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Bioenergy Publications

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

2016

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

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

Research Area: Bioseparations Bioenergy   

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Fiber-Based Monolithic Columns for Liquid Chromatography

2016

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

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

Research Area: Bioenergy    

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

2016

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

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

Research Area: Bioseparations Bioenergy   

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Liquid Hot Water Pretreatment Inhibitors

2016

Authors: E. Ximenes, Y. Kim, C. Farinas, M. R. Ladisch, 251st National ACS Meeting, Biofuel & Biobased Chemical Production: Biomass Pretreatment and Hydrolysis, San Diego, CA, March 14, 2016
Journal:
Book Chapter:

Abstract: Liquid hot water pretreatment enhances the rates and extents of cellulose hydrolysis for corn stover, sugar cane bagasse, switchgrass, hardwood, and other lignocellulosic materials as long as there is sufficient enzyme present to catalyze the reaction. The rationale that drives the use of pretreatment is the reduction in cost of enzyme and feedstock by increasing yields of fermentable sugars, principally glucose and xylose. Compared to untreated lignocellulose, pretreated feedstocks result in enhanced hydrolysis since pretreatment opens up the cell wall structure of the substrate, thereby enabling access of enzyme to the cellulose and disrupting the tightly packed cellulose structure. However, pretreatments also release inhibitors. More severe pretreatments are not always better since they can release greater amounts of inhibitors and deactivators which significantly reduce enzyme activity. Inhibitors include xylo-oligosaccharides, acetic acid, tannic acid, and phenolics. This effect is particularly noticeable as enzyme loading is decreased and the ratio of biomass derived inhibitors to added enzyme protein increases. Higher severity pretreatment may also expose more lignin as well as more cellulose in the cell wall structure. The lignin may unproductively adsorb proteins, including enzymes. Hence pretreatment can both help and hinder the enzyme hydrolysis of cellulose. This paper describes interactions between multiple enzyme components, inhibitors, and pretreated lignocellulosic substrates. Mitigation strategies are presented that reduce the amount of enzymes required to overcome inhibition due to pretreatment and achieve high conversion of lignocellulosic feedstocks to fermentable monosaccharides.

Research Area: Bioseparations Bioenergy Biofuels/Bioproducts Bioprocessing 

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

2016

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

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

Research Area: Bioenergy    

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Maleic Acid Treatment of Bioabated Corn Stover Liquors Improves Cellulose Conversion to Ethanol

2016

Authors: D. Kim, E. Ximenes, G. Cao, N. N. Nichols, S. Frazer, M. R. Ladisch, 38th Symposium on Biotechnology for Fuels and Chemicals, Poster Session 1: Bioprocessing, Reactor Design, and Separations Technology; Pretreatment and Fractionation; Microbial Science and Technology; Molecular Engineering, Synthetic Systems Biology, Poster M66, April 25, 2016, Baltimore, MD
Journal:
Book Chapter:

Abstract: Elimination of inhibitory compounds released during pretreatment of lignocellulose is critical for efficient cellulose conversion and ethanol fermentation. This study examined the effect of bioabated liquor from pretreated corn stover on enzyme hydrolysis of Solka Floc or pretreated corn stover solids. Xylo-oligosaccharides in the liquor were hydrolyzed by hemicellulose or maleic acid. Pretreatment was at 20% solids, 190 C, 45 min, and subsequent hydrolysis, after bioabatement was done with 5% corn stover, and ethanol fermentation by Saccharomyces cerevisiae. The fungus Coniochaeta ligniaria NRRL30616 removed inhibitory compounds in the liquor from LHW-pretreated corn stover. The conversion of cellulose to glucose in bioabated liquor was higher when the liquor was treated with maleic acid than with hemicellulose. For corn stover slurried in hemicellulose treated liquor, cellulose conversion was 39%, while corn stover in maleic acid treated liquor gave 68% yield. The observed lower glucose yield may be related to inhibition of beta-xylosidase caused by accumulation of xylo-oligomers, which in turn inhibited beta-glucosidase, leading to accumulation of cellobiose. The use of maleic acid alleviated the inhibitory effect on beta-glucosidase by hydrolyzing the xylo-oligomers to xylose. Ethanol production from Solka Floc hydrolysate or sugars from corn stover solids was 20 to 30% higher for bioabated liquor compared to non-bioabated liquor. Furthermore, the fermentation lag phase was decreased by 3 hours. Our results confirm bioabatement removes compounds that inhibit enzyme hydrolysis and fermentation. The treatment of bioabated samples with maleic acid improved overall cellulose conversion due to hydrolysis of xylo-oligomers to xylose, where xylose is much less inhibitory towards beta-glucosidase.

Research Area: Bioprocessing Bioenergy Bioseparations  

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Maleic Acid Treatment of Biologically Detoxified Corn Stover Liquor

2016

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

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

Research Area: Bioenergy Biofuels/Bioproducts Bioseparations Bioprocessing 

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

2016

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

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

Research Area: Bioenergy Bioprocessing Bioseparations  

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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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Adsorption of Enzyme Onto Lignins of Liquid Hot Water Pretreated Hardwoods

2015

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

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

Research Area: Bioenergy Bioprocessing Bioseparations  

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Bioabatement with Hemicellulase Supplementation to Reduce Enzymatic Hydrolysis Inhibitors

2015

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

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

Research Area: Bioenergy Bioprocessing Biofuels/Bioproducts  

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

2015

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

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

Research Area: Bioenergy Bioprocessing Biofuels/Bioproducts  

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

2015

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

Abstract: Lignin, one of the major components of lignocellulosic biomass, plays an inhibitory role on the enzymatic hydrolysis of cellulose. This work examines the role of lignin in pretreated hardwood, where extents of cellulose hydrolysis decrease, rather than increase with increasing severity of liquid hot water pretreatment. Hardwood pretreated with liquid hot water at severities ranging from log Ro = 8.25 to 12.51 resulted in 80-90% recovery of the initial lignin in the residual solids. The ratio of acid insoluble lignin (AIL) to acid soluble lignin (ASL) increased and the formation of spherical lignin droplets on the cell wall surface was observed as previously reported in the literature. When lignins were isolated from hardwoods pretreated at increasing severities and characterized based on glass transition temperature (Tg), the Tg of isolated lignins was found to increase from 171 to 180 C as the severity increased from log RoĽ10.44 to 12.51. The increase in Tg suggested that the condensation reactions of lignin molecules occurred during pretreatment and altered the lignin structure. The contribution of the changes in lignin properties to enzymatic hydrolysis were examined by carrying out Avicel hydrolysis in the presence of isolated lignins. Lignins derived from more severely pretreated hardwoods had higher Tg values and showed more pronounced inhibition of enzymatic hydrolysis.

Research Area: Bioenergy Bioprocessing   

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

2015

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

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

Research Area: Bioenergy Biofuels/Bioproducts Bioprocessing  

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Hydrolysis-Determining Substrate Characteristics in Liquid Hot Water Pretreated Hardwood

2015

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

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

Research Area: Bioenergy Biofuels/Bioproducts Bioprocessing  

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

2015

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

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

Research Area: Bioenergy Bioprocessing Biofuels/Bioproducts  

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

2015

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

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

Research Area: Bioenergy Bioprocessing   

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

2015

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

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

Research Area: Bioenergy Biofuels/Bioproducts   

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

2015

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

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

Research Area: Bioenergy    

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

2015

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

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

Research Area: Bioenergy    

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Tandem Mass Spectrometric Characterization of the Conversion of Xylose to Furfural

2015

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

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

Research Area: Bioenergy Biofuels/Bioproducts   

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Biological Engineering and the Emerging Cellulose Ethanol Industry

2014

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

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

Research Area: Biofuels/Bioproducts Bioenergy   

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

2014

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

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

Research Area: Bioenergy Bioprocessing   

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

2014

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

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

Research Area: Bioenergy    

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

2014

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

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

Research Area: Bioenergy Bioprocessing   

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

2014

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

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

Research Area: Bioenergy Bioprocessing Biofuels/Bioproducts  

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Liquefaction of Sugarcane Bagasse for Enzyme Production

2014

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

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

Research Area: Bioprocessing Bioenergy Biofuels/Bioproducts  

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

2014

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

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

Research Area: Bioenergy Bioprocessing   

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Modeling Water Quality Impacts of Cellulosic Biofuel Production from Corn Silage

2014

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

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

Research Area: Bioenergy Bioprocessing   

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

2014

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

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

Research Area: Bioenergy    

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

2013

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

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

Research Area: Bioprocessing Bioenergy Bioseparations  

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Biomass Chemistry

2013

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

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

Research Area: Bioprocessing Bioenergy   

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

2013

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

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

Research Area: Bioenergy Bioprocessing Bioseparations  

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Fractionation of Cellulase and Fermentation Inhibitors from Steam Pretreated Mixed Hardwood

2013

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

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

Research Area: Bioenergy    

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

2013

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

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

Research Area: Bioenergy    

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

2013

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

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

Research Area: Bioenergy Bioprocessing   

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

2012

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

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

Research Area: Bioenergy    

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

2008

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

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

Research Area: Bioenergy    

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

1996

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

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

Research Area: Bioenergy    

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Analysis of Plant Harvest Indices for Bioregenerative Life Support Systems

1995

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

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

Research Area: Bioenergy    

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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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Bioprocessing in Space

1992

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

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

Research Area: Bioenergy    

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

1986

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

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

Research Area: Bioenergy Bioprocessing   

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

1985

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

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

Research Area: Bioenergy Bioseparations   

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Preparation of Cellodextrins Using Sulfuric Acid

1984

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

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

Research Area: Bioenergy    

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Purification and Properties of Glucose Isomerase of Actinoplanes missouriensis

1980

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

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

Research Area: Bioenergy Bioprocessing Biofuels/Bioproducts  

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

1979

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

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

Research Area: Bioenergy Biofuels/Bioproducts   

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

1979

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

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

Research Area: Bioenergy Bioprocessing   

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