Presentations
L. Zhang, E. Ximenes, M. R. Ladisch, 38th Symposium on Biotechnology for Fuels and Chemicals, Session 3: Enzyme Science and Technology I - Modelling and Structure/Function, April 25, 2016, Baltimore, MD, ,
Abstract: Our previous work demonstrated that severe pretreatment not only opens up the structure for enzymatic hydrolysis, but also increases lignin surface area exposed to cellulases. Non-productive binding of cellulases onto lignin decreases their activity. Therefore, higher enzyme loading is required to compensate for loss of enzyme due to adsorption on lignin. Previous reports have shown that BSA is effective in adsorbing onto lignin and blocking exposed lignin surface against adsorption of cellulose enzymes, thus increasing the effectiveness of enzymatic hydrolysis. Further studies on competitive adsorption of BSA and enzyme are now being carried out to better understand the lignin blocking effects. The traditional method of determining adsorption parameters for enzyme-lignin interactions through batch-adsorption studies is time consuming and labor intensive. Therefore, an inverse liquid chromatography method was developed instead, in order to determine the protein adsorption characteristics of lignin and lignocellulosic solids packed in a chromatography column. In this study, sugarcane bagasse was the stationary phase. Preliminary results observed by injecting 500 uL of BSA (20 mg/mL) showed that BSA is retained in the column with a rretention time of 17.6 min at both 20 and 50 C, although sharper peaks were observed at 50 C, consistent with the Arrhenius definition of the temperature dependence of an adsorption constant. These results confirmed the expected adsorption behavior of BSA, but more importantly, illustrated the utility of inverse liquid chromatography to better understand the adsorption of cellulases and other proteins to lignin. Inverse chromatography is being developed further as a rapid screening process for potential lignin blocking proteins.
Research Area: Bioprocessing
X. Li, X. Ku, T. Kreke, K. Foster, E. Ximenes, J. Hardenstein, X. Liu, M. Ladisch, 251st National ACS Meeting, Biofuel & Biobased Chemical Production: Biomass Pretreatment and Hydrolysis, San Diego, CA, March 14, 2016, ,
Abstract: The detection and characterization of bacterial food pathogens from homogenates of meats and vegetables will benefit from methods that accelerate their concentration and recovery. We report an approach based on hollow fiber membrane microfiltration that enables small volumes of viable bacteria to be rapidly concentrated from a large volume of food extract. The resulting sample is in a form amenable to probing for presence of pathogens using PCR or antibody reagents. However, fouling at the membrane surface must be addressed since the bacteria are present in a background of proteins, colloidal particles, and macromolecules which can accumulate within, or at the surface, of the membrane pores. Otherwise, this becomes a major impediment to achieving sustainable fluxes across membranes even though the porosity, equivalent to a 0.2 to 0.45 micrometer cutoff, is relatively large. This paper presents a bioprocess model of microfluidic transport that describes transmembrane pressure, flux, and deposition of a fouling layer as a function of distance from the entrance of a hollow fiber membrane during crossflow filtration of aqueous protein homogenates. Applications of the model in identifying optima for the membrane's geometric configuration will be discussed in the context of an approach that combines enzyme pretreatment of the initial sample followed by pre-filtration and crossflow microfiltration. The model identifies conditions that control membrane fouling so that efficient and reproducible concentration and recovery of bacterial cells in a viable form is achieved. Wider application of this model to microfiltration of other biological media will also be presented.
Research Area: Food Safety
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, ,
Abstract: 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.
Research Area: Bioenergy
X. Liu, J. Hardenstein, S. Ku, T. Kreke, K. Foster, K. Jeffries, E. Ximenes, M. Ladisch, 17th Annual USDA-CFSE Meeting, Purdue University, November 16-17, 2015, ,
Abstract: Application of hollow fiber technology to food pathogen concentration and recovery has significant potential for reducing the time required to detect contamination in food. The Laboratory of Renewable Resources Engineering (LORRE) has designed and developed the Continuous Cell Concentration Device (C3D), which utilizes cross-flow microfiltration to rapidly separate and concentrate pathogens from liquid samples. The automated device consists of a 0.2um hollow fiber membrane module and two peristaltic pumps to recirculate flow, achieving large sample volume reduction and concentration of pathogenic populations such as E. coli O157:H7, Salmonella and Listeria. With a large hollow fiber surface area available for filtration, flow rates are increased for solutions with high-protein content, such as beef or chicken homogenates. However, microfiltration of food solution poses challenges due to its complex matrix of fat, proteins, colloids, and other macromolecules. To enable microfiltration, food solutions are usually pre-treated with enzyme and pre-filtered prior to C3D processing. Food solutions are then concentrated in the C3D and probed for potential pathogenic populations. Previous experimental results show 68% recovery of E. coli in ground beef and 70-80% recovery of Salmonella in chicken homogenates. This research aims at adapting a novel technology for detection of E. coli in post-processed C3D samples. Crystal Diagnostics' Xpress (CDx) system utilizes a liquid crystal biosensor for detection of E. coli. Antibodies added during sample preparation form microbial aggregates, which when placed on a BioCassette, distort the aligned liquid crystal matrix, enabling detection. With this system, both higher (107-108 CFU/mL) and lower (105-106) concentrations were detected.
Research Area: Food Safety
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, ,
Abstract: 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.
Research Area: Bioseparations
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, ,
Abstract: 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.
Research Area: Bioprocessing
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, ,
Abstract: 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.
Research Area: Bioenergy
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, ,
Abstract: 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.
Research Area: Bioprocessing
M. Ladisch, E. Ximenes, Bioenergy Short Course, UNSEP, San Jose do Rio Preto, SP Brazil, ,
Abstract: The emergence of bioenergy as a major source of low carbon footprint transportation fuels with potential to provide electricity and power for stationary applications will require agriculture to provide sustainable feedstocks for this emerging industry. In addition, advances in enzymes and microbial biotechnology, scale-up through bioprocess engineering, and carbon efficient utilization of renewable resources will be major factors if agriculture is able to provide food, feed, fiber, and bioprocess feedstocks. This intensive short course addressed the critical topics that define bioenergy. The topics to be addressed are: 1. Basic biomass biochemistry; 2. Mechanisms of enzyme hydrolysis of pretreated lignocellulosic feedstocks; 3. Bioprocess design at large-scale lignocellulose conversion processes to produce fuel alcohol and bioproducts; and 4. Analysis of approaches that integrate sustainability and food production. The course will be taught in 4 modules that will address each of these topics. Renewable feedstocks to be considered include sugarcane bagasse, corn stover, and hardwood. Enzymes to be discussed will include cellulases and hemicellulases derived from T. reesei and A. niger. These enzymes are capable of hydrolyzing both insoluble cellulose and hemicellulose, as well as soluble oligosaccharides to monosaccharides. Their efficiency in generating five and six carbon sugars is an important factor for large-scale cellulose conversion to ethanol in a cost effective manner. In addition, characteristics of yeast fermentations, and the role of lignocellulose derived inhibitors will also be addressed. Mapping of the key biotechnologies into equipment that is needed to facilitate large-scale production will be outlined. This short will conclude with a discussion of sustainability and the impacts of major trends in world population, energy use, transportation fuels, and economic factors on achieving a world with a manageable carbon footprint and a sustainable food and energy supply.
Research Area: Bioenergy
M. Ladisch, E. Ximenes, Bioenergy Short Course, UNSEP, San Jose do Rio Preto SP, Brazil, ,
Abstract: The emergence of bioenergy as a major source of low carbon footprint transportation fuels with potential to provide electricity and power for stationary applications will require agriculture to provide sustainable feedstocks for this emerging industry. In addition, advances in enzyme and microbial biotechnology, scale-up through bioprocess engineering, and carbon efficient utilization of renewable resources will be major factors if agriculture is able to provide food, feed, fiber, and bioprocess feedstocks. This intensive short course will address the critical topics of that define bioenergy. The topics to be addressed are: 1. Basic biomass biochemistry; 2. Mechanisms of enzyme hydrolysis of pretreated lignocellulosic feedstocks; 3. Bioprocess design at large-scale lignocellulose conversion processes to produce fuel alcohol and bioproducts; and 4. Analysis of approaches that integrate sustainability and food production.
Research Area: Bioenergy
G. Cao, E. Ximenes, N. N. Nishols, S. E. Frazer, D. Kim, M. A. Cotta, M. Ladisch, presented at 37th Symposium on Biotechnology for Fuel and Chemicals, San Diego, CA, April, 2015, ,
Abstract: Bioabatement, using the fungus Coniochaeta ligniaria NRRL30616 can effectively eliminate enzyme inhibitors from pretreated biomass hydrolysates. However, our recent research suggested that bioabatement had no beneficial effect on removing xylo-oligomers which are strong inhibitors to cellulase. Here, we evaluated bioabatement with xylanase supplementation to mitigate potential enzyme inhibitors observed in corn stover liquors after pretreatment with liquid hot water at 10% (w/v) solids. Resuslts showed that cellulose conversion in the presence of 10% (w/v) LHW-pretreated liquor reached 70.5% and 57.4%, for conversion of Solka Flock cellulose and pretreated corn stover solids, respectively, after bioabatement and xylanase supplementation. These represent an increase of 21.6% and 17.6%, respectively, in comparison with non-treated samples. The squence in which xylanase and cellulase are added affects cellulose conversion, possibly as a result of competition between xylanase cellulase binding to xylo-oligomers. Replacement of xylanase using maleic acid treatment to hydrolyze xylo-oligomers yielded equivalent increases in efficiency of cellualse hydrolysis.
Research Area: Bioenergy
M. R. Ladisch, E. Ximenes, T. Kreke, K. Foster, S. Ku, L. Liu, A. Deering, 16th Beijing Conference and Exhibition on Instrumental Analysis, Session G: Analytical Techniques in Life Sciences, Beijing, China, October 29, 2015 , ,
Abstract: The detection of pathogenic microorganisms in foods is an important component of food safety. The requirements for pathogen detection include obtaining a representative sample of the food being tested, and then amplifying or concentrating the microorganisms present so that the viable cell count is high enough to enable detection of pathogens, if present. The time that elapses between sampling and detection is preferably less than 8 hours, so that the result may be achieved within one work shift. Methods that address these goals will be addressed and include cross-flow microfiltration and enrichment culture coupled to PCR. The recovery and concentration of microorganisms from various types of foods, and the detection of Salmonella for purposes of food safety inspection will be discussed.
Research Area: Food Safety
M. Ladisch, Purdue University Borlaug Summer Institute 2015, Purdue University, June 16, 2015, ,
Abstract: This lecture asks the question of how fossil and bio-energy supply and demand might change if the world were to experience a leveling off of birthrate, a decreasing demand for petroleum, and in increasing demand for food, feed, fiber, and bio-products accompanied by global climage change. This open-ended presentation will discuss possible roles of technology, societal changes, water availability, and economics in allocating resources for sustainable production of food and energy, and for achieving a higher standard of living on a global basis.
Research Area: Bioenergy
M. R. Ladisch, Y. Kim, J. K. Ko, T. Kreke, E. Ximenes, 2015 AIChE Meeting, Paper 775b, Salt Lake City, Utah, November 13, 2015, ,
Abstract: A fundamental understanding of the combined factors that impact recalcitrance in enzyme hydrolysis of pretreated hardwood explains how cellulase loading may be decreased by a factor of 10 while maintaining 80% glucose yield when non-catalytic protein is added to the enzyme. Factors that impact enzyme hydrolysis of solid biomass include the interaction of the cellulase and beta-glucosidase components with solubilized phenolic inhibitors and the enhanced accessibility of lignin as a consequence of pretreatment. While the added protein decreases overall specific activity of the enzyme, it also reduces cellulase adsorption on lignin, thus making more enzyme available for cellulose hydrolysis. Consequently, 15 and 1.3 FPU cellulase/g total solids both give 80% yield, with the 1.3 FPU loading approaching the enzyme levels usually associated with amylases in starch hydrolysis. These results reinvigorate motivation for the search for other approaches that prevent enzyme adsorption on lignin and enable high glucose yields at low enzyme loadings. This paper presents measurements in our laboratory and prior reports from the literature to offer an explanation of how changes in the physical attributes of cellulosic biomass during liquid hot water pretreatment affect glucose yields and enzyme loading.
Research Area: Bioenergy
M. Michelin, E. Ximenes, M. L. T. m. Polizeli, M. R. Ladisch, presentd at 37th Symposium on Biotechnology for Fuel and Chemicals, San Diego, CA, April, 2015, ,
Abstract: Lignocellulosic residues, such as sugarcane bagasse (SCB), are a complex matrix composed by cellulose, hemicellulose and lignin that can be used for different biotechnological applications. These materials need to be pretreated to be accessible for enzymatic hydrolysis. Liquid hot water (LHW) pretreatment is an effective and cost-saving approach, since no catalyst is required, and an expensive reactor is avoided due to the low corrosive nature of this pretreatment. However, during the pretreatment phenolics derived from lignin are released, which are inhibitory of enzymes. Here, we evaluated the effect of phenolic compounds formed during the pretreatment of the SCB on cellulolytic activity. Two conditions for LHW pretreatment were used: 180 and 200 C for 30 min and two fractions were obtained: solid and liquid fractions enriched by cellulose/lignin and hemicellulose, respectively. The phenolics contained in the liquid and solid fractions were used for the experiments of enzymatic inhibition (cellulase and beta-glucosidase activities). The higher amount of phenolics (2.4 g/L) was observed in the liquid fraction of SCB pretreated at 200 C/30 min. This condition also resulted in the highest inhibition of the enzymatic activity. Phenolics extracted from solid fraction (0.86 g/L) were shown to be more inhibitory than liquid for the beta-glucosidase activity. This work shows the importance of the optimization of the pretreatment process in relation to maximize the production of sugars and minimize the formation of inhibitory compounds to achieve the maximal efficiency of an enzyme hydrolysis-based process.
Research Area: Bioenergy
D. Kim, N. Hengge, D. Orrego, E. Ximenes, M. R. Ladisch, 2015 AIChE Annual Meeting, Paper 257B, Salt Lake City, Utah, November 10, 2015, ,
Abstract: There are two sources of cellulosic feedstocks for corn to ethanol, dry grind facilities: corn pericarp (fiber) and corn stover. Both materials quality for D3 RINS (renewable identification numbers for cellulosic ethanol), and could be an attractive resource for re-purposing corn to ethanol facilities for producing cellulosic ethanol. This approach would employ mixtures of endo- and exo-cellulases, pectinases, xylanases, protease, beta-glucosidase, as well as other possible auxiliary enzymes to liquefy and/or hydrolyze the cellulosic substrates. Tests, both in our laboratory and other research centers have demonstrated successful conversion of pretreated corn stover and other lignocellulosic materials. Processes for conversion of pretreated cellulose-containing distillers' solids have been demonstrated and are being marketed. Reports on this technology, available in the literature, show that a $10 to $12 million capital investment enables a 6% increase in ethanol production in existing corn to ethanol plants. This process requires pretreatment prior to hydrolysis and fermentation of the cellulosic portion of the corn kernel. Reports by Scott, Wyman, Schell, Elander, Kumar, Saville, Lawson and others have discussed the impact of mixing and reactor configurations on increasing both rate and yield of enzyme hydrolysis of cellulose, and/or liquefaction of pretreated lignocellulosic biomass (corn stover and hardwood). One significant factor that impacts economic viability of cellulose ethanol is being able to process biomass at high solids concentration in order to reduce energy for heating and other processing steps and increase the concentration of the ethanol produced. We recently reported the liquefaction of steam-exploded (pretreated) sugar cane bagasse at concentrations of up to 300 g/L. We present here the impact of reactor operation on the liquefaction of untreated corn stover and pericarp (corn fiber), and compares this to our recently reported liquefaction of sugar cane bagasse. The objective is to prepare these materials in pumpable slurries, thereby simplifying subsequent hydrolysis procedures, and reducing the cost of equipment that would otherwise require introduction of solid materials into a pressure vessel (i.e., a pulping digester).
Research Area: Bioenergy
Jaycey Hardenstein, Alisha Tungare, Xingya Liu, Eduardo Ximenes, Michael Ladisch, presented at Posters on the Hill, Washington, DC, April, 2015, ,
Abstract: With a growing number of consumers in the American market and with food production at an all-time high, food safety is a huge priority for both consumers and corporations everywhere. Recently, the Laboratory of Renewable Resources Engineering (LORRE) at Purdue University, developed a Continuous Cell Concentration Device (C3D) that has the potential to reduce the amount of time required to detect foodborne pathogens. The C3D utilizes microfiltration to produce a smaller, concentrated sample, which facilitates the identification of microbial populations. Before cell concentration, food samples are subjected to a pretreatment process that utilizes enzymes to prevent the build-up of proteins and large molecules that can plug the hollow fibers used in the C3D. Pretreated samples are then run through the C3D to recover a solution with a higher concentration of microbial cells. Our research investigates the role of enzymes to enable microfiltration and ensure recovery of Escherichia coli (E. coli) in ground beef solutions. We are working to quantify the effect of enzyme pretreatment E. coli cell viability. Experiments are currently being conducted to determine the effect of enzyme treatment, if any, on microbial cell growth and to optimize the amount of enzyme used. Preliminary results show that enzyme pretreatment effectively breaks down large proteins and prevents fouling of the membrane, as enzyme-treated solutions filter four times faster than untreated food solutions and recover more than 90% of E. coli during the pretreatment process. Thus, enzyme pretreatment, coupled with C3D technology, begins to address the critical need for rapid pathogen detection.
Research Area: Food Safety
M. Ladisch, E. Ximenes, K. Foster, A. Deering, T. Kreke, X. Liu, Seockmo Ku, 5th Annual FDA Foods and Veterinary Medicine Science and Research Conference, Silver Spring, MD, August 13, 2015, ,
Abstract: The Centers for Disease Control and Prevention (CDC) report that viruses are major causative agents for foodborne illnesses, although the most severe cases are associated with bacteria including Salmonella species. Salmonella (non-typhoidal) and Toxoplasma gondii are the first and second most costly foodborne pathogens in the United States. Our concept addresses concentration, recovery and detection of pathogens, specifically Salmonella, starting with stomaching of food sample followed by pre-filtering through glass microfiber (2.7 um) or nylon (10 um) membranes to remove larger particulates. Enzyme is added to neutralize agents in the extract that foul microfiltration membranes. Next, cross-flow microfiltration with a commercial polyethersulfone hollow fiber membrane module (0.2 um cut-off) removes liquid and retains microorganisms in viable and concentrated form. The microfiltration module is an integrated component of an automated instrument, developed in our laboratory, for accelerating sample preparation to detect Salmonella in unprepared foods. The type of food determines which enzymes are selected to remove fouling agents. Cellulases, hemicellulases and pectinases are used for vegetables and fruits, while proteases are key enzymes for meat and egg samples. Since maintaining viability of the microorganisms is critical, we have selected and tested enzymes effective at conditions that maintain viability. Microfiltration of enzyme-treated extract is based on an automated Continuous Cell Concentration Device (C3D). This system is the result of laboratory research with a series of prototypes that were successively designed, constructed, tested and improved to validate materials of construction, operability, cleaning (sterility) cycles, automation, control of membrane fouling, and recovery of viable microorganisms by quickly processing a large sample volume into a small one. The C3D carries out automated, cross flow microfiltration of up to 500 mL of food extracts into a 0.5 to 2 mL sample containing viable microbial cells in a concentrated form. A short enrichment step (using selective medium such as Rappaport Vassiliadis (RV) broth for Salmonella) further increases cell numbers by 10%, and is particularly useful for a low initial number of pathogens (less than or equal to 1 CFU/g) and/or reduction of non-target naturally-occurring microorganisms. Recoveries of target microorganisms range from 50 to 100% in 0.5 to 2 mL sample volumes obtained from 50 to 500 mL extract. The entire process, including a short enrichment step of 1 hr and PCR analysis is completed in 8 hours. Sample handling, preparation, and instrument sterilization corresponds to an elapsed time of 3 hr. Subsequent concentration through C3D requires between 15 min and 1.5 hr, depending on the type and size of sample volume being processed. Detection through PCR adds 2 hrs. If a pathogen is detected, confirmation occurs by the next day by plating concentrate on selective medium. The system and the hollow fiber membranes are cleaned and sterilized for re-use through sequential application of sodium hydroxide, water, ethanol, and water. This procedure enables the microfiltration membranes to be re-used 15 times or more. Target pathogens are Salmonella sp and Listeria monocytogenes.
Research Area: Food Safety
B. B. Hewetson, A. Kreger, N. S. Mosier AIChE Meeting, Atlanta, GA, November 20, 2014, ,
Abstract: 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.
Research Area: Bioenergy
X. Zhang, B. Hewetson, N. S. Mosier AIChE Meeting, Atlanta, GA, November 20, 2014, ,
Abstract: 5-hydroxymethylfurfural (HMF) and levulinic acid are platform chemicals for producing a variety of fuels and polymers. However, undesirable humic substances can be generated in substantial amounts, lowering the yields of desired products. We report the use of hydrochloric acid and maleic acid separately and mixed with a Lewis acid (AlCl3) to catalyze the process of glucose isomerization, dehydration, and hydrolysis. Analysis of results between 130 and 180 C were used to develop a kinetic model for the glucose conversion to HMF and levulinic acid by these selected catalysts. Preliminary results show that after 6 minutes at 180 C, maleic acid combined with AlCl3 generated only 50% of total humins compared to hydrochloric acid combined with AlCl3. We report an analysis of this shift in selectivity of the reaction toward levulinate and describe possible mechanisms for interactions between maleic/maleate and the reactants and intermediates.
Research Area: Bioenergy
M. Ladisch, E. Ximenes, Y. Kim, J. K. Ko, BIO Pacific Rim Summit, San Diego, CA, December 8, 2014, ,
Abstract: This panel focuses on recent advances in leading pretreatment technologies that can be coupled with enzymatic hydrolysis to convert lignocellulosic biomass to sugars for fermentation to ethanol or other products. The low cost of lignocellulosic biomass coupled with widespread domestic abundance, ability to dramatically reduce greenhouse gas emissions, and potential to spawn new rural manufacturing jobs make it an attractive resource from which to produce fuels and chemicals. However, converting this low cost resource into commodity products is expensive, with recalcitrance to sugar release being the key obstacle to achieving low prices by biological conversion routes. Most forms of lignocellulosic biomass must be pretreated prior to biological conversion operations to realize the high yields vital to economic competitiveness, and effective pretreatments can also lower loadings of expensive enzymes to economic levels, reduce costs of downstream operations,and produce valuable co-products that can improve overall process economics and provide additional benefits. Various studies have shown that thermochemical pretreatments that employ chemicals in combination with heat are most effective in realizing high sugar yields from the coupled operations of pretreatment and enzymatic hydrolysis. This Panel will include a presentation of recent work at Purdue University on reducing the amount of enzyme required for hydrolysis and the fundamentals of pretreatment related to changes in cell wall structure and chemistry. Increased severity of pretreatment exposes both additional lignin and cellulose. However, lignin adsorbs cellulase, so more enzyme must be added if the additional exposed cellulose is to be effectively hydrolyzed. Conversely, cellulase loading may be decreased by a factor of 10 while maintaining 80% glucose yield by diluting the enzyme with non-catalytic protein (BSA) that binds to lignin and decreases cellulase adsorption on lignin. More enzyme is therefore available for cellulose hydrolysis resulting in enhanced hydrolysis. Michigan State University is advancing Ammonia Fiber Expansion (AFEX) pretreatment, now being commercialized, to produce cellulosic biomass that can be used either for animal feed or as biofuel feedstock, thereby largely eliminating the "food versus fuel" issue. The AFEX presentation will briefly describe AFEX science and technology and how it can be performed in distributed processing facilities called depots. These depots greatly improve the logistics of cellulosic biofuel systems and allow local communities to capture part of the added value of AFEX processing. A presentation by the University of California at Riverside will describe a novel Co-solvent Enhanced Lignocellulosic Fractionation CELF) pretreatment that removes nearly all the lignin from biomass, recovers most of the hemicellulose sugars, and produces glucan-enriched solids that can be almost completely enzymatically digested to glucose with about one tenth the enzyme loadings typically required. Furthermore, CELF has been found to be effective with a wide range of hardwoods, grasses, and agricultural residues. Following the fate of major biomass components, kinetic modeling and SEM imaging suggest that the high lignin removal afforded by CELF could play a key role in achieving such high sugar yields with extremely low enzyme loadings and lead to alternate strategies to improve pretreatment.
Research Area: Bioenergy
E. Ximenes, T. Kreke, R. Hendrickson, J. K. Ko, and M. Ladisch, Corn Utilization Technology Conference, Louisville, KY, June 2-4, 2014, ,
Abstract: Our work addresses the goal of developing new uses for corn and relating it to Indiana's development of a new concept in enzyme-based processing that deconstructs the kernel into its base components of starch, pericarp, germ (and oil), and sugars, and then transforms the sugars into value-added molecules using catalytic processing. High-value chemicals that can be produced from corn-derived sugars include furans, levulinic acid, biohydrocarbons, succinic acid and sugar alcohols. These add significant value through product diversification (Bozell et al., 2000; Hayes et al., 2005, Bozell and Peterson, 2010). Our experience with cellulolytic, hemicellulolytic, proteolytic, lipolytic, and amylolytic enzymes, as well as with the catalytic conversion of oligosaccharides to glucose and furans, motivated research on investigating the energy efficient recovery of starch slurries directly from corn kernels using selected enzymes in a low temperature process. After screening different enzymes and formulation of mixtures of activities, our results found the combined use of cellulase and protease could yield up to 80% of the starch in the kernel recoverable in a concentrated, particulate form, with intact pericarp being recovered, as well. This approach has the added benefit of yielding another identifiable component from the kernel which makes up 5% of the kernel, is rich in cellulose and hemicellulose, and may be hydrolyzed to sugars that are fermentable to ethanol or used to make other value-added products. Ethanol from pericarp has the potential of providing up to 100% more ethanol per bushel of corn with a lower carbon footprint (i.e., cellulosic ethanol) than ethanol from corn. Our work seeks to incorporate the principles of enzyme assisted deconstruction of corn kernels using cellulases, proteases, and other enzymes in order to obtain optimal fractionation of the corn kernel into components that may be separated into distinct components by physical means of centrifugation, filtration, or settling. The rationale for this approach is based on utilizing existing equipment in a dry grind facility, thereby minimizing additional capital investment, while at the same time introducing new co-product streams to the industry that will add value to corn and revenues to corn growers.
Research Area:
Hardenstein, Jaycey, Tungare, Alisha, Ladisch, Michael, Liu, Xingya, and Ximenes, Eduardo, ,
Abstract: With a growing number of consumers in the American market and with food production at an all-time high, food safety is a huge priority for both consumers and corporations everywhere. Recently, the Laboratory of Renewable Resources Engineering (LORRE), at Purdue University, developed a Continuous Cell Concentration Device (C3D) that has the potential to reduce the time required to detect food pathogens. In LORRE's research, food samples are subjected to enzyme pretreatment and pre-filtration to prevent protein aggregation and the subsequent plugging of hollow fiber membranes used in the C3D microfiltration process. The pretreated food samples can then be run through the C3D to recover a concentrated cell solution. Our research investigates the role of pre-filter materials and enzymes to enable microfiltration and ensure the recovery of non-pathogenic filter materials and enzymes to enable microfiltration and ensure the recovery of non-pathogenic Escherichia coli bacterial cells. The ideal pre-filter material would allow for a large cell recovery while also removing enough particles so that the sample will not plug the C3D. It was determined that the most effective pre-filter material for turkey extract samples was the Advantec 101 filter paper. Through quantifying the reduction of E. coli colonies, the Advantec 101 filter paper recovered 80-90% of cells. On the other hand, the GF/D filter currently used in the pre-filtration process resulted in only 40-60% bacterial cell recovery. In addition, experiments are currently being conducted to discover how enzyme treatment affects the characteristics of ground beef extract solutions, such as: pre-filtration speed, cell concentration time, and E. coli cell recovery. Ultimately, this research begins to address the critical need for rapid pathogen detection.
Research Area: Food Safety
I. Beheshti Tabar, P. T. Murphy, N. S. Mosier AIChE Meeting, Atlanta, GA, November 20, 2014, ,
Abstract: 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.
Research Area: Bioenergy
I. Emery, J. Dunn, J. Han, M. Wang, ASABE 2013, Kansas City, MO, July 24, 2013, ,
Abstract: Environmental assessments of biofuel production, including greenhouse gas inventories, rarely account for the full impacts of the feedstock supply chain. Dry matter losses, direct emissions of non-CO2 greenhouse gasses (CH4 and N2O) and material use in some feedstock production pathways can alter the emissions profile of cellulosic biofuels. Statistical distributions were fit to the frequency of reported losses during five biomass storage methods, including dry bales stored indoors, outdoors (covered), or outdoors (uncovered), bale silage, and bunker silage. Biomass losses during on-farm operations, handling, and transportation for each biomass format were integrated with expected storage losses into the Greenhouse Gases, Regulated Emissions, and Energy Use in Transportation (GREET) model, developed by Argonne National Laboratory. Direct emissions during anaerobic storage and plastic use for storage covers were also included for each relevant biomass pathway. Net greenhouse gas emissions from ethanol produced from switchgrass, Miscanthus, and corn stover increased by up to 9.8, 9.0, and 8.3 g CO2e/MJ, respectively, due to inclusion of dry matter loss in the biomass supply chain. Fossil energy use increased by up to 0.14, 0.13, and 0.14 MJ/MJ, respectively. Round bale silage had the greatest impact on greenhouse gasses and energy use. Bunker silage requires lower fossil energy use but results in similar levels of GHGs. Indoor storage minimizes all emissions and fossil energy use. Integration of supply chain pathways into GREET provides a novel and more accurate model of biofuel net emissions and allows side-by-side comparisons of multiple biomass supply chains for regulatory and environmental assessments.
Research Area: Bioenergy
N. S. Mosier, CUTC Conference, Indiana Corn Growers, Indianapolis, IN, June 4-6, 2012
Abstract: Biochemical and some thermochemical routes to producing advanced biofuels require the fractionation of lignocellulosic biomass into a sugar-rich stream. This requires the depolymerization of plant cell wall polysaccharides and is generally regarded as the major hurdle for cost-effective advanced biofuel production. To achieve the saccharification of cellulosics, a combination of pretreatment to enhance the reactivity of cellulose and catalysts (enzymes, etc.) are required. This task will describe advances in understanding how components of cellulosic biomass inhibit and deactivate cellulase enzymes. In addition, results will be presented from a novel approach using enzyme-mimicking acid catalysts to release and even convert cellulosic sugars to advanced biofuels and value-added compounds. These reseult suggest that technology approaches to control the ionic strength, ion character, and pH of aqueous solutions can control the selectivity of saccharification to favor sugar formation over undesired degradation products. A similar approach has application in starch conversion toward value-added chemicals.
Research Area: Biofuels/Bioproducts
M. Ladisch, CUTC Conference, Indiana Corn Growers, Indianapolis, IN, June 4, 2012
Abstract:
Research Area: Biofuels/Bioproducts
Y. Kim, R. Hendrickson, J. K. Ko, E. Ximenes, and M. R. Ladisch, CUTC Conference, Indiana Corn Growers, Indianapolis, IN, June 4-6, 2012
Abstract: Corn biorefineries with diversified product portfolios offer great potential for corn growers and sugar producers by providing new, high margin market opportunities to capture added value and a higher return on investment. Conventional dry grind utilizes starch to produce ethanol, while leaving all other components (germ, pericarp) unutilized and mixed together as in distillers' grains. Wet mill processes involve steeping at elevated temperatures. In this study, we present a new approach for disassembly rather than destruction of corn kernels into its components (starch, pericarp, and germ) by enzyme catalysis at at temperatures of 50 to 60 C. The enzymes are formulated to separate pericarp from endosperm while leaving germ floating on the reaction solution at the end of the process. The process involves no mechanical grinding and no chemical steeping of corn kernels prior to the enzymatic deconstruction and can be easily adapted to a conventional dry grind process. Fractionation of pericarp and germ, followed by washing will generate a starch stream which is subsequently hydrolyzed to glucose by amylases. The enzymes are specifically formulated for this task by screening numerous commercially available enzymes that will disassemble corn kernels. To facilitate the enzyme penetration, the tip caps of kernels are removed. This process provides an alternative approach to fractionate corn kernels into components that are suitable for production of chemical building blocks for polymers, chemicals, and liquid fuels.
Research Area: Biofuels/Bioproducts
I. Emery, N. Mosier, LCA XII, Tacoma, WA, September 25-27, 2012, ,
Abstract:
Research Area: Bioenergy
I. Emery, N. Mosier, 34th Symposium on Biotechnology for Fuels and Chemicals, New Orleans, LA, April 30-May 3, 2012, ,
Abstract: Accurate estimates of greenhouse gasa emissions from biofuel production are necessary to ensure the economic and environmental sustainability of the biofuels industry and to meet government mandates for low-carbon fuel production. Biomass storage represents a critical gap in many biofuel life cycle assessment (LCA) methodologies, and may have a large impact on production and transportation logistics for biofuel feedstocks, in addition to greenhouse gas emissions. In this study, 143 laboratory-scale bales made from switchgrass and Miscanthus grown at Purdue University were stored in insulated boxes. Initial moisture content and bulk density were varied among the bales (11.8 - 34.2% w.b., and 103 - 308 kg/m3, respectively) and dry matter loss was tracked for each treatment combination over three months of storage. 22 additional laboratory-scale switchgrass bales at 9.9%, 14.1% and 18.6% moisture (w.b.) were stored at 4 C, 23 C, and 40 C under controlled aeration to monitor the direct emissions of the greenhouse gases CO2, CH4, and N2O during storage. Relationships between biomass moisture and rates of dry matter loss, and between moisture, temperature, and direct greenhouse gas emissions, were used to model the potential impacts of biomass storage on biomass supply logistics and net global warming potential of ethanol from switchgrass and Miscanthus at the biorefinery scale.
Research Area: Bioenergy
E. Ximenes, Y. Kim, N. Mosier, and M. Ladisch, American Chemical Society Meeting, San Diego, CA, March 25, 2012
Abstract: Pretreatment is an important cost-driver of lignocellulose conversion to ethanol and a critical step prior to enzyme hydrolysis. It disrupts the plant cell wall network and partially separates the major polymer components (lignin, cellulose and hemicellulose). However, pretreatment of lignocellulosic materials may also result in the release of inhibitors and deactivators of the enzymatic hydrolysis of cellulose. Development of enzyme processes for hydrolysis of cellulose to glucose must reduce inhibition and deactivation effects in order to enhance hydrolysis and reduce enzyme usage. Here we report the identification of phenols with major inhibition and/or deactivation effect on enzymes used for conversion of cellulose to ethanol. The strength of the inhibition or deactivation effect depended on the type of enzyme, the microorganism from which the enzyme was derived, and the type of phenolic compounds present. The effects of inhibitors on enzyme hydrolysis of pretreated lignocellulosic materials are presented
Research Area: Biofuels/Bioproducts
Y. Kim, T. Kreke and M. R. Ladisch, 34th Society for Industrial Microbiology and Biotechnology Symposium, New Orleans, LA, May 1, 2012
Abstract: Hydrothermal pretreatment of lignocellulosic materials generates a liquid stream rich in pentose sugar oligomers. Cost-effective hydrolysis and utilization of these soluble sugar oligomers is an integral process of biofuel production. We report integrated rate equations for hydrolysis of xylo-oligomers derived from pretreated hardwood by dicarboxylic maleic and oxalic acids. The highest xylose yield observed with dicarboxylic acids was 96%, and compared to sulfuric acid, was 5–15% higher with less xylose degradation. Dicarboxylic acids showed an inverse correlation between xylose degradation rates and acid loadings unlike sulfuric acid for which less acid results in less xylose degradation to aldehydes and humic substances. A combination of high acid and low-temperature leads to xylose yield improvement. Hydrolysis time course data at three different acid concentrations and three temperatures between 140 and 180°C were used to develop a reaction model for the hydrolysis of xylo-oligosaccharides to xylose by dicarboxylic acids.
Research Area: Biofuels/Bioproducts
E. Casey, N. S. Mosier, J. Adamec, A. Jannasch, N. Ho and M. Sedlak, 34th Society for Industrial Microbiology and Biotechnology Symposium, New Orleans, LA, May 1, 2012
Abstract: The commercialization of cellulosic ethanol has faced a number of different technical hurdles. One major challenge is the negative impact of inhibitors on the fermentative performance of industrial microorganisms. One such inhibitory compound is acetic acid, liberated from hemicellulose during the pretreatment of the biomass. To study the effect of acetic acid on glucose/xylose co-fermentation by S. cerevisiae 424A(LNH-ST), a genetically engineered yeast strain that can effectively co-ferment both glucose and xylose to ethanol, we first determined the impact of the acetic acid on various yeast performance characteristics. Results showed acetic acid to be inhibitory to cell growth, substrate consumption (especially xylose), and ethanol productivity, and stimulatory to the metabolic ethanol yield. To further explore and understand these effects of acetic acid, we took a systems biology approach by analyzing intracellular metabolite levels and gene expression levels. Reverse-phase liquid chromatography-mass spectrometry and in vitro 13C labeling was used for the identification and quantification of key intracellular glycolytic and pentose phosphate pathway metabolites. Initial results show significant differences in the concentration of the selected intracellular metabolites between fermentations with and without acetic acid. Microarray technology was used to determine the expression levels of the full yeast genome (with the exception of the genes inserted to allow for xylose fermentation). Preliminary analysis shows minimal differences in the expression of central carbon metabolism genes during glucose fermentation; however, significant differences were seen during xylose fermentation. Relationships between metabolomic, transcriptomic, and fermentation performance will be presented. The commercialization of cellulosic ethanol has faced a number of different technical hurdles. One major challenge is the negative impact of inhibitors on the fermentative performance of industrial microorganisms. One such inhibitory compound is acetic acid, liberated from hemicellulose during the pretreatment of the biomass. To study the effect of acetic acid on glucose/xylose co-fermentation by S. cerevisiae 424A(LNH-ST), a genetically engineered yeast strain that can effectively co-ferment both glucose and xylose to ethanol, we first determined the impact of the acetic acid on various yeast performance characteristics. Results showed acetic acid to be inhibitory to cell growth, substrate consumption (especially xylose), and ethanol productivity, and stimulatory to the metabolic ethanol yield. To further explore and understand these effects of acetic acid, we took a systems biology approach by analyzing intracellular metabolite levels and gene expression levels. Reverse-phase liquid chromatography-mass spectrometry and in vitro 13C labeling was used for the identification and quantification of key intracellular glycolytic and pentose phosphate pathway metabolites. Initial results show significant differences in the concentration of the selected intracellular metabolites between fermentations with and without acetic acid. Microarray technology was used to determine the expression levels of the full yeast genome (with the exception of the genes inserted to allow for xylose fermentation). Preliminary analysis shows minimal differences in the expression of central carbon metabolism genes during glucose fermentation; however, significant differences were seen during xylose fermentation. Relationships between metabolomic, transcriptomic, and fermentation performance will be presented. The commercialization of cellulosic ethanol has faced a number of different technical hurdles. One major challenge is the negative impact of inhibitors on the fermentative performance of industrial microorganisms. One such inhibitory compound is acetic acid, liberated from hemicellulose during the pretreatment of the biomass. To study the effect of acetic acid on glucose/xylose co-fermentation by S. cerevisiae 424A(LNH-ST), a genetically engineered yeast strain that can effectively co-ferment both glucose and xylose to ethanol, we first determined the impact of the acetic acid on various yeast performance characteristics. Results showed acetic acid to be inhibitory to cell growth, substrate consumption (especially xylose), and ethanol productivity, and stimulatory to the metabolic ethanol yield. To further explore and understand these effects of acetic acid, we took a systems biology approach by analyzing intracellular metabolite levels and gene expression levels. Reverse-phase liquid chromatography-mass spectrometry and in vitro 13C labeling was used for the identification and quantification of key intracellular glycolytic and pentose phosphate pathway metabolites. Initial results show significant differences in the concentration of the selected intracellular metabolites between fermentations with and without acetic acid. Microarray technology was used to determine the expression levels of the full yeast genome (with the exception of the genes inserted to allow for xylose fermentation). Preliminary analysis shows minimal differences in the expression of central carbon metabolism genes during glucose fermentation; however, significant differences were seen during xylose fermentation. Relationships between metabolomic, transcriptomic, and fermentation performance will be presented. The commercialization of cellulosic ethanol has faced a number of different technical hurdles. One major challenge is the negative impact of inhibitors on the fermentative performance of industrial microorganisms. One such inhibitory compound is acetic acid, liberated from hemicellulose during the pretreatment of the biomass. To study the effect of acetic acid on glucose/xylose co-fermentation by S. cerevisiae 424(LNH-ST), a genetically engineered yeast strain that can effectively co-ferment both glucose and xylose to ethanol, we first determined the impact of the acetic acid on various yeast performance characteristics. Results showed acetic acid to be inhibitory to cell growth, substrate consumption (especially xylose), and ethanol productivity, and stimulatory to the metabolic ethanol yield. To further explore and understand these effects of acetic acid, we took a systems biology approach by analyzing intracellular metabolite levels and gene expression levels. Reverse-phase liquid chromatography-mass spectrometry and in vitro 13C labeling was used for the identification and quantification of key intracellular glycolytic and pentose phosphate pathway metabolites. Initial results show significant differences in the concentration of the selected intracellular metabolites between fermentations with and without acetic acid. Microarray technology was used to determine the expression levels of the full yeast genome (with the exception of the genes inserted to allow for xylose fermentation). Preliminary analysis shows minimal differences in the expression of central carbon metabolism genes during glucose fermentation; however, significant differences were seen during xylose fermentation. Relationships between metabolomic, transcriptomic, and fermentation performance will be presented.
Research Area:
M. Zeng, E. Ximenes, M. Ladisch, N. Mosier, W. Vermerris, C.-P. Huang and D. Sherman, 34th Society for Industrial Microbiology and Biotechnology Symposium, New Orleans, LA, April 30, 2012
Abstract: Lignin content, composition, distribution as well as cell wall thickness, structures, and type of tissue all have measurable effects on enzymatic hydrolysis of cellulose in lignocellulosic feedstocks. Our work combined compositional analysis, pretreatment, enzyme hydrolysis and SEM imaging for fractionated pith, rind, and leaf tissues from a hybrid stay-green corn, in order to identify the role of structural characteristics on enzyme hydrolysis of cell walls. Hydrolysis followed the sequence rind < leaves < pith, with 75% conversion to glucose achieved with 9 mg enzyme protein/g glucan or 3.6 mg protein/total solids and 90% with l08 mg protein/g glucan or 43.2 mg protein/total solids in 24 hours. Physical fractionation of corn stalks or other C4 grasses into soft and hard tissue types could reduce cost of cellulose conversion by enabling reduced enzyme loadings to hydrolyze soft tissue, and directing the hard tissue to other uses. The amount of lignin alone remaining after pretreatment of the different fractions is about the same, so differences in lignin content do not explain the differences in enzymatic hydrolysis. SEM images show sugar yields correlate with changes in plant cell wall structure both before and after liquid hot water pretreatment.
Research Area: Biofuels/Bioproducts
M. Ladisch and E. Ximenes, BECA, Medellin, Colombia, August 5, 2011
Abstract: Sugarcane and corn account for most of the world's current fuel ethanol production of 25 billion liters. Long-term growth of fuel ethanol and other biofuels will require utilization of the cellulosic feedstocks: wood, sugarcane bagasse, agricultural residues, and purposely grown energy crops such as switchgrass, energy cane and wood. These sources have the potential to substantially increase the amount of liquid biofuels, and especially cellulose ethanol, from fermentation processes, as well as to catalyze growth of new industries in Colombia and in the Americas. The rate and extent of adoption of cellulose-derived, lqiuid biofuels will depend on technology, feedstock availability, production costs, government policies and oil prices. Examples from emerging companies in the biofuels sector will illustrate how biotechnology is enabling the industry to evolve and produce both fuels and chemicals from renewable resources. This talk presents an overview of processes that are changing the world of biofuels, and moving the biofuels industry beyond corn and sugarcane.
Research Area: Bioenergy
E. Casey, N. S. Mosier, Z. Stockdale, N. Ho, J. Adamec and M. Sedlak , 33rd Symposium on Biotechnology for Fuels and Chemicals, Seattle, WA, May 2-5, 2011
Abstract: The commercialization of cellulosic ethanol has faced a number of different technical hurdles. One major challenge is the negative impact of inhibitors on the fermentative performance of industrial microorganisms. Most inhibition studies have focused on furan derivatives and weak acids; however, potential fermentation inhibitors also include cations and anions. Cations and anions are present in cellulosic biomass and are also used for pH adjustment prior to and during fermentation. To characterize the inhibitory effect of cations (potassium, sodium, ammonium) and anions (chloride and sulfate), a series of lab-scale fermentations were completed using S. cerevisiae 424A(LNH-ST), a recombinant yeast strain that can effectively co-ferment glucose and xylose. The concentration of the cations and anions tested ranged from 0.1M to 0.5M. Preliminary analysis of these fermentations showed xylose fermentation to be more sensitive to the presence of cations and anions than glucose fermentation. Results also found sodium to be the most inhibitory cation. To further explore the effect of sodium, a comprehensive analysis of intracellular metabolites involved in glycolysis and the pentose phosphate pathway was conducted. The Global Isotope-labeled Internal Standard (GILISA) MS quantization method was used for the identification and quantification of intracellular metabolites at key metabolic stages during fermentation.
Research Area: Biofuels/Bioproducts
N. S. Mosier, E. Kim, S. Liu, M. Abu-Omar , 2011 Annual Meeting of the American Institute of Chemical Engineers, Minneapolis, MN, October 18, 2011, ,
Abstract: Direct catalytic conversion of lignocellulosic biomass to biofuels could improve the carbon efficiency of biofuel production. We report the use of maleic acid, a dicarboxylic acid, to catalyze the fractionation of biomass into an aqueous solution of pentose (primarily xylose) and insoluble cellulose and lignin, followed by the conversion of the xylose to furfural under higher temperature and pressure. This method achieved 80-90% yield of xylose through hydrolysis of the hemicellulose from various biomass sources (switchgrass, poplar, pine) and achieved 54-61% yield of furfural (based on original biomass). We present a kinetic analysis of biomass hydrolysis and furfural formation and discuss application of results from pure sugars to results from biomass conversion.
Research Area: Bioenergy
M. Ladisch, E. Ximenes, H. Vibbert, L. Liu, A. Bhunia, R. Bashir, J. Shin, and R. Linton, AIMBE 20th Annual Event, Washington, DC, February 21, 2011
Abstract: The rapid sample processing of extracts from food matrices is an essential component for rapid detection of food pathogens and food safety. The objective of our research is to develop and integrate operational technologies that rapidly and effectively concentrate viable target cell from food matrices and to couple concentration with interrogation for the presence of pathogens. Rapid detection requires rapid sample concentration and amplification of the target population, interrogation of a concentrated sample of cells containing both non-pathogenic and pathogenic organisms, and identification of the type of pathogenic organism should a target population such as Salmonella sp., Listeria sp., or E. coli be detected. This work shows the development of an automated instrument to concentrate and recover cells from both natural flora and artificially spiked organisms from foods is possible using membrane separations. This work describes the properties of food extracts containing microbial cells with respect to fouling of membranes as well as methods that overcome the fouling issues so rapid concentration in less than 30 min may be achieved. A combination of pre- and post-filtration protocols are required so that the instrument itself can be automated, and membrane filtration devices cycled through repeated uses. The utility of this approach has been demonstrated with microorganisms recovered from food samples (specifically chicken rinse), where 500 x in less than 60 min.
Research Area: Food Safety
I. Emery, N. Mosier, 33rd Symposium on Biotechnology for Fuels and Chemicals, Seattle, WA, May 2-5, 2011, ,
Abstract: Life cycle assessment (LCA) of biofuel production is crucial in order to comply with regulations and to avoid or mitigate negative environmental impacts. Critical gaps in current LCA methodology, in particular a limited or absent consideration of biomass storage, may have dramatic impacts on net greenhouse gas (GHG) and other emissions. Our prior work shows that storage losses can increase the life-cycle GHG emissions of ethanol from corn stover by 20% to 100%. In this study, we examine the impact of multiple biomass storage and supply systems on life cycle GHG emissions from sweet sorghum and switchgrass grown in Tippecanoe County, Indiana. We assess potential dry matter losses, compositional changes, ethanol yield, and direct GHG emissions during storage of bales and silage in centralized and decentralized processing systems. Net emissions and energy use were calculated using the GREET model framework, into which we incorporated storage losses and direct emissions from biomass. Results highlight the impact of logistics, storage, and management decisions on the environmental impacts of second-generation biofuel feedstocks, and the potential benefits of dedicated energy crops for large-scale ethanol production.
Research Area: Bioenergy
A. Athmanathan and N. S. Mosier, AIChE 2011, Minneapolis, MN, October 18, 2011
Abstract:
Research Area: Bioenergy
Y. Kim, E. Ximenes, N. S. Mosier and M. R. Ladisch, 32nd Symposium on Biotechnology for Fuels and Chemicals, Clearwater Beach, FL, April 19-22, 2010
Abstract: Lignocellulose consists of various components which are released by pretreatment and the actions of cellulolytic enzymes. In the case of liquid hot water pretreatment (LHW) of lignocellulosic biomass, the preatreatment solubilizes oligomers and acetic acid from hemicellulose and phenolic compounds from both hemicellulose and lignin. The soluble compounds in the liquid fraction of LHW pretreated cellulosic biomass strongly inhibits the cellulolytic activities of enzymes. In this study, the inhibitory effects of the soluble components in the LHW pretreatment liquid were assessed using pretreated maple and corn stover as a source of inhibitors and Solka Floc as the reactant, Solka Floc at 1% solids loading was readily hydrolyzed at an enzyme loading of 15 FPU cellulase per g cellulose. However when inhibitors were introduced by adding pretreatment liquid to the Solka Floc and buffer, the glucose yield after 72 hrs was reduced by 50%. Among the soluble components in the pretreatment liquid, phenolic compounds were found to be the strongest inhibitors of cellulose hydrolysis. This was further verified by removal of phenolics from the pretreatment liquid which resulted in a significant yield improvement. The relationship between hydrolysis efficiency and the mass ratio of phenolic compounds to cellulase proteins was also measured. The mechanisms of cellulase inhibition/deactivation by sugar-oligomers and phenolics were further probed using individual inhibitor molecules. The combined effects were then studied through simultaneous saccharification and fermentation of Solka Floc and pretreated lignocellulosic substrates. The results show that phenolics are strong inhibitors whose effects may be moderated by washing them away from the lignocellulosic substrates.
Research Area: Biofuels/Bioproducts
Y. Kim, N. S. Mosier, M. R. Ladisch, 32nd Symposium on Biotechnology for Fuels and Chemicals, Clearwater Beach, FL, April 19-22, 2010
Abstract: Variability in feedstock quality as a function of cultivar, production location, and harvest time may have significant impacts on enzymatic saccharifaction and biofuels production. The Biomass Consortium for Applied Fundamentals and Innovation (CAFI) has examined several leading pretreatment technologies applied toward processing switchgrass (Panicum virgatum L.). Switchgrass varieties can be categorized into two different ecotypes primarily based on latitude of origin: upland and lowland. Upland varieties are more adapted to cold temperature and semi-arid climates and tend to grow shorter and less coarse than low land types. Southern-origin lowland curtivars tend to grow taller and be more bunchy and thicker-stemmed, producing more biomass than upland types. In this study, we report comparative saccharification yields of three different varieties of switchgrass, two upland types (Dacotah and Shawnee) and one lowland type (Alamo) switchgrass harvested in the fall or in the spring after standing in the field over winter. Comparisons were also made among the types of switchgrass before and after processed by pretreatment technologies as part of the CAFI project (ammonia fiber expansion, aqueous ammonia recycle, dilute sulfuric acid, lime, and neutral pH liquid hot water). These comparisons are of data obtained through identical experimental protocols and data analysis techniques using common supplies of switchgrass. The key features of different types of switchgrass and the effects these differences had on hydrolysis performance for the applied pretreatment methods are discussed.
Research Area: Bioenergy
N. S. Mosier, M. Sedlak, and N. Ho, AIChE Annual Meeting, Salt Lake City, Utah, November 9, 2010
Abstract: Efficient conversion of hemicellulose-derived sugars to ethanol at high yields and titers are goals toward commercializing cellulosic ethanol production. S. cerevisiae 424A (LNH-ST) developed at Purdue University can efficiently ferment glucose and xylose. However, inhibitors present in cellulosic feedstocks (acetic acid) and the desired fermentation product (ethanol) reduce yeast growth rate and fermentation rates, especially during xylose fermentation. Through adaptation we have developed new strains with improved xylose fermentation compared to the original strain. The new strain has 500% higher ethanol volumetric productivity on xylose in the presence of higher ethanol concentrations (above 6%) than the original strain. An acetic acid-resistant yeast strain co-fermenting glucose and xylose in the presence of acetic acid (10 g/L) when compared to the original strain has 3 times the rate of xylose utilization (1.05 g/L/h from 0.32 g/L/h) and results in a higher final ethanol titer (76.3 g/L from 61.2 g/L). We present the results from a system biology approach to analyzing differences between our original strain and newly developed strains. We focus not only on expression profiling (transcriptomics), but also report changes in metabolic intermediates and fluxes, and lipid membrane composition to elucidate the basis for improved yeast performance. Efficient conversion of hemicellulose-derived sugars to ethanol at high yields and titers are goals toward commercializing cellulosic ethanol production. S. cerevisiae 424A (LNH-ST) developed at Purdue University can efficiently ferment glucose and xylose. However, inhibitors present in cellulosic feedstocks (acetic acid) and the desired fermentation product (ethanol) reduce yeast growth rate and fermentation rates, especially during xylose fermentation. Through adaptation we have developed new strains with improved xylose fermentation compared to the original strain. The new strain has 500% higher ethanol volumetric productivity on xylose in the presence of higher ethanol concentrations (above 6%) than the original strain. An acetic acid-resistant yeast strain co-fermenting glucose and xylose in the presence of acetic acid (10 g/L) when compared to the original strain has 3 times the rate of xylose utilization (1.05 g/L/h from 0.32 g/L/h) and results in a higher final ethanol titer (76.3 g/L from 61.2 g/L). We present the results from a system biology approach to analyzing differences between our original strain and newly developed strains. We focus not only on expression profiling (transcriptomics), but also report changes in metabolic intermediates and fluxes, and lipid membrane composition to elucidate the basis for improved yeast performance. Efficient conversion of hemicellulose-derived sugars to ethanol at high yields and titers are goals toward commercializing cellulosic ethanol production. S. cerevisiae 424A(LNH-ST) developed at Purdue University can efficiently ferment glucose and xylose. However, inhibitors present in cellulosic feedstocks (acetic acid) and the desired fermentation product (ethanol) reduce yeast growth rate and fermentation rates, especially during xylose fermentation. Through adaptation we have developed new strains with improved xylose fermentation compared to the original strain. The new strain has 500% higher ethanol volumetric productivity on xylose in the presence of higher ethanol concentrations (above 6%) than the original strain. An acetic acid-resistant yeast strain co-fermenting glucose and xylose in the presence of acetic acid (10 g/L) when compared to the original strain has 3 times the rate of xylose utilization. (1.05 g/L/h from 0.32 g/.L/h) and results in a higher final ethanol titer (76.3 g/L from 61.2 g/L). We present the results from a system biology approach to analyzing differences between our original strain and newly developed strains. We focus not only on expression profiling (transcriptomics), but also report changes in metabolic intermediates and fluxes, and lipid membrane composition to elucidate the basis for improved yeast performance.
Research Area: Biofuels/Bioproducts
N. Mosier and W. Vermerris, 32nd Symposium on Biotechnology for Fuels and Chemicals, Clearwater Beach, FL, April 19-22, 2010
Abstract:
Research Area: Biofuels/Bioproducts
A. Athmanathan, P. Friedemann, and N. S. Mosier, AIChE Annual Meeting 2010, Salt Lake City, Utah, November 9, 2010
Abstract: Both corn grain and grain stover have been examined and utilized as biofuel feedstocks. Maize silage (wet stored, partially fermented maize stover plus immature grain) is an alternative that combines starch and cellulosic processing in a single feedstock. The commercial brown midrib (BMR marketed by Mycogen, wholly owned subsidiary of Dow AgroSciences) has lowered expression of caffeic acid O-methyl transferase, a key enzyme in the biosynthesis of S monolignols. We carried out a compositional analysis for two commercial varieties of maize silage (regular and brown midrib) for starch, cellulose, hemicellulose, and lignin content. Our results show that for the commercial varieties, the lignin content (Klason lignin plus acid soluble lignin) is indistinguishable. However, the BMR silage exhibits significantly higher cellulose enzymatic digestibility. Liquid hot-water pretreatment was optimized for each silage variant. Optimal pretreatment conditions were similar between BMR and regular silage, which was less severe than required for dry stover from similar maize varieties. Simultaneous saccharifications and fermentations were subsequently performed on pretreated whole silage and ground silage at 25% (w/v) total solids using Celluclast 1.5L and Novozyme 188 and the glucose/xylose co-fermenting yeast S. cerevisiae 424A(LNH-ST). The results show that the improved cellulose hydrolysis performance of BMR silage compared to regular silage is also seen in pretreated material, resulting in significantly higher yields of ethanol after SSF.
Research Area: Biofuels/Bioproducts
C-L Wu, N. S. Mosier, J. Adamec, N. Ho, and M. Sedlak, 32nd Symposium on Biotechnology for Fuels and Chemicals, Clearwater Beach, FL, April 19-22, 2010
Abstract: Bio-ethanol has gained much attention due to its economical and environmental benefits as a renewable fuel. Our lab had genetically engineered a yeast strain 424A(LNH-ST) that can co-ferment glucose and xylose, the two most abundant sugars in cellulosic biomass. However, several inhibitors such as acetic acid, furfural, and ethanol are created and accumulated during the process of cellulosic biomass pretreatment, hydrolysis, and/or during fermentation. Our previous work has shown that acetic acid under process relevant conditions do not significantly affect glucose fermentation. However, xylose utilization is significantly affected, especially at low pH environment (pH < 5.5) and high acetic acid concentration (> 10 g/L). An acetic acid-resistant yeast strain alternated from original 424A(LNH-ST) strain was developed by adaptation to acetic acid. Small-scale fermentation (100 ml YEP) containing 120 g glucose and 80 g xylose per L with 10 g acetic acid per L has shown more than triple the rate of xylose utilization (1.05 g/L/h from 0.32 g/L/h) and higher final ethanol titer (76.3 g/L from 61.2 g/L) by the new strain compared to the original strain. In this study, a system biology analysis including transcriptomic and metabolomic measurements were completed to understand gene expression and metabolic fluxes in this improved strain as compared to the original strain.
Research Area: Biofuels/Bioproducts
I. Emery, J. Park, E. M. Sajeev, and N. Mosier, 32nd Symposium on Biotechnology for Fuels and Chemicals, Clearwater Beach, FL, April 19-22, 2010
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Research Area: Bioenergy
E. Casey, M. Sedlak, N. Ho, and N. Mosier, 32nd Symposium on Biotechnology for Fuels and Chemicals, Clearwater Beach, FL, April 19-22, 2010
Abstract: Lignocellulosic biomass is a promising renewable feedstock for the microbial production of chemicals and fuels, especially ethanol. Processing lignocellulose for biofuel production results in the release of the major fermentable sugars glucose and xylose. However, the primary processing steps required for this conversion also produce a range of compounds that can inhibit the subsequent microbial fermentation. One such inhibitory compound is acetic acid, liberated from hemicelluloses during the pretreatment of the biomass. We previously reported acetic acid to be inhibitory to cell growth, substrate consumption (especially xylose), and ethanol productivity, and stimulatory to the metabolic yield of ethanol. To further explore the effect of acetic acid on a cellular level, a genome-wide analysis of gene expression levels over the course of a batch co-fermentation of glucose and xylose was conducted using microarray technology. RNA samples were extracted for analysis from S. cerevisiae 424A(LNH-ST) at various time points throughout the co-fermentation of glucose and xylose with either 0 or 10 g/L acetic acid at a controlled pH of 5.5. In this poster, we report the results of this transciptomic analysis, focusing on genes that are identified as differentially expressed when cells are inhibited by acetic acid.
Research Area: Biofuels/Bioproducts
E. A. Ximenes, SURF Program Presentation, Purdue University, July 21, 2009
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Research Area: Biofuels/Bioproducts
Y. Kim, N. Mosier, and M. Ladisch, 31st Symposium on Biotechnology for Fuels and Chemicals, San Francisco, CA, May 3-6, 2009
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Research Area: Biofuels/Bioproducts
Y. Kim, N. S. Mosier, M. R. Ladisch, 2009 AIChE Annual Meeting, Nashville, TN, Nov. 8-13, 2009
Abstract: Switchgrass (Panicum virgatum L.) is a promising dedicated bioenergy feedstock with numerous environmental benefits, due to its low fertility requirements, tolerance of poor soils and drought, and high biomass yield. Switchgrass varieties can be categorized into two different ecotypes primarily based on latitude of origin: upland and lowland. Upland varieties are more adapted to cold temperature and semi-arid climates and tend to grow shorter and less coarse than low land types. Southern-origin lowland curtivars tend to grow taller and be more bunchy and thicker-stemmed, producing more biomass than upland types. In this study, we report comparative saccharification yields of three different varieties of switchgrass, two upland types (Dacotah and Shawnee) and one lowland type (Alamo) switchgrass, pretreated by controlled pH, liquid hot water (LHW) pretreatment. Hydrolysis of LHW pretreated switchgrass at 15% w/v dry solids loading resulted in 80% glucose yield and 90% xylose yield at a total protein loading of 11 mg protein/g dry biomass where the total protein consists of cellulase combined with supplementary xylanase. Comparisons were also made among the types of switchgrass processed by other pretreatment technologies as part of the CAFI project (ammonia fiber expansion, aqueous ammonia recycle, dilute sulfuric acid, lime, and neutral pH liquid hot water). These comparisons are of data obtained through identical experimental protocols and data analysis techniques using common supplies of switchgrass. The key features of different types of switchgrass and the effects these differences had on hydrolysis performance for the applied pretreatment methods are briefly discussed.
Research Area: Bioenergy
H. Mohammad, N. S. Mosier, N. Ho, M. Sedlak, Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, IN, Poster 2-67, 31st Symposium on Biotechnology for Fuels and Chemicals, San Francisco, CA, May, 2009
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Research Area: Biofuels/Bioproducts
E. Casey, M. Sedlak, N. Ho, J. Adamec, A. Jannasch, and N. Mosier, 31st Symposium on Biotechnology for Fuels and Chemicals, San Francisco, CA, May 3-6, 2009
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Research Area: Biofuels/Bioproducts
E. A. Ximenes, Y. Kim, X. Li, H. Vibbert, P. Rubinelli, N. Bonawitz, R. Meilan, C. Chapple, and M. R. Ladisch, 31st Symposium on Biotechnology for Fuels and Chemicals, San Francisco, CA, May 3-6, 2009
Abstract: Plant genetic engineering is considered a potential approach to reduce costs for biofuel production from lignocellulosic material. However, the ability to control cell-wall composition without compromising plant performance jis a key objective of bioenergy crop improvements. Plants have been engineered for the production of enzymes within the crop biomass, with an aim to minimize the costs of catalyst production in bioreactors. Future research on the upregulation of cellulose and hemicellulose biosynthesis pathway enzymes for an increase in polysaccharides may also have the potential to improve cellullosic feedstocks. The most successful efforts to date have focused on the modification of lignin quantity and/or quality, in an effort to obviate the need for expensive pretreatment processes. Here we report a method for rapid detection of improved biodegradability in genetically modified plants that vary in lignin content and/or composition. For this purpose, only 50 mg of ground material is needed for liquid hot water pretreatment, and the method allows the pretreatment of up to 9 samples every 10 min per sandbath. Enzyme hydrolysis in the presence of commercial cellulases and beta-glucosidase is performed in a final volume of 1 mL for 30 min, at 50 C, and pH 4.8. The samples are then centrifuged, and the amount of glucose liberated is analyzed via a microplate assay. Using this approach, we have been able to rapidly and reproducibly identify genetically modified plants jwith improved biodegradability.
Research Area: Food Safety
Youngmi Kim, Michael R. Ladisch, Peter Friedemann, Darin W. Lickfeldt, Katherine Armstrong, and Nathan S. Mosier, Laboratory of Renewable Resources Engineering, Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN 47907 , and Dow AgroSciences, Indianapolis, IN, Poster 12-30, 31st Symposium on Biotechnology for Fuels and Chemicals, San Francisco, CA, May, 2009
Abstract: The processing characteristics of biofuel feedstocks are strongly affected by the quantity and quality of lignin in the cell wall structure. We present the effect of brown midrib mutations on rates and yields of cellulosic ethanol production from maize silage. Both raw silage and silage from commercial sources were pretreated using liquid hot water (160-180 C) and assessed by enzymatic hydrolysis and fermentation using the glucose/xylose fermenting Purdue recombinant S. cerevisiae 424A(LNH-ST). At 20% solids concentration (20 g/L), pretreated bmr silage achieved higher yields of sugars than non-bmr silage pretreated under the same conditions. At the optimal pretreatment conditions, bmr silage achieved 62% of theoretical yield of glucose after 24 hours of enzymatic hydrolysis (15 FPU cellulase per gram glucan) compared to 50% yield from non-bmr silage. Sugars from both silage varieties fermented to ethanol at high yields using the Purdue recombinant yeast strain.
Research Area: Biofuels/Bioproducts
Isaac Emery and Nathan Mosier, Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN 47907 , 2009 ESE Symposium, September, 2009
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Research Area: Bioenergy
A. Athmanathan, M. Sedlak, N. Ho, N. Mosier, 30th Symposium on Biotechnology for Fuels and Chemicals, New Orleans, Louisiana, May 4 - 7, 2008
Abstract: Ethanol toxicity could be a significant bottleneck in industrial ethanol fermentation of sugars from lignocellulose. To understand ethanol impact on xylose fermentation, batch fermentations were carried out using S. cerevisiae 424A (LNH-ST), an engineered strain capable of co-fermenting glucose and xylose. The fermentation of xylose was carried out in YEP growth media, using largely non-growing cells in the presence of initial ethanol concentrations between 4 - 8% (w/v). The effects of extraneously added ethanol (pure xylose fermentation) and ethanol generated from glucose equivalent (co-fermentation) are compared. This yeast strain was found to cease fermentation of xylose at an extraneously added ethanol concentration of 9% (w/v). However, co-fermentation of glucose and xylose was capable of achieving a final ethanol titer over 11% (w/v). A preliminary unstructured, Monod-type model of these batch fermentations that include ethanol inhibition is presented.
Research Area: Bioprocessing
E. Casey, M. Sedlak, N. Ho, and N. Mosier, 30th Symposium on Biotechnology for Fuels and Chemicals, New Orleans, Louisiana, May 4-7, 2008
Abstract: Lignocellulosic biomass, primarily comprised of cellulose, hemicellulose, and lignin, is a promising renewable feedstock for the microbial production of chemicals, especially ethanol. The major fermentable sugars (hydrolysates) released from the processing of the lignocellulose are glucose and xylose. However, the primary processing steps required for this conversion also produce a range of compounds that can inhibit the subsequent microbial fermentation. One such inhibitory compound is acetic acid, liberated during the pretreatment of the biomass. In this poster, we report the effect of acetic acid on glucose/xylose co-fermentation by the genetically modified S. cerevisiae 424A(LNH-ST). The co-fermentation of glucose and xylose was performed under acetic acid conditions of 5, 10, 15 g/L over a pH range of 5 – 6. To maintain the pH at the specified initial value, the fermentations were carried out in a 1L New Brunswick BioFlow 110 benchtop fermentor equipped with a pH controller. Results showed that the fermentation of both sugars was affected by the presence of acetic acid. The inhibitory effect of acetic acid increased as the pH decreased. The results also indicate that the utilization of xylose is more influenced by acetic acid concentration and pH than the utilization of glucose.
Research Area: Biofuels/Bioproducts
Y. Kim, N. S. Mosier, and M. R. Ladisch, 2008 Annual Meeting of the American Institute of Chemical Engineers, Philadelphia, PA, November 20, 2008
Abstract: The conversion of switchgrass to fermentable sugars and ethanol provides a cellulosic feedstock for production of fuel ethanol which may be grown on lands not suitable for food agriculture. Switchgrass itself consists of 33% cellulose, 25% hemicelluloses, 18% lignin, and 24% other. If switchgrass is processed without pretreatment, the maximal conversion achieved at an enzyme loading of 15 FPU/g glucan (5 FPU/g biomass) is less than 5%. When the switchgrass is pretreated in liquid hot water, the conversion increases by 25-fold, resulting in 80% glucose yield. The utilization of liquid hot water followed by enzyme hydrolysis and fermentation is described in this paper. The levels of enzyme loading and inhibition effects are briefly discussed as part of the overall CAFI research project.
Research Area: Biofuels/Bioproducts
Y. Lu and N. Mosier, 30th Symposium on Biotechnology for Fuels and Chemicals, New Orleans, Louisiana, May 4 - 7, 2008
Abstract: The degradation reaction routes of glucose and fructose under hydrothermal acidic conditions have been studied extensively; in contrast, xylose degradation has received less extensive study under similar conditions. In this study, we investigated the aqueous pH (0.5 – 7.0) impact on xylose degradation, and determined the kinetics of xylose disappearance rates at different pH conditions. The initial buffer system employed in this study was the McIlvaine buffer consisting of phosphate salt and citric acids (except for pH 0.5 – 1.5 buffers, where HCl/NaCl system was employed). It was observed that at pH 2.2, the xylose degradation rate was minimized (e.g. xylose disappearance rate at pH 4.2 is 9-times higher, and at pH 7.0 complete xylose disappearance occurred in 5-min reaction). In addition, the degradation reaction path changed from simple dehydration product (furfural) formation at lower pH range (0.5 – 3.0), to multiple complex liquid and polymerized products formation at higher pH range (4.5 – 7.0). In order to test the effect of buffering salt (phosphate, etc.), experiments at pH 1.0 with equivalent amount phosphate produced identical results to the same condition without phosphate addition. Therefore, the proton concentration in the aqueous solution may be the main controlling factor to which xylose degradation reactions occur. The degree of proton availability in the solution and potential protonation of the sugar –OH groups were analyzed to determine how the pH affects reaction path direction and products formation.
Research Area: Bioprocessing
M. R. Ladisch, 2008 Annual Meeting of the American Institute of Chemical Engineers, AIChE Centennial, Philadelphia, PA, November 19, 2008
Abstract: The production of ethanol from cellulose for use as a liquid transportation fuel requires a combination of process engineering, microbiology, and accessibility to feedstock. The feedstock must be available to supply the plant 24 hours / day, 7 days per week. Siting of the plant is key to ensuring feedstock supply. Conversion of the feedstock to sugars and to ethanol requires pretreatment, hydrolysis, and fermentation. Pretreatment softens up the plant cell wall structure and enables enzymes to access the cellulose so that they may catalyze the formation of monosaccharides. The monsaccahrides, in turn, may be converted to ethanol through microbial fermentation by yeast or bacteria that have been engineered to convert both glucose and xylose to ethanol. During the bioconversion steps, cascading molecular control of enzyme activity occurs due to inhibitors that are formed during the pretreatment and/or hydrolysis steps. This paper discusses the role of process engineering in addressing issues of inhibition, solids loading, and fermentation, and gives a review of fundamental mechanisms and future research needs for converting renewable resources to biofuels in a cost effective manner.
Research Area: Biofuels/Bioproducts
M. R. Ladisch, R. Bashir, A. Bhunia, Y. Kim, and N. Mosier, 2008 Annual Meeting of the American Institute of Chemical Engineers, Philadelphia, PA, Plenary Session I on Bioseparations: Celebrating 100 Years of Bioseparations, November 17, 2008
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Research Area: Food Safety
M. Ladisch, Guest Lecture, IAP Energy Ventures Minicourse, MIT Energy Club, , January 22, 2008
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Research Area: Biofuels/Bioproducts
R. Hendrickson, N. S. Mosier, and M. R. Ladisch, 29th Symposium on Biotechnology for Fuels and Chemicals, Denver, Colorado, April 29-May 2, 2007
Abstract: Commercial cellulasse preparations effectively hydrolyze cellulose present in liquid hot water pretreated distillers grains (DG) to glucose. However, commercial xylanase preparations yield approximately 25% of the xylose and arabinose from the hemicellulose fraction of the same hydrolysate. Since hemicellulose accounts for nearly 40% of the carbohydrate content of DG, optimizing enzyme activities is required to maximize total fermentatble sugar yields from this biomass material. In this paper we report the effect of supplementing commercial xylanase with additional enzyme activities. The addition of these enzymes to commercial cellulase significantly increased the yields of arabinose and xylose to 78%. Also presented is the effect of high solids concentrations on yield and rate of xylose and arabinose liberation.
Research Area: Biofuels/Bioproducts
M. Zeng, J. Goetz, R. Hendrickson, C. P. Huang, D. Sherman, N. S. Mosier, and M. R. Ladisch, 29th Symposium on Biotechnology for Fuels and Chemicals, Denver, Colorado, Poster 5B-25 , April 29-May 2, 2007
Abstract: Corn stover is a heterogeneous substrate consisting of different fractions including leaves, stalk fiber and stalk pith. Tissue types and proportions in these fractions are not uniform which result in different cell structures, average cell wall thickness and lignin distribution. These factors may have different impacts on enzyme digestion, since the lignin barrier (content/distribution) and cell wall thickness are believed to be substrate related factors that influence the effectiveness of enzymatic hydrolysis of cellulose in lignocellulosic feedstocks. The hypothesis being tested in this research addresses potential differences of intrinsic reactivity of different parts of stay green corn stover (leaves, stalk fiber and stalk pith). Carbohydrate analysis shows that pith is more readily hydrolyzed than leaves and fiber at cellulase level equivalent to 5 FPU Spezyme CP/g glucan. Hot water pretreatment at 190 C for 15 min removes 40% to 50% hemicellulose in these fractions, respectively, although structural changes in the cell wall are not evident when the residual material is imaged by scanning electron microscopy. Enzyme hydrolysis of pretreated and washed fractions of leaves and pith exhibit much higher glucose conversion than the fractions that have not been pretreated. Pretreated fibere (from the rind) is still resistant to hydrolysis and shows 2/3 lower glucose formation than pretreated leaf or pith at low enzyme loadings equivalent to about 5 FPU Spezyme CP/g glucan.
Research Area: Bioprocessing
R. Warner, M. Sedlak, N. W. Y. Ho, M. R. Ladisch, and N. S. Mosier, 29th Symposium on Biotechnology for Fuels and Chemicals, Denver, Colorado, April 29-May 2, 2007
Abstract: Furfural, the acid-catalyzed degradation product of pentoses, has been shown to decrease the fermentability and the ethanol yields from sugars derived from lignocellulose. This paper reports a systematic study of the effect of furfural on cell growth and fermentation of both glucose and xylose to ethanol by the recombinant yeast S. cerevisiae 424A(LNH-ST). Fermentations were run with furfural, HMF, or both in a control solution of YEP with glucose and xylose as co-substrates or xylose alone. Cell concentrations at the beginning of the fermentation varied between 0.1 and 9 g/L. Inhibitor concentrations were varied from 0 to 40 g/L. Batch fermentations were carried out for at least 48 hours in 300 mL sidearm flasks at 30 C and 200 rpm with periodic sampling for analysis by HPLC. Our results show that concentrations of either furfural below about 5 g/L cause negligible inhibition for yeast cells in early stationary phase while similar concentrations will lengthen the lag phase of lower innoculations of cells. Xylose fermentation to ethanol is more sensitive to furfural than glucose for fermentation to ethanol. These results are then compared to the fermentation of xylose obtained from pretreated corn stover and pretreated poplar hydrolyzates from the Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI) that contain varying concentrations of inhibitors.
Research Area: Biofuels/Bioproducts
Mosier, N. S., President's Council "Back to Class", Naples, Florida, February 10, 2007
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Research Area: Biofuels/Bioproducts
W. Vermerris, J. Zhao, M. R. Ladisch, and M. S. Mosier, 29th Symposium on Biotechnology for Fuels and Chemicals, Denver, Colorado, April 29-May 2, 2007
Abstract: We have recently shown that modification of lignin subunit composition can significantly increase the yield of fermentable sugars obtained from enzymatic saccharification of maize stover. The brown midrib1 (bmi) and bm3 mutations each increase the yield of glucose per gram dry stover by 50% relative to the wild-type control (inbred A619). When combined in a near-isogenic bm1-bm3 double mutant, the two mutations act in an additive manner, resulting in a doubling of the yield of glucose. Even though there was no apparent increase in cellulose content, based on kinetic studies both the rate of hydrolysis and the overall yield of glucose increased as a result of the mutations. In order to be able to generalize our results, we are investigating if this increased yield is consistent in different genetic backgrounds. In addition, we are investigating what the basis is of the enhanced hydrolysis in these bm mutants by in situ visualization of cellulases. We have designed recombinant proteins consisting of the cellulose binding domain (CBD) isolated from Trichoderma reesei endoglucanases labeled with green-fluorescent protein (GFP) to study how changes in cell wall composition and architecture impact the distribution of cellulolytic enzymes. These analyses will be performed in intact plant tissue as well as in ground stover using UV fluorescence microscopy. The resulting information will be valuable for designing plant cell wall composition in such a way that agronomic properties and biomass conversion are optimally balanced.
Research Area: Biofuels/Bioproducts
M. Ladisch, Richard Lugar Energy Summit, August 29, 2006
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Research Area: Bioenergy
Mosier, N. S., and Otto Doering, Seminar at Purdue College of Agriculture Workshop on Cropping Systems, Purdue University, April 7, 2006
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Research Area: Biofuels/Bioproducts
Ryan E. Warner, Miroslav Sedlak, Nancy Ho, and Nathan S. Mosier, 28th Symposium on Biotechnology for Fuels and Chemicals, Nashville, TN, April 30 to May 3, 2006
Abstract: Pretreatment of lignocellulosic biomass, while improving enzymatic digestibility, can also produce fermentation inhibitors. Two important inhibitors, furfural and HMF, are formed from the degradation of carbohydrates from lignocellulose. Thus, pretreated material may require conditioning to either remove or otherwise detoxify these inhibitors. This paper explores some conditioning methods on hydrolysates obtained from corn stover and poplar pretreated by dilute acid, controlled pH l iquid hot water, SO2 steam explosion, and others. The effects of these conditioning methods on the subsequent fermentation of both glucose and xylose by the recombinant yeast S. cerevisiae 424A(LNH-ST) is presented. Overliming the pretreated corn stover to pH 9 or higher removes 100% of the HMF and furfural present in corn stover hydrolysates. However, the fermentation is negatively affected, producing only 53% of theoretical ethanol yields as opposed to 82% yield from the unconditioned material. Hydrophobic resins (Amberlite XAD2, XAD4, and XAD7) were also examined for their ability to remove HMF and furfural. The resins were able to remove 100% of furfural and approximately 60% or more HMF. The yield from fermentation was 87%; slightly better than the unconditioned corn stover hydrolysate.
Research Area: Bioprocessing
Ryan E. Warner, Miroslav Sedlak, Nancy Ho, and Nathan S. Mosier, 28th Symposium on Biotechnology for Fuels and Chemicals, Nashville, TN, April 30 to May 3, 2006
Abstract: Pretreatment of lignocellulosic biomass, while improving enzymataic digestibility, can also produce fermentation inhibitors such as furfural and HMF. Both furfural and HMF can decrease the fermentability and the ethanol yields from sugars derived from lignocellulose. This paper reports a systematic study of the effect of furfural and HMF on the fermentation of both glucose and xylose to ethanol by the recombinant yeast S. cerevisiae 424A(LNH-ST). Fermentations were run with furfural, HMF, or both in a control solution of YEP with glucose and xylose as co-substrates. Inhibitor concentrations were varied and range from 0 to 40 g/L. Further experiments varied inhibitor concentrations in the presence of a single substrate, either glucose or xylose. Batch fermentations were carried out for 48 hours in 300 mL sidearm flasks at 30 C and 200 rpm with periodic sampling for anlaysis by HPLC. Our results show that concentrations of either furfural or HMF below about 5 g/L cause negligible inhibition for yeast cells in early stationary phase. We confirm that furfural is more inhibitory than HMF. Lastly, xylose fermentation to ethanol is more sensitive to these inhibitors than glucose for fermentation to ethanol.
Research Area: Bioprocessing
Mosier, N. S., Handout for Seminar at Indiana Farm Bureau Supper Series, Purdue University, May 8, 2006
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Research Area: Biofuels/Bioproducts
Tom T. Huang, David G. Taylor, Kwan Seop Lim, Miroslav Sedlak, Rashid Bashir, Nathan S. Mosier, Michael R. Ladisch, Pharmaceutical Technology and Education Workshop, Dauch Center, Purdue University, April 19, 2006
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Research Area: Bioprocessing
Richard Hendrickson, Youngmi Kim, Yulin Lu, Nathan Mosier, and Michael Ladisch, 28th Symposium on Biotechnology for Fuels and Chemicals, Nashville, TN, April 30 to May 3, 2006
Abstract: The aqueous pretreatment of corn fiber at a pH of 4 to 7, while being pumped through a hold coil is effective in increasing the rate of enzyme hydrolysis of the cellulose. However, scale-up of the pretreatment process depends on physical properties of the material to be pumped through the system. High concentrations of fermentable sugars require that aqueous biomass streams from which these sugars are derived have a high solids content. Since the corn fiber solids at high loading have characteristics that resemble a shear-thinning fluid, measurement of viscosity in the laboratory is difficult, particularly at temperatures above ambient. Consequently, we carried out measurements in a plant setting. Corn fiber at 150 to 200 g/L were pumped at rates of 1 to 10 gal/minute through sections of jacketed tubing having diameters ranging from 1 to 1.5 inches and a length of 17.25 feet. The temperatures and pressure drops were measured at the inlet and outlet of the tubes and recorded through a LabVIEW programmed data acquisition system. The pressure drop and flow rate enabled calculation of viscosity and determination of correlations that will be useful for scale-up.
Research Area: Bioprocessing
Bruce E. Dale, Richard T. Elander, Mark T. Holtzapple, Rajeev Kumar, Michael R. Ladisch, Yoon Y. Lee, Nate Mosier, Jack Saddler, Mohammed Moniruzzaman, Charles E. Wyman, CAFI BIO 2006, Annual International Convention, Chicago, Illinois, April 12, 2006
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Research Area: Bioprocessing
Bwatwa, J., N. S. Mosier, T. Huang, X. Liu, A. Stewart, C. M. Ladisch, and M. R. Ladisch, Division of Analytical Chemistry, Paper 349, 229th National ACS Meeting of the American Chemical Society, San Diego, CA, March 15, 2005
Abstract: 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.
Research Area: Bioseparations
Ladisch, Michael, on behalf of an interdisciplinary research team from the Schools of Agriculture, Engineering and Science, IN- ATAIN Network Event, Alternative Energy Research, Purdue University, Stewart Center, West Lafayette, IN, September 28, 2005
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Research Area: Bioenergy
Ladisch, Michael, Seminar for Purdue's Electrical and Computer Engineering Class, ECE694, Purdue University, West Lafayette, IN, October 6, 2005
Abstract: The price of gasoline has attracted our attention to the nature of our economy, lifestyle, and use of liquid fuels. Currently, gasoline is expensive, but marketplace economics have prevented shortages. Now, imagine a world with gasoline at $100/gallon and oil at $2000/barrel. We would probably use more bioenergy - particularly liquid fuel obtained from plants, wind and solar sources. Since bioenergy is already available, would this scenario translate into an energy boom? How would our lifestyle change? The answers will reflect availability, sustainability, and the ability of agriculture to generate renewable feedstocks for use in producing liquid fuels. Efficient transformation of renewable solid forms of carbon into liquid transportation fuels will require metabolic engineering of yeast, advanced macromolecular or nano-scale catalysts, efficient separations technology and engineering of crops and microorganisms for industrial use. If bioenergy is to be used forever (or at least as long as the sun shines) sustainability becomes a key issue. The toolbox for building our energy future will include biology, mathematics, biocatalysis, and bioprocess engineering, as well as the tehcnologies: bio-, nano-, info-, and eco-. The ultimate impact of bioenergy will depend on how it is integrated into a global society, where sources and uses of energy are distributed both geographically as well as technologically. More nuclear, coal, solar, wind, shale oil, and methane hydrolytes, and less oil and less NIMBY will be part of the alternate energy portfolio. We will discover that the future of bioenergy is not only about alternate energy and tehcnology, but also about the role of engineers as leaders of societal change. Arguments in support of these hypotheses will be presented.
Research Area: Bioenergy
Michael R. Ladisch, National Science Foundation Workshop on "Design of Catalyst Systems for Biorenewables, Washington, DC, June 23, 24, 2005
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Research Area: Bioprocessing
Wyman, C. E., Y. Y. Lee, B. E. Dale, T. Eggeman, R. T. Elander, M. R. Ladisch, N. W. Y. Ho, M. Sedlak, N. S. Mosier, M. T. Holtzapple, and J. N. Saddler, Bioprocessing of Agricultural Feedstocks: Report on Pretreatment for Biomass Refining, 2nd World Congress on Industrial Biotechnology and Bioprocessing, Orlando, Florida, April 20, 2005
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Research Area: Biofuels/Bioproducts
Mosier, N. S., Warner, R., Sedlak, M., Ho, N. W. Y., Hendrickson, R., and Ladisch, M. R., 2005 Annual Meeting of the American Institute of Chemical Engineers, Paper 183E, Cincinnati, OH, November 1, 2005
Abstract: Mid-severity dilute acid pretreatment liquor from Kramer corn stover pretreated in the Sunds reactor at NREL was analyzed, conditioned, and fermented by glucose/xylose co-fermenting yeast (S. cerevisiae 424A(LNH-ST). This yeast is currently being validated for large scale industrial cellulosic ethanol production. The pretreatment hydrolysate liquid contained 22.4 to 24.6 g/L glucose, 72.7 to 76.2 g/L xylose, 13 g/L acetic acid, 2.1 g/L furfural and 2.7 g/L HMF, and was conditioned by over-limiting contact with polymeric (XAD-4 resin), or a combination of the two steps before fermentation. The sugar compositions were similar to those for the untreated hydrolysate, although in all cases a significant fraction of the furfural was removed, and in the case of overliming, some HMF was also removed. XAD4 has been previously shown to selectively remove furfural and color from the aqueous sugar solutions. S. cerevisiae 424A(LNH-ST) completes the fermentation in 48 hours for media containing the same amounts of pure sugars as are found in the hydrolysates. However, high salt and acetic acid concentration in the dilute acid pretreatment liquor, and/or residual HMF, is known to decrease the fermentation rate, and this was found to be the cse here as well. When the different solutions were fermented by 424A(LNH-ST), glucose was consumed in 2 to 6 hours, but only 40% of the xylose was fermented to ethanol within 72 hours as compared to complete fermentation in 48 hours in the synthetic and other media. Research is continuing to optimize conditions and enhance rates and extents of ethanol fermentation from xylose in hydrolysates obtained from acid pretreated corn stover.
Research Area: Biofuels/Bioproducts
Ladisch, M. R., T. Huang, R. Armstrong, and N. Mosier, Division of Agricultural and Food Chemistry, Paper 205, 229th National ACS Meeting of the American Chemical Society, San Diego, CA, March 17, 2005
Abstract: Nanoscience is the fabrication, study, and modeling of principles of devices and structures for which at least one dimension is several 100 nanometers or smaller. Nanotechnology is the enabling component of the discovery and development process that assembles nano-structures into compact, portable devices that carry out sensing functions currently relgated to scientific laboratories. Some types of devices will integrate biotechnology with silicon or plastic surfaces to form biosensing systems that enhance detection and enable study of biomarkers generated in resonse to environmental stress and other biological conditions of importance to agriculture. When coupled with devices that have capabilities to give temporal and geograhic information, nanotechnology may contribute to tracking of agricultural commodities. This paper will discuss possible applications of very small, intelligent, sensing devices for monitoring products from a widely distributed, global agricultural enterprise, and their potential contribution to identify preservation.
Research Area: Food Safety
Zeng, M., N. S. Mosier, C. Huang, D. Sherman, J. Goetz, and M. R. Ladisch, Division of Biochemical Technology, Poster 335, 229th National ACS Meeting of the American Chemical Society, San Diego, CA, March 16, 2005
Abstract: 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.
Research Area: Bioseparations
Mosier, N. S., Division of Agricultural and Food Chemistry, 229th ACS Annual Meeting, San Diego, CA, March 16, 2005
Abstract: Developments in the understanding of the nanoscale structure and molecular mechanism of cellulolytic enzymes provide insights that may guide the development of nanoscale catalysts that efficiently hydrolyzes cellulose and hemicellulose from agriculturally derived feedstocks. Nanoscale, biomimetic catalysts may provide cost effective means for producing fermentable sugars from lignocellulosic biomass for renewable fuel and chemical production from agriculturally derived jplant biomass. This enzyme mimetic is composed of two functional domains: a catalytic domain and a cellulose binding domain. The cellulose binding domain selectively adsorbs the acid catalytic domain to the cellulose surface, thus concentrating the catalyst at the substrate surface. Maleic acid, a leading catalytic domain, effectively hydrolyzes cellulose with the glucose degradation when compared against mineral acids such as sulfuric acid. Maleic acid was found to be capable of yielding at least 50% more fermentable glucose from microcrystalline cellulose and corn stover compared to sulfuric acid at similar acid strength and hydrolysis conditions. When coupled with a cellulose binding domain, maleic acid may be concentrated near the cellulose surface. A number of cellulose binding domain candidates have been screened for adsroption to cellulose at hydrolysis conditions (>100 C, > 1 atm). Effective binding domain candidates have physiochemical properties similar to enzyme binding domains - planar, hydrophobic molecules capable of hydrogen bonding. Indole, the side chain of the amino acid tryptophan which is critical for enzymatic adsorption, has been showed to adsorb to cellulose at these conditions.
Research Area: Food Safety
Huang, T., D. Taylor, N. S. Mosier, M. Sedlak, and M. R. Ladisch, Novel Applications: Nanobiotechnology, 2nd World Congress on Industrial Biotechnology and Bioprocessing, Orlando, Florida, April 20, 2005
Abstract:
Research Area: Food Safety
Young Mi Kim, Mosier, Nathan, Hendrickson, Rick, and Ladisch, Michael R., 27th Symposium on Biotechnology for Fuels and Chemicals, Denver, CO, May 4, 2005
Abstract: Liquid hot water pretreatment of plant biomass produces a liquid stream with dissolved oligosaccharides which are usually converted to fermentable sugars by enzymatic hydrolysis. In previous work, strong cation exchanger, Amberlyst 35W, has shown to hydrolyze cellobiose and oligosaccharides in liquid from corn fiber pretreatment at high conversion rates. This paper reports the effects of particle size, degree of cross-linking, and temperature on hydrolysis of oligosaccharides and degradation of monosaccharides. High temperature and short residence times were required to minimize formation of aldehydes and other fermentation inhibitors formation while achieving high glucose yield. The catalysts, SK104 (4% corrlinked gell type) and Amberlyst 35 (macroreticular sulfonic acid resin) were tested for hydrolysis of maltooligosaccharides at various reaction conditions. Maltooligosaccharides were used as a model oligosaccharide since their activation energy for bond breakage is similar to that of xylo- or cello-oligosaccharides, and since malto-oligosaccharides are more readily obtainable compared to the other types of oligosaccharides. Results show that low percentage cross-linked gel-type cation exchange resins give a higher glucose yield than macroreticular-type resins. The hydrolysis was diffision limited in both resins. A mathematical model that quantifies diffusion and kinetic characteristics of this reaction is presented and potential application of plug flow reactors to hydrolysis of oligosaccharides obtained from pretreatment of cellulose is discussed.
Research Area: Bioprocessing
Mosier, N. S., Craig, B., Workshop on Overarching Issues in Risk Analysis, National Institute of Statistical Sciences, Ames, IA, October 28, 2005
Abstract:
Research Area: Food Safety
Huang, T., D. G. Taylor, X. Liu, M. Sedlak, N. S. Mosier, and M. R. Ladisch, Poster, Indiana Biosensor Conference, Indianapolis, Indiana, April 6, 2005
Abstract: We report a rapid microfluidic device construction technique which does not employ lithography or stamping methods. Device assembly physically combines a silicon wafer, an elastomer (polydimethylsiloxane (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 ina 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 minutes, the technique results in an operable microdevice with micron scale features of nanoliter volume. Microfiber-directed boundary flow is achieved by usse 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. E. coli cells that express green fluorescent protein (GFP) or mixtures of non-pathogenic and heat-killed E. coli O157:H7 cells incubated and labeled with fluorescein-conjugated antibodies were readily detected and counted with this device.
Research Area: Food Safety
Zang, Y., X. Liu, B. Tyner, A. Stewart, W.-T. Chen, M. Sedlak, N. S. Mosier, B. Craig, and M. R. Ladisch, Division of Biochemical Technology, Poster 341, 229th National ACS Meeting of the American Chemical Society, San Diego, CA, March 16, 2005
Abstract: The detection of low numbers of organisms in large volumes of liquids is a challenge for both the fermentation and food industries. The detection of microbial contamination or the presence of pathogens requires that the sample be processed, concentrated, and assayed to detect living cels. The rapid concentration and detection of the pathogen, Listeria monocytogenes, from liquid extract of meat is one application where sampling size to achieve adequate detection confidence levels is crucial. The prediction of the minimal sample volume required to enable detection of a specified microorganism must be carefully carried out so that the probability of detection meets pre-determined criteria. We show that detection of 10 to 50 living cells extracted from a 50 g meat sample into 250 mL of buffer can be calculated using the Poisson distribution equation. Using GFP expressing E. coli that can be individually visualized microscopically, we show that a random distribution model accurately represents the probability of detection as a function of sample volume and concentration. This work is generalized to the detection of bacteria in meat, vegetable, and fermentation broth. The significance of these results in the context of rapid detection of pathogens using microfluidic devices for purposes of bioprocess monitoring and control is discussed.
Research Area: Food Safety
Huang, T., N. S. Mosier, and M. R. Ladisch, Division of Biochemical Technology, Paper 63, 229th National ACS Meeting of the American Chemical Society, San Diego, CA, March 14, 2005
Abstract: 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.
Research Area: Bioseparations
Ladisch, M. R., N. Mosier, G. Welch, B. Dien, A. Aden, and P. Shane, U. S. DOE and EU, 1st International Biorefinery Workshop, Washington, DC, July 20 - 21, 2005
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Research Area: Biofuels/Bioproducts
Mosier, N. A., and M. R. Ladisch, 26th Symposium on Biotechnology for Fuels and Chemicals, Chattanooga, Tennessee, May 10, 2004
Abstract: Cellulolytic enzymes consist of a catalytic domain, a linking peptide, and a binding domain. This poster describes research on carboxylic acids that have potential as the catalytic domain and planar cellulose adsorbing molecules for constructing organic catalysts that mimic the action of enzymes in hydrolyzing cellulose by adsorbing the acid catalyst near the cellulose substrate. Glucose degradation, unlike cellulose hydrolysis, was shown to be independent of hydrogen ion concentration for carboxylic acids. Maleic acid, a dicarboxylic acid, effectively hydrolyzes cellobiose, the repeat unit of cellulose, by the relatively well-understood mechanism of acid hydrolysis. However, unlike sulfuric acid, maleic acid does not catalyze glucose degradation. Consequently, overall yields of glucose from cellulose were shown to be higher for maleic acid, when compared to sulfuric acid at equivalent solution pH. A number of organic, planar, molecules were screened for adsorption to cellulose at temperatures ranging from 30 - 140 C using a chromatogrpahic method. Trypan blue was shown to strongly adsorb to cellulose at high temperatures and possesses moieties that offer possibilities for linking acid catalysts to this cellulose adsortive compound.
Research Area: Biofuels/Bioproducts
Sedlak, M., A. Mukerji and N. W. Y. Ho, 26th Symposium on Biotechnology for Fuels and Chemicals, Abstract No. 2-21, Chattanooga, Tennessee, May 9-12, 2004
Abstract: Cellulosic biomass is known to be an ideal raw material for the production of chemicals by microbial processes, particularly those produced in large volumes such as ethanol. However, cellulosic biomass contains large amounts of xylose in addition to glucose. The naturally-occurring Saccharomyces yeasts used for large-scale ethanol production from starch (glucose) cannot metabolize xylose. In recent years, we have been able to genetically engineer the Saccharomyces yeasts to effectively co-metabolize glucose and xylose both aerobically and anaerobically. This was accomplished by cloning and overexpressing three major xylose-metabolizing genes - xylose reductase, xylitol dehydrogenase, and xylulokinase genes (KDR). The resulting genetically engineered yeast can metabolize xylose aerobically and anaerobically as well as effectively co-ferment both glucose and xylose simultaneously to ethanol. First, these three genes were cloned on a high copy number plasmid. Subsequently, we developed an effective and reliable system for integrating multiple copies of multiple genes into the yeast chromosome, and made it possible to effectively integrate the three genes into the chromosomes of any Saccharomyces yeast. In this paper, we compare the ability of haploid, diploid and tetraploid S. cerevisiae with identical genetic background to co-ferment glucose and xylose when transformed with multiple copies of KDR, either on high-copy-number plasmid or integrated on the host chromosomes.
Research Area: Biofuels/Bioproducts
Huang, T., D. Taylor, M. Sedlak, G. Gregori, D. Akin, R. Bashir, M. R. Ladisch, and P. Robinson, BIOT Division, Paper 10, 227th ACS National Meeting, Section: Biosensors and Bioprocess Monitoring and Control, Anaheim, CA, March 28, 2004
Abstract: We demonstrate a simple and rapid (1 hour) technique for fabricating microfluidic flow channels using microfibers positioned on a galss or silica surface, and covered with a preformed poly(dimethylsiloxane) (PDMS) that binds to the surface to give a liquid seal. We used this technique to construct hydrohobic microchannels with a microfiber at its center. This design allows a 5 micron wide stream of liquid to be focused along the side of the microfiber. This phenomenon, utilized in combination with a conventional epi-fluorescence microscope and a photometer allows us to count fluorescently labeled bacteria. A model that quantitates both bacterial motility and convective motion due to fluid movement predicts movement of cells in this microfluidic device. Application of the model, combined with the facile assembly of microfluidic channels, enables biosensors to be designed that integrate microfluidic transport, separation, and detection of pathogenic and non-pathogenic microbes.
Research Area: Food Safety
Lu, Y., and N. Mosier, 36th Great Lakes Regional Meeting of the American Chemical Society, Peoria, IL, October 17, 2004
Abstract:
Research Area: Biofuels/Bioproducts
Chen, W.-T. (Speaker), M. R. Ladisch, T. Geng, and A. K. Bhunia, BIOT Division Paper 140, 227th ACS National Meeting, Section: High Throughput Screening/Genomics and Proteomics, Anaheim, CA, March 30, 2004
Abstract: Rapid concentration and recovery of bacterial cells for the purpose of pathogen detection fluid, derived from meat, may be achieved in less than 30 min using polycarbonate membranes. Concentration of microbial cells accompanied by selective capture of a pathogenic microbe, such as Listeria monocytogenes, from non-pathogenic E. coli, requires a membrane that is functionalized with an antibody capable of selectively capturing the target microbe. We report immobilization techniques that enable attachment of an antibody that is specific to our target organisms, L. monocytogenes, based on Poly-L-Lysine activation of the membrane surface. Subsequent immobilization of the antibody in the presence of glutaraldehyde resulted in a functionalized membrane surface for selective capture of L. monocytogenes from a mixture containing E. coli. The selectivity of the membrane is demonstrated using both imaging and culture techniques. Retention characteristics are modeled based on equilibrium binding of microbe to membrane.
Research Area: Food Safety
Kim, Y., R. Hendrickson, N. S. Mosier, and M. R. Ladisch, BIOT Division, Paper 130, 227th ACS National Meeting, Section: Bioseparations for Primary Recovery, Anaheim, CA, March 30, 2004
Abstract: Extraction of fermentable substrates from biopolymers is a form of primary separatin. Pretreatment of corn fiber by pressure cooking a 15 g/L fiber slurry in water at controlled pH produces soluble oligosaccharides. Our quest for catalysts that mimic the selectivity of cellulolytic enzymes, but at a lower cost, led us to rediscover the utility of a packed bed of strong cation exchange resin for saccharification of these oligosaccharides. The combination of controlled residence time, high ratio of diffisivity of monosaccharides to oligosaccharide, pore structure of the resin, and reactivity of glycosidic bonds in dissolved oligosaccharides, enables hydrolysis to be achieved in a flow reactor while minimizing formation of aldehydes and fermentation inhibitors. We report hydrolysis and diffusional effects for Amberlyst 35W over a temperature range of 100 to 130 C, as well as approaches that minimize fouling of the catalyst by proteins, phenolics and minerals. Conversions of over 80% are achieved.
Research Area: Biofuels/Bioproducts
Sedlak, M., C. Chen and N. W. Y. Ho, 26th Symposium on Biotechnology for Fuels and Chemicals, Abstract No. 2-29, Chattanooga, Tennessee, May 9-12, 2004
Abstract: The naturally occurring Saccharomyces yeasts, particularly those capable of effectively fermenting glucose to ethanol, are unable to metabolize xylose aerobically or anaerobically. We succeeded in developing genetically engineered yeasts that effectively utilize xylose aerobically for growth, as well as effectively co-ferment glucose and xylose to ethanol. However, our genetically engineered yeasts still utilize glucose much faster than xylose. One reason is that the Saccharomyces yeasts deo not contain specific transporters for xylose but instead rely on glucose transporters to transport xylose. Unfortunately, the glucose transporters greatly favor glucose over xylose. Saccharomyces yeasts have at least 7 major glucose transporters (Hxt1-7) with varying affinities for glucose. We studied the affinity of each yeast Hxt transporters for xylose and found that Hxt 4 is one of the transporters with moderate affinity to glucose and xylose. We believe that converting such an Hxt transporter to solely transport xylose could lead to the development of yeast that ferments xylose more efficiently. It was reported that Phe431 is crucial for yeaqst Hxt 2 to transport glucose. In this presentation, we report our recent finding on the role of Phe431 in Hxt 4 for transporting glucose and xylose.
Research Area: Biofuels/Bioproducts
Chen, W.-T., (Poster presenter), M. R. Ladisch, T. Geng, and A. K. Bhunia, BMES Annual Meeting, Nashville, TN, October 2003
Abstract: Membrane filtration has been used widely in separation processes for a long time. It has the ability to sort out different substances based on size difference and also concentrate the target into a smaller area. Our previous study showed success in using polycarbonate membrane (PC) to recover our target organism Listeria monocytogenes, it gives us ideas how we can expand this membrane from simple separation tool to detection platform where separation and pathogen detection can be done simultaneously. PC has defined pore size and pore pathway has made PC a very good candidate for screen filter and our antibody immobilization. The microorganism of interest is Listeria monocytogenes. It is detrimental for immunocompromised people, like the elderly, infants and pregnant women. The average death rate is ~28%, but can be as high as 70% for these immunocompromised persons. L. monocytogenes usually occurs in ready-to-eat (RTE) dairy food, such as hotdogs, cheese and milk. All the facts above have led the U.S. Department of Agriculture (USDA) to set up a "zero tolerance" for L. monocytogenes.
Research Area: Food Safety
Huang, T., T. Geng, D. Akin, W. Chang, J. Sturgis, R. Bashir, A. K. Bhunia, J. P. Robinson, and M. R. Ladisch, BIOT Division Paper 97, 225th ACS National Meeting, Section: Novel Bioanalyses Using Lab-on-a-Chip Technologies, New Orleans, LA, March 24, 2003
Abstract: There has been a growing interest to combine microbeads-based surfaces with microfluidic devices to provide bead-based surfaces with microfluidic devices to provide bead-based separation, detection or analysis of specific biological species. This paper reports fabrication of functionalized particulate monolayer on a C18 coated SiO2 surface via bio-mediate self-assembly or adsorption. A microchip with C18 surface pre-adsorbed with biotinylated BSA enables rapid self-assembly of streptavidin coated microbeads through specific biotin-streptavidin interaction. When coated with BSA, this microchip surface immobilizes polystyrene or dimethylamino beads through possible non-specific hydrophobic or electrostatic interactions. Protein coated microbeads such as ones coated with anti-Listeria antibody cana lso be immobilized onto a bare C18 surface through hyrophobic interactions. A microbead patterned surface results where the only area capable of binding proteins or microbes is the microbead itself, since biotinylated BSA or BSA pre-adsorbed onto a C18 surface blocks non-specific adsorptions. Fluorescnece microscopy was used in this work to study the adsorption of Escherichia coli and the food pathogen Listeria monocytogenes onto various types of microbeads immobilized on microchip surfaces. Four types of microbead coated surfaces were used: (1) streptavidin microbeads surface pre-adsorbed with biotinylated anti-Listeria antibody; (2) anti-Listeria microbead surfaces pre-blocked with BSA; (3) polystyrene microbead surfaces; and (4) dimethylamino microbead surfaces. Streptavidin microbeads pre-adsorbed with biotinylated anti-Listeria antibody and anti-Listeria antibody coated microbeads showed specific capture of L. monocytogenes while polystyrene microbeads and dimethylamino microbeads captured E. coli and L. monocytogenes non-specifically. The combined use of functionalized microbeads for specific capture and biotinylated BSA or BSA for blocking non-specific adsorption enables development of fully functional microfluidic devices for separation, detection or analysis of specific biological species.
Research Area: Food Safety
Chen, W. T., M. R. Ladisch, T. Geng, and A. K. Bhunia, 225th ACS National Meeting, BIOT Division Paper 112, Section: Advances in Bioseparations, New Orleans, LA, March 25, 2003., 225th ACS National Meeting, Section: Advances in Bioseparations, New Orleans, March 25, 2003
Abstract: Membrane filtration has offered the advantage of concentrating substances into small volumes or areas. In this study, we utilized polycarbonate membrane filters with defined pore sizes, and paths for filtering innoculated samples and carried out membrane-based detection for Listeria monocytogenes, which is a foodborne pathogen related to several food recals and listeriosis outbreaks. Membranes immobilized with specific antibodies to L. monocytogenes were used to filter innoculates samples. The chemistry utilizes direction reaction of a spacer with the membrane surface, followed by reaction with a bifunctional cross-linker, glutaraldehyde. Polyclonal anti-Listeria antibody was reacted and covalently bound on this surface. Tests with L. monocytogenes showed capture of this bacteria, which is reduced when the blocking agent BSA is added to the mix. Mechanisms for bacterial capture during microfiltration will be discussed.
Research Area: Food Safety
Huang, T., W.-J. Chang, D. Akin, R. Gomez, R. Bashir, N. Mosier, and M. R. Ladisch, Poster Session: Advances in Biosensors and Biomedical Sensors I, Paper 193a, AIChE National Meeting, San Francisco Hilton & Towers, San Francisco, CA, November 17, 2003
Abstract:
Research Area: Food Safety
Hendrickson, R., N. S. Mosier, and M. R. Ladisch, 25th Symposium on Biotechnology for Fuels and Chemicals, Poster 6A-38, Breckenridge, CO, May 4, 2003
Abstract: Ethanol production utilizing five and six carbon sugars recovered from corn stover hydrolysate has been documented. Hot water pretreatment of corn stover has been shown to assist in the enzxymatic hydrolysis of the biomass to fermentable sugars. Corn stover contains carbon sources other than carbohydrates including lignin (17-18% dry mass) and crude fat (1-2% dry mass). The first objective of this study was to investigate the coproducts generated by modification of the hot water pretreatment method by the addition of varying concentrations of ethanol. Sample from this study were analyzed by GC/MS and contained free fatty-acids (Palmitic and Linoleic acids) and lignin derivatives (coniferyl alcohol, vanillin, etc.) that are soluble in ethanol-water mixtures. Phase two of this study involved passing the pretreatment liquid stream through a tubular reactor containing Amberlyst 35 catalyst. This catalyst is sulfonic acid-based and has an ion exchange capacity of 5.48 meq/gram. Analysis of this liquid stream by GC/MS found ethyl esters of Palmitic acid, Linoleic acid, Oleic acid and Steric acid which are components of bio-diesel. Phenolic compounds identified included 2 ethyl phenol and ethyl 3-(4-hydroxyphenol)-propenate. Solids remaining following pretreatment were hydrolyzed by enzyme with minimal difference in results as compared to water only pretreatment at up to 50% ethanol.
Research Area: Biofuels/Bioproducts
Sedlak, M., Z. Chen, Y. Pang, T. Applegate and N. W. Y. Ho, 215th Symposium on Biotechnology for Fuels and Chemicals, Abstract No. 5-26, Breckenridge, Colorado, May 4-7, 2003
Abstract: We should strive to make the cost for the production of cellulosic ethanol as low as possible. One way to reduce the overall cost for the production of cellulosic ethanol is to produc ehigh valued co-products or by-products during the production of ethanol. One class of co-products could be various industrial enzymes that are high priced products. One important industrial enzyme is phytase, which is used as a supplement in animal feed to improve phosphorus nutrition and to reduce phosphorus pollution of animal excreta. Saccharomyces yeast has the GRAS status and has been used for the preparation of food and drinks for human consumption for thousands of years. Thus, it can be used for the production of any enzyme or special protein including those for human and animal consumption. In this presentation we focus on the expression and secretion of a bacterial phytase in our glucose/xylose co-fermenting Saccharomyces yeast.
Research Area: Biofuels/Bioproducts
Vermerris, W., and M. R. Ladisch, Plant Biotechnology and Feedstock Session, Poster 6B-12, 25th Symposium on Biotechnology for Fuels and Chemicals, Breckenridge, CO, May 4-7, 2003
Abstract: We are looking at c hanging lignin composition as a way to improve the efficiency of bio-fuel production from maize stover. In secondary cell walls, carbohydrates are intimately associated with the hydrophobic polymer lignin. We hypothesize that the enzymatic or chemical hydrolysis of cell wall carbohydrates is impeded by the presence of lignin. Changing the content and subunit composition of lignin is expected to alter the interaction between lignin and carbohydrates and therefore affect the yield of fermentable sugars, ideally in a positive manner. Preliminary experiments with a set of near-isogenic maize mutants with altered lignin composition revealed that (1) changes in lignin composition could increase the yield of fermentable sugars by as much as 35%, and (2) lignin composition is a more important determinant of the yield of fermentable sugars than lignin content. We are currently using a deconvolution strategy to define a relationship between lignin subunit composition and the efficiency of hydrolysis of cell wall carbohydrates. This involves the analysis of a set of single and double cell wall mutants in terms of bio-fuel production, but, given the importance of lignin in the overall viability of the plant, also in terms of agronomic performance. This approach is expected to lead to the development of high-efficiency biofuel crops that still perform well agronomically.
Research Area: Biofuels/Bioproducts
Mosier, N., R. Hendrickson, Y. Kim, M. Zeng, B. Dien, G. Welch, C. E. Wyman, and M. R. Ladisch, Poster Session: Pretreatment of Lignocellulosic Biomass: Update on Biomass Refining CAFI Studies I, Paper 163d, AIChE Annual Meeting, San Francisco, CA , November 20, 2003
Abstract:
Research Area: Biofuels/Bioproducts
Sedlak, M. and N. W. Y. Ho, 25th Symposium on Biotechnology for Fuels and Chemicals, Abstract No. 2-44, Breckenridge, Colorado, May 4-7, 2003
Abstract: Recent studies have proven ethanol to be the ideal liquid fuel for transportation and renewable cellulosic biomass to be the attractive feedstocks for ethanol-fuel production by fermentation. The major fermentable sugars from hydrolysis of cellulosic biomass (such as rice stow, sugarcane bagasse, corn fiber, softwoods, hardwoods, and grasses) are D-glucose and D-xylose. The efficient fermentation of both glucose and xylose present in cellulosic biomass to ethanol is essential for these renewable resources to be used as feedstocks for bio-fuel production. The naturally-occurring Saccharomyces yeasts have proven to be safe, effective, and user-friendly microorganisms for the large-scale production of industrial ethanol from glucose-based feedstocks. However, these yeasts cannot metabolize xylose. Our group at Purdue University succeeded in the development of the genetically engineered Saccharomyces yeasts that can effectively co-ferment glucose and xylose to ethanol. This was accomplished by the cloning and over-expression of three major xylose-metabolizing genes; xylose reductase, xylitol dehydrogenase, and xylulokinase genes in yeast. In this presentation, we demonstrate that our stable recombinant Saccharomyces yeast can efficiently ferment glucose and xylose present in hydrolysates from different cellulosic biomass to ethanol.
Research Area: Biofuels/Bioproducts
Sedlak, M. and N. W. Y. Ho, 24th Symposium on Biotechnology for Fuels and Chemicals, poster 2-08, Gatlinburg, Tennessee, April 28 - May 1, 2002
Abstract: The naturally-occurring Saccharomyces yeasts, particularly those superior for fermenting gluvcose to ethanol, are unable to metabolize xylose aerobically or anaerobically. AWe succeeded in developing metabolically-engineered yeasts that not only metabolized xylose effectively, but could also effectively coferment both glucose and xylose simultaneously to ethanol by altering some of the control mechanisms present in microbial cells. However, our genetically engineered yeasts can still be further improved. For example, our engineered yeasts still utilize glucose much faster than xylose, particularly when the glucose concentration is high. The rate of xylose fermentation is further lowered after 5-6 hrs in glucose-depleted media or when xylose concentration is very low. One reason might be that the glucose transporters in yeast also transport xylose but greatly favor transporting glucose over xylose. It has been reported that at least 7 major glucose transporters (HXT1-7) are expressed under different growth conditions. Another reason might be that some of genes encoding major glycolytic enzymes do not express well in the absence of glucose. Using DNA microarray techniques, we are analyzing the expression of genes encoding the transport proteins and other key enzymes involved in ethanol fermentation in the presence and absence of glucose during co-fermentation of glucose and xylose to ethanol. We will report the results from such analyses.
Research Area:
Chen, W.-T. (Speaker), T. Geng, R. Hendrickson, A. K. Bhunia, and M. R. Ladisch, Paper 328A, AIChE Annual Meeting, Indianapolis, IN, November 7, 2002
Abstract: 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.
Research Area: Bioseparations
Huang, T., J. Sturgis, R. Gomez, T. Geng, R. Bashir, A. K. Bhunia, J. P. Robinson, and M. R. Ladisch, BIOT Division Paper 24, 224th ACS National Meeting, Section: Bioanalyses in the Micro to Nano-flow Regime, Boston, MA, August 18-22, 2002
Abstract: The design and fabrication of protein biochips requires characterization of blocking agents that minimize non-specific binding of proteins or organisms. Non-specific adsorption of E. coli, Listeria innocua, and Listeria monocytogenes is prevented by BSA or biotinylated BSA adsorbed on SiO2 surfaces of a biochip that had been modified with C18 coating. Biotinylated BSA forms a protein-based surface that in turn binds streptavidin. Since streptavidin has multiple binding sites for biotin, it in turn anchors other biotinylated proteins including antibodies. Henve, biotinylated BSA simultaneously serves as a blocking agent and a foundation for binding an interfcing protein, avidin or streptavidin, which in turn anchors biotinylated antibody, which in our case is antibody C11E9, that binds Listeria spp. Non-specific adsorption of another bacterium, E. coli, is minimized due to the blocking action of the BSA. Derivatization of the chip's surfaces and preparation of protein coated chips for anchoring of antibodies is discussed.
Research Area: Food Safety
Huang, T., J. Sturgis, R. Gomez, T. Geng, R. Bashir, A. K. Bhunia, J. P. Robinson, and M. R. Ladisch, Poster 22, International Society for BioMEMS and Biomedical Nanotechnology (ISBBN) Meeting, Columbus, OH, September 6-8, 2002
Abstract: This work describes a simple approach to immobilize functionalized colloidal microstructures onto a C18 coated SiO2 substrate via specific or non-specific bio-mediated interactions. Biotinylated bovine serum albumin pre-adsorbed onto a C18 surface was used to mediate the surface assembly of streptavidin coated microbeads (2.5 um), while a bare C18 surface was used to immobilize anti-Listeria antibody coated microbeads (2.5 um) through hydrophobic interactions. For a C18 surface pre-adsorbed wth bovine serum albumin, hydrophobic polystyrene microbeads (0.8 um) and positively charged dimethylamino microbeads (0.8 um) were allowed to be self-assembled onto the surface. A complete monolayer with high surface coverage was observed for both polystyrene and dimethylamino microbeads. The adsorption characteristics of E. coli and Listeria monocytogenes on these microbeads based surfaces were studied using fluorescence microscopy. Both streptavidin microbeads pre-adsorbed with biotinylated anti-Listeria antibody and anti-Listeria antibody coated microbeads showed specific capture of Listeria monocytogenes, while polystyrene and dimethylamino microbeads captured both E. coli and Listeria monocytogenes non-specifically. The preparation of microbeads based surfaces for the construction of microfluidic devices for separation, detection or analysis of specific biological species is discussed.
Research Area: Food Safety
Welch, G., M. R. Ladisch, R. Hendrickson, N. S. Mosier, and M. Brewer, Twenty-fourth Symposium for Biotechnology for Fuels and Chemicals, Gatlinburg, Tennessee, April 29-30, 2002
Abstract: A process was designed, based on experimental knowledge and industrial experience, to incorporate a corn fiber pretreatment/enzyme hydrolysis/ethanol fermentation system into an existing corn starch-fermenting ethanol plant. This process for corn residue pretreatment was incorporated into an existing corn starch-fermenting ethanol plant for a pilot-scale test of the design. The pretreatment process cnsists of several steps. The corn fiber enters a storage tank where it is mixed with stillage. The resulting slurr is pumped through two heat exchangers; the first heat exchanger transfers heat from the fiber stream leaving the pretreatment reactor to the fiber entering the pretreatment reactor, and the second heat exchanger transfers heat from steam to the fiber stream. The hot fiber stream passes through a snake-coil at 16 C for 20 minutes. It is during this time that the cellulose structure loses the crystalline structure. The fiber stream leaves the pretreatment reactor and exchanges heat with the incoming fiber stream. Finally, an economic analysis of the key process steps was conducted to generate a pro forma analysis for corn fiber/enzyme hydrolysis/ethanol fermentation.
Research Area: Biofuels/Bioproducts
Chen, W.-T., R. Hendrickson, and M. Ladisch, BioMEMS and Biomedical Nanotechnology, Poster 24, Columbus, OH, September 6-8, 2002
Abstract: Detection of foodborne pathogens requires that food samples have to be processed in order to remove interferring factors, including food particles, proteins and lipids, and concentration microorganisms that are to be probed for the presence of pathogens. Conventional involving culture stes may require up to 7 days. In the current study, membrane filtration is able to concentrate the foodborne pathogen, Listeria monocytogenes by a factor of 95 x, with 90% recovery of microorganisms by filtering 50 mL of food samples innoculated with Listeria monocytogenes using a syringe filter. Tween 20 was required to prevent irreversible adsorption of the microorganism to the membrane, due to hydrophobic interactions. Polycarbonate, cellulose, nylon and PVDF membranes were tested for their ability to retain Listeria monocytogenes and to separate proteins from microorganisms. The polycarbonate membrane filters with straight through, mono-radial pores were proved to be the most successful one. The results show that Listeria monocytogenes concentrated in this manner gives sufficient volume of sample for processing on a protein biochip where as little as 1 uL of sample is needed.
Research Area: Food Safety
Chen, W.-T., (Speaker), R. Hendrickson, M. R. Ladisch, T. Geng, and A. K. Bhunia, BIOT Division Paper 77, 224th ACS National Meeting, Section: Advances in Bioseparations, Boston, MA, August 18-22, 2002
Abstract: Biochips offer the promise of quickly detecting foodborne pathogens with time to result of 3 hours or less if the time-consuming microbial enrichment of samples can be avoided. The work addresses the concentration of microbial cells using membrane filtration that can be carried out in 15 minutes. Samples collected from foods are chemically and biologically complex and contain proteins, lipids and fine particles that must be removed to avoid fouling the surface of the biochip. PVDF, nucleopore, nylon and cellulose membranes have been studied for separation of contaminating components and for concentration of cells. The pore size ranges of 0.22 um and 0.45 um were examined. The surface charge of the membrane was negative and did not absorb the cells, since the pH of the samples gave the cells a negative charge, as well. However, capture of the cells by the membrane was also observed due to the membrane structure.
Research Area: Food Safety
Weil, J., B. Dien, R. Bothast, R. Hendrickson, N. S. Mosier, and M. R. Ladisch, Twenty-fourth Symposium for Biotechnology for Fuels and Chemicals, Gatlinburg, Tennessee, April 29-30, 2002
Abstract: The production of aldehydes that are microbial inhibitors may occur when hexoses and pentoses are exposed to temperatures above 150 C and acidic pH in water. These are common conditions encountered when biomass is pretreated. Concentrations of about 0.1% or higher of the degradation product, furfural, strongly inhibit fermentation as was confirmed for hydrolysate that contained 0.5% (w/o) furfural. This paper reports contacting of a polymeric adsorbent, XAD-4, with biomass hydrolysate that contains furfural. Liquid chromatographic analysis of the remaining effluent showed that furfural concentrations were less than 0.1 g/L in contrast to the initial concentrations, which were in the range jof 1 to 5 g/L. Fermentation of the resulting sugars with recombinant E. coli ethanologenic strain K011 confirmed that the concentration of furfural in the hydrolysate was at a low enough level that the inhibition effect was negligible. Fermentation of XAD-4 treated hydrolysate with E. coli K011 was near as rapid as the control medium, which was formulated with reagent grade sugars of the same concentration. Ethanol yields for both fermentations were 90% of theoretical. Modeling of the adsorptive properties of this styrene-based adsorbent indicates that it is suitable for on-off chromatography, and could be useful for removing small amounts of aldehydes that might otherwise inhibit fermentation.
Research Area: Biofuels/Bioproducts
Huang, T., J. Sturgis, R. Bashir, J. P. Robinson and M. R. Ladisch, Paper 340c, AIChE National Meeting, Indianapolis Convention Center, Indianapolis, IN, November 6, 2002
Abstract: Microwicks formed from a continuous strand of twisted threads of natural or synthetic fibers, such as cotton, silk, nylon or polyester, are capable of transporting nanoliter to microliter amounts of fluids. When dipped into a liquid, the microwick can draw the liquid through small flow channels formed bypatterns of fibers, by the action of capillary forces. This work presents the potential application of using microwick to transport one or more liquids into a microfluidic device. It was shown that microliter amounts of bovine serum albumin (BSA) solution can be transported using silk fibers in a matter of seconds. Microwicks formed by silk fibers were also shown to be able to transport bacterial cells (Listeria innocua) that were suspended in a carbonate bicarbonate buffer. By interfacing the silk fibers with a glass flow channel, a rapid and reliable introduction of bacterial cells (Listeria innocua) into the glass microchannel through the microwick was clearly demonstrated.
Research Area: Food Safety
Huang, T., W. Chen, T. Geng, R. Gomez, R. Bashir, A. Bhunia, and M. R. Ladisch, Invited Lecture, Paper 45-1, 2004 Institute of Food Technologies Annual Meeting, Symposium: Nanoscale Science, Engineering and Technology for Food Safety and Engineering ", Las Vegas, NV, July 14, 2004
Abstract: Nanoscience is the fabrication, study, and modeling of principles of devices and structures for which at least one dimension is several 100 nanometers or smaller. Nanotechnology is the enabling component of the discovery and development process that assembles nano-structures into compact, portable devices that carry out sensing functions currently relegated to scientific laboratories. Some types of devices will integrate biotechnology with silicon or plastic surfaces to form bio-sensing systems that enhance detection and enable study of biomarkers generated in response to environmental stress and other biological conditions of importance to agriculture. When coupled with devices that have capabilities to give temporal and geographic information, nanotechnology may contribute to tracking of agricultural commodities. This paper will discuss possible applications of very small, intelligent, sensing devices for monitoring products from a widely distributed, global agricultural enterprise, and their potential contribution to identity preservation.
Research Area: Food Safety