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Bioprocessing Publications

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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UNSEP Bioenergy Short Course

2016

Authors: M. Ladisch, E. Ximenes, Bioenergy Short Course, UNSEP, San Jose do Rio Preto, SP Brazil
Journal:
Book Chapter:

Abstract: The emergence of bioenergy as a major source of low carbon footprint transportation fuels with potential to provide electricity and power for stationary applications will require agriculture to provide sustainable feedstocks for this emerging industry. In addition, advances in enzymes and microbial biotechnology, scale-up through bioprocess engineering, and carbon efficient utilization of renewable resources will be major factors if agriculture is able to provide food, feed, fiber, and bioprocess feedstocks. This intensive short course addressed the critical topics that define bioenergy. The topics to be addressed are: 1. Basic biomass biochemistry; 2. Mechanisms of enzyme hydrolysis of pretreated lignocellulosic feedstocks; 3. Bioprocess design at large-scale lignocellulose conversion processes to produce fuel alcohol and bioproducts; and 4. Analysis of approaches that integrate sustainability and food production. The course will be taught in 4 modules that will address each of these topics. Renewable feedstocks to be considered include sugarcane bagasse, corn stover, and hardwood. Enzymes to be discussed will include cellulases and hemicellulases derived from T. reesei and A. niger. These enzymes are capable of hydrolyzing both insoluble cellulose and hemicellulose, as well as soluble oligosaccharides to monosaccharides. Their efficiency in generating five and six carbon sugars is an important factor for large-scale cellulose conversion to ethanol in a cost effective manner. In addition, characteristics of yeast fermentations, and the role of lignocellulose derived inhibitors will also be addressed. Mapping of the key biotechnologies into equipment that is needed to facilitate large-scale production will be outlined. This short will conclude with a discussion of sustainability and the impacts of major trends in world population, energy use, transportation fuels, and economic factors on achieving a world with a manageable carbon footprint and a sustainable food and energy supply.

Research Area: Bioenergy Bioprocessing   

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UNSEP Short Course in Basic Biomass Chemistry

2016

Authors: M. Ladisch, E. Ximenes, Bioenergy Short Course, UNSEP, San Jose do Rio Preto SP, Brazil
Journal:
Book Chapter:

Abstract: The emergence of bioenergy as a major source of low carbon footprint transportation fuels with potential to provide electricity and power for stationary applications will require agriculture to provide sustainable feedstocks for this emerging industry. In addition, advances in enzyme and microbial biotechnology, scale-up through bioprocess engineering, and carbon efficient utilization of renewable resources will be major factors if agriculture is able to provide food, feed, fiber, and bioprocess feedstocks. This intensive short course will address the critical topics of that define bioenergy. The topics to be addressed are: 1. Basic biomass biochemistry; 2. Mechanisms of enzyme hydrolysis of pretreated lignocellulosic feedstocks; 3. Bioprocess design at large-scale lignocellulose conversion processes to produce fuel alcohol and bioproducts; and 4. Analysis of approaches that integrate sustainability and food production.

Research Area: Bioenergy Bioprocessing Biofuels/Bioproducts  

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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. Nishols, S. E. Frazer, D. Kim, M. A. Cotta, M. Ladisch, presented at 37th Symposium on Biotechnology for Fuel and Chemicals, San Diego, CA, April, 2015
Journal: Bioresource Technology, 190, 412-415, 2015
Book Chapter:

Abstract: Bioabatement, using the fungus Coniochaeta ligniaria NRRL30616 can effectively eliminate enzyme inhibitors from pretreated biomass hydrolysates. However, our recent research suggested that bioabatement had no beneficial effect on removing xylo-oligomers which are strong inhibitors to cellulase. Here, we evaluated bioabatement with xylanase supplementation to mitigate potential enzyme inhibitors observed in corn stover liquors after pretreatment with liquid hot water at 10% (w/v) solids. Resuslts showed that cellulose conversion in the presence of 10% (w/v) LHW-pretreated liquor reached 70.5% and 57.4%, for conversion of Solka Flock cellulose and pretreated corn stover solids, respectively, after bioabatement and xylanase supplementation. These represent an increase of 21.6% and 17.6%, respectively, in comparison with non-treated samples. The squence in which xylanase and cellulase are added affects cellulose conversion, possibly as a result of competition between xylanase cellulase binding to xylo-oligomers. Replacement of xylanase using maleic acid treatment to hydrolyze xylo-oligomers yielded equivalent increases in efficiency of cellualse hydrolysis.

Research Area: Bioenergy Bioprocessing   

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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 on Enzyme Loading and Hydrolysis of Hardwood

2015

Authors: M. R. Ladisch, Y. Kim, J. K. Ko, T. Kreke, E. Ximenes, 2015 AIChE Meeting, Paper 775b, Salt Lake City, Utah, November 13, 2015
Journal: 2015 AIChE Meeting, Salt Lake City, Utah, Paper 775b, November 13, 2015
Book Chapter:

Abstract: A fundamental understanding of the combined factors that impact recalcitrance in enzyme hydrolysis of pretreated hardwood explains how cellulase loading may be decreased by a factor of 10 while maintaining 80% glucose yield when non-catalytic protein is added to the enzyme. Factors that impact enzyme hydrolysis of solid biomass include the interaction of the cellulase and beta-glucosidase components with solubilized phenolic inhibitors and the enhanced accessibility of lignin as a consequence of pretreatment. While the added protein decreases overall specific activity of the enzyme, it also reduces cellulase adsorption on lignin, thus making more enzyme available for cellulose hydrolysis. Consequently, 15 and 1.3 FPU cellulase/g total solids both give 80% yield, with the 1.3 FPU loading approaching the enzyme levels usually associated with amylases in starch hydrolysis. These results reinvigorate motivation for the search for other approaches that prevent enzyme adsorption on lignin and enable high glucose yields at low enzyme loadings. This paper presents measurements in our laboratory and prior reports from the literature to offer an explanation of how changes in the physical attributes of cellulosic biomass during liquid hot water pretreatment affect glucose yields and enzyme loading.

Research Area: Bioenergy Bioprocessing   

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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 Ro10.44 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 Derived from Pretreated Sugarcane Bagasse on Cellulolytic Activities

2015

Authors: M. Michelin, E. Ximenes, M. L. T. m. Polizeli, M. R. Ladisch, presentd at 37th Symposium on Biotechnology for Fuel and Chemicals, San Diego, CA, April, 2015
Journal:
Book Chapter:

Abstract: Lignocellulosic residues, such as sugarcane bagasse (SCB), are a complex matrix composed by cellulose, hemicellulose and lignin that can be used for different biotechnological applications. These materials need to be pretreated to be accessible for enzymatic hydrolysis. Liquid hot water (LHW) pretreatment is an effective and cost-saving approach, since no catalyst is required, and an expensive reactor is avoided due to the low corrosive nature of this pretreatment. However, during the pretreatment phenolics derived from lignin are released, which are inhibitory of enzymes. Here, we evaluated the effect of phenolic compounds formed during the pretreatment of the SCB on cellulolytic activity. Two conditions for LHW pretreatment were used: 180 and 200 C for 30 min and two fractions were obtained: solid and liquid fractions enriched by cellulose/lignin and hemicellulose, respectively. The phenolics contained in the liquid and solid fractions were used for the experiments of enzymatic inhibition (cellulase and beta-glucosidase activities). The higher amount of phenolics (2.4 g/L) was observed in the liquid fraction of SCB pretreated at 200 C/30 min. This condition also resulted in the highest inhibition of the enzymatic activity. Phenolics extracted from solid fraction (0.86 g/L) were shown to be more inhibitory than liquid for the beta-glucosidase activity. This work shows the importance of the optimization of the pretreatment process in relation to maximize the production of sugars and minimize the formation of inhibitory compounds to achieve the maximal efficiency of an enzyme hydrolysis-based process.

Research Area: Bioenergy Bioprocessing   

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Enzymatic Liquefaction of Corn Stover and Pericarp (Fiber)

2015

Authors: D. Kim, N. Hengge, D. Orrego, E. Ximenes, M. R. Ladisch, 2015 AIChE Annual Meeting, Paper 257B, Salt Lake City, Utah, November 10, 2015
Journal:
Book Chapter:

Abstract: There are two sources of cellulosic feedstocks for corn to ethanol, dry grind facilities: corn pericarp (fiber) and corn stover. Both materials quality for D3 RINS (renewable identification numbers for cellulosic ethanol), and could be an attractive resource for re-purposing corn to ethanol facilities for producing cellulosic ethanol. This approach would employ mixtures of endo- and exo-cellulases, pectinases, xylanases, protease, beta-glucosidase, as well as other possible auxiliary enzymes to liquefy and/or hydrolyze the cellulosic substrates. Tests, both in our laboratory and other research centers have demonstrated successful conversion of pretreated corn stover and other lignocellulosic materials. Processes for conversion of pretreated cellulose-containing distillers' solids have been demonstrated and are being marketed. Reports on this technology, available in the literature, show that a $10 to $12 million capital investment enables a 6% increase in ethanol production in existing corn to ethanol plants. This process requires pretreatment prior to hydrolysis and fermentation of the cellulosic portion of the corn kernel. Reports by Scott, Wyman, Schell, Elander, Kumar, Saville, Lawson and others have discussed the impact of mixing and reactor configurations on increasing both rate and yield of enzyme hydrolysis of cellulose, and/or liquefaction of pretreated lignocellulosic biomass (corn stover and hardwood). One significant factor that impacts economic viability of cellulose ethanol is being able to process biomass at high solids concentration in order to reduce energy for heating and other processing steps and increase the concentration of the ethanol produced. We recently reported the liquefaction of steam-exploded (pretreated) sugar cane bagasse at concentrations of up to 300 g/L. We present here the impact of reactor operation on the liquefaction of untreated corn stover and pericarp (corn fiber), and compares this to our recently reported liquefaction of sugar cane bagasse. The objective is to prepare these materials in pumpable slurries, thereby simplifying subsequent hydrolysis procedures, and reducing the cost of equipment that would otherwise require introduction of solid materials into a pressure vessel (i.e., a pulping digester).

Research Area: Bioenergy 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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Characteristics of Volatile Fatty Acids in Stored Dairy Manure Before and After Anaerobic Digestion

2014

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

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

Research Area: Bioprocessing    

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Effects of Lignin and Phenolic Inhibitors on Enzyme Loading

2014

Authors: M. Ladisch, E. Ximenes, Y. Kim, J. K. Ko, BIO Pacific Rim Summit, San Diego, CA, December 8, 2014
Journal:
Book Chapter:

Abstract: This panel focuses on recent advances in leading pretreatment technologies that can be coupled with enzymatic hydrolysis to convert lignocellulosic biomass to sugars for fermentation to ethanol or other products. The low cost of lignocellulosic biomass coupled with widespread domestic abundance, ability to dramatically reduce greenhouse gas emissions, and potential to spawn new rural manufacturing jobs make it an attractive resource from which to produce fuels and chemicals. However, converting this low cost resource into commodity products is expensive, with recalcitrance to sugar release being the key obstacle to achieving low prices by biological conversion routes. Most forms of lignocellulosic biomass must be pretreated prior to biological conversion operations to realize the high yields vital to economic competitiveness, and effective pretreatments can also lower loadings of expensive enzymes to economic levels, reduce costs of downstream operations,and produce valuable co-products that can improve overall process economics and provide additional benefits. Various studies have shown that thermochemical pretreatments that employ chemicals in combination with heat are most effective in realizing high sugar yields from the coupled operations of pretreatment and enzymatic hydrolysis. This Panel will include a presentation of recent work at Purdue University on reducing the amount of enzyme required for hydrolysis and the fundamentals of pretreatment related to changes in cell wall structure and chemistry. Increased severity of pretreatment exposes both additional lignin and cellulose. However, lignin adsorbs cellulase, so more enzyme must be added if the additional exposed cellulose is to be effectively hydrolyzed. Conversely, cellulase loading may be decreased by a factor of 10 while maintaining 80% glucose yield by diluting the enzyme with non-catalytic protein (BSA) that binds to lignin and decreases cellulase adsorption on lignin. More enzyme is therefore available for cellulose hydrolysis resulting in enhanced hydrolysis. Michigan State University is advancing Ammonia Fiber Expansion (AFEX) pretreatment, now being commercialized, to produce cellulosic biomass that can be used either for animal feed or as biofuel feedstock, thereby largely eliminating the "food versus fuel" issue. The AFEX presentation will briefly describe AFEX science and technology and how it can be performed in distributed processing facilities called depots. These depots greatly improve the logistics of cellulosic biofuel systems and allow local communities to capture part of the added value of AFEX processing. A presentation by the University of California at Riverside will describe a novel Co-solvent Enhanced Lignocellulosic Fractionation CELF) pretreatment that removes nearly all the lignin from biomass, recovers most of the hemicellulose sugars, and produces glucan-enriched solids that can be almost completely enzymatically digested to glucose with about one tenth the enzyme loadings typically required. Furthermore, CELF has been found to be effective with a wide range of hardwoods, grasses, and agricultural residues. Following the fate of major biomass components, kinetic modeling and SEM imaging suggest that the high lignin removal afforded by CELF could play a key role in achieving such high sugar yields with extremely low enzyme loadings and lead to alternate strategies to improve pretreatment.

Research Area: Bioenergy Bioprocessing Biofuels/Bioproducts  

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

2014

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

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

Research Area: Bioenergy Bioprocessing   

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

2013

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

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

Research Area: Bioprocessing Bioenergy Bioseparations  

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Biomass Chemistry

2013

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

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

Research Area: Bioprocessing Bioenergy   

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

2013

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

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

Research Area: Bioseparations Bioprocessing Bioenergy  

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

2013

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

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

Research Area: Bioenergy Bioprocessing Bioseparations  

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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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Maleic Acid Catalyzed Conversion of Hemicellulose to Furfural

2011

Authors: N. S. Mosier, E. Kim, S. Liu, M. Abu-Omar , 2011 Annual Meeting of the American Institute of Chemical Engineers, Minneapolis, MN, October 18, 2011
Journal:
Book Chapter:

Abstract: Direct catalytic conversion of lignocellulosic biomass to biofuels could improve the carbon efficiency of biofuel production. We report the use of maleic acid, a dicarboxylic acid, to catalyze the fractionation of biomass into an aqueous solution of pentose (primarily xylose) and insoluble cellulose and lignin, followed by the conversion of the xylose to furfural under higher temperature and pressure. This method achieved 80-90% yield of xylose through hydrolysis of the hemicellulose from various biomass sources (switchgrass, poplar, pine) and achieved 54-61% yield of furfural (based on original biomass). We present a kinetic analysis of biomass hydrolysis and furfural formation and discuss application of results from pure sugars to results from biomass conversion.

Research Area: Bioenergy Bioprocessing   

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The Role of Bioprocess Engineering in Biotechnology

2004

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

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

Research Area: Bioprocessing    

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

2002

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

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

Research Area: Bioprocessing    

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

1997

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

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

Research Area: Bioprocessing    

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Biosynthesis of Cephalosporin C: Regulation and Recombinant Technology

1995

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

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

Research Area: Bioprocessing    

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Cephalosporin C: Mode of Action and Biosynthetic Pathway

1995

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

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

Research Area: Bioprocessing    

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

1995

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

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

Research Area: Bioprocessing    

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

1995

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

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

Research Area: Bioprocessing    

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

1991

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

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

Research Area: Bioprocessing    

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In Situ Observation of Casein Micelle Coagulation

1991

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

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

Research Area: Bioprocessing    

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Production of Proteases by Psychotropic Microorganisms

1991

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

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

Research Area: Bioprocessing    

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Maintaining Constant Enzyme Activity in a Continuous Flow Reactor

1990

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

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

Research Area: Bioprocessing    

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Stoichiometry and Kinetics of Xylose Fermentation by Pichia stipitis

1990

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

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

Research Area: Biofuels/Bioproducts Bioprocessing   

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

1988

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

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

Research Area: Bioprocessing    

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Casein Micelles: Structure, Properties and Enzymatic Coagulation

1987

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

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

Research Area: Bioprocessing    

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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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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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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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Studies on Glucose Isomerase from a Streptomyces Species

1976

Authors: Chou, C. C., M. R. Ladisch, and G. T. Tsao
Journal: Appl. Envr. Microbiol. 32(40), 489-493 (1976)
Book Chapter:

Abstract: Production and properties of glucose isomerase from a Co2+-sensitive Streptomyces species were studied. After 4 days of shaking cultivation at 30o C and 200 rpm, a maximum of 1.1 enzyme units per ml of broth was obtained. Cell-free glucose isomerase, obtained from mycelia heat-treated in the presence of 0.5 mM untreated mycelia. The optimum pH and temperature for the glucose isomerase were 7 to 8 and 80o C, respectively. The Michaelis constant for fructose was 0.40 M. Mg2+ was found to enhance the glucose isomerase activity, whereas the effect of Co2+ on enzyme activity depended on the manner in which the enzyme was prepared. This glucose isomerase was quite heat stable, with a half-life of 120 h at 70oC.

Research Area: Bioprocessing    

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