Ethanol production from corn cob hydrolysates by <Emphasis Type="Italic">Escherichia coli</Emphasis> KO11

Corn cob hydrolysates, with xylose as the dominant sugar, were fermented to ethanol by recombinant Escherichia coli KO11. When inoculum was grown on LB medium containing glucose, fermentation of the hydrolysate was completed in 163 h and ethanol yield was 0.50 g ethanol/g sugar. When inoculum was grown on xylose, ethanol yield dropped, but fermentation was faster (113 h). Hydrolysate containing 72.0 g/l xylose and supplemented with 20.0 g/l rice bran was readily fermented, producing 36.0 g/l ethanol within 70 h. Maximum ethanol concentrations were not higher for fermentations using higher cellular concentration inocula. A simulation of an industrial process integrating pentose fermentation by E. coli and hexose fermentation by yeast was carried out. At the first step, E. coli fermented the hydrolysate containing 85.0 g/l xylose, producing 40.0 g/l ethanol in 94 h. Baker's yeast and sucrose (150.0 g/l) were then added to the spent fermentation broth. After 8 h of yeast fermentation, the ethanol concentration reached 104.0 g/l. This two-stage fermentation can render the bioconversion of lignocellulose to ethanol more attractive due to increased final alcohol concentration. Journal of Industrial Microbiology & Biotechnology (2002) 29, 124–128 doi:10.1038/sj.jim.7000287 Received 20 February 2002/ Accepted in revised form 04 June 2002     

Ethanol production from alfalfa fiber fractions by saccharification and fermentation

This work describes ethanol production from alfalfa fiber using separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) with and without liquid hot water (LHW) pretreatment. Candida shehatae FPL-702 produced 5 and 6.4 g/l ethanol with a yield of 0.25 and 0.16 g ethanol/g sugar respectively by SHF and SSF from alfalfa fiber without pretreatment. With LHW pretreatment using SSF, C. shehatae FPL-702 produced 18.0 g/l ethanol, a yield of 0.45 g ethanol/g sugar from cellulosic solids or ‘raffinate’. Using SHF, it produced 9.6 g/l ethanol, a yield of 0.47 g ethanol/g sugar from raffinate. However, the soluble extract fraction containing hemicelluloses was poorly fermented in both SHF and SSF due to the presence of inhibitors. Addition of dilute acid during LHW pretreatment of alfalfa fiber resulted in fractions that were poorly saccharified and fermented. These results show that unpretreated alfalfa fiber produced a lower ethanol yield. Although LHW pretreatment can increase ethanol production from raffinate fiber fractions, it does not increase production from the hemicellulosic and pectin fractions.     

Fed-batch Cultivation of Mucor indicus in Dilute-acid Lignocellulosic Hydrolyzate for Ethanol Production

Mucor indicus fermented dilute-acid lignocellulosic hydrolyzates to ethanol in fed-batch cultivation with complete hexose utilization and partial uptake of xylose. The fungus was tolerant to the inhibitors present in the hydrolyzates. It grew in media containing furfural (1 g/l), hydroxymethylfurfural (1 g/l), vanillin (1 g/l), or acetic acid (7 g/l), but did not germinate directly in the hydrolyzate. However, with fed-batch methodology, after initial growth of M. indicus in 500 ml enzymatic wheat hydrolyzate, lignocellulosic hydrolyzate was fermented with feeding rates 55 and 100 ml/h. The fungus consumed more than 46% of the initial xylose, while less than half of this xylose was excreted in the form of xylitol. The ethanol yield was 0.43 g/g total consumed sugar, and reached the maximum concentration of 19.6 g ethanol/l at the end of feeding phase. Filamentous growth, which is regarded as the main obstacle to large-scale cultivation of M. indicus, was avoided in the fed-batch experiments.     

Red oak wood derived inhibitors in the ethanol fermentation of xylose byPichia stipitis CBS 5776

Summary Xylose, the predominant sugar in red oak prehydrolysate, is fermented to ethanol byPichia stipitis CBS 5776. Toxic model compounds derived from red oak hemicelluloses, lignin, and extractives inhibited the fermentation. Treatment of the prehydrolysate with molecular sieve and mixed bed ion resins facilitated the ethanol fermentation giving about 10 g/l ethanol from 32 g/l initial xylose. Fermentation inhibitors derived from red oak lignin and extractives were identified.     

Fermentation of enzymatically saccharified sunflower stalks for ethanol production and its scale up

Pretreated sunflower stalks saccharified with a Trichoderma reesei Rut-C 30 cellulase showed 57.8% saccharification. Enzyme hydrolysate concentrated to 40 g/l reducing sugars was fermented under optimum conditions of fermentation time (24 h), pH (5.0), temperature (30 degrees C) and inoculum size (3% v/v) and, showed a maximum ethanol yield of 0.444 g/g ethanol. Ethanol production scaled up in a 1 l and a 15 l fermenter under optimum conditions revealed maximum ethanol yields of 0.439 and 0.437 g/g respectively.     

Fermentation of corn fibre sugars by an engineered xylose utilizing Saccharomyces yeast strain

The ability of a recombinant Saccharomyces yeast strain to ferment the sugars glucose, xylose, arabinose and galactose which are the predominant monosaccharides found in corn fibre hydrolysates has been examined. Saccharomyces strain 1400 (pLNH32) was genetically engineered to ferment xylose by expressing genes encoding a xylose reductase, a xylitol dehydrogenase and a xylulose kinase. The recombinant efficiently fermented xylose alone or in the presence of glucose. Xylose-grown cultures had very little difference in xylitol accumulation, with only 4 to 5g/l accumulating, in aerobic, micro-aerated and anaerobic conditions. Highest production of ethanol with all sugars was achieved under anaerobic conditions. From a mixture of glucose (80g/l) and xylose (40g/l), this strain produced 52g/l ethanol, equivalent to 85% of theoretical yield, in less than 24h. Using a mixture of glucose (31g/l), xylose (15.2g/l), arabinose (10.5g/l) and galactose (2g/l), all of the sugars except arabinose were consumed in 24h with an accumulation of 22g ethanol/l, a 90% yield (excluding the arabinose in the calculation since it is not fermented). Approximately 98% theoretical yield, or 21g ethanol/l, was achieved using an enzymatic hydrolysate of ammonia fibre exploded corn fibre containing an estimated 47.0g mixed sugars/l. In all mixed sugar fermentations, less than 25% arabinose was consumed and converted into arabitol.     

The production of ethanol from sucrose using Zymomonas mobilis

Summary A new single-batch fermentation process for the commercial production of ethanol from refined sucrose, raw sugar, sugar cane juice and sugar cane syrup has been developed using a highly adapted and efficient strain of Zymomonas mobilis. The process gives a 94–98% sucrose hydrolysis efficiency and a 95–98% ethanol conversion efficiency. Within 24–30 h, 200 g/l sucrose is converted to produce 95.5 g/l ethanol. Reinoculation is carried out from the fermented broth without the need for centrifugation or membrane filtration.     

Fermentation of sugar cane bagasse hemicellulosic hydrolysate and sugar mixtures to ethanol by recombinant Escherichia coli KO11

Escherichia coli KO11, carrying the ethanol pathway genes pdc (pyruvate decarboxylase) and adh (alcohol dehydrogenase) from Zymomonas mobilis integrated into its chromosome, has the ability to metabolize pentoses and hexoses to ethanol, both in synthetic medium and in hemicellulosic hydrolysates. In the fermentation of sugar mixtures simulating hemicellulose hydrolysate sugar composition (10.0 g of glucose/l and 40.0 g of xylose/l) and supplemented with tryptone and yeast extract, recombinant bacteria produced 24.58 g of ethanol/l, equivalent to 96.4% of the maximum theoretical yield. Corn steep powder (CSP), a byproduct of the corn starch-processing industry, was used to replace tryptone and yeast extract. At a concentration of 12.5 g/l, it was able to support the fermentation of glucose (80.0 g/l) to ethanol, with both ethanol yield and volumetric productivity comparable to those obtained with fermentation media containing tryptone and yeast extract. Hemicellulose hydrolysate of sugar cane bagasse supplemented with tryptone and yeast extract was also readily fermented to ethanol within 48 h, and ethanol yield achieved 91.5% of the theoretical maximum conversion efficiency. However, fermentation of bagasse hydrolysate supplemented with 12.5 g of CSP/l took twice as long to complete. This revised version was published online in November 2006 with corrections to the Cover Date.     

Optimization of fermentation conditions for ethanol production from whey

Summary Optimal conditions for ethanol production in 7% whey solutions by the yeast Candida pseudotropicalis ATCC 8619 included initial pH of 4.57 and 30°C. Complete fermentation of the available lactose took place without supplementary nutrients; additions of nitrogen or phosphorus salts, yeast extract or corn steep liquor resulted in increased yeast production and lower ethanol yields. A positive correlation was observed between increases in yeast inocula and lactose utilization and ethanol production rates; 8.35 g/l of ethanol was obtained within 22 h by using yeast inoculum of 13.9 g/l. No differences in fermentation rates or ethanol yields were observed when whole or deproteinized whey solutions were used. Concentrated whey permeates, obtained after removal of the valuable proteins from whey, can be effectively fermented for ethanol production.     

Production of ethanol from corn stover hemicellulose hydrolyzate using <Emphasis Type="Italic">Pichia stipitis</Emphasis>

Hemicellulose liquid hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol using Pichia stipitis CBS 6054. The fermentation rate increased with aeration but the pH also increased due to consumption of acetic acid by Pichia stipitis. Hemicellulose hydrolyzate containing 34 g/L xylose, 8 g/L glucose, 8 g/L Acetic acid, 0.73 g/L furfural, and 1 g/L hydroxymethyl furfural was fermented to 15 g/L ethanol in 72 h. The yield in all the hemicellulose hydrolyzates was 0.37–0.44 g ethanol/g (glucose + xylose). Nondetoxified hemicellulose hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol with high yields, and this has the potential to improve the economics of the biomass to ethanol process.     

Xylose fermentation by genetically modified Saccharomyces cerevisiae 259ST in spent sulfite liquor

Spent sulfite pulping liquor (SSL) is a high-organic content byproduct of acid bisulfite pulp manufacture which is fermented to make industrial ethanol. SSL is typically concentrated to 240 g/l (22% w/w) total solids prior to fermentation, and contains up to 24 g/l xylose and 30 g/l hexose sugars, depending upon the wood species used. The xylose present in SSL is difficult to ferment using natural xylose-fermenting yeast strains due to the presence of inhibitory compounds, such as organic acids. Using sequential batch shake flask experiments, Saccharomyces cerevisiae 259ST, which had been genetically modified to ferment xylose, was compared with the parent strain, 259A, and an SSL adapted strain, T2, for ethanol production during SSL fermentation. With an initial SSL pH of 6, without nutrient addition or SSL pretreatment, the ethanol yield ranged from 0.32 to 0.42 g ethanol/g total sugar for 259ST, compared to 0.15-0.32 g ethanol/g total sugar for non-xylose fermenting strains. For most fermentations, minimal amounts of xylitol (<1 g/l) were produced, and glycerol yields were approximately 0.12 g glycerol/g sugar consumed. By using 259ST for SSL fermentation up to 130% more ethanol can be produced compared to fermentations using non-xylose fermenting yeast.     

Effect of substrate concentration on ethanol production by Zymomonas mobilis on cellulose hydrolysate

Summary A cellulose hydrolysate from Aspen wood, containing mainly glucose, was fermented into ethanol by a thermotolerant strain MSN77 of Zymomonas mobilis. The effect of the hydrolysate concentration on fermentation parameters was investigated. Growth parameters (specific growth rate and biomass yield) were inhibited at high hydrolysate concentrations. Catabolic parameters (specific glucose uptake rate, specific ethanol productivity and ethanol yield) were not affected. These effects could be explained by the increase in medium osmolality. The results are similar to those described for molasses based media. Strain MSN77 could efficiently ferment glucose from Aspen wood up to a concentration of 60 g/l. At higher concentration, growth was inhibited.Nomenclature S glucose concentration (g/l) - X biomass concentration (g/l) - P ethanol concentration (g/l) - C conversion of glucose (%) - t fermentation time (h) - q_S specific glucose uptake rate (g/g.h) - q_p specific ethanol productivity (g/g.h) - YINX/S biomass yield (g/g) - Y_p/S ethanol yield (g/g) - specific growth rate (h^-1)     

Tower fermentation of sugar cane juice

Summary Sugar cane juice, of feed fermentables concentration of 100–180g/l, was simply and rapidly fermented to ethanol in a tower fermenter, using a naturally flocculant yeast. Stable cell densities of 65–80g dry wt/l were obtained. Conversion and ethanol productivity data are given as a function of throughput and initial feed concentration. The limitation to fermenter performance with this yeast was imposed by carryover of non-fermented sugars, principally fructose, rather than washout of the yeast flocs under high hydraulic loadings (residence times of less than 1.5 hours). Further improvement of the fermenter performance may be achieved by strain selection.     

Continuous-flow fermentation of banana fruit pulp sugar into ethanol by carrageenan-immobilized yeast

Summary The extracted juice of ripe Cavendish banana fruit pulp, which contained about 126 g/l total sugars, was fermented into ethanol by yeast immobilized on kappa-carrageenan. The volumetric productivity and fermentation efficiency were about 15 g/l-h and 94%, respectively. The concentrations of alcohol and residual sugar in the beer were 54 g/l and 12.8–14.5 g/l, respectively.     

Elimination of ethanol inhibition by perstraction

Perstraction (membrane-aided solvent extraction) was utilized for elimination of ethanol inhibition in continuous ethanol fermentation using high sugar concentrate. Hollow fibers for an artificial kidney were used as a permeable membrane, and their capacity to extract ethanol was examined by using several organic solvents. When tri-n-butylphosphate was used as an extractant, a 500 g/L feed glucose medium was successfully fermented by immobilized yeast cells. During this continuous fermentation a high ehtanol productivity of 48 g/h-L-gel was held, and the solvent requirement per consumed glucose was 6 L-solvent/kg-glucose.     

Fermentation of arabinose to ethanol by Sarcina ventriculi

Summary In the absence of oxygen, a strain of sarcina ventriculi, isolated from soil, could rapidly and completely ferment up to 20 g/l of arabinose. The principal products were ethanol, acetate, CO₂ and H₂. The yield of alcohol, up to 30% by weight of the sugar fermented, was not appreciably influenced by the pH of fermentation in the range 4–7. Sugar concentrations up to 100 g/l did not affect initial growth, but fermentation was incomplete at high sugar levels. This was probably due to the accumulation of end products other than ethanol, because the cells could grow in the presence of up to 25 g/l of added ethanol. Glucose, galactose and arabinose were sequentially utilized, in that order, when initially present as a mixed substrate. These sugars are major components of the hemicellulose from some agricultural residues. Practical implications for the general problem of pentose conversion to alcohol are discussed briefly.     

Bioconversion into ethanol of decorticated red sorghum (Sorghum bicolor L. Moench) supplemented with its phenolic extract or spent bran

The effect of extracted phenolics or spent bran added to decorticated red sorghum kernels during fuel ethanol production was studied and compared to maize and whole red and white sorghums. After liquefaction, free amino nitrogen ranged from 65 to 101 mg/l and at the end of saccharification all mashes had approx. 80 g glucose and 2–5 g maltose/100 g meal (dry basis). Saccharified worts were fermented giving 50–90 ml ethanol/l. The lowest fermentation efficiency (76%) was obtained in the white sorghum. Ethanol yields indicate that sorghum bran or its associated phenolics did not significantly affect the efficiency of the sequential steps involved in ethanol production. Red sorghum is a good alternative to maize to produce ethanol and the difference regarding white sorghum and maize was mainly due to endosperm protein structure and composition.     

Production of ethanol from straw and bamboo pulp by primary isolates of Clostridium thermocellum

Clostridium thermocellum strains SS8 and GS1 grew poorly on crude blopolymers but termented them easily after alkall treatment. With 1% alkall-extracted rice straw (AERS) and dellgnified bamboo pulp (DBP), the ethanol-to-substrate (E/S) ratios were almost the same as those obtained when using fillter paper. Increasing the substrate concentrations decreased the percentage substrate degraded and the E/S ratio and concomitantly increased the amount of reducing sugars accumulated. A maximum amount of 8.6 g ethanol/l was produced by strain SS8 out of 37.5 g DBP degraded. Strain GS1 accumulated reducing sugars at substrate concentrations >50 g/l, thereby accounting for about 70% of AERS degraded. This strain produced cellulase on both cellulose and cellobiose. Both the strains grew in the presence of 1.5% (v/v) ethanol. Strain SS8 fermented starch, but the ethanol yield was low compared to that from cellulose. About 75% of starch degraded accumulated as reducing sugars at a substrate concentration of 40 g/l. The Inhibitory effects of ethanol (2 to 4%) were less drastic when growing cultures were challenged than when they were formed in situ. The effect of ethanol depended upon the phase of the culture.The authors are with the Department of Microbiology, Osmania University, Hyderabad-500007, India.     

Improvement of direct ethanol fermentation from woody biomasses by the Antarctic basidiomycetous yeast, Mrakia blollopis, under a low temperature condition

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 can quite uniquely ferment various sugars under low temperature conditions. When strain SK-4 fermented lignocellulosic biomass using the direct ethanol fermentation (DEF) technique, approximately 30% to 65% of the theoretical ethanol yield was obtained without and with the addition of the non-ionic surfactant Tween 80, respectively. Therefore, DEF from lignocellulosic biomass with M. blollopis SK-4 requires the addition of a non-ionic surfactant to improve fermentation efficiency. DEF with lipase converted Eucalyptus and Japanese cedar to 12.6 g/l, and 14.6 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80 and 5 U/g-dry substrate lipase, ethanol concentration increased about 1.4- to 2.4-fold compared to that without Tween 80 and lipase. We therefore consider that the combination of M. blollopis SK-4 and DEF with Tween 80 and lipase has good potential for ethanol fermentation in cold environments.     

Ethanol production from dilute acid hydrolysate of rice hulls using genetically engineered Escherichia coli

Approximately 30% of rice hulls, which represent an abundant residue with little commercial value, was solubilized with 0.4 M H2SO4 acid to produce a syrup containing over 100 g monomer sugar/l. Toxins generated during hydrolysis were mitigated with Ca(OH)2. Treated hydrolysate plus additional nutrients was fermented with Escherichia coli KO11 to produce over 46 g ethanol/L in 72 h (92% of theoretical yield). © Rapid Science Ltd. 1998