US BioEnergy

Inver Grove Heights, MN, United States

US BioEnergy

Inver Grove Heights, MN, United States
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Saha B.C.,US BioEnergy | Yoshida T.,US BioEnergy | Yoshida T.,Kyushu University | Cotta M.A.,US BioEnergy | Sonomoto K.,Kyushu University
Industrial Crops and Products | Year: 2013

Corn stover used in this study contained 37.0 ± 0.4% cellulose, 31.3 ± 0.6% hemicellulose and 17.8 ± 0.2% lignin on dry basis. Hydrothermal pretreatment and enzymatic saccharification were evaluated for conversion of corn stover cellulose and hemicellulose to fermentable sugars. Under the optimum conditions of hydrothermal pretreatment of corn stover (10%, w/v; 200 °C; 5. min) and enzymatic saccharification (45 °C, pH 5.0, 72. h), a total of 550 ± 5. mg of fermentable sugars was obtained per g corn stover which is equivalent to 72% of theoretical sugar yield. The corn stover hydrolyzate was fermented without any detoxification by recombinant Escherichia coli strain FBR 5 at pH 6.5 and 37 °C for 74. h to produce 20.9 ± 0.5. g ethanol from 42.8 ± 1.7. g sugars per L with a yield of 0.49. g ethanol per g available sugars and 0.27. g ethanol per g corn stover which is equivalent to 68.7% of theoretical ethanol yield from corn stover. This is the first report on the production of ethanol from hydrothermally pretreated corn stover by the recombinant bacterium. © 2012.


Liu Z.L.,US BioEnergy
Applied Microbiology and Biotechnology | Year: 2011

Pretreatment of lignocellulose biomass for biofuel production generates inhibitory compounds that interfere with microbial growth and subsequent fermentation. Remediation of the inhibitors by current physical, chemical, and biological abatement means is economically impractical, and overcoming the inhibitory effects of lignocellulose hydrolysate poses a significant technical challenge for lower-cost cellulosic ethanol production. Development of tolerant ethanologenic yeast strains has demonstrated the potential of in situ detoxification for numerous aldehyde inhibitors derived from lignocellulose biomass pretreatment and conversion. In the last decade, significant progress has been made in understanding mechanisms of yeast tolerance for tolerant strain development. Enriched genetic backgrounds, enhanced expression, interplays, and global integration of many key genes enable yeast tolerance. Reprogrammed pathways support yeast functions to withstand the inhibitor stress, detoxify the toxic compounds, maintain energy and redox balance, and complete active metabolism for ethanol fermentation. Complex gene interactions and regulatory networks as well as co-regulation are well recognized as involved in yeast adaptation and tolerance. This review presents our current knowledge on mechanisms of the inhibitor detoxification based on molecular studies and genomic-based approaches. Our improved understanding of yeast tolerance and in situ detoxification provide insight into phenotype-genotype relationships, dissection of tolerance mechanisms, and strategies for more tolerant strain development for biofuels applications. © 2011 Springer-Verlag (outside the USA).


Liu Z.L.,US BioEnergy | Weber S.A.,US BioEnergy | Cotta M.A.,US BioEnergy
Bioenergy Research | Year: 2013

We previously reported on a new yeast strain of Clavispora sp. NRRL Y-50464 that is capable of utilizing cellobiose as sole source of carbon and energy by producing sufficient native β-glucosidase enzyme activity without further enzyme supplementation for cellulosic ethanol production using simultaneous saccharification and fermentation. Eliminating the addition of external β-glucosidase reduces the cost of cellulosic ethanol production. In this study, we present results on the isolation and identification of a β-glucosidase protein from strain Y-50464. Using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and blast search of the NCBInr database (National Center for Biotechnology Information nonredundant), the protein from Y-50464 was identified as a β-glucosidase (BGL1) with a molecular weight of 93. 3 kDa. The BGL1 protein was purified through multiple chromatographic steps to a 26-fold purity (Km = 0. 355 mM [pNPG]; Ki = 15. 2 mM [glucose]), which has a specific activity of 18. 4 U/mg of protein with an optimal performance temperature at 45 °C and pH of 6. 0. This protein appears to be intracellular although other forms of the enzyme may exist. The fast growth rate of Y-50464 and its capability to produce sufficient β-glucosidase activity for ethanol conversion from cellobiose provide a promising means for low-cost cellulosic ethanol production through a consolidated bioprocessing development. © 2012 Springer Science+Business Media, LLC (outside the USA).


Hamdan L.J.,U.S. Navy | Fulmer P.A.,US BioEnergy
Aquatic Microbial Ecology | Year: 2011

Hydrocarbon-degrading bacteria are important for controlling the fate of natural and anthropogenic hydrocarbons in the marine environment. In the wake of the Deepwater Horizon spill in the Gulf of Mexico, microbial communities will be important for the natural attenuation of the effects of the spill. The chemical dispersant COREXIT® EC9500A was widely deployed during the response to the Deepwater Horizon incident. Although toxicity tests confirm that COREXIT® EC9500A does not pose a significant threat to invertebrate and adult fish populations, there is limited information on its effect on microbial communities. We determined the composition of the microbial community in oil that had been freshly deposited on a beach in Louisiana, USA, as a result of the Deepwater Horizon spill. The metabolic activity and viability in cultures obtained from oil samples were determined in the absence and presence of COREXIT® EC9500A at concentrations ranging from 0.001 to 100 mg ml-1. In length heterogeneity PCR (LH-PCR) fingerprints of oil samples, the most abundant isolates were those of Vibrio, followed by hydrocarbon-degrading isolates affiliated with Acine - tobacter and Marinobacter. We observed significant reductions in production and viability of Acineto - bacter and Marinobacter in the presence of the dispersant compared to controls. Of the organisms examined, Marinobacter appears to be the most sensitive to the dispersant, with nearly 100% reduction in viability and production as a result of exposure to concentrations of the dispersant likely to be encountered during the response to the spill (1 to 10 mg ml-1). Significantly, at the same concen tration of dispersant, the non-hydrocarbon-degrading Vibrio isolates proliferated. These data suggest that hydro-carbon-degrading bacteria are inhibited by chemical dispersants, and that the use of dispersants has the potential to diminish the capacity of the environment to bioremediate spills. © Inter-Research 2011.


Avci A.,US BioEnergy | Avci A.,Sakarya University | Saha B.C.,US BioEnergy | Kennedy G.J.,US BioEnergy | Cotta M.A.,US BioEnergy
Bioresource Technology | Year: 2013

A pretreatment strategy for dilute H2SO4 pretreatment of corn stover was developed for the purpose of reducing the generation of inhibitory substances during pretreatment so that a detoxification step is not required prior to fermentation while maximizing sugar yield. The optimal conditions for pretreatment of corn stover (10%, w/v) were: 0.75% H2SO4, 160°C, and 0-5min holding time. The conditions were chosen based on maximum glucose release after enzymatic hydrolysis, minimum loss of pentose sugars and minimum formation of sugar degradation products such as furfural and hydroxymethyl furfural. The pretreated corn stover after enzymatic saccharification generated 63.2±2.2 and 63.7±2.3g total sugars per L at 0 and 5min holding time, respectively. Furfural production was 0.45±0.1 and 0.87±0.4g/L, respectively. The recombinant Escherichia coli strain FBR5 efficiently fermented non-detoxified corn stover hydrolyzate if the furfural content is <0.5g/L. © 2013 Elsevier Ltd.


Jordan D.B.,US BioEnergy | Braker J.D.,US BioEnergy
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2011

Conformational inversion occurs 7-8 kcal/mol more readily in furanoses than pyranoses. This difference is exploited here to probe for active-site residues involved in distorting pyranosyl substrate toward reactivity. Spontaneous glycoside hydrolysis rates are ordered 4-nitrophenyl-α-l-arabinofuranoside (4NPA) > 4-nitrophenyl-β-d-xylopyranoside (4NPX) > xylobiose (X2). The bifunctional β-d-xylosidase/α-l-arabinofuranosidase exhibits the opposite order of reactivity, illustrating that the enzyme is well equipped in using pyranosyl groups of natural substrate X2 in facilitating glycoside hydrolysis. Probing the roles of all 17 active-site residues by single-site mutation to alanine and by changing both moieties of substrate demonstrates that the mutations of subsite - 1 residues decrease the ratio kcat 4NPX/4NPA, suggesting that the native residues support pyranosyl substrate distortion, whereas the mutations of subsite + 1 and the subsite - 1/+1 interface residues increase the ratio kcat 4NPX/4NPA, suggesting that the native residues support other factors, such as C1 migration and protonation of the leaving group. Alanine mutations of subsite - 1 residues raise kcat X2/4NPX and alanine mutations of subsite + 1 and interface residues lower kcat X2/4NPX. We propose that pyranosyl substrate distortion is supported entirely by native residues of subsite - 1. Other factors leading to the transition state are supported entirely by native residues of subsite + 1 and interface residues. © 2011 Elsevier B.V. All Rights Reserved.


Avci A.,US BioEnergy | Avci A.,Sakarya University | Saha B.C.,US BioEnergy | Dien B.S.,US BioEnergy | And 2 more authors.
Bioresource Technology | Year: 2013

Dilute H3PO4 (0.0-2.0%, v/v) was used to pretreat corn stover (10%, w/w) for conversion to ethanol. Pretreatment conditions were optimized for temperature, acid loading, and time using central composite design. Optimal pretreatment conditions were chosen to promote sugar yields following enzymatic digestion while minimizing formation of furans, which are potent inhibitors of fermentation. The maximum glucose yield (85%) was obtained after enzymatic hydrolysis of corn stover pretreated with 0.5% (v/v) acid at 180°C for 15min while highest yield for xylose (91.4%) was observed from corn stover pretreated with 1% (v/v) acid at 160°C for 10min. About 26.4±0.1g ethanol was produced per L by recombinant Escherichia coli strain FBR5 from 55.1±1.0g sugars generated from enzymatically hydrolyzed corn stover (10%, w/w) pretreated under a balanced optimized condition (161.81°C, 0.78% acid, 9.78min) where only 0.4±0.0g furfural and 0.1±0.0 hydroxylmethyl furfural were produced. © 2012 Elsevier Ltd.


Ma M.,US BioEnergy | Ma M.,Sichuan Agricultural University | Liu Z.L.,US BioEnergy | Moon J.,US BioEnergy
Bioenergy Research | Year: 2012

For economical lignocellulose-to-ethanol production, a desirable biocatalyst should tolerate inhibitors derived from preteatment of lignocellulose and be able to utilize heterogeneous biomass sugars of hexoses and pentoses. Previously, we developed an inhibitor-tolerant Saccharomyces cerevisiae strain NRRL Y-50049 that is able to in situ detoxify common aldehyde inhibitors such as 2-furaldehyde (furfural) and 5-(hydroxymethyl)-2-furaldehyde (HMF). In this study, we genetically engineered Y-50049 to enable and enhance its xylose utilization capability. A codon-optimized xylose isomerase gene for yeast (YXI) was synthesized and introduced into a defined chromosomal locus of Y-50049. Two newly identified xylose transport related genes XUT4 and XUT6, and previously reported xylulokinase gene (XKS1), and xylitol dehydrogenase gene (XYL2) from Scheffersomyces stipitis were also engineered into the yeast resulting in strain NRRL Y-50463. The engineered strain was able to grow on xylose as sole carbon source and a minimum ethanol production of 38.6 g l -1 was obtained in an anaerobic fermentation on mixed sugars of glucose and xylose in the presence of furfural and HMF. © 2012 Springer Science+Business Media, LLC. (outside the USA).


Hector R.E.,US BioEnergy | Dien B.S.,US BioEnergy | Cotta M.A.,US BioEnergy | Qureshi N.,US BioEnergy
Journal of Industrial Microbiology and Biotechnology | Year: 2011

Saccharomyces' physiology and fermentation-related properties vary broadly among industrial strains used to ferment glucose. How genetic background affects xylose metabolism in recombinant Saccharomyces strains has not been adequately explored. In this study, six industrial strains of varied genetic background were engineered to ferment xylose by stable integration of the xylose reductase, xylitol dehydrogenase, and xylulokinase genes. Aerobic growth rates on xylose were 0.04-0.17 h -1. Fermentation of xylose and glucose/xylose mixtures also showed a wide range of performance between strains. During xylose fermentation, xylose consumption rates were 0.17-0.31 g/l/h, with ethanol yields 0.18-0.27 g/g. Yields of ethanol and the metabolite xylitol were positively correlated, indicating that all of the strains had downstream limitations to xylose metabolism. The better-performing engineered and parental strains were compared for conversion of alkaline pretreated switchgrass to ethanol. The engineered strains produced 13-17% more ethanol than the parental control strains because of their ability to ferment xylose. © 2010 Society for Industrial Microbiology (outside the USA).


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