Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli ATCC 9637(pLO1297), ATCC 11303(pLO1297), and ATCC 15224(pLO1297) were selected for further development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productivities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3 g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose (8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.     

Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli.

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli ATCC 9637(pLO1297), ATCC 11303(pLO1297), and ATCC 15224(pLO1297) were selected for further development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productivities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3 g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose (8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.     

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).     

Parametric studies of ethanol production form xylose and other sugars by recombinant Escherichia coli

The conversion of xylose to ethanol by recombinant Escherichia coli has been investigated in pH-controlled batch fermentations. Chemical and environmental parameters were varied to determine tolerance and to define optimal conditions. Relatively high concentrations of ethanol (56 g/L) were produced from xylose with excellent efficiencies. Volumetric productivities of up to 1.4 g ethanol/L h were obtained. Productivities, yields, and final ethanol concentrations achieved from xylose with recombinant E. coli exceeded the reported values with other organisms. In addition to xylose, all other sugar constituents of biomass (glucose, mannose, arabinose, and galactose) were efficiently converted to ethanol by recombinant E. coli. Unusually low inocula equivalent to 0.033 mg of dry cell weight/L were adequate for batch fermentations. The addition of small amounts of calcium, magnesium, and ferrous ions stimulated fermentation. The inhibitory effects of toxic compounds (salts, furfural, and acetate) which are present in hemicellulose hydrolysates were also examined.     

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.     

Continuous Ethanol Fermentation of Pretreated Lignocellulosic Biomasses,Waste Biomasses,Molasses and Syrup Using the Anaerobic,Thermophilic Bacterium Thermoanaerobacter italicus Pentocrobe 411

Lignocellosic ethanol production is now at a stage where commercial or semi-commercial plants are coming online and, provided cost effective production can be achieved, lignocellulosic ethanol will become an important part of the world bio economy. However, challenges are still to be overcome throughout the process and particularly for the fermentation of the complex sugar mixtures resulting from the hydrolysis of hemicellulose. Here we describe the continuous fermentation of glucose, xylose and arabinose from non-detoxified pretreated wheat straw, birch, corn cob, sugar cane bagasse, cardboard, mixed bio waste, oil palm empty fruit bunch and frond, sugar cane syrup and sugar cane molasses using the anaerobic, thermophilic bacterium Thermoanaerobacter Pentocrobe 411. All fermentations resulted in close to maximum theoretical ethanol yields of 0.47–0.49 g/g (based on glucose, xylose, and arabinose), volumetric ethanol productivities of 1.2–2.7 g/L/h and a total sugar conversion of 90–99% including glucose, xylose and arabinose. The results solidify the potential of Thermoanaerobacter strains as candidates for lignocellulose bioconversion.     

Fermentation of xylose into ethanol by a new fungus strain <Emphasis Type="Italic">Pestalotiopsis</Emphasis> sp. XE-1

A new fungus, Pestalotiopsis sp. XE-1, which produced ethanol from xylose with yield of 0.47 g ethanol/g of consumed xylose was isolated. It also produced ethanol from arabinose, glucose, fructose, mannose, galactose, cellobiose, maltose, and sucrose with yields of 0.38, 0.47, 0.45, 0.46, 0.31, 0.25, 0.31, and 0.34 g ethanol/g of sugar consumed, respectively. It produced maximum ethanol from xylose at pH 6.5, 30°C under a semi-aerobic condition. Acetic acid produced in xylose fermenting process inhibited ethanol production of XE-1. The ethanol yield in the pH-uncontrolled batch fermentation was about 27% lower than that in the pH-controlled one. The ethanol tolerance of XE-1 was higher than most xylose-fermenting, ethanol-producing microbes, but lower than Saccharomyces cerevisiae and Hansenula polymorpha. XE-1 showed tolerance to high concentration of xylose, and was able to grow and produce ethanol even when it was cultivated in 97.71 g/l xylose.     

Repeated-batch fermentations of xylose and glucose-xylose mixtures using a respiration-deficient Saccharomyces cerevisiae engineered for xylose metabolism

Xylose-fermenting Saccharomyces strains are needed for commercialization of ethanol production from lignocellulosic biomass. Engineered Saccharomyces cerevisiae strains expressing XYL1, XYL2 and XYL3 from Pichia stipitis, however, utilize xylose in an oxidative manner, which results in significantly lower ethanol yields from xylose as compared to glucose. As such, we hypothesized that reconfiguration of xylose metabolism from oxidative into fermentative manner might lead to efficient ethanol production from xylose. To this end, we generated a respiration-deficient (RD) mutant in order to enforce engineered S. cerevisiae to utilize xylose only through fermentative metabolic routes. Three different repeated-batch fermentations were performed to characterize characteristics of the respiration-deficient mutant. When fermenting glucose as a sole carbon source, the RD mutant exhibited near theoretical ethanol yields (0.46 g g(-1)) during repeated-batch fermentations by recycling the cells. As the repeated-batch fermentation progressed, the volumetric ethanol productivity increased (from 7.5 to 8.3 g L(-1)h(-1)) because of the increased biomass from previous cultures. On the contrary, the mutant showed decreasing volumetric ethanol productivities during the repeated-batch fermentations using xylose as sole carbon source (from 0.4 to 0.3 g L(-1)h(-1)). The mutant did not grow on xylose and lost fermenting ability gradually, indicating that the RD mutant cannot maintain a good fermenting ability on xylose as a sole carbon source. However, the RD mutant was capable of fermenting a mixture of glucose and xylose with stable yields (0.35 g g(-1)) and productivities (0.52 g L(-1)h(-1)) during the repeated-batch fermentation. In addition, ethanol yields from xylose during the mixed sugar fermentation (0.30 g g(-1)) were higher than ethanol yields from xylose as a sole carbon source (0.21 g g(-1)). These results suggest that a strategy for increasing ethanol yield through respiration-deficiency can be applied for the fermentation of lignocellulosic hydrolyzates containing glucose and xylose.     

Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2, and XKS1 in mineral medium chemostat cultures

For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. Anaerobic ethanol formation from xylose by recombinant S. cerevisiae was demonstrated for the first time. However, the strain grew on xylose only in the presence of oxygen. Ethanol yields of 0.45 to 0.50 mmol of C/mmol of C (0.35 to 0.38 g/g) and productivities of 9.7 to 13.2 mmol of C h(-1) g (dry weight) of cells(-1) (0.24 to 0.30 g h(-1) g [dry weight] of cells(-1)) were obtained from xylose-glucose mixtures in anaerobic chemostat cultures, with a dilution rate of 0.06 h(-1). The anaerobic ethanol yield on xylose was estimated at 0.27 mol of C/(mol of C of xylose) (0.21 g/g), assuming a constant ethanol yield on glucose. The xylose uptake rate increased with increasing xylose concentration in the feed, from 3.3 mmol of C h(-1) g (dry weight) of cells(-1) when the xylose-to-glucose ratio in the feed was 1:3 to 6.8 mmol of C h(-1) g (dry weight) of cells(-1) when the feed ratio was 3:1. With a feed content of 15 g of xylose/liter and 5 g of glucose/liter, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux.     

Carbohydrate utilization patterns and substrate preferences in Bacteroides thetaiotaomicron

Specific growth rates of Bacteroides thetaiotaomicron NCTC 10582 with either glucose, arabinose, mannose, galactose or xylose as sole carbon sources were 0.42/h, 0.10/h, 0.38/h, 0.38/h and 0.16/h respectively, suggesting that hexose metabolism was energetically more efficient than pentose fermentation in this bacterium. Batch culture experiments to determine whether carbohydrate utilization was controlled by substrate-induced regulatory mechanisms demonstrated that mannose inhibited uptake of glucose, galactose and arabinose, but had less effect on xylose. Arabinose and xylose were preferentially utilized at high dilution rates (D > 0.26/h) in carbon-limited continuous cultures grown on mixtures of arabinose, xylose, galactose and glucose. When mannose was also present, xylose was co-assimilated at all dilution rates. Under nitrogen-limited conditions, however, mannose repressed uptake of all sugars, showing that its effect on xylose utilization was strongly concentration dependent. Studies with individual D-ZU-14C]-labelled substrates showed that transport systems for glucose, galactose, xylose and mannose were inducible. Measurements to determine incorporation of these sugars into trichloroacetic acid-precipitable material indicated that glucose and mannose were the principal precursor monosaccharides. Xylose was only incorporated into intracellular macromolecules when it served as growth substrate. Phosphoenolpyruvate:phosphotransferase systems were not detected in preliminary experiments to elucidate the mechanisms of sugar uptake, and studies with inhibitors of carbohydrate transport showed no consistent pattern of inhibition with glucose, galactose, xylose and mannose. These results indicate the existence of a variety of different systems involved in sugar transport in B. thetaiotaomicron.     

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.     

Genetic engineering of Zymobacter palmae for production of ethanol from xylose

Its metabolic characteristics suggest that Zymobacter palmae gen. nov., sp. nov. could serve as a useful new ethanol-fermenting bacterium, but its biotechnological exploitation will require certain genetic modifications. We therefore engineered Z. palmae so as to broaden the range of its fermentable sugar substrates to include the pentose sugar xylose. The Escherichia coli genes encoding the xylose catabolic enzymes xylose isomerase, xylulokinase, transaldolase, and transketolase were introduced into Z. palmae, where their expression was driven by the Zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase promoter. When cultured with 40 g/liter xylose, the recombinant Z. palmae strain was able to ferment 16.4 g/liter xylose within 5 days, producing 91% of the theoretical yield of ethanol with no accumulation of organic acids as metabolic by-products. Notably, xylose acclimation enhanced both the expression of xylose catabolic enzymes and the rate of xylose uptake into recombinant Z. palmae, which enabled the acclimated organism to completely and simultaneously ferment a mixture of 40 g/liter glucose and 40 g/liter xylose within 8 h, producing 95% of the theoretical yield of ethanol. Thus, efficient fermentation of a mixture of glucose and xylose to ethanol can be accomplished by using Z. palmae expressing E. coli xylose catabolic enzymes.     

一株中型假丝酵母发酵木糖产乙醇的特性研究

本研究对自然界中筛选得到的1株可以发酵木糖产乙醇的中型假丝酵母(Candida intermedia)的特性进行了研究.该菌株在28℃、120 r/min、72 h条件下,发酵3%木糖的乙醇产率最高,达到理论值的43.70%,发酵7%木糖得到的乙醇产量最高,为6.480 g/L.发酵时间延长到156 h,可以利用8%木糖产乙醇21.225 g/L,产率为理论值的72.87%.该菌株还可以在同样条件下,发酵13%葡萄糖得到乙醇50.965 g/L,达到理论值的76.90%.以3% 2% 3%分批补加糖,比一次性发酵8%木糖的乙醇产量提高9.91%.在葡萄糖和木糖的混合培养基中,优先利用葡萄糖,同时还表现出葡萄糖对木糖发酵的促进作用,当2%的木糖与6%葡萄糖混合时,乙醇产量比两者单独发酵的加和提高了25%.     

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。     

Succinic acid production by <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis> from batch fermentation of mixed sugars

Succinic acid production from the monosaccharides xylose, arabinose, glucose, mannose and galactose was studied using the bacterium Actinobacillus succinogenes. In Duran bottle cultures, containing 10 g/L of each of sugar, succinic acid was produced from all sugars except for galactose. The highest succinate yield, 0.56 g/g, was obtained with glucose, whereas the succinate yield was 0.42, 0.38 and 0.44 g/g for xylose, mannose and arabinose, respectively. The specific succinate productivity was 0.7 g/g h for glucose, but below 0.2 g/g h for the other sugars. Batch bioreactor fermentations were carried out using a sugar mixture of the five sugars giving a total concentration of 50 g/L, mimicking the distribution of sugars in spent sulfite liquor (SSL) from Eucalyptus which is rich in xylose. In this mixture, an almost complete conversion of all sugars (except galactose) was achieved resulting in a final succinate concentration of 21.8–26.8 g/L and a total yield of 0.59–0.68 g/g. There was evidence of co-consumption of glucose and xylose, whereas mannose was consumed after glucose. The main by-products were acetate 0.14–0.20 g/g and formate 0.08–0.13 g/g. NADH balance calculations suggested that NADH required for succinate production was not met solely from formate and acetate production, but other means of NADH production was necessary. Results from mixed sugar fermentations were verified using SSL as substrate resulting in a succinate yield of 0.60 g/g. In addition, it was found that CO₂ sparging could replace carbonate supply in the form of MgCO₃ without affecting the succinate yield.     

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.     

休哈塔假丝酵母乙醇发酵性能的研究

木糖的高效发酵是制约纤维素燃料乙醇生产的技术瓶颈之一,高性能发酵菌种的开发是本领域研究的重点。以木糖发酵的典型菌株休哈塔假丝酵母为材料,研究氮源配比、葡萄糖和木糖初始浓度、葡萄糖添加及典型抑制物等因素对其木糖利用和乙醇发酵性能的影响规律。结果表明,硫酸铵更适宜于木糖和葡萄糖发酵产乙醇。在摇瓶振荡发酵条件下,该酵母可发酵164.0 g/L葡萄糖生成61.9 g/L乙醇,糖利用率和乙醇得率分别为99.8%和74.0%;受酵母细胞膜上转运体系的限制,对木糖的最高发酵浓度为120.0 g/L,可生成45.7 g/L乙醇,糖利用率和乙醇得率分别达到94.8%和87.0%。休哈塔假丝酵母发酵木糖的主要产物为乙醇,仅生成微量的木糖醇;添加葡萄糖可促进木糖的利用;休哈塔假丝酵母在葡萄糖发酵时的乙酸和甲酸的耐受浓度分别为8.32和2.55 g/L,木糖发酵时的乙酸和甲酸的耐受浓度分别为6.28和1.15 g/L。     

On-line estimation of sugar concentration for control of fed-batch fermentation of lignocellulosic hydrolyzates by Saccharomyces cerevisiae

A feed control strategy, based on estimated sugar concentrations, was developed with the purpose of avoiding severe inhibition of the yeast Saccharomyces cerevisiae during fermentation of spruce hydrolyzate. The sum of the fermentable hexose sugars, glucose and mannose, was estimated from on-line measurements of carbon dioxide evolution rate and biomass concentration by use of a simple stoichiometric model. The feed rate of the hydrolyzate was controlled to maintain constant sugar concentration during fed-batch fermentation, and the effect of different set-point concentrations was investigated using both untreated and detoxified hydrolyzates. The fed-batch cultivations were evaluated with respect to cellular physiology in terms of the specific ethanol productivities, ethanol yields, and viability of the yeast. The simple stoichiometric model used resulted in a good agreement between estimated sugar concentrations and off-line determinations of sugar concentrations. Furthermore, the control strategy used made it possible to maintain a constant sugar concentration without major oscillations in the feed rate or the sugar concentration. For untreated hydrolyzates the average ethanol productivity could be increased by more than 130% compared to batch fermentation. The average ethanol productivity was increased from 0.12 to 0.28 g/g h. The productivity also increased for detoxified hydrolyzates, where an increase of 16% was found (from 0.50 to 0.58 g/g h).     

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.     

Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae

Bioconversion of xylose—the second most abundant sugar in nature—into high-value fuels and chemicals by engineered Saccharomyces cerevisiae has been a long-term goal of the metabolic engineering community. Although most efforts have heavily focused on the production of ethanol by engineered S. cerevisiae, yields and productivities of ethanol produced from xylose have remained inferior as compared with ethanol produced from glucose. However, this entrenched focus on ethanol has concealed the fact that many aspects of xylose metabolism favor the production of nonethanol products. Through reduced overall metabolic flux, a more respiratory nature of consumption, and evading glucose signaling pathways, the bioconversion of xylose can be more amenable to redirecting flux away from ethanol towards the desired target product. In this report, we show that coupling xylose consumption via the oxidoreductive pathway with a mitochondrially-targeted isobutanol biosynthesis pathway leads to enhanced product yields and titers as compared to cultures utilizing glucose or galactose as a carbon source. Through the optimization of culture conditions, we achieve 2.6 g/L of isobutanol in the fed-batch flask and bioreactor fermentations. These results suggest that there may be synergistic benefits of coupling xylose assimilation with the production of nonethanol value-added products.