Abstract: The present invention is directed to a system and method for producing an organic compound using fermentation wherein multiple components of the system are recycled within the system. The system and method allow for extraction of a high concentration of the organic compound from the fermentation broth in a continuous system that allows recycling of the biomass, aqueous fermentation broth and extraction solvents. The system and method are particularly well adapted for producing and extracting ethanol.

Abstract: Methods of identifying genes conferring ethanol tolerance in yeasts, genes that confer ethanol tolerance, and mutant strains used to identify such genes are described. A gene herein designated HpETT1 was isolated from the yeast Hansenula polymorpha. Expression of HpETT1 in an ethanol sensitive mutant H. polymorpha strain designated 7E complimented ethanol sensitivity of the mutant. When multiple copies of the HpETT1 were integrated into the genome and overexpressed, the transformed strain demonstrated approximately 10-fold greater resistance to ethanol and resistance to the protein misfolding agent AZC. Expression of HpETT1 also increased ethanol tolerance in Saccharomyces cerevisiae. HpEtt1 has 39% sequence identity to a previously identified protein from S. cerevisiae denoted MPE1, however, the MPE1 gene does not confer ethanol resistance to the 7E mutant.

Abstract: The present invention is directed to a system and method for producing an organic compound using fermentation wherein multiple components of the system are recycled within the system. The system and method allow for extraction of a high concentration of the organic compound from the fermentation broth in a continuous system that allows recycling of the biomass, aqueous fermentation broth and extraction solvents. The system and method are particularly well adapted for producing and extracting ethanol.

Abstract: Methods of identifying genes conferring ethanol tolerance in yeasts, genes that confer ethanol tolerance, and mutant strains used to identify such genes are described. A gene herein designated HpETT1 was isolated from the yeast Hansenula polymorpha. Expression of HpETT1 in an ethanol sensitive mutant H. polymorpha strain designated 7E complimented ethanol sensitivity of the mutant. When multiple copies of the HpETT1 were integrated into the genome and overexpressed, the transformed strain demonstrated approximately 10-fold greater resistance to ethanol and resistance to the protein misfolding agent AZC. Expression of HpETT1 also increased ethanol tolerance in Saccharomyces cerevisiae. HpEtt1 has 39% sequence identity to a previously identified protein from S. cerevisiae denoted MPE1, however, the MPE1 gene does not confer ethanol resistance to the 7E mutant.

Abstract: A truncated version of Saccharomyces cerevisiae IVL2 gene encoding a cytosolic form of acetolactate synthase was cloned into an expression cassette under control of a strong constitutive alcohol dehydrogenase (ADH1) promoter. The plasmid was introduced into the S. cerevisiae strain and the recombinant strain was tested for ability to overproduce glycerol under anaerobic conditions. It was shown that the recombinant strain was characterized by increased glycerol production and decreased ethanol production under anaerobic conditions.

Abstract: Methods of identifying genes conferring ethanol tolerance in yeasts, genes that confer ethanol tolerance, and mutant strains used to identify such genes are described. A gene herein designated HpETT1 was isolated from the yeast Hansenula polymorpha. Expression of HpETT1 in an ethanol sensitive mutant H. polymorpha strain designated 7E complimented ethanol sensitivity of the mutant. When multiple copies of the HpETT1 were integrated into the genome and overexpressed, the transformed strain demonstrated approximately 10-fold greater resistance to ethanol and resistance to the protein misfolding agent AZC. Expression of HpETT1 also increased ethanol tolerance in Saccharomyces cerevisiae. HpEtt1 has 39% sequence identity to a previously identified protein from S. cerevisiae denoted MPE1, however, the MPE1 gene does not confer ethanol resistance to the 7E mutant.

Abstract: A method to increase ethanol production from a corn dry-mill process is described that comprises adding an enzyme preparation derived from Trichoderma reesei having cellulolytic activity to a saccharification process that includes conventional alpha amylase and glucoamylase. The addition of the cellulolytic enzyme decreases viscosity of the saccharified mash and can increase ethanol yield from a dry grind fermentation by as much as 10% or more. Specific characteristics are provided to show surprising and advantageous results of one particular preparation of cellulolytic enzymes from T. reesei.

Abstract: Genes SWA2 and GAM1 from the yeast, Schwanniomyces occidentalis, encoding ?-amylase and glucoamylase, respectively, were cloned and expressed in H. polymorpha. The expression was achieved by integration of the SWA2 and GAM1 genes into the chromosome of H. polymorpha under operably linked to a strong constitutive promoter of the H. polymorpha-glyceraldehyde-3-phosphate dehydrogenase gene (HpGAP. Resulting transformants acquired the ability to grow on a minimal medium containing soluble starch as a sole carbon source and can produce Ethanol at high-temperature fermentation from starch up to 10 g/L. A XYN2 gene encoding endoxylanase was obtained from the fungus Trichoderma resee, and a xlnD gene coding for ?-xylosidase was obtained from the fungus Aspergillus niger. Co-expression of these genes was also achieved by integration into the H. polymorpha chromosome under control of the HpGAP promoter.

Abstract: Disclosed herein are methods of treating an edible fiber source to make an animal feed with increased digestible energy. An exemplary method includes hydrolyzing the edible fiber source with an inorganic fiber hydrolyzing agent in a twin screw mixer that shears the edible fiber to a size of between 0.5 to 25 mm. The hydrolysis in the mixer occurs at pressure of about 14 psig or higher with a temperature about 100° C. to 110° C. The inorganic hydrolysis liberates a first portion of soluble carbohydrates from the edible fiber source. The inorganically hydrolyzed material is also treated (before or after) with a fiber degrading enzyme to solubilize a second portion of carbohydrates. The dually hydrolyzed material is dried to form an animal feed or feed ingredient having a soluble and insoluble carbohydrate fraction with the amount of soluble carbohydrate being at least 45% wt/wt of the total carbohydrates obtained from the edible fiber source.

Abstract: Recombinant genetic constructs and strains of H. polymorpha having significantly increased ethanol productivity with a simultaneous decreased production of xylitol during high-temperature xylose fermentation are disclosed. The constructs include a H. polymorpha XYL1 gene encoding xylose reductase mutated to decrease affinity of the enzyme toward NADPH. The modified version of XYL1 gene under control of a strong constitutive HpGAP promoter was overexpressed in a ?xyl1 background. A recombinant H. polymorpha strain overexpressing the mutated enzyme together with native xylitol dehydrogenase and xylulokinase in the ?xyl1 background was also constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were evaluated during high-temperature xylose fermentation (48° C.). A significant increase in ethanol productivity (up to 7.4 times) was shown in the recombinant strain as compared with the wild type strain.

Abstract: A method to increase ethanol production from a corn dry-mill process is described that comprises adding an enzyme preparation derived from Trichoderma reesei having cellulolytic activity to a saccharification process that includes conventional alpha amylase and glucoamylase. The addition of the cellulolytic enzyme decreases viscosity of the saccharified mash and can increase ethanol yield from a dry grind fermentation by as much as 10% or more. Specific characteristics are provided to show surprising and advantageous results of one particular preparation of cellulolytic enzymes from T. reesei.

Abstract: Methods of identifying genes conferring ethanol tolerance in yeasts, genes that confer ethanol tolerance, and mutant strains used to identify such genes are described. A gene herein designated HpETT1 was isolated from the yeast Hansenula polymorpha. Expression of HpETT1 in an ethanol sensitive mutant H. polymorpha strain designated 7E complimented ethanol sensitivity of the mutant. When multiple copies of the HpETT1 were integrated into the genome and overexpressed, the transformed strain demonstrated approximately 10-fold greater resistance to ethanol and resistance to the protein misfolding agent AZC. Expression of HpETT1 also increased ethanol tolerance in Saccharomyces cerevisiae. HpEtt1 has 39% sequence identity to a previously identified protein from S. cerevisiae denoted MPE1, however, the MPE1 gene does not confer ethanol resistance to the 7E mutant.

Abstract: A process for production of C5 and C6 sugar enriched syrups from lignocellulosic biomass and fermentation products therefrom is described. A lignocellulosic biomass is treated with a C1-C2 acid (e.g., acetic acid) with washing thereof with a C1-C2 acid miscible organic solvent, (e.g., ethyl acetate). A soluble hemicellulose and lignin enriched fraction is obtained separately from a cellulose pulp enriched fraction and lignin is removed from the soluble hemicellulose fraction. These fractions contain acylated (e.g., acetylated) cellulose and hemicellulose, which are deacylated by treatment with an alkali and/or with an acetyl esterase enzyme. The deacylated fractions are then digested with suitable cellulolytic and/or hemicellulolytic enzymes, preferably in the presence of non-ionic detergent to yield the C5 and C6 enriched syrups.

Abstract: A process for production of C5 and C6 sugar enriched syrups from lignocellulosic biomass and fermentation products therefrom is described. A lignocellulosic biomass is treated with a C1-C2 acid (e.g., acetic acid) with washing thereof with a C1-C2 acid miscible organic solvent, (e.g., ethyl acetate). A soluble hemicellulose and lignin enriched fraction is obtained separately from a cellulose pulp enriched fraction and lignin is removed from the soluble hemicellulose fraction. These fractions contain acylated (e.g., acetylated) cellulose and hemicellulose, which are deacylated by treatment with an alkali and/or with an acetyl esterase enzyme. The deacylated fractions are then digested with suitable cellulolytic and/or hemicellulolytic enzymes, preferably in the presence of non-ionic detergent to yield the C5 and C6 enriched syrups.

Abstract: A process for production of C5 and C6 sugar enriched syrups from lignocellulosic biomass and fermentation products therefrom is described. A lignocellulosic biomass is treated with a C1-C2 acid (e.g., acetic acid) with washing thereof with a C1-C2 acid miscible organic solvent, (e.g., ethyl acetate). A soluble hemicellulose and lignin enriched fraction is obtained separately from a cellulose pulp enriched fraction and lignin is removed from the soluble hemicellulose fraction. These fractions contain acylated (e.g., acetylated) cellulose and hemicellulose, which are deacylated by treatment with an alkali and/or with an acetyl esterase enzyme. The deacylated fractions are then digested with suitable cellulolytic and/or hemicellulolytic enzymes, preferably in the presence of non-ionic detergent to yield the C5 and C6 enriched syrups.

Abstract: Methods and compositions for the production of ethanol from lignocellulosic starting materials are provided herein. Embodiments provide yeast cells of the genus H. polymorpha with one or more modifications, including, for example, an inactive acid trehalase gene, overexpression of xylulokinase, and/or overexpression of heat-shock protein 104.

Abstract: The present invention relates to the use of “green” or relatively benign solvents such as ethanol, ethanol/water, isopropyl alcohol, isopropyl alcohol/water, ethyl lactate, acetone, butanol, isoamyl alcohol, or ethyl acetate to extract phytosterols from wet corn fiber. The resulting oil product contains free phytosterols and free fatty acids.

Abstract: Methods and compositions for the production of ethanol from lignocellulosic starting materials are provided herein. Embodiments of the invention provide methods of manipulating the carbon flux of a host cell transformed with plasmids of the invention. Plasmids of the invention may include nucleotides that encode pyruvate decarboxylase. In one embodiment, a strain of the thermotolerant yeast Hansenula polymorpha that has been transformed with plasmids and polynucleotides of the invention is provided.

Abstract: A method for the production of ethanol and a modified animal feed is provided. The method replaces the starch in known corn-based animal feed with biomass fiber treated to make it more digestible by animals. The process includes wherein the pericarp and germ are removed from the corn kernel and processed for by-products. The starch and protein are also removed and separated. The starch is then fermented and distilled to ethanol and stillage. The bioavailable modified animal feed comprises the pericarp and germ removed from corn kernels and optionally by-products of the pericarp and germ processing, and lignocellulosic materials. The modified animal feed may optionally include energy materials such as animal and vegetable fats, vegetable soapstocks, or glycerin, and combinations thereof.

Abstract: Recombinant genetic constructs and strains of H. polymorpha having significantly increased ethanol productivity with a simultaneous decreased production of xylitol during high-temperature xylose fermentation are disclosed. The constructs include a H. polymorpha XYL1 gene encoding xylose reductase mutated to decrease affinity of the enzyme toward NADPH. The modified version of XYL1 gene under control of a strong constitutive HpGAP promoter was overexpressed in a ?xyll background. A recombinant H. polymorpha strain overexpressing the mutated enzyme together with native xylitol dehydrogenase and xylulokinase in the ?xyll background was also constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were evaluated during high-temperature xylose fermentation (48° C.). A significant increase in ethanol productivity (up to 7.4 times) was shown in the recombinant strain as compared with the wild type strain.