monolithic Columns for Liquid Chromatography

2016

Authors: M. Ladisch, L. Zhang
Journal: Analytical and Bioanalytical Chemistry, 408(25), 6871-6883
Book Chapter: 

Abstract: Fiber-based monoliths for use in liquid chromatographic separations are defined by columns packed with aligned fibers, woven matrices, or contiguous fiber structures capable of achieving rapid separations of proteins, macromolecules, and low molecular weight components. A common denominator and motivating driver for this approach, first initiated 25 years ago, was reducing the cost of bioseparations in a manner that also reduced residence time of retained components while achieving a high ratio of mass to momentum transfer. This type of medium, when packed into a liquid chromatography column, minimized the fraction of stagnant liquid and resulted in a constant plate height for non-adsorbing species. The uncoupling of dispersion from eluent flow rate enabled the surface chemistry of the stationary phase to be considered separately from fluid transport phenomena and pointed to new ways to apply chemistry for the engineering of rapid bioseparations. This paper addresses developments and current research on fiber-based monoliths and explains how the various forms of this type of chromatographic stationary phase have potential to provide new tools for analytical and preparative scale separations. The different stationary phases are discussed, and a model that captures the observed constant plate height as a function of mobile phase velocity is reviewed. Methods that enable hydrodynamically stable fiber columns to be packed and operated over a range of mobile phase flow rates, together with the development of new fiber chemistries, are shown to provide columns that extend the versatility of liquid chromatography using monoliths, particularly at the preparative scale.

Research Area: Bioenergy    

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Identifying Conditions to Optimize Lactic Acid Production from Food Waste Co-Digested with Primary Sludge

2016

Authors: R. Red Corn, A. S. Engelberth
Journal: Biochemical Engineering Journal, 105, 205-213
Book Chapter: 

Abstract: Lactic acid is a platform chemical useful for the production of polymers, oxychemicals, solvents, and for biological nutrient removal in wastewater streams. Food waste offers a renewable feedstock to produce lactic acid, but the co-digestion with sludge has not been suitably studied. In this study, response surface methodology was used to identify the pH, temperature, loading rate, and retention time for co-digestion of foodwaste and primary sludge that optimized lactic acid production. The optimum conditions occur at pH 5.5 and temperature 41 C. A loading rate of 150 gL-1 volatile solids food waste maximizes lactate yield while 250 gL-1 volatile solids maximizes lactate concentration, resulting in 48 gL-1 and 58 gL-1 lactate, respectively. Optical purity and ammonium concentration were evaluated to inform end uses. This research indicates that the co-digestion can achieve 97% of theoretical yield while requiring less pH adjustment and retention time than experiments that did not co-digest with primary sludge.

Research Area: Bioseparations Bioenergy   

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Maleic Acid and Aluminum Chloride Catalyzed Conversion of Glucose to 5-(hydroxymethyl) furfural and Levulinic Acid in Aqueous Media

2016

Authors: X. Zhang, P. Murria, Y. Jiang, W. Xiao, H. I. Kenttamaa, M. M. Abu-Omar and N. S. Mosier, 18, 5219-5229
Journal: Green Chemistry
Book Chapter: 

Abstract: Maleic acid (MA) and AlCl3 self-assemble into catalytic complexes (Al-(MA)2-(OH)2(aq)) with improved selectivity for converting glucose to HMF, and levulinic acid. The calculated activation energy (Ea) of the MA-aluminum catalyzed glucose-to-fructose isomerization is 95 kJ mol-1 compared to 149 kJ mol-1 for HCl and AlCl3 alone. Furthermore, conversion of fructose to HMF is enhanced. The catalytic conversion of fructose to HMF by MA and AlCl3 at 180 C is 1.7 x faster with 3.3x higher selectivity when compared to HCl with AlCl3. Liquid 13C NMR spectra results indicate that glucose undergoes a ring-opening process in aqueous solution when maleic acid is introduced, which we hypothesize facilitates the hydride shift in glucose for isomerization leading to enhanced rates and selectivity. Improved selectivity of glucose conversion to HMF and levulinic acid could improve the economics of producing these value-added chemicals for use in renewable, sustainable polymers.

Research Area: Bioenergy    

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Maleic Acid Treatment of Biologically Detoxified Corn Stover Liquor

2016

Authors: D. Kim, E. A. Ximenes, N. N. Nichols, G. Cao, S. E. Frazer, M. R. Ladisch
Journal: Bioresource Technology, 216, 437-445
Book Chapter: 

Abstract: Elimination of microbial and enzyme inhibitors from pretreated lignocellulose is critical for effective cellulose conversion and yeast fermentation of liquid hot water (LHW) pretreated corn stover. In this study, xylan oligomers were hydrolyzed using either maleic acid or hemicellulases, and other soluble inhibitors were eliminated by biological detoxification. Corn stover at 20% (w/v) solids was LHW pretreated LHW (severity factor: 4.3). The 20% solids (w/v) pretreated corn stover derived liquor was recovered and biologically detoxified using the fungus Coniochaeta ligniaria NRRL30616. After maleic acid treatment, and using 5 filter paper units of cellulose/g glucan (8.3 mg protein/g glucan), 73% higher cellulose conversion from corn stover was obtained for biodetoxified samples compared to undetoxified samples. This corresponded to 87% cellulose to glucose conversion. Ethanol production by yeast of pretreated corn stover solids hydrolysate was 1.4 times higher than undetoxified samples, with a reduction of 3 h in the fermentation lag phase.

Research Area: Bioenergy Biofuels/Bioproducts Bioseparations Bioprocessing 

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Microfiltration of Enzyme Treated Egg Whites for Accelerated Detection of Viable Salmonella

2016

Authors: S. Ku, E. Ximenes, T. Kreke, K. Foster, A. J. Deering, M. R. Ladisch
Journal: Biotechnology Progress, 323(6), 1464-1471
Book Chapter: 

Abstract: We report detection of <13 CFU of Salmonella per 25 g egg white within 7 h by concentrating the bacteria using microfiltration through 0.2-um cutoff polyethersulfone hollow fiber membranes. A combination of enzyme treatment, controlled cross-flow on both sides of the hollow fibers, and media selection were key to controlling membrane fouling so that rapid concentration and the subsequent detection of low numbers of microbial cells were achieved. We leveraged the protective effect of egg white proteins and peptone so that the proteolytic enzymes did not attack the living cells while hydrolyzing the egg white proteins responsible for fouling. The molecular weight of egg white proteins was reduced from about 70 kDa to 15 kDa during hydrolysis. This enabled a 50-fold concentration of the cells when a volume of 525 mL of peptone and egg white, containing 13 CFU of Salmonella, was decreased to a 10 mL volume in 50 min. A 10-min microcentrifugation step further concentrated the viable Salmonella cells by 10 x. The final cell recovery exceeded 100%, indicating that microbial growth occurred during the 3 h processing time. The experiments leading to rapid concentration, recovery, and detection provided further insights on the nature of membrane fouling enabling fouling effects to be mitigated. Unlike most membrane processes where protein recovery is the goal, recovery of viable microorganisms for pathogen detection is the key measure of success, with modification of cell-free proteins being both acceptable and required to achieve rapid microfiltration of viable microorganisms.

Research Area: Food Safety    

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Secretome Analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis

2016

Authors: C. Florencio, F. M. Cunha, A. C. Badino, C. S. Farinas, E. Ximenes, M. R. Ladisch
Journal: Enzyme and Microbial Technology, 90, 53-60 (2016)
Book Chapter: 

Abstract: Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and beta-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulose activity/g glucan was surprisingly effective when compared to the 5-15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei.

Research Area: Bioenergy Bioprocessing Bioseparations  

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Techno-economic Analysis for Incorporating a Liquid-Liquid Extraction System to Remove Acetic Acid into a Proposed Commercial Scale Biorefinery

2016

Authors: M. Aghazadeh, A. S. Engelberth
Journal: Biotechnology Progress, 32(4), 971-977
Book Chapter: 

Abstract: Mitigating the effect of fermentation inhibitors in bioethanol plants can have a great positive impact on the economy of this industry. Liquid-liquid extraction (LLE) using ethyl acetate is able to remove fermentation inhibitors, chiefly acetic acid, from an aqueous solution used to produce bioethanol. The fermentation broth resulting from LLE has higher performance for ethanol yield and its production rate. Previous techno-economic analyses focused on second-generation biofuel production did not address the impact of removing the fermentation inhibitors on the economic performance of the biorefinery. A comprehensive analysis of applying a separation system to mitigate the fermentation inhibition effect and to provide an analysis on the economic impact of removal of acetic acid from corn stover hydrolysate on the overall revenue of the biorefinery is necessary. This study examines the pros and cons associated with implementing LLE column along with the solvent recovery system into a commercial scale bioethanol plant. Using details from the NREL-developed model of corn stover biorefinery, the capital costs associated with the equipment and the operating cost for the use of solvent were estimated and the results were compared with the profit gain due to higher ethanol production. Results indicate that the additional capital will add 1% to the total capital and manufacturing cost will increase by 5.9%. The benefit arises from the higher ethanol production rate and yield as a consequence of inhibitor extraction and results in a $0.35 per gallon reduction in the minimum ethanol selling price (MESP).

Research Area: Bioseparations    

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A Synergistic Biorefinery Based on Catalytic Conversion of Lignin Prior to Cellulose Starting from Lignocellulosic Biomass

2015

Authors: T. Parsell, S. Yohe, J. Degenstein, T. Jarrell, I. Klein, E. Gencer, B. Hewetson, M. Hurt, J. I. Kim, H. Choudhari, B. Saha, R. Meilan, N. Mosier, F. Ribeiro, W. N. Delgass, C. Chapple, H. I. Kenttamaa, R. Agrawal, M. M. Abu-Omar
Journal: Green Chemistry, 17, 1492-1499 (2015)
Book Chapter: 

Abstract: Current biomass utilization processes do not make use of lignin beyond its heat value. Here we report on a bimetallic Zn/Pd/C catalyst that converts lignin in intact lignocellulosic biomass directly into two methoxyphenol products, leaving behind the carbohydrates as a solid residue. Genetically modified poplar enhanced in syringyl (S) monomer content yields only a single product, dihydroeugenol. Lingin-derived methoxyphenols can be deoxygenated further to propylcyclohexane. The leftover carbohydrate residue is hydrolyzed by cellulases to give glucose in 95% yield, which is comparable to lignin-free cellulose (solka floc). New conversion pathways to useful fuels and chemicals are proposed based on the efficient conversion of lignin into intact hydrocarbons.

Research Area: Bioenergy Biofuels/Bioproducts Bioprocessing  

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Accelerating Sample Preparation Through Enzyme-Assisted Microfiltration of Salmonella in Chicken Extract

2015

Authors: H. B. Vibbert, S. Ku, X. Li, X. Liu, E. Ximenes, T. Kreke, M. R. Ladisch
Journal: Biotechnology Progress, 31(6), 1551-1562 (2015).
Book Chapter: 

Abstract: Microfiltration of chicken extracts has the potential to significantly decrease the time required to detect Salmonella, as long as the extract can be efficiently filtered and the pathogenic microorganisms kept in a viable state during this process. We present conditions that enable microfiltration by adding endopeptidase from Bacillus amyloliquefaciens to chicken extracts or chicken rinse, prior to microfiltration with fluid flow on both retentate and permeate sides of 0.2 um cutoff polysulfone and polyethersulfone hollow fiber membranes. After treatment with this protease, the distribution of micron, submicron, and nanometer particles in chicken extracts changes so that the size of the remaining particles corresponds to 0.4-1 um. Together with alteration of dissolved proteins, this change helps to explain how membrane fouling might be minimized because the potential foulants are significantly smaller or larger than the membrane pore size. At the same time, we found that the presence of protein protects Salmonella from protease action, thus maintaining cell viability. Concentration and recovery of 1-10 CFU Salmonella/mL from 400 mL chicken rinse is possible in less than 4 h, with the microfiltration step requiring less than 25 min at fluxes of 0.028-0.32 mL/cm2 min. The entire procedure - from sample processing to detection by polymerase chain reaction - is completed in 8 h.

Research Area: Food Safety    

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Adsorption of Enzyme Onto Lignins of Liquid Hot Water Pretreated Hardwoods

2015

Authors: J. K. Ko, E. Ximenes, Y. Kim, M. R. Ladisch
Journal: Biotechnology and Bioengineering, 112(3), 447-456, 2014
Book Chapter: 

Abstract: The adsorption of cellulase enzymes onto lignin is shown to be non-productive and therefore reduces enzymatic hydrolysis of liquid hot water pretreated cellulose. Among the enzyme components of Trichoderma reesei cellulase cocktail, beta-glucosidase showed the strongest adsorption onto lignin. Only 2-18% of the initial beta-glucosidase activity remained in the supernatant while 50-60% of cellobiohydrolase and endoglucanase activities werre recovered after incubation with lignin. By increasing the pH to 5.5 and adding NaCl to a 200 mM, the free enzymes in the supernatant were increased but hydrolysis was not enhanced since optimal pH for enzymatic hydrolysis is at 4.8. Electrostatic interactions contributed to enzyme adsorption and their effect was most pronounced for T. reesei beta-glucosidase which had high molecular weights (78-94 kDa) and high isoelectric points (pI 5.7-6.4). Since the enzyme components which are required to synergistically hydrolyze cellulose have different profiles (molecular weight, hydrophobicity and pI), they exhibit different adsorption behaviors with lignin, and thereby change the ratio of enzyme activities needed for synergism during cellulose hydrolysis. Beta-glucosidase from Aspergillus niger exhibits less adsorption than beta-glucosidase from T. reesei. Supplemental addition of A. niger beta-glucosidase to the enzyme mixture increases hydrolysis of pretreated hardwood by a factor of two. The analysis presented in this paper shows that lignins with higher guaiacyl content adsorb more cellulase enzymes, particularly beta-glucosidase, and that adsorption of beta-glucosidase onto lignin indirectly suppresses enzymatic hydrolysis of cellulose in pretreated hardwoods due to decreased hydrolysis of cellobiose which in turn accumulates and inhibits CBH. 

Research Area: Bioenergy Bioprocessing Bioseparations  

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Bioabatement with Hemicellulase Supplementation to Reduce Enzymatic Hydrolysis Inhibitors

2015

Authors: G. Cao, E. Ximenes, N. N. Nichols, S. E. Frazer, D. Kim, M. A. Cotta, M. Ladisch
Journal: Bioresource Technology, 190 412-415, 2015
Book Chapter: 

Abstract: A stepwise removal of inhibitory comounds by bioabatement combined with hemicellulase supplementation was conducted to enhance cellulose hydrolysis of liquid hot water-pretreated corn stover. Results showed that the fungus Coniochaeta ligniaria NRRL30616 eliminated most of the enzyme and fermentation inhibitors from liquid hot water-pretreated corn stover hydrolysates. Moreover, addition of hemicellulases after bioabatement and before enzymatic hydrolysis of cellulose achieved 20% higher glucose yields compared to non-treated samples. This work presents the mechanisms by which supplementation of the fungus with hemicellulase enzymes enables maximal conversion, and confirms the inhibitory effect of xylo-oligosaccharides in corn stover hydrolysates once the dominant inhibitory effect of phenolic compounds is removed.

Research Area: Bioenergy Bioprocessing Biofuels/Bioproducts  

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Direct Emission of Methane and Nitrous Oxide from Switchgrass and Corn Stover: Implications for Large-Scale Biomass Storage

2015

Authors: I. Emery and Nathan Mosier
Journal: Global Change Biology Bioenergy, 7(4), 865-876
Book Chapter: 

Abstract: Little is known about the contributions of biomass feedstock storage to the net greenhouse gas emissions from cellulosic biofuels. Direct emissions of methane and nitrous oxide during decomposition in storage may contribute substantially to the global warming potential of biofuels. In this study, laboratory-scale bales of switchgrass and corn stover were stored under a range of moisture (13.0-32.9%) and temperature (5-35 C) conditions and monitored for O2 consumption and CO2, CH4, and N2O production over 8 weeks. Gas concentrations and emissions rates were highly variable within and between experimental groups. Stover bales produced higher CO2 concentrations (P=0.0002) and lower O2 (P<0.0001) during storage than switchgrass bales. Methane concentrations (1.8-2100 ppm) were inversely correlated with bale moisture (P < 0.05), with emissions rates ranging from 4.4-914.9 ug kg-1 DM day-1. Nitrous oxide concentrations ranged from 0 to 31 ppm, and emissions from switchgrass bales inversely correlated with temperature and moisture (P < 0.0001). Net global warming potential from each treatment (0-2.4 gCO2e kg-1 DM) suggests that direct emission of methane and nitrous oxide from aerobically stored feedstocks have a small effect on net global warming potential of cellulosic biofuels.

Research Area: Bioenergy Bioprocessing Biofuels/Bioproducts  

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Effect of Liquid Hot Water Pretreatment Severity on Properties of Hardwood Lignin and Enzymatic Hydrolysis of Cellulose

2015

Authors: Ja Kyong Ko, Youngmi Kim, Eduardo Ximenes, Michael R. Ladisch
Journal: Biotechnology and Bioengineering, 112(2), 252-262, 2014
Book Chapter: 

Abstract: Lignin, one of the major components of lignocellulosic biomass, plays an inhibitory role on the enzymatic hydrolysis of cellulose. This work examines the role of lignin in pretreated hardwood, where extents of cellulose hydrolysis decrease, rather than increase with increasing severity of liquid hot water pretreatment. Hardwood pretreated with liquid hot water at severities ranging from log Ro = 8.25 to 12.51 resulted in 80-90% recovery of the initial lignin in the residual solids. The ratio of acid insoluble lignin (AIL) to acid soluble lignin (ASL) increased and the formation of spherical lignin droplets on the cell wall surface was observed as previously reported in the literature. When lignins were isolated from hardwoods pretreated at increasing severities and characterized based on glass transition temperature (Tg), the Tg of isolated lignins was found to increase from 171 to 180 C as the severity increased from log Ro¼10.44 to 12.51. The increase in Tg suggested that the condensation reactions of lignin molecules occurred during pretreatment and altered the lignin structure. The contribution of the changes in lignin properties to enzymatic hydrolysis were examined by carrying out Avicel hydrolysis in the presence of isolated lignins. Lignins derived from more severely pretreated hardwoods had higher Tg values and showed more pronounced inhibition of enzymatic hydrolysis.

Research Area: Bioenergy Bioprocessing   

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Effect of Phenolic Compounds from Pretreated Sugarcane Bagasse on Cellulolytic and Hemicellulolytic Activities

2015

Authors: M. Michelin, E. Ximenes, M. de Lourdes Teixeira de Moraes Polizeli, M. R. Ladisch
Journal: Bioresource Technology
Book Chapter: 

Abstract: This work shows both cellulases and hemicellulases are inhibited and deactivated by water-soluble and acetone extracted phenolics from sugarcane bagasse pretreated at 10% (w/v) for 30 min in liquid hot water at 180 or 20 C. The dissolved phenolics in vacuum filtrate increased from 1.4 to 2.4 g/L as temperature increased from 180 to 20 C. The suppression of cellulose and hemicellulose hydrolysis by phenolics is dominated by deactivation of the beta-glucosidase or beta-xylosidase components of cellulase and hemicellulase enzyme by acetone extraqct at 0.2 - 0.65 mg phenolics/mg enzyme protein and deactivation of cellulases and hemicellulases by the water soluble components in vacuum filtrate at 0.05 - 2 mg/mg. Inhibition was a function of the type of enzyme and the manner in which the phenolics were extracted from the bagasse.

Research Area: Bioenergy Biofuels/Bioproducts Bioprocessing  

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Hydrolysis-Determining Substrate Characteristics in Liquid Hot Water Pretreated Hardwood

2015

Authors: Y. Kim, T. Kreke, J. K. Ko, M. R. Ladisch
Journal: Biotechnology & Bioengineering, 112(4), 677-687, 2015
Book Chapter: 

Abstract: Fundamental characterization of pretreated hardwood and its interactions with cellulolytic enzymes has confirmed that a pathway exists for dramatically reducing the loading of cellulase required for hydrolysis of pretreated biomass. We demonstrate that addition of protein effecting a seven-fold decrease in the specific activity of cellulases enables a ten-fold reduction in enzyme loading while maintaining a high level of cellulose hydrolysis in pretreated hardwood. While use of protein and other additives that adsorb on lignin have been reported previously, the current work demonstrates the effect in a dramatic manner and brings the rationale for this change into clear focus. The key to this result is recognizing and mitigating the pretreatment conundrum where increasingly severe pretreatment conditions enhance accessibility of the enzymes not only to cellulose, but also to lignin. The lignin adsorbs enzyme protein causing loss of cellulase activity. More enzyme, added to compensate for this lost activity, results in a higher cellulase loading. The addition of a different protein, such as BSA, prevents cellulase adsorption on lignin and enables the enzyme itself to better target its glucan substrate. This effect dramatically reduces the amount of cellulase for a given level of conversion with enzyme loadings of 15 FPU and 1.3 FPU/g solids both achieving 80% conversion. The understanding of this phenomenon reinvigorates motivation for the search for other approaches that prevent cellulase adsorption on lignin in order to achieve high glucose yields at low enzyme loadings for pretreated lignocellulose.

Research Area: Bioenergy Biofuels/Bioproducts Bioprocessing  

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Impact of Temperature, Moisture, and Storage Duration on the Chemical Composition of Switchgrass, Corn Stover, and Sweet Sorghum Bagasse

2015

Authors: A. Athmanathan, I. R. Emery, T. Kuczek, N. S. Mosier
Journal: BioEnergy Research, 8(2), 843-856
Book Chapter: 

Abstract: Packaged samples of three bioenergy feedstocks - sweet sorghum, corn stover, and switchgrass - were stored indoors under aerobic conditions to determine the change in chemical composition, track loss of specific chemical constituents, and determine the impact of dry matter loss on saccharification yields with and without pretreatment. Biomass samples were stored under controlled temperature conditions at varying stable biomass moisture contents (10-34 % w/w), temperatures (8-35 C), and durations up to 16 weeks. Total dry matter losses were measured and sample compositions determined to develop a material balance of storage losses for free sugars, glucan, xylan, and lignin. Maximal losses (24-30 %) were observed for sweet sorghum bagasse at high moisture, while minimal losses (0%) were observed with switchgrass below the highest tested moisture. Sorghum losses predominantly consisted of free sugars, while switchgrass and stover losses consisted of structural carbohydrates - cellulose and hemicellulose. The mass fraction (% dry weight) of lignin was observed to increase in samples showing dry matter loss, as a result of carbohydrate consumption.

Research Area: Bioenergy Bioprocessing Biofuels/Bioproducts  

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In situ Micro-spectroscopic Investigation of Lignin in Poplar Cell Walls Pretreated by Maleic Acid

2015

Authors: Y. Zeng, S. Zhao, H. Wei, M. P. Tucker, M. E. Himmel, N. S. Mosier, R. Meilan, S-Y. Ding
Journal: Biotechnology for Biofuels, 8:126
Book Chapter: 

Abstract: In higher plant cells, lignin provides necessary physical support for plant growth and resistance to attack by microorganisms. For the same reason, lignin is considered to be a major impediment to the process of deconstructing biomass to simple sugars by hydrolytic enzymes. The in situ variation of lignin in plant cell walls is important for better understanding of the roles lignin plays in biomass recalcitrance.

Research Area: Bioenergy Bioprocessing   

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Kinetics of Maleic Acid and Aluminum Chloride Catalyzed Dehydration and Degradation of Glucose

2015

Authors: X. Zhang, B. B. Hewetson, N. S. Mosier
Journal: Energy & Fuels, 29, 2387-2393, 2015
Book Chapter: 

Abstract: We report the positive effect of maleic acid, a dicarboxylic acid, on the selectivity of hexose dehydration to 5-hydroxymethyfurfural (HMF), and subsequent hydrolysis to levulinic and formic acids. We also describe the kinetic analysis of a Lewis acid (AlCl₃) alone and in combination with HCl or maleic acid to catalyze the isomerization of glucose to fructose, dehydration of fructose to HMF, hydration of HMF to levulinic and formic acids, and degradation of these compounds to humins. Results show that AlCl3 significantly enhances the rate of glucose conversion to HMF and levulinic acid in the presence of both maleic acid and HCl. In addition, the degradation of HMF to humins, rather than levulinic and formic acids, is reduced by 50% in the presence of maleic acid and AlCl₃ compared to hydrochloric acid combined with AlCl₃. The results suggest a different reaction mechanism for the dehydration of glucose and rehydration of HMF between maleic acid and HCl.

Research Area: Bioenergy Biofuels/Bioproducts   

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Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl Alcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall Structure

2015

Authors: N. A. Anderson, Y. Tobimatsu, P. N. Ciesielski, E. Ximenes, J. Ralph, B. S. Donohoe, M. Ladisch, C. Chapple
Journal: The Plant Cell
Book Chapter: 

Abstract: Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabisopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content.

Research Area: Bioenergy    

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Reduction of Volatile Fatty Acids and Odor Offensiveness by Anaerobic Digestion and Solid Separation of Dairy Manure During Manure Storage

2015

Authors: L. H. Page,J. Q. Ni, H. Zhang, A. J. Heber, N. S. Mosier, X. Liu, H. S. Joo, P. M. Ndegwa, J. H. Harrison
Journal: Journal of Environmental Management, 152, 91-98, 2015
Book Chapter: 

Abstract: Volatile fatty acids (VFA) play an important role in the biodegradation of organic wastes and production of bioenergy under anaerobic digestion, and are related to malodors. However, little is known about the dynamics of VFA during dairy manure storage. This study evaluated the characteristics of VFA in dairy manure before and after anaerobic co-digestion in a laboratory experiment using eight lab-scale reactors. The reactors were loaded with four different types of dairy manure: (1) liquid dairy manure from a freestall, barn, (2) mixture of dairy manure and co-digestion food processing wastes at the inlet of an anaerobic digester, (3) effluent from the digester outlet, and (4) the liquid fraction of effluent from a solid separator. Four VFA (acetic, propionic, butyric, and 2-methylbutyric acids) were identified and quantified in weekly manure samples from all reactors. Results showed that the dominant VFA was acetic acid in all four manure sources. The off-farm co-digestion wastes significantly increased the total VFA concentrations and the proportions of individual VFA in the influent. The dairy manure under storage demonstrated high temporal and spatial variations in pH and VFA concentrations. Anaerobic digestion reduced the total VFA by 86-96%; but solid-liquid separation did not demonstrate a significant reduction in total VFA in this study. Using VFA as an indicator, anaerobic digestion exhibited an effective reduction of dairy manure odor offensiveness.

Research Area: Bioenergy    

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Tandem Mass Spectrometric Characterization of the Conversion of Xylose to Furfural

2015

Authors: N. R. Vinueza, E. S. Kim, V. A. Gallardo, N. S. Mosier, M. M. Abu-Omar, N. C. Carpita, H. I. Kenttamaa
Journal: Biomass and Bioenergy, 74, 1-5 (2015)
Book Chapter: 

Abstract: Thermal decomposition of xylose into furfural under acidic conditions has been studied using tandem mass spectrometry. Two different Bronsted acids, maleic and sulfuric acids, were used to demonstrate that varying the Bronsted acid does not affect the mechanism of the reaction. Two selectively labeled xylose molecules, 1-¹³C and 5-¹³C xyloses, were examined to determine which carbon atom is converted to the aldehyde carbon in furfural. This can be done by using tandem mass spectrometry since collision-activated dissociation (CAD) of protonated unlabeled furfural results in the loss of CO from the aldehyde moiety. The loss of a neutral molecule with MW of 29 Da (¹³CO) was observed for protonated furfural derived from 1-¹³C-labeled xylose while the loss of a neutral molecule with MW of 28 Da (CO) was observed for protonated furfural derived from 5-¹³C labeled xylose. These results support the hypothesis that the mechanism of formation of furfural under mildly hot acidic conditions involves an intramolecular rearrangement of protonated xylose into the pyranose form rather than into an open-chain form.

Research Area: Bioenergy Biofuels/Bioproducts   

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Biological Engineering and the Emerging Cellulose Ethanol Industry

2014

Authors: M. R. Ladisch, E. Ximenes, A. S. Engelberth, N. S. Mosier
Journal: Chemical Engineering Progress, Supplement November, 2014
Book Chapter: 

Abstract: Biochemical and thermochemical processes effectively convert lignocellulosic biomass, such as wood, sugarcane bagasse, corn stover, and dedicated energy crops (particularly grasses), to liquid transportation fuels and chemicals, while reducing the generation of greenhouse gases by 60% or more. An SBE Supplement in the March 2010 issue of CEP looked at cellulosic biofuels. One of the articles described the technologies being developed to convert lignocellulosic feedstocks to liquid fuels, and discussed some of the factors that must be considered in devising economical processes. This article provides an update on the significant progress and developments in cellulosic biofuels over the past four years.

Research Area: Biofuels/Bioproducts Bioenergy   

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Characteristics of Volatile Fatty Acids in Stored Dairy Manure Before and After Anaerobic Digestion

2014

Authors: L. H. Page, J.-Q. Ni, A. J. Heber, N. S. Mosier, X. Liu, H.-S. Joo, P. M. Ndegwa, J. H. Harrison
Journal: Biosystems Engineering, 118, 16-28
Book Chapter: 

Abstract: Volatile fatty acids (VFA) are among the most abundant volatile organic compounds in dairy manure and are associated with odour nuisance. This paper presents research results of VFA production during a three-month storage of dairy manure from four different sources: a dairy barn (raw), the inlet of an anaerobic digester (influent), the digester outlet (effluent), and the effluent after solid separation (effluent SS). Manure from each source was studied in two lab-scale reactors that were continuously ventilated with fresh air in the manure headspace to simulate manure storage conditions. Two manure samples were taken weekly in the top and bottom manure layers from each reactor for VFA analysis. Five VFA (formic acid, acetic acid, propionic acid, butyric acid, and 2-methylbutyric acid) were identified in all reactors using high performance liquid chromatography (HPLC). The dominant VFA was formic acid for the influent and acetic acid for the other three manure sources. The overall average concentrations of the five VFA were 1963 plus or minus 685 (mean plus or minus standard deviation), 14,175 plus or minus 4825, 286 plus or minus 98, and 169 plus or minus 80 mg1-1 for the raw, influent, effluent, and effluent SS< respectively. The "pre-consumer" organic wastes mixed with dairy manure in the influent significantly increased the total VFA concentrations and the proportion of individual VFA. Concentrations of VFA demonstrated highly temporal and spatial variations. Anaerobic digestion significantly reduced formation of VFA in the effluent and effluent SS. However, the complexity of VFA characteristics made it difficult to reliably model and predict the concentrations and compositions of VFA in dairy manure.

Research Area: Bioprocessing    

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Disruption of Mediator Rescues the Stunted Growth of a Lignin-deficient Arabidopsis Mutant

2014

Authors: N. C. Bonawitz, J. I. Kim, Y. Tobimatsu, P. N. Cieslelski, N. A. Anderson, E. Ximenes, J. Maeda, J. Ralph, B. S. Donohoe, M. Ladisch, C. Chapple
Journal: Nature, 509, 376-380, 2014
Book Chapter: 

Abstract: Lignin is a phenylpropanoid-derived heteropolymer important for the strength and rigidity of the plant secondary cell wall. Genetic disruption of lignin biosynthesis has been proposed as a means to improve forage and bioenergy crops, but frequently results in stunted growth and developmental abnormalities, the mechanisms of which are poorly understood. Here we show that the phenotype of a lignin-deficient Arabidopsis mutant is dependent on the transcriptional co-regulatory complex, Mediator. Disruption of the Mediator complex subunits MED5a (also known as REF4) and MED5b (also known as RFP1) rescues the stunted growth, lignin deficiency and widespread changes in gene expression seen in the phenylpropanoid pathway, mutant ref8, without restoring the synthesis of guaiacyl and syringyl lignin subunits. Cell walls of rescued med5a/5b ref8 plants instead contain a novel lignin consisting almost exclusively of p-hydroxyphenyl lignin subunits, and moreover exhibit substantially facilitated polysaccharide saccharification. These results demonstrate that quaiacyl and syringyl lignin subunits are largely dispensable for normal growth and development, implicate Mediator in an active transcriptional process responsible for dwarfing and inhibition of lignin biosynthesis, and suggest that the transcription machinery and signalling pathways responding to cell wall defects may be important targets to include in efforts to reduce biomass recalcitrance.

Research Area: Biofuels/Bioproducts    

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Engineering Plant Cell Walls: Tuning Lignin Monomer Composition for Deconstructable Biofuel Feedstocks or Resilient Biomaterials

2014

Authors: P. N. Ciesielski, M. G. Resch, B. Hewetson, J. P. Killgore, A. Curtin, N. Anderson, A. N. Chiaramonti, D. C. Hurley, A. Sanders, M. E. Himmel, C. Chapple, N. Mosier, B. S.Donohoe
Journal: Green Chemistry
Book Chapter: 

Abstract: Advances in genetic manipulation of the biopolymers that compose plant cell walls will facilitate more efficient production of biofuels and chemicals from biomass and lead to specialized biomaterials with tailored properties. Here we investigate several genetic variants of Arabidopsis: the wild type, which makes a lignin polymer of primarily guaiacyl (G) and syringyl (S) monomeric units, the fah1 mutant, which makes lignin from almost exclusively G subunits, and a ferulate 5-hydroxylase (F5H) overexpressing line (C4H:F5H) that makes lignin from S subunits. We employ multiscale, multimodal imaging techniques that reveal the biomass of the C4H:F5H transgenic to be more susceptible to deconstruction by maleic acid treatment than the other variants. Enzymatic saccharification assays of the treated materials show that C4H:F5H transgenic tissue is significantly more digestible than the wild type, while the fah1 mutant is clearly the least digestible of these materials. Finally, we show by contact resonance force microscopy, an atomic force microscopy technique, that F5H overexpression in C4H:F5H transgenic plants significantly reduces the stiffness of the cell walls in the region of the compound middle lamella relative to wild type and fah1. 

Research Area: Bioenergy Bioprocessing   

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Evaluation of a Kinetic Model for Computer Simulation of Growth and Fermentation by Scheffersomyces (Pichia) stipitis Fed D-Xylose

2014

Authors: Slininger, P.J., B. S. Dien, J. M. Lomont, R. J. Bothast, M. R. Ladisch, M. R. Okos
Journal: Biotechnology and Bioengineering, 111(8), 1532-1540, 2014
Book Chapter: 

Abstract: Scheffersomyces (formerly Pichia) stipitis is a potential biocatalyst for converting lignocelluloses to ethanol because the yeast natively ferments xylose. An unstructured kinetic model based upon a system of linear differential equations has been formulated that describes growth and ethanol production as functions of ethanol, oxygen, and xylose concentrations for both growth and fermentation stages. The model was validated for various growth conditions including batch, cell recycle, batch with in situ ethanol removal and fed-batch. The model provides a summary of basic physiological yeast properties and is an important tool for simulating and optimizing various culture conditions and evaluating various bioreactor designs for ethanol production. 

Research Area: Bioenergy    

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Genetic Determinants for Enzymatic Digestion of LIgnocellulosic Biomass Are Indendent of Those for Lignin Abundance in a Maize Recombinant Inbred Population

2014

Authors: B. W. Penning, R. W. Sykes, N. C. Babcock, C. K. Dugard, M. A. Held, J. F. Klimek, J. T. Shreve, M. Fowler, A. Ziebell, M. F. Davis, S. R. Decker, G. B. Turner, N. S. Mosier, N. M. Springer, J. Thimmapuram, C. F. Weil, M. C. McCann, N. C. Carpita
Journal: Plant Physiology, 165(4), 1475-1487
Book Chapter: 

Abstract: Biotechnological approaches to reduce or modify lignin in biomass crops are predicated on the assumption that it is the principal determinant of the recalcitrance of biomass to enzymatic digestion for biofuels production. We defined quantitative trait loci (QTL) in the Intermated B73 x Mol7 recombinant inbred maize (Zea mays) population using pyrolysis molecular-beam mass spectrometry to establish stem lignin content and an enzymatic hydrolysis assay to measure glucose and xylose yield. Among five multiyear QTL for lignin abundance, two for 4-vinylphenol abundance, and four for glucose and/or xylose yield, not a single QTL for aromatic abundance and sugar yield was shared. A genome-wide assocation study for lignin abundance and sugar yield of the 282-member maize association panel provided candidate genes in the 11 QTL of the B73 and Mol7 patents but showed that many other alleles impacting these traits exist among this broader pool of maize genetic diversity. B73 and Mo17 genotypes exhibited large differences in gene expression in developing stem tissues independent of allelic variation. Combining these complementary genetic approaches provides a narrowed list of candidate genes. A cluster of SCARECROW-LIKE9 and SCARECROW-LIKE14 transcription factor genes provides exceptionally strong candidate genes emerging from the genome-wide association study. In addition to these and genes associated with cell wall metabolism, candidates inclde several other transcription factors associated with vascularization and fiber formation and components of cellular signaling pathways. These results provide new insights and strategies beyond the modification of lignin to enhance yields of biofuels from genetically modified biomass.

Research Area: Bioenergy Bioprocessing   

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Impact of Temperature, Moisture, and Storage Duration on the Chemical Composition of Switchgrass, Corn Stover, and Sweet Sorghum Bagasse

2014

Authors: A. Athmanathan, I. R. Emery, AT. Kuczek, N. S. Mosier
Journal: Bioenergy Research, 8(2), 843-856, 2014
Book Chapter: 

Abstract: Packaged samples of three bioenergy feedstocks - sweet sorghum, corn stover, and switchgrass - were stored indoors under aerobic conditions to determine the change in chemical composition, track loss of specific chemical constituents, and determine the impact of dry matter loss on saccharification yields with and without pretreatment. Biomass samples were stored under controlled temperature conditions at varying stable biomass moisture contents (10-34% w/w), temperatures (8-35 C), and durations up to 16 weeks. Total dry matter losses were measured and sample compositions determined to develop a material balance of storage losses for free sugars, glucan, xylan, and lignin. Maximal losses (24-30%) were observed for sweet sorghum bagasse at high moisture, while minimal losses (0%) were observed with switchgrass below the highest tested moisture. Sorghum losses predominantly consisted of free sugars, while switchgrass and stover losses consisted of structural carbohydrates - cellulose and hemicellulose. The mass fraction (% dry weight) of lignin was observed to increase in samples showing dry matter loss, as a result of carbohydrate consumption.

Research Area: Bioenergy Bioprocessing Biofuels/Bioproducts  

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Investigation of Fatty Acid Methyl Esters in Jet Fuel

2014

Authors: G. Kilaz, S. Bist, D. W. Lopp, D. L. Stanley, B. Y. Tao
Journal: International Journal of Sustainable Aviation, 1(1), 103-118
Book Chapter: 

Abstract: Sustainable aviation fuels research has considerable momentum in efforts led by government, academia and industry. Environmentally sound domestic fuels allow significant benefits, while also creating some challenges due to their novelty. One of these challenges is the cross contamination of fatty acid methyl esters (FAME) in biodiesel with jet fuels. It was suspected that sharing the same supply chain caused FAME to contaminate jet fuels which led to aircraft malfunction. Consequently, in 2010, aero engine original equipment manufacturers (OEMs) mandated an immediate allowable limit of 5 ppm FAME in jet fuels. Civil Aviation Authority later increased the limit to 30 ppm (2012). This study finds that the presence of FAME in Jet-A at a much higher concentration of 2 vol% does not have an adverse impact on the ASTM D1655 specifications (2013). Therefore, it is recommended that the current limit of 30 ppm be revised.

Research Area: Biofuels/Bioproducts    

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Liquefaction of Sugarcane Bagasse for Enzyme Production

2014

Authors: F. M. Cunha, T. Kreke, A. C. Badino, C. S. Farinas, E. Ximenes, M. R. Ladisch
Journal: Bioresource Technology, 172, 249-252, 2014
Book Chapter: 

Abstract: The objective of this paper is to report liquefaction of pretreated and sterilized sugarcane bagasse for enhancing endoglucanase production through submerged fermentation by Aspergillus niger. After initial solid state fermentation of steam pretreated bagasse solids by A. Niger, fed-batch addition of the substrate to cellulase in buffer over a 12 h period, followed by 36 h reaction, resulted in a liquid slurry with a viscosity of 0.30 ± 0.07 Pa s at 30% (w/v) solids. Addition of A. niger for submerged fermentation of sterile liquefied bagasse at 23% w/v solids resulted in an enzyme teter of 2.5 IU mL−1 or about 15 x higher productivity than solid-state fermentation of non-liquefied bagasse (final activity of 0.17 IU mL−1). Bagasse not treated by initial solid-state fermentation but liquefied with enzyme gave 2 IU mL-1). These results show the utility of liquefied bagasse as a culture medium for enzyme production in submerged fermentations.

Research Area: Bioprocessing Bioenergy Biofuels/Bioproducts  

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Liquid Hot Water and Steam Explosion Pretreatment of Sugarcane Bagassse for Enzyme Production by a Sequential Solid-State and Submerged Method

2014

Authors: F. M. Cunha, A. Badino, C. S. Farinas, E. Ximenes, M. R. Ladisch
Journal: Annals of XX Brazilian Congress of Chemical Engineering, Congress Brasileiro de Engenharia Quimica, COBEQ, Biotechnological Processes, Florianopolis, SC, Brazil, Pages 1-8
Book Chapter: 

Abstract: The use of sugarcane bagasse on enzyme production is a promising alternative for reducing the costs of second generation ethanol. However, a pretreatment step is required to increase cellulose and hemicellulose accessibility. Here, the influence of Liquid Hot Water (LHW) and steam explosion (SE) pretreatments in cultivations with three Aspergillus strains were investigated. A new sequential method was carried out with a first step in solid-state for 24h, followed by the transition to submerged cultivation and enzyme production in the presence of 1% (w/v) of sugarcane for 72h. For both A. niger strains, the endoglucanase production was 20 to 50% higher in cultivations with steam exploded sugarcane bagasee. The xylanase and beta-glucosidase production, however, were higher in LHW pretreated sugarcane bagasse, with xylanase production around 23% higher and beta-glucosidase up to 4-fold higher. The A. niger A12 strain produced the higher titers of all enzymes evaluated, resulting in 1.26; 26.25; 3.70 and 0.58 IU.mL-1 of endoglucanase, xylanase, beta-glucosidase, and beta-xylosidase, respectively, in LHW bagasse. Pretreated bagasse is not suitable for enzyme production by A. oryzae P27C3, indicating that this strain may be more sensitive to possible inhibitory products released from both pretreatments

Research Area: Bioenergy Bioprocessing   

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Modeling Water Quality Impacts of Cellulosic Biofuel Production from Corn Silage

2014

Authors: M. A. Thomas, L. M. Ahiablame, B. A. Engel, I. Chaubey, N. Mosier
Journal: Bioenergy Research, 7, 636-653
Book Chapter: 

Abstract: The growing interest in the use of alternative biomass products for fuel production requires a thorough understanding of the environmental impacts associated with the production of these bioenergy crops. Corn silage is a potential bioenergy feedstock; however, water quality implications for its utilization as a biofeedstock are not understood. The objective of this work was to evaluate water quality impacts associated with corn silage production. The GLEAMS-NAPRA model was used to quantify runoff, percolation, erosion, nitrate-nitrogen, total phosphorus, and pesticide losses attributed to the production of corn silage with and without winter cover crops for two tillage options (conventional tillage and no till) on three Indiana soils. Results revealed that corn silage would generate greater annual surface runoff (1 to 6 mm) and percolation (1 to 20 mm) compared with corn grain and grain plus stover cropping systems. Silage/winter cereal rye cover crop reduced annual surface runoff and percolation and was strongly influenced by ncreases in evapotranspiration, when compared with continuous silage production. Silage managed with winter cereal rye cover crop influenced water quality by reducing annual nitrate losses with runoff from a low of 14% to a high of 27%, with relatively no effect because of tillage management. No-till practice on silage system produced significantly greater phosphorus losses (7.46 to 18.07 kg/ha) in comparison to silage/cereal rye, corn grain, and grain plus stover harvest (p < 0.05). For every 1,000 l of ethanol produced from corn silage, erosion losses ranged from 0.07 to 0.95 t/ha for conventional tillage practices and from 0.6 to 0.83 t/ha for no-till practices. The feasibility of cropping systems such as corn silage/cereal rye could contribute to large-scale biomass production but should be further investigated.

Research Area: Bioenergy Bioprocessing   

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Nano/Micro and Spectroscopic Approaches to Food Pathogen Detection

2014

Authors: I.-H. Cho, A. D. Radadia, K. Farrokhzad, E. Ximenes, E. Bae, A. K. Singh, H. Oliver, M. Ladisch, A. Bhunia, B. Applegate, L. Mauer, R. Bashir, J. Irudayaraj
Journal: Annual Review of Analytical Chemistry, 7, 65-88, 2014
Book Chapter: 

Abstract: Despite continuing research efforts, timely and simple pathogen detection with a high degree of sensitivity and specificity remains an elusive goal. Given the recent explosion of sensor technologies, significant strides have been made in addressing the various nuances of this important global challenge that affects not only the food industry but also human health. In this review, we provide a summary of the various ongoing efforts in pathogen detection and sample preparation in areas related to Fourier transform infrared and Raman spectroscopy, light scattering, phage display, micro/nanodevices, and nanoparticle biosensor. We also discuss the advantages and potential limitations of the detection methods and suggest next steps for further consideration.

Research Area: Food Safety    

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Understanding ASTM Turbine Fuel Standard Specifications

2014

Authors: R. F. Brender, G. Kilaz
Journal: International Review of Aerospace Engineering, 7(4), 2014
Book Chapter: 

Abstract: An overview is presented of the three primary ASTM International standards regarding aviation turbine fuels; D1655 Standard Specification for Aviation Turbine Fuels, D4054 Standard Practice for Qualification and Approval of New Aviation Turbine Fuels and Fuel Additives, and D7566 Standard Specification for Aviation Turbine Fuel Containing Synthesized Hydrocarbons. Emphasis is on how the three standards relate to and interact with each other. The discussion draws out some less obvious implications of these documents and addresses the question whether the focus on source and manufacturing process rather than composition is still appropriate.

Research Area: Biofuels/Bioproducts    

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Validation of PyMBMS as a HIgh-throughput Screen for Lignin Abundance in Lignocellulosic Biomass of Grasses

2014

Authors: B. W. Penning, R. W. Sykes, N. C. Babcock, C. K. Dugard, J. E. Klimek, D. Gamblin, M. Davis, T. R. Filley, N. S. Mosier, C. F. Weil, M. C. M cCann, N. C. Carpita
Journal: Bioenergy Research, 7, 899-908
Book Chapter: 

Abstract: Pyrolysis molecular-beam mass spectrometry (PyMBMS) was tested as a high-throughput method for relative abundance of guaiacyl and syringyl lignin in lignocellulosic cell-wall materials from stems of a popularion of maize intermated B73 x Mo17 (IBM) recombinant inbred lines. Variations of up to twofold across the population in phenylpropanoid abundance were observed. Several hitochemical and quantitative biochemical assays were used to validate the mass spectrometric data for lignin, hydroxycinnamic acids, crystalline cellulose, non-cellulosic glucans, and xylans. We demonstrate PyMBMS to be a valid high-throughput screen suitable for analysis of lignin abundance in large populations of bioenergy grasses. Pentose from xylans and hexose from cellulosic and non-cellulosic glucans also varied substantially across the population, but abundance of diagnostic fragments for these monosaccharides were not well correlated with the abundance of cell-wall polysaccharides.

Research Area: Bioenergy    

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Biological Abatement of Cellulase Inhibitors

2013

Authors: Guangli Cao, Eduardo Ximenes, Nancy N. Nichols, Leyu Zhang, and Michael Ladisch
Journal: Bioresource Technology, 146, 604-610, 2013
Book Chapter: 

Abstract: Removal of enzyme inhibitors released during lignocellulose pretreatment is essential for economically feasible biofuel production. We tested bio-abatement to mitigate enzyme inhibitor effects observed in corn stover liquors after pretreatment with either dilute acid or liquid hot water at 10% (w/v) solids. Bio-abatement of liquors was followed by enzymatic hydrolysis of cellulose. To distinguish between inhibitor effects on enzymes and recalcitrance of the substrate, pretreated corn stover solids were removed and replaced with 1% (w/v) Solka Floc. Cellulose conversion in the presence of bio-abated liquors from dilute acid pretreatment was 8.6% (0.1x enzyme) and 16% (1x enzyme) higher than control (non-abated) samples. In the presence of bio-abated liquor from liquid hot water pretreated corn stover, 10% (0.1x enzyme) and 13% (1x enzyme) higher cellulose conversion was obtained compared to control. Bio-abatement yielded improved enzyme hydrolysis in the same range as that obtained using a chemical (overliming) method for mitigating inhibitors. 

Research Area:     

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Biological Conversion of Plants to Fuels and Chemicals and the Effects of Inhibitors

2013

Authors: C. E. Wyman, E. Ximenes, Y. Kim, M. R. Ladisch
Journal: 
Book Chapter: Aqueous Pretreatment of Plant Biomass for Biological and Chemical Conversion to Fuels and Chemicals, Wiley, Ed. C. V. Stevens

Abstract: Pretreatments have the potential to both enhance the rates and extents of cellulose conversion by biological catalysts including cell-free enzymes, enzymes produced during consolidated bioprocessing, and simultaneous saccharification and fermentation. The efficiency of the enzymes that hydrolyze either hemicellulose or cellulose to monosaccharides (principally glucose and xylose) is affected by inhibitors released during pretreatment and hydrolysis. The inhibitory class of inhibitors and deactivators has been rediscovered and their effects studied with respect to enzymatic cellulose hydrolysis. Phenolics (e.g, vanillin, p-coumaric, ferulic, gallic and tannic acids) can reduce enzyme activity by over 50% and de-activate beta-glucosidase, principally through precipitation. Phenolic inhibitors may be more potent than the hydrolysis products derived from cellulose itself. In addition, xylo-oligosaccharides also inhibit cellulase. Consequently, removing xylo-oligosaccharides either through enzymatic hydrolysis or washing after pretreatment has been considered numerous times. However, once xylo-oligosaccharides are washed away from the solid material, they still must be hydrolyzed to monosaccharides that can be fermented to ethanol, and thereby increase yield. To achieve this, one method is to use a solid-acid catalytic bed (i.e., ion exchange resin) over which the oligosaccharide solution is passed. At temperaturs 150 C, hydrolysis is rapid and the formation of degradation products is minimized. This chapter provides an overview of biological processing of cellulosic biomass followed by a discussion of the important inhibitory impacts of lignin-derived phenolics and xylo-oligosacchraides on cellulolytic enzymes. In addition, the effect of major inhibitors on ethanol fermentation (furans and acetic acid) will also be discussed. Possible strategies are discussed for removing phenolics and xylo-oligosaccharides.

Research Area: Bioprocessing Bioenergy Bioseparations  

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Biomass Chemistry

2013

Authors: M. Ladisch, E. Ximenes, Y. Kim, N. S. Mosier
Journal: 
Book Chapter: Catalysis for the Conversion of Biomass and Its Derivatives, Wiley, Ed. Charles Wyman

Abstract: The pretreatment of biomass materials for subsequent biological processing requires an understanding of the chemistry of biomass which makes up the feedstock for such processes. The combination of pretreatment and enzyme hydrolysis is a key step in deriving fermentable sugars for the subsequent transformation to ethanol or other fermentation products by either yeast or bacteria. Pretreatment can also impact the chemical processing of biomass materials to synthesis gas containing CO, methane, and other organic molecules. The chemical structurre of biomass (lignocellulosic) materials determines the most appropriate combinations of pretreatment and hydrolysis. The types and sources of biomass, their structure and the overall impact of chemistry on pretreatment approaches are presented in this chapter. Recent developments in pretreatment, using water only approaches, as well as the effects of inhibitors on cellulases are also discussed.

Research Area: Bioprocessing Bioenergy   

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Comparative Performance of Leading Pretreatment Technologies for Biological Conversion of Corn Stover, Poplar Wood, and Switchgrass to Sugars

2013

Authors: C. E. Wyman, B. E. Dale, V. Balan, R. T. Elander, M. T. Holtzapple, R. S. Ramirez, M. R. Ladisch, N. Mosier, Y. Y. Lee, R. Gupta, S. R. Thomas, B. R. Hames, R. Warner, R. Kumar
Journal: 
Book Chapter: Aqueous Prretreatment of Plant Biomass for Biological and Chemical Conversion to Fuels and Chemicals, Wiley, Ed., Charles E. Wyman

Abstract: The Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI), formed in early 2000, completed its last study in 2010 to determine comparative sugar yields from application of leading pretreatments to shared sources of cellulosic feedstocks followed by enzymatic hydrolysis of the resulting solids with a common source of enzymes. This chapter highlights key findings jover the 10-year life of the CAFI team on the enzymatic hydrolysis of corn stover, poplar wood, and switchgrass that had been subjected to the following leading pretreatments: ammonia fiber expansion (AFEX), ammonia recycle percolation (ARP), dilute sulfuric acid, liquid hot water (LHW), lime, soaking in aqueous ammonia (SAA), and sulfur dioxide steam explosion. First, compositions are reported for each of the three baseline CAFI feedstocks. For all three feedstocks, the highest yields of xylose, glucose, and soluble oligomers are then reported for pretreatment coupled with subsequent enzymatic hydrolysis with baseline loadings of cellulase and beta-glucosidase. In all cases, material balances were performed. Differrences in yields are then reported for application of the same pretreatments to a second source of poplar and two other varieties of switchgrass. Following pretreatment of each feedstock, the compositions of the solids are compared to demonstrate that high yields can be realized even though the different pretreatments left different proportions of xylan and lignin in the pretreated solids. Temperatures,times, and catalyst types and loadings that resulted in the highest xylose and glucose yields in solution are summarized for each feedstock and pretreatment. The results show that a wide range of pretreatment conditions can realize high yields of sugars from cellulosic biomass, and that different types of biomass and even different varieties of the same biomass perform differently and can require modification of pretreatment conditions to increase yields.

Research Area: Bioseparations Bioprocessing Bioenergy  

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Effect of Salts on the Co-fermentation of Glucose and Xylose by a Genetically Engineered Strain of Saccharomyces cerevisiae

2013

Authors: E. Casey, N. S. Mosier, J. Adamec, Z. Stockdale, N. Ho, M. Sedlak
Journal: Biotechnology for Biofuels, 6, 83
Book Chapter: 

Abstract: A challenge currently facing the cellulosic biofuel industry is the efficient fermentation of both C5 and C6 sugars in the presence of inhibitors. To overcome this challenge, microorganisms that are capable of mixed-sugar fermentation need to be further developed for increased inhibitor tolerance. However, this requires an understanding of the physiological impact of inhibitors on the microorganism. This paper investigates the effect of salts on Saccharomyces cerevisiae 424A(LNH-ST), a yeast strain capable of effectively co-fermenting glucose and xylose.

Research Area: Bioenergy Bioprocessing Bioseparations  

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Fractionation of Cellulase and Fermentation Inhibitors from Steam Pretreated Mixed Hardwood

2013

Authors: Youngmi Kim, Thomas Kreke, Rick Hendrickson, Josh Patenti, and Michael R. Ladisch
Journal: Bioresource Technology, 135, 30-38 2013
Book Chapter: 

Abstract: The purpose of liquid hot water and steam pretreatment of wood is to fractionate hemicelluloses, partially solubilize lignin, and enhance enzyme hydrolysis of cellulose. The pretreatment also solubilizes sugar oligomers, lignin-derived phenolic compounds, acetic acid, and furan derivatives that inhibit cellulase enzymes and/or impede fermentation of hydrolysates by yeasts. This work extends knowledge of the relative contribution of identified inhibitors, and the effect of temperature on their release when pretreated materials are washed and filtered with hot water. Dramatic yield improvements occur when polymeric or activated carbon adsorbs and removes inhibitors. By desorbing, recovering, and characterizing adsorbed molecules we found phenolic compounds were strong inhibitors of enzyme hydrolysis and fermentation of concentrated filtrates by Saccharomyces cerevisiae wine yeast NRRL Y-1536 or xylose fermenting yeast 424A (LNH-ST). These data show that separation of inhibitors from pretreatment liquid will be important in achieving maximal enzyme activity and efficient fermentations. 

Research Area: Bioenergy    

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Rapid Sample Processing for Detection of Food-Borne Pathogens via Cross-Flow Microfiltration

2013

Authors: Xuan Li, Eduardo Ximenes, Mary Anne Roshni Amalaradjou, Hunter B. Vibbert, Kirk Foster, Jim Jones, Xingya Liu, Arun K. Bhunia, and Michael R. Ladisch
Journal: Applied and Environmental Microbiology, 79(22), 7048-7054, 2013
Book Chapter: 

Abstract: This paper reports an approach to enable rapid concentration and recovery of bacterial cells from aqueous chicken homogenates as a preanalytical step of detection. This approach includes biochemical pretreatment and prefiltration of food samples, and development of an automated cell concentration instrument based on cross-flow microfiltration. A polysulfone hollow-fiber membrane module having a nomimal pore size of 0.2 um constitutes the core of the cell concentration instrument. The aqueous chicken homogenate samples were circulated within the cross-flow system achieving 500- to 1,000-fold concentration of innoculated Salmonella enterica serovar Enteritidis and naturally occurring microbiota with 70% recovery of viable cells as determined by plate counting and quantitative PCR (qPCR) within 35 to 45 min. These steps enabled 10 CFU/ml microorganisms in chicken homogenates or 102 CFU/g chicken to be quantified. Cleaning and sterilizing the instrument and membrane module by stepwise hydraulic and chemical cleaning (sodium hydroxide and ethanol) enabled reuse of the membrane 15 times before replacement. This approach begins to address the critical need for the food industry for detecting food pathogens within 6 h or less.

Research Area: Food Safety    

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Reaction Mechanisms and Kinetics of Xylo-Oligosaccharide Hydrolysis by Dicarboxylic Acids

2013

Authors: Youngmi Kim, Thomas Kreke and Michael R. Ladisch
Journal: AIChE Journal, 59(1), 188-199, 2013
Book Chapter: 

Abstract: Hydrothermal pretreatment of lignocellulosic materials generates a liquid stream rich in pentose sugar oligomers. Cost-effective hydrolysis and utilization of these soluble sugar oligomers is an integral process of biofuel production. We report integrated rate equations for hydrolysis of xylo-oligomers derived from pretreated hardwood by dicarboxylic maleic and oxalic acids. The highest xylose yield observed with dicarboxylic acids was 96%, and compared to sulfuric acid, was 5-15% higher with less xylose degradation. Dicarboxylic acids showed an inverse correlation between xylose degradation rates and acid loadings, unlike sulfuric acid for which less acid results in less xylose degradation to aldehydes and humic substances. A combination of high acid and low-temperature leads to xylose yield improvement. Hydrolysis time course data at three different acid concentrations and three temperatures between 140 and 180 C were used to develop a reaction model for the hydrolysis of xylo-oligosaccharides to xylose by dicarboxylic acids.

Research Area: Bioenergy    

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Severity Factor Coefficients for Subcritical Liquid Hot Water Pretreatment of Hardwood Chips

2013

Authors: Y. Kim, T. Kreke, N. S. Mosier, M. R. Ladisch
Journal: Biotechnology and Bioengineering, 111(2), 254-263, 2013 
Book Chapter: 

Abstract: Single stage and multi-stage liquid hot water pretreatments of mixed hardwood pinchips were investigated at various severities (log Ro=3.65-4.81) to assess the efficiencies of the pretreatments with respect to achieving high pentose sugar yields and improved enzymatic digestibility of pretreated cellulose. We investigate the effect of pretreatment parameters that is, temperature, and time, as expressed in the severity factor, on the recovery of sugars and hydrolyzability of pretreated cellulose. We find the severity factor, in its widely used form, is an incomplete measure for evaluating the pretreatment efficiencies and predicting overall sugar yields when pretreatment temperatures above 200 C are used. Correctins to the severity factor and its correlation to the measured pretreatment responses (% xylan solubilization, xylan recovery as fermentable sugars, cellulose enzymatic digestibility) indicate a greater influence of temperature on the pretreatment efficiencies than predicted by the commonly used severity factor. A low temperature, long residence time is preferred for hemicellulose dissolution during the pretreatment since the condition favors oligosaccharide and monomeric sugar formation overe sugar degradation. On t he contrary, high cellulose hydrolyzability is achieved with a high temperature (>200 C), high severity pretreatment when pretreatment is followed by enzyme hydrolysis. In multi-stage pretreatment, the first low-severity pretreatment is optimized for solubilizing fast-hydrolyzing hemicellulose while minimizing formation of furans. The subsequent pretreatment is carried out at over 200 C to recover the difficult-to-hydrolyze hemicellulose fraction as well as to increase susceptibility of pretreated cellulose to enzymes. High recovery (>92%) of hemicellulose-derived pentose sugars and enhanced enzymatic hydrolysis of pretreated cellulose (where >80% glucose yield results with 20 FPU = 32 mg, protein/g glucan or 10-13 mg/g initial hardwood) are achieved by applying a multi-stage pretreatment. This work shows how the severity equation may be used to obtain a single characteristic curve that correlate xylan solubilization and enzymatic cellulose hydrolysis as a function of severity at pretreatment temperatures up to 230 C.

Research Area: Bioenergy Bioprocessing   

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Adding Value to the Integrated Forest Biorefinery with Co-Products from Hemicellulose-Rich Pre-Pulping Extract

2012

Authors: C. Bergeron, D. J. Carrier and S. Ramaswamy (Book Chapter authors Abigail Engelberth and G. Peter van Walsum)
Journal: 
Book Chapter: Wiley, ISBN: 978-0-470-97357-8

Abstract: The only source for sustainable and renewable organic carbon for use in chemicals and transportation fuels is plant biomass (Huber, Iborra, and Corma, 2006). Forestry biomass potential in the US is around 368 million dry tons annually (Perlack et al. 2005), with around 108 million tons used for pulp production (Ragauskas et al., 2006a). Combining existing pulp production with new technologies for production of biofuels and other bioproducts can leverage existing biomass collection methods, conversion infrastructure and technical know-how to advance the development of new bio-based products. The idea of an integrated biorefinery is to optimize the use of all fractions of biomass for the production of biofuels, bioenergy and biomaterials (Ragauskas et al., 2006b). In an integrated forest biorefinery (IFBR), the three major components of wood can be allocated to different uses that make best use of the component characteristics; the cellulose would be used for the production of pulp, the hemicellulose would act as a precursor to sugar-based chemicals and the lignin could be used for production of high-value products such as carbon fibers, chemicals, or polymers, or simply relegated to boiler fuel (van Heiningen, 2006; Marinova et al., 2009). In current pulp mill operations, the cellulose is allocated to pulp, but most of the hemicellulose is allocated to boiler fuel, where it delivers low net value. In an IFBR, some of the hemicellulose could be removed prior to pulping, thus enabling the possibility of adding greater value to this stream. With current extraction technologies and pulp production, around 14 million tons of hemicellulose could be recovered annually (Ragauskas et al., 2006a). Figure 13.1 is a depiction of the integration of the IFBR concept with woody biomass used in pulping. This particular design envisions using pre-pulping extraction and gasification to derive a greater variety of materials from the starting materials, all the while maintaining pulp production. An IFBR would take advantage of the "know-how" of the pulping industry to effectively transport and process lignocellulosic biomass (Kautto et al., 2010). 

Research Area: Biofuels/Bioproducts    

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The Impact of Dry Matter Loss During Herbaceous Biomass Storage on Net Greenhouse Gas Emissions from Biofuels Production

2012

Authors: I.Emery, N. Mosier
Journal: Biomass and Bioenergy, 39, 237-246, 2012
Book Chapter: 

Abstract: Life cycle inventory models of greenhouse gas emissions from biofuel production have become tightly integrated into government mandates and other policies to encourage biofuel production. Current models do not include life cycle impacts of biomass storage or reflect current literature on emissions from soil and biomass decomposition. In this study, the GREET model framework was used to determine net greenhouse gas emissions during ethanol production from corn and switchgrass via three biomass storage systems: wet ensiling of whole corn, and indoor and outdoor dry bale storage of corn stover and switchgrass. Dry matter losses during storage were estimated from the literature and used to modify GREET inventory analysis. Results showed that biomass stability is a key parameter affecting fuel production per farmed hectare and life cycle greenhouse gas emissions. Corn silage may generate 5358 L/ha of ethanol at 26.5 g CO2 eq/MJ, relative to 5654 L/ha at 52.3 g CO2 eq/MJ from combined corn stover and conventional grain corn ethanol production, or 3919 L/ha at 21.3 g CO2 eq/MJ from switchgrass. Dry matter losses can increase net emissions by 3-25% (ensiling), 5-53% (bales outdoors), or 1-12% (bales indoors), decreasing the net GHG reduction of ethanol over gasoline by up to 10.9%. Greater understanding of biomass storage losses and greenhouse gas fluxes during storage is necessary to accurately assess biomass storage options to ensure that the design of biomass supply logistics systems meet GHG reduction mandates for biofuel production. 

Research Area: Bioenergy    

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Application of Cellulase and Hemicellulase to Pure Xylan, Pure Cellulose, and Switchgrass Solids from Leading Pretreatments

2011

Authors: Shi, J., M. A. Ebrik, B. Yang, R. J. Garlock, V. Balan, B. E. Dale, V. R. Pallapolu, Y. Y. Lee, Y. Kim, N. S. Mosier, M. R. Ladisch, M. T. Holtzapple, M. Falls, R. Sierra, B. S. Donohoe, T. B. Vinzant, R. T. Elander, B. Hames, S. Thomas, R. E. Warner, and C. E. Wyman 
Journal: Bioresource Technology, 102(24), 11080-11088 (2011) 
Book Chapter: 

Abstract: Accellerase 1000 cellulase, Spezyme CP cellulase, Beta-glucosidase, Multifect xylanase, and beta-xylosidase were evaluated for hydrolysis of pure cellulose, pure xylan, and switchgrass solids from leading pretreatments of dilute sulfuric acid, sulfur dioxide, liquid hot water, lime, soaking in aqueous ammonia, and ammonia fiber expansion. Distinctive sugar release patterns were observed from Avicel, phosphoric acid swollen cellulose (PASC), xylan, and pretreated switchgrass solids, with accumulation of significant amounts of xylooligomers during xylan hydrolysis. The strong inhibition of cellulose hydrolysis by xylooligomers could be partially attributed to the negative impact of xylooligomers on cellulase adsorption. The digestibility of pretreated switchgrass varied with pretreatment but could not be consistently correlated to xylan, lignin, or acetyl removal. Initial hydrolysis rates did correlate well with cellulase adsorption capacities for all pretreatments except lime, but more investigation is needed to relate this behavior to physical and compositional properties of pretreated switchgrass.

Research Area: Biofuels/Bioproducts    

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Biofuels from Cellulosic Feedstocks

2011

Authors: Ho, N. W. Y., M. R. Ladisch, M. Sedlak, N. Mosier, and E. Casey 
Journal: 
Book Chapter: Comprehensive Biotechnology, Second Edition, 1, 51-62 (2011). 

Abstract: 

Research Area: Biofuels/Bioproducts    

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Cassava Starch Pearls as a Desiccant for Drying Ethanol

2011

Authors: Y. Kim, R. Hendrickson, N. Mosier, A. Hilaly, and M. R. Ladisch 
Journal: Industrial & Engineering Chemistry Research, 50(14), 8678-8685 (2011) 
Book Chapter: 

Abstract: The fuel ethanol industry uses corn grits packed in fixed bed adsorption towers to dry hydrous ethanol vapors in an energy efficient manner. Spherical micropearl cassava starch exhibits a higher adsorption capacity than corn grits of the same size and may be a viable replacement for ground corn. Adsorption equilibrium curves, BET surface area measurements, and SEM images provide an explanation for the enhanced performance of cassava micropearls based on particle architecture and the surface area available to water molecules. The SEM images show that the micropearls form a core-shell structure with pregel starch acting as the scaffold that holds starch granules in an outer layer. This layer determines the BET surface area and the measured equilibrium adsorption capacity. The core-shell microstructure results in a shortened diffusion pathlength and enhanced adsorption rates. These microstructural and operational characteristics provide a template for microfabrication of enhanced capacity starch-based spherical adsorbents that could replace ground corn for the drying of ethanol. 

Research Area: Biofuels/Bioproducts    

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Comparative Data on Effects of Leading Pretreatments and Enzyme Loadings and Formulations on Sugar Yields from Different Switchgrass Sources

2011

Authors: Wyman, C. E., V. Balan, B. E. Dale, R. T. Elander, M. Falls, B. Hames, M. T. Holtzapple, M. R. Ladisch, Y. Y. Lee, N. Mosier, V. R. Pallapolu, J. Shi, S. R. Thomas, and R. E. Warner
Journal: Bioresource Technology, 102(24), 11052-11062 (2011)
Book Chapter: 

Abstract: Dilute sulfuric acid (DA), sulfur dioxide (SO2), liquid hot water (LHW), soaking in aqueous ammonia (SAA), ammonia fiber expansion (AFEX), and lime pretreatments were applied to Alamo, Dacotah, and Shawnee switchgrass. Application of the same analytical methods and material balance approaches facilitated meaningful comparisons of glucose and xylose yields from combined pretreatment and enzymatic hydrolysis. Use of a common supply of cellulase, beta-glucosidase, and xylanase also eased comparisons. All pretreatments enhanced sugar recovery from pretreatment and subsequent enzymatic hydrolysis substantially compared to untreated switchgrass. Adding beta-glucosidase was effective early in enzymatic hydrolysis while cellobiose levels were high but had limited effect on longer term yields at the enayme loadings applied. Adding xylanase improved yields most for higher pH pretreatments where more xylan was left in the solids. Harvest time had more impact on performance than switchgrass variety, and microscopy showed changes in different features could impact performance by different pretreatments.

Research Area: Biofuels/Bioproducts    

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Comparative Material Balances Around Pretreatment Technologies for the Conversion of Switchgrass to Soluble Sugars

2011

Authors: Garlock, R. J., B. Balan, B. E. Dale, V. R. Pallapolu, Y. Y. Lee, Y. Kim, N. S. Mosier, M. R. Ladisch, M. T. Holtzapple, M. Falls, R. Sierra-Ramirez, J. Shi, M. A. Ebrik, T. Redmond, B. Yang, C. E. Wyman, B. S. Donohoe, T. B. Vinzant, R. T. Elander, B. hames, S. Thomas, and R. E. Warner 
Journal: Bioresource Technology, 102(24), 11063-11071 (2011) 
Book Chapter: 

Abstract: For this project, six chemical pretreatments were compared for the Consortium for Applied Fundamentals and Innovation (CAFI): ammonia fiber expansion (AFEX), dilute sulfuric acid (DA), lime, liquid hot water (LHW), soaking in aqueous ammonia (SAA), and sulfur dioxide (SO2). For each pretreatment, a material balance was analyzed around the pretreatment, optional post-washing step, and enzymatic hydrolysis of Dacotah switchgrass. All pretreatments + enzymatic hydrolysis solubilized over two-thirds of the available glucan and xylan. Lime, post-washed LHW, and SO2 achieved >83% total glucose yields. Lime, post-washed AFEX, and DA achieved >83% total xylose yields. Alkaline pretreatments, except AFEX, solubilized the most lignin and a portion of the xylan as xylo-oligomers. As pretreatment pH decreased, total solubilized xylan and released monomeric xylose increased. Low temperature-long time or high temperature-short time pretreatments are necessary for high glucose release from late-harvest Dacotah switchgrass but high temperatures may cause xylose degradation. 

Research Area: Biofuels/Bioproducts    

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Deactivation of Cellulases by Phenols

2011

Authors: Ximenes, E., Kim, Y., Mosier, N., Dien, B., and Ladisch, M.
Journal: Enzyme & Microbial Technology, 48(2011), 54-60 (2010)
Book Chapter: 

Abstract: Pretreatment of lignocellulosic materials may result in the release of inhibitors and deactivators of cellulose enzyme hydrolysis. We report the identification of phenols with major inhibition and/or deactivation effect on enzymes used for conversion of cellulose to ethanol. The inhibition effects were measured by combining the inhibitors (phenols) with enzyme and substrate immediately at the beginning of the assay. The deactivation effects were determined by pre-incubating phenols with cellulases or beta-glucosidases for specified periods of time, prior to the respective enzyme assays. Tannic, gallic, hydroxy-cinnamic, and 4-hydroxybenzoic acids, together with vanillin caused 20-80% deactivation of cellulases and/or beta-glucosidases after 24 h of pre-incubation while enzymes pre-incubated in buffer alone retained all of their activity. The strength of the inhibition or deactivation effect depended on the type of enzyme, the microorganism from which the enzyme was derived, and the type of phenolic compounds present, beta-glucosidase from Aspergillus niger was the most resistant to inhibition and deactivation, requiring about 5 and 10-fold higher concentrations, respectively, for the same levels of inhibition or deactivation as observed for enzymes from Trichoderma reesei. Of the phenol molecules tested, tannic acid was the single, most damaging aromatic compound that caused both deactivation and reversible loss (inhibition) of all of enzyme activities tested. 

Research Area: Biofuels/Bioproducts    

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Dry Grind Coproducts as Cellulosic Ethanol Feedstock

2011

Authors: N. Mosier 
Journal: 
Book Chapter: Distillers Grains - Production, Properties, and Utilization, CRC Press, New York, New York, Liu, K., and Rosentrater, K. A. (Eds.), 475-486 (2011). 

Abstract: Corn grain is the staple feedstock for fuel ethanol production in the United States, accounting for more than 95% of fuel ethanol production. First-generation ethanol biofuel production from corn breaks down the starch portion of the grain into glucose, which is then fermented to ethanol. While improved efficiencies in the U.S. fuel ethanol industry have increased yields of ethanol near the theorttical maximum for corn starch, converting residual biomass possesses the opportunity for further increasing ethanol yields from a bushel of corn by as much as an additional 10%-14%.

Research Area: Biofuels/Bioproducts    

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Effect of Product Inhibition on Xylose Fermentation to Ethanol by Saccharomyces cerevisae 424A (LHN-ST)

2011

Authors: Athmanathan, A., M. Sedlak, N. W. Y. Ho, and N. S. Mosier 
Journal: Biological Engineering Transactions (ASABE), 3(2), 111-124 (2011)
Book Chapter: 

Abstract: Commercially viable manufacturing of cellulosic ethanol requires high titers of product from both hexose and pentose fermentation. This article reports inhibition of initial specific xylose consumption rates and ethanol generation in batch fermentations of xylose using S. cerevisiae 424A (LNH-ST), a genetically modified strain capable of co-fermenting glucose and xylose. The fermentations were carried out in yeast extract peptone (YEP) medium in the presence of varying concentrations of ethanol (0% to 9% w/v) either added in a single dose or generated in situ by the yeast from glucose prior to xylose fermentation. The Levenspiel inhibition function was used to model inhibition of initial specific xylose consumption rates by ethanol. When ethanol was added in a single dose, the yeast ceased xylose fermentation when ethanol concentration reached 90 g L-1. However, when ethanol was generated in situ from glucose, the maximum final titer of ethanol was 110 g L-1. Comparing the effect of ethanol on xylose fermentation to the effect of ethanol on HXT transport of xylose in non-xylose-fermenting yeast suggests that inhibition of xylose transport into the cell is rate-limiting for fermentation.

Research Area: Biofuels/Bioproducts    

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Effects of Enzyme Loading and Beta-Glucosidase Supplementation on Enzymatic Hydrolysis of Switchgrass Processed by Leading Pretreatment Technologies

2011

Authors: Pallapolu, V. R., Y. Y. Lee, R. J. Garlock, V. Balan, B. E. Dale, Y. Kim, N. S. Mosier, M. R. Ladisch, M. Falls, M. T. Holtzapple, R. Sierra, J. Shi, M. A. Ebrik, T. Redmond, B. Yang, C. E. Wyman, B. S. Donohoe, T. B. Vinzant, R. T. Elander, B. Hames, S. Thomas, and R. E. Warner 
Journal: Bioresource Technology, 102(24), 11115-11120 (2011) 
Book Chapter: 

Abstract: The objective of this work is to investigate the effects of cellulase loading and beta-glucosidase supplementation on enzymatic hydrolysis of pretreated Dacotah switchgrass. To assess the difference among various pretreatment methods, the profiles of sugars and intermediates were determined for differently treated substrates. For all pretreatments, 72 h glucan/xylan digestibilities increased sharply with enzyme loading up to 25 mg protein/g-glucan, after which the response varied depending on the pretreatment method. For a fixed level of enzyme loading, dilute sulfuric acid (DA), SO2, and Lime pretreatments exhibited higher digestibility than the soaking in aqueous ammonia (SAA) and ammonia fiber expansion (AFEX). Supplementation of Novozyme-188 to Spezyme-CP improved the 72 h glucan digestibility only for the SAA treated samples. The effect of ?-glucosidase supplementation was discernible only at the early phase of hydrolysis where accumulation of cellobiose and oligomers is significant. Addition of Beta-glucosidase increased the xylan digestibility of alkaline treated samples due to the Beta-xylosidase activity present in Novozyme-188.

Research Area: Biofuels/Bioproducts    

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Investigation of Enzyme Formulation on Pretreated Switchgrass

2011

Authors: Falls, M., J. J. Shi, M. A. Ebrik, T. Redmond, B. Yang, C. E. Wyman, R. Garlock, V. Balan, B. E. Dale, V. R. Pallapolu, Y. Y. Lee, Y. Kim, N. S. Mosier, M. R. Ladisch, B. Hames, S. Thomas, B. S. Donohoe, T. B. Vinzant, R. T. Elander, R. Sierra, and M. T. Holtzapple 
Journal: Bioresource Technology, 102(24), 11072-11079 (2011) 
Book Chapter: 

Abstract: For this project, six chemical pretreatments were compared for the Consortium for Applied Fundamentals and Innovation (CAFI): ammonia fiber expansion (AFEX), dilute sulfuric acid (DA), lime, liquid hot water (LHW), soaking in aqueous ammonia (SAA), and sulfur dioxide (SO2). For each pretreatment, a material balance was analyzed around the pretreatment, optional post-washing step, and enzymatic hydrolysis of Dacotah switchgrass. All pretreatments + enzymatic hydrolysis solubilized over two-thirds of the available glucan and xylan. Lime, post-washed LHW, and SO2 achieved >83% total glucose yields. Lime, post-washed AFEX, and DA achieved >83% total xylose yields. Alkaline pretreatments, except AFEX, solubilized the most lignin and a portion of the xylan as xylo-oligomers. As pretreatment pH decreased, total solubilized xylan and released monomeric xylose increased. Low temperature-long time or high temperature-short time pretreatments are necessary for high glucose release from late-harvest Dacotah switchgrass but high temperatures may cause xylose degradation. 

Research Area: Biofuels/Bioproducts    

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Soluble Inhibitors/Deactivators of Cellulase Enzymes from Lignocellulosic Biomass

2011

Authors: Y. Kim, E. Ximenes, N. S. Mosier and M. R. Ladisch 
Journal: Enzyme & Microbial Technology, 48(2011), 408-415 (2011) 
Book Chapter: 

Abstract: Liquid hot water, steam explosion, and dilute acid pretreatments of lignocellulose general soluble inhibitors which hamper enzymatic hydrolysis as well as fermentation of sugars to ethanol. Toxic and inhibitory compounds will vary with pretreatment and include soluble sugars, furan derivatives (hydroxymethyl fulfural, furfural), organic acids (acetic, formic and, levulinic acid), and phenolic compounds. Their effect is seen when an increase in the concentration of pretreated biomass in a hydrolysis slurry results in decreased cellulose conversion, even though the ratio of enzyme to cellulose is kept constant. We used lignin-free cellulose, Solka Floc, combined with mixtures of soluble compounds released during pretreatment of wood, to prove that the decrease in the rate and extent of cellulose hydrolysis is due to a combination of enzyme inhibition and deactivation. The causative agents were extracted from wood pretreatment liquid using PEG surfactant, activated charcoal or ethyl acetate and then desorbed, recovered, and added back to a mixture of enzyme and cellulose. At enzyme loadings of either 1 or 25 mg protein/g glucan, the most inhibitory components, later identified as phenolics, decreased the rate and extent of cellulose hydrolysis by half due to both inhibition and precipitation of the enzymes. Full enzyme activity occurred when the phenols were removed. Hence detoxification of pretreated woods through phenol removal is expected to reduce enzyme loadings, and therefore reduce enzyme costs, for a given level of cellulose conversion. 

Research Area: Biofuels/Bioproducts    

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Surface and Ultrastructural Characterization of Raw and Pretreated Switchgrass

2011

Authors: Donohoe, B. S., T. B. Vinzant, R. T. Elander, V. R. Pallopolu, Y. Y. Lee, R. J. Garlock, V. Balan, B. E. Dale, Y. Kim, N. S. Mosier, M. R. Ladisch, M. Falls, M. T. Holtzapple, R. Sierra, J. Shi, M. A. Ebrik, T. Redmond, B. Yang, C. E. Wyman, B. Hames, S. Thomas, and R. E. Warner 
Journal: Bioresource Technology, 102(24), 11097-11104 (2011) 
Book Chapter: 

Abstract: The US Department of Energy-funded Biomass Refining CAFI (Consortium for Applied Fundamentals and Innovation) project has developed leading pretreatment technologies for application to switchgrass and has evaluated their effectiveness in recovering sugars from the coupled operations of pretreatment and enzymatic hydrolysis. Key chemical and physical characteristics have been determined for pretreated switchgrass samples. Several analytical microscopy approaches utilizing instruments in the Biomass Surface Characterization Laboratory (BSCL) at the National Renewable Energy Laboratory (NREL) have been applied to untreated and CAFI-pretreated switchgrass samples. The results of this work have shown that each of the CAFI pretreatment approaches on switchgrass result in different structural impacts at the plant tissue, cellular, and cell wall levels. Some of these structural changes can be related to changes in chemical composition upon pretreatment. There are also apparently different structural mechanisms that are responsible for achieving the highest enzymatic hydrolysis sugar yields.

Research Area: Biofuels/Bioproducts    

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Bioenergy: Renewable Liquid Fuels

2010

Authors: Michael Ladisch 
Journal: 
Book Chapter: In "Bio-Inspired Innovation and National Security", R. E. Armstrong, Mark D. Drapeau, Cheryl A. Loeb, James J. Valdes (Eds.), Center for Technology and National Security Policy, National Defense University, pp. 119-138 (2010). 

Abstract: Becoming independent of crude oil imports, mainly from the Middle East, is an urgent concern for many countries all over the world. In order to secure a sustainable energy supply, especially in the transportation sector, governments need to apply policies that promote the use of renewable energy technologies. The dependence on crude oil imports decreases as the production of total energy from renewable sources (renewable liquid fuels) increases. There are two major factors that will influence the amount of final energy production from renewable sources. 

Research Area: Biofuels/Bioproducts    

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Comparing Extraction Methods to Recover Ginseng Saponins from American Ginseng (Panax quinquefolium), Followed by Purification Using Fast Centrifugal Partition Chromatogrpahy with HPLC Verification`

2010

Authors: A. S. Engelberth, E. C. Clausen, D. J. Carrier
Journal: Separation and Purification Technology, 72, 1-6
Book Chapter: 

Abstract: A series of experiments were carried out to compare the extraction of ginseng saponins, ginsenosides, from powdered American ginseng (Panax quinquefolium) using pressurized hot water and the more conventional ultrasonic-assisted extraction. Three solvents were tested, water, n-butanol-saturated water, and water-saturated n-butanol. Each resulting extraction was further purified using fast centrifugal partition chromatography (FCPC) in order to better quantify the contents of the crude plant extract. The pressurized hot water system extracted a greater yield of saponins, 11.2 mg/g (extraction at 110 C and 440 kPa), than the ultrasonic-assisted method, 7.2 mg/g (extraction at variable temperature with no external pressure). The difference in solvent system for either extraction methods was not significant, and the results gave credence for the use of water as the extraction solvent, n-Butanol-saturated water yielded the most saponins (10.1 mg/g), while water yielded 9.8 mg/g, and water-saturated n-butanol yielded 7.8 mg/g. Since water is an environmentally benign solvent, this result is quite attractive for future work.

Research Area: Bioseparations    

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Converting Cellulose to Biofuels

2010

Authors: Ladisch, M., Mosier, N. S., Kim, Y., Ximenes, E., and Hogsett, D. 
Journal: Chemical Engineering Progress (CEP), SBE Special Supplement Biofuels, 106(3), 56-63 (March 2010) 
Book Chapter: 

Abstract: The recent National Academies report "America's Energy Future" concluded that alternative liquid fuels have the potential to reduce dependence on imported oil, enhance energy security, and potentially reduce greenhouse gas emissions. It identified renewable cellulosic biomass as a major resource (as well as nonrenewable coal, which is outside the scope of this article) and biochemical and thermochemical processing as two major conversion approaches on the critical path to attaining about a 20% reduction in oil used for light-duty transportation at current consumption levels. Biochemical and thermochemical processes that transform cellulosic biomass into liquid fuels have a common denominator: Both requuire preprocessing to break down the polymers in the biomass into small molecules (sugars, CO, H2, CO2) followed by a catalytic step to form a fuel. The bioprocessing of cellulose to ethanol is conceptually simpler than a thermal route in which the biomass is first gasified and cleaned up before it is converted to a biofuel (diesel) through Fisher-Tropsch (FT) synthesis. The tradeoff is selectivity vs. conversion rate - selectivity is high for bioprocesses and low for thermal, whereas rates are low for bioprocesses and high for thermal. Feedstock, catalyst robustness, and costs are key factors that must be addressed to achieve economical processes for both biochemical and thermochemical technologies. Bioprocesses for making ethanol from cellulose have a long history. The potential of cellulose to produce ethanol using mineral oil catalysts was demonstrated prior to World War II. Thermochemical and acid routes for obtaining fermentable sugars were mature technologies more than 70 years ago, whereas enzyme biocatalysts that perform similar functions were identified since then. These enzymes have been purified and characterized, and the genes that encode them have been sequenced. Current production methods use genetically modified fungal and bacterial microorganisms to produce cellulolytic enzymes that are used in the food and consumer products industries, as well as in the emerging biofuels industry.

Research Area: Biofuels/Bioproducts    

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Effect of Acetic Acid and pH on the Cofermentation of Glucose and Xylose to Ethanol by a Genetically Engineered Strain of Saccharomyces cerevisiae

2010

Authors: Casey E., Sedlak, M., Ho, N.W.Y., and Mosier, N. S. 
Journal: Yeast Research, 10(4), 385-393 (2010). 
Book Chapter: 

Abstract: A current challenge of the cellulosic ethanol industry is the effect of inhibitors present in biomass hydrolysates. Acetic acid is an example of one such inhibitor that is released during the pretreatment of hemicellulose. This study examined the effect of acetic acid on the cofermentation of glucose and xylose under controlled pH conditions by Saccharomyces cerevisiae 424A(LNH-ST), a genetically engineered industrial yeast strain. Acetic acid concentrations of 7.5 and 15 gL-1, representing the range of concentrations expected in actual biomass hydrolysates, were tested under controlled pH conditions of 5, 5.5, and 6. The presence of acetic acid in the fermentation media led to a significant decrease in the observed maximum cell biomass concentration. Glucose- and xylose-specific consumption rates decreased as the acetic acid concentration increased, with the inhibitory effect being more severe for xylose consumption. The ethanol production rates also decreased when acetic acid was present, but ethanol metabolic yields increased under the same conditioons. The results also revealed that the inhibitory effect of acetic acid could be reduced by increasing media pH, thus confirming that the undissociated form of acetic acid is the inhibitory form of the molecule.

Research Area: Biofuels/Bioproducts    

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Effect of Compositional Variability of Distillers' Grains on Cellulosic Ethanol Production

2010

Authors: Kim, Y., Hendrickson, R., Mosier, N. S., Ladisch, M. R., Bals, B., Balan, V., Dale, B. E., Dien, B. S. and Cotta, M. A. 
Journal: Bioresource Technology, 101(14), 5385-5393 (2010) 
Book Chapter: 

Abstract: In a dry grind ethanol plant, approximately 0.84 kg of dried distillers' grains with solubles (DDGS) is produced per liter of ethanol. The distillers' grains contain the unhydrolyzed and unprocessed cellulosic fraction of corn kernels, which could be further converted to ethanol or other valuable bioproducts by applying cellulose conversion technology. Its compositional variability is one of the factors that could affect the overall process design and economics. In this study, we present compositional variability of distillers' grains collected from four different dry grind ethanol plants and its effect on enzymatic digestibility and fermentability. We then selected two sources of distillers' grains based on their distinctive compositional difference. These were pretreated by either controlled pH liquid hot water (LHW) or ammonia fiber expansion (AFEX) and subjected to enzymatic hydrolysis and fermentation. Fermentation of the pretreated distillers' grains using either industrial yeast or genetically engineered glucose and xylose co-fermenting yeast, yielded 70-80% of theoretical maximum ethanol concentration, which varied depending on the batch of distillers' grains used. Results show that cellulose conversion and ethanol fermentation yields are affected by the compositions of distillers' grains. Distillers' grains with a high extractives content exhibit a lower enzymatic digestibility but a higher fermentability. 

Research Area: Biofuels/Bioproducts    

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Establishment of L-arabinose Fermentation in Glucose/Xylose Co-Fermenting Recombinant Saccharomyces cerevisiae 424A(LNH-ST) by Genetic Engineering

2010

Authors: A. K. Bera, M. Sedlak, A. Khan and N. W. Y. Ho 
Journal: Applied Genetics and Molecular Biotechnology (Online) 
Book Chapter: 

Abstract: Cost-effective and efficient ethanol production from lignocellulosic materials requires the fermentation of all sugars recovered from such materials including glucose, xylose, mannose, galactose, and L-arabinose. Wild-type strains of Saccharomyces cerevisia used in industrial ethanol production cannot ferment D-xylose and L-arabinose. Our genetically engineered recombinant S. cerevisiae yeast 424A(LNH-ST) has been made able to efficiently ferment xylose to ethanol, which was achieved by integrating multiple copies of three xylose-metabolizing genes. This study reports the efficient anaerobic fermentation of L-arabinose by the derivative of 424A(LNH-ST). The new strain was constructed by over-expression of two additional genes from fungi L-arabinose utilization pathways. The resulting new 424A(LNH-ST) strain exhibited production of ethanol from L-arabinose, and the yield was more than 40%. An efficient ethanol production, about 72.5% yield from five-sugar mixtures containing glucose, galactose, mannose, xylose, and arabinose was also achieved. This co-fermentation of five-sugar mixture is important and crucial for application in industrial economical ethanol production using lignocellulosic biomass as the feedstock .

Research Area: Biofuels/Bioproducts    

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Inhibition of Cellulases by Phenols

2010

Authors: Ximenes, E., Y. Kim, N. Mosier, B. Dien, and M. Ladisch 
Journal: Enzyme and Microbial Technology , 46(3), 170-176 (2010) 
Book Chapter: 

Abstract: Enzyme hydrolysis of pretreated cellulosic materials slows as the concentration of solid biomass material increases, even though the ratio of enzyme to cellulose is kept constant. This form of inhibition is distinct from substrate and product inhibition, and has been noted for lignocellulosic materials including wood, corn stover, switch grass, and corn wet cake at solids concentrations greater than 10 g/L. Identification of enzyme inhibitors and moderation of their effectsjis of considerable practical importance since favorable ethanol production economics require that at least 200 g/L of cellulosic substrates be used to enable monosaccharide concentrations of 100 g/L, which result in ethanol titers of 50 g/L. Below about 45 g/L ethanol, distillation becomes energy inefficient. This work confirms that the phenols: vanillin, syringaldehyde, trans-cinnamic acid, and hydroxybenzoic acid, inhibit cellulose hydrolysis in wet cake by endo- and exo-cellulases, and cellobiose hydrolysis by beta-glucosidase. A ratio of 4 mg of vanillin to 1 mg protein (0.5 FPU) reduces the rate of cellulose hydrolysis by 50%. Beta-glucosidases from Trichoderma reesei and Aspergillus niger are less susceptible to inhibition and require about 10 x and 100 x higher concentrations of phenols for the same levels of inhibition. Phenols introduced with pretreated cellulose must be removed to maximize enzyme activity. 

Research Area: Biofuels/Bioproducts    

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Lignin Monomer Composition Affects Arabidopsis Cell-Wall Degradability After Liquid Hot Water Pretreatment

2010

Authors: Li, X., Ximenes, E., Kim, Y., Slininger, M., Meilan, R., Ladisch, M., and Chapple, C. 
Journal: Biotechnology for Biofuels, doi:10.1186/1754-6834-3-27 (2010) 
Book Chapter: 

Abstract: Lignin is embedded in the plant cell wall matrix, and impedes the enzymatic saccharification of lignocellulosic feedstocks. To investigate whether enzymatic digestibility of cell wall materials can be improved by altering the relative abundance of the two major lignin monomers, guaiacyl (G) and syringyl (S) subunits, we compared the degradability of cell wall material from wild-type Arabidopsis thaliana with a mutant line and a genetically modified line, the lignins of which are enriched in G and S subunits. Arabidopsis tissue containing G- and S-rich lignins had the same saccharification performance as the wild type when subjected to enzyme hydrolysis without pretreatment. After a 24-hour incubation period, less than 30% of the total glucan was hydrolyzed. By contrast, when liquid hot water (LHW) pretreatment was included before enzyme hydrolysis, the S-lignin-rich tissue gave a much higher glucose yield than either the wild-type or G-lignin-rich tissue. Applying a hot-water washing step after the pretreatment did not lead to a further increase in final glucose yield, but the initial hydrolytic rate was doubled. Our analyses using the model plant A. thaliana revealed that lignin composition affects the enzymatic digestibility of LHW pretreated plant material. Pretreatment is more effective in enhancing the saccharification of A. thaliana cell walls that contain S-rich lignin. Increasing lignin S monomer content through genetic engineering may be a promising approach to increase the efficiency and reduce the cost of biomass to biofuel conversion. 

Research Area: Biofuels/Bioproducts    

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Purification of Resveratrol, Arachidin-1, and Arachidin-3 from Hairy Root Cultures of Peanut (Arachis hypogaea) and Determination of Their Antioxidant Activity and Cytotoxicity

2010

Authors: J. A. Abbott, F. Medina-Bolivar, E. M. Martin, A. S. Engelberth, H. Villagarcia
Journal: Biotechnology Progress, 26(5), 1344-1351
Book Chapter: 

Abstract: Antioxidant stilbenoids, such as resveratrol, arachidin-1, and arachidin-3, have demonstrated beneficial effects on human health. Although resveratrol is commercially available, arachidin-1 and arachidin-3 are not, resulting in an opportunity to explore purification methods and to confirm biological activity. Recently, Arachis hypogaea hairy root cultures (produced via Agrobacterium rhizogenes-mediated transformation) were reported to secrete stilbenoids into liquid growth media upon elicitation in quantities sufficient for commercial production. The purpose of this study was to purify substantial quantities of resveratrol, arachidin-1, and arachidin-3 from A. hypogaea hairy root cultures using centrifugal partition chromatography (CPC), determine the antioxidant activity of these compounds using the thiobarbituric acid reactive substances (TBARS) assay, and determine the cytotoxicity of the compounds using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In a single run of CPC, resveratrol, arachidin-1, and arachidin-3 were separated to a purity of 97.1%, 97.0%, and 91.8%, respectively. Lipid oxidation was inhibited by a 27 and 7 uM dose for reference standards of resveratrol and arachidin-1, respectively, while oxidation was not inhibited up to a 27 uM dose for reference standard of arachidin-3. Oxidation was inhibited at a 14, 7, and 14 uM doses for CPC-purified resveratrol, arachidin-1, and arachidin-3, respectively. Arachidin-1 and arachidin-3 demonstrated cytotoxicity at 27 and 55 uM in RAW 264.7 and HeLa cell lines, respectively, while resveratrol exhibited no cytotoxicity to either cell line. These results demonstrate the integration of a production and purification system for the manufacturing of A. hypogaea-derived stilbenoids.

Research Area: Bioseparations    

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Two-Dimensional Particle Focusing: Sheath Flow on Two Sides

2010

Authors: J. Shin, M. Ladisch
Journal: 
Book Chapter: From "The Microflow Cytometer", Frances S. Ligler and Jason S. Kim, Pan Stanford Publishing Pte. Ltd., Singapore (2010) 

Abstract: The ability to obtain precise information from the particles traveling through a cytometer requires adequate focus of the sample stream. One approach to obtaining a focused stream is the induction of sheathed flow and utilization of the hydrodynamic characteristics of 10 to 100 micron-wide channels to obtain a flow inequality that focuses particles into a narrow band, i.e. sheathed flow. While there have been many examples of microcytometry in the literature, only a few have succeeded in completely sheathing the stream. This chapter reviews several recent approaches to achieving focused sample introduction in a manner that may be suitable for the microflfow channels associated with flow cytometers. The fabrication of these channels shares fabrication techniques based on two-dimensional networks in microelectronics. Appropriate design, characterization of surface properties, and optimization of channel geometry that enhances stable sheath flow is discussed.

Research Area: Bioseparations    

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Comparison of Glucose/Xylose Cofermentation of Poplar Hydrolysates Processed by Different Pretreatment Technologies

2009

Authors: Lu, Y., Warner, R., Sedlak, M., Ho, N., Mosier, N. S.
Journal: Biotechnology Progress 25(2), 349-356 (2009) Abstract
Book Chapter: 

Abstract: The inhibitory effects of furfural and acetic acid on the fermentation of xylose and glucose to ethanol in YEPDX medium by a recombinant Saccharomyces cerevisiae strain (LNH-ST 424A) were investigated. Initial furfural concentrations below 5 g/L caused negligible inhibition to glucose and xylose consumption rates in batch fermentations with high inoculum (4.5-6.0 g/L). At higher initial furfural concentrations (10-15 g/L) the inhibition became significant with xylose consumption rates especially affected. Interactive inhibition between acetic acid and pH were observed and quantified, and the results suggested the importance of conditioning the pH of hydrolysates for optimal fermentation performance. Poplar biomass pretreated by various CAFI processes (dilute acid, AFEX, ARP, SO2-catalyzed steam explosion, and controlled-pH) under respective optimal conditions was enzymatically hydrolyzed, and the mixed sugar streams in the hydrolysates were fermented. The 5-hydroxymethyl furfural (HMF) and furfural concentrations were low in all hydrolysates and did not pose negative effects on fermentation. Maximum ethanol productivity showed that 0-6.2 g/L initial acetic acid does not substantially affect the ethanol fermentation with proper pH adjustment, confirming the results from rich media fermentations with reagent grade sugars.

Research Area: Biofuels/Bioproducts    

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Differential Effects of Mineral and Organic Acids on the Kinetics of Arabinose Degradation Under Lignocellulose Pretreatment Conditions

2009

Authors: A.M.J. Kootstra, N.S. Mosier, E.L. Scott, H.H. Beeftink, and J.P.M. Sanders 
Journal: Biochemical Engineering Journal, 43(1), 92-97 (2009) 
Book Chapter: 

Abstract: Sugar degradation occurs during acid-catalyzed pretreatment of lignocellulosic biomass at elevated temperatures, resulting in degradation products that inhibit microbial fermentation in the ethanol production process. Arabinose, the second most abundant pentose in grasses like corn stover and wheat straw, degrades into furfural. This paper focuses on the first-order rate constants of arabinose (5 g/L) degradation to furfural at 150 and 170 °C in the presence of sulfuric, fumaric, and maleic acid and water alone. The calculated degradation rate constants (kd) showed a correlation with the acid dissociation constant (pKa), meaning that the stronger the acid, the higher the arabinose degradation rate. However, de-ionized water alone showed a catalytic power exceeding that of 50 mM fumaric acid and equaling that of 50 mM maleic acid. This cannot be explained by specific acid catalysis and the shift in pKw of water at elevated temperatures. These results suggest application of maleic and fumaric acid in the pretreatment of lignocellulosic plant biomass may be preferred over sulfuric acid. Lastly, the degradation rate constants found in this study suggest that arabinose is somewhat more stable than its stereoisomer xylose under the tested conditions.

Research Area: Biofuels/Bioproducts    

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Enzymatic Digestion of Liquid Hot Water Pretreated Hybrid Poplar

2009

Authors: Kim, Y., Mosier, N. S., and Ladisch, M. R. 
Journal: Biotechnology Progress 25(2), 340-348 (2009) 
Book Chapter: 

Abstract: Liquid hot (LHW) water pretreatment (LHW) of lignocellulosic material enhances enzymatic conversion of cellulose to glucose by solubilizing hemicellulose fraction of the biomass, while leaving the cellulose more reactive and accessible to cellulase enzymes. Within the range of pretreatment conditions tested in this study, the optimized LHW pretreatment conditions for a 15% (wt/vol) slurry of hybrid poplar were found to be 200oC, 10 min, which resulted in the highest fermentable sugar yield with minimal formation of sugar decomposition products during the pretreatment. The LHW pretreatment solubilized 62% of hemicellulose as soluble oligomers. Hot-washing of the pretreated poplar slurry increased the efficiency of hydrolysis by doubling the yield of glucose for a given enzyme dose. The 15% (wt/vol) slurry of hybrid poplar, pretreated at the optimal conditions and hot-washed, resulted in 54% glucose yield by 15 FPU cellulase per gram glucan after 120 h. The hydrolysate contained 56 g/L glucose and 12 g/L xylose. The effect of cellulase loading on the enzymatic digestibility of the pretreated poplar is also reported. Total monomeric sugar yield (glucose and xylose) reached 67% after 72 h of hydrolysis when 40 FPU cellulase per gram glucan were used. An overall mass balance of the poplar-to-ethanol process was established based on the experimentally determined composition and hydrolysis efficiencies of the liquid hot water pretreated poplar.

Research Area: Biofuels/Bioproducts    

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Ethanol Production from Maize (book chapter)

2009

Authors: S. Schwietzke, Y. Kim, E. Ximenes, N. S. Mosier 
Journal: 
Book Chapter: Molecular Genetics Approaches to Maize Improvement, Biotechnology in Agriculture and Forestry, Springer-Verlag, Berlin Heidelberg, 63, 347-364 (2009) 

Abstract: The production of fuel ethanol from corn grain is widely carried out in the US, with total current production at 7 billion gallons. This may soon reach 10 billion gallons or more. This chapter addresses the potential of fuel ethanol as an additional source of product based on utilization of the cellulosic (non-food) portions of maize, and in particular, the pericarp, cobs, stalks, and leaves of the corn plant. An analysis of the composition of corn, and possible processing schemes that transform the cellulosic portions to ethanol are addressed. Technologies for the bioprocessing of cellulose to ethanol, as well as the impact of cellulose utilization on supplementing corn ethanol, are presented.

Research Area: Biofuels/Bioproducts    

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Extraction of Co-Products from Biomass: Example of Thermal Degradation of Silymarin Compounds in Subcritical Water

2009

Authors: L. Duan, S. N. Wallace, A. Engelberth, J. K. Lovelady, E. C. Clausen, J. W. King, D. J. Carrier
Journal: Applied Biochemistry and Biotechnology, 158(2), 362-373
Book Chapter: 

Abstract: In an effort to increase revenues from a given feedstock, valuable co-products could be extracted prior to biochemical or thermochemical conversion with subcritical water. Although subcritical water shows significant promise in replacing organic solvents as an extraction solvent, compound degradation has been observed at elevated extraction temperatures. First order thermal degradation kinetics from a model system, silymarin extracted from Silybum marianum, in water at pH 5.1 and 100, 120, 140, and 160 C were investigated. Water pressure was maintained slightly above its vapor pressure. Silymarin is a mixture of taxifolin, silichristin, silidianin, silibinin, and isosilibinin. The degradation rate constants ranged from 0.0104 min-1 at 100 C for silichristin to a maximum of 0.0840 min-1 at 160 C for silybin B. Half-lives, calculated from the rate constants, ranged from a low of 6.2 min at 160 C to a high of 58.3 min at 100 C, both for silichristin. The respective activation energies for the compounds ranged from 37.2 kJ/gmole for silidianin to 45.2 kJ/gmole for silichristin. In extracting the silymarin with pure ethanol at 140 C, no degradation was observed. However, when extracting with ethanol/water mixtures at 140 C, degradation increased exponentially as the concentration of water increased. 

Research Area: Bioseparations    

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Liquid Hot Water Pretreatment of Cellulosic Biomass

2009

Authors: Y. Kim, R. Hendrickson, N. S. Mosier, M. R. Ladisch, Methods in Molecular Biology: Biofuels, ed Mielenz, J. R. (The Humana Press, Totowa), 581:93-102
Journal: 
Book Chapter: Biofuels: Methods and Protocols, Ed. J. R. Mielenz (The Humana Press, Totowa), 581:93-102

Abstract: Lignocellulosic biomass is an abundant and renewable resource for fuel ethanol production. However, the lignocellulose is recalcitrant to enzymatic hydrolysis because of its structural complexity. Controlled-pH liquid hot water (LHW) pretreatment of cellulosic feedstock improves its enzymatic digestibility by removing hemicellulose and making the cellulose more accessible to cellulase enzymes. The removed hemicellulose is solubilized in the liquid phase of the pretreated feedstock as oligosaccharides. Formation of monomeric sugars during the LHW pretreatment is minimal. The LHW pretreatment is carried out by cooking the feedstock in process water at temperatures between 160 and 190°C and at a pH of 4–7. No additional chemicals are needed. This chapter presents the detailed procedure of the LHW pretreatment of lignocellulosic biomass.

Research Area: Bioenergy Biofuels/Bioproducts Bioprocessing  

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Switchgrass Water Extracts: Extraction, Separation, and Biological Activity of Rutin and Quercitrin

2009

Authors: N. Uppugundla, A. Engelberth, S. V. Ravindranath, E. C. Clausen, J. O. Lay, J. Giddens, D. J. Carrier
Journal: Journal of Agricultural and Food Chemistry, 57(17), 7763-7770
Book Chapter: 

Abstract: Switchgrass (Panicum virgatum L.) has recently received significant attention as a possible feedstock for the production of liquid fuels such as ethanol. In addition, switchgrass may also be a source of valuable co-products, such as antioxidants, and our laboratory recently reported that switchgrass contains policosanols and a-aocopherol. Motivation for this work began when a switchgrass sample was extracted with water at 50 C and was then tested for low-density lipoprotein (LDL) oxidation inhibition activity using the Thiobarbituric Acid Reactive Substances (TBARS) assay. The TBARS results showed that the switchgrass water extracts inhibited LDL oxidation by as much as 70% in comparison to the control. Liquid chromatography coupled with mass spectrometry (LC-MS) and high performance liquid chromatography (HPLC) were used to identify the compounds that were responsible for LDL oxidation inhibition activity as flavonoids: quercitrin (quercetin-3-O-rhamnoside) and rutin (quercetin-3-O-rutinoside). To maximize flavonoid concentrations, switchgrass was then extracted with water and 60% methanol at different temperatures. The 60% methanol treatment resulted in higher rutin and quercitrin yields when compared to water-only extraction; however, the use of this solvent would not be practical with current biorefinery technology. Centrifugal partition chromatography (CPC) was then used to purify rutin and quercitrin from the switchgrass water extract, which were then tested via the TBARS assay and shown to exhibit lipid peroxidation inhibition activity similar to that obtained with pure flavonoid standards. This is the first report on the presence of rutin and quercitrin in switchgrass. The results support the extraction of viable co-products from switchgrass prior to conversion to liquid fuel.

Research Area: Bioseparations    

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Targeted Capture of Pathogenic Bacteria Using a Mammalian Cell Receptor Coupled with Dielectrophoresis on a Biochip

2009

Authors: O. K. Koo, Y.S. Liu, S. Shuaib, S. Bhattacharyall, M. R. Ladisch, R. Bashir and A. K. Bhunia 
Journal: Journal of Analytical Chemistry, 81(8), 3094-3101 (2009)
Book Chapter: 

Abstract: Efficient capture of target analyte on biosensor platforms is a prerequisite for reliable and specific detection of pathogenic microorganisms in a microfluidic chip. Antibodies have been widely used as ligands, however, because of their occasional unsatisfactory performance, a search for alternative receptors is underway. Heat shock protein 60 (Hsp60), a eukaryotic mitochondrial chaperon protein is a receptor for Listeria adhesion protein (LAP) during Listeria monocytogenes infection. This paper reports application of biotinylated Hsp60 as a capture molecule for living (viable) L. monocytogenes in a microfluidic environment. Hsp60, immobilized on the surface of streptavidin-coated silicon dioxide exhibited specific capture of pathogenic Listeria against a background of other Listeria species, Salmonella, Eschericia, Bacillus, Psuedomonas, Serratia, Hafnia, Enterobacter, Citrobacter, and Lactobacillus. The capture efficiency of L. monocytogenes was 83 times greater than another Listeria receptor, the monoclonal antibody, mAb-C11E9. Additionally, the capture rate was further increased on a Hsp60-coated biochip by 60% when a dielectrophoresis force was applied for 5 min at the beginning of the final 1 h incubation step. Our data show that Hsp60 could be used for specific detection of L. monocytogenes on a biochip sensor platform.

Research Area: Bioseparations    

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Cellulose Conversion in Dry Grind Ethanol Plants

2008

Authors: Michael Ladisch, Bruce Dale, Wally Tyner, Nathan Mosier, Youngmi Kim, Michael Cotta, Bruce Dien, Hans Blaschek, Edmund Laurenas, Brent Shanks, John Verkade, Chad Schell, Gene Petersen
Journal: Bioresource Technology, 99, 5157-5159 (2008). 
Book Chapter: 

Abstract: The expansion of the dry grind ethanol industry provides a unique opportunity to introduce cellulose conversion technology to existing grain to ethanol plants, while enhancing ethanol yields by up to 14%, and decreasing the volume while increasing protein content of distillers' grains. The technologies required are cellulose pretreatment, enzyme hydrolysis, fermentation, and drying. Laboratory data combined with compositional analysis and process simulations are used to present a comparative analysis of a dry grind process to a process with pretreatment and hydrolysis of cellulose in distillers' grains. The additional processing steps are projected to give a 32% increase in net present value if process modifications are made to a 100 million gallon/year plant. 

Research Area: Biofuels/Bioproducts    

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Composition of Corn Dry-Grind Ethanol By-Products: DDGS, Wet Cake, and Thin Stillage

2008

Authors: Youngmi Kim, Nathan S. Mosier, Rick Hendrickson, Thaddeus Ezeji, Hans Blaschek, Bruce Dien, Michael Cotta, Bruce Dale, Michael R. Ladisch 
Journal: Bioresource Technology, 99, 5165-5176 (2008). 
Book Chapter: 

Abstract: DDGS and wet distillers' grains are the major co-products of the dry grind ethanol facilities. As they are mainly used as animal feed, a typical compositional analysis of the DDGS and wet distillers' grains mainly focuses on defining the feedstock's nutritional characteristics. With an increasing demand for fuel ethanol, the DDGS and wet distillers' grains are viewed as a potential bridge feedstock for ethanol production from other cellulosic biomass. The introduction of DDGS or wet distillers' grains as an additional feed to the existing dry grind plants for increased ethanol yield requires a different approach to the compositional analysis of the material. Rather than focusing on its nutritional value, this new approach aims at determining more detailed chemical composition, especially on polymeric sugars such as cellulose, starch and xylan, which release fermentable sugars upon enzymatic hydrolysis. In this paper we present a detailed and complete compositional analysis procedure suggested for DDGS and wet distillers' grains, as well as the resulting compositions completed by three different research groups. Polymeric sugars, crude protein, crude oil and ash contents of DDGS and wet distillers' grains were accurately and reproducibly determined by the compositional analysis procedure described in this paper.

Research Area: Biofuels/Bioproducts    

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Distillers Grains: On the Pathway to Cellulose Conversion (Editorial)

2008

Authors: M. Ladisch and B. Dale 
Journal: Bioresource Technology, 99(12), 5155-5156 (2008) 
Book Chapter: 

Abstract: The papers in this special edition report results from a project that integrates multiple institutional capabilities to help solve a crucial problem: the proliferation of low value, fiber rich distillers' grains (DC) now being produced in the corn dry milling industry. The dry milling industry is growing rapidly, particularly in the Midwest. This proliferation has the potential to depress the market for this by-product and decrease the profitability of dry mills. Our coordinated efforts show it is possible to add value to DG by further processing them into additional fermentable sugars and ethanol, while leaving a solid that is reduced in weight and rich in protein. The project involves and integrates focused research carried out in a coordinated manner among members of a consortium of 4 universities, 2 government laboratories, and industry.

Research Area: Bioenergy    

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Economic Analysis of a Modified Dry Grind Ethanol Process with Recycle of Pretreated and Enzymatically Hydrolyzed Distillers' Grains

2008

Authors: David Perkis, Wallace Tyner, and Rhys Dale
Journal: Bioresource Technology, 99, 5243-5249 (2008). 
Book Chapter: 

Abstract: A modification of the conventional dry grind process for producing ethanol from yellow dent corn is considered with respect to its economic value. Process modifications include recycling distillers' grains, after being pretreated and hydrolyzed, with the ground corn and water to go through fermentation again and increase ethanol yields from the corn starch. A dry grind financial model, which has been validated against other financial models in the industry, is utilized to determine the financial impact of the process changes. The hypothesis was that the enhanced process would yield higher revenues through additional ethanol sales, and higher valued dried distillers' grains (DDGS), due to its higher protein content, to mitigate the drop in DDGS yields. A 32% increase in net present value (NPV) for the overall operation is expected when applying the process modifications to a 100 million gallon ethanol plant, and an enyzme cost of $0.20 for each additional gallon of ethanol produced. However, there may be no value added to the enhanced dried distillers' grains (eDDGS), even in light of its higher protein levels, as current pricing is expected to be more sensitive to the amino acid profile than the total protein level, and the eDDGS has lower lysine levels, a key amino acid. Thus, there is a decrease in revenue from eDDGS due to the combination of no price change and loss of DDGS yield to ethanol. The financial improvements are a result of the increased revenue from higher ethanol yields outpacing the sum of all added costs, which include higher capital costs, larger loan payments, increased operating costs, and decreased revenues from dried distillers' grains. 

Research Area: Biofuels/Bioproducts    

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Enzyme Characterization for Hydrolysis of AFEX and Liquid Hot-Water Pretreated Distillers' Grains and Their Conversion to Ethanol

2008

Authors: Bruce S. Dien, Eduardo A. Ximenes, Patricia J. O'Bryan, Mohammed Moniruzzaman, Xin-Liang Li, Venkatesh Balan, Bruce Dale, and Michael A. Cotta 
Journal: Bioresource Technology, 99, 5216-5225 (2008) 
Book Chapter: 

Abstract: Dried distillers' grains with solubles (DDGS), a co-product of corn ethanol production, was investigated as a feedstock for additional ethanol production. DDGS was pretreated with liquid hot-water (LHW) and ammonia fiber explosion (AFEX) processes. Cellulose was readily converted to glucose from both LHW and AFEX treated DDGS using a mixture of commercial cellulase and ß-glucosidase; however, these enzymes were ineffective at saccharifying the xylan present in the pretreated DDGS. Several commercial enzyme preparations were evaluated in combination with cellulase to saccharify pretreated DDGS xylan and it was found that adding commercial grade (e.g. impure) pectinase and feruloyl esterase (FAE) preparations were effective at releasing arabinose and xylosse. The response of sugar yields for pretreated AFEX and LHW DDGS (6 wt%/solids) were determined for different enzyme loadings of FAE and pectinase and modeled as a reponse surfaces. Arabinose and xylose yields rose with increasing FAE and pectinase enzyme dosages for both pretreated materials. When hydrolyzed at 20 wt%/solids with the same blend of commercial enzymes, the yields were 278 and 261 g sugars (i.e. total o f arabinose, xylose, and glucose) per kg of DDGS (dry basis, db) for AFEX and LHW pretreated DDGS, respectively. The pretreated DDGS's were also evaluated for fermentation using Saccharomyces cerevisiae at 15 wt%/solids. Pretreated DDGS were readily fermented and were converted to ethanol at 89-90% efficiency based upon total gluucans; S. cerevisiae does not ferment arabinose or xylose.

Research Area: Biofuels/Bioproducts    

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Enzyme Hydrolysis and Ethanol Fermentation of Liquid Hot Water and AFEX Pretreated Distillers' Grains at High-Solids Loadings

2008

Authors: Youngmi Kim, Rick Hendrickson, Nathan S. Mosier, Michael R. Ladisch, Bryan Bals, Venkatesh Balan, Bruce E. Dale 
Journal: Bioresource Technology, 99, 5206-5215 (2008). 
Book Chapter: 

Abstract: The dry milling ethanol industry produces distillers' grains as major co-products, which are composed of unhydrolyzed and unfermented polymeric sugars. Utilization of the distillers' grains as an additional source of fermentable sugars has the potential to increase overall ethanol yields in current dry grind processes. In this study, controlled pH liquid hot water pretreatment (LHW) and ammonia fiber expansion (AFEX) treatment have been applied to enhance enzymatic digestibility of the distillers' grains. Both pretreatment methods significantly increased the hydrolysis rate of distillers' dried grains with solubles (DDGS) over unpretreated material, resulting in 90% cellulose conversion to glucose within 24 h of hydrolysis at an enzyme loading of 15 FPU cellulase and 40 IU ß-glucosidase per gram of glucan and a solids loading of 5% DDGS. Hydrolysis of the pretreated wet distillers' grains at 13-15% (wt of dry distillers' grains per wt of total mixture) solids loading at the same enzyme reduced cellulose conversion to 70% and increased conversion time to 72 h for both LHW and AFEX pretreatments. However, when the cellulase was supplemented with xylanase and feruloyl esterase, the pretreated wet distillers' grains at 15% or 20% solids (w/w) gave 80% glucose and 50% xylose yields. The rationale for supplementation of cellulases with non-cellulolytic enzymes is given by Dien et al., later in this journal volume. Fermentation of the hydrolyzed wet distillers' grains by glucose fermenting Saccharomyces cerevisiae ATCC 4124 strain resulted in 100% theoretical ethanol yields for both LHW and AFEX pretreated wet distillers' grains. The solids remaining after fermentation had significantly higher protein content and are representative of a protein-enhanced wet DG that would result in enhanced DDGS. Enhanced DDGS refers to the solid product of a modified dry grind process in which the distillers' grains are recycled and processed further to extract the unutilized polymeric sugars. Compositional changes of the laboratory generated enhanced DDGS are also presented and discussed. 

Research Area: Biofuels/Bioproducts    

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Fermentation of Dried Distillers' Grains and Solubles (DDGS) Hydrolysates to Solvents and Value-Added Products by Solventogenic Clostridia

2008

Authors: Thaddeus Ezeji, Hans P. Blaschek
Journal: Bioresource Technology, 99, 5232-5242 (2008). 
Book Chapter: 

Abstract: Pretreatment and hydrolysis of lignocellulosic biomass using either dilute acid, liquid hot water (LHW), or ammonium fiber expansion (AFEX) results ina complex mixture of sugars such as hexoses (glucose, galactose, mannose), and pentoses (xylose, arabinose). A detailed description of the utilization of representative mixed sugar streams (pentoses and hexoses) and their sugar preferences by the solventogenic clostridia (Clostridium beijerinckii BA101, C. acetobutylicum 260, C. acetobutylicum 824, Clostridium saccharobutylicum 262, and C. butylicum 592) is presented. In these experiments, all the sugars were utilized concurrently throughout the fermentation, although the rate of sugar utilization was sugar specific. For all clostridia tested, the rate of glucose utilization was higher than for the other sugars in the mixture. In addition, the availability of excess fermentable sugars in the bioreactor is necessary for both the onset and the maintenance of solvent production otherwise the fermentation will become acidogenic leading to premature termination of the fermentation process. During an investigation on the effect of some of the known lignocellulosic hydrolysate inhibitors on the growth and ABE production by clostridia, ferulic and p -coumaric acids were found to be potent inhibitors of growth and ABE production. Interestingly, furfural and HMF were not inhibitory to the solventogenic clostridia; rather they had a stimulatory effect on growth and ABE production at concentrations up to 2.0 g/L. 

Research Area: Biofuels/Bioproducts    

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Hydrolysis of Oligosaccharides from Distillers' Grains Using Organic-Inorganic Hybrid Mesoporous Silica Catalysts

2008

Authors: Jason A. Bootsma, Matthew Entorf, Judd Eder, Brent H. Shanks 
Journal: Bioresource Technology, 99, 5226-5231 (2008). 
Book Chapter: 

Abstract: The use of propylsulfonic acid-functionalized mesoporous silica as a catalyst for the hydrolysis of oligosaccharides released by hydrothermal pretreatment of distillers' grains was examined in batch reactor studies. The effectiveness of the catalyst system for oligosaccharide hydrolysis was found to improve significantly with increased reaction temperature. This higher temperature operation allowed for more selective recovery of glucose, but was detrimental to arabinose recovery since significant degradation occurred. Xylose recovery efficiency improved with increasing temperature, but the higher temperature led to increased degradation. Using a model feed, solubilized proteins were found to deactivate the organic-inorganic hybrid catalyst, but a simple pretreatment with activated silica was found to alleviate the deactivation. 

Research Area: Biofuels/Bioproducts    

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Kinetic Modeling Analysis of Maleic Acid Catalyzed Hemicellulose Hydrolysis in Corn Stover

2008

Authors: Y. Lu , N. S. Mosier
Journal: Biotechnology and Bioengineering, 101(6), 1170-1181 (2008). 
Book Chapter: 

Abstract: Maleic acid-catalyzed hemicellulose hydrolysis reaction in corn stover was analyzed by kinetic modeling. Kinetic constants for Saeman and biphasic hydrolysis models were analyzed by an Arrhenius-type expansion which includes activation energy and catalyst concentration factors. The activation energy for hemicellulose hydrolysis by maleic acid was determined to be 83.3± 10.3 kJ/mol, which is significantly lower than the reported Ea values for sulfuric acid catalyzed hemicellulose hydrolysis reaction. Model analysis suggests that increasing maleic acid concentrations from 0.05 to 0.2 M facilitate improvement in xylose yields from 40% to 85%, while the extent of improvement flattens to near-quantitative by increasing catalyst loading from 0.2 to 1 M. The model was confirmed for the hydrolysis of corn stover at 1 M maleic acid concentrations at 150 C, resulting in a xylose yield of 96% of theoretical. The refined Saeman model was used to evaluate the optimal condition for monomeric xylose yield in the maleic acid-catalyzed reaction: low temperature reaction conditions were suggested, however, experimental results indicated that bi-phasic behavior dominated at low temperatures, which may be due to the insufficient removal of acetyl groups. A combination of experimental data and model analysis suggests that around 80-90% xylose yields can be achieved at reaction temperatures between 100 and 150 C with 0.2 M maleic acid. 

Research Area: Biofuels/Bioproducts    

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Life Cycle Assessment of Fuel Ethanol Derived from Corn Grain Via Dry Milling

2008

Authors: Seungdo Kim, Bruce E. Dale 
Journal: Bioresource Technology, 99, 5250-5260 (2008). 
Book Chapter: 

Abstract: Life cycle analysis enables us to investigate environmental performance of fuel ethanol used in an E10 fueled compact passenger vehicle. Ethanol is derived from corn grain via dry milling. This type of analysis is an important component for identifying practices that will help to ensure that a renewable fuel, such as ethanol, may be produced in a sustainable manner. Based on data from eight counties to seven Corn Belt states as corn farming sites, we show ethanol derived from corn grain as E10 fuel would reduce nonrenewable energy and greenhouse gas emissions, but would increase acidification, eutrophication and photochemical smog, compared to using gasoline as liquid fuel. The ethanol fuel systems considered in this study offer economic benefits, namely more money returned to society than the investment for producing ethanol. The environmental perfromance of ethanol fuel system varies significantly with corn farming sites because of different crop management practices, soil properties, and climatic conditions. The dominant factor determining most environmental impacts considered here (i.e., greenhouse gas emissions, acidification, eutrophication, and photochemical smog formation) is soil related nitorgen losses (e.g., N2O, NOx, and NO3-). The sources of soil nitrogen include nitrogen fertilizer, crop residues, and air deposition. Nitrogen fertilizer is probably the primary source. Simulations using an agro-ecosystem model predict that planting winter cover crops would reduce soil nitrogen losses and increase soil organic carbon levels, thereby greatly improving the environmental performance of the ethanol fuel system. 

Research Area: Biofuels/Bioproducts    

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Pathways for Development of a Biorenewables Industry

2008

Authors: C. Schell, C. Riley, and G. R. Petersen 
Journal: Bioresource Technology, 99(12), 5160-5164 (2008) 
Book Chapter: 

Abstract: The advanced energy initiative to reduce the nation's future demand for oil has resulted in the definition of a number of pathways for the development of the bio-renewables industry. This paper gives an overview of the pathways which could lead to both ethanol and other types of bio-products. The methods that would be used for cellulose conversion also apply to adding value for the co-products of ethanol production. Process milestones and pathways for research that would enable corn dry mill operations to improve are described. A corn dry mill improvement pathway is outlined, and introduces the topics that are covered in this particular special volume.

Research Area: Biofuels/Bioproducts    

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Process simulation of modified dry grind ethanol plant with recycle of pretreated and enzymatically hydrolyzed distillers’ grains

2008

Authors: Y. Kim, N. Mosier, M. R. Ladisch 
Journal: Bioresource Technology, 99, 5177-5192 (2008). 
Book Chapter: 

Abstract: Distillers’ grains (DG), a co-product of a dry grind ethanol process, is an excellent source of supplemental proteins in livestock feed. Studies have shown that, due to its high polymeric sugar contents and ease of hydrolysis, the distillers’ grains have potential as an additional source of fermentable sugars for ethanol fermentation. The benefit of processing the distillers’ grains to extract fermentable sugars lies in an increased ethanol yield without significant modification in the current dry grind technology. Three different potential configurations of process alternatives in which pretreated and hydrolyzed distillers’ grains are recycled for an enhanced overall ethanol yield are proposed and discussed in this paper based on the liquid hot water (LHW) pretreatment of distillers’ grains. Possible limitations of each proposed process are also discussed. This paper presents a compositional analysis of distillers’ grains, as well as a simulation of the modified dry grind processes with recycle of distillers’ grains. Simulated material balances for the modified dry grind processes are established based on the base case assumptions. These balances are compared to the conventional dry grind process in terms of ethanol yield, compositions of its co-products, and accumulation of fermentation inhibitors. Results show that 14% higher ethanol yield is achievable by processing and hydrolyzing the distillers’ grains for additional fermentable sugars, as compared to the conventional dry grind process. Accumulation of fermentation by-products and inhibitory components in the proposed process is predicted to be 2–5 times higher than in the conventional dry grind process. The impact of fermentation inhibitors is reviewed and discussed. The final eDDGS (enhanced dried distillers’ grains) from the modified processes has 30–40% greater protein content per mass than DDGS, and its potential as a value-added process is also analyzed. While the case studies used to illustrate the process simulation are based on LHW pretreated DG, the process simulation itself provides a framework for evaluation of the impact of other pretreatments.

Research Area: Biofuels/Bioproducts    

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Separation of Silymarins from Milk Thistle (Silybum marianum L.) Extracted with Pressurized Hot Water Using Fast Centrifugal Partition Chromatography

2008

Authors: A. S. Engelberth, D. J. Carrier, E. C. Clausen
Journal: Journal of Liquid Chromatography & Related Technologies, 31 (19), 3001-3011
Book Chapter: 

Abstract: Fast centrifugal partition chromatography was used to separate a class of flavonolignans called silymarins from both a purchased silymarin powder and a crude pressurized hot water extract of milk thistle (Silybum marianum L.). Initially, a purchased power of a mixture of the six silymarin compounds was separated with a two-phase solvent system consisting of heptane/ethyl acetate/methanol/water (1:4:3:4 v/v/v/v) in order to verify elution times of the compounds by fast centrifugal partition chromatography. Next, a crude pressurized hot water extract from 10 g of ground seeds of Silybum marianum was separated with the same solvent system. The separation from the hot water extract gave yields of silychristin at 70.2% purity, silydianin at 93.7% purity, and a mixture of silybinin and isosilybinin at 96.1% purity.

Research Area: Bioseparations    

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Simultaneous Quantification of Metabolites Involved in Central Carbon and Energy Metabolism Using Reverse Phase Liquid Chromatography - Mass Spectrometry and In Vitro 13C Labeling

2008

Authors: Yang, W.-C., Sedlak, M., Regnier, F., Mosier, N., Ho, N. and Adamec, J. 
Journal: Analytical Chemistry, 80(24), 9508-9516 (2008) 
Book Chapter: 

Abstract: Comprehensive analysis of intracellular metabolites is a critical component of elucidating cellular processes. Although the resolution and flexibility of reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) makes it one of the most powerful analytical tools for metabolite analysis, the structural diversity of even the simplest metabolome provides a formidable analytical challenge. Here we describe a robust RPLC-MS method for identification and quantification of a diverse group of metabolites ranging from sugars, phosphosugars, and carboxylic acids to phosphocarboxylic acids, nucleotides, and coenzymes. This method is based on in vitro derivatization with a C-13 labeled tag that allows internal standard based quantification and enables separation of structural isomer pairs like glucose 6-phosphate and fructose 6-phosphate in a single chromatographic run. Calibration curves for individual metabolites showed linearity ranging over more than 2 orders of magnitude with correlation coefficients of R-2 > 0.9975. The detection limits at a signal-to-noise ratio of 3 were below 1.0 mu M (20 pmol) for most compounds. Thirty common metabolites involved in glycolysis, the pentose phosphate pathway, and tricarboxylic acid cycles were identified and quantified from yeast lysate with a relative standard deviation of less than 10%. 

Research Area: Biofuels/Bioproducts    

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Water Solubilization of DDGS via Derivatization with Phosphite Esters

2008

Authors: Oshel, R. E., M. V. Nandakumar, S. Urgaonkar, D. G. Hendricker, and J. G. Verkade 
Journal: Bioresource Technology, 99(12), 5193-5205 (2008) 
Book Chapter: 

Abstract: Ethanol production from corn starch in the corn dry milling process leaves Distillers' Dry Grains and Solubles (DDGS) as a major by-product from which additional ethanol may be economically obtained from its glucan content. A challenge in processing the cellulose content of this material lies in its extensive inter-cellulose chain hydrogen bonding, which inhibits access of enzymes capable of cleaving glycosidic bonds, a transformation required for providing fermentable sugars. The phosphitylation of cellulosic OH groups using a reactive bicyclic phosphite ester is utilized to disrupt cellulosic hydrogen bonds, thus providing access to cellulose chains for further processing. We describe a method of pretreating DDGS with commercially available trimethylolpropane phosphite [P(OCH2)3CEt] in the presence of a slight molar excess of water to afford greater than 90% DDGS solubility in the reaction mixture in methanol and in water. Preliminary results using a model compound [D-(+)-permethylated cellobiose] indicate that glycosidic bonds are cleaved as a consequence of this pretreatment.

Research Area: Biofuels/Bioproducts    

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Biomimetic Catalysis for Hemicellulose Hydrolysis in Corn Stover

2007

Authors: Yulin Lu and Nathan S. Mosier 
Journal: Biotechnology Progress, 23, 116-123 (2005) 
Book Chapter: 

Abstract: Efficient and economical hydrolysis of plant cell wall polysaccharides into monomeric sugars is a significant technical hurdle in biomass processing for renewable fuels and chemicals. One possible approach to overcoming this hurdle is a biomimetic approach with dicarboxylic acid catalyst mimicking the catalytic core microenvironment in natural enzymes. This paper reports developments in the use of a dicarboxylic acid catalyst, maleic acid, for hemicellulose hydrolysis in corn stover. Hemicellulose hydrolysis and xylose degradation kinetics in the presence of maleic acid was compared to sulfuric acid. At optimized reaction conditions for each acid, maleic acid hydrolysis results in minimal xylose degradation, whereas sulfuric acid causes 3 - 10 times more xylose degradation. These results formed the basis for optimizing the hydrolysis of hemicellulose from corn stover using maleic acid. At 40 g/L dry corn stover solid-loading, both acid catalysts can achieve near-quantitative monomeric xylose yield. At higher solids loadings (150-200 g dry stover per liter), sulfuric acid catalyzed hydrolysis results in more than 30% degradation of the xylose, even under the previously reported optimal condition. However, as a result of minimized xylose degradation, optimized biomimetic hydrolysis of hemicellulose by maleic acid can reach ~95% monomeric xylose yields with trace amounts of furfural. Fermentation of the resulting unconditioned hydrolysate by recombinant S. cerevisiae results in 87% of theoretical ethanol yield. Enzyme digestibility experiments on the residual corn stover solids show that >90% yields of glucose can be produced in 160 h from the remaining cellulose with cellulases (15 FPU/g-glucan). 

Research Area: Biofuels/Bioproducts    

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Lignin Modification Improves Fermentable Sugar Yields for Biofuel Production

2007

Authors: Fang Chen and Richard A. Dixon 
Journal: Nature Biotechnology , 25(7), 759-761 (2007) 
Book Chapter: 

Abstract: Recalcitrance to saccharification is a major limitation for conversion of lignocellulosic biomass to ethanol. In stems of transgenic alfalfa lines independently downregulated in each of six lignin biosynthetic enzymes, recalcitrance to both acid pretreatment and enzymatic digestion is directly proportional to lignin content. Some transgenics yield nearly twice as much sugar from cell walls as wild-type plants. Lignin modification could bypass the need for acid pretreatment and thereby facilitate bioprocess consolidation. 

Research Area: Biofuels/Bioproducts    

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Loosening Lignin's Grip on Biofuel Production

2007

Authors: Clint Chapple, Michael Ladisch, & Rick Meilan 
Journal: Nature Biotechnology , 25(7), 746-748 (2007)
Book Chapter: 

Abstract: Concerns about global warming, the soaring cost of gasoline and national security issues have rekindled interest in producing l iquid transportation fuels from renewable resources, particularly those derived from cellulose. But, in addition to cellulose, plant cell walls contain lignin, a phenolic polymer that hinders the degradation of cell wall polysaccharides to simple sugars destined for fermentation to ethanol. In this issue, Chen and Dixon use antisense-mediated down-regulation of lignin biosynthesis in alfalfa to demonstrate the potential of transgenic approaches to reduce or eliminate the need for pretreatment of lignocellulosic biomass. 

Research Area: Biofuels/Bioproducts    

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Microscopic Examination of Changes of Plant Cell Structure in Corn Stover Due to Hot Water Pretreatment and Enzymatic Hydrolysis

2007

Authors: Meijuan Zeng, Nathan S. Mosier, Chia-Ping Huang, Debra M. Sherman, and Michael R. Ladisch 
Journal: Biotechnology and Bioengineering, 97(2), 265-278 (2007) 
Book Chapter: 

Abstract: Particle size associated with accessible surface area has a significant impact on the saccharification of plant cell walls by cellulolytic enzymes. Small particle sizes of untreated cellulosic substrate are more readily hydrolyzed than large ones because of higher specific surface area. Pretreatment enlarges accessible and susceptible surface area leading to enhanced cellulose hydrolysis. These hypotheses were tested using ground corn stover in the size ranges of 425-710 and 53-75 µm. Ultrastructural changes in these particles were imaged after treatment with cellulolytic enzymes before and after liquid hot water pretreatment. The smaller 53-75 µm corn stover particles are 1.5 x more susceptible to hydrolysis than 425-710 µm corn stover particles. This difference between the two particle size ranges is eliminated when the stover is pretreated with liquid hot water pretreatment at 190 C for 15 min, at pH between 4.3 and 6.2. This pretreatment causes ultrastructural changes and formation of micron-sized pores that make the cellulose more accessible to hydrolytic enzymes. 

Research Area: Biofuels/Bioproducts    

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Molecular Breeding to Enhance Ethanol Production from Corn and Sorghum Stover

2007

Authors: Wilfred Vermerris, Ana Saballos, Gebisa Ejeta, Nathan S. Mosier, Michael R. Ladisch, and Nicholas C. Carpita 
Journal: Crop Science , 47(S3), S142-S153 (2007) 
Book Chapter: 

Abstract: Political and ennvironmental concerns have resulted in a growing interest in renewable energy, especially transportation fuels. In the United States the majority of fuel ethanol is currently produced from corn (Zea mays L.) starch, but grain supplies will be insufficient to meet anticipated demands. Enzymatic hydrolysis of lignocellulosic biomass such as corn and sorghum [Sorghum bicolor (L.) Moench] stover can provide an abundant alternative source of fermentable sugars. While production of cellulosic ethanol from stover is feasible from an energy-balance perspective, its production is currently not economically competitive. Along with improvements in bioprocessing, enhancing the yield and composition of the biomass has the potential to make ethanol production considerably more cost effective. This requires (i) a better understanding of how cell wall composition and structure affect the efficiency of enzymatic hydrolysis, (ii) the development of traits that enhance biomass conversion efficiency and increase biomass yield, and (iii) the development of rapid screening protocols to evaluate biomass conversion efficiency. Several genetic resources are available to improve maize and sorghum as sources of lignocellulosic biomass. This includes the use of existing mutants, forward and reverse genetics to obtain novel mutants, and transgenic approaches in which the expression of genes of interest is modified. Plant breeding can be implemented to improve biomass yield, biomass quality, and biomass conversion efficiency, either through selection among progeny obtained by crossing parents with desirable traits, or as a way to enhance the agronomic performance of promising mutants and transgenics. Examples from current research will be used to illustrate progress in these different areas. 

Research Area: Biofuels/Bioproducts    

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A Multifunctional Micro-Fluidic System for Dielectrophoretic Concentration Coupled with Immuno-Capture of Low Numbers of Listeria monocytogenes

2006

Authors: Yang, L., P. P. Banada, M. R. Chatni, K. S. Lim, A. K. Bhunia, M. Ladisch, and R. Bashir
Journal: Lab on a Chip , 6, 896-905 (2006)
Book Chapter: 

Abstract: In this study, we demonstrated a micro-fluidic system with multiple functions, including concentration of bacteria using dielectrophoresis (DEP) and selective capture using antibody recognition, resulting in a high capture efficiency of bacterial cells. The device consisted of an array of oxide covered interdigitated electrodes on a flat silicon substrate and a ~ 16 µm high and ~ 260 µm wide micro-channel within a PDMS cover. For selective capture of Listeria monocytogenes from the samples, the channel surface was functionalized with a biotinylated BSA-streptavidin-biotinylated monoclonal antibody sandwich structure. Positive DEP (at 20 Vpp and 1 MHz) was used to concentrate bacterial cells from the fluid flow. DEP could collect ~90% of the cells in a continuous flow at a flow rate of 0.2µl min-1 into the micro-channel with concentration factors between 102 - 103 , in sample volumes of 5-20µl. A high flow rate of 0.6 µl min-1 reduced the DEP capture efficiency to ~65%. Positive DEP attracts cells to the edges of the electrodes where the field gradient is the highest. Cells concentrated by DEP were captured by the antibodies immobilized on the channel surface with efficiencies of 18 to 27% with bacterial cell numbers ranging from 101 to 103 cells. It was found that DEP operation in our experiments did not cause any irreversible damage to bacterial cells in terms of cell viability. In addition, increased antigen expression (antigens to C11E9 monoclonal antibody) on cell membranes was observed following the exposure to DEP.

Research Area: Bioseparations    

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Anomalous Resonance in a Nanomechanical Biosensor

2006

Authors: Gupta, A. K., P. R. Nair, D. Akin, M. R. Ladisch, S. Broyles, M. A. Alam, and R. Bashir 
Journal: Proceedings of the National Academy of Sciences, 103, 13362-13367 (2006)
Book Chapter: 

Abstract: The decrease in resonant frequency of a classical cantilever provides a sensitive measure of the mass of entities attached on its surface. This elementary phenomenon has been the basis of a new class of bio-nanomechanical devices as sensing components of integrated microsystems that can perform rapid, sensitive, and selective detection of biological and biochemical entities. Based on classical analysis, there is a widespread perception that smaller sensors are more sensitive, and this notion has motivated scaling of biosensors to nanoscale dimensions. In this work, we show that the response of a nanomechanical biosensor is far more complex than previously anticipated. Indeed, in contrast to classical microscale sensors, the resonant frequencies of the nanosensor may actually decrease or increase after attachment of protein molecules. We demonstrate theoretically and experimentally that the direction of the frequency change arises from a size-specific modification of diffusion and attachment kinetics of biomolecules on the cantilevers. This work may have broad impact on microscale and nanoscale biosensor design, especially when predicting the characteristics of bio-nanoelectromechanical sensors functionalized with biological capture molecules.

Research Area: Bioseparations    

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Surface Engineering of Microchannel Walls for Protein Separation and Directed Microfluidic Flow

2006

Authors: T. T. Huang, N. S. Mosier, and M. R. Ladisch 
Journal: Journal of Separation Science, 29(12), 1733-1742 (2006) 
Book Chapter: 

Abstract: The preparation of surfaces in microfluidic devices that selectively retain proteins may be difficult to implement due to the incompatibility of derivatization methods with microdevice fabrication techniques. This review describes recently reported developments in simple and rapid methods for engineering the surface chemistries of microchannels bassed on construction of press-fit microdevices. These devices are fabricated by placing a glass fiber on a PDMS film and pressing the film on a silicon wafer or a microscope slide that has been derivatized with octadecyltrichlorosilane (ODS). The film adheres to the slide and forms an elliptically shaped channel around the fiber. The combination of surface wettability of a hydrophilic glass microfiber and the surrounding hydrophobic microchannel surfaces directs a narrow boundary layer of liquid next to the fiber in order to bring the sample in contact with the separation media and results in selective retention of proteins. This phenomenon may be exploited to enable microscale separation applications since there are a wide variety of fibers available with different chemistries. These may be used to rapidly fabricate microchannels that serve as stationary phases for separation at a microscale. The fundamental properties of such devices are discussed. 

Research Area: Bioseparations    

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Surface-Directed Boundary Flow in Microfluidic Channels

2006

Authors: Huang, T. T., D. G. Taylor, K. S. Lim, M. Sedlak, R. Bashir, N. S. Mosier, and M. R. Ladisch 
Journal: Langmuir, 22, 6429-6437 (2006)
Book Chapter: 

Abstract: Channel geometry combined with surface chemistry enables a stable liquid boundary flow to be attained along the surfaces of a 12 µm diameter hydrophilic glass fiber in a closed semi-elliptical channel. Surface free energies and triangular corners formed by PDMS/glass fiber or OTS/glass fiber surfaces are shown to be responsible for the experimentally observed wetting phenomena and formation of liquid boundary layers that are 20-50 µm wide and 12 µm high. Viewing this stream through a 20 µm slit results in a virtual optical window with a 5 pL liquid volume suitable for cell counting and pathogen detection. The geometry that leads to the boundary layer is a closed channel that forms triangular corners where glass fiber and the OTS coated glass slide or PDMS touch. The contact angles and surfaces direct positioning of the fluid next to the fiber. Preferential wetting of corner regions initiates the boundary flow, while the elliptical cross-section of the channel stabilizes the microfluidic flow. The Young-Laplace equation, solved using fluid dynamic simulation software, shows contact angles that exceed 105° will direct the aqueous fluid to a boundary layer next to a hydrophilic fiber with a contact angle of 5°. We believe this is the first time that an explanation has been offered for the case of a boundary layer formation in a closed channel directed by a triangular geometry with two hydrophobic wetting edges adjacent to a hydrophilic surface. 

Research Area: Bioseparations    

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Comparative Sugar Recovery Data from Laboratory Scale Application of Leading Pretreatment Technologies to Corn Stover

2005

Authors: Charles E. Wyman, Bruce E. Dale, Richard T. Elander, Mark Holtzapple, Michael R. Ladisch, Y. Y. Lee 
Journal: Bioresource Technology, 96, 2026-2032 (2005). 
Book Chapter: 

Abstract: Biological processing of cellulosic biomass to fuels and chemicals would open up major new agricultural markets and provide powerful societal benefits, but pretreatment operations essential to economically viable yields have a major impact on costs and performance of the entire system. However, little comparative data is available on promising pretreatments. To aid in selecting appropriate systems, leading pretreatments based on ammonia explosion, aqueous ammonia recycle, controlled pH, dilute acid, flowthrough, and lime were evaluated in a coordinated laboratory program using a single source of corn stover, the same cellulase enzyme, shared analytical methods, and common data interpretation approaches to make meaningful comparisons possible for the first time. Each pretreatment made it possible to subsequently achieve high yields of glucose from cellulose by cellulase enzymes, and the cellulase formulations used were effective in solubilizing residual xylan left in the solids after each pretreatment. Thus, overall sugar yields from hemicellulose and cellulose in the coupled pretreatment and enzymatic hydrolysis operations were high for all of the pretreatments with corn stover. In addition, high-pH methods were found to offer promise in reducing cellulase use provided hemicellulase activity can be enhanced. However, the substantial differences in sugar release patterns in the pretreatment and enzymatic hydrolysis operations have important implications for the choice of process, enzymes, and fermentative organisms. 

Research Area: Biofuels/Bioproducts    

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Comparison of Two Adsorbents for Sugar Recovery from Biomass Hydrolyzate

2005

Authors: Xie, Y., Diana Phelps, Chong-Ho Lee, Miroslav Sedlak, Nancy Ho, and N.-H. L. Wang
Journal: Ind. Eng. Chem. Res., 44, 6816-6823 (2005) 
Book Chapter: 

Abstract: Two polymeric adsorbents, Dowex99 and poly(4-vinyl pyridine) (PVP), have been studied for the recovery of sugars from a corn-stover hydrolyzate. The major components of the hydrolyzate are five sugars, glucose, xylose, mannose, arabinose, and galactose, and four impurities, sulfuric acid, acetic acid, hydroxymethyl furfural (HMF), and furfural. In elution chromatography in a column packed with Dowex99, the five sugars are the "center-cut", whereas sulfuric acid elutes earlier and the other three impurities elute later than the sugars. For a column packed with PVP, the sugars elute earlier than all the impurities. The intrinsic adsorption and mass-transfer parameters of the sugars and the major impurities were obtained from elution and frontal chromatography tests of single components. The experimental elution chromatograms of the hydrolyzate are in close agreement with the simulations based on a detailed rate model and the single-component intrinsic parameters. The results indicate that other unidentified impurities in the hydrolyzate do not affect the adsorption of the identified components. The hydrolyzate sugars recovered from the batch elution chromatography processes were fermented with genetically engineered yeast. The fermentation results show that the hydrolyzate sugars recovered from the PVP columns have the highest fermentability, compared with those for an overlimed hydrolyzate and the sugars recovered from the Dowex99 columns.

Research Area: Biofuels/Bioproducts    

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Coordinated Development of Leading Biomass Pretreatment Technologies

2005

Authors: Charles E. Wyman, Bruce E. Dale, Richard T. Elander, Mark Holtzapple, Michael R. Ladisch, and Y. Y. Lee 
Journal: Bioresource Technology, 96, 1959-1966 (2005). 
Book Chapter: 

Abstract: For the first time, a single source of cellulosic biomass was pretreated by leading technologies using identical analytical methods to provide comparative performance data. In particular, ammonia explosion, aqueous ammonia recycle, controlled pH, dilute acid, flowthrough, and lime approaches were applied to prepare corn stover for subsequent biological conversion to sugars through a Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI) among Auburn University, Dartmouth College, Michigan State University, the National Renewable Energy Laboratory, Purdue University, and Texas A&M University. An Agricultural and Industrial Advisory Board provided guidance to the project. Pretreatment conditions were selected based on the extensive experience of the team with each of the technologies, and the resulting fluid and solid streams were characterized using standard methods. The data were used to close material balances, and energy balances were estimated for all processes. The digestibilities of the solids by a controlled supply of cellulase enzyme and the fermentability of the liquids were also assessed and used to guide selection of optimum pretreatment conditions. Economic assessments were applied based on the performance data to estimate such pretreatment cost on a consistent basis. Through this approach, comparative data were developed on sugar recovery from hemicellulose and cellulose by the combined pretreatment and enzymatic hydrolysis operations when applied to corn stover. This paper introduces the project and summarizes the shared methods for papers reporting results of this research in this special edition of Bioresource Technology. 

Research Area: Biofuels/Bioproducts    

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Dielectrophoresis and Antibody Mediated Selective Capture of Microorganisms in Micro-Fluidic Biochips

2005

Authors: H. Li, L. Yang, D. Akin, T. Geng, A. Bhunia, T. T. Huang, M. Ladisch, R. Bashir 
Journal: The 13th International Conference on Solid-State Sensors, Actuators and Microsystems, Seoul, Korea, June 5-9, 2005, Vol. 2, Papers 3A1.1-4D3.4, pp. 1103-2162.
Book Chapter: 

Abstract: A dielectrophoretic (DEP) filter device was fabricated for antibody mediated specific capture of microorganisms. The device consists of a network of fluidic inlet/outlet ports and chamber etched into silicon substrate with bonded glass on the top and interdigitated electrodes at the bottom of the chamber. The whole electrode array was covered with a thin silicon oxide layer for preventing electroosmotic currents at the electrodes. For selective capture of Listeria monocytogenes from the mixture of L. monocytogenes and Escherichia coli, the channel surface of the DEP filter device was functionalized with biotinylated BSA-streptavidin-biotinylated monoclonal antibody sandwich structure. Postive DEP (at 20Vpp and 1 MHz) was used to attract, capture and concentrate all bacteria in the sample from the fluid flow. About 8% Listeria bacteria were captured while no E. coli was captured with 40 min DEP. We demonstrated that this novel method combining dielectrophoresis, micro-fluidics, and antibody-antigen recognition can be used to selectively capture L. monocytogenes or other target bacteria in the microfluidic device with good efficiency and selectivity. 

Research Area: Bioseparations    

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Features of Promising Technologies for Pretreatment of Lignocellulosic Biomass

2005

Authors: N. Mosier, C. Wyman, B. Dale, R. Elander, Y. Y. Lee, M. Holtzapple, M. R. Ladisch 
Journal: Bioresource Technology, 96, 673-686 (2005)
Book Chapter: 

Abstract: Cellulosic plant material represents an as-of-yet untapped source of fermentable sugars for significant industrial use. Many physio-chemical structural and compositional factors hinder the enzymatic digestibility of cellulose present in lignocellulosic biomass. The goal of any pretreatment technology is to alter or remove structural and compositional impediments to hydrolysis in order to improve the rate of enzyme hydrolysis and increase yields of fermentable sugars from cellulose or hemicellulose. These methods cause physical and/or chemical changes in the plant biomass in order to achieve this result. Experimental investigation of physical changes and chemical reactions that occur during pretreatment is required for the development of effective and mechanistic models that can be used for the rational design of pretreatment processes. Furthermore, pretreatment processing conditions must be tailored to the specific chemical and structural composition of the various, and variable, sources of lignocellulosic biomass. This paper reviews process parameters and their fundamental modes of action for promising pretreatment methods. 

Research Area: Biofuels/Bioproducts    

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Industrial Scale-Up of pH-Controlled Liquid Hot Water Pretreatment of Corn Fiber for Fuel Ethanol Production

2005

Authors: N. S. Mosier, R. Hendrickson, M. Brewer, N. W. Y. Ho, M. Sedlak, R. Dreshel, G. Welch, B. S. Dien, A. Aden, and M. R. Ladisch 
Journal: Applied Biochemistry and Biotechnology, 125, 77-85 (2005)
Book Chapter: 

Abstract: The pretreatment of cellulose in corn fiber by liquid hot water at 160 C and a pH above 4.0 dissolved 50% of the fiber in 20 min. The pretreatment also enabled the subsequent complete enzymatic hydrolysis of the remaining polysaccharides to monosaccharides. The carbohydrates dissolved by the pretreatment were 80% soluble oligosaccharides and 20% monosaccharides with <1% of the carbohydrates lost to degradation products. Only a minimal amount of protein was dissolved, thus enriching the protein content of the undissolved material. Replication of laboratory results in an industrial trial at 43 gallons per minute (163 L/min) of fiber slurry with a residence time of 20 min illustrates the utility and practicality of this approach for pretreating corn fiber. The added costs owing to pretreatment, fiber, and hydrolysis are equivalent to less than $0.84/gal of ethanol produced from the fiber. Minimizing monosaccharide formation during pretreatment minimized the formation of degradation products; hence, the resulting sugars were readily fermentable to ethanol by the recombinant hexose and by pentose-fermenting Saccharomyces cerevisiae 424A(LNH-ST) and ethanologenic Escherichia coli at yields >90% of theoretical based on the starting fiber. This cooperative effort and first successful trial opens the door for examining the robustness of the pretreatment system under extended run conditions as well as pretreatment of other celluylose-containing materials using water at controlled pH.

Research Area: Biofuels/Bioproducts    

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Mechanistic Study of Membrane Concentration and Recovery of Listeria monocytogenes

2005

Authors: Wan-Tzu Chen, Richard L. Hendrickson, Chia-Ping Huang, Deb Sherman, Tao Geng, Arun K. Bhunia, Michael R. Ladisch
Journal: Biotechnology and Bioengineering, 89, 3, 263-273 (2005)
Book Chapter: 

Abstract: Detection of the foodborne pathogen Listeria monocytogenes requires that food samples be processed to remove proteins and lipids, concentrate microorganisms to a detectable concentration, and recover the concentrated cells in a small volume compatible with micron-scale biochips. Mechanistic considerations addressed in this research include the roles of membrane structure, pore size, and detergents in maximizing recovery of cells from a complex biological fluid. The fluid in this case was a food sample (hotdog extract) innoculated with L. monocytogenes. This study showed how membrane filtration using a syringe filter is able to concentrate L. monocytogenes by 95 x with up to 95% recovery of living microorganisms by concentrating 50 mL of food samplejinto a volume of 500 uL. Tween 20 was added to the sample to prevent irreversible adsorption of the microorganism to the membrane and thereby help to ensure high recovery. Comparison of polycarbonate, mixed cellulose, nylon, and PVDF membranes with 0.2 to 0.45 um pores showed the 0.2 um polycarbonate membrane with straight through, mono-radial pores gives the highest recovery of living microorganisms. The mixed cellulose, nylon, and PVDF membranes have a fibrous structure whose characteristic openings are much larger than their effective pore size cut-offs of 0.22 or 0.45 um. We define conditions for rapid membrane-based cell concentration and recovery that has the potential to supplant enrichment steps that require a day or more. This approach has the added benefit of facilitating examination of a large amount of fluid volume by reducing its volume to a range that is compatible with the microliter scales of biochip or other biosensor detection systems. 

Research Area: Food Safety     

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Membrane for Selective Capture of the Microbial Pathogen Listeria monocytogenes

2005

Authors: Wan-Tzu Chen, Michael R. Ladisch, Tao Geong, Arun K. Bhunia
Journal: AIChE Journal, 51(12), 3305-3308, 2005
Book Chapter: 

Abstract: Membrane filtration is widely used for sterile filtration, flowthrough immuno-filtration, virus removal, and microbiological analysis.1,2,3,4,5,6 Prior work has described membranes that retain one protein over others.7,8 The purpose of this work is to capture a target species of living cells from a mixed population of microorganisms being concentrated in the retentate of solution being pushed through a membrane. We report selective capture of one living microorganism from another by an antibody immobilized on a lysine-modified polycarbonate membrane. Capture of a relatively large microbial entity (L. monocytogenes at 1 m) by a 75 to 150 fold smaller ligand (the antibody) occurs with high efficiency, when a spacer (poly-Llysine) reacted to the membrane’s surface is derivatized with an antibody (P66) specific to Listeria. The results show that isolation of the food pathogen L. monocytogenes from E. coli in less than 2 h is possible.

Research Area: Food Safety    

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Microfiber-Directed Boundary Flow in Press-Fit Microdevices Fabricated from Self-Adhesive Hydrophobic Surfaces

2005

Authors: T. T. Huang, D. G. Taylor, M. Sedlak, N. S. Mosier, and M. R. Ladisch 
Journal: Analytical Chemistry, 77, 3671-3675 (2005). 
Book Chapter: 

Abstract: We report a rapid microfluidic device construction technique which does not employ lithography or stamping methods. Device assembly physically combines a silicon wafer, an elastomer (poly(dimethylsiloxane) (PDMS)), and microfibers to form patterns of hydrophobic channels, wells, elbows, or orifices that direct fluid flow into controlled boundary layers. Tweezers are used to place glass microfibers in a defined pattern onto an elastomeric (PDMS) hydrophobic film. The film is then manually pressed onto a hydrophobic silicon wafer, causing it to adhere to the silicon wafer and form a liquid-tight seal around the fibers. Completed in 15 min, the technique results in an operable microdevice with micrometer-scale features of nanoliter volume. Microfiber-directed boundary flow is achieved by use of the surface wetting properties of the hydrophilic glass fiber and the hydrophobicity of surrounding surfaces. The simplicity of this technique allows quick prototyping of microfluidic components, as well as complete biosensor systems, such as we describe for the detection of pathogenic bacteria. 

Research Area: Bioseparations    

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Optimization of pH Controlled Liquid Hot Water Pretreatment of Corn Stover

2005

Authors: Nathan Mosier, Richard Hendrickson, Nancy Ho, Miroslav Sedlak, Michael R. Ladisch
Journal: Bioresource Technology, 96, 1986-1993 (2005)
Book Chapter: 

Abstract: Controlled pH, liquid hot water pretreatment of corn stover has been optimized for enzyme digestibility with respect to processing temperature and time. This processing technology does not require the addition of chemicals such as sulfuric acid, lime, or ammonia that add cost to the process because these chemicals must be neutralized or recovered in addition to the significant expense of the chemicals themselves. Second, an optimized controlled pH, liquid hot water pretreatment process maximizes the solubilization of the hemicellulose fraction as liquid soluble oligosaccharides while minimizing the formation of monomeric sugars. The optimized conditions for controlled pH, liquid hot water pretreatment of a 16% slurry of corn stover in water was found to be 190 C for 15 min. At the optimal conditions, 90% of the cellulose was hydrolyzed to glucose by 15 FPU of cellulase per gram of glucan. When the resulting pretreated slurry, in undiluted form, was hydrolyzed by 11 FPU of cellulase per gram of glucan, a hydrolyzate containing 32.5 g/L glucose and 18 g/L xylose was formed. Both the xylose and the glucose in this undiluted hydrolyzate were shown to be fermented by recombinant yeast 424A(LNH-ST) to ethanol at 88% of theoretical yield.

Research Area: Biofuels/Bioproducts    

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Plug-Flow Reactor for Continuous Hydrolysis of Glucans and Xylans from Pretreated Corn Fiber

2005

Authors: Young-mi Kim, Rick Hendrickson, Nathan Mosier, and Michael R. Ladisch 
Journal: Energy & Fuels, 19, 2189-2200 (2005). 
Book Chapter: 

Abstract: Pressure cooking of corn fiber in liquid water at 160 C and a pH maintained at 4-7 produces an aqueous stream of dissolved glucans, xylans, proteins, phenolics, and minerals. We report hydrolysis of these oligosaccharides to glucose and xylose in a fixed-bed reactor packed with a macroreticular strong cation exchanger. The aqueous stream is first contacted with the cation exchanger at room temperature where proteins, phenolics, minerals, and other catalyst fouling components are removed. The material is then passed over a packed-bed of the same catalyst at 130 C to give 88% hydrolysis for a space time of 105 min. Comparison of cation exchanger in a plug-flow versus a batch reactor for hydrolysis of oligosaccharides as well as for hydrolysis of the disaccharide cellobiose shows that yields at 110 - 160 C are greatest for a plug-flow reactor. Maximum glucose yield increases as hydrolysis temperature increases and reaches 90% at 160 C, which was the highest temperature tested in this study. A model of reactor performance based on first-order kinetics with diffusion resistance fit the data for cellobiose with an observed hydrolysis yield of 90% at a residence time of 3.5 min at 160 C. A preliminary economic analysis shows 1 lb of catalyst that generates 1000 lb of glucose will give incremental costs of between $0.01 and $0.18/gal of ethanol, depending on catalyst cost. Further improvements in catalyst life and selectivity could result in an alternative or complimentary approach to enzyme hydrolysis for biomass pretreatment processes that generate water-soluble glucans and xylans from corn fiber and other cellulosic residues. Ultimately a sequential continuous pretreatment and hydrolysis system is envisioned that has the added benefit of minimizing reactor volumes in large-scale cellulose to ethanol plants.

Research Area: Biofuels/Bioproducts    

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Separation of Denatured Proteins in Free Solution on a Microchip Based on Differential Binding of Alkyl Sulfates with Different Carbon Chain Lengths

2005

Authors: Chang Lu, Aaron E. Smith and Harold G. Craighead
Journal: Chemical Communication, 183-185 (2005).
Book Chapter: 

Abstract: Electrophoresis of polyelectrolytes such as DNA and denatured proteins is usually performed in chemical or physical gels instead of in free solution, except in some special cases. It has been generally accepted that proteins with molecular weights more than 10 kDa have a constant free solution mobility that is independent of their molecular weights, after they are fully denatured by sodium dodecyl sulfate (SDS) and a reducing agent. This phenomenon is generally attributed to the constant charge density along the polypeptide chain. The coating of the negatively charged surfactant makes the intrinsic charge of the proteins insignificant. For similar reasons, DNA fragments longer than 10–20 bp have the same free solution mobilities regardless of fragment size or base composition. In this report, we demonstrate that denatured proteins have different electrophoretic mobilities in free solution after the denaturation is carried out using a mixture of alkyl sulfates with different carbon chain lengths. Furthermore, the free solution mobilities are not correlated with the molecular weights of the proteins. In this work, the free solution electrophoresis was carried out on a glass microchip. 

Research Area: Bioseparations    

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Bioseparation Techniques in Microfluidic Devices Using Micro-Bead

2004

Authors: W.-J. Chang, K. W. Ro, T. T. Huang, Y.-M. Koo, J. H. Hahn, M. R. Ladisch, D. Akin, R. Bashir 
Journal: Theories and Applications of Chemical Engineering, 10, 1, 203-206, (2004) 
Book Chapter: 

Abstract: There has been a significant increase of interest on microfluidic device as miniaturized analytical system, recently. Micron-size fluidic paths and other components are integrated in microfluidic device, performing essential procedures for the analysis of chemical and/or biological materials (Harrison et al., 1993, Jacobson et al., 1994). The device that has dimensions of a few centimeters is capable of providing rapid identification of molecules and enhanced sensitivity with reduced consumption of reagents and samples (Stone and Kim, 2001). Various separation processes have been applied to microfluidic device such as zone electrophoresis, gel electrophoresis, isoelectric focusing, micellar electrokinetic chromatography (MEKC) and electrochromatography, resulted in the increase of its applications. The applications using polymer-based microfluidic devices are increasing due to their ease of fabrication, inexpensive fabrication costs and increasing versatility (Becker and Locascio, 2002). These devices have been fabricated from various polymers including poly(methyl- methacrylate), polycarbonate, polystyrene, and poly(dimethylsiloxane) (PDMS). The usages of PDMS are increasing among these polymers because of relatively lower expense and simpler procedures for fabrication than others. The control of minute volume of liquid in PDMS microfluidic device using electroosmotic flow (EOF) enhanced the increase of its application. In this work, capillary electrochromatography (CEC) and preconcentration of neutral compounds have been realized on poly(dimethylsiloxane) (PDMS) microfluidic devices. The micro-structures of PDMS were fabricated in micro-channel for the packing of micro-beads. In addition, rapid prototyping technique for the fabrication of micro-channels using micro-fiber and PDMS slab was developed. The flexible characteristic of PDMS makes simple fabrication of micro-channel possible, by directed placement of glass micro-fibers on solid substrate. The applications of developed microfluidic device are tested.

Research Area: Bioseparations    

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Characterization of the Effectiveness of Hexose Transporters for Transporting Xylose During Glucose and Xylose Co-Fermentation by a Recombinant Saccharomyces Yeast

2004

Authors: Miroslav Sedlak and Nancy W. Y. Ho
Journal: Yeast, 21, 671-684 (2004) 
Book Chapter: 

Abstract: We have developed recombinant Saccharomyces yeasts that can effectively co-ferment glucose and xylose to ethanol. However, these yeasts still ferment glucose more efficiently than xylose. The transport of xylose could be one of the steps limiting the fermentation of xylose. In this study, we characterized the changes in the expression pattern of the hexose transporter and related genes during co-fermentation of glycose and xylose using one of our recombinant yeasts, Saccharomyces cerevisiae 424A(LNH-ST). The transcription of the hexose transporter and related genes was strongly influenced by the presence of glucose; HXT1, HXT2 and HXT3 were greatly activated by glucose and HXT5, HXT7 and AGT1 were significantly repressed by glucose. We also examined the effectiveness of individual transporters encoded by HXT1, HXT2, HXT4, HXT5, HXT7 and GAL2 genes for transporting xylose during co-fermentation of glucose and xylose in a Saccharomyces hxt mutant (RE700A). We compared these hxt derivatives to RE700A wild-type strain (S. cerevisiae MC996A) where all of them contained the same xylose metabolizing genes present in our xylose-fermenting yeasts such as 424A(LNH-ST). Our results showed that recombinant RE700A containing the cloned HXT7 or HXT5 were substantially more effective for fermenting xylose to ethanol. In addition, we found that the efficiency of transporters for intracellular accumulation of xylose was as follows: HXT7 > HXT5 > GAL2 > WT > HXT1 > HXT4 >>> RE700A. Furthermore, we provided evidence that the Saccharomyces galactose transporter system could be a highly effective xylose transporter. The information reported here should be of great importance for improving the Saccharomyces yeast transport of xylose.

Research Area: Biofuels/Bioproducts    

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Detection of Low Levels of Listeria monocytogenes Cells by Using a Fiber-Optic Immunosensor

2004

Authors: Geng, T., Morgan, M. T., and Bhunia, A. K. 
Journal: Applied and Environmental Microbiology, 70, 10, 6138-6146 (2004) 
Book Chapter: 

Abstract: Biosensor technology has a great potential to meet the need for sensitive and nearly real-time microbial detection from foods. An antibody-based fiber-optic biosensor to detect low levels of Listeria monocytogenes cells following an enrichment step was developed. The principle of the sensor is a sandwich immunoassay where a rabbit polyclonal antibody was first immobilized on polystyrene fiber waveguides through a biotin-streptavidin reaction to capture Listeria cells on the fiber. Capture of cells on the fibers was confirmed by scanning electron microscopy. A cyanine 5-labeled murine monoclonal antibody, C11E9, was used to generate a specific fluorescent signal, which was acquired by launching a 635-nm laser light from an Analyte 2000 and collected by a photodetector at 670 to 710 nm. This immunosensor was specific for L. monocytogenes and showed a significantly higher signal strength than for other Listeria species or other microorganisms, including Escherichia coli, Enterococcus faecalis, Salmonella enterica, Lactobacillus plantarum, Carnobacterium gallinarum, Hafnia alvei, Corynebacterium glutamicum, Enterobacter aerogenes, Pseudomonas aeruginosa, and Serratia marcescens, in pure or in mixed-culture setup. Fiber-optic results could be obtained within 2.5 h of sampling. The sensitivity threshold was about 4.3 x 103 CFU/ml for a pure culture of L. monocytogenes grown at 37°C. When L. monocytogenes was mixed with lactic acid bacteria or grown at 10°C with 3.5% NaCl, the detection threshold was 4.1 x 104 or 2.8 x 107 CFU/ml, respectively. In less than 24 h, this method could detect L. monocytogenes in hot dog or bologna naturally contaminated or artificially inoculated with 10 to 1,000 CFU/g after enrichment in buffered Listeria enrichment broth.

Research Area: Food Safety    

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Heat Shock Protein 60 Acts as a Receptor for the Listeria Adhesion Protein in Caco-2 Cells

2004

Authors: J. L. Wampler, K.-P. Kim, Z. Jaradat, and A. K. Bhunia 
Journal: Infection and Immunity, 72, 2, 931-936 (2004) 
Book Chapter: 

Abstract: The 104-kDa Listeria adhesion protein (LAP) in Listeria monocytogenes is involved in binding to various mammalian cell lines. However, the receptor that interacts with LAP in eukaryotic cells is unknown. In this study, scanning immunoelectron microscopy qualitatively demonstrated greater binding capacity of wild-type (WT) L. monocytogenes strain (F4244) than a LAP-deficient mutant strain (KB208) to Caco-2 cells. The goal of this study was identification of the host cell receptor for LAP. Using a Western blot ligand overlay assay, we identified a protein of 58 kDa to be the putative receptor for LAP from Caco-2 cells. N-terminal sequencing and subsequent database search identified this protein as heat shock protein 60 (Hsp60). Modified immunoseparation with protein A-Sepharose beads bound to the LAP-specific monoclonal antibody H7 (MAb-H7) and a sequential incubation with LAP preparation and Caco-2 lysate confirmed the receptor to be the same 58-kDa protein. Western blot analysis with anti-Hsp60 MAb of whole-cell adhesion between Caco-2 and WT also revealed the receptor protein to be a 58-kDa protein, thus corroborating the identification of Hsp60 as a host cell receptor for LAP. Furthermore, the anti-Hsp60 antibody also caused approximately 74% reduction in binding of L. monocytogenes WT to Caco-2 cells, whereas a control antibody, C11E9, had no effect on binding. The adhesion mechanism of L. monocytogenes to eukaryotic cells is a complex process, and identification of Hsp60 as a receptor for LAP adds to the list of previously discovered ligand-receptor modules that are essential to achieve successful adhesion.

Research Area: Food Safety     

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Impedance Microbiology-on-a-Chip: Microfluidic Bioprocessor for Rapid Detection of Bacterial Metabolism

2004

Authors: R. Gomez-Sjoberg, D. T. Morisette, and R. Bashir 
Journal: Journal of Microelectromechanical Systems, 1-8, (2004) 
Book Chapter: 

Abstract: Detection of a few live bacterial cells in many industrial or clinical samples is a very important technological problem. We have developed a microscale technique for concentrating bacterial cells from a dilute sample, by factors on the order of 10 to the 4th power to 10 to the 5th power, and detecting their metabolic activity by purely electrical means. The technique was implemented on a silicon-based microfluidic chip where the cells are concentrated and incubated in a chamber with a volume of 400 pl. Concentration and capture are obtained by the use of dielectrophoresis on the bacterial cells, and metabolism detection is achieved by means of impedance measurements of the medium in which the bacteria are incubated. Performing impedance-based detection at the microscale results in drastically reduced detection times for dilute bacterial samples, thanks to the ability to efficiently concentrate and capture the cells in an extremely small volume. Such concentration eliminates the need to amplify the bacterial population by long culture steps. This detection technique can be used for a wide variety of applications.

Research Area: Food Safety     

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Investigating Pathogen-Specific TLR Signaling of Innate Immune Cells for Biosensor Applications

2004

Authors: A. Lottes, H. Oh, H. HogenEsch, M. Ladisch, J. Hutchcroft, A. Rundell 
Journal: 30th NE Bioengineering Conference, April 17-18, 2004, Western New England College
Book Chapter: 

Abstract: The goal of this project is to develop a real-time cell-based biosensor using Toll-like receptors (TLRs) for pathogen detection. Existing biosensors rely on technologies that recognize only specific target analytes, requiring prior knowledge of the possible contaminating agents. Innate immune cells express TLRs that recognize conserved pathogen-associated molecular patterns on bacteria, viruses, parasites and fungi. Using TLRs as the receptor element in this biosensor will eliminate the need for a priori knowledge of the threat. At least 10 different members of the TLR family are expressed on cells of the innatejimmune system, each responding to different attributes of pathogenic organisms. Through flow cytometry, TLRs 2, 4 and 9 have been identified on THP-1 cells, and TLRs 2, 3, 4, 5 and 9 have been detected on J774 cells. Western blotting has identified Erk activation upon lipopolysacharide (LPS), E. coli and Poly(I):(C) exposure in J774 cells, and upon LPS and E. coli exposure in THP-1 cells. Cellular model systems are being developed to distinguish between bacteria and virus by selective stimulation of TLR3 and TLR5 (TLR3 specifically recognizes double-stranded viral RNA and TLR5 detects bacterial flagellin). A target application of this technology is point--of-care diagnostics. Real-time detection of viruses in nasal or throat swabs could help decrease the inappropriate use of antibiotics. 

Research Area: Food Safety    

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Optimization of a Rapid Dot-Blot Immunoassay for Detection of Salmonella entrica serovar Enteritidis in Poultry Products and Environmental Samples

2004

Authors: Z. W. Jaradat, J. H. Bzikot, J. Zawistowski, A. K. Bhunia
Journal: Food Microbiology, 21, 761-769, (2004) 
Book Chapter: 

Abstract: An immunoassay was developed for the detection of Salmonella serovar enteritidis in poultry and environmental samples. This assay consisted of a two-step procedure that involved an enrichment step using whole egg homogenate (EH) as the enrichment medium and detection by a monoclonal antibody (MAb)-based dot-blot assay. Egg homogenate enriched Salmonella enteritidis was heated to 100 C for 10 min in the presence of cholic acid, a detergent, to liberate the lipopolysaccharide (LPS) antigen in gelled egg matrix. This was subsequently transferred onto a nitrocellulose membrane for detection with MAb 2F11. Several commercially available media were compared with egg homogenate for their relative ability to resuscitate and propagate Salmonella enterititis to detectable levels. Incubation in EH, trypticase soy broth (TSB), and lactose broth (LB) resulted in comparable levels of Salmonella Enteritidis as demonstrated by viable plate counts. Salmonella enteritidis grown in TSB exhibited the greatest visual intensity showing a positive test when tested by the dot-blot assay. Incubation time necessary to detect one cfu of Salmonella enteritidis was reduced from 20 to 10 h using TSB as the enrichment broth. Addition of ferrous sulphate or ferrioxamine E or cholic acid in the enrichment broth had negligible negative effects on the growth of Salmonella. Salmonella enteritidis when incubated with a mixture of naturally contaminated or artificially innoculated competitive micro-organisms in environmental samples at a ratio of 1:10 to the 2nd power, was able to reproduce to detectable numbers for the immunoassay. This method was able to detect all phage types (PT 1, 6, 7, 8, 13, 13a, 14b, 21 and 28) with unique ribopatterns. The results demonstrated that Salmonella enteritidis, when pre-enriched in a medium containing ferrous sulphate or cholic acid, could be readily detected in the presence of 100-fold higher competition of other microorganisms.

Research Area: Food Safety     

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Production of Ethanol from Cellulosic Biomass Hydrolysates Using Genetically Engineered Saccharomyces Yeast Capable of Cofermenting Glucose and Xylose

2004

Authors: Miroslav Sedlak and Nancy W. Y. Ho
Journal: Applied Biochemistry and Biotechnology, 113-116, 403-405 (2004) 
Book Chapter: 

Abstract: Recent studies have proven ethanol to be the ideal liquid fuel for transportation, and renewable lignocellulosic materials to be the attractive feedstocks for ethanol fuel production by fermentation. The major fermentable sugars from hydrolysis of most cellulosic biomass are D-glucose and D-xylose. The naturally occurring Saccharomyces yeasts that are used by industry to produce ethanol from starches and cane sugar cannot metabolize xylose. Our group at Purdue University succeeded in developing genetically engineered Saccharomyces yeasts capable of effectively cofermenting glucose and xylose to ethanol, which was accomplished by cloning three xylose-metabolizing genes into the yeast. In this study, we demonstrated that our stable recombinant Saccharomyces yeast, 424A(LNH-ST), which contains the cloned xylose-metabolizing genes stably integrated into the yeast chromosome in high copy numbers, can efficiently ferment glucose and xylose present in hydrolysates from different cellulosic biomass to ethanol.

Research Area: Biofuels/Bioproducts    

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Rapid Chromatography for Evaluating Adsorption Characteristics of Cellulase Binding Domain Mimetics

2004

Authors: Nathan S. Mosier, Jonathan J. Wilker, Michael R. Ladisch
Journal: Wiley InterScience, 86, 7, 756-764 (2004) 
Book Chapter: 

Abstract: The cost of cellulolytic enzymes is one barrier to the economic production of fermentable sugars from lignocellulosic biomass for the production of fuels and chemicals. One functional characteristic of cellulolytic enzymes that improves reaction kinetics over mineral acids is a cellulose binding domain that concentrates the catalytic domain to the substract surface. We have identified maleic acid as an attractive catalytic domain with pKa and dicarboxylic acid structure properties that hydrolyze cellulose while producing minimal degradation of the glucose formed. In this study we report results of a rapid chromatographic method to assess the binding characteristics of potential cellulose binding domains for the construction of a synthetic cellulase over a wide range of temperatures (20 deg to 120 deg C). Aromatic, planar chemical structures appear to be key indicators of cellulose adsorption. Indole, the sidechain of the amino acid tryptophan, has been shown to reversibly adsorb to cellulose at temperatures between 30 deg and 120 deg C. Trypan blue, a polyarometic, planar molecular, was shown to be irreversibly adsorbed to cotton cellulose at temperatures of <120 deg C on the time scale of the experiments. These results confirm the importance of hydrophobic cellulose and the cellulose-binding component of cellulolytic enzymes and cellulolytic enzyme mimetics.

Research Area: Biofuels/Bioproducts    

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The Role of Bioprocess Engineering in Biotechnology

2004

Authors: Michael R. Ladisch 
Journal: "The Bridge", The National Academy of Engineering, 34, 3, 26-31, (Fall 2004)
Book Chapter: 

Abstract: Biotechnology involves using organisms, tissues, cells, or their molecular comonents to act on living things and to intervene in the workings of cells or the molecular components of cells, including thier genetic material. Biotechnology evolved as a means of producing food, beverages, and medicines. More than 8.00 years ago, it was used to make leavened bread. Some 5,000 years ago, moldy soybean curd was used to treat skin infections in China. The malting of barley and fermentation of beer was ussed in Egypt in 2500 BC. The benefits of biotechnology might be an anomaly if it were not for engineering, specifically bioprocess engineering, the discipline that puts biotechnology to work.

Research Area: Bioprocessing    

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Analysis of the Logistic Function Model: Derivation and Applications Specific to Batch Cultured Microorganisms

2003

Authors: Daniel E. Wachenheim, John A. Patterson, and Michael R. Ladisch
Journal: Bioresource Technology, 86, 157-164 (2003) 
Book Chapter: 

Abstract: Mathematical models are useful for describing microbial growth, both in natural ecosystems and under research conditions. To this end, a rate expression that accounted for depletion of nutrients was used to derive the logistic function model for batch cultures. Statistical analysis was used to demonstrate the suitability of this model for growth curve data. Two linear forms of the model and two procedures for calculating growth rate constants were derived to facilitate statistical evaluation of growth curves. The procedures for calculating growth rate constants were found to be useful for calculation of growth rate constants at each time point, or for estimating growth rate constants from early growth curve data. The utility of the logistic function model and its alternative forms is discussed with respect to planning experiments, analyzing growth curves for the effects of factors other than nutrient limitation, and developing more complete descriptions of cell proliferation.

Research Area: Biofuels/Bioproducts    

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Biotextiles - Monoliths with Rolled Geometrics

2003

Authors: Jeremiah Bwatwa, Yiqi Yang, Chenghong Li, Craig Keim, Christine Ladisch, and Michael Ladisch
Journal: Journal of Chromatography, 67, 235-253, 2003
Book Chapter: 

Abstract: Stationary phases that are formed from textiles are a continuous, interconnected fibrous matrix in the form of yarns and fabric. The fibers are assembled into yarns and the yarns are woven into fabric. Since individual fibers have exhibited poor flow properties when used as stationary phases, rolled fabric stationary phases have been developed. Rolled stationary phases enable a long bed length to be attained while retaining food flow properties [1,2]. This kind of stationary phase orients the fabric into a three-dimensional structure through contact between adjacent layers of fabric where the fabric [1,3,4] supports the fibers (Fig. 11.1(a)) [5] assembled into the yarns (Fig. 11.1(b)) [6], and the woven fabric (Fig. 11.1(c)) [6]. This is a type of monolithic material since there are no distinct or individual particles packed into the column. Further, since the material is a textile, and it is used to fractionate biomolecules, we have called this material a biotextile.

Research Area: Bioseparations    

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Building a Bridge to the Ethanol Industry - Follow-Up Project

2003

Authors: Ladisch, M. R., G. Welch, N. Mosier, and B. Dien 
Journal: National Renewable Energy Laboratory, April 2003 
Book Chapter: 

Abstract: The first trial of the corn fiber pretreatment process has been completed. The data and operating experience for the pump, heat exchanger, coil, and centrifuge show the pretreatment of corn fiber in water is technically achievable and economically feasible. The goals of this trial include showing that the pretreatment process is scalable to fit the existing process at Williams Bioenergy, that pilot-size equipment achieves the goals of pretreatment - at least 75% recovery of starch from the corn fiber, and testing the performance of pilot-scale equipment at or near operational conditions. These goals were met. Cellulose conversion was also significantly enhanced by the pretreatment process.

Research Area: Biofuels/Bioproducts    

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Characterization and Application of a Listeria monocytogenes Reactive Monoclonal Antibody C11E9 in a Resonant Mirror Biosensor

2003

Authors: Amanda A. Lathrop, Ziad W. Jaradat, Tim Haley, Arun K. Bhunia 
Journal: Journal of Immunological Methods, 281, 119-128, (2003) 
Book Chapter: 

Abstract: Typical detection of Listeria monocytogenes involves selective enrichment, isolation and biochemical testing. Development of antibodies to Listeria species has improved detection; however, most antibodies detect all species of Listeria. A previously developed monoclonal antibody (MAb)-C11E9 was examined for its reaction to 13 L. innocua and 40 L. monocytogenes strains representing all 13 serotypes by ELISA. Absorbance values for L. monocytogenes strains were 0.44–3.58 and for L. innocua 0.22–1.44. ELISA reactions were divided into three arbitrary groups of high (Abs 1.0 or higher), intermediate (0.6–0.99) and low (0.18–0.59). Most L. monocytogenes strains (32/41, 78%) were in the high group while only 23% (3/13) of L. innocua were in the same group. In the Western blot assay, antibody reacted with phosphate-buffered saline (PBS) extracted protein preparations of 52, 66 and 97 kDa. Ribopattern of all strains was analyzed and no clear relationship was observed for antibody reaction and ribotype of a given strain. MAb C11E9 was used in a resonant mirror biosensor (IAsys sensor), but failed to detect any viable intact L. monocytogenes cells at levels as high as 108 cells/ml; however, it showed binding (85–150 arc/s) with the surface protein preparations containing the 97-, 66- and 52-kDa proteins at 208 Ag/ml. Binding kinetics of L. monocytogenes and L. innocua surface protein extracts showed significantly ( p<0.05) higher responses than the three other Listeria species (L. ivanovii, L. welshimeri and L. grayi), which could be detected in 10–20 min. These data corroborate with ELISA results. In summary, this study suggest that MAb-C11E9 is suitable for detection of all serotypes of L. monocytogenes despite crossreaction with L. innocua and could be used for detection of soluble protein extracts in the resonant mirror (IAsys) biosensor.

Research Area: Food Safety     

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Composite Surface for Blocking Bacterial Adsorption on Protein Biochips

2003

Authors: Huang, T. T., J. Sturgis, R. Gomez, T. Geng, R. Bashir, A. K. Bhunia, J. P. Robinson, M. R. Ladisch
Journal: Biotechnology and Bioengineering, 81(5), 618-624, (2003)
Book Chapter: 

Abstract: The design and fabrication of protein biochips requires characterization of blocking agents that minimize nonspecific binding of proteins or organisms. Nonspecific adsorption of Escherichia coli, Listeria innocua, and Listeria monocytogenes is prevented by bovine serum albumin (BSA) or biotinylated BSA adsorbed on SiO2 surfaces of a biochip that had been modified with a C18 coating. Biotinylated BSA forms a protein-based surface that in turn binds streptavidin. Because streptavidin has multiple binding sites for biotin, it in turn anchors other biotinylated proteins, including antibodies. Hence, biotinylated BSA simultaneously serves as a blocking agent and a foundation for binding an interfacing protein, avidin or streptavidin, which in turns anchors biotinylated antibody. In our case, the antibody is C11E9, an IgG-type antibody that binds Listeria spp. Nonspecific adsorption of another bacterium, Escherichia coli, is also minimized due to the blocking action of the BSA. The blocking characteristics of BSA adsorbed on C18-derivatized SiO2 surfaces for construction of a protein biochip for electronic detection of pathogenic organisms is investigated.

Research Area: Food Safety     

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DNA Microarray Analysis of the Expression of the Genes Encoding the Major Enzymes in Ethanol Production During Glucose and Xylose Co-fermentation by Metabolically Engineered Saccharomyces Yeast

2003

Authors: Miroslav Sedlak, Howard J. Edenberg, Nancy W.Y. Ho 
Journal: Enzyme and Microbial Technology, 33, 19-28 (2003) 
Book Chapter: 

Abstract: Lignocellulosic biomass, which contains large amounts of glucose and xylose, is the new ideal feedstock for ethanol production used as renewable liquid fuel for transportation. The naturally occurring Saccharomyces yeasts traditionally used for industrial ethanol production are unable to ferment xylose. We have successfully developed genetically engineered Saccharomyces yeasts that can effectively co-ferment both glucose and xylose simultaneously to ethanol. Our engineered yeast contains three xylose metabolizing genes, the xylose reductase (XR), xylitol dehydrogenase (XD) and xylulokinase (XK) genes, fused to glycolytic promoters, on high copy plasmids or integrated into the yeast chromosome in multiple copies. Although our glucose/xylose co-fermenting yeasts are currently the most effective yeast for producing ethanol from cellulosic biomass, they still utilize glucose more efficiently than xylose. We believe that carefully analyzing gene expression during co-fermentation of glucose and xylose to ethanol, using our genetically modified strains, will reveal ways to optimize xylose fermentation. In this paper, we report our results on analyzing the expression of genes in the glycolitic and alcoholic fermentation pathways using microarray technology. We also report the results on the analysis of the activities of the selected enzymes for ethanol production during co-fermentation of glucose and xylose to ethanol by one of our effective glucose/xylose co-fermenting yeasts 424A(LNH-ST).

Research Area: Biofuels/Bioproducts Bioprocessing   

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Expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies C11E9 and EM-7G1 under acid-, salt- or temperature-induced stress environments

2003

Authors: T. Geng, K. P. Kim, R. Gomez, D. M. Sherman, R. Bashir, M. R. Ladisch, A. K. Bhunia
Journal: Journal of Applied Microbiology, 95, 762-777 (2003) 
Book Chapter: 

Abstract: To study the expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies (MAbs) C11E9 and EM-7G1 under acid-, salt- or temperature-induced stress environments.

Research Area: Food Safety     

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Lysozyme for Capture of Microorganisms on Protein Biochips

2003

Authors: Tom Huang, Tao Geng, Jennifer Sturgis, Haibo Li, Rafael Gomez, Rashid Bashir, Arun K. Bhunia, J. Paul Robinson, Michael R. Ladisch
Journal: Enzyme and Microbial Technology, 33, 958-966 (2003) 
Book Chapter: 

Abstract: Lysozyme placed on the SiO2 surfaces that have previously been derivatized with C18 coating will capture both Escherichia coli and Listeria monocytogenes cells from PBS buffer at pH 7.2. This phenomenon is of significance for the design and fabrication of protein biochips that are designed to capture bacteria from buffer or water so that these can be further interrogated with respect to possible pathogenicity. Fluorescent microscopy shows that two types of bacteria (gram-negative E. coli and gram-positive Listeria spp.) will be adsorbed by lysozyme placed on the surface of the biochip but that strong adsorption of the bacteria is reduced but not eliminated when Tween 20 is present (at 0.5%) in the PBS buffer in which the cells are suspended. In comparison, Tween 20 and Bovine Serum Albumin (BSA) almost completely block adsorption of these bacteria on C18 coated surfaces. The combination of a lysozyme surface with Tween 20 gives a greater degree of adsorption of L. monocytogenes than E. coli, and hence suggests selectivity for the more hydrophobic E. coli may be reduced by the Tween 20. This paper presents protocols for preparing protein-coated, SiO2 surfaces and the effect of buffer containing Tween 20 on adsorption of bacteria by SiO2 surfaces coated with C18 to which BSA, lysozyme or C11E9 antibody is immobilized at pH 7.2 and ambient temperature.

Research Area: Bioseparations    

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Micro-assembly of Functionalized Particulate Monolayer on C18-Derivatized SiO2 Surfaces

2003

Authors: Tom T. Huang, Tao Geng, Demir Akin, Woo-Jin Chang, Jennifer Sturgis, Rashid Bashir, Arun K. Bhunia, J. Paul Robinson, and Michael R. Ladisch
Journal: Biotechnology and Bioengineering, 83, 4, 416-427, 2003.
Book Chapter: 

Abstract: This work describes a simple approach to immobilize functionalized colloidal microstructures onto a C18-coated SiO2 substrate via specific or non-specific biomediated interactions. Biotinylated bovine serum albumin pre-adsorbed onto a C18 surface was used to mediate the surface assembly of streptavidin-coated microbeds (2.8 um), while a bare C18 surface was used to immobilize anti-Listeria antibody-coated microbeads (2.8 um) through hydrophobic interactions. For a C18 surface pre-adsorbed with bovine serum albumin, hydrophobic polystyrene microbeads (0.8 um) and positively charged dimethylamino microbeads (0.8 um) and positively charged dimethylamino microbeads (0.8 um) were allowed to self-assemble onto the surface. A monolayer with high surface coverage was observed for both polystyrene and dimethylamino microbeads. The adsorption characteristics of Escherichia coli and Listeria monocytogenes on these microbead-based surfaces were studied using fluorescence microscopy. Both streptavidin microbeads preadsorbed with biotinylated anti-Listeria antibody and anti-Listeria antibody-coated microbeads showed specific capture of L. monocytogenes, while polystyrene and dimethylamino microbeads captured both E. coli and L. monocytogenes non-specifically. The preparation of microbead-based surfaces for the construction of microfluidic devices for separation, detection, or analysis of specific biological species is discussed.

Research Area: Bioseparations    

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Microfiber Assisted Fabrication of Microfluidic Channels Using Poly(dimethylsiloxane)

2003

Authors: Tom T. Huang, Woo-Jin Chang, Demir Akin, Rafael Gomez, Rashid Bashir, Nathan Mosier, Michael R. Ladisch
Journal: AIChE Journal, 49, 11, 2984-2987 (2003)
Book Chapter: 

Abstract: A microfluidic device is typically formed using bulk silicon etching techniques on a silicon substrate (Kovacs et al., 1998). A photolithography step defines the desired pattern on the silicon substrate with photoresist. Etching using acids or gases, followed by a solvent and acid cleaning process to remove residual photoresist, leaves micron-scale features. Such devices are capable of providing rapid identification of nucleic acids, proteins, drugs, or other important biological compounds with enhanced sensitivity and time-to-result, while reducing consumption of expensive reagents compared to microtiter plates or test tube scale analyses (Stone and Kim, 2001; Khandurina and Guttman, 2002; Meldrum and Holl, 2002). Polydimethylsiloxane (PDMS), created by mixing a silicone elastomer base and a curing agent in a 10:1 ratio, gives an alternate material for fabricating microfluidic devices (McDonald and Whitesides, 2002). The liquid pre-polymer is poured over a master generated either from photolithography using a high resolution transparency as a photomask, or by laser ablation or Solid-Object Printing to form the device (Grzybowski et al., 1998; McDonald et al., 2002; McDonald and Whitesides, 2002). We report formation of a master by directed placement of glass microfibers on silicon or glass substrates, followed by pressing a preformed PDMS sheet onto the substrate to form microfluidic channels. Wells are formed by criss-cross fiber patterns, while functionalized microbeads coated onto fibers result in microscale channels that separate proteins. We believe this approach is an attractive research tool, because it places rapid prototyping capability within the reach of laboratories that have access to glass slides, an optical microscope, digital camera, tweezers, and PDMS.

Research Area: Food Safety     

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Model for Temperature Profiles in Large Diameter Electrochromatography Columns

2003

Authors: Craig Keim and Michael Ladisch
Journal: AIChE Journal, 49, 2, 402-410 (2003)
Book Chapter: 

Abstract: Scale-up of electrochromatographic separations has been problematic due to electrically induced heating. A two-dimensional transient temperature model for electrochromatography was developed, which accounts for physical properties of the stationary and mobile phase, and the column wall. The model also accounts for both the temperature effect on the electrical conductivity and a nonuniform, radially variant current density. This model was compared to experimental data from two electrochromatography systems with different cylindrical-column dimensions, packing materials, and operating conditions. In all cases, the model predicts the temperature to within 3 C of the actual temperature, both for column heatup and cooldown. Separation of a mixture of model proteins on the 3.81-cm-ID scale was used as the basis for scale-up calculations. The model identifies equipment parameters that control heating characteristics and can be scaled up to process 75 mL of sample per run.

Research Area: Bioseparations    

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Pathogen Detection, Food-Borne

2003

Authors: R. Bashir 
Journal: McGraw-Hill Yearbook of Science and Technology, 1-3 (2003)
Book Chapter: 

Abstract: The presence of microorganisms in food is a natural and unavoidable occurrence. Cooking generally destroys most harmful bacteria, but undercooked foods, processed ready-to-eat foods, and minimally processed foods can contain harmful bacteria that are serious health threats. Meat, dairy, and poultry products are important reservoirs for many of the food-borne pathogens, including Salmonella, Campylobacter, Listeria, and Escherichia coli O157:H7. Animal by-products, such as feed supplements, may also transmit pathogens to food animals (for example, Salmonella and bovine spongiform encephalopathy). Seafood is another potential source of food-borne pathogens, such as Vibrio, Listeria, and Hepatitis A. Infectious doses of many of these pathogens are very low (~10 bacterial cells), increasing the vulnerability of the elderly, infants, and people with immunological deficiencies or organ transplants. Researchers are continuously searching for sensitive tools that are fast, accurate, and ultrasensitive. In recent years, there has been much research activity in the area of sensor development for detecting pathogenic microorganisms.

Research Area: Food Safety    

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Poly(dimethylsiloxane) (PDMS) and Silicon Hybrid Biochip for Bacterial Culture

2003

Authors: Woo-Jin Chang, Demir Akin, Miroslav Sedlak, Michael R. Ladisch and Rashid Bashir
Journal: Biomedical Microdevices, 5, 4, 281-290 (2003) 
Book Chapter: 

Abstract: In this study, a novel PDMS/silicon hybrid microfluidic biochip was fabricated and tested for the long-term batch culture of bacterial cells. The PDMS (poly(dimethylsiloxane)) cover with 3-dimensional micro-channels for flow was fabricated using Teflon tubing and hole-punch techniques, without photolithographic methods. The PDMS/silicon hybrid biochip was prepared by bonding of PDMS cover and a silicon chip that had electrodes and micro-fluidic channels defined. The absorption of liquid into PDMS cover was characterized and conditions to prevent drying of nutrient media within the micro-chamber were shown. The absorption of liquid from micro-chambers into the PDMS cover was reduced up to 2.5 times by changing the mixing ratio of PDMS and curing agent from 10:1 to 2.5:1. In addition, pre-saturation of the PDMS cover with media prior to the incubation resulted in the preservation of liquid in the micro-chambers for up to 22 hours. Optimization of the mixing ratio and pre-saturation of the PDMS cover reduced the drying time 10 times when compared to the unsaturated PDMS cover composed of 10:1 ratio of PDMS and curing agent. Listeria innocua and a strain of Escherichia coli, expressing green fluorescent protein (GFP), were successfully cultured in batch mode within the PDMS/silicon hybrid biochip.

Research Area: Food Safety    

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Biotechnology for Future Army Applications

2002

Authors: Ladisch, M. R., J. J. Valdes, and LTC R. J. Love
Journal: Army AL&T, July-August, 36-37, (2002) 
Book Chapter: 

Abstract: The September 11, 2001, attacks against the United States and the spate of civilian anthrax casualties provided a painful wake-up call to the Nation. Clearly U.S. adversaries donot need large armies or intercontinental missiles to threaten ordinary citizens, and asymmetrical warfare can potentially negate traditional military strengths. The Army's challenge is to use science and technology to consistently transform itself with the expanding spectrum of threats.

Research Area: Bioseparations    

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Characterization of Acid Catalytic Domains for Cellulose Hydrolysis and Glucose Degradation

2002

Authors: Mosier, N. S., C. M. Ladisch, and M. R. Ladisch
Journal: Biotechnology and Bioengineering, 79, 610-618, (2002) 
Book Chapter: 

Abstract: Cellulolytic enzymes consist of a catalytic domain, a linking peptide, and a binding domain. The paper describes research on carboxylic acids that have potential as catalytic domains for constructing organic macromolecules for use in cellulose hydrolysis that mimic the action of enzymes. The tested domains consist of the series of mono-, di-, and tricarboxylic acids with a range of pKa’s. This paper systematically characterizes the acids with respect to hydrolysis of cellobiose, cellulose in biomass, and degradation of glucose and compares these kinetics data to dilute sulfuric acid. Results show that acid catalyzed hydrolysis is proportional to H+ concentration. The tested carboxylic acids did not catalyze the degradation of glucose while sulfuric acid catalyzed the degradation of glucose above that of water alone. Consequently, overall yields of glucose obtained from cellobiose and cellulose are higher for the best carboxylic acid tested, maleic acid, when compared to sulfuric acid at equivalent solution pH.

Research Area: Biofuels/Bioproducts    

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Ganoderma lucidum Suppresses Motility of Highly Invasive Breast and Prostate Cancer Cells

2002

Authors: Daniel Sliva, Carlos Labarrere, Veronika Slivova, Miroslav Sedlak, Frank P. Lloyd Jr., and Nancy W.Y. Ho
Journal: Biochemical and Biophysical Research Communications, 298, 603-612 (2002)
Book Chapter: 

Abstract: A dried powder from basidiomycetous fungi, Ganoderma lucidum, has been used in East Asia in therapies for several different diseases, including cancer. However, the molecular mechanisms involved in the biological actions of Ganoderma are not well understood. We have recently demonstrated that phosphatidylinositol 3-kinase (PI 3-kinase) and nuclear factor-j B (NF-jB) regulate motility of highly invasive human breast cancer cells by the secretion of urokinase-type plasminogen activator (uPA). In this study, we investigated the effect of G. lucidum on highly invasive breast and prostate cancer cells. Here we show that spores or dried fruiting body of G. lucidum inhibit onstitutively active transcription factors AP-1 and NF-jB in breast MDA-MB-231 and prostate PC-3 cancer cells. Furthermore, Ganoderma inhibition of expression of uPA and uPA receptor (uPAR), as well secretion of uPA, resulted in the suppression of the migration of MDA-MB-231 and PC-3 cells. Our data suggest that spores and unpurified fruiting body of G. lucidum inhibit invasion of breast and prostate cancer cells by a common mechanism and could have potential therapeutic use for cancer treatment. 

Research Area: Bioprocessing    

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Genetic Homogeneity Among Listeria monocytogenes Strains from Infected Patients and Meat Products from Two Geographic Locations Determined by Phenotyping, Ribotyping and PCR Analysis of Virulence Genes

2002

Authors: Jaradat, Z. W., G. E. Schutze, and A. K. Bhunia
Journal: International Journal of Food Microbiology, 76, 1-10, (2002)
Book Chapter: 

Abstract: Thirty Listeria monocytogenes isolates from human patients and foods originated from two different geographic locations without any epidemiological relations were analyzed for their genotypic and phenotypic virulence gene expressions and genetic relatedness. All strains contained virulence genes, inlA, inlB, actA, hlyA, plcA and plcB, with expected product size in PCR assay except for the actA gene. Some strains produced actA gene product of 268 and others 385 bp. Phenotypically, all were hemolytic but showed variable expressions of phospholipase activity. Ribotyping classified isolates into 12 different groups based on the similarity to DuPont Identification numbers (DID), which consisted primarily of clinical or food isolates or both. Cluster analysis also indicated possible existence of clones of L. monocytogenes that are found in food or human hosts or are evenly distributed between these two. Two isolates (F1 from food and CHL1250 from patient) had unique ribotype patterns that were not previously reported in the RiboPrinterR database. This study indicates distribution of diverse L. monocytogenes strains in clinical and food environments. The isolates showed 92–99% genetic homogeneity, in spite of their origins from two different geographic locations and environments.

Research Area: Food Safety     

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Microfabricated Device for Impedance-Based Detection of Bacterial Metabolism

2002

Authors: Gomez, R., M. R. Ladisch, A. K. Bhunia, and R. Bashir
Journal: Materials Research Society Symposium Proceedings, 729 (2002)
Book Chapter: 

Abstract: We present the use of a microfabricated device for impedance-based detection of a few live bacterial cells. Impedance-based detection relies on measuring changes in the AC impedance of two electrodes immersed in a liquid where the bacteria are cultured, caused by the release of ionic species by metabolizing bacterial cells. Rapid detection of a few cells (1 to 10) is possible if the cells are confined into a volume on the order of nanoliters. A microfluidic biochip prototype has been fabricated to test this miniaturized assay. The conductance of the bacterial suspensions is extracted from measuring their complex impedance in a 5.27 nl chamber in the biochip, at several frequencies between 100 Hz and 1 MHz. Measurements on suspensions of the bacteria Listeria innocua, Listeria monocytogenes, and Escherichia coli in a low conductivity buffer demonstrate that, under the current experimental conditions, the minimum detection level is between 50 and 200 live cells, after two hours of off-chip incubation. Work is in progress to develop techniques for selective capture of bacteria inside the chip, and to minimize background changes in impedance during on-chip incubation. 

Research Area: Food Safety     

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Microscale Electronic Detection of Bacterial Metabolism

2002

Authors: Rafael Gomez, Rashid Bashir, and Arun K. Bhunia 
Journal: Sensors and Actuators B, 86, 198-208 (2002)
Book Chapter: 

Abstract: In this paper, we present a microscale impedance-based technique for detecting the metabolic activity of a few live bacterial cells. Impedance-based detection relies on measuring changes in the ac impedance of two electrodes in contact with a liquid where the bacteria are cultured, caused by the release of ionic species by metabolizing cells. Rapid detection of a few live cells (1-10) is, in theory, possible if the cells are confined into a volume on the order of nanoliters. A microfluidic biochip prototype has been fabricated to explore this technique, consisting of a network of channels and chambers etched in a crystalline silicon substrate. The complex impedance of bacterial suspensions is measured with interdigitated platinum electrodes in a 5.27 nl chamber in the biochip at frequencies between 100 Hz and 1 MHz. After 2 h of off-chip incubation, the minimum number of live cells suspended in a low conductivity buffer that could be easily distinguished from the same number of heat-killed cells was on the order of 100 Listeria innocua, 200 L. monocytogenes, and 40 Escherichia coli cells, confined into the 5.27 nl chamber. A number on the order of 100 live L. innocua cells suspended in Luria-Bertani (LB) broth produced a significantly higher signal than the same number of heat-killed cells, and a difference is evident even down to ~5-20 cells. To the best of our knowledge, this is the first demonstration of microscale impedance-based detection of bacterial metabolism. 

Research Area: Food Safety     

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Modeling Pore Size Distribution in Cellulose Rolled Stationary Phases

2002

Authors: Keim, C., C. Li, C. M. Ladisch, and M. R. Ladisch
Journal: Biotechnology Progress, 18, 317-321, (2002)
Book Chapter: 

Abstract: Rolled stationary phases are fabrics (i.e., nonparticulate phases) that rapidly separate proteins from salts on the basis of size exclusion. Pore size and pore size distributions in the stationary phase determine how different size molecules distribute between the stationary and mobile phases in liquid chromatography columns. The potential for size exclusion chromatography by fabrics is not initially obvious because their interlaced structures are atypical for size exclusion supports. A simple logistic model fits the pore size distribution of a rolled stationary phase when pore sizes were measured using PEG, Dextran, D2O, glucose, and NaCl probes. When the fabric is treated with cellulase enzymes, the water-accessible pores uniformly decrease and peak retention is lower. The logistic function model captures this result and enables comparison of pore size distribution curves between enzyme-treated and untreated fabrics in rolled stationary phase columns.

Research Area: Bioseparations    

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Optimal Packing Characteristics of Rolled, Continous Stationary-Phase Columns

2002

Authors: Li, C., C. M. Ladisch, Y. Yang, R. Hendrickson, C. Keim, N. Mosier, M. R. Ladisch
Journal: Biotechnology Progress, 18, 309-316, (2002)
Book Chapter: 

Abstract: Rolled, continuous stationary phases were constructed by tightly rolling and inserting a whole textile fabric into a chromatography column. This work reports the column performance, in terms of plate height, void fraction, and resolution, of 10 cellulose based fabrics. The relation between fabric structural properties of yarn diameter, fabric count, fabric compressibility, and column performance are quantitated. General requirements, including reproducibility of packing, for choosing fabrics to make a good SEC column are identified. This research showed that the packed columns have an optimal mass of fabric that minimizes plate height and maximizes resolution, in a manner that is consistent with chromatography theory. Mass of material packed is then an important column parameter to consider when optimizing columns for the rapid desalting of proteins. Proteins were completely separated from salt and glucose in less than 8 min at a pressure drop less than 500 psi on the rolled, continuous stationary-phase columns. These results, together with stability and reproducibility, suggest potential industrial applications for cellulose-based rolled, continuous stationary-phase columns where speed is a key parameter in the production process.

Research Area: Bioseparations    

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Removal of Fermentation Inhibitors Formed during Pretreatment of Biomass by Polymeric Adsorbents

2002

Authors: Joseph R. Weil, Bruce Dien, Rodney Bothast, Richard Hendrickson, Nathan S. Mosier, and Michael R. Ladisch
Journal: Ind. Eng. Chem. Res. , 41, 6132-6138 (2002)
Book Chapter: 

Abstract: The production of aldehydes that are microbial inhibitors may occur when hexoses and pentoses in an aqueous solution are exposed to temperatures above 150 °C under acidic conditions common to acid-catalyzed lignocellulose biomass pretreatment. Concentrations greater than 0.1% of the degradation product, furfural, strongly inhibit fermentation, as was confirmed for hydrolysate that contained 0.5% (w/o) furfural. Methods of furfural removal that have been reported include sulfite or alkali addition to achieve chemical reduction, ion exchange, hydrophobic adsorption, and irreversible adsorption on activated carbon. This paper reports the removal of furfural from biomass hydrolysate by a polymeric adsorbent, XAD-4, and desorption of the furfural to regenerate the adsorbent using ethanol. Liquid chromatographic analysis showed that furfural concentrations were less than 0.01 g/L compared to the initial concentrations that were in the range of 1-5 g/L. Fermentation of the resulting biomass hydrolysate with recombinant Escherichia coli ethanologenic strain K011 confirmed that the concentration of furfural in the hydrolysate caused negligible inhibition. Fermentation of XAD-4-treated hydrolysate with E. coli K011 was nearly as rapid as the control medium that was formulated with reagent-grade sugars of the same concentration. Ethanol yields for both fermentations were 90% of theoretical. Modeling of the adsorptive properties of this styrene-based adsorbent indicates that it is suitable for on-off chromatography and could be useful in a continuous processing system for removing small amounts of aldehydes that might otherwise inhibit fermentation.

Research Area: Biofuels/Bioproducts    

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Adsorption of Avidin on Microfabricated Surfaces for Protein Biochip Applications

2001

Authors: Bashir, R., R. Gomez, A. Sarikaya, M. R. Ladisch, J. Sturgis, and J. P. Robinson
Journal: Biotechnology and Bioengineering, 73(4), 325-328 (2001)
Book Chapter: 

Abstract: The adsorption of the protein avidin from hen egg white on patterns of silicon dioxide and platinum surfaces on a microchip and the use of fluorescent microscopy to detect binding of biotin are described. A silicon dioxide microchip was formed using plasma-enhanced chemical vapor deposition while platinum was deposited using radio frequency sputtering. After cleaning using a plasma arc, the chips were placed into solutions containing avidin or bovine serum albumin. The avidin was adsorbed onto the microchips from phosphate-buffered saline (PBS) or from PBS to which ammonium sulfate had been added. Avidin was also adsorbed onto bovine serum albumin (BSA)-coated surfaces of oxide and platinum. Fluorescence microscopy was used to confirm adsorption of labeled protein, or the binding of fluorescently labeled biotin onto previously adsorbed, unlabeled avidin. When labeled biotin in PBS was presented to avidin adsorbed onto a BSA-coated microchip, the fluorescence signal was significantly higher than for avidin adsorbed onto the biochip alone. The results show that simple, low-cost adsorption process can deposit active protein onto a chip in an approach that has potential application in the development of protein biochips for the detection of biological species.

Research Area: Food Safety     

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Adsorption of Water from Liquid-Phase Ethanol-Water Mixtures at Room Temperature Using Starch-Based Adsorbents

2001

Authors: Beery, K. E., M. R. Ladisch
Journal: Ind. Eng. Chem. Res., 40, 2112-2115, (2001)
Book Chapter: 

Abstract: The kinetically controlled, selective removal of water from ethanol vapors by desiccants is well documented. However, studies on the removal of water by liquid-phase contacting of water- ethanol mixtures with starch-based material are limited. This research presents a screening study that shows that starch-based adsorbents remove liquid-phase water between 1 and 20 wt% from ethanol without the adsorbent being dissolved. The mass of water adsorbed per gram of dry adsorbent increases with increasing water content. Side by side comparisons of these starch-based adsorbents to silica gel and molecular sieves show that, in a kinetically controlled adsorption scheme below 10 wt % water, the inorganic desiccants have a greater operational (nonequilibrium) adsorption capacity per gram. At water concentrations at or above 10% water, however, the operational adsorptive capacity per gram of the starch-based adsorbents is roughly equivalent to the inorganic adsorbents, when using the same regeneration and adsorption conditions. The starch-based adsorbents adsorb water by forming hydrogen bonds between the hydroxyl groups on the surface of the adsorbent and the water molecules.

Research Area: Bioseparations    

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Characterization of Dicarboxylic Acids for Cellulose Hydrolysis

2001

Authors: Mosier, Nathan S.; Sarikaya, Ayda; Ladisch, Christine M., and Ladisch, Michael R.
Journal: Biotechnol. Prog., 17(3), 474-480, (2001)
Book Chapter: 

Abstract: In this paper, we show that dilute maleic acid, a dicarboxylic acid, hydrolyzes cellobiose, the repeat unit of cellulose, and the microcrystalline cellulose Avicel as effectively as dilute sulfuric acid but with minimal glucose degradation. Maleic acid, superior to other carboxylic acids reported in this paper, gives higher yields of glucose that is more easily fermented as a result of lower concentrations of degradation products. These results are especially significant because maleic acid, in the form of maleic anhydride, is widely available and produced in large quantities annually.

Research Area: Biofuels/Bioproducts    

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Chemistry and Properties of Starch Based Dessicants

2001

Authors: Kyle E. Beery, Michael R. Ladisch
Journal: Enzyme and Microbial Technology, 28, 573-581 (2001)
Book Chapter: 

Abstract: Desiccants currently used in industry include molecular sieves, lithium chloride, silica gel, and corn grits. Of these, only corn grits (a form of ground corn) are biodegradable and derived from a renewable resource. A major component of the corn grits, starch, is the primary adsorptive material in the corn grits. Other polysaccharides, including cellulose and hemicellulose also have adsorptive properties. The use of alpha-amylase (EC 3.2.1.1) to modify porosity and surface properties of starch resulted in materials with enhanced water sorption properties compared to the native material. This paper reviews the chemical and structural properties of starch, corn grits, and cellulose-based scaffolds on which starch can be affixed, in order to attain structures that might someday find uses in a range of desiccant applications for industrial, commercial, and residential processes.

Research Area: Bioseparations    

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Expression of E. coli araBAD Operon Encoding Enzymes for Metabolizing L-arabinose in Saccharomyces cerevisiae

2001

Authors: Miroslav Sedlak, Nancy W.Y. Ho
Journal: Enzyme and Microbial Technology, 28, 16-24 (2001) 
Book Chapter: 

Abstract: The Escherichia coli araBAD operon consists of three genes encoding three enzymes that convert L-arabinose to D-xylulose-5 phosphate. In this paper we report that the genes of the E. coli araBAD operon have been expressed in Saccharomyces cerevisiae using strong promoters from genes encoding S. cerevisiae glycolytic enzymes (pyruvate kinase, phosphoglucose isomerase, and phosphoglycerol kinase). The expression of these cloned genes in yeast was demonstrated by the presence of the active enzymes encoded by these cloned genes and by the presence of the corresponding mRNAs in the new host. The level of expression of L-ribulokinase (araB) and L-ribulose-5-phosphate 4-epimerase (araD) in S. cerevisiae was relatively high, with greater than 70% of the activity of the enzymes in wild type E. coli. On the other hand, the expression of L-arabinose isomerase (araA) reached only 10% of the activity of the same enzyme in wild type E. coli. Nevertheless, S. cerevisiae, bearing the cloned L-arabinose isomerase gene, converted L-arabinose to detectable levels of L-ribulose during fermentation. However, S. cerevisiae bearing all three genes (araA, araB, and araD) was not able to produce detectable amount of ethanol from L-arabinose. We speculate that factors such as pH, temperature, and competitive inhibition could reduce the activity of these enzymes to a lower level during fermentation compared to their activity measured in vitro. Thus, the ethanol produced from L-arabinose by recombinant yeast containing the expressed BAD genes is most likely totally consumed by the cell to maintain viability.

Research Area: Biofuels/Bioproducts    

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Impedance Spectroscopy and Biochip Sensor for Detection of Listeria monocytogenes

2001

Authors: Bhunia, A. K., Z. W. Jaradat, K. Naschansky, M. Shroyer, M. Morgan, R. Gomez, R. Bashir, and M. Ladisch
Journal: Proceedings of SPIE, (4206) 32-39 (2001)
Book Chapter: 

Abstract: Listeria monocytogenes is a deadly foodborne human pathogen. Its ubiquitous nature and its ability to grow at refrigeration temperatures makes this organism a difficult one to control. High-volume processed, ready-to-eat (RTE) foods. Improved processing along with real-time detection could reduce the incidence of this pathogen. Conventional methods can detect this pathogen accurately, but take several days (2-7d) to complete, which is not practical considering the short shelf-life and cost fiber optic and microelectrical-mechanical system (MEMS) biochips were designed and examined for direct detection of L. monocytogenes from liquid samples. Also, interdigitated microsensor electrode (IME) chip and spectrofluorometer were used to measure L. monocytogenes interaction with mammalian cells (cytopathogenic activities) for indirect detection. Preliminary data generated using laboratory cultures of Listeria species indicated that L. monocytogenes could be detected in 30 min to 1 h 30 min depending on the techniques used.

Research Area: Food Safety     

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Microfluidic Biochip for Impedance Spectroscopy of Biological Species

2001

Authors: Gomez, R., R. Bashir, A. Sarikaya, M. Ladisch, J. Sturgis, J. P. Robinson, T. Geng, A. Buhnia, H. Apple, S. Wereley
Journal: Biomedical Microdevices, 3(3), 201-209, (2001).
Book Chapter: 

Abstract: This paper describes the fabrication and characterization of a microelectronic device for the electrical interrogation and impedance spectroscopy of biological species. Key feature of the device include an all top-side processing for the formation of fluidic channels, planar fluidic interface ports, integrated metal electrodes for impedance measurements, and a glass cover sealing the nonplanar topography of the chip using spin-on-glass as an intermediate bonding layer. The total fluidic path volume in the device is on the order of 30 nl. Flow fields in the closed chip were mapped by particle image velocimetry. Electrical impedance measurements of suspensions of the live microorganism Listeria innocua injected into the chip demonstrate an easy method for detecting the viability of a few bacterial cells. By-products of the bacterial metabolism modify the ionic strength of a low conductivity suspension medium, significantly altering its electrical characteristics.

Research Area: Food Safety     

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Opportunities in Biotechnology for Future Army Applications

2001

Authors: Committee on Opportunities in Biotechnology for Future Army Applications, Board on Army Science and Technology, National Research Council 
Journal: published by The National Academies Press, 500 Fifth St., N.W., Washington, DC 20001, 118 pages (2001).
Book Chapter: 

Abstract: This report surveys opportunities for future Army applications in biotechnology, including sensors, electronics and computers, materials, logistics, and medical therapeutics, by matching commercial trends and developments with enduring Army requirements. Several biotechnology areas are identified as important for the Army to exploit, either by direct funding of research or by indirect influence of commercial sources, to achieve significant gains in combat effectiveness before 2025.

Research Area: Biofuels/Bioproducts    

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Genetically Engineered Saccharomyces Yeasts for Conversion of Cellulosic Biomass to Environmentally Friendly Transportation Fuel Ethanol

2000

Authors: Nancy W. Y. Ho, Zhendao Chen, Adam P. Brainard, and Miroslav Sedlak
Journal: American Chemical Society Symposium Series (2000) 
Book Chapter: 

Abstract: Ethanol is an effective, environmentally friendly, nonfossil, transportation biofuel that produces far less pollution than gasoline and contributes essentially no net carbon dioxide to the atmosphere. Furthermore, unlike crude oil for the production of gasoline, ethanol can be produced from plentiful, domestic, renewable, cellulosic biomass feedstocks. However, a major obstacle in this process is that cellulosic biomass contains two major sugars, glucose and xylose. Saccharomyces yeasts, traditionally used for large scale industrial production of ethanol from glucose, is unable to ferment xylose to ethanol. This makes the use of the safest, most effective microorganism for conversion of cellulosic biomass to ethanol economically unfeasible. In the fall of 1993, we achieved a historic breakthrough in the successful development of genetically engineered recombinant Saccharomyces yeast that can effectively ferment both glucose and xylose to ethanol. This paper provides an up-to-date overview of the design, development, and continuous innovative perfection of our recombinant Saccharomyces yeast that is widely regarded as the microorganism which will make the conversion of cellulosic biomass to ethanol commercially possible.

Research Area: Biofuels/Bioproducts    

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New System for Preparative Electrochromatography of Proteins

2000

Authors: Keim, C., and M. R. Ladisch
Journal: Biotechnology and Bioengineering, 70, 72-81, (2000)
Book Chapter: 

Abstract: Electrochromatography employs an axial electric field across a chromatographic stationary phase to separate proteins and other molecules based on differences in electrophoretic mobility. Because the separation is electrically driven, the need for additional chemical reagents is reduced. Two major impediments to scale-up of electrochromatography columns, removal of heat and electrolysis gases, have historically limited the diameter of packed columns to 2.5 cm ID with volumes of approximately 55 mL. We report a novel electrochromatography column that effectively removes electrolysis gases and minimizes heating. A vital component of this system is a new electrode design that couples a platinum gauze with an ultrafiltration membrane across both ends of the column. Use of a methacrylate base stationary phase enabled axial voltage gradients of 10 to 20 V/cm. Thermocouples inserted radially in the column at four axial positions showed that the flow of a 4 °C mobile phase coupled with heat conduction through the column walls controlled the temperature to 28 °C. The new column design, with dimensions of 3.81 cm ID x 38.1 cm long and bed volume of 400 mL, was demonstrated by separating mixtures of BSA and myoglobin. The column was operated in a horizontal position with radial sample injection and withdrawal at the ends of the packed bed. These experiments are a first step in demonstrating that scale up of electrochromatography columns can be achieved by choosing appropriate flow rates, voltage gradients, and stationary phase.

Research Area: Bioseparations    

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Analysis of Aluminum-26 Labeled Aluminum Chlorohydrate

1999

Authors: Flarend, R., C. Keim, T. Bin, D. Elmore, S. Hem, M. Ladisch
Journal: J. Inorgnaic Biochemistry, 76, 149-152 (1999)
Book Chapter: 

Abstract: A small quantity of aluminum chlorohydrate (ACH), the active ingredient in antiperspirants, was labeled with the radioisotope Al. This labeled drug will be used in future studies to measure the absorption of aluminum from antiperspirant use. The purpose of this study was to demonstrate that the technique was used to make the 26Al-labeled ACH resulted in a uniformly labeled complex, and therefore any measurement of 26Al would be indicative of total aluminum. The labeled ACH was fractionated using gel filtration chromatography into 72 evenly spaced samples. The fractions were then measured for 26Al and total aluminum content using accelerator mass spectrometry (AMS) and inductively coupled plasma atomic emission spectroscopy (ICP-AES). Results indicate that the ACH is uniformly labeled.

Research Area:     

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Ethanol Production from Renewable Resources

1999

Authors: C.S. Gong, N.J. Cao, J. Du, and G.T. Tsao
Journal: Advances in Biochemical Engineering / Biotechnology, 65 (1999)
Book Chapter: 

Abstract: Vast amounts of renewable biomass are available for conversion to liquid fuel, ethanol. In order to convert biomass to ethanol, the efficient utilization of both cellulose-derived and hemicellulose-derived carbohydrates is essential. Six-carbon sugars are readily atilized for this purpose. Pentoses, on the other hand, are more difficult to convert. Several metabolic factors limit the efficient utilization of pentoses (xylose and zrabinosc). Recent developments in the improvement of microbial cultures provide the versatility of conversion of both hexoses and peatoses to ethanol more efficiently. In addition , novel bioprocess technologies offer a promising prospective for the efficient conversion of biomass and recovery of ethanol.

Research Area: Biofuels/Bioproducts    

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Fermentation Kinetics of Ethanol Production from Glucose and Xylose by Recombinant Saccharomyces 1400(pLNH33)

1999

Authors: Krishnan, M. S., N. W. Y. Ho, and G. T. Tsao
Journal: Appl. Biochem. Biotechnol., Part A: Enzyme Engineering and Biotechnology, 77-79, 373-388 (1999)
Book Chapter: 

Abstract: Fermentation kinetics of ethanol production from glucose, xylose, and their mixtures using a recombinant Saccharomyces 1400 (pLNH33) are reported. Single-substrate kinetics indicate that the specific growth rate of the yeast and the specific ethanol productivity on glucose as the substrate was greater than on xylose as a substrate. Ethanol yields from glucose and xylose fermentation were typically 95 and 80% of the theoretical yield, respectively. The effect of ethanol inhibition is more pronounced for xylose fermentation than for glucose fermentation. Studies on glucose-xylose mixtures indicate that the recombinant yeast co-ferments glucose and xylose. Fermentation of aq 52.8 g/L glucose and 56.3 g/L xylose mixture gave an ethanol concentration of 47.9 g/L after 36 h. Based on a theoretical yield of 0.51 g ethanol/g sugars, the ethanol yield from this experiment (for data up to 24 h) was calculated to be 0.46 g ethanol/g sugar or 90% of the theoretical yield. The specific growth rate of the yeast on glucose-xylose mixtures wasa found to lie between the specific growth rate on glucose and the specific growth rate on xylose. Kinetic studies were used to develop a fermentation model incorporating the effects of substrate inhibition, product innhibition, and innoculum size. Good agreements were obtained betwen model predictions and experimental data from batch fermentation of glucose, xylose, and their mixtures.

Research Area: Biofuels/Bioproducts    

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Kinetic Studies of TAME Formation from Methyl Butenes Using a Strong Acid

1999

Authors: Ladisch, M. R., R. Hendrickson, M. T. Vandersall, S. G. Maroldo
Journal: 10th Congresso Brasileiro Catalise (1999)
Book Chapter: 

Abstract: The industrial synthesis of gasoline oxygenates such as methyl tertiary-butyl ether (MTBE) and tertiary-amylmethyl ether (TAME) is presently carried out using strong acid ion exchange catalysts, such as Amberlyst 15. Both the MTBE and TAME synthesis reactions involve the acid-catalyzed addition of an alcohol (methanol) to an oldfin, but the TAME reaction offers the additional complication that isomerization can occur between the two reactive methylbutenes, namely 2-methyl-1-butene (2M1B) and 2-methyl-2-butene (2M2B). Previously published results have shown that a new catalyst introduced by Rohm and Haas Company, Amberlyst 35 Wet catalyst, has substantially higher activity in the MTBE reaction that Amberlyst 15, and can be used to facilitate isobutene conversions of 98%, or higher, in some cases. Results presented at a previous IBP Instituto Brasileiro de Petroleo Seminar showed that Amberlyst 35 is also substantially more active than Amberlyst 15 for the TAME reaction. This work has been extended by using differential reactor studies to determine the initial kinetic rate constants and the Anhenius activation energies for both catalysts. These results are presented here.

Research Area: Biofuels/Bioproducts    

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Production of Multifunctional Organic Acids from Renewable Resources

1999

Authors: G. T. Tsao, N. J. Cao, J. Du, C. S. Gong
Journal: Adv. Bio. Eng. / Biotech., 65, 243-280 (1999)
Book Chapter: 

Abstract: Recently, the microbial production of multifunctional organic acids has received interest due to their increased use in the food industry and their potential as raw materials for the manufacture of biodegradable polymers. Certain species of microorganisms produce significant quantities of organic acids in high yields under specific cultivation conditions from biomass-derived carbohydrates. The accumulation of some acids, such as fumaric, malic and succinic acid, are believed to involve CO2 fixation which gives high yields of products. The application of special fermentation techniques and the methods for downstream processing of products are described. Techniques such as simultaneous fermentation and product recovery and downstream processing are likely to occupy an important role in the reduction of production costs. Finally, some aspects of process design and current industrial production processes are discussed.

Research Area: Biofuels/Bioproducts    

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Reaction Kinetics, Molecular Action, and Mechanisms of Cellulolytic Proteins

1999

Authors: Mosier, N. S., P. Hall, C. M. Ladisch, and M. R. Ladisch
Journal: Adv. in Biochemical Engineering/Biotechnology, 65, 23-40, (1999)
Book Chapter: 

Abstract: Cellulolytic proteins form a complex of enzymes that work together to depolymerize cellulose to the soluble products cellobiose and glucose. Fundamental studies on their molecular mechanisms have been facilitated by advances in molecular biology. These studies have shown homology between cellulases from different microorganisms, and common mechanisms between enzymes whose modes of action have sometimes been viewed as being different, as suggested by the distribution of soluble products. A more complete picture of the Cellulolytic action of these proteins has emerged and combines the physical and chemical characteristics of solid cellulose substrates with the specialized structure and function of the cellulases that break it down. This chapter combines the fundamentals of cellulose structure with enzyme function in a manner that relates the cellulose binding and biochemical kinetics at the catalytic site of the proteins to the macroscopic behavior of cellulose enzyme systems.

Research Area: Biofuels/Bioproducts    

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Rolled Stationary Phases: Dimensionally Structured Textile Adsorbents for Rapid Liquid Chromatography of Proteins

1999

Authors: Hamaker, K., S-L. Rau, R. Hendrickson, J. Liu, C. M. Ladisch, and M. R. Ladisch
Journal: Ind. Eng. Chem. Res., 38, 865-872 (1999)
Book Chapter: 

Abstract: A woven textile fabric, consisting of 60% cotton/40% polyester, tightly rolled in a cylindrical configuration, has a three-dimensional structure with sufficient hydrodynamic stability to withstand interstitial eluent velocities of up to 300 cm/min when packed into standard liquid chromatography column assemblies. Demonstration of the pressure stability of the cotton/polyester fabric was followed up with experiments in which the cotton (cellulose) portion was derivatized and the fabric evaluated for chromatography of proteins. When derivatized to give a (diethylamino) ethyl (DEAE) anion exchanger, a velocity independent plate height of 2 mm, a static capacity of 115 mg of bovine serum albumin/g of stationary phase, and a dynamic protein loading capacity which decreases only 25% over an 800% increase in mobile-phase velocity from 6.7 to 54 cm/min was achieved. The fibers that make up the stationary phase have a relatively nonporous structure which minimizes pore diffusional effects. A protein separation of Cytochrome C from B-lactoglobulin a is shown to be completed by ion-exchange chromatography in less than 10 min using an NaCl step gradient. Gradient chromatography of a hen egg white shows resolution of the proteins into two major components (lysozyme and ovalbumin) as well as two minor ones. A size exclusion separation of PEG 20000 from glucose requires only 90 s. These characteristics, together with the ability of the cellulose-based stationary phase to withstand rapid flow rates, indicate that this type of stationary phase has potential for applications where chromatography using DEAE-cellulose particles has proven successful.

Research Area: Bioseparations    

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Solid State Fermentation of Lignocellulosic Plant Residues from Brassica napus by Pleurotus ostreatus

1999

Authors: Sarikaya, A. and M. R. Ladisch
Journal: Appl. Biochem. Biotechnol., Part A: Enzyme Engineering and Biotechnology, 82(1), 1-15 (1999)
Book Chapter: 

Abstract: Solid-state fermentation (SSF) of inedible parts of rapeseed was carried out using a white-rot fungus, Pleurotus ostreatus, to degrade lignocellulosic material for mycelial-single cell protein (SCP) production. This SSF system has the potential to be adapted to a controlled ecological life support system in space travel owing to the lack of storage space. The system for converting lignocellulosic material to SCP by P. ostreatus is simple; it can be carried out in a compact reactor. The fungal vegetative growth was better with a particle size of plant material ranging from 0.42 to 10 mm, whereas lignin degradation of the lignocellulose was the highest with particle sizes ranging from 0.42 to 0.84 mm. The addition of veratryl alcohol (3, 4-dimethoxybenzyl alcohol), hydrogen peroxide, and glycerol promotes lignocellulose degradation by P. ostreatus. The enhancement of bioconversion was also observed when a gas-blow bioreactor was used to supply oxygen and to maintain the constant moisture of the reactor. With this reactor, approx 85% of the material was converted to fungal and other types of biomass after 60 d of incubation.

Research Area: Biofuels/Bioproducts    

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Successful Design and Development of Genetically Engineered Saccharomyces Yeasts for Effective Cofermentation of Glucose and Xylose from Cellulosic Biomass to Fuel Ethanol

1999

Authors: Nancy W. Y. Ho, Zhengdao Chen, Adam P. Brainard, Miroslav Sedlak
Journal: Advances in Biochemical Engineering/Biotechnology, 65, 163-192 (1999) 
Book Chapter: 

Abstract: Ethanol is an effective, environmentally friendly, nonfossil, transportation biofuel that produces far less pollution than gasoline. Furthermore, ethanol can be produced from plentiful, domestically available, renewable, cellulosic biomass. However, cellulosic biomass contains two major sugars, glucose and xylose, and a major obstacle in this process is that Saccharomyces yeasts, traditionally used and still the only microorganisms currently used for large scale industrial production of ethanol from glucose, are unable to ferment xylose to ethanol. This makes the use of these safest, most effective Saccharomyces yeasts for conversion of biomass to ethanol economically unfeasible. Since 1980, scientists worldwide have actively been trying to develop genetically engineered Saccharomyces yeasts to ferment xylose. In 1993, we achieved a historic breakthrough to succeed in the development of the first genetically engineered Saccharomyces yeasts that can effectively ferment both glucose and xylose to ethanol. This was accomplished by carefully redesigning the yeast metabolic pathway for fermenting xylose to ethanol, including cloning three xylose-metabolizing genes, modifying the genetic systems controlling gene expression, changing the dynamics of the carbon flow, etc. As a result, our recombinant yeasts not only can effectively ferment both glucose and xylose to ethanol when these sugars are present separately in the medium, but also can effectively coferment both glucose and xylose present in the same medium simultaneously to ethanol. This has made it possible because we have genetically engineered the Saccharomyces yeasts as such that they are able to overcome some of the natural barriers present in all microorganisms, such as the synthesis of the xylose metabolizing enzymes not to be affected by the presence of glucose and by the absence of xylose in the medium. This first generation of genetically engineered glucose-xylose-cofermenting Saccharomyces yeasts relies on the presence of a high-copy-number 2u-based plasmid that contains the three cloned genetically modified xylose-metabolizing genes to provide the xylose-metabolizing capability. In 1995, we achieved another breakthrough by creating the super-stable genetically engineered glucose-xylose-cofermenting Saccharomyces yeasts which contain multiple copies of the same three xylose-metabolizing genes stably integrated on the yeast chromosome. This is another critical development which has made it possible for the genetically engineered yeasts to be effective for cofermenting glucose and xylose by continuous fermentation. It is widely believed that the successful development of the stable glucose-xylose-cofermenting Saccharomyces yeasts has made the biomass-to-ethanol technology a step much close to commercialization. In this paper, we present an overview of our rationales and strategies as well as our methods and approaches that led to the ingenious design and successful development of our genetically engineered Saccharomyces yeasts for effective coferermentation of glucose and xylose to biofuel ethanol.

Research Area: Biofuels/Bioproducts    

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Bioseparations

1998

Authors: M. Ladisch
Journal: Encyclopedia of Chemical Technology, John Wiley & Sons, p89-122 (1998)
Book Chapter: 

Abstract: The large-scale purification of proteins and other bioproducts is the final production step, prior to product packaging, in the manufacture of therapeutic proteins, specialty enzymes, diagnostic products, and value-added products from agriculture. These separation steps, taken to purify biological molecules or compounds obtained from biological sources, are referred to as bioseparations. Large-scale bioseparations combine art and science. bioseparations often evolve from laboratory-scale techniques, adapted and scaled up to satisfy the need for larger amounts of extremely pure test quantities of the product. Uncompromising standards for product quality, driven by commercial competition, applications, and regulatory oversight, provide many challenges to the scale-up of protein purification. The rigorous quality control embodied in current good manufacturing practices, and the complexity and lability of the macromolecules being processed provide other practical issues to address.

Research Area: Bioseparations    

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Continuous pH Monitoring During Pretreatment of Yellow Poplar Wood Sawdust by Pressure Cooking in Water

1998

Authors: Weil, J. R., M. Brewer, R. Hendrickson, A. Sarikaya, and M. R. Ladisch
Journal: Appl. Biochem. Biotechnol., 70-72, 99-111 (1998)
Book Chapter: 

Abstract: Yellow poplar wood sawdust consists of 41% cellulose and 19% hemicellulose. The goal of pressure cooking this material in water is to hydrate the more chemically resistive regions of cellulose in order to enhance enzymatic conversion to glucose. Pretreatment can generate organic acids through acid-catalyzed degradation of monosaccharides formed because of acids released from the biomass material or the inherent acidity of the water at temperatures above 160oC. The resulting acids will further promote the acid-catalyzed degradation of monomers that cause both a reduction in the yield and the formation of fermentation inhibitors such as hydroxymethyl furfural and furfural. A continuous pH-monitoring system was developed to help characterize the trends in pH during pretreatment and to assist in the development of a base (2.0 M KOH) addition profile to help keep the pH within a specified range in order to reduce any catalytic degradation and the formation of any monosaccharide degradation products during pretreatment. The results of this work are discussed.

Research Area: Biofuels/Bioproducts    

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Effect of Enzyme Modification of Corn Grits on Their Properties As An Adsorbent in Skarstrom Pressure Swing Cycle Drier

1998

Authors: Beery, K., M. Gulati, E. P. Kvam, and M. R. Ladisch
Journal: Adsorption, 4, 321-335 (1998) 
Book Chapter: 

Abstract: Corn grits have been tested as a desiccant in a pressure swing adsorption (PSA) system to produce dry air. Two sizes of unmodified corn grits were tested in the PSA system: 2.16 and 0.978 mm in diameter, which dried corn grits that gives an increase in the operational adsorptive capacity in a pressure swing, adsorption system, so that grits dry moist air to a – 56 °C dew point and the 0.978 mm corn grits dry air to a – 80 °C dew point. The modification process creates surface modifications on the corn grits apparently making more adsorption sites easily available. The modification procedure increases the specific surface area of the grits and possibly decreases the crystallinity, which would make more hydroxyl groups available for adsorption of water. Possible applications of using corn grits in the pressure swing adsorption system include industrial gas dryers, sorptive cooling air conditioners, and recycling equipment for industrial solvents.

Research Area: Bioseparations    

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Genetically Engineered Saccharomyces Yeast Capable of Effective Cofermentation of Glucose and Xylose

1998

Authors: Nancy W. Y. Ho, Zhengdao Chen, and Adam P. Brainard
Journal: Applied and Environmental Microbiology, 64, 5, 1852-1859 (1998) 
Book Chapter: 

Abstract: Xylose is one of the major fermentable sugars present in cellulosic biomass, second only to glucose. However, Saccharomyces spp., the best sugar-fermenting microorganisms, are not able to metabolize xylose. We developed recombinant plasmids that can transform Saccharomyces spp. into xylose-fermenting yeasts. These plasmids, designated pLNH31, -32, -33, and -34, are 2mm-based high-copy-number yeast-E. coli shuttle plasmids. In addition to the geneticin resistance and ampicillin resistance genes that serve as dominant selectable markers, these plasmids also contain three xylose-metabolizing enes, a xylose reductase gene, a xylitol dehydrogenase gene (both from Pichia stipitis), and a xylulokinase gene (from Saccharomyces cerevisiae). These xylose-metabolizing genes were also fused to signals controlling gene expression from S. cerevisiae glycolytic genes. Transformation of Saccharomyces sp. strain 1400 with each of these plasmids resulted in the conversion of strain 1400 from a non-xylose-metabolizing yeast to a xylose-metabolizing yeast that can effectively ferment xylose to ethanol and also effectively utilizes xylose for aerobic growth. Furthermore, the resulting recombinant yeasts also have additional extraordinary properties. For example, the synthesis of the xylose-metabolizing enzymes directed by the cloned genes in these recombinant yeasts does not require the presence of xylose for induction, nor is the synthesis repressed by the presence of glucose in the medium. These properties make the recombinant yeasts able to efficiently ferment xylose to ethanol and also able to efficiently coferment glucose and xylose present in the same medium to ethanol simultaneously.

Research Area: Biofuels/Bioproducts    

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Pretreatment of Corn Fiber By Pressure Cooking in Water

1998

Authors: Weil, J. R., A. Sarikaya, S-L. Rau, J. Goetz, C. M. Ladisch, M. Brewer, R. Hendrickson, and M. R. Ladisch
Journal: Appl. Biochem. Biotechnol, 73, 1-17 (1998)
Book Chapter: 

Abstract: The pretreatment of corn fiber using liquid water at temperatures between 220 and 260oC enhances enzymatic hydrolysis. This paper describes the laboratory reactor system currently in use for cooking of corn fiber at temperatures ranging from 200 to 260o C. The corn fiber at approx 4.4% solid/liquid slurry was treated in a 2-L, 304 SS, Parr reactor with three turbine propeller agitators and a Proportional-Integral-Derivative (PID), controller that controlled temperature within +-1oC. Heat-up times to the final temperatures of 220, 240, or 260oC were achieved in 50 to 60 min. Hold time at the final temperature was less than 10 s. A serpentine cooling coil, through which tap water was circulated at the completion of the run, cooled the reactor’s contents to 180oC within 2 min after the maximum temperature was attained. Ports in the reactor’s head plate facilitated sampling of the slurry and monitoring the pH. A continuous pH monitoring system was developed to help observe trends in pH during pretreatment and to assist in the development of a base (2.0 M KOH) addition profile to help keep the pH within the range of 5.0 to 7.0. Enzymatic hydrolysis gave 33 to 84% conversion of cellulose in the pretreated fiber to glucose compared to 17% for untreated fiber.

Research Area: Biofuels/Bioproducts    

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Transport Properties of Rolled, Continuous Stationary Phase Columns

1998

Authors: Hamaker, K., J. Liu, C. Ladisch, and M. R. Ladisch
Journal: Biotechnol. Progr., 14(1), 21-30 (1998) 
Book Chapter: 

Abstract: Continuous stationary phase columns consist of woven textile matrixes of fibers rolled into a cylindrical configuration and inserted into a liquid chromatography column. This configuration allows separations to be carried out at interstitial mobile phase velocities in excess of 100 cm/min and pressures of up to 700 psig for stationary phases based on cellulose. Ordinarily, these conditions would cause compaction of a Cellulosic stationary phase to the point where flow is no longer possible. The packing of the column with cellulose as a continuous stationary phase enables these linear velocities to be achieved. Most importantly, this type of column allows the study of momentum transport and mass transfer in a media in which the mobile phase explores almost all of the void volumes in the column. The analysis of flow patterns in these columns has been modeled using elution patterns of both retained and unretained components, and plate height has been correlated as a function of velocities in the range of 1-100 cm/min. Engineering analysis of this type of chromatography column based on visual representation of the packed fibers by scanning electron microscopy, analysis of porosities using unretained (nonadsorbing) molecular probes, and application of momentum and mass transport equations is discussed.

Research Area: Bioseparations    

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An Unstructured Mathematical Model for Growth of Pleurotus ostreatus on Lignocellulosic Material in Solid-State Fermentation Systems

1997

Authors: Sarikaya, A. and M. R. Ladisch
Journal: Appl. Biochem. Biotechnol., 62, 71-85 (1997)
Book Chapter: 

Abstract: Inedible plant material, generated in a Controlled Ecological Life Support System (CELSS), should be recycled preferably by bioregenerative methods that utilize enzymes or micro-organisms. This material consists of hemicellulose, cellulose, and lignin with the lignin fraction representing a recalcitrant component that is not readily treated by enzymatic methods. Consequently, the white-rot fungus, Pleurotus ostreatus, is attractive since it effectively degrades lignin and produces edible mushrooms. This work describes an unstructured model for the growth of P. ostreatus in a solid-state fermentation system using lignocellulosic plant materials from Brassica napus (rapeseed) as a substrate at three different particle sizes. A logistic function model based on area was found to fit the surface growth of the mycelium on the solid substrate with respect to time, whereas a model based on diameter, alone, did not fit the data as well. The difference between the two measures of growth was also evident for mycelial growth in a bioreactor designed to facilitate a slow flowrate of air through the 1.5 cm thick mat of lignocellulosic biomass particles. The result is consistent with the concept of competition of the mycelium for the substrate that surrounds it, rather than just substrate that is immediately available to single cells. This approach provides a quantitative measure of P. ostreatus growth on lignocellulosic biomass in a solid-state fermentation system. The experimental data show that the best growth is obtained for the largest particles (1 cm) of the lignocellulosic substrate.

Research Area: Bioprocessing    

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Biobased Adsorbents for Drying of Gases

1997

Authors: Ladisch, M. R.
Journal: Enzyme Microb. Tech., 20, 162-164 (1997)
Book Chapter: 

Abstract: Fundamental, structural and compositional studies on the properties of corn grits and their ability to selectively adsorb water from organic vapors have resulted in new bio-based adsorbents. Structure/function relationships of these bio-based adsorbents are reviewed.

Research Area: Bioseparations    

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Enhanced Cofermentation of Glucose and Xylose by Recombinant Saccharomyces Yeast Strains in Batch and Continuous Operating Modes

1997

Authors: Susan T. Toon, George P. Philippidis, Nancy W. Y. Ho, ZhengDao Chen, Adam Brainard, Robert E. Lumpkin, and Cynthia J. Riley
Journal: Applied Biochemistry and Biotechnology, 63-65, 243-255 (1997) 
Book Chapter: 

Abstract: Agricultural residues, such as grain by-products, are rich in the hydrolyzable carbohydrate polymers hemicellulose and cellulose; hence, they represent a readily available source of the fermentable sugars xylose and glucose. The biomass-to-ethanol technology is now a step closer to commercialization because a stable recombinant yeast strain has been developed that can efficiently ferment glucose and xylose simultaneously (coferment) to ethanol. This strain, LNH-ST, is a derivative of Saccharomyces yeast strain 1400 that carries the xylose-catabolism encoding genes of Pichia stipitis in its chromosome. Continuous pure sugar cofermentation studies with this organism resulted in promising steady-state ethanol yields (70.4% of theoretical based on available sugars) at a residence time of 48 h. Further studies with corn biomass pretreated at the pilot scale confirmed the performance characteristics of the organism in a simultaneous saccharificatin and cofermentation (SSCF) process: LNH-ST converted 78.4% of the available glucose and 56.1% of the available xylose within 4 d, despite the presence of high levels of metabolic inhibitors. These SSCF data were reproducible at the bench scale and verified in a 9000-L pilot scale bioreactor.

Research Area: Biofuels/Bioproducts    

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Fermentation of Corn Fibre Sugars by an Engineered Xylose Utilizing Saccharomyces Yeast Strain

1997

Authors: M. Moniruzzaman, B.S. Dien, C.D. Skory, Z.D. Chen, R.B. Hespell, N.W.Y. Ho, B.E. Dale and R.J. Bothast
Journal: World Journal of Microbiology & Biotechnology, 13, 341-346 (1997) 
Book Chapter: 

Abstract: The ability of a recombinant Saccharomyces yeast strain to ferment the sugars glucose, xylose, arabinose and galactose which are the predominant monosaccharides found in corn bre hydrolysates has been examined. Saccharomyces strain 1400 (pLNH32) was genetically engineered to ferment xylose by expressing genes encoding a xylose reductase, a xylitol dehydrogenase and a xylulose kinase. The recombinant efficiently fermented xylose alone or in the presence of glucose. Xylose-grown cultures had very little difference in xylitol accumulation, with only 4 to 5 g/l accumulating, in aerobic, micro-aerated and anaerobic conditions. Highest production of ethanol with all sugars was achieved under anaerobic conditions. From a mixture of glucose (80 g/l) and xylose (40 g/l), this strain produced 52 g/l ethanol, equivalent to 85% of theoretical yield, in less than 24 h. Using a mixture of glucose (31 g/l), xylose (15.2 g/l), arabinose (10.5 g/l) and galactose (2 g/l), all of the sugars except arabinose were consumed in 24 h with an accumulation of 22 g ethanol/l, a 90% yield (excluding the arabinose in the calculation since it is not fermented). Approximately 98% theoretical yield, or 21 g ethanol/l, was achieved using an enzymatic hydrolysate of ammonia bre exploded corn ®bre containing an estimated 47.0 g mixed sugars/l. In all mixed sugar fermentations, less than 25% arabinose was consumed and converted into arabitol. 

Research Area: Biofuels/Bioproducts    

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Mechanism and Potential Applications of Bio-lignolytic Systems in a CELSS

1997

Authors: Sarikaya, A. and M. R. Ladisch
Journal: Appl. Biochem. and Biotechnol., 62 (213), 131-149 (1997)
Book Chapter: 

Abstract: A large amount of inedible plant material, generated as a result of plant growth in a Controlled Ecological Life Support System (CELSS), should be pretreated and converted into forms that can be recycled on earth as well as in space. The main portion of the inedible biomass is lignocellulosic material. Enzymatic hydrolysis of this cellulose would provide sugars for many other uses by recycling carbon, hydrogen, oxygen, and nitrogen through formation of carbon dioxide, heat, and sugars, which are potential foodstuffs. To obtain monosaccharides from cellulose, the protective effect of lignin should be removed. White-rot fungi degrade lignin more extensively and rapidly than other microorganisms. Pleurotus ostreatus degrades lignin effectively, and produces edible and flavorful mushrooms that increase the quality and nutritional value of the diet. This mushroom is also capable of metabolizing hemicellulose, thereby providing a food use of this pentose containing polysaccharide. This study presents the current knowledge of physiology and biochemistry of primary and secondary metabolisms of basidiomycetes, and degradation mechanism of lignin. A better understanding of the ligninolytic activity of white-rot fungi will impact the CELSS Program by providing insights on how edible fungi might be used to recycle the inedible portions of the crops.

Research Area: Biofuels/Bioproducts    

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Pretreatment of Yellow Poplar Sawdust by Pressure Cooking in Water

1997

Authors: Weil, J., A. Sarikaya, S-L. Rau, J. Goetz, C. Ladisch, M. Brewer, R. Hendrickson, and M. R. Ladisch
Journal: Appl. Biochem. Biotechnol., 68 (1-2), 21-40 (1997) 
Book Chapter: 

Abstract: The pretreatment of yellow poplar wood sawdust using liquid water at temperatures above 220 C enhanced enzyme hydrolysis. This paper reviews our prior research and describes the laboratory reactor system currently in use for cooking wood sawdust at temperatures ranging from 220 to 260 C. The wood sawdust at a 6-6.6% solid/liquid slurry was treated in a 2 L, 304 SS, Parr reactor with three turbine propeller agitators and a proportional integral derivative (PID) controller, which controlled temperature within a plus or minus 1 C. Heat-up times to the final temperatures of 220, 240 or 260 C were achieved in 60-70 min. Hold time at the final temperature was less than 1 min. A serpentine cooling coil, through which tap water was circulated at the completion of the run, cooled the reactor's contents within 3 min after the maximum temperature was attained. A bottoms port, as well as ports in the reactor's head plate, facilitated sampling of the slurry and measuring the pH, which changes from an initial value of 5 before cooking to a value of approx 3 after cooking. Enzyme hydrolysis gave 80-90% conversion of cellulose in the pretreated wood to glucose. Simultaneous saccharification and fermentation of washed, pretreated lignocellulose gave an ethanol yield that was 55% of theoretical. Untreated wood sawdust gave less than 5% hydrolysis under the same conditions.

Research Area: Biofuels/Bioproducts    

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Production of Ethanol From Recycled Paper Sludge Using Cellulase and Yeast, Kluveromyces markianus

1997

Authors: N. Lark, Y. Xia, C.-G. Qin, C.S. Gong, and G. T. Tsao
Journal: Biomass and Bioenergy, 12, No 2. pp. 135-143, (1997)
Book Chapter: 

Abstract: Paper recycling is expected to increase by an average of 10% annually for the next few years. Likewise, the recycled paper sludge (RPS) generated during repulping will increase accordingly. The typical RPS has an average content of 60% moisture and 50% cellulose on a dry basis. Simultaneous saccharification and fermentation (SSF) with fungal cellulase and yeast, Kluveromyces marxianus, were used to convert cellulose fibers of RPS samples to ethanol. The cellulase loading was 8 filter paper units (FPU)/g dry RPS. About 32 and 35 g/l of ethanol were produced from 180 and 190 g/l dry materials, respectively, after 72 h of incubation. This indicates that at least 72% of cellulose in the RPS was converted into ethanol. During incubation, the thick slurry of RPS was liquefied within 24 h, resulting in the reduction of water-holding capacity (WHC) of RPS to 30-35% of the original.

Research Area: Biofuels/Bioproducts    

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Assessment of Ethanol Production Options for Corn Products

1996

Authors: Gulati, M., K. L. Kohlmann, M. R. Ladisch, R. Hespell, and R. J. Bothast
Journal: Bioresource Technol., 58, 253-264 (1996)
Book Chapter: 

Abstract: The production of ethanol from corn fiber has the potential to increase ethanol yields by a maximum of 0.3 gallbushed in a wet-milling process. Incremental yields would be 0.13 gallbushed from hexose, 0.1 from D-xylose and 0.07 from L-arabinose, at 100% hydrolysis and fermentation efficiency. At 80% efficiency for hexose hydrolysis and fermentation, and 70% for pentose, an incremental yield of 0.22 gallons/bushel of corn is expected. Of this total, 0.1 gal/bushel would be from hexoses, 0.07 from D-xylose, and 0.05 from L-arabinose. A maximum practical incremental yield would probably fall between 0.22 and 0.3 gallons/bushel. These calculations are based on published compositional analyses of cellulose, starch, mono-saccharides, hemicellulose, protein and oil as distributed between the compartmentalized components of the corn kernel and published yield factors for hexose and pentose fermentations. Experimental yield factors for xylose (0.36 g ethanol/g xylose) and arabinose (0.34) fermenting microorganisms are lower than that for glucose (0.45-0.50), and significantly less than the theoretical yield of 0.51 g ethanol/g pentose. Nonetheless, we estimate that a wet-milling facility which currently produces 100 million gallons/year of ethanol from starch could generate an additional $4-8 million of annual income if the fiber components were processed into ethanol. Hence, advances in fiber pretreatment and pentose fermentation are likely to have a major impact on enhancing productivity of corn ethanol plants. An engineering framework for assigning economic consequences of the additional utilization of fiber is presented.

Research Area: Biofuels/Bioproducts    

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Chromatography for Rapid Buffer Exchange and Refolding of Secretory Leukocyte Protease Inhibitor

1996

Authors: Hamaker, K. H., J. Liu, R. J. Seely, C. M. Ladisch, and M. R. Ladisch
Journal: Biotechnol. Progr., 12, 184-189 (1996) 
Book Chapter: 

Abstract: A DEAE-cellulose stationary phase in a rolled configuration was used to separate recombinant secretory leukocyte protease inhibitor (rSLPI) from denaturants and reducing agents (3 M guanidine-HCl and 5 mM DTT) in less than 5 min to promote refolding of the protein to an active form. The mobile phase consisted of buffer and 500 mM NaCl, where NaCl suppressed binding of protein to this stationary phase. Separation of an initial concentration of 2 mg/mL protein from the other constituents resulted in 96% recovery of the rSLPI at an average concentration of 1.28 mg/mL. When incubated for 4 h at 20oC, the fractionated rSLPI gave a 46% yield of properly refolded protein. The protein concentration was 6.4 times higher than that reported in a previously published method, where refolding was carried out by diluting the mixture of protein, denaturants, and reducing agents by a factor of 10. The results show that a combination of rapid chromatographic separation over a cellulosic stationary phase followed by protein refolding will significantly enhance process throughput by minimizing tankage, water requirements, and process time.

Research Area: Bioseparations    

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Earth Benefits of Interdisciplinary Celss-Related Research by The NSCORT in Bioregenerative Life Support

1996

Authors: Mitchell, C., L. Sherman, S. Nielsen, P. Nelson, P. Trumbo, T. Hodges, P. Hasegawa, R. Bressan, M. Ladisch, and D. Auslander
Journal: Adv. Space Res.,(18) 23-31 (1996)
Book Chapter: 

Abstract: Earth benefits of research from the NSCORT in Bioregenerative Life Support will in clued the following: development of active control mechanisms for light, CO2, and temperature to maximize photosynthesis of crop plants during important phases of crop development; creation of value-added crops with superior nutritional, yield, and waste-process characteristics; environmental control of food and toxicant composition of crops; new process technologies and novel food products for safe, nutritious, palatable vegetarian diets; creation of menus for healthful vegetarian diets with psychological acceptability; enzymatic procedures to degrade recalcitrant crop residues occurring in municipal waste; control-system strategies to ensure sustainability of a CELSS that will enable management of diverse complex systems on earth.

Research Area: Bioenergy    

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Enzyme Conversion of Lignocellulosic Plant Materials for Resource Recovery in a Controlled Ecological Life Support System

1996

Authors: Kohlmann, K. L., P. J. Westgate, A. Velayudhan, J. Weil, A. Sarikaya, M. A. Brewer, R. L. Hendrickson, and M. R. Ladisch
Journal: Advances in Space Research, 18(1/2), 251-265 (1996) 
Book Chapter: 

Abstract: A large amount of inedible plant material composed primarily of the carbohydrate materials cellulose, hemicellulose, and lignin is generated as a result of plant growth in a Controlled Ecological Life-Support System (CELSS). Cellulose is a linear homopolymer of glucose, which when properly processed with yield glucose, a valuable sugar because it can be added directly to human diets. Hemicellulose is a heteropolymer of hexoses and pentoses that can be treated to give a sugar mixture that is potentially a valuable fermentable carbon source. Such fermentations yield desirable supplements to the edible products from hydroponically-grown plants such as rapeseed, soybean, cowpea, or rice. Lignin is a three-dimensionally branched aromatic polymer, comprised of phenyl propane units, which is susceptible to bioconversion through the growth of the white rot fungus, Pluerotus ostreatus. Processing conditions, that include both a hot water pretreatment and fungal growth and that lead to the facile conversion of plant polysaccharides to glucose, are presented.

Research Area: Biofuels/Bioproducts    

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Intraparticle Flow and Plate Height Effects in Liquid Chromatography Stationary Phases

1996

Authors: Hamaker, K. H. and M. R. Ladisch
Journal: Separation and Purification Methods, 25(1), 47-83 (1996)
Book Chapter: 

Abstract: Velocity independent plate heights were apparently first recognized for hydrodynamic chromatography columns, packed with nonporous, 115 micron glass beads which were run at reduced mobile phase velocities of 10 to 10,000. Hydrodynamic chromatography separates based on the tendency of small molecules (or particles) to associate with slower moving fluid streamlines near the surfaces of particles, compared to larger molecules which seek faster streamlines. Consequently, the larger molecules elute first. Velocity independent plate heights in liquid chromatography have also been observed for nonadsorbed solutes in particulate and fibrous stationary phases. These stationary phases have pores which exceed 10-4 to 10-5 cm in dimension. The flat plate height is attributed to flow in the channels formed by these large intraparticle spaces. The development of plate height expressions which represent dispersion at interstitial velocities above 10 cm/min are discussed. Explanations of the uncoupling of dispersion from eluent flow rate in continuous stationary phases, membranes, and gigaporous particles is shown to have their origins in the studies of distribution of particles and molecules in hydrodynamic chromatography columns, and to be adequately described by modifications of the van Deemter equation.

Research Area: Bioseparations    

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Simultaneous Production and Recovery of Fumaric Acid from Immobilized Rhizopus oryzae with a Rotary Biofilm Contactor and an Adsorption Column

1996

Authors: N. Cao, J. Du, C. S. Gong, and G. T. Tsao
Journal: Applied and Environmental Microbiology, p. 2926-2931 (Aug. 1996)
Book Chapter: 

Abstract: An integrated system of simultaneous fermentation-adsorption for the production and recovery of fumaric acid from glucose by Rhizopus oryzae was investigated. The system was constructed such that growing Rhizopus mycelia were self-immobilized on the plastic discs of a rotary biofilm contactor during the nitrogen-rich growth phase. During the nongrowth, production phase, the biofilm was alternately exposed to liquid medium and air upon rotation of the discs in the horizontal fermentation vessel. The production of fermentation, fumaric acid, was removed simultaneously and continuously by a coupled adsorption column, thereby moderating inhibition, enhancing the fermentation rate, and sustaining cell viability. Another beneficial effect of the removal of fumaric acid is release of hydroxyl ions from a polyvinyl pyridine adsorbent into the circulating fermentation broth. This moderates the decrease in pH that would otherwise occur. Polyvinyl pyridine and IRA-900 gave the highest loading for this type of fermentation. This fermentation system is capable of producing fumaric acid with an average yield of 85 g/liter from 100 g of glucose per liter within 20 h under repetitive fed-batch cycles. On a weight yield basis, 91% of the theoretical maximum was obtained with a productivity of 4.25 g/liter/h. This is in contrast to stirred-tank fermentation supplemented with calcium carbonate, whose average weight yield was 65% after 72 h with a productivity of 0.9 g/liter/h. The immobilized reactor was operated repetitively for 2 weeks without loss of biological activity.

Research Area: Biofuels/Bioproducts    

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Sorptive Recovery of Dilute Ethanol from Distillation Column Bottom Stream

1996

Authors: Gulati, M., P. J. Westgate, M. Brewer, R. Hendrickson, and M. R. Ladisch
Journal: Appl. Biochem. and Biotechnol., 103-119 (1996) 
Book Chapter: 

Abstract: Modern ethanol distillation processes are designed to ensure removal of all ethanol from the column bottoms, i.e., to levels <100 ppm ethanol, and utilize substantial stripping steam to achieve this result. An alternate approach using sorption was attempted as a a means to reduce energy requirements in the stripping section, and thereby reduce cost. Adsorbents tested for use in such an application showed that carbonaceous supports, in particular Ambersorb XEN 572, gave alcohol-free water as effluent when a 1% (w/w) starting ethanol concentration was passed downflow at 1 bed vol/h over a fixed-bed adsorber at 70° C. Regeneration was readily achieved at 70-90° C using hot air, vacuum, superheated steam, or hot water to strip the ethanol from the column, and yielded ethanol streams containing a maximum of 5.9% alcohol, with average concentrations of 2.5-3.5% depending on the regeneration method used. These experimentally determined operating conditions combined with distillation energy calculations have enabled development of a process concept for sorptive concentration of dilute ethanol which is more energy efficient than distillation alone. The combination of existing distillation and corn grit drying technologies, with sorptive recovery of dilute ethanol (from the column bottoms) shows promise of recovering a fuel grade, 99.4% ethanol product from a 4.5% ethanol broth with an energy requirement of 23,100 BTU/gal. The potential energy saving of 3600 BTU/gal over distillation alone corresponds to 1.8 cents/gal, and provide motivation for further examination of this approach in reducing costs of ethanol production from biomass.

Research Area: Biofuels/Bioproducts    

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Synthesis and Optimization of a New Starch Based Adsorbent for Dehumidification of Air in a Pressure Swing Drier

1996

Authors: Anderson, L., M. Gulati, P. Westgate, E. Kvam, K. Bowman, and M. R. Ladisch
Journal: Ind. & Eng. Chem. Res., 35, 1180-1187 (1996)
Book Chapter: 

Abstract: Corn grits selectively adsorb water from many types of organic vapors and are used commercially to dry 2.8 billion L of fuel-grade fermentation ethanol annually. Evaluation of grits in a pressure-swing dryer at 308 kPa, combined with analyses of their physical properties, showed that the specific surface of the grits (0.5 m2/g) limited steady-state drying of air to a dewpoint of -20 °C. By selectively taking advantage of the best features of the natural material, a new class of natural adsorbents with a higher affinity for water was then synthesized using material derived from corn: starch and cob flour. The chemical composition of the synthesized adsorbent was determined, as well as specific physical properties. Scanning electron microscopy showed the synthesized adsorbent surface had a large number of macropores (10-25 um in diameter) unlike corn grits which have limited porosity. This material gave reasonable and reproducible results, similar to those obtained with molecular sieves using a commercially available pressure-swing air dryer. After 70 h of operation at 30 psi, the new adsorbent provided air at a dewpoint of -63 °C. The methods for preparing this material and an explanation of its performance in terms of macroscopic and microscopic structural characteristics are described.

Research Area: Bioseparations    

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Analysis of Plant Harvest Indices for Bioregenerative Life Support Systems

1995

Authors: Velayudhan, A., K. L. Kohlmann, P. J. Westgate and Ladisch, M. R.
Journal: Enz. Microb. Technol., 17, 907-910 (1995)
Book Chapter: 

Abstract: Harvest indices, which are measures of the ratio of edible to total plant weight, are redefined to include edible sugars derived from enzymatic hydrolysis of the cellulose content of inedible plant components. Compositional analysis and carbohydrate contents of rapeseed, rice, soybeans, cowpea, wheat, sweet potato, white potato, and lettuce were analyzed to develop such generalized harvest indices. Cellulose conversion is shown to extend considerably the food available from plants otherwise grown for their oil and protein content in a bioregenerative life support system.

Research Area: Bioenergy    

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Biosynthesis of Cephalosporin C: Regulation and Recombinant Technology

1995

Authors: J. Weil, J. Miramonti, and M. R. Ladisch
Journal: Enzyme Microb. Technol., 7, 88-90 (1995). 
Book Chapter: 

Abstract: The regulation of the pathway in C. acremonium involves both enzyme inhibition and repression. The first enzyme in the pathway, aminoadipyl cysteinyl valine synthetase (ACVS), has optimal activity if all three substrates, aminoadipic acid, cysteine, and valine are present. ACVS regulation is most likely to occur at the transcriptional level. Repression of ACVS is caused by ammonium or phosphate ions. Methionine induces ACVS.

Research Area: Bioprocessing    

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Cephalosporin C: Mode of Action and Biosynthetic Pathway

1995

Authors: Weil, J., J. Miramonti, and M. R. Ladisch
Journal: Enz. Microb. Technol., 17, 85-87 (1995)
Book Chapter: 

Abstract: The world market for antibiotics is large with sales estimated at $16 billion in 1987, of which B-lactam antibiotics accounted for $9 billion. Cephalosporin C is a B-lactam produced by a fungus discovered off the coast of Sardinia by Giuseppe Brotzu in 1948 which was given the name Cephalosporium sp. Five years later, the active compound was identified as cephalosporin C, a secondary metabolite derived from aminoadipic acid, valine, and cysteine and presumably secreted as a defense mechanism against attacking bacteria. Cephalosporin-derived antibiotics are part of the large class of B-lactam antibiotics that include penicillins, norcardicins, monohactams, and thienemycins. Cephalosporin is one of the few B-lactams, however, that show activity against gram-negative as well as gram-positive bacteria.

Research Area: Bioprocessing    

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Correlation of Electrophoretic Mobilities of Proteins and Peptides with Their Physicochemical Properties

1995

Authors: Basak, S. and M. R. Ladisch
Journal: Anal. Biochem., 226, 51-58 (1995) 
Book Chapter: 

Abstract: Electrophoretic mobilites, u, of nine proteins (Mr 14,200 to 70,000) in 28 mM Tris/47 mM glycine buffer at pH 8.77 and 5 mM ionic strength were measured by laser Doppler velocimetry and correlated to ratios of charge (q) to molecular weight (Mr) and shape factor (f/f0) by the equation u(f/f0) = (Aq/Mr –B). This correlation was previously reported for peptides and proteins for u measured at 100 mM ionic strength. When A = 6.048 x 10-3, B = 1.13 x 10-5, and p = 2/3, the correlation fitted 51 measured and literature values over the molecular weight range of 178 to 140,000 for components whose electrophoretic Mobilities ranged from + 13.35 x 10-5 to -19.7 x 10-5 cm2/ (V.s). The experimental measurements confirm the general suitability of p=2/3 and show that the familiar charge/mass relation for electrophoresis is applicable to proteins in low-ionic-strength buffers which are typical of electrochromatography systems. Extrapolation of the correlation to different ionic strengths indicates that a low-ionic-strength buffer amplifies differences of electrophoretic mobility as a function of charge/mass, while high ionic strength diminishes such differences.

Research Area: Bioseparations    

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Effect of Modulator Sorption on Gradient Shape in Ion Exchange Chromatography

1995

Authors: Velayudhan, A. and M. R. Ladisch
Journal: Ind. Eng. Chem. Res., 34(8), 2805-2810 (1995)
Book Chapter: 

Abstract: Mobile phase additives, or modulators, are used in gradient elution chromatography to facilitate separation and reduce separation time. The modulators are usually assumed to be linearly adsorbed or unadsorbed. Here, the consequences of nonlinear modulator adsorption are examined for ion-exchange gradient elution through a series of simulations. Even when the buffer salt is identical to the modulator salt, gradient deformation is observed; the extent of deformation increases as the volume of the feed is increased. When the modulator salt is different from the buffer salt, unusual effects are observed, and the chromatograms are quite different from those predicted by classical gradient elution theory. In particular, local increases in the buffer concentration are found between feed bands, and serve to improve the separation. These effects become more pronounced as the feed volume increases, and could therefore prove valuable in preparative applications.

Research Area: Bioseparations    

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Electrochromatographic Separations of Proteins

1995

Authors: Basak, S., A. Velayudhan, K. Kohlmann and M. R. Ladisch
Journal: Journal of Chromatography A., 707, 69-76 (1995)
Book Chapter: 

Abstract: We have developed a modified electrochromatography system which minimizes Joule heating at electric field strengths up to 125 V/cm. A non-linear equilibrium model is described which incorporates electrophoretic mobility, hydrodynamic flow velocity, and an electrically induced concentration polarization at the surface of the stationary phase. This model is able to provide useful estimates of protein retention time and velocity in a column packed with Sephadex gel and subjected to an electric field. A correlation of electrophoretic mobility of peptide and proteins with respect to their charge, molecular mass and asymmetry enables the selection of solute target molecules for electrochromatographic separations. Good separation of protein mixtures have been obtained.

Research Area: Bioseparations    

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Enhanced Enzyme Activities on Hydrated Lignocellulosic Substrates

1995

Authors: K. L. Kohlmann, A. Sarikaya, P. J. Westgate, J. Weil, A. Velayudhan, R. Hendrickson, M. R. Ladisch
Journal: 
Book Chapter: In Enzymatic Degradation of Insoluble Carbohydrates, Saddler, J., et al.; ACS Symposium Series, American Chemical Society, Washington, DC

Abstract: Enzyme and substrate factors which limit hydrolysis include cellulose crystallinity and lignocellulose morphology, as well as enzyme activity, stability and inhibition. Brassica napus (rapeseed) is a biomass having large amounts of inedible material proposed for use in a controlled ecological life support system (CELSS) for human space flight. Mechanistic descriptions between morphological, chemical, and surface properties of this lignocellulose and enzyme hydrolysis are being developed. The goal is to define conditions for a cost effective pretreatment based on biological lignin removal followed by pressure cooking of the remaining cellulose in water at 180 to 220 C. Liquid water treatment of plant stems has resulted in a 6-fold improvement in cellulose hydrolysis during a 24 h incubation with commercial cellulases. When the water treatment is preceded by mycelial growth of the mushroom, Pleurotus ostreatus, further enhancement of enzymatic hydrolysis is achieved. Enzyme hydrolysis of plant material will be analyzed for its ability to sustain a CELSS.

Research Area: Bioenergy Biofuels/Bioproducts Bioseparations  

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Framework for Correlating Composition Dependent Equilibrium Conversion in Methyl tert-Butyl Ether Formation by Ion-Exchange Catalysts

1995

Authors: Ladisch, M., P. Westgate, R. Hendrickson, and M. Brewer
Journal: Ind. Eng. Chem. Res., 34(8), 2811-2816 (1995) 
Book Chapter: 

Abstract: Catalyst performance for the reaction of methanol and isobutylene (IB) to form methyl tert-butyl ether can be assessed based on maximum conversion. The equilibrium conversion attainable for this reaction is of practical interest since separation of products from reactants downstream of the reactor can be simplified as the extent of conversion increases. A framework is presented by which different catalysts can be compared on an internally consistent basis for different temperatures, isobutylene concentrations, and/or methanol/isobutylene mole ratios. An equilibrium expression which accounts for the presence of nonreacting components is presented to correlate the effect of methanol/IB ratios and IB concentrations with observed conversions for different catalysts. Assumptions inherent in this method are discussed and data for several types of ion-exchange catalysts illustrate use of this framework. 

Research Area: Bioprocessing    

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Mechanistic Description and Experimental Studies of Electrochromatography of Proteins

1995

Authors: Basak, S. and M. R. Ladisch
Journal: AIChE J., 41(11), 2499-2507, (1995) 
Book Chapter: 

Abstract: Electrochromatography is a form of gradient liquid chromatography in which an axial electric potential is applied to columns packed with gel-filtration media. Experimental methodology and a mechanistic model are further developed for a system that minimizes Joule heating at electric field strengths of 100 V/cm by dissipating heat through a cooling jacket and use of a cooled, low ionic strength eluting buffer. Focusing of proteins can be achieved in a 15-mm-dia. Column by the interplay of eluent velocity, electrophoretic migration rate, and electrically induced concentration polarization when the stationary phase is more conductive than the mobile phase. Voltage gradients of up to 125 V/cm for eluent velocities at 18-25 cm/h separate binary protein mixtures of Bhb-a–lactalbumin, BSA-myoglobin, and a-lactalbumin-myoglobin over Sephadex G-100 and G-50. Retention times are consistent with values obtained from a mechanistic nonlinear model.

Research Area: Bioseparations    

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Scale-up Techniques in Bioseparation Processes

1995

Authors: Ladisch, M. R., and A. Velayudhan
Journal: Bioseparation Processes in Foods, Dekker, (1995)
Book Chapter: 

Abstract: Chromatography plays a major role in the downstream processing of biological materials encountered in the manufacture of food, pharmaceutical, and biotechnology products. Here, we offer approaches to scaling up linear chromatography in chromatographic separations based on isocratic elution, as well as illustrate a nonideal, gradient-induced peak deformation effect, which may occur upon scale-up of gradient chromatography.

Research Area: Bioseparations    

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Simulation of Diauxic Production of Cephalosporin C by Cephalosporium acremonium: Lag Model for Fed Batch Fermentation

1995

Authors: Basak, S., A. Velayudhan, and M. R. Ladisch
Journal: Biotechnol. Progr., 11, 626-631 (1995) 
Book Chapter: 

Abstract: We extend a previously reported model( Chu, W.B.; Constantinides, A. Biotechnol. Bioeng. 1988, 32, 277-288) for the batch fermentation of cephalosporin C under the diauxic growth of Cephalosporium acremonium on glucose and sucrose to a fed-batch system. For this purpose, a novel lag model is proposed for diauxie, which has two functional forms, each embodying the dependence of lag on total cell mass and secondary substrate concentration. This lag model is applicable for batch simulations for arbitrary initial glucose and sucrose concentrations. We used the previously reported batch data to perform locally optimized fed-batch simulations. When applied to fed-batch fermentations, multiple lag times were accounted for. These studies showed that fed-batch fermentations (under the restriction that cell mass concentration did not exceed 25 g/L) could be more productive than simple batch runs. A representative result for a glucose-pulse fed-batch run at optimal cephalosporin production is a productivity of 4.22 mg of cephalosporin C/(L*h) and a production yield of 9.25 mg of cephalosporin C/g of total sugar used.

Research Area: Bioprocessing    

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Simultaneous Concentration and Purification Through Gradient Deformation in Gradient Elution Chromatography

1995

Authors: Velayudhan, A., R. L. Hendrickson, and M. R. Ladisch
Journal: AIChE J., 41(5), 1184-1193 (1995)
Book Chapter: 

Abstract: Mobile-phase additives, commonly used to modulate absorbate retention in gradient elution chromatography, are usually assumed to be either linearly retained or unretained. Previous theoretical work from our laboratory has shown that these modulators, such as salts in ion-exchange and hydrophobic interaction chromatography and organic modifiers in reversed-phase chromatography, can absorb nonlinearly, giving rise to gradient deformation. Consequently, adsorbate peaks that elute in the vicinity of the head of the deformed gradient may exhibit unusual shapes, form shoulders, and/or be concentrated. These effects for a reversed-phase sorbent with aqueous acetonitrile (CAN) as the modulator are verified experimentally. Gradient deformation is demonstrated experimentally and agrees with simulations based on CAN isotherm parameters that are independently determined from batch equilibrium studies using the layer model. Unusual adsorbate peak shapes were found experimentally for single-component injections of phenylalanine, similar to those calculated by the simulations. A binary mixture of tryptophan and phenylalanine is used to demonstrate simultaneous concentration and separation, again in agreement with simulations. The possibility of gradient deformation in ion-exchange and hydrophobic interaction chromatography is discussed.

Research Area: Bioseparations    

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Synthesis of TAME Using Solid Acid Catalysts

1995

Authors: Vandersall, M. T., and M. R. Ladisch
Journal: Rohm and Haas Brasil Ltdr. (1995)
Book Chapter: 

Abstract: The formation of tertiary amyl methyl ether (TAME) from 2-methyl-2-butene and methanol has been studied in fixed-bed tubular reactors, using the strong acid functionalized polymeric catalysts Amberlyst 15 and Amberlyst 35. The results of experiments to compare the kinetics and equilibria for the reaction are presented. The effect of methanol to isoamylene ratio, reactor space velocity, temperature, and catalyst, on the conversion and selectivity to TAME are described. Amberlyst 35 catalyst is shown to have a higher activity than Amberlyst 15 catalyst and can therefore by effectively used at lower temperatures and higher production rates.

Research Area: Biofuels/Bioproducts    

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Cellulose Pretreatments of Lignocellulosic Substrates

1994

Authors: Weil, J., P. J. Westgate, K. L. Kohlmann, and M. R. Ladisch
Journal: Enz. Microb. Technol., 16, 1002-1004 (1994)
Book Chapter: 

Abstract: Cellulose is a linear polymer of glucose in plant and woody materials. It is associated with hemicellulose and other structural polysaccharides, and surrounded by a lignin seal. Lignin, a complex 3-dimensional polyaromatic matrix, forms a seal around cellulose microfibrils and exhibits limited covalent association with hemicellulose. This prevents enzymes and acids from accessing some regions of the cellulose polymers.

Research Area: Biofuels/Bioproducts    

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Characterization of Buffers for Electrokinetic Separations

1994

Authors: Basak, S. K., A. Velayudhan, and M. R. Ladisch
Journal: Appl. Biochemistry Biotechnol., 44, 243-261 (1994)
Book Chapter: 

Abstract: Buffers used in electrophoresis and electrochromatography must have a relatively low ionic strength in order to minimize ohmic heating in the presence of an applied potential. Calculation of pH, ionic strength, and the van Slyke buffer capacity, B, is therefore important. This paper describes the a priori calculation of these parameters for tris buffer made up with either glycine (a zwitterions) or HCl. A quadratic expression for pH, valid over wide ranges, is obtained for both buffer systems. The calculated values of pH, ionic strength, and buffer capacity are shown to agree with experimental results as a function of tris, HCl, and glycine concentrations ranging from 1 to 50 mM. A new parameter, the electrokinetic buffer effectiveness factor, is introduced to characterize buffers being considered for use in electrokinetic systems such as electrochromatography, and is used to determine the appropriate composition ranges for the buffer components.

Research Area: Bioseparations    

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Increasing MTBE Production Without Increasing Capital Costs

1994

Authors: Chavez, R., R. Olsen, and M. R. Ladisch
Journal: Ind. Chem. Res., 32, 1888-1894 (1994)
Book Chapter: 

Abstract: As a result of the motor vehicle emissions standards imposed by the Clean Air Act Amendments of 1990, there is a potential for a large increase in demand for oxygenates such as MTBE. According to the Oxygenated Fuels Association’s Report, “Benefits of a National Oxygenated Fuels Policy,” oxygenates are now added to more than 30% of the U.S. gasoline pool, which represents about 4% of the total amount of gasoline consumed. By the year 2000, oxygenates are expected to be added to 70% of the U.S. gasoline pool, and will represent nearly 10% of the total amount of gasoline consumed. With increasing demand for MTBE, there is interest in finding a cost effective way to increase production from existing process units. One way is to use a catalyst with increased activity. Rohm and Haas has developed Amberlyst 35 Wet polymeric catalyst as its “next generation” catalyst for enhancing oxygenate production.

Research Area: Biofuels/Bioproducts    

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Air Drying Using Corn Grits as the Sorbent in a Pressure Swing Adsorber

1993

Authors: Westgate, P. J., and M. R. Ladisch
Journal: AIChE J., 39(4), 720-723 (1993)
Book Chapter: 

Abstract: Vapor streams containing organics and water can be dried in an energy-efficient manner by being passed over a cellulose or starch adsorbent such as corn grits. The success of this dehydration method appears to be related to differences in the rates of adsorption, as well as differences in the strength of interaction between each species and the adsorbent. Hence, compounds which exhibit either weak interactions or slow rates of adsorption are expected to be readily separated from those that exhibit strong, relatively fast interactions with the adsorbent.

Research Area: Bioseparations    

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Biological-Based Systems for Waste Processing

1993

Authors: Kohlman, K., P. Westgate, J. Weil, and M. R. Ladisch
Journal: SAE Technical Paper Series, (1993)
Book Chapter: 

Abstract: Inedible plant materials are a valuable resource in a controlled ecological life support system (CELSS). These plant “wastes” yield the sugars which facilitate the microbial-based recycle of C, H, O, and N. Conversion of these wastes to carbon dioxide and heat while also generating nutritious foodstuffs requires that: 1) the recalcitrance of cellulose in these materials be understood, and 2) ways be found to efficiently overcome the protective effect of lignin and other components closely associated with the cellulose. Means must be found to cost effectively increase the bioavailability of the cellulose which are intrinsically safe and environmentally compatible. The pretreatment of Cellulosic materials in liquid water temperatures about 200o C can give a hydrated, swollen cellulose. The resulting enhancements in surface area increase the rate of enzyme hydrolysis. However, this pretreatment involves complex phenomena due to auto-catalytic degradation of cellulose which occurs at pH levels below 5. A model is currently being developed to aid in selection of conditions which minimize chemical degradation of cellulose while maximizing disruption of its physical structure. The goal is to maximize cellulose surface area. Pretreatment studies are being coupled with measurements and modeling of changes in cellulose properties in order to relate the effect of pretreatments on hydrolysis (using various Cellulolytic enzymes) and microbial use of lignocellulosics. The fundamental modeling and experimental studies are being complemented by analyses of lignocellulosic materials which may be grown in a CELSS including rapeseed, cowpea, and rice.

Research Area: Biofuels/Bioproducts    

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Catalyst-Induced Yield Enhancement in a Tubular Reactor

1993

Authors: M. R. Ladisch, R. L. Hendrickson, M. A. Brewer, and P. J. Westgate
Journal: Industrial & Engineering Chemistry Research, 32, 9, 1888-1894 (1993) 
Book Chapter: 

Abstract: A macroreticular cation-exchange resin with an acid capacity of 5.5 mequiv/g and a higher acid group density was evaluated for MTBE formation from isobutylene and methanol using a 10-ft-long nonisothermal tubular reactor system with the methanol/isobutylene feed at close to stoichiometric ratio. A side-by-side comparison to a standard sulfonated catalyst (4.7 mequiv/g) shows this catalyst has a higher activity and increases maximum conversion by 2-5% and liquid hourly space yields by 10-25%. Selectivities for both catalysts were close to 1. Concentration-based equilibrium constants (Kx) for the enhanced catalyst were 870-2500 at temperatures ranging from 343 to 313 K compared to 300-850 for the standard sulfonated catalyst over the same temperature range. The catalyst with the higher acid group density enhances the maximum conversion of MTBE and increases the rate of reaction relative to the sulfonated catalyst currently in wide use in the industry.

Research Area: Biofuels/Bioproducts    

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Liquid Chromatography Using Cellulosic Continuous Stationary Phases

1993

Authors: Yang, Y., A. Velayudhan, C. M. Ladisch, and M. R. Ladisch
Journal: Advances in Biochemical Engineering and Biotechnology, 49, 147-160, (1993)
Book Chapter: 

Abstract: A novel type of continuous stationary phase based on fabric materials is described. This column packing utilizes the continuous character of a cellulose (cotton) stationary phase, and the chemistry of the derivatized forms of the adsorbent, to obtain separations of proteins and small molecules based on cation and anion exchange, hydrophobic interactions, and size. The mechanical stability of the stationary phase facilitates chromatographic velocities in excess of 70 cm min-1. The influence of eluent properties on the adsorption of sample proteins is discussed in this chapter. Sequential stepwise desorption is used to separate 100 ul mixtures of BSA, IgG, B-galac10sidease, and insulin in 10 minutes or less, using 10 mm i.d. x 500 mm length columns.

Research Area: Bioseparations    

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Modulator Sorption in Gradient Elution Chromatography

1993

Authors: Velayudhan, A., and M. R. Ladisch
Journal: Bioproducts and Bioprocesses, 2, 217-232, (1993)
Book Chapter: 

Abstract: Gradient elution chromatography is widely used to separate both small molecules and macromolecules. The mobile phase additive (modulator) used to modify adsorbate retention is usually considered to be either unretained or linearly retained. In both cases, the shape of the gradient does not change as it moves down the column. However, the high mobile phase concentrations at which such a modulator is commonly used makes it likely to adsorb according to its nonlinear sorption isotherm. Here the quantitative consequences of such nonlinear modulator sorption are reviewed. Nonlinear sorption deforms the shape of the gradient during its passage through the column; ultimately a shock (or shock layer) could be formed. The condition for shock formation is discussed, and numerical simulations using representative parameter illustrate the magnitude of gradient deformation and the consequences for separation.

Research Area: Bioseparations    

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Plates Models in Chromatography: Analysis and Implications for Scale up

1993

Authors: Velayudhan, A., and M. R. Ladisch
Journal: Advances in Biochem. Engr. Biotech., 49, 123-145, (1993)
Book Chapter: 

Abstract: Detailed chromatography rate theories from the literature can be used to determine the appropriate plate count for a plate model of linear chromatography so that the bandspreading generated by the detailed rate model is reproduced by the plate model. This process provides a link between the plate count and the physical parameters that cause bandspreading. Each sample component can be assigned an appropriate plate count, thus allowing the accurate simulation of multicomponent separations even for widely differing adsorbates. Analytical solutions are presented for the Craig distribution and the continuous plate model for both finit-pulse elution and frontal chromatography. The Craig model is widely considered unsuitable because it assumes discontinuous flow; it is shown that, for a suitably corrected plate count, the Craig model is as accurate as the continuous-flow plate theory (expect for the case of an unretained solute). Direct calculation of effluent histories from these plate models show excellent agreement between themselves and with results from complex rate models available in the literature. Reasonable agreement is also found when the plate models are used a priori to predict experimental scale-up results.

Research Area: Bioseparations    

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Solute Retention in Electrochromatography by Electrically Induced Sorption

1993

Authors: Rudge, S. R., S. Basak and M. R. Ladisch
Journal: AIChE J., 39(5), 797-808 (1993)
Book Chapter: 

Abstract: Column chromatography and electrophoresis are combined in electrochromatography, where an electric potential is applied to a chromatography column in the axial direction. These studies utilized a dextran gel stationary phase and an eluent of low ionic strength, which were chosen to minimize electric current and therefore column heating and undesirable dispersion effects. The gel, with a small ion exchange capacity of several microequivalents per mL, turned out to be more conductive than the eluent and was able to concentrate macromolecules in the presence of combined electric and flow fields. The model presented describes solute retention due to electrically induced concentration polarization of solute on the resin surfaces, as well as electrophoresis in the mobile and stationary phases. The polarization effect explains differences between retention of high-molecular-weight solutes with exclusion coefficients of less than 1 and that of a charged low-molecular-weight solute, which is hypothesized to pass through the gel matrix in the presence of an electric field and does not experience concentration polarization. It also shows the application of this effect for protein separation in a liquid chromatography system with a superimposed electric potential.

Research Area: Bioseparations    

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Sorption of Organics and Water on Starch

1993

Authors: Westgate P., and M. R. Ladisch
Journal: Ind. Eng. Chem. Res , 32(8), 1676-1680 (1993)
Book Chapter: 

Abstract: Starch is a well-established adsorption agent for drying ethanol. This work examines its potential for other gas-phase drying applications. Results from gas chromatography studies confirm that starch separates water from organic acids, alcohols, ketones, ethers, and aromatics, many of which from azeotropes with water. Trends in organics with respect to size and functional group show that the efficiency of this separation is related to both transport properties and strength of interaction between the organic components and starch. Small, polar molecules such as methanol and formic acid that have rapid mass-transfer characteristics and relatively strong interactions with starch are retained to a greater degree and are more difficult to separate from water than either compounds of higher molecular weight or decreased polarity. The large number of possible separations indicates that starch is a versatile material for use in sorbents for vapor-phase separations.

Research Area: Bioseparations    

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A New Approach to the Study of Textile Properties by Liquid Chromatography, Comparison of Void Volume and Surface area of Cotton and Ramie Using a Rolled Fabric Stationary Phase

1992

Authors: Ladisch, C. M., Y. Yang, A. Velayudhan, and M. R. Ladisch
Journal: Textile Res. J., 62(6), 361-369 (1992)
Book Chapter: 

Abstract: A novel rolled stationary phase using whole fabric has been developed for liquid chromatography. This paper describes the column and its properties and use in characterizing pore volumes of the whole fabric. The logistic model gives a good fit of the measured void volume data obtained by size exclusion chromatography. With the help of this function, both pore size and surface area distribution of cotton and ramie fabrics can be obtained. The relationship between pore shape and surface area is discussed. Cotton has 100 to 200% more void volume and surface area than ramie (for all sizes of pores). Increasing the temperature from 30 to 60 °C does not significantly influence either the total void volume or surface area of cotton and ramie. The void volume and surface area of cotton as determined by this rolled fabric column method are comparable to previously reported data.

Research Area: Bioseparations    

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Bioprocessing in Space

1992

Authors: Westgate, P. J., K. Kohlmann, R. L. Hendrickson, and M. R. Ladisch
Journal: Enzyme Microb. Technol., 14(1), 76-79 (1992)
Book Chapter: 

Abstract: The potential role of separations, hydrolysis, and fermentations in a Controlled Ecological Life Support System (CELSS) environment presents challenging opportunities for bioprocessing in outer space. Introduction Manned missions may soon attempt to establish bases on the moon and Mars. Hence, manned space flights will be of longer duration and distances from earth, and a Controlled Ecological Life Support System (CELSS) will be needed to provide a constant supply of food, air, and water through bioregenerative means. CELSS is defined as a closed system with only energy crossing the outer boundary. All raw materials are provided by recycling wastes.

Research Area: Bioenergy    

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Effect of Modulator Sorption in Gradient Elution Chromatography: Gradient Deformation

1992

Authors: A. Velayudhan, and M. R. Ladisch
Journal: Chemical Eng. Sci. J., 47. No. 1, 233-239 (1992)
Book Chapter: 

Abstract: In gradient elution chromatography, the mobile phase composition at the column inlet is a function of time. The consequences of accounting for the adsorption of the mobile phase components themselves are investigated for the experimentally common situation of a linear inlet gradient. Surface excess adsorption data from the literature for water-acetonitrile mobile phases in reversed-phase chromatography using different octadecyl stationary phases are shown to be in good internal agreement. The resulting individual isotherms as calculated by Tani and Suzuki (1989) are fitted to Langmuir and BET forms; it is shown that the Langmuirian form fits the data well except at extremely high acetonitrile concentrations. Using these isotherm parameter, it is found that a linear inlet gradient could suffer significant distortion while moving down the column. In the extreme case, a shock front of the mobile phase modulator could result. Analytical expressions are derived describing the conditions under which such a shock could occur within the column and its subsequent path, assuming Langmoirian sorption.

Research Area: Bioseparations    

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Effect of pH on Subunit Association and Heat Protection of Soybean a-Galactosidase

1992

Authors: Porter, J., A. Sarikaya, K. M. Hermann, and M. R. Ladisch
Journal: Enz. Microb. Technol., 14, 609-613 (1992)
Book Chapter: 

Abstract: Soybeans contain the enzyme a-galactosidase, which hydrolyzes a-1.6 linkages in stachyose and raffinose to give sucrose and galactose. We have found that galactose, a competitive product inhibitor of a-galactosidase, strongly promotes the heat stability of the tetrameric form of the enzyme at pH 4.0 and at temperatures of up to 70oC for 60 min. Stachyose and raffinose also protect a-galactosidase from denaturation at pH 4.0, although to a lesser extent. Glucose and mannose have little effect. At the absence of heat protection of the enzyme by added sugars, a series deactivation mechanism was found to describe the deactivation data. In comparison, a unimolecular, non-first order deactivation model applies at pH 4.0, where heat protection effects were observed. At a temperature above 60oC, simple deactivation is a suitable model. The results suggest that a-galactosidase conformation and heat stability are directly related.

Research Area: Biofuels/Bioproducts    

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Intercalation in the Pretreatment of Cellulose

1992

Authors: Michael R. Ladisch, Lori Waugh, Paul Westgate, Karen Kohlmann, Rick Hendrickson, Yiqi Yang, and Chris Ladisch
Journal: American Chemical Society, 1992
Book Chapter: 

Abstract: The structural features of cellulose are known to profoundly influence the kinetics of cellulose hydrolysis. Cellulose in biomass is resistant to hydrolysis due to hydrophobic interactions between cellulose sheets, hydrogen bonding between adjacent cellulose chains, and cellulose's close association with lignin. A useful pretreatment disrupts hydrophobic and hydrogen bonds, as well as the lignin seal, in a manner which minimizes chemical change of the cellulose and formation of undesirable degradation products. The resulting polysaccharide structure must be stabilized against spontaneous recrystallization, once pretreatment conditions are removed. Otherwise the benefit of enhanced hydrolysis is lost. This work reports the intercalating effects and mechanisms of sulfate esters, and the role of water in altering the physical properties of pretreated cellulose. A mechanism is proposed which leads to a leveling off in particle size (LOPS) during enzyme hydrolysis of lignin free, microcrystalline cellulose.

Research Area: Biofuels/Bioproducts    

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Modeling of Equilibrium Sorption of Water Vapor on Starch Materials

1992

Authors: Westgate, P. J., J. Y. Lee, and M. R. Ladisch
Journal: Transactions ASAE, 35(1), 213-219 (1992)
Book Chapter: 

Abstract: The equilibrium behavior of corn grits and corn starch at temperatures above 70o C, a region for which limited data and theory are available, are compared and found to be similar. Sircar’s model and potential theory accurately represent isotherm data for both adsorption systems as well as data for desorption from corn. Values of the model parameters indicate that physical properties of these starch-based sorption materials exhibit a temperature dependence that is likely related to the breaking of hydrogen bonds as water interacts with the sorbent. Modification of the two models with an exponential temperature relation is proposed to account for the experimentally measured temperature dependence of model parameters. The resulting modified Sircar’s model and potential theory equations are shown to fit the data for both starch and corn grits in the high temperature range.

Research Area: Bioseparations    

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Modeling of Non-Linear Elution Chromatography for Preparative-Scale Separations

1992

Authors: Velayudhan, A., M. R. Ladisch, and J. Porter
Journal: AIChE Symp. Ser., 88, (1992)
Book Chapter: 

Abstract: Many models are available for describing elution profiles in preparative-scale overloaded chromatography. These range from empirical to theoretical and describe experimental data with varying success. This chapter reviews models and approaches to analyzing isocratic and gradient elution. When the loading is low-to-moderate in nonlinear isocratic elution, analytical solutions are available from which productivity can be calculated as a function of operating parameters. For higher loadings, optimization studies have been carried out on binary mixtures obeying Langmuirian isotherms. Gradient elution separations have been successfully scaled-up using a simplified theory.

Research Area: Bioseparations    

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Protein Chromatography Using a Continuous Stationary Phase

1992

Authors: Yang, Y., A. Velayudhan, C. M. Ladisch, and M. R. Ladisch
Journal: J. Chromatogr., 598, 169-180 (1992)
Book Chapter: 

Abstract: A continuous stationary phase consisting of yarns woven into a fabric is rolled and packed into mechanically stable liquid chromatography columns. This work utilized yarns have a characteristic width of 200 400-um, made from 10 20-um fibers consisting of 95% poly(m-phenylene isophthalamide) and 5% poly(p-phenylene terephthalamide). Although loadings on this stationary phase were low at 4 mg/g for bovine serum albumin and 6 mg/g for B-galactosidase, this material shows the interesting characteristic of a leveling off of plate height at mobile phase velocities of 30 80-cm/min. This phenomenon is explained on the basis of a coupling argument whereby a fraction of the mobile phase flows through the intramatrix pore space, and convective transport through the pore space dominates transport by diffusion. A modified Van Deemter expression is derived and shown to lit plate height data for polyethylene glycol standards having molecular weights of 200 and 20 000. The characteristic of this continuous stationary phase at high eluent velocities are discussed and conditions which give separation of immunoglobulin G, bovine serum albumia, insulin and B-galactosidase in 12 min are described.

Research Area: Bioseparations    

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Recombinant Human Insulin

1992

Authors: Ladisch, M. R., and K. Kohlmann
Journal: Biotechnol. Prog., 8(6), 469-478 (1992)
Book Chapter: 

Abstract: Insulin is a well-characterized peptide that can be produced by recombinant DNA technology for human therapeutic use. A brief overview of insulin production from both traditional mammalian pancreatic extraction and recombinant bacterial and yeast systems is presented, and detection techniques, including electrophoresis, are reviewed. Analytical systems for insulin separation are principally based on reversed-phase chromatography, which resolves the deamidation product(s) (desamido insulin) of insulin, proinsulin, and insulin. Process-scale separation is a multistep process and includes ion exchange, reversed-phase, and size exclusion chromatography. Advantages and/or disadvantages of various separation approaches, as described by the numerous literature references on insulin purification, are presented.

Research Area: Bioseparations    

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Analytical and Preparative Scale Chromatography of Phenylalanine from Aspartame Using a New Polymeric Sorbent

1991

Authors: Ladisch, M. R., R. L. Hendrickson, and E. Firouztale
Journal: J. Chromatogr. (540), 85-101 (1991)
Book Chapter: 

Abstract: A new, large-pore, cross-linked, polymethacrylate stationary phase separates Phe from Aspartame in 10% aqueous ethanol by reversed-phase chromatography. Batch equilibrium data at 30, 50, and 70 °C, obtained with 165-um particle size material showed linear sorption at loadings of up to 140 mg/g stationary phase, and corresponded to a mobile phase adsorbate concentration approaching the solubility limits. Column runs with 40-, 60-, 117-, and 165-um particle size materials at 30-70 °C showed retention behavior that was predictable from batch equilibrium data, and was independent of particle size at sample volumes as high as 80% of the column void volume and at outlet concentrations of 5-10 mg/ml. The relatively large pores (250 A) of the stationary phase allowed free access of small molecules, with the methacrylate structure promoting strong sorption of aromatic amino acids. These characteristics permitted the ready calculation of column retention times, and facilitated extrapolation of analytical-scale results obtained with small particle size material to reparative-scale separations carried out under volume overload conditions with a larger particle size stationary phase.

Research Area: Bioseparations    

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Effects of a Low Concentration of Added Plasmin to Ultra-High Temperature Processed Milk

1991

Authors: Kohlmann, K. L., S. S. Nielsen, and M. R. Ladisch
Journal: J. Dairy Sci., 74, 1151-1156 (1991)
Book Chapter: 

Abstract: The relationship between proteolysis and gelation was studied in UHT-processed milk following the aseptic addition of the enzyme plasmin at a concentration of .15 mg/L. Individual 250-ml containers of commercially processed (direct steam injection, 134.4oC for 14.2 s) milk were used. The milk was injected with plasmin 1 wk after processing and stored at room temperature (-23oC). Over a 6-mo period, the milk was examined for changes in appearance, pH, apparent viscosity, gel formation, enzymatic activity, and casein breakdown. Control milk samples did not gel during the test period. The milk containers that received the plasmin addition began to form a gel at 90 d of storage, and this gelation was accompanied by an increase in apparent viscosity. In the samples with added plasmin, enzyme activity was detected using the chromogenic substrate, H-D-valyl-L-leucyl-L-lysyl-4nitroanilide (S-2251), and casein breakdown was apparent as examined by SDS-Page. It appeared that the added plasmin preferentially attacked B- and a-caseins over k-casein. The evidence supports a relationship between a low level of plasmin activity and the gelation of UHT milk.

Research Area: Bioprocessing    

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Ethanol Production and the Cost of Fermentable Sugars from Biomass

1991

Authors: Ladisch, M. R. and J. A. Svarczkopf
Journal: Bioresource Technol. 36, 83-95 (1991) 
Book Chapter: 

Abstract: The current fermentation alcohol industry in the US is based on utilization of glucose and/or starch derived principally from corn. Biomass materials including wood and agricultural residues, newspaper, and other sources of cellulose could provide a source of fermentable sugars for expanding fuel ethanol production. Prospects for the utilization of biomass for conversion to fermentable hexoses and pentoses are continually improving with advances in enzyme technology, specially engineered microorganisms which can ferment pentoses, and improvement in cellulose pretreatments. Technical and economic factors which affect utilization of sugars from biomass are summarized, and the key steps in wet- and dry-milling of corn are described for purposes of comparison. An approach for estimating fermentable sugar costs is presented to gauge the impact of technical improvements on reducing fermentable sugar costs. An analytical framework resulting from this approach facilitates comparison of effects of feedstock costs, by-product credits, differences in technology, and process costs on the cost of fermentable sugars. A systematic strategy for evaluating differences in cost is presented as a tool for making a first comparison of different technologies and feed stock materials for ethanol production. This analysis suggest that fermentable sugars from enzymatic hydrolysis of cellulose must cost no more than 4-5c lb-1 (88-11c kg-1), at current conditions, if they are to be economically competitive with fermentable sugars derived from corn.

Research Area: Biofuels/Bioproducts    

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Fermentation Derived Butanol and Scenarios for its Uses in Energy Related Applications

1991

Authors: Ladisch, M. R.
Journal: Enz. Microb. Technol. 13(3), 280-283 (1991)
Book Chapter: 

Abstract: The production of acetone, normal butanol, and ethanol by anaerobic fermentation using Clostridium acetobutylicum, i.e. the Weizmann process, is a well-known technology. However, due to economic considerations, commercial scale acetone/butanol/ethanol (ABE) fermentations have been limited in recent times.

Research Area: Biofuels/Bioproducts    

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Growth, Death and Oxygen Uptake Kinetics of Pichia stipitis on Xylose

1991

Authors: Slininger, P. J., L. E. Branstator, R. J. Bothast, M. R. Okos, and M. R. Ladisch
Journal: Biotechnol. Bioeng., 37(10), 973-980 (1991) 
Book Chapter: 

Abstract: Pichia stipitis NRRL Y-7124 has potential application in the fermentation of xylose-rich waste streams produced by wood hydrolysis. Kinetic models of cell growth, death, and oxygen uptake were investigated in batch and oxygen-limited continuous cultures fed a rich synthetic medium. Variables included rates of dilution (D) and oxygen transfer (K, a) and concentrations of xylose (X), ethanol (E), and dissolved oxygen (Cox). Sustained cell growth required the presence of oxygen. Given excess xylose, specific growth rate (u) was a Monod function of Cox. Specific oxygen uptake rate was proportional to u by a yield coefficient relating biomass production to oxygen consumption; but oxygen uptake for maintenance was negligible. Thus steady-state Cox depended only on D, while steady-state biomass concentration was controlled by both D and Ka. Given excess oxygen, cells grew subject to Monad limitation by xylose, which became inhibitory above 40 g/L. Ethanol inhibition was consistent with Luong’s model, and 64.3 g/L was the maximum ethanol concentration allowing growth. Actively growing cells died at a rate that was 20% of u. The dying portion increased with E and X.

Research Area: Biofuels/Bioproducts    

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In Situ Observation of Casein Micelle Coagulation

1991

Authors: Ruettimann, K. W., and M. R. Ladisch
Journal: J. Colloid. Interface Sci., 146(1), 276-287 (1991)
Book Chapter: 

Abstract: Milk proteins occur in aqueous media as colloidal particles which are calcium-dependent, water-containing structures referred to as casein micelles. In situ studies of casein micelles using dark-field microscopy allow direct observation of micellar motion and interaction during coagulation induced by the enzymatic hydrolysis of the k-casein component of the micelles. The evolution of floc structure can be clearly visualized with the dark-field method. It is seen that flocs form irreversibly from only hydrolyzed micelles during the coagulation in a stagnant fluid. Coagulation patterns show that flocs from filamentous clusters incorporating micelles which may have several surface reaction sites. The techniques which make it possible to directly observe the coagulation reaction as it is occurring in its aqueous environment in a manner which provides insights into the mechanisms of the reaction are described.

Research Area: Bioprocessing    

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Ion-Exchange and Affinity Chromatography Costs in a-Galactosidase Purification

1991

Authors: Jill E. Porter and Michael R. Ladisch
Journal: Biotechnology and Bioengineering, 39, 717-724 (1991)
Book Chapter: 

Abstract: The purification of a-galactosidase from soybean seeds is a five to six-step procedure consisting of cryoprecipitation, acid precipitation and ammonium sulfate fractionation followed by two or three chromatography steps. The procedures, while not optimized, were carried out in a manner that resulted in 414-515-fold purification, as reported previously. The costs of two purification sequences were compared. In the best case, the preparative-scale costs of stationary phase, reagents, and hardware were $790 per million enzyme units, excluding labor. Stationary phase costs predominated over extraction, chromatography reagent and eluent costs when the stationary phase is replaced after 10-40 cycles of use. However, if stationary phase life exceeds 50-200 cycles, stationary phase costs become similar in magnitude to eluent and reagent costs. Labor costs, which are process-specific and difficult to estimate, exceed all other costs by a factor of 10-50 at a small scale of operation and constitute a major cost, regardless of scale. This case study provides equations and a framework for carrying out a first comparison of costs for multistep purification sequences. Column life, throughput, and scale of operation were found to determine not only the magnitude, but also the relative contributions of the different components that make up purification costs. This analysis shows that there are major opportunities for reducing purification costs through the development of less expensive stationary phases and the implementation of intelligent process control and automation for process scale chromatography.

Research Area: Bioseparations    

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Production of Proteases by Psychotropic Microorganisms

1991

Authors: Kohlmann, K. L., S. S. Nielsen, L. R. Steenson, and M. R. Ladisch
Journal: J. Dairy Sci., 74, 3275-3283 (1991) 
Book Chapter: 

Abstract: Six milk-derived psychorotrophic microbial cultures were screened for the ability to grow at refrigerated temperatures and produce proteases in reconstituted skim milk. Of these, two cultures, Pseudomonas fluorescens M3/6 and Pseudomonas fragi K122, produced extracellular protease(s) beginning 7 d postinoculation when the cultures had entered late log or early stationary phases of growth. Further work with these tow cultures showed that intracellular proteases were present after only 20-h incubation, before detection of the extracellular proteases. Using H-D-valyl-L-leucyl-L-lysy1-4-nitroanilide (S-2251), a sensitive substrate for plasmin activity, P. fluorescens was shown to have greater intracellular proteolytic activity than extracellular activity at 20 h of incubation. The intracellular enzyme activity remained constant while the extracellular and periplasmic activities increased over the remaining 6-d incubation period. The proteases in crude extracellular extracts from both cultures were characterized and were heat stable with broad temperature (7 to 52 °C) and pH (pH 5.5 to 8.5) ranges for activity and were inhibited by the metal chelator, EDTA, indicating that they were metalloproteases.

Research Area: Bioprocessing    

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Purification and Characterization of an Extracellular Protease Produced by Pseudomonas fluorescens M3/6

1991

Authors: Kohlmann, K. L., S. S. Nielsen, and M. R. Ladisch
Journal: J. Dairy Sci., 74, 4125-4136 (1991) 
Book Chapter: 

Abstract: Pseudomonas fluorescens strain M3/6 was inoculated into reconstituted NDM and incubated at 7 °C for 46 d. A significant amount of extracellular protease was produced, mainly during the latter part of the culture’s life cycle. The protease was purified using ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The isolated protease had activity on azocasein, a-, B-, and k-caseins and a plasmin substrate but did not have plasminogen activator activity. The protease had a molecular weight of 45 kDa, an isoelectric point of pH 8.25, a broad temperature and pH range for activity, and was less heat stable in the isolated form than in the cellfree extract.

Research Area: Bioseparations    

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Role of Modulator in Gradient Elution Chromatography

1991

Authors: Velayudhan, A. and M. R. Ladisch
Journal: Anal. Chem., 63(18), 2028-2032 (1991)
Book Chapter: 

Abstract: Mobile-phase additives are frequently used in gradient elution chromatography to modulate adsorbate retention. These additives are known to adsorb themselves onto the stationary phase, resulting in solvent dernixing. In this paper, the concentration of the mobile-phase additive in the mobile phase is assumed to be high enough for it to lie in the nonlinear region of the its own adsorption isotherm. Then the shape of the gradient would become deformed while passing down the column and could ultimately form a shock layer. A series of numerical simulations of a binary feed mixture is presented under conditions where a shock layer is formed, and the possible consequences are discussed. Depending on the location of the adsorbate peak with respect to the mobile-phase shock layer, leading or trailing shoulders can result, along with significant peak sharpening. The implications of these effects on separation are presented, and conditions under which the present analysis might be tested experimentally are indicated.

Research Area: Bioseparations    

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Water and Ethanol Sorption Phenomena on Starch

1991

Authors: Lee, J. Y., P. J. Westgate, and M. R. Ladisch
Journal: AIChE J., 8(37), 1187-1195 (1991)
Book Chapter: 

Abstract: The sorption behavior of water and ethanol on starch material has been investigated in relation to the adsorptive separation of water from ethanol. The adsorption isotherms of water-starch, ethanol-starch and water-ethanol-starch were measured using a Cahn electrobalance. Careful examination of the many sorption isotherm models resulted in selection of Sircar’s model and the potential theory to best represent the isotherm data of water-starch and ethanol-starch adsorption. Experimental results showed that ethanol as well as water can adsorb on starch. The adsorption rate of ethanol, however, is much slower than that of water. This suggests that the selective removal of water from ethanol vapor in a packed-bed adsorber is likely a rate-dependent, not equilibrium-dependent, process.

Research Area: Bioseparations    

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Alcohol Adsorption on Softwood Lignin from Aqueous Solutions

1990

Authors: Yang, Y., M. R. Ladisch, and C. M. Ladisch
Journal: Biotechnol. Bioeng., 35, 268-278 (1990)
Book Chapter: 

Abstract: Lignin prepared by acid and enzyme hydrolysis of a softwood mixture adsorbs acetone, butanol, and other alcohols while showing only a slight uptake of glucose. Adsorption of butanol is independent of temperature in the range of 30-65° C. The Polanyi theory fits adsorption for the linear alcohols methanol through hexanol with values of S and u ranging from 2.6 to 26 J mol-1 K –1 and –0.8 to –8 kJ/mol. The adsorption capacity is given by Q (g alcohol/g lignin) = KC*, where C* is the equilibrium alcohol concentration (g/mL), K = Ew exp, and Ew is the porosity of the lignin (0.23 – 0.42 mL/g). The value of the adsorption capacity constant K for n-butanol ranges from 1.3 to 2.7 mL/g on sorbent containing 26-72% lignin, while ethanol is 0.5-0.73, acetone is 0.62-1.0, and glucose is 0.35. Adsorption is shown to occur through combined hydrophobic and hydrophilic interactions of the alkyl and hydroxyl groups, respectively, of the adsorbate with the lignin. Consequently, for the alcohols methanol to hexanol, we present the capacity constant K[=K(R) + K(OH)] as a sum of an alkyl adsorption constant (0.1-9.5 mL/g) and a hydrophilic (0.40-0.50 mL/g) contribution. This approach may be applicable to organic acids. Lignin’s sorbent properties have potential to moderate product inhibition in the anaerobic acetone-butanol-ethanol (ABE) fermentation.

Research Area: Biofuels/Bioproducts    

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Analysis of Sub-microgram Quantities of Cellodextrins by Aqueous Liquid Chromatography Using a Differential Refractometer

1990

Authors: A. N. Pereira, K. L. Kohlmann, and M. R. Ladisch
Journal: Biomass, 23, 307-317 (1990)
Book Chapter: 

Abstract: The analysis of water-soluble cellodextrins using liquid chromatography is readily achieved with a variety of packings. Direct injection of enzyme incubation mixtures allows quantitation of 10 mM cellodextrins in hydrolysis mixtures, resulting in a method which is useful for kinetic studies. Reported here are operating procedures for a 4% cross-linked, styrene-divinyl benzene cation exchanger (Aminex 50W-X4 (Bio Rad Lab., Griffin, CA, USA), 20-30 um particle size) in the Ca++ form, packed in a column of dimensions 6 mm i.d. x 60 cm long. Using this column, resolution of the cellodextrins, celloheptaose through cellobiose and glucose was possible with 91 mM HsSO4 as the eluent. Requirements of the separation system included use of a pulsation free syringe pump to minimize baseline fluctuations, the use of Ca++ as the counterion to give a column operational life of 500-1000 injections, and injection of sample volumes of up to 25 uL. Cellodextrins were quantified at sub-microgram (nmole) levels using a differential refractometer as the detector. Examples of this technique for analysis of the acid hydrolysis of cellodextrins and enzymatic hydrolysis of cellodextrins and carboxymethylcellulose are described.

Research Area: Bioseparations    

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Characterization of the Swelling of a Size Exclusion Gel

1990

Authors: Monke, K., A. Velayudhan, and M. R. Ladisch
Journal: Biotechnol. Progress, 6(5), 376-382 (1990)
Book Chapter: 

Abstract: The swelling of a dextran gel, Sephadex G-75, was observed in an aqueous environment at room temperature by a noninvasive technique that uses light microscopy coupled to an image analysis system via video camera. The rate of swelling was found to follow the Tanaka and Fillmore theory, from which the overall gel diffusion coefficient was estimated at 6.3x10-7cm2/s. In addition to giving a quantitative measure of gel swelling that could be useful in the mechanical design of liquid chromatography columns, this approach provides data on wet particle size and particle size range, which is needed for the modeling of diffusional and mass transfer effects in size-exclusion chromatography. In this context, key observations are that the gel particles are nearly spherical with an elliptical shape factor of 0.98 (perfect sphere = 1) and that there is little difference between sizes of particles obtained in water, 50 mM Tris-glycine buffer (pH 10.2), and buffer containing 1 mg/mL protein. The diameter of the dry material ranged from 20 to 100 um, while the hydrated particles had diameters of 40-350 um. The rate of swelling is rapid, with 50% swelling occurring in about 10 s and swelling to 99% of the final wet particle size being obtained in less than 90 s.

Research Area: Bioseparations    

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Displacement Effect in Multicomponent Chromatography

1990

Authors: Gu, T., G. T. Tsao, G-J. Tsai, and M. R. Ladisch
Journal: AIChE J. 36(8), 1156-1162 (1990)
Book Chapter: 

Abstract: The study of interference effects is of fundamental importance in understanding multicomponent chromatography. In this work, a displacement effect is examined and shown to be able to explain the dominating interference effects in three major modes of chromatography-frontal, elution, and displacement-involving competitive isotherms. It is concluded that the concentration profile of a component usually becomes sharper due to the displacement effect from another component, while the concentration from of the displacer is usually diffused. Five factors that escalate the displacement effect in multicomponent elution were investigated. A binary elution with a competing modifier in the mobile phase was also discussed. This study was carried out using computer simulations based on a general nonlinear multicomponent rate equation model that considers axial dispersion, external mass transfer intraparticle diffusion, and Langmuir isotherms. The use of the general model helps the visualization of the multicomponent interactions in chromatography under mass transfer conditions.

Research Area: Bioseparations    

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Freeze Concentration of Dyes

1990

Authors: Yang, Y., C. M. Ladisch, and M. R. Ladisch
Journal: Textile Res. J., 12(60) 744-753 (1990)
Book Chapter: 

Abstract: Concentration of water soluble direct, acid, basic, and reactive dyes occurs when dilute solutions are frozen at a temperature below the melting point and above the eutectic point of the solution. When freezing is done in still solution, a concentration of up to 500% is achieved in one step, with the dye solution collecting in an oblately spheroidal liquid pocket surrounded by clear ice containing voids, formed from air pockets, radiating outward. Three repetitive freezing cycles concentrate the dye during freezing. The freezing rate of four acid and direct dyes had no close relationship with the size of the dyes studied. Over a larger molecular weight range, an effect was observed for other kinds of molecules. Freeze concentration of salt solution (MW = 58.5) gives almost a 700% concentration, detergent solution (sodium dodecyl sulfate, MW = 288.4) gives 400%, and bovine serum albumin, a large water soluble macro-molecule (MW = 66,200), gives 160%. A theory is presented suggesting that the concentration effect and the formation of the central sphere are consistent with minimizing of the free energy of the overall system. This simple technique may find application in the concentration of heat sensitive, labile dyes for analytical purposes, as well as in the recovery of dyes and other chemicals on a bench scale.

Research Area: Bioseparations    

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Ion Exchange and Affinity Chromatography in the Scaleup of the Purification of a-Galactosidase from Soybean Seeds

1990

Authors: Jill E. Porter, Michael R. Ladisch, and Klaus M. Herrmann
Journal: Biotechnology and Bioengineering, 37, 356-363 (1990)
Book Chapter: 

Abstract: Soybeans (Glycine max) contain an a-galactosidase that makes up a small fraction of the total protein of the seed. The properties of this enzyme are of interest because of its potential to convert the galactooligosaccharides, stachyose and raffinose, in soybean meal to sugars digestible in the human gastro intestinal tract and thereby increase potential uses of this vegetable protein source in human and animal foods. Study of this enzyme required the isolation of milligram quantities of electrophoretically pure protein from ground soybeans and therefore, scaleup of laboratory procedures by a factor of 300 times. Large scale acid precipitation, ammonium sulfate precipitation, and centrifugal recovery of the precipitated protein allowed a-galactosidase to be isolated from 45.5 kg soybean meal containing 17.1 kg protein, to obtain an enzyme extract with a specific activity of 90 to 100. A novel combination of strong anion exchange and cation exchange chromatography followed by Concanavalin-A affinity chromatography with a methyl a-D mannoside gradient gave a-galactosidase with an average specific activyt of 56,000. Ion exchange chromatography preceding Concanavalin-A affinity chromatography allowed elimination of a relatively costly melibiose affinity chromatography step (which followed the Concanavalin-A column in the laboratory procedure) thereby making scaleup practical.

Research Area: Bioseparations    

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Maintaining Constant Enzyme Activity in a Continuous Flow Reactor

1990

Authors: Lee, J. Y., A. Velayudhan, and M. R. Ladisch
Journal: Chemical Eng. J., 45. B1-B4 (1990)
Book Chapter: 

Abstract: The feed rate of make-up enzyme into a continuous flow enzyme reactor is theoretically determined to offset exactly the loss of activity through enzyme deactivation. An expression for the freed rate is developed for arbitrary deactivation kinetics, and then applied to two realistic deactivation mechanisms for which analytical solutions are developed. When a first-order, deactivation mechanism is used, it is found that the make-up enzyme must be fed into the reactor at a constant rate to maintain constant enzyme activity. When a two-step deactivation scheme applies, the corresponding feed rate is an increasing function of time. These results can be used to simplify the study of enzyme kinetics as well as to specify conditions for generating a useful product at a constant rate from an enzyme reactor for which enzyme deactivation cannot be neglected.

Research Area: Bioprocessing    

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Stoichiometry and Kinetics of Xylose Fermentation by Pichia stipitis

1990

Authors: Slininger, P., L. Branstrator, J. Lomont, B. Dien, M. Okos, M. Ladisch, and R. Bothast
Journal: Annals of the New York Acandemy of the Sciences, 589, 25-39, (1990) 
Book Chapter: 

Abstract: Conclusions of previous investigations have led us to focus on Pichia stipitis as a yeast with high potential for producing ethanol from xylose-rich, wood-processing wastes. Given 150 g/L xylose in complex medium, strain Y-7124 functions optimally at 25-26 °C and pH 4-7 to accumulate 56 g/L ethanol with negligible xylitol by-production. In a past report, we cited the need for an optimal bioreactor system; toward this end, we put oxygen uptake, growth, and death kinetics into mathematical form. The present report builds on our previous work as the pathways and stoichiometry of xylose metabolism are examined and models of xylose uptake and ethanol production are identified.

Research Area: Biofuels/Bioproducts Bioprocessing   

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Xylulokinase Activity in Various Yeasts Including Saccharomyces cerevisiae Containing the Cloned Xylulokinase Gene

1990

Authors: Xue Xing Deng and Nancy W. Y. Ho
Journal: Applied Biochemistry and Biotechnology, 24/25, 193-199 (1990) 
Book Chapter: 

Abstract: D-Xylose is a major constituent of hemicellulose, which makes up 20-30% of renewable biomass in nature. D-Xylose can be fermented by most yeasts, including Saccharomyces cerevisiae, by a two-stage process. In this process, xylose is first converted to xylulose in vitro by the enzyme xylose (glucose) isomerase, and the latter sugar is then fermented by yeast to ethanol. With the availability of an inexpensive source jof xylose isomerase produced by recombinant E. coli, this process of fermenting xylose to ethanol can become quite effective. In this paper, we report that yeast xylose and xylulose fermentation can be further improved by cloning and overexpression of the xylulokinase gene. For instance, the level of xylulokinase activity in S. cerevisiae can be increased 230fold by cloning its xylulokinase gene on a high copy-number plasmid, coupled with fusion of the gene with an effective promoter. The resulting genetically-engineered yeasts can ferment xylose and xylulose more than twice as fast as the parent yeast.

Research Area: Biofuels/Bioproducts    

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Bioseparations of Milk Proteins

1989

Authors: Ladisch, M. R., S. R. Rudge, K. W. Ruettimann, and J. K. Lin
Journal: Bioproducts and Processes (1989)
Book Chapter: 

Abstract: Milk is a complex biological fluid consisting of lipids, phospholipids, carbohydrates, proteins, sugars, salts and vitamins. Casein proteins exist in the milk serum (non-fat milk) as micellar structures stabilized by colloidal calcium phosphate and protein surfactant. Raw milk also contains microorganisms and somatic cells, which contribute small amounts of protcolytic enzymes and nucleic acids to milk in the course of processing. Since milk is a suitable carrier for a diverse range of biological molecules, its fractionation is considerable interest. Knowledge of these fractionation methods may further facilitate recovery of proteins of therapeutic value or of enhanced nutritional qualities from milk. The purpose of this chapter is to show separation strategies aimed at recovering selected molecules found in milk serum, and to indicate possible approaches for larger scale fractionations.

Research Area: Bioseparations    

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Cellulosic Adsorbents for Treating Textile Mill Effluents

1989

Authors: Yang, Y., C. M. Ladisch, and M. R. Ladisch
Journal: Enzyme Microb. Technol., 10(10), 632-636 (1988)
Book Chapter: 

Abstract: The textile industry is one of the top 10 water consuming industries with much being used in chemical treatment of textiles. Effluent treatment for pollution control is important due to the color and chemistry of the dyes and additives used during dyeing. Textile effluents vary in composition, and, thus, numerous approaches have been developed for treatment of dyeing waste water with several steps being combined to obtain effluent suitable for discharge. For example, treatment of typical dyes used on synthetic fibers entails four stages: calcium chloride reaction, electrolysis, activated carbon decolorization, and filtration.

Research Area: Bioseparations    

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Cloning of Yeast Xylulokinase Gene by Complementation of E. coli and Yeast Mutations

1989

Authors: Nancy W. Y. Ho and Sue-Fen Chang
Journal: Enzyme Microbial Technology, 11, 417-421 (1989)
Book Chapter: 

Abstract: The gene encoding yeast (Saccharomyces cerevisiae) xylulokinase has been isolated by complementation of E. coli xylulokinase mutations. Through subcloning, the gene has been localized on two HindIII fragments (1.2 and 2.4 kb). Within these HindIII fragments, there lies a 2.2-kb Xho fragment which contains the structural gene of yeast xylulokinase. Upon insertion of a selectable gene into the XhoI fragment, the resulting recombinant fragment has been used to construct a yeast xylulokinase mutant by the gene disruption technique. The cloned xylulokinase gene was found to be able to complement such a xylulokinase mutant.

Research Area: Biofuels/Bioproducts    

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Construction of Yeast Xylulokinase Mutant by Recombinant DNA Techniques

1989

Authors: Panayiotis E. Stevis and Nancy W. Y. Ho
Journal: Applied Biochemistry and Biotechnology, 20/21, 327-334 (1989)
Book Chapter: 

Abstract: A Saccharomyces cerevisiae xylulokinase mutant was constructed by using the cloned yeast xylulokinase gene, XYK-Sc, and the gene disruption technique. The S. cerevisiae LEU2 gene was used to disrupt the XYK-Sc gene cloned on pLSK4 by insertion into the unique HindIII site of the gene. The disrupted gene was liberated from the remainder of the plasmid with Xhol digestion, yielding a 4.4 kb DNA fragment. Transformation of a S. cerevisiae leu2 mutant with this fragment and selection for Leu+ complementation resulted in the isolation of transformants that were unable to grow in pure xylulose medium. The ability to grow in xylulose medium and increased xylulokinase activity were obtained by transforming the mutant with a plasmid-borne wild-type XYK-Sc gene. Insertional inactivation of the chromosomal XYK-Sc gene was also demonstrated by xululokinase assays.

Research Area: Biofuels/Bioproducts    

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Hydrolysis

1989

Authors: Ladisch, M. R.
Journal: Biomass Handbook, 434-451 (1989)
Book Chapter: 

Abstract: Forests make up 80% of the phytomass worlwide. The total annual biomass productivity in the U.S., alone, may be as high as 3.5 billion tons/yr, of which wood is the major fraction. The woody biomass exists in a highly concentrated form and can be shipped to a biomass plant within a 50-mile radius at $20/ton, dry basis. Utilization of mixed stands of uneven age for biomass conversion could hasten improved forest management, as well. Hence, development of innovative and economical conversion processes will aid more efficient utilization of forest resources, as well as expand wood utilization.

Research Area: Biofuels/Bioproducts    

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Hydrophobic Interaction Chromatography

1989

Authors: Roettger, B. F., and M. R. Ladisch
Journal: Biotech. Adv., Vol. 7, 15-29 (1989)
Book Chapter: 

Abstract: Hydrophobic interaction chromatography (HIC) is emerging as a useful technique for the separation of biological compounds. Advances in the past two years in HIC applications, stationary phase, eluents, and theory are reviewed. Recent applications of HIC processes include analytical and semi-preparative separations of a variety of proteins, such as isolectins, hemoglobins, calmodulin, and cardiotoxins. Additionally, HIC is being employed as a tool to investigate protein properties and mechanisms. Advances in HIC stationary phases include development of non-porous, microparticulate supports as well as supports with pore sizes up to 1000 Angstroms. Studies of HIC eluents have further shown the effects of mobile phase pH, water-structuring characterization, and surface tension increments on retention. Various retention mechanisms which have been presented are reviewed; and a correlation relating resolution to column and solute parameters is presented. Protein conformational effects at specific sites have been shown to have a significant impact on retention and specific examples illustrating such effects are discussed.

Research Area: Bioseparations    

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Mechanisms of Protein Retention in Hydrophobic Interaction Chromatography

1989

Authors: Roettger, B., J. Myers, M. R. Ladisch, and F. Regnier
Journal: American Chemical Society, 80-92, (1989)
Book Chapter: 

Abstract: Protein retention in hydrophobic interaction chromatography (HIC) depends on surface hydrophobicity of the support and solute and the kosmotropic nature and concentration of the salt used in the mobile phase. Wyman’s linkage theory, extended to provide a unifying model of HIC retention, relates protein retention to the preferential interactions of the mobile phase salt with the support and protein. Preferential interactions of ammonium salts with HIC supports were determined by extremely sensitive densimetric measurements. Chromatographic retention of lysozyme was also determined on a column packed with hydrophilic polymoric supports and retention of myoglobin was determined on butyl-derivatized polymeric sobenis. Mobile phases containing ammonium salts of SO4, C2H3O2, Cl-, and I- at several concentrations were used to probe retention behavior of lysozyme and myoglobin with respect to these supports. Preferential interactions of the salts with the supports and proteins were found to explain adsorption behavior in hydrophobic interaction chromatography, and results in an equation which predicts capacity factor as a function of lyotropic number and salt molality.

Research Area: Bioseparations    

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Effect of Serine Proteolytic Enzymes (Trypsin and Plasmin), Trypsin Inhibitor, and Plasminogen Activator Addition to Ultra-High Temperature Processed Milk

1988

Authors: Kohlmann, K. L., S. S. Nielsen, and M. R. Ladisch
Journal: J. Dairy Science, 71, 1728-1739 (1988) 
Book Chapter: 

Abstract: Proteolysis and gelation were investigated in single strength, 2% fat, UHT-processed milk following aseptic addition of combinations of plasmin, plasminogen, trypsin, trypsin inhibitor (Kunitz), and urokinase (plasminogen activator). Individual 250-ml milk containers processed by direct or indirect methods were examined for the following attributes over 10 mo: growth on slants, appearance, pH, apparent viscosity, gel formation, enzymatic activity, and casein breakdown. Control milk samples in the study did not gel. Addition of trypsin at 1.5 or 7.5 mg protein/L of milk or addition of plasmin at .3 or 1.5 mg protein/L did not result in gelation. However, containers with plasminogen at .3 mg protein/ L began forming a gel at 5.5 mo. Enzyme activity in plasminogen-treated samples was not detected spectrophotometrically using an L-lysine-p-nitroanilide substrate, but extensive casein breakdown was apparent by SDS-PAGE. The evidence suggests plasmin-ogen-derived activity promotes UHT milk gelation.

Research Area: Bioprocessing    

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Electrochromatography

1988

Authors: Rudge, S. R., and M. R. Ladisch
Journal: Biotechnol. Prog., 4(3), 123-133 (1988)
Book Chapter: 

Abstract: Chromatography is a separation process that exploits different chemical and/or physical affinities between the different components of a mixture and a fixed solid sorbent or gel matrix. The solid or gel may be chemically modified to enhance its affinity for a particular component. A source of energy, in the form of a temperature or chemical gradient, is often used to drive or improve the separation. Chemical gradients are usually used to specifically desorb a component, or to focus that component in a small volume of eluent. The scale-up of chromatography of protein mixtures has progressed along the lines of other fixed-bed operations, with special attention being paid to sorbent chemistry, sorbent particle size, and bed dimensions.

Research Area: Bioseparations    

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Liquid Chromatography of Carbohydrate Monomers and Oligomers

1988

Authors: Lin, J. K., B. J. Jacobson, A. N. Pereira, and M. R. Ladisch
Journal: Biomass Handbook: Academic Press, Inc., Methods in Enzymology 160, (1988)
Book Chapter: 

Abstract: Cellodextrins, water-soluble B-1,4 oligomers of glucose with degree of polymerization (DP) between 2 and 7 (Table 1), are important substrates for the characterization of cellulases. There contribution to understanding the kinetics of cellulose hydrolysis has been reviewed. Cellodextrins can be used to infer features of the active site of carbohydrates and to study induction and repression of enzyme synthesis during fermentation.

Research Area: Bioseparations    

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Casein Micelles: Structure, Properties and Enzymatic Coagulation

1987

Authors: Ruettimann, K. W., and M. R. Ladisch
Journal: Enzyme Microb. Technol., 9(10), 578-589 (1987)
Book Chapter: 

Abstract: The enzymatic hydrolysis of K-casein by chymosin causes the spherical protein agglomerates, known as casein micelles, to coagulate. This forms the basis of the cheese-making process. The literature shows the enzymatic coagulation of casein micelles to be dependent on the concentration of the micelles in the milk, and on the temperature, pH and ionic strength at which the hydrolysis occurs and coagulation is initiated. These factors, in turn, affect the physical and chemical interactions that occur when micelles containing substantial amounts of hydrolyzed casein approach each other. It is necessary to understand the effects of micelle size, chemical and physical structure and the electrostatic, hydrophobic and steric forces acting between micelles if realistic explanation of the coagulation process are to be developed. This paper reviews the developments in the literature that contribute to such an understanding. The literature suggests that prediction of coagulation should be possible once the forces that impact micelle coagulation are understood.

Research Area: Bioprocessing    

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Cause and Correction of Baseline Interruptions Observed for Small-Bore Liquid Chromatography Columns Using Cation Exchange Resin in the H+ Form

1987

Authors: Lin, J. K., S. J. Karn, and M. R. Ladisch
Journal: Biotechnol. Bioeng, 30, 331-333 (1987)
Book Chapter: 

Abstract: Liquid chromatography of oligo- and monosaccharides using cation exchange resin in the H+ form with water or aqueous buffer as the eluent has proven utility in the analysis of hydrolysates obtained from biomass. As smaller diameter (2 mm i.d.) micro-columns come into use, greater sensitivities of sugar analysis and faster analysis times will result. Experience with such columns in our laboratory has suggested several procedures which minimize operating instabilities unique to 2 mm i.d. columns and not observed when larger bore (3.2 to 8 mm i.d.) columns are used. While the causes and solutions for these problems seem almost trivial, in retrospect, the details are briefly mentioned here to aid other biomass researchers who may have observed similar phenomena.

Research Area: Bioseparations    

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Cloning of the Pachysolen tannophilus Xylulokinase Gene by Complementation in Escherichia coli

1987

Authors: Panayiotis E. Stevis, James J. Huang, and Nancy W. Y. Ho
Journal: Applied and Environmental Microbiology, 53, 12, 2975-2977 (1987)
Book Chapter: 

Abstract: The gene coding for xylulokinase has been isolated from the yeast Pachysolen tannophilus by complementation of Escherichia coli xylulokinase (xylB) mutants. Through subcloning, the gene has been localized at one end of a 3.2-kilobase EcoRI-PstI fragment. Expression of the cloned gene was insensitive to glucose inhibition. Furthermore, the cloned gene did not cross-hybridize with E. coli and Saccharomyces cerevisiae xylulokinase genes.

Research Area: Biofuels/Bioproducts    

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Correlation of Glucose (Dextrose) Degradation at 90 to 190°C in 0.4 to 20% Acid

1987

Authors: Bienkowski, P. R., M. R. Ladisch, R. Narayan, G. T. Tsao, and R. Eckert
Journal: Chem. Eng. Comm. 51, 179-192 (1987)
Book Chapter: 

Abstract: Decomposition of glucose is described over a wide range of acid concentrations and temperatures by a correlation based on an Arrhenius type model combined with a modified Deby-Huckel equation. Degradation depends on temperature and acid concentration, represented by hydrogen ion activity. Kinetic data from our laboratory for 4 and 12 wt% glucose were combined with literature data to establish this model. Alternative non-linear mechanisms were compared by regression analysis of the published data as well as data developed in this study. Values of the degradation constant vary from 0.109 hr-1 (at 20 wt% acid and 190 C) to 4.18 x 10-7 (at 0.4 wt% acid and 1000 C). This result will be useful to model glucose degradation in acid hydrolysis of celluloses, sterilization of fermentor feeds, and stripping of fermentation ethanol.

Research Area: Biofuels/Bioproducts    

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Determining Pore Size Distribution in Wet Cellulose by Measuring Solute Exclusion Using a Differential Refractometer

1987

Authors: Lin, J. K., M. R. Ladisch, J. A. Patterson, and C. H. Noller
Journal: Biotechnol. Bioeng., 29, 976-981 (1987)
Book Chapter: 

Abstract: Solute exclusion was used to determine the pore volume and micropore size distribution of wet Cellulosic materials. Glucose, cellobiose, and polyethylene glycol (PEG) (8 to 130 A in diameter) were used as molecular probes. Four replicates of Cellulosic samples, with each sample being analyzed 4 to 8 times, gave the concentrations of each molecular probe before and after contact with cellulose. Sugar concentrations were determined by the DNS method and PEG concentrations by a differential Refractometer. Deviations arising from sample-to-sample variability result in variations of solute uptake from which the pore size distribution was determined. The need for replicate samples and a statistical approach to data analysis is indicated. Consequently, the data were fitted to an empirical logistic model function based on the minimum of the residual sum of squares using the finite-difference, Levenberg-Marquardt algorithm. A smooth increasing function resulted. We report experimental methodology employing a differential Refractometer, common in many laboratories having a liquid chromatograph instrument, combined with statistical treatment of the data. This method may also find application in determining pore size distribution in wet, hydrophilic polymers used in some types of membranes, chromatographic supports, and gel-type resins.

Research Area: Biofuels/Bioproducts    

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Polysaccharides as Adsorbents- An Update on Fundamental Properties and Commercial Prospects

1987

Authors: Lee, J. Y., and M. R. Ladisch
Journal: Annals of the New York Academy of Sciences, 506, 491-498, (1987) 
Book Chapter: 

Abstract: Ethanol is produced by fermentation of starch or sugars using yeast. The concentration of ethanol produced by the fermentation process is usually low (less than 10%). Thus, economically feasible separation processes to remove water from ethanol are important. Distillation has been widely used to separate ethanol from water. Because water forms an azeotropic mixture with ethanol, an additional step to break the azeotrope is required. This step needs a third solvent such as benzene. The energy consumption in this conventional process is about 22,000 Btu/gallon ethanol, in which 10,000 Btu/gallon ethanol is used to break the azeotrope and to recover the benzene. An alternative, energy-efficient way of breaking the azeotrope is with an adsorption process. Previous studies have indicated that a polysaccharide material such as corn is an excellent absorbent for this purpose. Selective adsorption of water is carried out by feeding water-ethanol vapor to a fixed bed column packed with corn grits. After the adsorption, regeneration is carried out by allowing hot inert gas to pass through the column. A schematic representation of this process is shown in Figure 2. Advantages of this process are: (1) corn is less expensive (10 cents/lb) than other commercial adsorbents; (2) regeneration is possible at a relatively low temperature (100 °C); and (3) the heat of adsorption is retained inside the column during the adsorption so that the heat can be utilized for the regeneration.

Research Area: Bioseparations    

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Separation by Adsorption

1987

Authors: Ladisch, M. R.
Journal: Advanced Biochemical Engineering, H. R. Bungay and G. Belfort, eds., J. Wiley & Sons, NY (1987)
Book Chapter: 

Abstract: Many processes for manufacturing biochemicals have costs that are dominated by the expense of purification. Fermentation products are diluted by water and contaminated by debris, salts, proteins, and a variety of compounds that may have properties quite similar to those of the desired material. Purification usually starts with some way to increase the concentration of the product so that large volumes of water need not be handled during the more selective steps. Processes such as solvent extraction and ion exchange can accomplish severalfold concentration and considerable purification. Separation of the product from molecules with similar properties can be very difficult. This chapter will cover two methods that are in large-scale use: column procedures for vapor-phase adsorption of water and liquid chromatography.

Research Area: Bioseparations    

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Sulfuric Acid-Sugar Separation by Ion Exclusion

1987

Authors: Neuman, R. P., S. R. Rudge, and M. R. Ladisch
Journal: Reactive Polymers J., 5, 55-61 (1987)
Book Chapter: 

Abstract: Biomass conversion continues to have significant potential in the production of fuel ethanol by fermentation. A major cost in acid hydrolysis of biomass to fermentable sugars is the acid itself. Separation and recycle of the acid could reduce ethanol production costs by $0.10/gallon or more. In this context, a process that separates sulfuric acid from glucose using ion-exclusion technology is presented. A 61 cm long, fixed-bed of Rohm and Haas Amberlite IR-118, strong cation-exchange resin in the hydrogen form was used. Samples containing 7.7% H2SO4 and 1.0% glucose, at sample volumes of 10% to 50% of the column void volume gave separation of H2SO4 from glucose at column temperatures ranging from 27 to 81 °C with water as eluent at a superficial velocity of 0.6 cm/min. Skewing of the H2SO4 peak was observed and traced to a sharp density gradient between the acid and the water eluent while the glucose peak was sufficiently symmetric to be fitted by an axial dispersion model. This work shows that a chromatographic resin having a particle size range of 300 to 1200 micrometers can give complete recovery of sulfuric acid with 94% recovery of glucose. This case study has interesting implications for both the practice of process chromatography, using a resin with large particle size at gross loading conditions, and the prospects of further reducing fermentable sugar costs in biomass conversion.

Research Area: Bioseparations    

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Engineering and Economics of Cellulose Saccharification Systems

1986

Authors: Ladisch, M. R. and G. T. Tsao
Journal: Enzyme Microb. Technol., 8, 66-69 (1986)
Book Chapter: 

Abstract: The design of cellulose saccharification systems will govern the economics of biomass conversion to ethanol and other oxygenated compounds. Solids handling of bulky Cellulosic materials, chemical processing of a physically and chemically heterogeneous substrate, cellulose pretreatment and product recovery present formidable engineering challenges. Marketing strategy must also be carefully formulated given the variety of hexoses, pentoses, organic acids, as well as lignin which result from biomass processing. Since the intrinsic cost of the biomass is $0.015 to $0.03/lb, and the processing costs are $0.03 to $0.10/lb, the key is to identify products having a value in excess of $0.10/lb which are uniquely suited for production from biomass-derived sugars. Competitive pressures from other carbohydrate sources such as corn and sugar cane must also be considered in the economic analysis. Process concepts and associated costs are presented in a comparison of corn and biomass saccharification routes.

Research Area: Biofuels/Bioproducts    

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Gas Chromatographic Determination of Dimethyl Ether in a Mixture Containing Propane, C4 Hydrocarbons, n-Pentane, Methyl tert.-Butyl Ether and Methanol

1986

Authors: Lin, J. K., R. L. Hendrickson, and M. R. Ladisch
Journal: J. Chromatogr., 367, 195-200 (1986)
Book Chapter: 

Abstract: The reduction of lead use in gasoline mandated by the U.S. Environmental Protection Agency has greatly increased the demand for both methyl tert.-butyl ether (MTBE) and other octane boosters. Since then MTBE production has grown quickly and output totaled 1.5*109 of lbs. In 1984. During MTBE synthesis, a trace amount of dimethyl ether (DME) may be formed. Since DME decomposition leads to polymer formation, the presence of DME could impact refinery operation. Thus, precise determination of DME concentration is needed. A number of gas chromatographic (GC) procedures have been reported in the literature for DME analysis. Stockinger used (1) a column (2.4 m x 3.2 mm ).D.) packed with Porapak Q (80-100 mesh) with nitrogen and helium as the carrier gas with a thermal conductivity detector; and (2) a support (squalane)-coated open-tubular column(61 m x 0.8 mm O.D. x 0.5 mm I.D.) with helium as the carrier gas and flame ionization detector. Hayashi and Moffat separated methanol, DME, carbon monoxide, carbon dioxide and C2 – C3 hydrocarbons on a Porapak Q column (3 m x 6.4 mm O.D.) at 70 °C. Muja and co-workers used a C22 Celite column (5 ft. long) packed with 19% tri(cyanoethoxy)propane for detecting DME in the reaction mixture from MTBE synthesis. However, the DME peak as reported in the literature is closely adjacent to methanol or C4 fractions. Thus, it is difficult to determine small DME concentrations when methanol or C4 fractions are present. This paper describes a DME analysis using a column packed with Porapak N/Q (Porapak N-Porapak Q, 80;20).

Research Area: Biofuels/Bioproducts    

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In Vitro Anaerobic Fermentation of Alkali-Treated Corn Stover by Rumen Microbes

1986

Authors: K. W. Lin, D. M. Schaefer, M. R. Ladisch, J. A. Patterson, C. H. Noller
Journal: Journal of Animal Science, 62, 822-829, 1986
Book Chapter: 

Abstract: Individual and combined effects of sodium hydroxide (NaOH), ferrous chloride (FeCl2), ferric nitrate (Fe(NO3)3), and tartrate components of the cellulose solvent, iron sodium tartrate (FeTNa) on anaerobic fermentation of corn stover were investigated using a semi-continuous culture procedure. Ruminal fluid inocula were obtained from a ruminal-cannulated steer fed an alfalfa hay diet. The in vitro neutral detergent fiber digestibility (IVNFD) and total volatile fatty acid (VFA) concentration for non-treated corn stover (CS), NaOH-treated CS and FeTNa-treated CS were: 25.3%, 58.2% and 47.2%: and 41.3 mM, 64.5 mM and 70.2 mM, respectively. Reponse of ruminal microbes to Fe in NaOH-treated corn stover indicated that FeCl2 limited ammonia-N (NH3-N) availability. Addition of NH3 alloeviated the depression in digestibility by FeCL2. Tartrate in the solvent was metabolized to VFA and CO2 without apparent chemical inhibition. An apparent beneficial disruptive swelling action exerted by the ferric FeTNa appeared to be offset by a ferrous ion-induced NH3-N limitation. The FeTNa-treated residue may be washed before fermentation to remove Fe. Because nutrients solubilized by the solvent are removed during washing, there is little advantage to using FeTNa over NaOH alone to increase substrate digestion by ruminal microbes.

Research Area: Bioenergy Bioprocessing   

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Leaching: A Separation Process for the Production of Fuels and Chemicals from Biomass

1986

Authors: Ladisch, M. R., G. T. Tsao, and K. Lin
Journal: Biotechnol. Bioeng. Symp. Ser., No. 15, 723-736 (1986)
Book Chapter: 

Abstract: The application of biotechnology to the production of fuels and chemicals from biomass must include consideration of separation processes. One objective is to recover products of biomass processing in a concentrated form, if possible. The ability of biomass to absorb water will result in low fermentable sugar concentrations. A low moisture hydrolysis, coupled with leaching, can give fermentable sugar concentrations above 15%, and hence, circumvents this problem. Calculations required to design a sugar recovery process in a systematic manner are outlined.

Research Area: Bioseparations    

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Methyl t-Butyl Ether (MTBE) Process Catalyst Parameters

1986

Authors: Voloch, M., M. R. Ladisch, and G. T. Tsao
Journal: Reactive Polymers, 4, 91-98 (1986)
Book Chapter: 

Abstract: One billion pounds per year of methyl t-butyl ether (MTBE) are currently produced from acid-catalyzed, liquid phase reaction of methanol with isobutene (IB). We report results for Amberlyst 15 which is currently used as a catalyst for MTBE production. The evaluation of this strong-acid macroreticular resin was carried out in a bench scale, plug flow reactor system which was specially developed in our laboratory to evaluate MTBE catalysts under conditions believed to be compatible with those in industry. Factors considered include temperature, flow rate and feed composition. An integrated rate expression is used to estimate kinetic constants at 26, 51, and 67 °C, using data obtained from the plug flow reactor. Conditions which maximize selectivity and productivity, together with other catalyst characteristics, are briefly discussed.

Research Area: Biofuels/Bioproducts    

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Process Considerations for Scale-up of Liquid Chromatography and Electrophoresis

1986

Authors: Rudge, S., and M. R. Ladisch
Journal: American Chemical Society symp. 314, 122-152, (1986)
Book Chapter: 

Abstract: Chromatography is an important preparative and industrial process. Scale-up of chromatographic processes requires computation of mass transfer characteristics as a function of column area, support particle size and feed volume. In this context, an analytical solution for longitudinal diffusion in packed beds developed by Lapidus and Amundson, is used to demonstrate the characteristics of a typical size exclusion separation of proteins, including estimation of maximum sample size as allowed by support properties. Electrophoresis is also a powerful fractionation technique for proteins, but is subject to many microscopic effects. These include electric double layers, hydrodynamic drag, and electrical relaxation. In addition, macroscopic effects, such as electroosmosis and thermal gradients, also impact separation efficiency. These effects are discussed in relation to elution processes using selected examples. The combination of an electric field with a chromatographic process has recently been proposed to extend the power of electrophoresis separations. Analysis of such a process, referred to as electrochromatography, is also present.

Research Area: Bioseparations    

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Water Sorption Properties of a Polysaccharide Adsorbent

1986

Authors: Neuman, R., M. Voloch, P. Bienkowski, and M. R. Ladisch
Journal: I&EC Fundamentals, 25, 422-425 (1986)
Book Chapter: 

Abstract: A flow method was used to determine equilibrium isotherms of the water-corn grit system in the temperature range of 343-373 K. A modified Henderson’s equilibrium equation was used to fit the experimental data. Calculated values of the heat of adsorption (10.8-14.6 kcal/g-mol) and surface area (170 m2/g) are similar to those previously reported based on data taken at lower temperatures. These data extend knowledge of the equilibrium behavior of water with respect to corn grits to the temperature range used for the selective adsorption of water from alcohol vapor by corn.

Research Area: Bioseparations    

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2, 3-Butanediol

1985

Authors: Voloch, M., N. Jansen, M. Ladisch, G. Tsao, R. Narayan, and V. Rodwell
Journal: Comprehensive Biotechnology, (1985)
Book Chapter: 

Abstract: Biomass conversion gives both pentoses and hexoses as products. While the hexoses (primarily glucose) are readily fermented, routes for pentose fermentation are still being developed. Hence, pentoses represent a potentially significant source of sugars with xylose being the major product. Fermentation of xylose as well as glucose by Klebsiella oxytoca, ATCC 8724 (formerly known as Klebsiella pneumoniae and Aerobacter aerogenes) yields 2,3-butanediol as the major product. Other microorganisms capable of producing 2,3-butanediol (abbreviated 2,3-BD) include Bacillus subtilis (Ford strain), Aeromonas hydrophilia and several species of Serratia (Ledingham and Neish, 1954). Secondary products formed include acetoin, ethanol, lactic acid and glycerol. While K. oxytoca is able to yield high concentrations of 2,3 BD as mixtures of stereoisomers from monosaccharides, it is unable to utilize polysaccharides (Ledingham and Neish, 1954). In comparison, B. polymyxa is able to ferment starch directly giving L-2,3-butanediol and ethanol in almost equal amounts (Long and Patrick, 1963; Prescott and Dunn, 1959). However, B. polymyxa is unstable and difficult to maintain (Long and Patrick, 1963). Both K. oxytoca and B. polymyxa have been used in pilot scale fermentation (Ledingham and Neish, 1954; Blackwood et al., 1949), especially during World War II, as a possible means of producing 2,3-BD and subsequently 1,3-butadiene, an organic intermediate for rubber production. In the 1940's process development was carried out through the pilot plant stage at the National Research Laboratories in Ottawa, Canada. A 90% fermentation efficiency was attained on a 750-gallon scale for sugars obtained from whole wheat (Blackwood et al., 1949). Process evaouation with barley as a feedstock indicated a 2,3-BD cost of 13 to 18 cents per pound (Tomkins et al., 1948). Development was discontinued because less expensive routes for chemically producing 1,3-butadiene from petroleum became available. In recent times, the long-term prospects of rising petroleum prices have revived significant interest in producing alcohols, including 2,3-BD, from biomass.

Research Area: Biofuels/Bioproducts    

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Anaerobic Fermentation: Microbes from Ruminants

1985

Authors: Lin, K. W., J. A. Patterson, and M. R. Ladisch
Journal: Enz. Microbiol. Technol., 7, 98-107 (1985)
Book Chapter: 

Abstract: Fed-hatch fermentation of biomass could provide a route for direct conversion of renewable resources to commercially significant chemicals. The ecosystem in the forestomach (rumen) of ruminants provides a highly reduced environment (oxidation-reduction potential of -250 to -450 mV) in which anaerobic bacteria directly utilize cellulose, hemicellulose, and other fermentable biomass constituents to produce acetate, butyrate, propionate, methane and carbon dioxide at pH 5.7 to 7.3. The cellulose fermentation in the rumen is impacted by the physically and chemically heterogeneous character of the insoluble substrate, as well as the properties of the mixed culture responsible for fibre hydrolysis and carbohydrate utilization. The rumen system provides an interesting case study in the context of possible process concepts for direct fermentation of biomass to commercially important chemicals such as acetate, propionate, succinate, lactate and ethanol. The role of the chemical and physical characteristics of the substrate, the microbes in the rumen system and the metabolic pathways of soluble carbohydrates are discussed in the context of cellulose and hemicellulose fermentation.

Research Area: Biofuels/Bioproducts    

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Breakthrough Behavior of 17.5 mol % Water in Methanol, Ethanol, Isopropanol, and t-Butanol Vapors Passed over Corn Grits

1985

Authors: Bienkowski, P., A. Barthe', M. Voloch, R. N. Neuman, and M. R. Ladisch
Journal: Biotechnol. Bioeng., 28(7), 960-964 (1985)
Book Chapter: 

Abstract: Ground corn is now used in industry as an adsorbent to remove water from ethanol vapors. It is stable and inexpensive at 10 cents/lb (22 cents/kg). For regeneration it requires less than 2000 Btu/gal of 190 proof ethanol processed. If necessary, it could be readily saccharified and fermented into ethanol after use. This renewable resource has further exciting potential as an inexpensive adsorbent for water removal from other alcohols, including methanol, isopropanol, and t-butanol. Water sorption capacity in a fixed bed, nonisothermal adsorption column appears to be a function of the heat capacity of the non-adsorbed alcohol vapor, relative to the heat capacity of the corn adsorbent. Methanol, ethanol, isopropanol, and t-butanol containing 17.5 mol% water gave 105, 151, 284, and 358 g anhydrous product/kg adsorbent, respectively, per adsorption cycle. This adsorbent, having operational temperature ranges between 80 and 100 degrees C, is indicated to be of potential utility in solvent recycle processes using these industrially important alcohols. Observed adsorption characteristics are discussed in terms of the alcohol properties of molecular size, heat capacity, and diffusivity. The adsorption mechanism is hypothesized to include transport of water molecules into the structure of adjacent starch molecules present in small spherical bodies (diameter of several microns) immobilized on the surface of the corn grit particles.

Research Area: Bioenergy Bioseparations   

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Comparative Evaluation of Ethanol Production by Xylose Fermenting Yeasts Presented High Xylose Concentrations

1985

Authors: Slininger, P. J., R. J. Bothast, M. R. Okos, and M. R. Ladisch
Journal: Biotechnol. Letters, 7(3), 197-202 (1985)
Book Chapter: 

Abstract: Three strains of Pichia stipitis and three of the Candida shehatae were compared with Pachysolen tannophilus in their abilities to ferment xylose at concentrations as high as 200 g/L when subjected to both aerobic and microaerophilic conditions. Evaluations based on accumulated ethanol concentrations, ethanol productivities, xylose consumption, and ethanol and xylitol yields were determined from batch culture time courses. Of the strains considered, P. stipitis NRRL Y-7124 seemed most promising since it was able to utilize all but 7 g/L of 150 g/L xylose supplied aerobically to produce 52 g/L ethanol at a yield of 0.39 g per gram xylose (76% of theoretical yield) and at a rate comparable to the fastest shown by C. shehatae NRRL Y-12878. For all strains tested, fermentation results from aerobic cultures were more favorable than those from microaerophilic cultures.

Research Area: Biofuels/Bioproducts    

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Effect of Pretreatments and Fermentation on Pore Size in Cellulosic Materials

1985

Authors: Lin, K. W., M. R. Ladisch, M. Voloch, J. A. Patterson, and C. H. Noller
Journal: Biotechnol. Bioeng., 27, 1427-1433 (1985)
Book Chapter: 

Abstract: Surface area has been proposed as a major factor determining the extent of enzymatic hydrolysis of cellulose. We used cornstalk residue (CR) and Solka Floc BW-300 (SF) as substrates and NaOH (a cellulose swelling agent) and iron sodium tartrate (FeTNa, intercolates between cellulose microfibrils) as pretreatments to study the effect of surface area on extent of fermentation. Micropore sizes (8-130 A) were determined by a solute exclusion technique using glucose, cellobiose, and polyethylene glycols as molecular probes. The pore size distributions follow the logistic model function: I = a/[1 + exp(b-cX)] where I is pore volume; X = log D; D is the molecular probe diameter; and a, b, and c are constants. The pore volumes of CR (1.9 mL/g) and SF (1.6 mL/g) are increased to 2.1 mL/g by pretreatment with NaOH.

Research Area: Biofuels/Bioproducts    

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Overproduction of D-xylose Isomerase in Escherichia coli by Cloning the D-xylose Isomerase Gene

1985

Authors: Panagiotis E. Stevis and Nancy W. Y. Ho
Journal: Enzyme Microbial Technology, 7, 592-596 (1985)
Book Chapter: 

Abstract: The Escherichia coli D-xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) gene, xylA, has been cloned on various E. coli plasmids. However, it has been found that high levels of overproductin of the D-xylose isomerase, the protein product of the xylA gene, cannot be accomplished by cloning the intact gene on high copy-number plasmids alone. This is believed to be due to the fact that the expression of the gene through its natural promoter is highly regulated in E. coli. In order to overcome this, the xylA structural gene has been fused with other strong promoters such as tac and lac, resulting in the construction of a number of fused genes. Analysis of the E. coli transformants containing the fused genes, cloned on high copy-number plasmids, indicated that a 20-fold overproduction of the enzyme can now be obtained. It is expected that overproduction of the enzyme in E. coli can still be substantially improved through additional manipulation with recombinant DNA techniques.

Research Area: Biofuels/Bioproducts    

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Acid Hydrolysis of Pretreated Lignocellulose from Corn Residue

1984

Authors: Bienkowski, P., M. R. Ladisch, M. Voloch, and G. T. Tsao
Journal: Biotechnol. Bioeng. Symp. Ser., 14, 512-524 (1984)
Book Chapter: 

Abstract: The lignocellulose (LIC) derived from the hemicellulose hydrolysis of corn residue was steeped in 15 to 25% sulfuric acid at 40 to 103°C, filtered to recover solids, and then dried in a fluidized-bed dryer to concentrate the acid. Acid concentration, steeping temperature, drying time, and temperature effects are described by the current work. Hydrolysis of the pretreated LICs gave 90% cellulose conversion with acid consumption corresponding to 1.50 g H2SO4/ g glucose and sugar concentrations in the hydrolyzate of up to 6.5 wt% in the best cases. Kinetic parameters are presented which describe the observed rates and extent of hydrolysis.

Research Area: Biofuels/Bioproducts    

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Bioutilization of Cereal Lignocellulose

1984

Authors: Voloch, M., M. R. Ladisch, P. Bienkowski, and G. T. Tsao
Journal: Cereal Polysaccharides in Technology and Nutrition (ed. V. F. Rasper) The American Association of Cereal Chemists, Inc., St. Paul, MN 103-125 (1984)
Book Chapter: 

Abstract: Agricultural residues have a great potential as a chemical resource. Cereal residues make up a large portion of these agricultural residues. In this paper, we describe three different aspects of cereal bioutilization. The first aspect involves acid hydrolysis of corn stover. A “low temperature percolation” process is described which combines hemicellulose and cellulose hydrolysis through a sugar/acid recycle. The second part of this paper deals with the potential use of corn fiber derived from wet-milling processes. If the carbohydrate portion of the fiber is hydrolyzed under mild conditions without damaging the protein content, it may be possible to increase ethanol production together with producing a higher protein material. The last portion of the paper deals with a new development in biotechnology: the use of polysaccharides as selective sorbents. It involves the use of corn (or other biomass materials) to dehydrate ethanol to anhydrous product. This technology has the potential of being relatively more energy efficient than conventional azeotropic/extractive distillations, and it may be easier to implement in small or medium size plants.

Research Area: Biofuels/Bioproducts    

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Cloning and Characterization of the xyl Genes from Escherichia coli

1984

Authors: Stuart A. Rosenfeld, Panayiotis E. Stevis, and Nancy W. Y. Ho
Journal: Mol. Gen. Genet., 194, 410-415 (1984)
Book Chapter: 

Abstract: Specific xylose utilization mutants of Escherichia coli were isolated that had altered xylose isomerase (xylA), xylulokinase (xylB), and regulatory (xylR) or transport (xylT) activities. We screened the Clarke and Carbon E. coli gene bank and one clone, pLC10-15 was found to complement the xyl mutants we had characterized. Subclosing and DNA restriction mapping allowed us to locate the xylA and xylB genes on a 1.6 kbp Bg/II fragment and a 2.6 kbp HindIII-Sa/I fragment, respectively. The identification and mapping of xyl gene promoters suggest that the xylA and xylB genes are organized as an operon having a single xylose inducible promoter preceding the xylA gene.

Research Area: Biofuels/Bioproducts    

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Cornmeal Adsorber for Dehydrating Ethanol Vapors

1984

Authors: Ladisch, M. R., M. Voloch, J. Hong, P. Bienkowski and G. T. Tsao
Journal: Ind. Eng. Chem. Process Des. Dev., 23(3), 437-443 (1984) 
Book Chapter: 

Abstract: Ground corn selectively adsorbs water to give anhydrous product from ethanol vapors containing between 1.6 and 50.9% water. An adiabatic corn adsorbere system is characterized by formation of a combined wave front (the temperature and concentration waves elute from the column at the same time). Corn is stable under cyclic use when regeneration is carried out with air or N2 at 80 to 120 C. The energy required to regenerate the adsorbent is less than 1500 Btu/gal (528 kJ/kg) of anhydrous product obtained from ethanol vapor containing 7.4% water. Anhydrous product also results when water-containing vapors of n-propyl, isopropyl, or tert-butyl alcohol are passed over corn. Corn and other polysaccharide materials have potential as low-cost adsorbents for energy efficient water removal from alcohols.

Research Area: Bioseparations    

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Effect of Ferric Tartrate/Sodium Hydroxide Solvent Pretreatment on Enzyme Hydrolysis of Cellulose in Corn Residue

1984

Authors: Hamilton, T. J., B. E. Dale, M. R. Ladisch, and G. T. Tsao
Journal: Biotechnol. Bioeng., 26, 781-787 (1984)
Book Chapter: 

Abstract: Lignocellulose containing 62% cellulose was prepared from corn residue by dilute acid hydrolysis using 5% H2SO4 at 90°C. The lignocellulose was then treated with a cellulose solvent consisting of a ferric sodium tartrate complex in 1.5N sodium hydroxide at levels ranging from 4:1 to 12:1 (solvent volume: corn residue lignocellulose) or a 1.5N sodium hydroxide solution alone. Subsequent hydrolysis with cellulase enzymes from Trichoderma reesei gave cellulose conversions which were two to three times higher than untreated lignocellulose (30%) and approached 90% conversion after 24 h in the best cases. It was found that increasing cellulase enzyme levels from 3.74 IU/g lignocellulose to 7.71 IU/g lignocellulose increased cellulose conversion by 50% at all pretreatment conditions, while an increase from 7.71 to 10.1 IU/g gave only an additional 5-10% increase. Pretreatment with sodium hydroxide resulted in 5-25% lower conversions than observed for cellulose treated with the solvent, depending on enzyme levels and treatment levels. At high enzyme levels, sodium hydroxide pretreatment is almost as effective in enhancing cellulose conversion after 24 h as is pretreatment using the cellulose solvent.

Research Area: Biofuels/Bioproducts    

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Preparation of Cellodextrins Using Sulfuric Acid

1984

Authors: Voloch, M., M. R. Ladisch, M. Cantarella, and G. T. Tsao
Journal: Biotechnol. Bioeng., 26, 557-559 (1984) 
Book Chapter: 

Abstract: Cellodextrins are valuable substrates for the study of cellulose hydrolysis. They may also find use in the screening of cell cultures for specific cellulase activities and induction studies in microbial cellulasae production. The preparation of cellodextrins is often carried out using fuming hydrochloric acid as developed by Miller, Dean, and Blum to yield cellodextrins which are then separated into individual components using a charcoal-celite bed. Cellodextrins may also be prepared by acetolysis of cellulose followed by deacetylation and a separation step. We report a method for making cellodextrins by strong sulfuric acid hydrolysis, where the cellodextrins are precipitated from the acid with ethanol. This avoids formation of large quantities of salts due to the neutralization step used in the other methods, and hence, expedites separation of the cellodextrins into pure components by aqueous liquid chromatography (LC).

Research Area: Bioenergy    

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Effect of Solvent Treatments on Intake and Digestibiity of Corn Stover

1983

Authors: Schaefer, D. M., M. R. Ladisch, C. H. Noller, and V. L. Lechtenberg
Journal: An. Sci. Ag. Engr & Agronomy, (1983)
Book Chapter: 

Abstract: Treatment of crop residues with low levels of sodium hydroxide has been shown to improve organic-matter digestibility and animal performance. One objective of these experiments was to evaluate the effects of three treatments – water, sodium hydroxide, and chelating metal cellulose-swelling solution-on solubilization of cell-wall components in corn (Zea mays) stover. The objective was to determine the effects of these treatments on in-vitro and in-vivo digestibility as well as on consumption of corn stover. Twelve growing ram lambs were used in an intake and digestion trial composed of three periods.

Research Area: Biofuels/Bioproducts    

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Process Considerations in Enzymatic Hydrolysis of Biomass

1983

Authors: Ladisch, M. R., K. W. Lin, M. Voloch, and G. T. Tsao
Journal: Enz. Microb. Technol., 5, 82-102 (1983) 
Book Chapter: 

Abstract: The processes by which cellulases hydrolyse cellulose are a function of substrate reactivity as well as enzyme activity. The two must be considered together if an accurate description of biomass saccharification is to be developed. To accomplish this, cellulolytic systems must first be modeled using realistic, but well-defined, substrates so that optimum cellulolysis conditions can be formulated. This, together with reduction of enzyme cost, total utilization of biomass, and an efficient pretreatment are key elements for the economical conversion of biomass to sugars and fermentation products.

Research Area: Biofuels/Bioproducts    

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Reduction of Acetoin to 2,3-Butanediol in Klebsiella pneumoniae, A New Model

1983

Authors: Voloch, M., M. R. Ladisch, V. W. Rodwell, and G. T. Tsao
Journal: Biotechnol. Bioeng., 25, 173-183 (1983)
Book Chapter: 

Abstract: Fermentation of xylose by Klebsiella pneumoniae (ATCC 8724, formerly known as Aerobacter aerogenes) carried out in our laboratory yields 2.3-butanediol as the major product. Experimental data obtained in this work cannot be explained by the model presently in the literature for the formation of 2.3-butanediol isomers from acetoin isomers. A new model is proposed with the existence of two acetoin reductases and an acetoin racemase. The two reductases were separated and their stereospecificity determined. Extension of the model of other microorganisms is discussed.

Research Area: Biofuels/Bioproducts    

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Symposium on Fuels and Chemicals From Biomass

1983

Authors: Ladisch, M. R., B. Dale, and G. Tsao
Journal: Biotechnology and Bioengineering, XXV, 1-2, (1983)
Book Chapter: 

Abstract: The production of ethanol from biomass may be accomplished by a variety of approaches. The fiber components of biomass, hemicellulose and cellulose, can be hydrolyzed into fermentable sugars by either acid of enzyme catalysts. The sugars obtained from hemicellulose may include xylose, arabinose, galactose, glucose, and mannose, as well as other components such as glucuronic acid and acetic acid. The predominant component usually observed is xylose which typically makes up 70-80% of the sugars obtained.

Research Area: Biofuels/Bioproducts    

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The Effects of Oxygen and Temperature on Gas Composition from Gasification of Corn Cobs

1983

Authors: Voloch, M., R. Neuman, M. Ladisch, R. Peart, and G. Tsao
Journal: ASAE (1983)
Book Chapter: 

Abstract: The gasification of corn cobs in a furnace at temperatures ranging from 500oC to 1300oC is described. Equilibrium and heat-transfer characteristics are described. Preliminary results indicate that in absence of external oxygen, up to 23% of the initial cob weight can be obtained as CO.

Research Area: Biofuels/Bioproducts    

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Adsorption of Ethanol/Water Mixtures by Biomass Materials

1982

Authors: Hong, J., M. Voloch, M. R. Ladisch, and G. T. Tsao
Journal: Biotechnol. Bioeng. 24, 725-730 (1982)
Book Chapter: 

Abstract: The commercial production of biomass-derived ethanol is dependent on the energy balance which is defined as the ratio of the combustible energy obtained from the product to the energy necessary to its production. The recovery of ethanol in an anhydrous form from an 8-12% solution (wt. %) can be energy intensive.

Research Area: Bioseparations    

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Calcium Sulfate as a Selective Adsorbent of Water

1982

Authors: Pocium, D., M. Ladisch, G. Tsao, and P. Wankat
Journal: AIChE Annual Meeting (1982)
Book Chapter: 

Abstract: The removal of water from fermentation ethanol in an energy efficient manner is of practical interest for a biomass conversion/fermentation process. The use of CaSO4 to adsorb water has been suggested and investigated many years ago. Our work has focused on removing water from 90 to 95.6% ethanol to give an anhydrous product. It is in this range that the separation becomes challenging because of energy considerations and the ethanol/water azeotrope. We examined the properties of CaSO4 and found that while it is a selective adsorbent of water from ethanol vapor (at 80 to 90 °C) and can be regenerated at 140 to 150 °C, it can also rapidly lose its capacity. Careful fundamental research in our laboratory shows that this loss of capacity is probably related to changes in the crystalline structure of CaSO4 which occur at 150o C when the partial pressure of the water vapor is relatively high. We found that increasing the temperature form 80 to 140 °C gradually during regeneration (instead of a step change to 140 °C) minimizes changes in crystalline structure. Hence, CaSO4 is quite stable in repetitive use. Using these conditions, CaSO4 was examined in a bench-scale adsorber. Breakthrough and temperature profiles were measured and then applied to characterize adsorber performance.

Research Area: Bioseparations    

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Production of Ethanol and Chemicals from Cellulosic Materials

1982

Authors: Tsao, G. T., M. R. Ladisch, M. Voloch, and P. Bienkowski
Journal: Process Biochemistry, 34-38 (1982) 
Book Chapter: 

Abstract: The economic production of fuels and chemicals from renewable resources requires and integrated biochemical, chemical, and microbiological approach. Recent advances in these areas as relates to an acid hydrolysis process are discussed. 

Research Area: Biofuels/Bioproducts    

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Cellulase Kinetics: Trends in the Biology of Fermentations for Fuel and Chemicals

1981

Authors: Ladisch, M. R., J. Hong, M. Voloch, and G. Tsao
Journal: Basic Life Sciences, (18) 55-83 (1981)
Book Chapter: 

Abstract: The production of fermentable sugar from biomass is the first step in obtaining liquid fuels and chemicals from renewable resources by fermentation processes. Biomass materials include corn residue, small grain residues (straws), sugarcane bagasse, forages and forestry residues. It is estimated that these sources alone could yield up to 40 billion gallons of ethanol/year.

Research Area: Biofuels/Bioproducts    

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Combined Product and Substrate Inhibition Equation for Cellobiase

1981

Authors: Hong, J., M. R. Ladisch, C. S. Gong, P. C. Wankat, and G. T. Tsao
Journal: Biotechnol. Bioeng.,23, 2779-2788 (1981) 
Book Chapter: 

Abstract: Cellobiase ( EC 3.2.1.21) is a B-glucosidase which hydrolyzes cellobiose to glucose and is known to be subject to both product and substrate inhibition. This work reports a model which combines both product and substrate inhibition effects for cellobiase isolated from a commercial preparation of Trichoderma viride from Miles Laboratories (Elkhart, IN). An integrated rate equation is presented which predicts the trends of time courses for hydrolyses of cellobiose at concentrations ranging from 14.6 – 146mM cellobiose. The constants used in the model (determined from initial rate data) are compared to those reported for cellobiase obtained from other source of T. viride. Most notable in this comparison is the apparently higher activity and reduced inhibition of this enzyme compared to other sources of cellobiase.

Research Area: Biofuels/Bioproducts    

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Production of Ethanol from Wood Hemicellulose Hydrolyzates by a Xylose-Fermenting Yeast Mutant Candida sp. XF 217

1981

Authors: Gong, C. S., M. R. Ladisch, and G. T. Tsao
Journal: Biotechnol. Lett., 3(11), 657-662 (1981) 
Book Chapter: 

Abstract: Ethanol was produced from wood chip hemicellulose hydrolyzate by a xylose-fermenting yeast mutant, Candida sp. XF 217. The rates of D-xylose consumption and ethanol production were greater under aerobic than fermentative conditions. The slow rate of fermentation under fermentative conditions could be overcome by supplementing the broth with D-xylose isomerase (glucose isomerase). The ethanol yield, as based on the sugar consumed, was approximately 90% of the theoretical value.

Research Area: Biofuels/Bioproducts    

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Review on Effect of Pretreatment on Digestibility of Cellulosic Materials

1981

Authors: Lin, K. W., M. R. Ladisch, D. Schaefer, C. H. Noller, V. Lechtenberg, and G. T. Tsao
Journal: AIChE Symp. Ser., 77, 102-106 (1981)
Book Chapter: 

Abstract: Solvent pretreatments at cellulosic materials by agents which disrupt the physical structure of the cellulose as well as the associated lignin seal are known to improve the rate and extent of cellulose hydrolysis by cellulase enzymes. The use of pretreatment to improve digestibility of cellulosic materials by rumen micro-organisms has also been reported in the literature.

Research Area: Biofuels/Bioproducts    

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Separation of Meso and Racemic 2,3-Butanediol by Aqueous Liquid Chromatography

1981

Authors: Voloch, M., M. R. Ladisch, V. W. Rodwell, and G. T. Tsao
Journal: Biotechnol. Bioeng., 23, 1289-1296 (1981) 
Book Chapter: 

Abstract: Fermentation of xylose by Klebsiella pneumoniae (ATCC 8724) produces meso and non-meso, 2,3-butanediol. The enzyme kinetics of 2,3-butanediol stereoisomer formation from acetoin is currently under study in our laboratory. Modeling of these kinetics requires resolution of meso and racemic 2,3-butanediol and positive identification of these resolved components. We report their resolution by aqueous liquid chromatography on both an analytical and a preparative scale. The resolved stereoisomers were identified by a combination of gas chromatography, gas chromatography/mass spectroscopy, 13C-NMR spectroscopy, optical activity, and melting points of the m-dinitrobenzoyl esters of meso and racemic 2,3-butanediol. An aqueous liquid chromatographic technique for resolving and qualifying major components of a butanediol fermentation mixture in 40 min is presented.

Research Area: Bioseparations Biofuels/Bioproducts   

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Vapor-Liquid Equilibria of the Water-Ethanol System at Low Alcohol Concentration

1981

Authors: Hong, J., M. R. Ladisch, and G. T. Tsao
Journal: J. Chem. Eng. Data, 26, 305-307 (1981)
Book Chapter: 

Abstract: Vapor-liquid equilibrium data for the ethanol-water system at 760 mmHg are collected by using a Gillespie-type still. Data in the range of ethanol concentration from 0.01 to 1.0 wt % are scarce, and yet they are much needed in the design of efficient ethanol recovery systems.

Research Area: Bioseparations    

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Alcohol from Cellulose

1980

Authors: Hsu, T. A., M. R. Ladisch, and G. T. Tsao
Journal: Chemtech. 10(5), 315-319 (1980)
Book Chapter: 

Abstract: Biomass is a term which encompasses Cellulosic residues, including agricultural residues, municipal wastes, livestock wastes, paper wastes, and forestry residues. Conversion of biomass into fermentable sugars could help to stretch petroleum resources. Sugar fermentation gives ethanol, which when used in gasohol (a 10% ethanol/90% gasoline mixture) extends gasoline supplies. Other sugar fermentations yield chemical intermediates such as butanol, acetaldehyde, acetic acid, acetone, and butanediol.

Research Area: Biofuels/Bioproducts    

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Cellobiase Hydrolysis by Endoglucanase (Glucan-Glucanohydrolase) from Trichoderma reesei: Kinetics and Mechanism

1980

Authors: Ladisch, M. R., C. S. Gong, and G. T. Tsao
Journal: Biotechnol. Bioeng. 22, 1107-1126 (1980) 
Book Chapter: 

Abstract: Glucanohydrolase from Trichoderma reesei, having a molecular weight of 52,000, was evaluated for kinetic properties with respect to cellobiose. Results from this work include: 1) initial rate studies that show that glucanohydrolase hydrolyzes cellobiose by a competitive mechanism and that the product, glucose, inhibits the enzyme; 2) low-pressure aqueous liquid chromatography that shows that formation of a reversion product, cellotriose, is minor and occurs in detectable amounts only at very high (90mM) cellobiose concentrations; 3) development of an equation based on the mechanism of glucanohydrolase action as determined by initial rate kinetics, which accurately predicts the time course of cellobiose hydrolysis; 4) derivation of an initial rate expression for the combined activity of cellobiase and glucanohydrolase on cellobiose. Based on data in this paper it is shown that the difference in inhibition patterns of the two enzymes could be used for determining the contamination of one enzyme by small quantities of the other.

Research Area: Biofuels/Bioproducts    

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New Approach to Aqueous Gel Permeation Chromatography of Nonderivatized Cellulose

1980

Authors: Yen T. Bao, Arindam Bose, Michael R. Ladisch, and George T. Tsao
Journal: Journal of Applied Polymer Science, 25, 263-275 (1980) 
Book Chapter: 

Abstract: A novel approach to the gel permeation chromatography (GPC) of nonderivatized cellulose is reported using Sepharose CL-6B as the column packing material, 0.5 N NaOH as the eluent, and cadoxen as the cellulose solvent. The traditional approach to GPC of cellulose has been to convert the cellulose to its nitrate thereby making it soluble in the solvent tetrahydrofuran. The circumvention of the need to derivatize the cellulose in the new system results in considerable saving of time. The new system gives good fractionation for cellulose. It also provides excellent separation of polystyrene sulfonate and dextran standards thereby making the system amenable to calibration. The effect of the particle size distribution of the column packing material on the efficiency of separation is discussed. Potential applications for this new method include studies on both acidic and enzymatic hydrolysis as well as fine structure of cellulose, starch, and other polymers capable of forming stable alkaline solutions.

Research Area: Biofuels/Bioproducts    

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Purification and Properties of Glucose Isomerase of Actinoplanes missouriensis

1980

Authors: C. S. Gong, L. F. Chen, G. T. Tsao
Journal: Biotechnology Bioengineering
Book Chapter: 

Abstract: Actinoplanes missouriensis produces an extracellular soluble glucose isomerase. The soluble enzyme can be purified by a DEAE-cellulose beads column with a one-step salt solution. The purified enzyme exhibited a molecular weight of approximately 80,000 daltons, being composed of two identical subunits of about 42,000 daltons each. The Km for glucose is 1.33M, the Km for fructose is 1.67M. The enzyme has an optimal pH of 7.0. The presence of the cobalt ion is not required to produce optimal activity of the enzyme if the proper amount of magnesium ion is present.

Research Area: Bioenergy Bioprocessing Biofuels/Bioproducts  

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Separation of Methanol Derivatives of Imidazolidinines, Urea and Carbonates by Aqueous Liquid Chromatography

1980

Authors: Beck, K. R., B. J. Leibowitz, and M. R. Ladisch
Journal: J. Chromatogr. 190, 226-232 (1980)
Book Chapter: 

Abstract: Agents used to crosslink cellulose and produce durable press cotton fabrics include bis(1,3-hydroxymethyl) 4,5-dihydroxyimidazolidinone (1), hydroxymethyl derivatives of urea (II), carbamates (III), and other nitrogenous compounds. Since 100 million pounds of these chemicals are used annually by the U.S.A. textile industry, the analysis for these water soluble compounds is of significant interest. Previously reported methods of analysis include gas chromatography (GC), thinlayer chromatography (TLC), gel permeation chromatography (GPC) and nuclear magnetic resonance (NMR).

Research Area: Bioseparations    

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Biosynthesis, Purification, and Mode of Action of Cellulases of Trichoderma reesei

1979

Authors: C.-S. Gong, M. R. Ladisch, G. T. Tsao
Journal: 
Book Chapter: Book: Hydrolysis of Cellulose: Mechanisms of Enzymatic and Acid Catalysis, Advances in Chemistry Series, Authors: R. D. Brown, Jr. and L Jurasek, ACS Publication, Washington, DC

Abstract: Enhanced cellulase producing strains of Trichodeerma reesei QM 9414 were used to study the biosynthesis of cellulases by using soluble (lactose) and insoluble (crystalline cellulose) substrates as the sole carbon source. The major cellulase components were isolated from culture filtrates of T. reesei and purified (to homogeneity) by chromatography on ion-exchange resins, by affinity chromatography and by gel-filtration. These are beta-glucosidase, 1,4-beta-glucan cellobiohydrolase, and 1,4-beta-glucan glucanohydrolase. The latter components were found to form little glucose upon hydrolysis of cellulose. The beta-glucosidase had high glucose forming activity upon incubation with cellobiose. Other properties examined with these purified components include molecular weight, specificity of action, and effect of culture media on enzyme activity.

Research Area: Bioenergy Biofuels/Bioproducts   

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Current Biological Research in Conversion of Cellulosic Carbohydrates Into Liquid Fuels: How Far Have We Come?

1979

Authors: M. C. Flickinger
Journal: Biotechnology Bioengineering, 22 (Supplement 1), 24-48
Book Chapter: 

Abstract: Current developments in the conversion of cellulosic carbohydrates into liquid fuels are reviewed. Four processes using mixed microbial cultures are described that directly convert cellulose and hemicellulose to ethanol. The production of sugars, which are converted to liquid fuels by fermentation, by dilute acid hydrolysis are described together with yeast fermentation of starch-derived glucose to ethanol. It is predicted that the most significant advances towards biological production of liquid fuels from cellulosic carbohydrates will occur through the discovery of new microorganisms with expanded genetic versatilities and altered membrane compositions.

Research Area: Bioenergy Bioprocessing   

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Dehydration of Ethanol: New Approach Gives Positive Energy Balance

1979

Authors: Ladisch, M. R. and K. Dyck
Journal: Science, 205(4409), 898-900 (1979)
Book Chapter: 

Abstract: Water was removed from aqueous ethanol by using cellulosic materials, starch, corn, and other agents. The combustion energy of the ethanol product can exceed the energy needed to carry out the dehydration by a factor of 10.

Research Area: Bioseparations    

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Fermentable Sugars from Cellulosic Residues

1979

Authors: Ladisch, M. R.
Journal: Process Biochem., 14(1), 21-25 (1979) 
Book Chapter: 

Abstract: The production of fermentable sugars from cellulosic residues is reviewed. Acid and enzyme hydrolysis as well as new developments in solvent pretreatment are summarized including some discussion of economics.

Research Area: Biofuels/Bioproducts    

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Fuels and Chemicals from Biomass

1979

Authors: Ladisch, M. R., M. C. Flickinger, and G. T. Tsao
Journal: Energy, The International Journal, 4(20), 135-164, (1979) 
Book Chapter: 

Abstract: Increasing energy consumption, coupled with decreased petroleum supplies, has made development of alternate energy sources a pressing national problem. One of the alternatives presently being examined is obtaining fuels from biomass. Biomass, which is a form of stored solar energy (sunlight having been converted by photosynthesis to cellulosic materials) is an abundant, renewable, domestically available energy resource. Although techniques of converting cellulosic materials in biomass to sugars and then to alcohol have been available for over one hundred years, it is only recently that the efficiency of this type of process has been improved to the point where the economics look potentially attractive. A major processing step responsible for this improvement is the conversion of cellulose to glucose in high yield. While the yield of sugars from cellulose was on the order of 50% previously, this has been improved to 90% or greater by a process in which cellulosic material is solvent-pretreated to make it readily accessible to hydrolysis to sugars by either acid or enzyme. This approach to hydrolysis, together with the fermentation of the resulting sugars to alcohol, is known as the Purdue Process. This process is discussed in the context of prior work done in this field.

Research Area: Biofuels/Bioproducts    

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Measurement of Cellulolytic Activity by Low Pressure Liquid Chromatography

1979

Authors: Ladisch, M. R., A. W. Anderson, and G. T. Tsao
Journal: J. Liq. Chromatogr., 2(5), 745-760 (1979) 
Book Chapter: 

Abstract: The application of aqueous, low pressure liquid chromatography, to the assay of cellulolytic enzyme activity is discussed. The advantages of this method are speed of analysis (less than 20 min.), small sample size (20 ul), good resolution, and a tolerance of the system to the presence of extraneous salts and proteins. Examples showing the use of this tool are given.

Research Area: Biofuels/Bioproducts    

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Cellulose to Sugars: New Path Gives Quantitative Yield

1978

Authors: Ladisch, M. R., C. M. Ladisch, and G. T. Tsao
Journal: Science, 201, 743-745 (1978)
Book Chapter: 

Abstract: Cellulosic residues that had been treated with a small amount of chemical solvent under room conditions were quantitatively saccharified on enzyme hydrolysis. This treatment can be used to obtain simple sugars for the production of alcohol and other chemicals.

Research Area: Biofuels/Bioproducts    

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Fermentation Substrates from Cellulosic Materials

1978

Authors: Tsao, G. T., M. Ladisch, C. Ladisch, T. A. Hsu, B. Dale, T. Chou
Journal: Annual Reports on Fermentation Processes, (2) (1978)
Book Chapter: 

Abstract: In this chapter, the availability and the economy of utilization of cellulosic wastes as an alternative natural resource are first compared to those of petroleum crude oil which is the major raw material source of current chemical industries. Next, the technical background of the related subject is described in considerable details from which two factors, (1) highly ordered cellulose structure and (2) lignin seal surrounding cellulose fibers, will emerge as the major obstacles of hydrolysis of cellulose contained in cellulose materials. The main subject matter of this chapter will then be introduced, namely the use of selective solvent extraction to fractionate cellulosic wastes into three individual components: cellulose, hemicellulose, and lignin. Once cellulose is dissolved in a solution, it is no longer protected by a crystalline structure nor lignin seal. Experimental results indicate that the re-precipitated cellulose can be easily hydrolyzed by either acids or enzymes to give high yield of glucose.

Research Area: Biofuels/Bioproducts    

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Fuels and Chemicals from Biomass

1978

Authors: M. R. Ladisch, M. C. Flickinger, and G. T. Tsao
Journal: Energy, 4, 263-275 (1978) 
Book Chapter: 

Abstract: Increasing energy consumption, coupled with decreased petroleum supplies, has made development of alternate energy sources a pressing national problem. One of the alternatives presently being examined is obtaining fuels from biomass. Biomass, which is a form of stored solar energy (sunlight having been converted by photosynthesis to cellulosic materials) is an abundant, renewable, domestically available energy resource. Although techniques of converting cellulosic materials in biomass to sugars and then to alcohol have been available for over one hundred years, it is only recently that the efficiency of this type of process has been improved to the point where the economics look potentially attractive. A major processing step responsible for this improvement is the conversion of cellulose to glucose in high yield. While the yield of sugars from cellulose was on the order of 50% previously, this has been improved to 90% or greater by a process in which cellulosic material is solvent-pretreated to make it readily accessible to hydrolysis to sugars by either acid or enzyme. This approach to hydrolysis, together with the fermentation of the resulting sugars to alcohol, is known as the Purdue Process. This process is discussed in the context of prior work done in this field.

Research Area: Biofuels/Bioproducts    

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High-Speed Liquid Chromatography of Cellodextrins and Other Saccharide Mixtures Using Water as the Eluent

1978

Authors: Michael R. Ladisch, Aronson L. Huebner, and George T. Tsao
Journal: Journal of Chromatography, 147, 185-193 (1978)
Book Chapter: 

Abstract: Ion-exchange resin AG 50W-X4 (Ca2+) separates the oligomers celloheltaose through glucose within 30 min when water is used as the eluent. The fractionation capabilities of this resin are a function of the procedures used in its preparation, packing, and operation. These procedures are described in detail. In addition to chromatograms showing separations obtained for cellodextrins and other saccharide mixtures, quantitative data relating concentrations of individual cellodextrin components to their respective peak areas are also presented.

Research Area: Bioseparations    

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Preparation of Cellodextrins: An Engineering Approach

1978