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Biofuels/Bioproducts Publications

Maleic Acid Treatment of Biologically Detoxified Corn Stover Liquor

2016

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

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

Research Area: Bioenergy Biofuels/Bioproducts Bioseparations Bioprocessing 

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

2015

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

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

Research Area: Bioenergy Biofuels/Bioproducts Bioprocessing  

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

2015

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

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

Research Area: Bioenergy Biofuels/Bioproducts Bioprocessing  

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Hydrolysis-Determining Substrate Characteristics in Liquid Hot Water Pretreated Hardwood

2015

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

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

Research Area: Bioenergy Biofuels/Bioproducts Bioprocessing  

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

2015

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

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

Research Area: Bioenergy Biofuels/Bioproducts   

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Tandem Mass Spectrometric Characterization of the Conversion of Xylose to Furfural

2015

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

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

Research Area: Bioenergy Biofuels/Bioproducts   

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Biological Engineering and the Emerging Cellulose Ethanol Industry

2014

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

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

Research Area: Biofuels/Bioproducts Bioenergy   

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

2014

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

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

Research Area: Biofuels/Bioproducts    

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Effect of Maleic Acid on the Selectivity of Glucose and Fructose Dehydration and Degradation

2014

Authors: X. Zhang, B. Hewetson, N. S. Mosier AIChE Meeting, Atlanta, GA, November 20, 2014
Journal:
Book Chapter:

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 Biofuels/Bioproducts Bioprocessing  

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Investigation of Fatty Acid Methyl Esters in Jet Fuel

2014

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

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

Research Area: Biofuels/Bioproducts    

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Understanding ASTM Turbine Fuel Standard Specifications

2014

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

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

Research Area: Biofuels/Bioproducts    

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

2012

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

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

Research Area: Biofuels/Bioproducts    

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

2011

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

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

Research Area: Biofuels/Bioproducts    

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Biofuels from Cellulosic Feedstocks

2011

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

Abstract:

Research Area: Biofuels/Bioproducts    

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Cassava Starch Pearls as a Desiccant for Drying Ethanol

2011

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

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

Research Area: Biofuels/Bioproducts    

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

2011

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

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

Research Area: Biofuels/Bioproducts    

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

2011

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

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

Research Area: Biofuels/Bioproducts    

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Deactivation of Cellulases by Phenols

2011

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

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

Research Area: Biofuels/Bioproducts    

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Dry Grind Coproducts as Cellulosic Ethanol Feedstock

2011

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

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

Research Area: Biofuels/Bioproducts    

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

2011

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

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

Research Area: Biofuels/Bioproducts    

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

2011

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

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

Research Area: Biofuels/Bioproducts    

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Investigation of Enzyme Formulation on Pretreated Switchgrass

2011

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

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

Research Area: Biofuels/Bioproducts    

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Soluble Inhibitors/Deactivators of Cellulase Enzymes from Lignocellulosic Biomass

2011

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

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

Research Area: Biofuels/Bioproducts    

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Surface and Ultrastructural Characterization of Raw and Pretreated Switchgrass

2011

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

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

Research Area: Biofuels/Bioproducts    

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Bioenergy: Renewable Liquid Fuels

2010

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

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

Research Area: Biofuels/Bioproducts    

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Converting Cellulose to Biofuels

2010

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

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

Research Area: Biofuels/Bioproducts    

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

2010

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

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

Research Area: Biofuels/Bioproducts    

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

2010

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

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

Research Area: Biofuels/Bioproducts    

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

2010

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

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

Research Area: Biofuels/Bioproducts    

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Inhibition of Cellulases by Phenols

2010

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

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

Research Area: Biofuels/Bioproducts    

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

2010

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

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

Research Area: Biofuels/Bioproducts    

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

2009

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

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

Research Area: Biofuels/Bioproducts    

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

2009

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

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

Research Area: Biofuels/Bioproducts    

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Enzymatic Digestion of Liquid Hot Water Pretreated Hybrid Poplar

2009

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

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

Research Area: Biofuels/Bioproducts    

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Ethanol Production from Maize (book chapter)

2009

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

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

Research Area: Biofuels/Bioproducts    

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Liquid Hot Water Pretreatment of Cellulosic Biomass

2009

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

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

Research Area: Bioenergy Biofuels/Bioproducts Bioprocessing  

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Cellulose Conversion in Dry Grind Ethanol Plants

2008

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

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

Research Area: Biofuels/Bioproducts    

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

2008

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

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

Research Area: Biofuels/Bioproducts    

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

2008

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

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

Research Area: Biofuels/Bioproducts    

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

2008

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

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

Research Area: Biofuels/Bioproducts    

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

2008

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

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

Research Area: Biofuels/Bioproducts    

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

2008

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

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

Research Area: Biofuels/Bioproducts    

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

2008

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

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

Research Area: Biofuels/Bioproducts    

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

2008

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

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

Research Area: Biofuels/Bioproducts    

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

2008

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

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

Research Area: Biofuels/Bioproducts    

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Pathways for Development of a Biorenewables Industry

2008

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

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

Research Area: Biofuels/Bioproducts    

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

2008

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

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

Research Area: Biofuels/Bioproducts    

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

2008

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

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

Research Area: Biofuels/Bioproducts    

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Water Solubilization of DDGS via Derivatization with Phosphite Esters

2008

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

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

Research Area: Biofuels/Bioproducts    

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Biomimetic Catalysis for Hemicellulose Hydrolysis in Corn Stover

2007

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

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

Research Area: Biofuels/Bioproducts    

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Lignin Modification Improves Fermentable Sugar Yields for Biofuel Production

2007

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

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

Research Area: Biofuels/Bioproducts    

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Loosening Lignin's Grip on Biofuel Production

2007

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

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

Research Area: Biofuels/Bioproducts    

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

2007

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

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

Research Area: Biofuels/Bioproducts    

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Molecular Breeding to Enhance Ethanol Production from Corn and Sorghum Stover

2007

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

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

Research Area: Biofuels/Bioproducts    

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

2005

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

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

Research Area: Biofuels/Bioproducts    

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Comparison of Two Adsorbents for Sugar Recovery from Biomass Hydrolyzate

2005

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

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

Research Area: Biofuels/Bioproducts    

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Coordinated Development of Leading Biomass Pretreatment Technologies

2005

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

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

Research Area: Biofuels/Bioproducts    

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Features of Promising Technologies for Pretreatment of Lignocellulosic Biomass

2005

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

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

Research Area: Biofuels/Bioproducts    

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

2005

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

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

Research Area: Biofuels/Bioproducts    

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Optimization of pH Controlled Liquid Hot Water Pretreatment of Corn Stover

2005

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

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

Research Area: Biofuels/Bioproducts    

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

2005

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

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

Research Area: Biofuels/Bioproducts    

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

2004

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

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

Research Area: Biofuels/Bioproducts    

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

2004

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

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

Research Area: Biofuels/Bioproducts    

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Rapid Chromatography for Evaluating Adsorption Characteristics of Cellulase Binding Domain Mimetics

2004

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

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

Research Area: Biofuels/Bioproducts    

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

2003

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

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

Research Area: Biofuels/Bioproducts    

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

2003

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

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

Research Area: Biofuels/Bioproducts    

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

2003

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

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

Research Area: Biofuels/Bioproducts Bioprocessing   

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Characterization of Acid Catalytic Domains for Cellulose Hydrolysis and Glucose Degradation

2002

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

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

Research Area: Biofuels/Bioproducts    

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

2002

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

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

Research Area: Biofuels/Bioproducts    

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Characterization of Dicarboxylic Acids for Cellulose Hydrolysis

2001

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

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

Research Area: Biofuels/Bioproducts    

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

2001

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

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

Research Area: Biofuels/Bioproducts    

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Opportunities in Biotechnology for Future Army Applications

2001

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

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

Research Area: Biofuels/Bioproducts    

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

2000

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

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

Research Area: Biofuels/Bioproducts    

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Ethanol Production from Renewable Resources

1999

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

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

Research Area: Biofuels/Bioproducts    

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

1999

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

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

Research Area: Biofuels/Bioproducts    

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

1999

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

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

Research Area: Biofuels/Bioproducts    

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Production of Multifunctional Organic Acids from Renewable Resources

1999

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

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

Research Area: Biofuels/Bioproducts    

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Reaction Kinetics, Molecular Action, and Mechanisms of Cellulolytic Proteins

1999

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

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

Research Area: Biofuels/Bioproducts    

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

1999

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

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

Research Area: Biofuels/Bioproducts    

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

1999

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

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

Research Area: Biofuels/Bioproducts    

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

1998

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

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

Research Area: Biofuels/Bioproducts    

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

1998

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

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

Research Area: Biofuels/Bioproducts    

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Pretreatment of Corn Fiber By Pressure Cooking in Water

1998

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

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

Research Area: Biofuels/Bioproducts    

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

1997

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

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

Research Area: Biofuels/Bioproducts    

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

1997

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

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

Research Area: Biofuels/Bioproducts    

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

1997

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

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

Research Area: Biofuels/Bioproducts    

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Pretreatment of Yellow Poplar Sawdust by Pressure Cooking in Water

1997

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

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

Research Area: Biofuels/Bioproducts    

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

1997

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

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

Research Area: Biofuels/Bioproducts    

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Assessment of Ethanol Production Options for Corn Products

1996

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

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

Research Area: Biofuels/Bioproducts    

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

1996

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

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

Research Area: Biofuels/Bioproducts    

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

1996

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

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

Research Area: Biofuels/Bioproducts    

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Sorptive Recovery of Dilute Ethanol from Distillation Column Bottom Stream

1996

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

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

Research Area: Biofuels/Bioproducts    

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Enhanced Enzyme Activities on Hydrated Lignocellulosic Substrates

1995

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

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

Research Area: Bioenergy Biofuels/Bioproducts Bioseparations  

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Synthesis of TAME Using Solid Acid Catalysts

1995

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

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

Research Area: Biofuels/Bioproducts    

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Cellulose Pretreatments of Lignocellulosic Substrates

1994

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

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

Research Area: Biofuels/Bioproducts    

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Increasing MTBE Production Without Increasing Capital Costs

1994

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

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

Research Area: Biofuels/Bioproducts    

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Biological-Based Systems for Waste Processing

1993

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

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

Research Area: Biofuels/Bioproducts    

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Catalyst-Induced Yield Enhancement in a Tubular Reactor

1993

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

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

Research Area: Biofuels/Bioproducts    

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

1992

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

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

Research Area: Biofuels/Bioproducts    

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Intercalation in the Pretreatment of Cellulose

1992

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

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

Research Area: Biofuels/Bioproducts    

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Ethanol Production and the Cost of Fermentable Sugars from Biomass

1991

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

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

Research Area: Biofuels/Bioproducts    

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

1991

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

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

Research Area: Biofuels/Bioproducts    

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

1991

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

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

Research Area: Biofuels/Bioproducts    

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Alcohol Adsorption on Softwood Lignin from Aqueous Solutions

1990

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

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

Research Area: Biofuels/Bioproducts    

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Stoichiometry and Kinetics of Xylose Fermentation by Pichia stipitis

1990

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

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

Research Area: Biofuels/Bioproducts Bioprocessing   

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

1990

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

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

Research Area: Biofuels/Bioproducts    

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

1989

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

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

Research Area: Biofuels/Bioproducts    

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Construction of Yeast Xylulokinase Mutant by Recombinant DNA Techniques

1989

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

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

Research Area: Biofuels/Bioproducts    

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Hydrolysis

1989

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

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

Research Area: Biofuels/Bioproducts    

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

1987

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

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

Research Area: Biofuels/Bioproducts    

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

1987

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

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

Research Area: Biofuels/Bioproducts    

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

1987

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

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

Research Area: Biofuels/Bioproducts    

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Engineering and Economics of Cellulose Saccharification Systems

1986

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

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

Research Area: Biofuels/Bioproducts    

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

1986

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

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

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Methyl t-Butyl Ether (MTBE) Process Catalyst Parameters

1986

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

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

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2, 3-Butanediol

1985

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

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

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Anaerobic Fermentation: Microbes from Ruminants

1985

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

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

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

1985

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

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

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Effect of Pretreatments and Fermentation on Pore Size in Cellulosic Materials

1985

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

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

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

1985

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

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

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Acid Hydrolysis of Pretreated Lignocellulose from Corn Residue

1984

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

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

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Bioutilization of Cereal Lignocellulose

1984

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

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

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Cloning and Characterization of the xyl Genes from Escherichia coli

1984

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

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

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

1984

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

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

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Effect of Solvent Treatments on Intake and Digestibiity of Corn Stover

1983

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

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

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Process Considerations in Enzymatic Hydrolysis of Biomass

1983

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

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

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

1983

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

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

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Symposium on Fuels and Chemicals From Biomass

1983

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

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

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

1983

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

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

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Production of Ethanol and Chemicals from Cellulosic Materials

1982

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

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

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

1981

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

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

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Combined Product and Substrate Inhibition Equation for Cellobiase

1981

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

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

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

1981

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

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

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Review on Effect of Pretreatment on Digestibility of Cellulosic Materials

1981

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

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

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

1981

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

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

Research Area: Bioseparations Biofuels/Bioproducts   

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Alcohol from Cellulose

1980

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

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

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

1980

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

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

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New Approach to Aqueous Gel Permeation Chromatography of Nonderivatized Cellulose

1980

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

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

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

1979

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

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

Research Area: Bioenergy Biofuels/Bioproducts   

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Fermentable Sugars from Cellulosic Residues

1979

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

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

Research Area: Biofuels/Bioproducts    

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Fuels and Chemicals from Biomass

1979

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

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

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Measurement of Cellulolytic Activity by Low Pressure Liquid Chromatography

1979

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

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

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Cellulose to Sugars: New Path Gives Quantitative Yield

1978

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

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

Research Area: Biofuels/Bioproducts    

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Fermentation Substrates from Cellulosic Materials

1978

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

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

Research Area: Biofuels/Bioproducts    

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Fuels and Chemicals from Biomass

1978

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

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

Research Area: Biofuels/Bioproducts    

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Preparation of Cellodextrins: An Engineering Approach

1978

Authors: Huebner, A., M. R. Ladisch, and G. T. Tsao
Journal: Biotechnol. Bioeng., 20, 1669-1677 (1978)
Book Chapter:

Abstract: Cellodextrins are linear polymers of two to seven B-1-4 linked glucose molecules. Properties of these oligosaccharides include a decreasing water solubility with increasing molecular weight limited solubility in nonaqueous or partially aqueous solvents, and a melting point which increases with increasing weight. These and other properties are summarized in Table I.

Research Area: Biofuels/Bioproducts    

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Protein Determination in the Presence of Cellulose

1978

Authors: Ladisch, M. R., C. M. Ladisch, and G. T. Tsao
Journal: Biotechnol. Bioeng., 20, 461-462 (1978)
Book Chapter:

Abstract: Protein may not be assayed spectrophotometrically in the presence of cellulose using cadoxen, a solvent in which both cellulose and protein are soluble. This solvent is a solution of 5% cadmium oxide in 28oC aqueous ethylene diamine, which dissolves small quantities of cellulose, hemicellulose, and other polysaccharides quickly and easily at room temperature conditions. It is presently used in the textile field for viscometric studies on cellulose. Since cadoxen also dissolves protein, it appears that this solvent may also be applicable to protein determination in the manner described below.

Research Area: Biofuels/Bioproducts    

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Cellobiase from Trichoderma viride: Purification Properties, Kinetics and Mechanism

1977

Authors: Gong, C. S., M. R. Ladisch, and G. T. Tsao
Journal: Biotechnol. Bioeng., 19, 959-981 (1977)
Book Chapter:

Abstract: Three distinct cellobiose components were isolated from a commercial Trichoderma viride cellulase preparation by repeated chromatography on DEAE cellulose eluting by a salt gradient. The purified cellobiose preparations were evaluated for physical properties, kinetics, and mechanism. Results from this work include: 1) development of a one step enzyme purification procedure using DEAE-cellulose; 2) isolation of three chromatographically distinct, yet kinetically similar, cellobiose fractions of molecular weight of ~ 76,000; 3) determination of kinetics which shows that cellobiose hydrolyzes cellobiose by a noncompetitive mechanism and that the product, glucose, inhibits the enzyme, and 4) development of an equation, based on the mechanism of cellobiose action, which accurately predicts the time course of cellobiose hydrolysis over an eightfold range of substrate concentration and conversions of up to 90%. Based on the data presented in the paper, it is shown that product inhibition of cellobiose significantly retards the rate of cellobiose hydrolysis.

Research Area: Biofuels/Bioproducts    

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Corn Crop Residues as a Potential Source of Single Cell Protein: Kinetics of T. viride Cellobiase Action

1977

Authors: Ladisch, M. R., C. S. Gong, and G. T. Tsao
Journal: Dev. Ind. Microbiology (18) 157-168 (1977)
Book Chapter:

Abstract: There are 67 to 167 million tons of corn-crop residues generated each year in the United States. A significant portion of these is available for use for purposes other than ground cover. One future major use of corn-crop residues will be as animal feed, an application which requires it to be fortified with protein. Cultivation of a microorganism, such as Trichoderma viride, on the residue to serve as a source of single-cell protein might obviate the need for at least part of the protein supplement. In this report considerations are discussed in practical terms affecting the availability and utilization of corn-crop residues. Also treated are the kinetics and the effect of product inhibition (by glucose) of cellobiase, one of several enzymes utilized by cellulolytic microorganisms to degrade cellulose.

Research Area: Biofuels/Bioproducts    

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Economic Implications of Purification of Glucose Isomerase Prior to Immobilizations

1977

Authors: Ladisch, M. R., A. Emery, and V. W. Rodwell
Journal: Ind. Eng. Chem.Process Des. and Dev. 16(3), 309-313 (1977)
Book Chapter:

Abstract: We examined the impact on total process cost of purifying enzymes prior to immobilization for use in industrial processes. Glucose isomerase extracted from Bacillus cells was fractionated with acetone and ammonium sulfate yielding preparations of three different specific activities. These were immobilized on porous alkylamine glass beads. Catalyst activity and stability, monitored in a plug-flow reactor, all increased with specific activity of the soluble isomerase immobilized. To examine economic consequences of enzyme purification, we compared reactors producing 106 lb of fructose per year. Total process cost fell dramatically with enzyme purity. Our analysis suggests economic gain may accompany purification prior to enzyme immobilization.

Research Area: Biofuels/Bioproducts    

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