Acetic Acid Removal from Corn Stover Hydrolysate Using Ethyl Acetate and the Impact on Saccharomyces cerevisiae Bioethanol FermentationAuthors
M. Aghazadeh, M. R. Ladisch, A. S. Engelberth
Biotechnology Progress, 32(4), 929-937
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.
Enhanced Sugarcane Bagasse Conversion to Sugars by Ozonolysis and Liquid Hot Water PretreatmentsAuthors
S. Bordignon, R. da Silva, E. Ximenes, H. Roos, M. Ladisch, 38th Symposium on Biotechnology for Fuels and Chemicals, Poster Session 1: Bioprocessing, Reactor Design, and Separations Technology; Pretreatment and Fractionation; Microbial Science and Technology; Molecular Engineering, Synthetic Systems Biology, Poster M68, April 25, 2016, Baltimore, MD
Cellulose hydrolysis is achieved by a complex multi-enzymatic system that works more effectively when hemicellulose, lignin and their derived compounds are decreased in lignocellulosic substrates. In order to achieve this, we studied a combined approach by combining ozonolysis with liquid hot water (LHW) pretreatment of sugarcane bagasse. Under these conditions there was a 100% increase in available cellulose accompanied by an 80% decrease in hemicellulose, and 40% of lignin was oxidized. The double-pretreated material was further hydrolyzed in 50mM Sodium Citrate Buffer pH 5.0 at 10% (w/v) of solids loading using Cellic CTEC2 and HTEC2 (0.9 mg protein/g glucan) at 50 C. HPLC analysis showed that more than 40 g/L of glucose was released after 96 hours of hydrolysis, reaching 59% of conversion of the glucan. Single pretreatments (ozonolysis and LHW) were also performed separately and both gave 21 g/L of glucose, respectively. We showed that LHW pretreatment helps to remove partially the oxidized phenols after ozone attack, and also to solubilize the hemicellulose portion under high temperature, resulting in a more accessible glucan to the enzymes. The resultant liquor contains about 30 g/L of xylose and a large amount of phenolics (2.28 mg/L of Gallic Acid Equivalent). Conversion in the presence of this liquor is only 8% due to the strong inhibitory effect of phenols and carboxylic acids present in significant amounts in this fraction. Combining ozonolysis and LHW pretreatments is effective in separating cellulose from lignin and hemicellulose in bagasse, thereby generating fractions rich in sugars and phenolic compounds.
Identifying Conditions to Optimize Lactic Acid Production from Food Waste Co-Digested with Primary SludgeAuthors
R. Red Corn, A. S. Engelberth
Biochemical Engineering Journal, 105, 205-213
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.
Liquid Hot Water Pretreatment InhibitorsAuthors
E. Ximenes, Y. Kim, C. Farinas, M. R. Ladisch, 251st National ACS Meeting, Biofuel & Biobased Chemical Production: Biomass Pretreatment and Hydrolysis, San Diego, CA, March 14, 2016
Liquid hot water pretreatment enhances the rates and extents of cellulose hydrolysis for corn stover, sugar cane bagasse, switchgrass, hardwood, and other lignocellulosic materials as long as there is sufficient enzyme present to catalyze the reaction. The rationale that drives the use of pretreatment is the reduction in cost of enzyme and feedstock by increasing yields of fermentable sugars, principally glucose and xylose. Compared to untreated lignocellulose, pretreated feedstocks result in enhanced hydrolysis since pretreatment opens up the cell wall structure of the substrate, thereby enabling access of enzyme to the cellulose and disrupting the tightly packed cellulose structure. However, pretreatments also release inhibitors. More severe pretreatments are not always better since they can release greater amounts of inhibitors and deactivators which significantly reduce enzyme activity. Inhibitors include xylo-oligosaccharides, acetic acid, tannic acid, and phenolics. This effect is particularly noticeable as enzyme loading is decreased and the ratio of biomass derived inhibitors to added enzyme protein increases. Higher severity pretreatment may also expose more lignin as well as more cellulose in the cell wall structure. The lignin may unproductively adsorb proteins, including enzymes. Hence pretreatment can both help and hinder the enzyme hydrolysis of cellulose. This paper describes interactions between multiple enzyme components, inhibitors, and pretreated lignocellulosic substrates. Mitigation strategies are presented that reduce the amount of enzymes required to overcome inhibition due to pretreatment and achieve high conversion of lignocellulosic feedstocks to fermentable monosaccharides.
Maleic Acid Treatment of Bioabated Corn Stover Liquors Improves Cellulose Conversion to EthanolAuthors
D. Kim, E. Ximenes, G. Cao, N. N. Nichols, S. Frazer, M. R. Ladisch, 38th Symposium on Biotechnology for Fuels and Chemicals, Poster Session 1: Bioprocessing, Reactor Design, and Separations Technology; Pretreatment and Fractionation; Microbial Science and Technology; Molecular Engineering, Synthetic Systems Biology, Poster M66, April 25, 2016, Baltimore, MD
Elimination of inhibitory compounds released during pretreatment of lignocellulose is critical for efficient cellulose conversion and ethanol fermentation. This study examined the effect of bioabated liquor from pretreated corn stover on enzyme hydrolysis of Solka Floc or pretreated corn stover solids. Xylo-oligosaccharides in the liquor were hydrolyzed by hemicellulose or maleic acid. Pretreatment was at 20% solids, 190 C, 45 min, and subsequent hydrolysis, after bioabatement was done with 5% corn stover, and ethanol fermentation by Saccharomyces cerevisiae. The fungus Coniochaeta ligniaria NRRL30616 removed inhibitory compounds in the liquor from LHW-pretreated corn stover. The conversion of cellulose to glucose in bioabated liquor was higher when the liquor was treated with maleic acid than with hemicellulose. For corn stover slurried in hemicellulose treated liquor, cellulose conversion was 39%, while corn stover in maleic acid treated liquor gave 68% yield. The observed lower glucose yield may be related to inhibition of beta-xylosidase caused by accumulation of xylo-oligomers, which in turn inhibited beta-glucosidase, leading to accumulation of cellobiose. The use of maleic acid alleviated the inhibitory effect on beta-glucosidase by hydrolyzing the xylo-oligomers to xylose. Ethanol production from Solka Floc hydrolysate or sugars from corn stover solids was 20 to 30% higher for bioabated liquor compared to non-bioabated liquor. Furthermore, the fermentation lag phase was decreased by 3 hours. Our results confirm bioabatement removes compounds that inhibit enzyme hydrolysis and fermentation. The treatment of bioabated samples with maleic acid improved overall cellulose conversion due to hydrolysis of xylo-oligomers to xylose, where xylose is much less inhibitory towards beta-glucosidase.
Maleic Acid Treatment of Biologically Detoxified Corn Stover LiquorAuthors
D. Kim, E. A. Ximenes, N. N. Nichols, G. Cao, S. E. Frazer, M. R. Ladisch
Bioresource Technology, 216, 437-445
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.
Mechanisms of Lignin Derived Inhibition in Hydrolysis of Pretreated Biomass at Low Enzyme LoadingsAuthors
M. Ladisch, E. Ximenes, C. S. Farinas, Y. Kim, J. K. Ko, T. Kreke, 38th Symposium on Biotechnology for Fuels and Chemicals, Session 12: Enzyme Science and Technology II - Assays, Characterization and Application, April 27, 2016, Baltimore, MD
The recalcitrance of lignocellulosic biomass materials with respect to enzyme hydrolysis is caused by structural factors and the interrelated effects of enzyme inhibitors. While liquid hot water, dilute acid, steam explosion, ionic fluid or alkaline pretreatments result in high conversion, these are insufficient for achieving low enzyme loadings due to inhibition effects. The products of cellulose hydrolysis - cellobiose and glucose - are known to inhibit cellulases and beta-glucosidases, with lignin-derived phenolics amplifying the overall inhibition effects. Further, lignin exposed through pretreatment interferes with hydrolysis by adsorbing cellulases and beta-glucosidases. The combined effects result in a conundrum: increasing severity of pretreatment, whether by chemical addition or hydrothermal conditions, results in significantly enhanced enzyme hydrolysis but also requires higher enzyme loadings. Excess enzymes, i.e, high enzyme loadings, are therefore needed if high yields from pretreated lignocellulosic substrates are to be achieved. We report mechanisms by which lignin derived inhibitors negatively affect enzyme activity and show how the interactions between insoluble and soluble enzyme inhibitors mask the mechanisms involved in enzyme hydrolysis of pretreated biomass. The identity of the inhibitors and the manner in which these molecules interact with cellulases, hemicellulases and beta-glucosidases will be discussed, together with approaches that show how enzyme loadings of 1 to 2 FPU/g total solids (after pretreatment) are sufficient to achieve 80% hydrolysis. The current work utilizes results from our laboratory and other leading research facilities to define an integrated mechanistic framework for the complex interactions that both limit and enhance enzyme hydrolysis of cellulose.
Secretome Analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysisAuthors
C. Florencio, F. M. Cunha, A. C. Badino, C. S. Farinas, E. Ximenes, M. R. Ladisch
Enzyme and Microbial Technology, 90, 53-60 (2016)
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.
Techno-economic Analysis for Incorporating a Liquid-Liquid Extraction System to Remove Acetic Acid into a Proposed Commercial Scale BiorefineryAuthors
M. Aghazadeh, A. S. Engelberth
Biotechnology Progress, 32(4), 971-977
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).
The Effect of Lignin in Enzymatic Saccharification of Bred Sugarcane BagasseAuthors
R. L. Azar, T. Morgan, M. Barbosa, V. Guimaraes, E. Ximenes, M. Ladisch, 38th Symposium on Biotechnology for Fuels and Chemicals, Poster Session 2: Feedstocks; Enzyme Science and Technology; Renewable Fuels, Chemicals, and Bio-Based Products, Poster T20, April 26, 2018, Baltimore, MD
Lignin, one of the major components of lignocellulosic biomass, plays an important functional and structural role in plants. Lignin is also known as a major contributor to the recalcitrance of lignocellulosic biomass, and has been a target for feedstock improvement through genetic engineering. This work examines the influence of lignin in conventional breeded (clones 260 and 204) sugarcane bagasse after liquid hot water pretreatment. In conventional breeding, large differences in lignin are not expected because the plant does not easily lose this trait from one generation to the next. Moreover, we evaluate the enzyme-lignin interactions of lignins isolated from LHW pretreated sugarcane bagasse with and without BSA. FTIR analysis was used to investigate differences among the chemical composition of lignins studied.
Adsorption of Enzyme Onto Lignins of Liquid Hot Water Pretreated HardwoodsAuthors
J. K. Ko, E. Ximenes, Y. Kim, M. R. Ladisch
Biotechnology and Bioengineering, 112(3), 447-456, 2014
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.
Biomimetic Catalyst: Maximizing Yields of Hydroxymethylfurfural from Whole BiomassAuthors
B. B. Hewetson, A. Kreger, N. S. Mosier AIChE Meeting, Atlanta, GA, November 20, 2014
Achieving high yields of HMF requires effective hydrolysis, isomerization, and dehydration of glucose from cellulose. We report the use of a cellulose solvent (85% w/w phosphoric acid) to remove and then recover cellulose from several plant biomasses (corn stover, switchgrass, and poplar) and microcrystalline cellulose (Avicel). The resultant amorphous cellulose is subjected to a conversion process where maleic acid hydrolyzes the cellulose to glucose, AlCl3 isomerizes the resultant glucose to fructose, and both acid catalysts dehydrate the fructose to HMF in a single reactor bi-phasic reactor where HMF is continuously extracted into MTHF. The results confirm yields of HMF (35 to 40%) can be increased by cellulose dissolution in concentrated phosphoric acid followed by hydrolysis of the reprecipitated amorphous cellulose. The increase in HMF yields is dependent upon the type of biomass. The total sugar conversion (C5 and C6 sugars) from the whole intact lignocellulosic starting biomass reaches >90% in the best case.
Ozonolysis as a Pre-Pretreatment for Compacted Bioenergy FeedstockAuthors
I. Beheshti Tabar, P. T. Murphy, N. S. Mosier AIChE Meeting, Atlanta, GA, November 20, 2014
Ozone pretreatment has been shown to improve the enzymatic digestibility of cellulose. In this study, the chemical pretreatment of highly compacted switchgrass with ozone was carried out in a fixed bed reactor. Material density in the reactor, ozone concentration, and biomass particle size simulated large scale in-farm or conversion facility treatment of biomass bales. An industrially viable ozone concentration of 22.5 mg/l (15% w/w) was used to treat the samples for 24 hours. The results showwed that a significant amount of soluble sugars (about 10% of total sugars) was generated from ozone-catalyzed hydrolysis of the hemicellulose. Despite visible changes in color, compositional analysis showed no significant change in glucan content and insignificant changes in total lignin content after treatment. Nonetheless, digestibility of treated material increased by more than 5-fold. Enzymatic hydrolysis of the materials with a relatively low loading of 10 FPU/g glucan resulted in yields of glucose of 59% for water washed samples and 27% for unwashed, compared to 11 and 9% for non-treated samples, respectively. The significant improvement in hydrolysis yields for washed samples suggest that water-soluble inhibitors generated from lignin degradation may be present after ozone pretreatment.
Biological Conversion of Plants to Fuels and Chemicals and the Effects of InhibitorsAuthors
C. E. Wyman, E. Ximenes, Y. Kim, M. R. Ladisch
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.
Comparative Performance of Leading Pretreatment Technologies for Biological Conversion of Corn Stover, Poplar Wood, and Switchgrass to SugarsAuthors
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
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.
Effect of Salts on the Co-fermentation of Glucose and Xylose by a Genetically Engineered Strain of Saccharomyces cerevisiaeAuthors
E. Casey, N. S. Mosier, J. Adamec, Z. Stockdale, N. Ho, M. Sedlak
Biotechnology for Biofuels, 6, 83
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.
Comparing Extraction Methods to Recover Ginseng Saponins from American Ginseng (Panax quinquefolium), Followed by Purification Using Fast Centrifugal Partition Chromatogrpahy with HPLC Verification`Authors
A. S. Engelberth, E. C. Clausen, D. J. Carrier
Separation and Purification Technology, 72, 1-6
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.
Purification of Resveratrol, Arachidin-1, and Arachidin-3 from Hairy Root Cultures of Peanut (Arachis hypogaea) and Determination of Their Antioxidant Activity and CytotoxicityAuthors
J. A. Abbott, F. Medina-Bolivar, E. M. Martin, A. S. Engelberth, H. Villagarcia
Biotechnology Progress, 26(5), 1344-1351
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.
Two-Dimensional Particle Focusing: Sheath Flow on Two SidesAuthors
J. Shin, M. Ladisch
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.
Extraction of Co-Products from Biomass: Example of Thermal Degradation of Silymarin Compounds in Subcritical WaterAuthors
L. Duan, S. N. Wallace, A. Engelberth, J. K. Lovelady, E. C. Clausen, J. W. King, D. J. Carrier
Applied Biochemistry and Biotechnology, 158(2), 362-373
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.
Switchgrass Water Extracts: Extraction, Separation, and Biological Activity of Rutin and QuercitrinAuthors
N. Uppugundla, A. Engelberth, S. V. Ravindranath, E. C. Clausen, J. O. Lay, J. Giddens, D. J. Carrier
Journal of Agricultural and Food Chemistry, 57(17), 7763-7770
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.
Targeted Capture of Pathogenic Bacteria Using a Mammalian Cell Receptor Coupled with Dielectrophoresis on a BiochipAuthors
O. K. Koo, Y.S. Liu, S. Shuaib, S. Bhattacharyall, M. R. Ladisch, R. Bashir and A. K. Bhunia
Journal of Analytical Chemistry, 81(8), 3094-3101 (2009)
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.
Separation of Silymarins from Milk Thistle (Silybum marianum L.) Extracted with Pressurized Hot Water Using Fast Centrifugal Partition ChromatographyAuthors
A. S. Engelberth, D. J. Carrier, E. C. Clausen
Journal of Liquid Chromatography & Related Technologies, 31 (19), 3001-3011
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.
A Multifunctional Micro-Fluidic System for Dielectrophoretic Concentration Coupled with Immuno-Capture of Low Numbers of Listeria monocytogenesAuthors
Yang, L., P. P. Banada, M. R. Chatni, K. S. Lim, A. K. Bhunia, M. Ladisch, and R. Bashir
Lab on a Chip , 6, 896-905 (2006)
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 Âµm high and ~ 260 Âµm 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 0.2Âµl min-1 into the micro-channel with concentration factors between 102 - 103 , in sample volumes of 5-20Âµl. A high flow rate of 0.6 Âµl 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.
Anomalous Resonance in a Nanomechanical BiosensorAuthors
Gupta, A. K., P. R. Nair, D. Akin, M. R. Ladisch, S. Broyles, M. A. Alam, and R. Bashir
Proceedings of the National Academy of Sciences, 103, 13362-13367 (2006)
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.
Surface Engineering of Microchannel Walls for Protein Separation and Directed Microfluidic FlowAuthors
T. T. Huang, N. S. Mosier, and M. R. Ladisch
Journal of Separation Science, 29(12), 1733-1742 (2006)
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.
Surface-Directed Boundary Flow in Microfluidic ChannelsAuthors
Huang, T. T., D. G. Taylor, K. S. Lim, M. Sedlak, R. Bashir, N. S. Mosier, and M. R. Ladisch
Langmuir, 22, 6429-6437 (2006)
Channel geometry combined with surface chemistry enables a stable liquid boundary flow to be attained along the surfaces of a 12 Âµm 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 Âµm wide and 12 Âµm high. Viewing this stream through a 20 Âµm 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 105Â° will direct the aqueous fluid to a boundary layer next to a hydrophilic fiber with a contact angle of 5Â°. 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.
Bacterial Transport in Rolled Stationary Phase MonolithsAuthors
Bwatwa, J., N. S. Mosier, T. Huang, X. Liu, A. Stewart, C. M. Ladisch, and M. R. Ladisch
Rolled cotton monoliths enable rapid desalting of proteins in 1 to 10 minutes, and constitute an excelent hydrophilic chromatography support. The monoliths display rigidity and robustness at mobile phase linear velocities of 100 cm/min, unlike beds of cellulose particles which collapse at these conditions. This stationary phase is able to pass microorganisms without plugging. This has led to investigation of rolled stationary phase for rapid preprocessing of homogenized meat broths to separate and recover microbial cells. We report results fro the fractionation of microorganisms from a broth of lipids, protein and other colloidal particles and innoculated with GFP expressing E. coli. The location of GFP expressing E. coli during their passage through the monlith is readily monitored using fluorescence microscopy. The overall characteristics of the rolled monolith having a 2.5 cm diameter ar emodeled, and the probable trajectory of microbial cells, based on work with particle flow over single fibers, is estimated. The passage of the bacteria entails both tangential and radial flow. Application of rolled stationary phase monoliths to rapid filtering, fractionation, and detection of both proteins and bacteria using microfluidic devices is presented with examples.
Dielectrophoresis and Antibody Mediated Selective Capture of Microorganisms in Micro-Fluidic BiochipsAuthors
H. Li, L. Yang, D. Akin, T. Geng, A. Bhunia, T. T. Huang, M. Ladisch, R. Bashir
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.
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.
Microfiber-Directed Boundary Flow in Press-Fit Microdevices Fabricated from Self-Adhesive Hydrophobic SurfacesAuthors
T. T. Huang, D. G. Taylor, M. Sedlak, N. S. Mosier, and M. R. Ladisch
Analytical Chemistry, 77, 3671-3675 (2005).
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.
Microscopic Examination of Changes of Plant Cell Structure in Corn Stover Due to Cellulase Activity and Hot Water PretreatmentAuthors
Zeng, M., N. S. Mosier, C. Huang, D. Sherman, J. Goetz, and M. R. Ladisch
Particle size has a significant impact on the saccharification of plant cell walls by cellulolytic enzymes. It is believed that small particle sizes of a cellulosic substrate are more readily hydrolyzed than large ones and that pretreatment enlarges accessible and susceptible surface area. These hypotheses are being tested using ground corn stover (stalks and leaves) in the size range of 425 to 710 um and 53 to 75 um. Scanning electron microscopy shows that enzyme treatment induces pore formation in the surface of the corn stover. Corn stover pretreated at 190 C for 15 min generates a few pores on the surface. When followed by enzyme hydrolysis, pretreated stover exhibits greater porosity than the enzyme hydrolyzed stover that has not been pretreated. Comparison of the microscopic changes to macroscopic features of hydrolysis suggests that mechanism of enzyme action is more complex than would be suggested by particle size or surface area. This paper correlates microscopic change in structure to the activity of enzyme hydrolysis before and after pretreatment. The objective is to understand the changes that occur at cellular level, compared to a particulate or macroscopic level. In this manner, a specific understanding of enzyme activity on a cellular level can be developed and ultimately translated to pretreatment processes that impove hydrolysis.
Rapid Prototyping of Purification PlatformsAuthors
Huang, T., N. S. Mosier, and M. R. Ladisch
The development of protein products, particularly monoclonal antibodies, for pharmaceutical applicatins requires rapid development of purification methods. Previously small analytical columns, and advance scale systems have been used to evaluate different types of stationary phase, and to quickly evaluation whether or not its separation characteristics are compatible with the molecules to be fractionated. This particular paper presents an approach which utilizes rapid prototyping of microchips in order to rapidly evaluate different types of stationary phases. These chips are based on fibers to which particles of different ion exchange groups or antibodies are anchored. The labeled proteins are then microscopically observed with respect to the retention behavior. This work describes the rapid assembly of different types of stationary phases required for separation, and methodologies for the rapid evaluation of the observed fractionation. Examples are based on lgG class antibody interactions with affinity base stationary phases such as Protein A. The methods show how the observed properties can be used to quickly define the most appropriate stationary phase, and then begin rapid evaluation with respect to scale parameters. Since the methodology is based on path lengths that are less than 10 microns from the liquid to the surface of the stationary phase, difficusion control is the limiting factor. Consequently, close observation of separation chracteristics can be quickly conceived, reduced to practice, and be evaluated.
Separation of Denatured Proteins in Free Solution on a Microchip Based on Differential Binding of Alkyl Sulfates with Different Carbon Chain LengthsAuthors
Chang Lu, Aaron E. Smith and Harold G. Craighead
Chemical Communication, 183-185 (2005).
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 10â€“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.
Bioseparation Techniques in Microfluidic Devices Using Micro-BeadAuthors
W.-J. Chang, K. W. Ro, T. T. Huang, Y.-M. Koo, J. H. Hahn, M. R. Ladisch, D. Akin, R. Bashir
Theories and Applications of Chemical Engineering, 10, 1, 203-206, (2004)
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.
Biotextiles - Monoliths with Rolled GeometricsAuthors
Jeremiah Bwatwa, Yiqi Yang, Chenghong Li, Craig Keim, Christine Ladisch, and Michael Ladisch
Journal of Chromatography, 67, 235-253, 2003
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))  assembled into the yarns (Fig. 11.1(b)) , and the woven fabric (Fig. 11.1(c)) . 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.
Lysozyme for Capture of Microorganisms on Protein BiochipsAuthors
Tom Huang, Tao Geng, Jennifer Sturgis, Haibo Li, Rafael Gomez, Rashid Bashir, Arun K. Bhunia, J. Paul Robinson, Michael R. Ladisch
Enzyme and Microbial Technology, 33, 958-966 (2003)
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.
Micro-assembly of Functionalized Particulate Monolayer on C18-Derivatized SiO2 SurfacesAuthors
Tom T. Huang, Tao Geng, Demir Akin, Woo-Jin Chang, Jennifer Sturgis, Rashid Bashir, Arun K. Bhunia, J. Paul Robinson, and Michael R. Ladisch
Biotechnology and Bioengineering, 83, 4, 416-427, 2003.
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.
Model for Temperature Profiles in Large Diameter Electrochromatography ColumnsAuthors
Craig Keim and Michael Ladisch
AIChE Journal, 49, 2, 402-410 (2003)
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.
Bioseparations for BiochipsAuthors
Chen, W.-T. (Speaker), T. Geng, R. Hendrickson, A. K. Bhunia, and M. R. Ladisch
Biochip technology has opened the door to rapid detection of pathogens compared to conventional methods. Detection of some pathogens, notably Listeria monocytogenes, takes up to a week. It is costly both economically and in terms of food safety. Biochips offer detection times in hours, not in days. However, sample procesisng is required in order to remove the interferring substances, ideally leaving only the microorganisms. Biological samples, especially food products are complex substances containing crabohydrates, proteins, lipids, and salts, which interfere with the selectivity of the binding sites on the chips. This paper describes preparation for food samples by various chromatographic resins, including cationic, anionic ion exchangers, hydrophobic, bifunctional and reverse-phase resins. Among these, Amberlite 35, which is a strong cationic ion exchanger, gave the highest adsorption of proteins.
Biotechnology for Future Army ApplicationsAuthors
Ladisch, M. R., J. J. Valdes, and LTC R. J. Love
Army AL&T, July-August, 36-37, (2002)
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.
Modeling Pore Size Distribution in Cellulose Rolled Stationary PhasesAuthors
Keim, C., C. Li, C. M. Ladisch, and M. R. Ladisch
Biotechnology Progress, 18, 317-321, (2002)
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.
Optimal Packing Characteristics of Rolled, Continous Stationary-Phase ColumnsAuthors
Li, C., C. M. Ladisch, Y. Yang, R. Hendrickson, C. Keim, N. Mosier, M. R. Ladisch
Biotechnology Progress, 18, 309-316, (2002)
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.
Adsorption of Water from Liquid-Phase Ethanol-Water Mixtures at Room Temperature Using Starch-Based AdsorbentsAuthors
Beery, K. E., M. R. Ladisch
Ind. Eng. Chem. Res., 40, 2112-2115, (2001)
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.
Chemistry and Properties of Starch Based DessicantsAuthors
Kyle E. Beery, Michael R. Ladisch
Enzyme and Microbial Technology, 28, 573-581 (2001)
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 18.104.22.168) 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.
New System for Preparative Electrochromatography of ProteinsAuthors
Keim, C., and M. R. Ladisch
Biotechnology and Bioengineering, 70, 72-81, (2000)
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 Â°C mobile phase coupled with heat conduction through the column walls controlled the temperature to 28 Â°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.
Rolled Stationary Phases: Dimensionally Structured Textile Adsorbents for Rapid Liquid Chromatography of ProteinsAuthors
Hamaker, K., S-L. Rau, R. Hendrickson, J. Liu, C. M. Ladisch, and M. R. Ladisch
Ind. Eng. Chem. Res., 38, 865-872 (1999)
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.
Encyclopedia of Chemical Technology, John Wiley & Sons, p89-122 (1998)
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.
Effect of Enzyme Modification of Corn Grits on Their Properties As An Adsorbent in Skarstrom Pressure Swing Cycle DrierAuthors
Beery, K., M. Gulati, E. P. Kvam, and M. R. Ladisch
Adsorption, 4, 321-335 (1998)
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 â€“ 56 Â°C dew point and the 0.978 mm corn grits dry air to a â€“ 80 Â°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.
Transport Properties of Rolled, Continuous Stationary Phase ColumnsAuthors
Hamaker, K., J. Liu, C. Ladisch, and M. R. Ladisch
Biotechnol. Progr., 14(1), 21-30 (1998)
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.
Chromatography for Rapid Buffer Exchange and Refolding of Secretory Leukocyte Protease InhibitorAuthors
Hamaker, K. H., J. Liu, R. J. Seely, C. M. Ladisch, and M. R. Ladisch
Biotechnol. Progr., 12, 184-189 (1996)
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.
Intraparticle Flow and Plate Height Effects in Liquid Chromatography Stationary PhasesAuthors
Hamaker, K. H. and M. R. Ladisch
Separation and Purification Methods, 25(1), 47-83 (1996)
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.
Synthesis and Optimization of a New Starch Based Adsorbent for Dehumidification of Air in a Pressure Swing DrierAuthors
Anderson, L., M. Gulati, P. Westgate, E. Kvam, K. Bowman, and M. R. Ladisch
Ind. & Eng. Chem. Res., 35, 1180-1187 (1996)
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 Â°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 Â°C. The methods for preparing this material and an explanation of its performance in terms of macroscopic and microscopic structural characteristics are described.
Correlation of Electrophoretic Mobilities of Proteins and Peptides with Their Physicochemical PropertiesAuthors
Basak, S. and M. R. Ladisch
Anal. Biochem., 226, 51-58 (1995)
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 â€“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.
Effect of Modulator Sorption on Gradient Shape in Ion Exchange ChromatographyAuthors
Velayudhan, A. and M. R. Ladisch
Ind. Eng. Chem. Res., 34(8), 2805-2810 (1995)
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.
Electrochromatographic Separations of ProteinsAuthors
Basak, S., A. Velayudhan, K. Kohlmann and M. R. Ladisch
Journal of Chromatography A., 707, 69-76 (1995)
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.
Enhanced Enzyme Activities on Hydrated Lignocellulosic SubstratesAuthors
K. L. Kohlmann, A. Sarikaya, P. J. Westgate, J. Weil, A. Velayudhan, R. Hendrickson, M. R. Ladisch
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.
Mechanistic Description and Experimental Studies of Electrochromatography of ProteinsAuthors
Basak, S. and M. R. Ladisch
AIChE J., 41(11), 2499-2507, (1995)
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-aâ€“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.
Scale-up Techniques in Bioseparation ProcessesAuthors
Ladisch, M. R., and A. Velayudhan
Bioseparation Processes in Foods, Dekker, (1995)
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.
Simultaneous Concentration and Purification Through Gradient Deformation in Gradient Elution ChromatographyAuthors
Velayudhan, A., R. L. Hendrickson, and M. R. Ladisch
AIChE J., 41(5), 1184-1193 (1995)
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.
Characterization of Buffers for Electrokinetic SeparationsAuthors
Basak, S. K., A. Velayudhan, and M. R. Ladisch
Appl. Biochemistry Biotechnol., 44, 243-261 (1994)
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.
Liquid Chromatography Using Cellulosic Continuous Stationary PhasesAuthors
Yang, Y., A. Velayudhan, C. M. Ladisch, and M. R. Ladisch
Advances in Biochemical Engineering and Biotechnology, 49, 147-160, (1993)
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.
Modulator Sorption in Gradient Elution ChromatographyAuthors
Velayudhan, A., and M. R. Ladisch
Bioproducts and Bioprocesses, 2, 217-232, (1993)
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.
Plates Models in Chromatography: Analysis and Implications for Scale upAuthors
Velayudhan, A., and M. R. Ladisch
Advances in Biochem. Engr. Biotech., 49, 123-145, (1993)
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.
Solute Retention in Electrochromatography by Electrically Induced SorptionAuthors
Rudge, S. R., S. Basak and M. R. Ladisch
AIChE J., 39(5), 797-808 (1993)
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.
Sorption of Organics and Water on StarchAuthors
Westgate P., and M. R. Ladisch
Ind. Eng. Chem. Res , 32(8), 1676-1680 (1993)
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.
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 PhaseAuthors
Ladisch, C. M., Y. Yang, A. Velayudhan, and M. R. Ladisch
Textile Res. J., 62(6), 361-369 (1992)
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 Â°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.
Effect of Modulator Sorption in Gradient Elution Chromatography: Gradient DeformationAuthors
A. Velayudhan, and M. R. Ladisch
Chemical Eng. Sci. J., 47. No. 1, 233-239 (1992)
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.
Modeling of Equilibrium Sorption of Water Vapor on Starch MaterialsAuthors
Westgate, P. J., J. Y. Lee, and M. R. Ladisch
Transactions ASAE, 35(1), 213-219 (1992)
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. Sircarâ€™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 Sircarâ€™s model and potential theory equations are shown to fit the data for both starch and corn grits in the high temperature range.
Modeling of Non-Linear Elution Chromatography for Preparative-Scale SeparationsAuthors
Velayudhan, A., M. R. Ladisch, and J. Porter
AIChE Symp. Ser., 88, (1992)
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.
Protein Chromatography Using a Continuous Stationary PhaseAuthors
Yang, Y., A. Velayudhan, C. M. Ladisch, and M. R. Ladisch
J. Chromatogr., 598, 169-180 (1992)
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.
Recombinant Human InsulinAuthors
Ladisch, M. R., and K. Kohlmann
Biotechnol. Prog., 8(6), 469-478 (1992)
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.
Analytical and Preparative Scale Chromatography of Phenylalanine from Aspartame Using a New Polymeric SorbentAuthors
Ladisch, M. R., R. L. Hendrickson, and E. Firouztale
J. Chromatogr. (540), 85-101 (1991)
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 Â°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 Â°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.
Ion-Exchange and Affinity Chromatography Costs in a-Galactosidase PurificationAuthors
Jill E. Porter and Michael R. Ladisch
Biotechnology and Bioengineering, 39, 717-724 (1991)
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.
Purification and Characterization of an Extracellular Protease Produced by Pseudomonas fluorescens M3/6Authors
Kohlmann, K. L., S. S. Nielsen, and M. R. Ladisch
J. Dairy Sci., 74, 4125-4136 (1991)
Pseudomonas fluorescens strain M3/6 was inoculated into reconstituted NDM and incubated at 7 Â°C for 46 d. A significant amount of extracellular protease was produced, mainly during the latter part of the cultureâ€™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.
Role of Modulator in Gradient Elution ChromatographyAuthors
Velayudhan, A. and M. R. Ladisch
Anal. Chem., 63(18), 2028-2032 (1991)
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.
Water and Ethanol Sorption Phenomena on StarchAuthors
Lee, J. Y., P. J. Westgate, and M. R. Ladisch
AIChE J., 8(37), 1187-1195 (1991)
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 Sircarâ€™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.
Analysis of Sub-microgram Quantities of Cellodextrins by Aqueous Liquid Chromatography Using a Differential RefractometerAuthors
A. N. Pereira, K. L. Kohlmann, and M. R. Ladisch
Biomass, 23, 307-317 (1990)
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.
Characterization of the Swelling of a Size Exclusion GelAuthors
Monke, K., A. Velayudhan, and M. R. Ladisch
Biotechnol. Progress, 6(5), 376-382 (1990)
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.
Displacement Effect in Multicomponent ChromatographyAuthors
Gu, T., G. T. Tsao, G-J. Tsai, and M. R. Ladisch
AIChE J. 36(8), 1156-1162 (1990)
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.
Freeze Concentration of DyesAuthors
Yang, Y., C. M. Ladisch, and M. R. Ladisch
Textile Res. J., 12(60) 744-753 (1990)
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.
Ion Exchange and Affinity Chromatography in the Scaleup of the Purification of a-Galactosidase from Soybean SeedsAuthors
Jill E. Porter, Michael R. Ladisch, and Klaus M. Herrmann
Biotechnology and Bioengineering, 37, 356-363 (1990)
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.
Bioseparations of Milk ProteinsAuthors
Ladisch, M. R., S. R. Rudge, K. W. Ruettimann, and J. K. Lin
Bioproducts and Processes (1989)
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.
Cellulosic Adsorbents for Treating Textile Mill EffluentsAuthors
Yang, Y., C. M. Ladisch, and M. R. Ladisch
Enzyme Microb. Technol., 10(10), 632-636 (1988)
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.
Hydrophobic Interaction ChromatographyAuthors
Roettger, B. F., and M. R. Ladisch
Biotech. Adv., Vol. 7, 15-29 (1989)
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.
Mechanisms of Protein Retention in Hydrophobic Interaction ChromatographyAuthors
Roettger, B., J. Myers, M. R. Ladisch, and F. Regnier
American Chemical Society, 80-92, (1989)
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. Wymanâ€™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.
Rudge, S. R., and M. R. Ladisch
Biotechnol. Prog., 4(3), 123-133 (1988)
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.
Liquid Chromatography of Carbohydrate Monomers and OligomersAuthors
Lin, J. K., B. J. Jacobson, A. N. Pereira, and M. R. Ladisch
Biomass Handbook: Academic Press, Inc., Methods in Enzymology 160, (1988)
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.
Cause and Correction of Baseline Interruptions Observed for Small-Bore Liquid Chromatography Columns Using Cation Exchange Resin in the H+ FormAuthors
Lin, J. K., S. J. Karn, and M. R. Ladisch
Biotechnol. Bioeng, 30, 331-333 (1987)
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.
Polysaccharides as Adsorbents- An Update on Fundamental Properties and Commercial ProspectsAuthors
Lee, J. Y., and M. R. Ladisch
Annals of the New York Academy of Sciences, 506, 491-498, (1987)
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 Â°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.
Separation by AdsorptionAuthors
Ladisch, M. R.
Advanced Biochemical Engineering, H. R. Bungay and G. Belfort, eds., J. Wiley & Sons, NY (1987)
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.
Sulfuric Acid-Sugar Separation by Ion ExclusionAuthors
Neuman, R. P., S. R. Rudge, and M. R. Ladisch
Reactive Polymers J., 5, 55-61 (1987)
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 Â°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.
Leaching: A Separation Process for the Production of Fuels and Chemicals from BiomassAuthors
Ladisch, M. R., G. T. Tsao, and K. Lin
Biotechnol. Bioeng. Symp. Ser., No. 15, 723-736 (1986)
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.
Process Considerations for Scale-up of Liquid Chromatography and ElectrophoresisAuthors
Rudge, S., and M. R. Ladisch
American Chemical Society symp. 314, 122-152, (1986)
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.
Water Sorption Properties of a Polysaccharide AdsorbentAuthors
Neuman, R., M. Voloch, P. Bienkowski, and M. R. Ladisch
I&EC Fundamentals, 25, 422-425 (1986)
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 Hendersonâ€™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.
Breakthrough Behavior of 17.5 mol % Water in Methanol, Ethanol, Isopropanol, and t-Butanol Vapors Passed over Corn GritsAuthors
Bienkowski, P., A. Barthe', M. Voloch, R. N. Neuman, and M. R. Ladisch
Biotechnol. Bioeng., 28(7), 960-964 (1985)
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.
Adsorption of Ethanol/Water Mixtures by Biomass MaterialsAuthors
Hong, J., M. Voloch, M. R. Ladisch, and G. T. Tsao
Biotechnol. Bioeng. 24, 725-730 (1982)
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.
Calcium Sulfate as a Selective Adsorbent of WaterAuthors
Pocium, D., M. Ladisch, G. Tsao, and P. Wankat
AIChE Annual Meeting (1982)
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 Â°C) and can be regenerated at 140 to 150 Â°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 Â°C gradually during regeneration (instead of a step change to 140 Â°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.
Separation of Meso and Racemic 2,3-Butanediol by Aqueous Liquid ChromatographyAuthors
Voloch, M., M. R. Ladisch, V. W. Rodwell, and G. T. Tsao
Biotechnol. Bioeng., 23, 1289-1296 (1981)
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.
Vapor-Liquid Equilibria of the Water-Ethanol System at Low Alcohol ConcentrationAuthors
Hong, J., M. R. Ladisch, and G. T. Tsao
J. Chem. Eng. Data, 26, 305-307 (1981)
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.
Separation of Methanol Derivatives of Imidazolidinines, Urea and Carbonates by Aqueous Liquid ChromatographyAuthors
Beck, K. R., B. J. Leibowitz, and M. R. Ladisch
J. Chromatogr. 190, 226-232 (1980)
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).
Dehydration of Ethanol: New Approach Gives Positive Energy BalanceAuthors
Ladisch, M. R. and K. Dyck
Science, 205(4409), 898-900 (1979)
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.
High-Speed Liquid Chromatography of Cellodextrins and Other Saccharide Mixtures Using Water as the EluentAuthors
Michael R. Ladisch, Aronson L. Huebner, and George T. Tsao
Journal of Chromatography, 147, 185-193 (1978)
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.
Theory and Practice of Rapid Liquid Chromatography at Moderate Pressures Using Water as EluentAuthors
Ladisch, M. R. and G. T. Tsao
J. Chromatogr. 166, 85-100 (1978)
The rapid separation of oligo- and monosaccharides on long, narrow columns packed with easily compressed, 4% cross-linked, cation-exchange resin adds a new dimension to the chromatography of carbohydrates on gel type supports: speedy analysis at low pressure. The discovery of a pressure-critical diameter effect in the packing of Aminex 50W-X4 (Ca2+) makes it possible to pack 60-cm long columns capable of separating malto- and cellodextrins in 12-15 min using water as the sole eluent. The surprising phenomena of a 50-fold increase in pressure due to an increase in column diameter from 6 to 8 mm is reported and reasons for this effect are explained. Equally noteworthy is the stability of the 6-mm columns. One column described in this report has been in continuous operation for over 2600 h. The application of low-pressure liquid chromatography to enzyme kinetics as well as to separation of oligo- and monosaccharides is also discussed.