Enzymatic digestion of liquid hot water pretreated hybrid poplar

Liquid hot (LHW) water pretreatment (LHW) of lignocellulosic material enhances enzymatic conversion of cellulose to glucose by solubilizing hemicellulose fraction of the biomass, while leaving the cellulose more reactive and accessible to cellulase enzymes. Within the range of pretreatment conditions tested in this study, the optimized LHW pretreatment conditions for a 15% (wt/vol) slurry of hybrid poplar were found to be 200^oC, 10 min, which resulted in the highest fermentable sugar yield with minimal formation of sugar decomposition products during the pretreatment. The LHW pretreatment solubilized 62% of hemicellulose as soluble oligomers. Hot‐washing of the pretreated poplar slurry increased the efficiency of hydrolysis by doubling the yield of glucose for a given enzyme dose. The 15% (wt/vol) slurry of hybrid poplar, pretreated at the optimal conditions and hot‐washed, resulted in 54% glucose yield by 15 FPU cellulase per gram glucan after 120 h. The hydrolysate contained 56 g/L glucose and 12 g/L xylose. The effect of cellulase loading on the enzymatic digestibility of the pretreated poplar is also reported. Total monomeric sugar yield (glucose and xylose) reached 67% after 72 h of hydrolysis when 40 FPU cellulase per gram glucan were used. An overall mass balance of the poplar‐to‐ethanol process was established based on the experimentally determined composition and hydrolysis efficiencies of the liquid hot water pretreated poplar. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Ethanol production from wood hydrolysate using genetically engineered Zymomonas mobilis

An ethanologenic microorganism capable of fermenting all of the sugars released from lignocellulosic biomass through a saccharification process is essential for secondary bioethanol production. We therefore genetically engineered the ethanologenic bacterium Zymomonas mobilis such that it efficiently produced bioethanol from the hydrolysate of wood biomass containing glucose, mannose, and xylose as major sugar components. This was accomplished by introducing genes encoding mannose and xylose catabolic enzymes from Escherichia coli. Integration of E. coli manA into Z. mobilis chromosomal DNA conferred the ability to co-ferment mannose and glucose, producing 91 % of the theoretical yield of ethanol within 36 h. Then, by introducing a recombinant plasmid harboring the genes encoding E. coli xylA, xylB, tal, and tktA, we broadened the range of fermentable sugar substrates for Z. mobilis to include mannose and xylose as well as glucose. The resultant strain was able to ferment a mixture of 20 g/l glucose, 20 g/l mannose, and 20 g/l xylose as major sugar components of wood hydrolysate within 72 h, producing 89.8 % of the theoretical yield. The recombinant Z. mobilis also efficiently fermented actual acid hydrolysate prepared from cellulosic feedstock containing glucose, mannose, and xylose. Moreover, a reactor packed with the strain continuously produced ethanol from acid hydrolysate of wood biomass from coniferous trees for 10 days without accumulation of residual sugars. Ethanol productivity was at 10.27 g/l h at a dilution rate of 0.25 h(-1).     

Co-fermentation of a mixture of glucose and xylose to ethanol by Zymomonas mobilis and Pachysolen tannophilus

This research was designed to maximize ethanol production from a glucose-xylose sugar mixture (simulating a sugar cane bagasse hydrolysate) by co-fermentation with Zymomonas mobilis and Pachysolen tannophilus. The volumetric ethanol productivity of Z. mobilis with 50 g glucose/l was 2.87 g/l/h, giving an ethanol yield of 0.50 g/g glucose, which is 98% of the theoretical. P. tannophilus when cultured on 50 g xylose/l gave a volumetric ethanol productivity of 0.10 g/l/h with an ethanol yield of 0.15 g/g xylose, which is 29% of the theoretical. On optimization of the co-fermentation with the sugar mixture (60 g glucose/l and 40 g xylose/l) a total ethanol yield of 0.33 g/g sugar mixture, which is 65% of the theoretical yield, was obtained. The co-fermentation increased the ethanol yield from xylose to 0.17 g/g. Glucose and xylose were completely utilized and no residual sugar was detected in the medium at the end of the fermentation. The pH of the medium was found to be a good indicator of the fermentation status. The optimum conditions were a temperature of 30°C, initial inoculation with Z. mobilis and incubation with no aeration, inactivation of bacterium after the utilization of glucose, followed by inoculation with P. tannophilus and incubation with limited aeration.     

L-lactate production from xylose employingLactococcus lactis IO-1

Summary The growth, substrate utilisation and L-lactate production ofLactococcus lactis IO-1 were examined on xylose, and glucose and xylose media. The yield of lactate on xylose was 0.47 g lactate/g xylose at an initial xylose concentration of 51.2 g/l and the _max was 0.72 h^–1. Xylose cultures were more susceptible to lactate inhibition than were glucose cultures but showed similar kinetic behaviour. The organism was capable of complete sugar utilisation when grown on a mixture of 20 g/l xylose and 20 g/l glucose and synthesised 0.66 g lactate/g sugar.     

Optimization of pH controlled liquid hot water pretreatment of corn stover

Controlled pH, liquid hot water pretreatment of corn stover has been optimized for enzyme digestibility with respect to processing temperature and time. This processing technology does not require the addition of chemicals such as sulfuric acid, lime, or ammonia that add cost to the process because these chemicals must be neutralized or recovered in addition to the significant expense of the chemicals themselves. Second, an optimized controlled pH, liquid hot water pretreatment process maximizes the solubilization of the hemicellulose fraction as liquid soluble oligosaccharides while minimizing the formation of monomeric sugars. The optimized conditions for controlled pH, liquid hot water pretreatment of a 16% slurry of corn stover in water was found to be 190 degrees C for 15 min. At the optimal conditions, 90% of the cellulose was hydrolyzed to glucose by 15FPU of cellulase per gram of glucan. When the resulting pretreated slurry, in undiluted form, was hydrolyzed by 11FPU of cellulase per gram of glucan, a hydrolyzate containing 32.5 g/L glucose and 18 g/L xylose was formed. Both the xylose and the glucose in this undiluted hydrolyzate were shown to be fermented by recombinant yeast 424A(LNH-ST) to ethanol at 88% of theoretical yield.     

High-solids enzymatic hydrolysis of steam-exploded willow without prior water washing

A laboratory reactor equipped with a screw press was used for the hydrolysis of steam-SO₂-exploded willowSalix caprea by a composition ofTrichoderma reesei andAspergillus foetidus enzyme preparations at high substrate concentration. Optimal conditions providing the maximal volume of hydrolysis syrup with maximal sugar concentrations were determined. Two different hydrolysis procedures were developed in order to exclude the initial washing of steam-pretreated plant raw material by large volumes of water, which was necessary to eliminate the inhibitory effect of explosion byproducts on enzymatic hydrolysis. The first procedure included enzymatic prehydrolysis of the substrate for 1 h; separation of sugar syrup containing 40–60 g/l glucose, 20–25 g/l xylose, and up to 10 g/l disaccharides, as well as up to 35% of the initial enzymatic activity; then addition of a diluted acetate buffer (pH 4.5); and subsequent hydrolysis of the substrate by the adsorbed enzymes leading to the final accumulation of up to 140 g/l glucose and up to 15 g/l of xylose. In the second scenario, the exploded willow was initially adjusted by alkali to pH 4.5 and then hydrolyzed directly by the added enzymes over 24 h. This procedure resulted in a nearly total polysaccharide hydrolysis and accumulation of up to 170 g/l glucose and 20 g/l xylose. The reasons for inhibition of enzymatic hydrolysis are discussed. Deceased.     

Continuous fermentation of feed streams containing D-glucose and D-xylose in a two-stage process utilizing immobilized Saccharomyces cerevisiae and Pachysolen tannophilus

In the U.S., forest and crop residues contain enough glucose and xylose to supply 10 times the country's usage of ethanol and ethylene, but an efficient fermentation scheme is lacking,(1,2,3) To develop a strategy for process design, specific ethanol productivities and yields of Pachysolen tannophilus NRRL Y-2460 and Saccharomyces cerevisiae NRRL Y-2235 were compared. Batch cultures and continuous stirred reactors (CSTR) loaded with immobilized cells were fed glucose and xylose. As expected from previous reports, Y-2235 fermented glucose but not xylose. Y-2460 consumed both sugars but fermented glucose inefficiently relative to Y-2235, and it suffered a diauxic lag lasting 10-20 h when given a sugar mixture. Immobilized Y-2235 exhibited increasing productivity but constant yield with in creasing glucose concentration. In contrast, Y-2460 exhibited an optimum productivity at 30-40 g/L xylose and a declining yield with increasing xylose concentration. Immobilized Y-2235 tolerated more than 100 g/L ethanol while the productivity and yield of Y-2460 fell by 80 and 58%, respectively, as ethanol reached 50 g/L. A 38.8-g/L ethanol stream could be produced as 103 g/L xylose was continuously fed to Y-2460. If it was blended with a 274 g/L glucose stream to give a composite of 23.7 g/L ethanol and 107 g/L glucose, Y-2235 could en rich the ethanol to 75 g/L. Taken together these results suggest use of a two-stage continuous reactor for pro cessing xylose and glucose from lignocellulose. An immobilized Y-2460 CSTR (or cascade) would convert the hemicellulose hydrolyzate. Then downstream, an immobilized Y-2235 plug flow reactor would enrich the hemicellulose-derived ethanol to more than 70 g/L upon addition of cellulose hydrolyzate.     

Enzymatic hydrolysis of willow treated with a steam burst without preliminary water extraction with a high concentration of substrate

A laboratory reactor equipped with a screw press was used for hydrolysis of steam-SO2 exploded willow Salix caprea by a composition of Trichoderma reesei and Aspergillus foetidus enzyme preparations at high substrate concentrations. Optimal conditions providing the maximal volume of hydrolysis syrup with maximal sugar concentrations were determined. Two different hydrolysis procedures were developed in order to exclude initial washing of steam-pretreated plant raw material by large volumes of water, which is necessary to eliminate the inhibitory effect of explosion by-products on enzymatic hydrolysis. The first procedure included a one-hour-long enzymatic prehydrolysis of the substrate, then separation of sugar syrup containing 40-60 g/l of glucose, 20-25 g/l of xylose, and up to 10% of disaccharides, as well as up to 35% of the initial enzymatic activity, then addition of a diluted acetate buffer (pH 4.5), and subsequent hydrolysis of the substrate by the adsorbed enzymes leading to the final accumulation of up to 140 g/l glucose and up to 15 g/l xylose. In the second scenario, the exploded willow was initially adjusted by alkali to pH 4.5 and then hydrolyzed directly by added enzymes for 24 hours. This procedure resulted in a nearly total polysaccharide hydrolysis and accumulation of up to 170 g/l glucose and 20 g/l xylose. The reasons of inhibition of enzymatic hydrolysis are discussed.     

The influence of aeration and hemicellulosic sugars on xylitol production by Candida tropicalis

The influence of other hemicellulosic sugars (arabinose, galactose, mannose and glucose), oxygen limitation, and initial xylose concentration on the fermentation of xylose to xylitol was investigated using experimental design methodology. Oxygen limitation and initial xylose concentration had considerable influences on xylitol production by Canadida tropicalis ATCC 96745. Under semiaerobic conditions, the maximum xylitol yield was 0.62 g/g substrate, while under aerobic conditions, the maximum volumetric productivity was 0.90 g/l h. In the presence of glucose, xylose utilization was strongly repressed and sequential sugar utilization was observed. Ethanol produced from the glucose caused 50% reduction in xylitol yield when its concentration exceeded 30 g/l. When complex synthetic hemicellulosic sugars were fermented, glucose was initially consumed followed by a simultaneous uptake of the other sugars. The maximum xylitol yield (0.84 g/g) and volumetric productivity (0.49 g/l h) were obtained for substrates containing high arabinose and low glucose and mannose contents.     

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.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid from a mixture of xylose and glucose by co-cultivation of lactic acid bacteria

The production of optically pure lactic acid in a high yield from xylose or a mixture of xylose and glucose, which is a model hydrolysate of lignocellulose, is described. In a single cultivation, Enterococcus casseliflavus produced 38 g/l of lactic acid with an optical purity of 96% enantiomeric excess (ee) and 6.4 g/l of acetic acid from 50 g/l of xylose when MRS medium was used. When a mixture of 50 g/l of xylose and 100 g/l of glucose was used as the carbon source in a cultivation of E. casseliflavus alone, glucose was converted to lactic acid in the early phase of the cultivation but xylose was hardly consumed. In a co-cultivation where E. casseliflavus and Lactobacillus casei specific for glucose were simultaneously inoculated, little or no lactic acid was produced after the glucose was almost consumed. A co-cultivation with two-stage inoculation (in which E. casseliflavus was added at a cultivation time of 40 h after L. casei cells were inoculated) resulted in complete consumption of 50 g/l of xylose and 100 g/l of glucose. In the co-cultivation, 95 g/l of lactic acid with a high optical purity of 96% ee was obtained at 192 h. Such a co-cultivation using two microorganisms specific for each sugar is considered to be one promising cultivation technique for the efficient production of lactic acid from a sugar mixture derived from lignocellulose.     

One-dimensional model of the acid hydrolysis of cellulosic substrates in a solid-liquid cyclone reactor

The use of rotating flow in an annulus is investigated as a means of enhancing the yield of glucose and xylose in the acid hydrolysis of cellulosic slurries. A one-dimensional model of such a cyclone reactor is developed for flow cases, co-current and counter-current flow. For the case of 250°C, 1% w/w acid, the one-dimensional model indicates an increase in the maximum glucose yield from 48.1% in a plug flow reactor to 69.3% in a co-current cyclone reactor, and up to 81.0% in a countercurrent cyclone reactor. The corresponding xylose yields are 91.6% for co-current operation and 97.7% for countercurrent operation. In the co-current case the maximum glucose and xylose yields do not occur at the same location in the reactor; however, in the countercurrent case they do. Although product yields are dramatically improved over those obtained in a plug flow reactor, the product concentrations are lower than would typically be obtained in a plug flow reactor.List of Symbols A cm² cross sectional area perpendicular to radial flow - A _c cm² cross sectional area of slurry inlet - A _c cm² cross sectional area of steam inlet - A _w cm² cross sectional area of water inlet - C _c concentration of cellulose as potential glucose (grams of potential glucose/cm³ of total stream) - C _c ^* grams cellulose/cm³ of solids concentration of cellulose as potential glucose - C _ginitial ^* grams glulose/cm³ of solids concentration of cellulose entering reactor - C _g grams glucose/cm³ of total stream concentration of glucose - C _g ^* grams glucose/cm³ of liquid stream concentration of glucose - C _cinitial ^* grams cellulose/cm³ of liquid concentration of glucose entering reactor - C _xn concentration of xylan as potential xylose (grams of potential xylose/cm³ of total stream) - C _xs grams xyclose/cm³ of total stream concentration of nylose - d _f dilution factor - dr cm radial increment - g cm/s² gravitational acceleration - g ^* centrifugal acceleration proportionality constant - h cm height of cyclone reactor - j cm/s flux - K constant in general equation for vortex flow, Eq. (4.9) - k ₁ 1/s kinetic rate constant of cellulose hydrolysis - k _a 1/s kinetic rate constant of xylan hydrolysis - k ₂ 1/s kinetic rate constant of glucose decomposition - k _2a 1/s kinetic rate constant of xylose decomposition - m vortex exponent - M _steam g/s mass rate of steam addition at outer radius - M _water g/s mass rate of cold water addition at outer radius - n cm³/s empirically determined settling parameter - Q cm³/s net volumetric flow in outward radial direction - Q _tot cm³/s total volumetric flow through reactor - q _c cm³/s volumetric flow of slurry feed - q _s cm³/s volumetric flow of stream feed - q _water cm³/s volumetric flow of cold water feed - r cm radial position - r _c 1/s rate of cellulose hydrolysis - r _g 1/s rate of glucose decomposition - r _i cm inner radius - r _o cm outer radius - r _xn 1/s rate of xylan hydrolysis - r _xs 1/s rate of xylose decomposition - s _mom cm g/s² inlet steam momentum - T _bulk s bulk residence time in reactor - T °C reactor temperature - v _c cm³/g specific volume of slurry feed - v _s cm³/g specific volume of steam - v _w cm³/g specific volume of water - V _f cm/s velocity of liquid as a function of radius - V _i cm/s inlet velocity - V _s cm/s velocity of solids as a function of radius - V _steam cm/s inlet steam velocity to cyclone - V cm/s terminal settling velocity - V _q cm/s tangential velocity - w _mom cm g/s² water inlet momentum - Y grams product out/grams reactant in yield of product - solids volumetric fraction - _f solids volumetric fraction in slurry feed - _i initial solids volumetric fraction of slurry - Pi     

Production of xylitol by recombinant Saccharomyces cerevisiae containing xylose reductase gene in repeated fed-batch and cell-recycle fermentations

Xylitol is a well-known sugar substitute with low-calorie and anti-cariogenic characteristics. An effort of biological production of xylitol from xylose was made in repeated fed-batch and cell-recycle fermentations of recombinant Saccharomyces cerevisiae BJ3505/δXR harboring the xylose reductase gene from Pichia stipitis. Batch fermentation with 20 g/l xylose and 18 g/l glucose resulted in 9.52 g/l dry cell mass, 20.1 g/l xylitol concentration and approximately 100% conversion yield. Repeated fed-batch operation to remove 10% of culture broth and to supplement an equal volume of 200 g/l xylose was designed to improve xylitol production. In spite of a sudden drop of cell concentration, an increase in dry cell mass led to high accumulation of xylitol at 48.7 g/l. To overcome loss of xylitol-producing biocatalysts in repeated fed-batch fermentation, cell-recycle equipment of hollow fiber membrane was implemented into a xylitol production system. Cell-recycle operation maintained concentration of the recombinant cells high inside a bioreactor. Final dry cell mass of 22.0 g/l, 116 g/l xylitol concentration, 2.34 g/l h overall xylitol productivity were obtained in cell-recycle fermentation supplemented with xylose and yeast extract solution, which were equivalent to 2.3-, 5.8- and 3.8-fold increases compared with the corresponding values of batch-type xylitol production parameters.     

亚硫酸盐甘蔗渣浆酶解液发酵制备乙醇

以亚硫酸盐甘蔗渣浆酶解液作为原料,利用C. shehatae发酵制取燃料乙醇。结果表明：还原糖最适初始质量浓度为葡萄糖140 g/L、木糖60 g/L、酶解液总糖80 g/L。利用初始葡萄糖55.06 g/L、木糖11.18 g/L、纤维二糖4.51 g/L的亚硫酸盐甘蔗渣浆酶解液发酵,经18 h获得乙醇22.98 g/L。乙醇得率为67.23%,葡萄糖利用率为99.27%,木糖利用率为32.96%,C. shehatae适合作为蔗渣为原料的乙醇发酵菌株。     

Production of butanol from glucose and xylose with immobilized cells of Clostridium acetobutylicum

Pretreated cotton towels were used as carriers to immobilize Clostridium acetobutylicum CGMCC 5234 cells for butanol or ABE production from glucose and xylose. Results showed that cell immobilization was a promising method to increase butanol concentration, yield and productivity regardless of the sugar sources compared with cell suspension. In this study, a high butanol concentration of 10.02 g/L with a yield of 0.20 g/g was obtained from 60 g/L xylose with 9.9 g/L residual xylose using immobilized cells compared with 8.48 g/L butanol and a yield of 0.141 g/g with 20.2 g/L residual xylose from 60 g/L xylose using suspended cells. In mixed-sugar fermentation (30 g/L glucose plus 30 g/L xylose), the immobilized cultures produced 11.1 g/L butanol with a yield of 0.190 g/g, which were 28.3% higher than with suspended cells (8.65 g/L) during which 30 g/L glucose was utilized completely using both immobilized and suspended cells while 3.46 and 13.1 g/L xylose maintained untilized for immobilized and suspended cells, respectively. Based on the results, we speculated that immobilized cells showed enhanced tolerance to butanol toxicity and the cultures preferred glucose to xylose during ABE fermentation. Moreover, the cultures showed obvious difference when grown between high initial concentrations of glucose and those of xylose. Repeated-batch fermentations from glucose with immobilized cells showed better long-term stability than from xylose. At last, the morphologies of free and immobilized cells adsorbed on pretreated cotton towels during the growth cycle were examined by SEM.     

Xylitol Production by a Culture ofCandida guilliermondii2581

The yeast strain Candida guilliermondii2581 was chosen for its ability to produce xylitol in media with high concentrations of xylose. The rate of xylitol production at a xylose concentration of 150 g/l is 1.25 g/l per h; the concentration of xylitol after three days of cultivation is 90 g/l; and the relative xylitol yield is 0.6 g per g substrate consumed. The growth conditions were found that resulted in the maximum relative xylitol yield with complete consumption of the sugar: xylose concentration, 150 g/l; pH 6.0; and shaking at 60 rpm. It was shown that the growth under conditions of limited aeration favors the reduction of xylose.     

Novel endophytic yeast Rhodotorula mucilaginosa strain PTD3 I: production of xylitol and ethanol

An endophytic yeast, Rhodotorula mucilaginosa strain PTD3, that was isolated from stems of hybrid poplar was found to be capable of production of xylitol from xylose, of ethanol from glucose, galactose, and mannose, and of arabitol from arabinose. The utilization of 30 g/L of each of the five sugars during fermentation by PTD3 was studied in liquid batch cultures. Glucose-acclimated PTD3 produced enhanced yields of xylitol (67% of theoretical yield) from xylose and of ethanol (84, 86, and 94% of theoretical yield, respectively) from glucose, galactose, and mannose. Additionally, this yeast was capable of metabolizing high concentrations of mixed sugars (150 g/L), with high yields of xylitol (61% of theoretical yield) and ethanol (83% of theoretical yield). A 1:1 glucose:xylose ratio with 30 g/L of each during double sugar fermentation did not affect PTD3's ability to produce high yields of xylitol (65% of theoretical yield) and ethanol (92% of theoretical yield). Surprisingly, the highest yields of xylitol (76% of theoretical yield) and ethanol (100% of theoretical yield) were observed during fermentation of sugars present in the lignocellulosic hydrolysate obtained after steam pretreatment of a mixture of hybrid poplar and Douglas fir. PTD3 demonstrated an exceptional ability to ferment the hydrolysate, overcome hexose repression of xylose utilization with a short lag period of 10 h, and tolerate sugar degradation products. In direct comparison, PTD3 had higher xylitol yields from the mixed sugar hydrolysate compared with the widely studied and used xylitol producer Candida guilliermondii.     

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     

Continuous production of ethanol from a glucose,xylose and arabinose mixture by a flocculent strain ofPichia stipitis

Summary Enhanced rates of continuous ethanol production by a flocculent strain ofPichia stipitis from a sugar mixture (xylose 75%, glucose 20%, arabinose 5%) were attained using a single-stage gas lift tower fermentor. With a substrate feed of 50g/l, the biomass accumulated at a level near 50g/l, showed a maximum and stable ethanol productivity of 10.7 g/l.h, with a substrate conversion of 80%; the ethanol yield reached 0.41g/g. In these operating conditions, similar performances were obtained when D.xylose alone was supplied.     

Sequential Targeting of Xylose and Glucose Conversion in <Emphasis Type="Bold">F</Emphasis>ed-Batch Simultaneous Saccharification and <Emphasis Type="Bold">C</Emphasis>o-fermentation of Steam-Pretreated Wheat Straw for Improved Xylose Conversion to Ethanol

Efficient conversion of both glucose and xylose in lignocellulosic biomass is necessary to make second-generation bioethanol from agricultural residues competitive with first-generation bioethanol and gasoline. Simultaneous saccharification and co-fermentation (SSCF) is a promising strategy for obtaining high ethanol yields. However, with this method, the xylose-fermenting capacity and viability of yeast tend to decline over time and restrict the xylose utilization. In this study, we examined the ethanol production from steam-pretreated wheat straw using an established SSCF strategy with substrate and enzyme feeding that was previously applied to steam-pretreated corn cobs. Based on our findings, we propose an alternative SSCF strategy to sustain the xylose-fermenting capacity and improve the ethanol yield. The xylose-rich hydrolyzate liquor was separated from the glucose-rich solids, and phases were co-fermented sequentially. By prefermentation of the hydrolyzate liquor followed fed-batch SSCF, xylose, and glucose conversion could be targeted in succession. Because the xylose-fermenting capacity declines over time, while glucose is still converted, it was advantageous to target xylose conversion upfront. With our strategy, an overall ethanol yield of 84% of the theoretical maximum based on both xylose and glucose was reached for a slurry with higher inhibitor concentrations, versus 92% for a slurry with lower inhibitor concentrations. Xylose utilization exceeded 90% after SSCF for both slurries. Sequential targeting of xylose and glucose conversion sustained xylose fermentation and improved xylose utilization and ethanol yield compared with fed-batch SSCF of whole slurry.