Rodney J. Bothast

Conversion of D-xylose to ethanol by the yeast Pachysolen tannophilus.

The yeast Pachysolen tannophilus was found to be capable of converting D‐xylose to ethanol. Batch cultures initially containing 50 g/L D‐xylose yielded 0.34 g of ethanol per gram of pentose consumed. Aerobic conditions were required for cell growth but not for ethanol production. Both alcohol formation and growth were optimum when incubation temperature was 32°C, when pH was near 2.5, and when D‐xylose and ethanol concentrations did not exceed 50 and 20 g/L, respectively.

Use of catabolite repression mutants for fermentation of sugar mixtures to ethanol

Abstract. Use of agricultural biomass, other than cornstarch, to produce fuel ethanol requires a microorganism that can ferment the mixture of sugars derived from hemicellulose. Escherichiacoli metabolizes a wide range of substrates and has been engineered to produce ethanol in high yield from sugar mixtures. E. coli metabolizes glucose in preference to other sugars and, as a result, utilization of the pentoses in hemicellulose-derived sugar mixtures is delayed and may be incomplete. Residual sugar lowers the ethanol yield and is problematic for downstream processing of fermentation products. Therefore, a catabolite repression mutant that simultaneously utilizes glucose and pentoses would be useful for fermentation of complex substrate mixtures. We constructed ethanologenic E. coli strains with a glucose phosphotransferase (ptsG) mutation and used the mutants to ferment glucose, arabinose, and xylose, singly and in mixtures, to ethanol. Yields were 87–94% of theoretical for both the wild type and mutants, but the mutants had an altered pattern of mixed sugar utilization. Phosphotransferase mutants metabolized the pentoses simultaneously with glucose, rather than sequentially. Based upon fermentations of sugar mixtures, a catabolite-repression mutant of ethanologenic E. coli is expected to provide more efficient fermentation of hemicellulose hydrolysates by allowing direct utilization of pentoses.

Pretreatment and enzymatic saccharification of corn fiber

Corn fiber consists of about 20% starch, 14% cellulose, and 35% hemicellulose, and has the potential to serve as a low-cost feedstock for production of fuel ethanol. Several pretreatments (hot water, alkali, and dilute, acid) and enzymatic saccharification procedures were evaluated for the conversion of corn fiber starch, cellulose, and hemicellulose to monomeric sugars. Hot water pretreatment (121°C, 1 h) facilitated the enzymatic sacch arification of starch and cellulose but not hemicellulose. Hydrolysis of corn fiber pretreated with alkali un dersimilar conditions by enzymatic means gave similar results. Hemicellulose and starch components were converted to monomeric sugars by dilute H2SO4 pretreatment (0.5–1.0%, v/v) at 121°C. Based on these findings, a method for pretreatment and enzymatic saccharification of corn fiber is presented. It in volves the pretreatment of corn fiber (15% solid, w/v) with dilute acid (0.5% H2SO4, v/v) at 121°C for 1 h, neutralization to pH 5.0, then saccharification of the pretreated corn fiber material with commercial cellulase and β-glucosidase preparations The yield of monomeric sugars from corn fiber was typically 85–100% of the theoretical yield.

Fermentations with New Recombinant Organisms

United States fuel ethanol production in 1998 exceeded the record production of 1.4 billion gallons set in 1995. Most of this ethanol was produced from over 550 million bushels of corn. Expanding fuel ethanol production will require developing lower‐cost feedstocks, and only lignocellulosic feedstocks are available in sufficient quantities to substitute for corn starch. Major technical hurdles to converting lignocellulose to ethanol include the lack of low‐cost efficient enzymes for saccharification of biomass to fermentable sugars and the development of microorganisms for the fermentation of these mixed sugars. To date, the most successful research approaches to develop novel biocatalysts that will efficiently ferment mixed sugar syrups include isolation of novel yeasts that ferment xylose, genetic engineering of Escherichia coli and other gram negative bacteria for ethanol production, and genetic engineering of Saccharoymces cerevisiae and Zymomonas mobilis for pentose utilization. We have evaluated the fermentation of corn fiber hydrolyzates by the various strains developed. E. coliK011, E. coli SL40,E. coli FBR3, Zymomonas CP4 (pZB5), and Saccharomyces 1400 (pLNH32) fermented corn fiber hydrolyzates to ethanol in the range of 21–34 g/L with yields ranging from 0.41 to 0.50 g of ethanol per gram of sugar consumed. Progress with new recombinant microorganisms has been rapid and will continue with the eventual development of organisms suitable for commercial ethanol production. Each research approach holds considerable promise, with the possibility existing that different “industrially hardened” strains may find separate applications in the fermentation of specific feedstocks.

Development of New Ethanologenic Escherichia coli Strains for Fermentation of Lignocellulosic Biomass

Two new ethanologenic strains (FBR4 and FBR5) of Escherichia coli were constructed and used to ferment corn fiber hydrolysate. The strains carry the plasmid pLOI297, which contains the genes from Zymomonas mobilis necessary for efficiently converting pyruvate into ethanol. Both strains selectively maintained the plasmid when grown anaerobically. Each culture was serially transferred 10 times in anaerobic culture with sugar-limited medium containing xylose, but no selective antibiotic. An average of 93 and 95% of the FBR4 and FBR5 cells, respectively, maintained pLOI297 in anaerobic culture. The fermentation performances of the repeatedly transferred cultures were compared with those of cultures freshly revived from stock in pH-controlled batch fermentations with 10% (w/v) xylose. Fermentation results were similar for all the cultures. Fermentations were completed within 60 h and ethanol yields were 86-92% of theoretical. Maximal ethanol concentrations were 3.9-4.2% (w/v). The strains were also tested for their ability to ferment corn fiber hydrolysate, which contained 8.5% (w/v) total sugars (2.0% arabinose, 2.8% glucose, and 3.7% xylose). E. coli FBR5 produced more ethanol than FBR4 from the corn fiber hydrolysate. E. coli FBR5 fermented all but 0.4% (w/v) of the available sugar, whereas strain FBR4 left 1.6% unconsumed. The fermentation with FBR5 was completed within 55 h and yielded 0.46 g of ethanol/g of available sugar, 90% of the maximum obtainable.

Comparative evaluation of ethanol production by xylose-fermenting yeasts presented high xylose concentrations

SummaryThree strains ofPichiastipitis and three ofCandidashehatae were compared withPachysolentannophilus in their abilities to ferment xylose at concentrations as high as 200 g/L when subjected to both aerobic and microaerophilic conditions. Evaluations based on accumulated ethanol concentrations, ethanol productivities, xylose consumption, and ethanol and xylitol yields were determined from batch culture time courses. Of the strains considered,P.stipitis NRRL Y-7124 seemed most promising since it was able to utilize all but 7 g/L of 150 g/L xylose supplied aerobically to produce 52 g/L ethanol at a yield of 0.39 g per gram xylose (76% of theoretical yield) and at a rate comparable to the fastest shown byC.shehatae NRRL Y-12878. For all strains tested, fermentation results from aerobic cultures were more favorable than those from microaerophilic cultures.

Ethanol production from hemicellulose hydrolysates of agricultural residues using genetically engineeredEscherichia coli strain KO11

Hemicellulose hydrolysates of the agricultural residues bagasse, corn stover, and corn hulls plus fibers were readily fermented to ethanol by recombinantEscherichia coli strain KO11. Corn steep liquor and crude yeast autolysate served as excellent nutrients. Fermentations were substantially complete within 48 h, often achieving over 40 g ethanol L−1. Ethanol yields ranged from 86% to over 100% of the maximum theoretical yield (0.51 g ethanol g sugar−1.

Assessment of ethanol production options for corn products

Abstract The production of ethanol from corn fiber has the potential to increase ethanol yields by a maximum of 0.3 gal/bushel in a wet-milling process. Incremental yields would be 0.13 gal/bushel from hexose, 0.1 from d -xylose and 0.07 from l -arabinose, at 100% hydrolysis and fermentation efficiency. At 80% efficiency for hexose hydrolysis and fermentation, and 70% for pentose, an incremental yield of 0.22 gallons/bushel of corn is expected. Of this total, 0.1 gal/bushel would be from hexoses, 0.07 from d -xylose, and 0.05 from l arabinose. A maximum practical incremental yield would probably fall between 0.22 and 0.3 gallons/bushel. These calculations are based on published compositional analyses of cellulose, starch, mono-saccharides, hemicellulose, protein and oil as distributed between the compartmentalized components of the corn kernel and published yield factors for hexose and pentose fermentations. Experimental yield factors for xylose (0.36 g ethanol/g xylose) and arabinose (0.34) fermenting microorganisms are lower than that for glucose (0.45–0.50), and significantly less than the theoretical yield of 0.51 g ethanol/g pentose. Nonetheless, we estimate that a wet-milling facility which currently produces 100 million gallons/year of ethanol from starch could generate an additional

Recombinant Escherichia coli engineered for production of L-lactic acid from hexose and pentose sugars

4–8 million of annual income if the fiber components were processed into ethanol. Hence, advances in fiber pretreatment and pentose fermentation are likely to have a major impact on enhancing productivity of corn ethanol plants. An engineering framework for assigning economic consequences of the additional utilization of fiber is presented.

Fermentation of sugar mixtures using Escherichia coli catabolite repression mutants engineered for production of L-lactic acid

Recombinant Escherichia coli have been constructed for the conversion of glucose as well as pentose sugars into L-lactic acid. The strains carry the lactate dehydrogenase gene from Streptococcus bovis on a low copy number plasmid for production of L-lactate. Three E. coli strains were transformed with the plasmid for producing L-lactic acid. Strains FBR9 and FBR11 were serially transferred 10 times in anaerobic cultures in sugar-limited medium containing glucose or xylose without selective antibiotic. An average of 96% of both FBR9 and FBR11 cells maintained pVALDH1 in anaerobic cultures. The fermentation performances of FBR9, FBR10, and FBR11 were compared in pH-controlled batch fermentations with medium containing 10% w/v glucose. Fermentation results were superior for FBR11, an E. coli B strain, compared to those observed for FBR9 or FBR10. FBR11 exhausted the glucose within 30 h, and the maximum lactic acid concentration (7.32% w/v) was 93% of the theoretical maximum. The other side-products detected were cell mass and succinic acid (0.5 g/l). Journal of Industrial Microbiology & Biotechnology (2001) 27, 259–264.