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Bioprocessing Presentations

Adsorption Characteristics of Enzymes on Lignocellulosic Material by Liquid Chromatography

L. Zhang, E. Ximenes, M. R. Ladisch, 38th Symposium on Biotechnology for Fuels and Chemicals, Session 3: Enzyme Science and Technology I - Modelling and Structure/Function, April 25, 2016, Baltimore, MD, , 


Abstract: Our previous work demonstrated that severe pretreatment not only opens up the structure for enzymatic hydrolysis, but also increases lignin surface area exposed to cellulases. Non-productive binding of cellulases onto lignin decreases their activity. Therefore, higher enzyme loading is required to compensate for loss of enzyme due to adsorption on lignin. Previous reports have shown that BSA is effective in adsorbing onto lignin and blocking exposed lignin surface against adsorption of cellulose enzymes, thus increasing the effectiveness of enzymatic hydrolysis. Further studies on competitive adsorption of BSA and enzyme are now being carried out to better understand the lignin blocking effects. The traditional method of determining adsorption parameters for enzyme-lignin interactions through batch-adsorption studies is time consuming and labor intensive. Therefore, an inverse liquid chromatography method was developed instead, in order to determine the protein adsorption characteristics of lignin and lignocellulosic solids packed in a chromatography column. In this study, sugarcane bagasse was the stationary phase. Preliminary results observed by injecting 500 uL of BSA (20 mg/mL) showed that BSA is retained in the column with a rretention time of 17.6 min at both 20 and 50 C, although sharper peaks were observed at 50 C, consistent with the Arrhenius definition of the temperature dependence of an adsorption constant. These results confirmed the expected adsorption behavior of BSA, but more importantly, illustrated the utility of inverse liquid chromatography to better understand the adsorption of cellulases and other proteins to lignin. Inverse chromatography is being developed further as a rapid screening process for potential lignin blocking proteins.

Research Area: Bioprocessing

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

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


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

Research Area: Bioprocessing

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The Effect of Lignin in Enzymatic Saccharification of Bred Sugarcane Bagasse

R. L. Azar, T. Morgan, M. Barbosa, V. Guimaraes, E. Ximenes, M. Ladisch, 38th Symposium on Biotechnology for Fuels and Chemicals, Poster Session 2: Feedstocks; Enzyme Science and Technology; Renewable Fuels, Chemicals, and Bio-Based Products, Poster T20, April 26, 2018, Baltimore, MD, , 


Abstract: Lignin, one of the major components of lignocellulosic biomass, plays an important functional and structural role in plants. Lignin is also known as a major contributor to the recalcitrance of lignocellulosic biomass, and has been a target for feedstock improvement through genetic engineering. This work examines the influence of lignin in conventional breeded (clones 260 and 204) sugarcane bagasse after liquid hot water pretreatment. In conventional breeding, large differences in lignin are not expected because the plant does not easily lose this trait from one generation to the next. Moreover, we evaluate the enzyme-lignin interactions of lignins isolated from LHW pretreated sugarcane bagasse with and without BSA. FTIR analysis was used to investigate differences among the chemical composition of lignins studied.

Research Area: Bioprocessing

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An Analysis of Ethanol Impact on Xylose Fermentation in S. cerevisaie 424A (LNH-ST)

A. Athmanathan, M. Sedlak, N. Ho, N. Mosier, 30th Symposium on Biotechnology for Fuels and Chemicals, New Orleans, Louisiana, May 4 - 7, 2008


Abstract: Ethanol toxicity could be a significant bottleneck in industrial ethanol fermentation of sugars from lignocellulose. To understand ethanol impact on xylose fermentation, batch fermentations were carried out using S. cerevisiae 424A (LNH-ST), an engineered strain capable of co-fermenting glucose and xylose. The fermentation of xylose was carried out in YEP growth media, using largely non-growing cells in the presence of initial ethanol concentrations between 4 - 8% (w/v). The effects of extraneously added ethanol (pure xylose fermentation) and ethanol generated from glucose equivalent (co-fermentation) are compared. This yeast strain was found to cease fermentation of xylose at an extraneously added ethanol concentration of 9% (w/v). However, co-fermentation of glucose and xylose was capable of achieving a final ethanol titer over 11% (w/v). A preliminary unstructured, Monod-type model of these batch fermentations that include ethanol inhibition is presented.

Research Area: Bioprocessing

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pH and Buffer Effects on Xylose Degradation Rates and Products

Y. Lu and N. Mosier, 30th Symposium on Biotechnology for Fuels and Chemicals, New Orleans, Louisiana, May 4 - 7, 2008


Abstract: The degradation reaction routes of glucose and fructose under hydrothermal acidic conditions have been studied extensively; in contrast, xylose degradation has received less extensive study under similar conditions. In this study, we investigated the aqueous pH (0.5 – 7.0) impact on xylose degradation, and determined the kinetics of xylose disappearance rates at different pH conditions. The initial buffer system employed in this study was the McIlvaine buffer consisting of phosphate salt and citric acids (except for pH 0.5 – 1.5 buffers, where HCl/NaCl system was employed). It was observed that at pH 2.2, the xylose degradation rate was minimized (e.g. xylose disappearance rate at pH 4.2 is 9-times higher, and at pH 7.0 complete xylose disappearance occurred in 5-min reaction). In addition, the degradation reaction path changed from simple dehydration product (furfural) formation at lower pH range (0.5 – 3.0), to multiple complex liquid and polymerized products formation at higher pH range (4.5 – 7.0). In order to test the effect of buffering salt (phosphate, etc.), experiments at pH 1.0 with equivalent amount phosphate produced identical results to the same condition without phosphate addition. Therefore, the proton concentration in the aqueous solution may be the main controlling factor to which xylose degradation reactions occur. The degree of proton availability in the solution and potential protonation of the sugar –OH groups were analyzed to determine how the pH affects reaction path direction and products formation.

Research Area: Bioprocessing

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Differentiation in Activity of Fractions of Stay Green Corn Stover for Hot Water Pretreatment and Cellulase Saccharification

M. Zeng, J. Goetz, R. Hendrickson, C. P. Huang, D. Sherman, N. S. Mosier, and M. R. Ladisch, 29th Symposium on Biotechnology for Fuels and Chemicals, Denver, Colorado, Poster 5B-25 , April 29-May 2, 2007


Abstract: Corn stover is a heterogeneous substrate consisting of different fractions including leaves, stalk fiber and stalk pith. Tissue types and proportions in these fractions are not uniform which result in different cell structures, average cell wall thickness and lignin distribution. These factors may have different impacts on enzyme digestion, since the lignin barrier (content/distribution) and cell wall thickness are believed to be substrate related factors that influence the effectiveness of enzymatic hydrolysis of cellulose in lignocellulosic feedstocks. The hypothesis being tested in this research addresses potential differences of intrinsic reactivity of different parts of stay green corn stover (leaves, stalk fiber and stalk pith). Carbohydrate analysis shows that pith is more readily hydrolyzed than leaves and fiber at cellulase level equivalent to 5 FPU Spezyme CP/g glucan. Hot water pretreatment at 190 C for 15 min removes 40% to 50% hemicellulose in these fractions, respectively, although structural changes in the cell wall are not evident when the residual material is imaged by scanning electron microscopy. Enzyme hydrolysis of pretreated and washed fractions of leaves and pith exhibit much higher glucose conversion than the fractions that have not been pretreated. Pretreated fibere (from the rind) is still resistant to hydrolysis and shows 2/3 lower glucose formation than pretreated leaf or pith at low enzyme loadings equivalent to about 5 FPU Spezyme CP/g glucan.

Research Area: Bioprocessing

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Conditioning and Glucose/Xylose Co-fermentation of Pretreated Lignocellulosic Biomass

Ryan E. Warner, Miroslav Sedlak, Nancy Ho, and Nathan S. Mosier,  28th Symposium on Biotechnology for Fuels and Chemicals, Nashville, TN, April 30 to May 3, 2006


Abstract: Pretreatment of lignocellulosic biomass, while improving enzymatic digestibility, can also produce fermentation inhibitors. Two important inhibitors, furfural and HMF, are formed from the degradation of carbohydrates from lignocellulose. Thus, pretreated material may require conditioning to either remove or otherwise detoxify these inhibitors. This paper explores some conditioning methods on hydrolysates obtained from corn stover and poplar pretreated by dilute acid, controlled pH l iquid hot water, SO2 steam explosion, and others. The effects of these conditioning methods on the subsequent fermentation of both glucose and xylose by the recombinant yeast S. cerevisiae 424A(LNH-ST) is presented. Overliming the pretreated corn stover to pH 9 or higher removes 100% of the HMF and furfural present in corn stover hydrolysates. However, the fermentation is negatively affected, producing only 53% of theoretical ethanol yields as opposed to 82% yield from the unconditioned material. Hydrophobic resins (Amberlite XAD2, XAD4, and XAD7) were also examined for their ability to remove HMF and furfural. The resins were able to remove 100% of furfural and approximately 60% or more HMF. The yield from fermentation was 87%; slightly better than the unconditioned corn stover hydrolysate.

Research Area: Bioprocessing

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Effects of Furfural and HMF on the Co-fermentation of Glucose and Xylose from Pretreated Lignocellulosic Biomass by Recombinant Yeast

Ryan E. Warner, Miroslav Sedlak, Nancy Ho, and Nathan S. Mosier, 28th Symposium on Biotechnology for Fuels and Chemicals, Nashville, TN, April 30 to May 3, 2006


Abstract: Pretreatment of lignocellulosic biomass, while improving enzymataic digestibility, can also produce fermentation inhibitors such as furfural and HMF. Both furfural and HMF can decrease the fermentability and the ethanol yields from sugars derived from lignocellulose. This paper reports a systematic study of the effect of furfural and HMF on the fermentation of both glucose and xylose to ethanol by the recombinant yeast S. cerevisiae 424A(LNH-ST). Fermentations were run with furfural, HMF, or both in a control solution of YEP with glucose and xylose as co-substrates. Inhibitor concentrations were varied and range from 0 to 40 g/L. Further experiments varied inhibitor concentrations in the presence of a single substrate, either glucose or xylose. Batch fermentations were carried out for 48 hours in 300 mL sidearm flasks at 30 C and 200 rpm with periodic sampling for anlaysis by HPLC. Our results show that concentrations of either furfural or HMF below about 5 g/L cause negligible inhibition for yeast cells in early stationary phase. We confirm that furfural is more inhibitory than HMF. Lastly, xylose fermentation to ethanol is more sensitive to these inhibitors than glucose for fermentation to ethanol.

Research Area: Bioprocessing

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Flow Control and Surface Engineering of Microfluidics for Advanced Detection of Pathogens

Tom T. Huang, David G. Taylor, Kwan Seop Lim, Miroslav Sedlak, Rashid Bashir, Nathan S. Mosier, Michael R. Ladisch, Pharmaceutical Technology and Education Workshop, Dauch Center, Purdue University, April 19, 2006


Abstract:

Research Area: Bioprocessing

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Pilot Scale Measurement of Viscosity for a Biomass Slurry Composed of 15-20% Corn Fiber in Light Stillage

Richard Hendrickson, Youngmi Kim, Yulin Lu, Nathan Mosier, and Michael Ladisch, 28th Symposium on Biotechnology for Fuels and Chemicals, Nashville, TN, April 30 to May 3, 2006


Abstract: The aqueous pretreatment of corn fiber at a pH of 4 to 7, while being pumped through a hold coil is effective in increasing the rate of enzyme hydrolysis of the cellulose. However, scale-up of the pretreatment process depends on physical properties of the material to be pumped through the system. High concentrations of fermentable sugars require that aqueous biomass streams from which these sugars are derived have a high solids content. Since the corn fiber solids at high loading have characteristics that resemble a shear-thinning fluid, measurement of viscosity in the laboratory is difficult, particularly at temperatures above ambient. Consequently, we carried out measurements in a plant setting. Corn fiber at 150 to 200 g/L were pumped at rates of 1 to 10 gal/minute through sections of jacketed tubing having diameters ranging from 1 to 1.5 inches and a length of 17.25 feet. The temperatures and pressure drops were measured at the inlet and outlet of the tubes and recorded through a LabVIEW programmed data acquisition system. The pressure drop and flow rate enabled calculation of viscosity and determination of correlations that will be useful for scale-up.

Research Area: Bioprocessing

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Pretreatment Fundamentals

Bruce E. Dale, Richard T. Elander, Mark T. Holtzapple, Rajeev Kumar, Michael R. Ladisch, Yoon Y. Lee, Nate Mosier, Jack Saddler, Mohammed Moniruzzaman, Charles E. Wyman, CAFI BIO 2006, Annual International Convention, Chicago, Illinois, April 12, 2006


Abstract:

Research Area: Bioprocessing

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Catalysis for Biorenewables Conversion to Transportation Fuels and Bioproducts

Michael R. Ladisch, National Science Foundation Workshop on "Design of Catalyst Systems for Biorenewables, Washington, DC, June 23, 24, 2005


Abstract:

Research Area: Bioprocessing

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Oligosaccharide Hydrolysis in Plug Flow Reactor Using Strong Acid Catalyst

Young Mi Kim, Mosier, Nathan, Hendrickson, Rick, and Ladisch, Michael R., 27th Symposium on Biotechnology for Fuels and Chemicals, Denver, CO, May 4, 2005


Abstract: Liquid hot water pretreatment of plant biomass produces a liquid stream with dissolved oligosaccharides which are usually converted to fermentable sugars by enzymatic hydrolysis. In previous work, strong cation exchanger, Amberlyst 35W, has shown to hydrolyze cellobiose and oligosaccharides in liquid from corn fiber pretreatment at high conversion rates. This paper reports the effects of particle size, degree of cross-linking, and temperature on hydrolysis of oligosaccharides and degradation of monosaccharides. High temperature and short residence times were required to minimize formation of aldehydes and other fermentation inhibitors formation while achieving high glucose yield. The catalysts, SK104 (4% corrlinked gell type) and Amberlyst 35 (macroreticular sulfonic acid resin) were tested for hydrolysis of maltooligosaccharides at various reaction conditions. Maltooligosaccharides were used as a model oligosaccharide since their activation energy for bond breakage is similar to that of xylo- or cello-oligosaccharides, and since malto-oligosaccharides are more readily obtainable compared to the other types of oligosaccharides. Results show that low percentage cross-linked gel-type cation exchange resins give a higher glucose yield than macroreticular-type resins. The hydrolysis was diffision limited in both resins. A mathematical model that quantifies diffusion and kinetic characteristics of this reaction is presented and potential application of plug flow reactors to hydrolysis of oligosaccharides obtained from pretreatment of cellulose is discussed.

Research Area: Bioprocessing

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