Peoria, IL, United States
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Dien B.S.,Bioenergy Research Unit | Miller D.J.,DuPont Pioneer | Hector R.E.,Bioenergy Research Unit | Dixon R.A.,Samuel Roberts Noble Foundation | And 5 more authors.
Bioresource Technology | Year: 2011

Alfalfa (Medicago sativa L.) biomass was evaluated for biochemical conversion into ethanol using dilute-acid and ammonia pretreatments. The two alfalfa lines compared were a reduced S-lignin transgenic cultivar generated through down regulation of the caffeic acid O-methyltransferase gene and a wild-type control. Both were harvested at two maturities. All the samples had similar carbohydrate contents including a mean composition of 316. g glucan and 497. g total neutral carbohydrates per kg dry biomass, which corresponds to a theoretic ethanol yield of 382. l/ton. Ethanol yields for alfalfa stems pretreated with dilute-acid were significantly impacted by harvest maturity and lignin composition, whereas when pretreated with dilute-ammonia, yield was solely affected by lignin composition. Use of a recombinant xylose-fermenting Saccharomyces strain, for converting the ammonia pretreated alfalfa samples, further increased ethanol yields. Ethanol yields for the xylose-fermenting yeast were 232-278. l/ton and were significantly enhanced for the reduced S lignin cultivars. © 2011.


News Article | April 19, 2016
Site: phys.org

Polyphenol oxidase, an enzyme found in all plants, causes undesirable reactions such as browning in sliced apples, black spots in cut avocados, and dark marks on banana peels. It's also responsible for gray discoloration in wheat products such as fresh noodles, fresh and frozen breads, and refrigerated biscuits—all made from hard white wheat. Hard white wheat products such as white whole-grain breads are becoming more popular in the U.S. marketplace. Hard white wheat is the wheat of choice in select export markets, especially in Asia, where it is used to make a variety of fresh noodle products. High levels of polyphenol oxidase in the grain make U.S. hard white wheats undesirable and place them at a disadvantage relative to wheats from Australia in the Asian markets, says Bob Graybosch, plant geneticist and research leader of the Agricultural Research Service's (ARS) Grain, Forage, and Bioenergy Research Unit in Lincoln, Nebraska. "Polyphenol oxidase is a big deal for Asian markets, because they don't want to see gray noodles when they hang them up to dry and then sell." For 15 years, Graybosch has been studying the polyphenol oxidase trait in wheat, investigating numerous samples of white wheat obtained from the ARS National Small Grains Collection (NSGC), in Aberdeen, Idaho. "A lot of U.S. white wheats still have high levels of polyphenol oxidase," Graybosch says. "To have a successful white wheat for both the export market and the domestic market, milling companies want low or no polyphenol oxidase." Some low-polyphenol oxidase hard winter white wheats have been developed, but complete removal of this enzyme trait is more desirable. In 2000, working with the University of Nebraska and Montana State University, Graybosch screened more than 3,000 wheats from the NSGC for the presence of polyphenol oxidase. The team then mated wheats that had low levels of the enzyme. Wheat breeding lines with very low or even near-zero levels of polyphenol oxidase were generated from these crosses. "And lo and behold, we did it," he says. "We developed a wheat with practically no polyphenol oxidase." Graybosch produced the wheat line, called "070R1074," by crossing two Australian wheats that were entered into the small-grains germplasm collection in the 1930s."For 70 years, these two Australian wheats have been in the germplasm collection with this trait of interest and economic importance that the milling industry and exporters need and want," Graybosch says. "This demonstrates the value of this diversified wheat collection. You don't always know what you have until you do something with it."


Huang H.,University of Illinois at Urbana - Champaign | Qureshi N.,Bioenergy Research Unit | Qureshi N.,1815 North University Street | Chen M.-H.,University of Illinois at Urbana - Champaign | And 2 more authors.
Journal of Agricultural and Food Chemistry | Year: 2015

Ethanol production from food wastes does not only solve environmental issues but also provides renewable biofuels. This study investigated the feasibility of producing ethanol from food wastes at high solids content (35%, w/w). A vacuum recovery system was developed and applied to remove ethanol from fermentation broth to reduce yeast ethanol inhibition. A high concentration of ethanol (144 g/L) was produced by the conventional fermentation of food waste without a vacuum recovery system. When the vacuum recovery is applied to the fermentation process, the ethanol concentration in the fermentation broth was controlled below 100 g/L, thus reducing yeast ethanol inhibition. At the end of the conventional fermentation, the residual glucose in the fermentation broth was 5.7 g/L, indicating incomplete utilization of glucose, while the vacuum fermentation allowed for complete utilization of glucose. The ethanol yield for the vacuum fermentation was found to be 358 g/kg of food waste (dry basis), higher than that for the conventional fermentation at 327 g/kg of food waste (dry basis). © 2015 American Chemical Society.


Chen M.-H.,University of Illinois at Urbana - Champaign | Kaur P.,University of Illinois at Urbana - Champaign | Dien B.,BioEnergy Research Unit | Below F.,University of Illinois at Urbana - Champaign | And 2 more authors.
World Journal of Microbiology and Biotechnology | Year: 2013

Tropical maize is an alternative energy crop being considered as a feedstock for bioethanol production in the North Central and Midwest United States. Tropical maize is advantageous because it produces large amounts of soluble sugars in its stalks, creates a large amount of biomass, and requires lower inputs (e.g. nitrogen) than grain corn. Soluble sugars, including sucrose, glucose and fructose were extracted by pressing the stalks at dough stage (R4). The initial extracted syrup fermented faster than the control culture grown on a yeast extract/phosphate/sucrose medium. The syrup was subsequently concentrated 1.25-2.25 times, supplemented with urea, and fermented using Saccharomyces cerevisiae for up to 96 h. The final ethanol concentrations obtained were 8.1 % (v/v) to 15.6 % (v/v), equivalent to 90.3-92.2 % of the theoretical yields. However, fermentation productivity decreased with sugar concentration, suggesting that the yeast might be osmotically stressed at the increased sugar concentrations. These results provide in-depth information for utilizing tropical maize syrup for bioethanol production that will help in tropical maize breeding and development for use as another feedstock for the biofuel industry. © 2013 Springer Science+Business Media Dordrecht.


Saathoff A.J.,Forage and Bioenergy Research Unit | Sarath G.,Forage and Bioenergy Research Unit | Chow E.K.,Genomics and Gene Discovery Research Unit | Dien B.S.,Bioenergy Research Unit | Tobias C.M.,Genomics and Gene Discovery Research Unit
PLoS ONE | Year: 2011

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in monolignol biosynthesis and genetic evidence indicates CAD deficiency in grasses both decreases overall lignin, alters lignin structure and increases enzymatic recovery of sugars. To ascertain the effect of CAD downregulation in switchgrass, RNA mediated silencing of CAD was induced through Agrobacterium mediated transformation of cv. "Alamo" with an inverted repeat construct containing a fragment derived from the coding sequence of PviCAD2. The resulting primary transformants accumulated less CAD RNA transcript and protein than control transformants and were demonstrated to be stably transformed with between 1 and 5 copies of the TDNA. CAD activity against coniferaldehyde, and sinapaldehyde in stems of silenced lines was significantly reduced as was overall lignin and cutin. Glucose release from ground samples pretreated with ammonium hydroxide and digested with cellulases was greater than in control transformants. When stained with the lignin and cutin specific stain phloroglucinol-HCl the staining intensity of one line indicated greater incorporation of hydroxycinnamyl aldehydes in the lignin.


Hector R.E.,Bioenergy Research Unit | Mertens J.A.,Bioenergy Research Unit | Bowman M.J.,Bioenergy Research Unit | Nichols N.N.,Bioenergy Research Unit | And 2 more authors.
Yeast | Year: 2011

Saccharomyces strains engineered to ferment xylose using Scheffersomyces stipitis xylose reductase (XR) and xylitol dehydrogenase (XDH) genes appear to be limited by metabolic imbalances, due to differing cofactor specificities of XR and XDH. The S. stipitis XR, which uses both NADH and NADPH, is hypothesized to reduce the cofactor imbalance, allowing xylose fermentation in this yeast. However, unadapted S. cerevisiae strains expressing this XR grow poorly on xylose, suggesting that metabolism is still imbalanced, even under aerobic conditions. In this study, we investigated the possible reasons for this imbalance by deleting genes required for NADPH production and gluconeogenesis in S. cerevisiae. S. cerevisiae cells expressing the XR-XDH, but not a xylose isomerase, pathway required the oxidative branch of the pentose phosphate pathway (PPP) and gluconeogenic production of glucose-6-P for xylose assimilation. The requirement for generating glucose-6-P from xylose was also shown for Kluyveromyces lactis. When grown in xylose medium, both K. lactis and S. stipitis showed increases in enzyme activity required for producing glucose-6-P. Thus, natural xylose-assimilating yeast respond to xylose, in part, by upregulating enzymes required for recycling xylose back to glucose-6-P for the production of NADPH via the oxidative branch of the PPP. Finally, we show that induction of these enzymes correlated with increased tolerance to the NADPH-depleting compound diamide and the fermentation inhibitors furfural and hydroxymethyl furfural; S. cerevisiae was not able to increase enzyme activity for glucose-6-P production when grown in xylose medium and was more sensitive to these inhibitors in xylose medium compared to glucose. Published in 2011 by John Wiley & Sons, Ltd.


Dunlap C.A.,Crop Bioprotection Unit | Bowman M.J.,Bioenergy Research Unit | Schisler D.A.,Crop Bioprotection Unit | Rooney A.P.,Crop Bioprotection Unit
International Journal of Systematic and Evolutionary Microbiology | Year: 2016

Bacillus axarquiensis and Bacillus malacitensis were previously reported to be later heterotypic synonyms of Bacillus mojavensis, based primarily on DNA–DNA relatedness values. We have sequenced draft genomes of Bacillus axarquiensis NRRL B-41617T and Bacillus malacitensis NRRL B-41618T. Comparative genomics and DNA–DNA relatedness calculations showed that while Bacillus axarquiensis and Bacillus malacitensis are synonymous with each other, they are not synonymous with Bacillus mojavensis. In addition, a draft genome was completed for Brevibacterium halotolerans, a strain long suspected of being a Bacillus subtilis group member based on 16S rRNA similarities (99.8 % with Bacillus mojavensis). Comparative genomics and DNA–DNA relatedness calculations showed that Brevibacterium halotolerans is synonymous with Bacillus axarquiensis and Bacillus malacitensis. The pairwise in silico DNA–DNA hybridization values calculated in comparisons between the three conspecific strains were all greater than 92 %, which is well above the standard species threshold of 70 %. While the pairwise in silico DNA–DNA hybridization values calculated in comparisons of the three conspecific strains with Bacillus mojavensis were all less than 65 %. The combined results of our genotype and phenotype studies showed that Bacillus axarquiensis, Bacillus malacitensis and Brevibacterium halotolerans are conspecific and distinct from Bacillus mojavensis. Because the valid publication of the name Bacillus axarquiensis predates the publication of the name Bacillus malacitensis, we propose that Bacillus malacitensis be reclassified as a synonym of Bacillus axarquiensis. In addition, we propose to reclassify Brevibacterium halotolerans as a synonym of Bacillus axarquiensis. An amended description of Bacillus axarquiensis is provided. © 2016 IUMS.


Dien B.S.,Bioenergy Research Unit | Casler M.D.,U S WEST | Hector R.E.,Bioenergy Research Unit | Iten L.B.,Bioenergy Research Unit | And 3 more authors.
International Journal of Low-Carbon Technologies | Year: 2012

Reed canarygrass is a temperate perennial grass of interest as a bioenergy crop. The canarygrass was evaluated for conversion to bioethanol using liquid hot water and dilute ammonia pretreatments prior to fermentation. The resulting hydrolysates were evaluated for production of ethanol, xylose and soluble xylans. Dilute ammonia gave higher yield efficiencies than liquid hot water. The optimal condition for dilute ammonia (4% w/v) pretreatment was 170°C for 20 min. Hydrolysates were converted to ethanol using Saccharomyces in the presence of a blend of commercial cellulases and additional carbohydrases. The final ethanol conversion efficiency was 84% based upon total hexosans, with 72% of the xylan converted to soluble xylan oligomers.


PubMed | Bioenergy Research Unit, Mycotoxin Prevention and Applied Microbiology Research Unit and Michigan State University
Type: | Journal: Journal of visualized experiments : JoVE | Year: 2016

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.


PubMed | Bioenergy Research Unit
Type: Journal Article | Journal: Biotechnology for biofuels | Year: 2013

Saccharomyces cerevisiae strains expressing D-xylose isomerase (XI) produce some of the highest reported ethanol yields from D-xylose. Unfortunately, most bacterial XIs that have been expressed in S. cerevisiae are either not functional, require additional strain modification, or have low affinity for D-xylose. This study analyzed several XIs from rumen and intestinal microorganisms to identify enzymes with improved properties for engineering S. cerevisiae for D-xylose fermentation.Four XIs originating from rumen and intestinal bacteria were isolated and expressed in a S. cerevisiae CEN.PK2-1C parental strain primed for D-xylose metabolism by over expression of its native D-xylulokinase. Three of the XIs were functional in S. cerevisiae, based on the strains ability to grow in D-xylose medium. The most promising strain, expressing the XI mined from Prevotella ruminicola TC2-24, was further adapted for aerobic and fermentative growth by serial transfers of D-xylose cultures under aerobic, and followed by microaerobic conditions. The evolved strain had a specific growth rate of 0.23 h-1 on D-xylose medium, which is comparable to the best reported results for analogous S. cerevisiae strains including those expressing the Piromyces sp. E2 XI. When used to ferment D-xylose, the adapted strain produced 13.6 g/L ethanol in 91 h with a metabolic yield of 83% of theoretical. From analysis of the P. ruminicola XI, it was determined the enzyme possessed a Vmax of 0.81 mole/min/mg protein and a Km of 34 mM.This study identifies a new xylose isomerase from the rumen bacterium Prevotella ruminicola TC2-24 that has one of the highest affinities and specific activities compared to other bacterial and fungal D-xylose isomerases expressed in yeast. When expressed in S. cerevisiae and used to ferment D-xylose, very high ethanol yield was obtained. This new XI should be a promising resource for constructing other D-xylose fermenting strains, including industrial yeast genetic backgrounds.

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