Fermentative hydrogen production from glucose and starch using pure strains and artificial co-cultures of Clostridium spp.

Background

Pure bacterial strains give better yields when producing H₂ than mixed, natural communities. However the main drawback with the pure cultures is the need to perform the fermentations under sterile conditions. Therefore, H₂ production using artificial co-cultures, composed of well characterized strains, is one of the directions currently undertaken in the field of biohydrogen research.

Results

Four pure Clostridium cultures, including C. butyricum CWBI1009, C. pasteurianum DSM525, C. beijerinckii DSM1820 and C. felsineum DSM749, and three different co-cultures composed of (1) C. pasteurianum and C. felsineum, (2) C. butyricum and C. felsineum, (3) C. butyricum and C. pasteurianum, were grown in 20?L batch bioreactors. In the first part of the study a strategy composed of three-culture sequences was developed to determine the optimal pH for H₂ production (sequence 1); and the H₂-producing potential of each pure strain and co-culture, during glucose (sequence 2) and starch (sequence 3) fermentations at the optimal pH. The best H₂ yields were obtained for starch fermentations, and the highest yield of 2.91?mol?H₂/ mol hexose was reported for C. butyricum. By contrast, the biogas production rates were higher for glucose fermentations and the highest value of 1.5?L biogas/ h was observed for the co-culture (1). In general co-cultures produced H₂ at higher rates than the pure Clostridium cultures, without negatively affecting the H₂ yields. Interestingly, all the Clostridium strains and co-cultures were shown to utilize lactate (present in a starch-containing medium), and C. beijerinckii was able to re-consume formate producing additional H₂. In the second part of the study the co-culture (3) was used to produce H₂ during 13?days of glucose fermentation in a sequencing batch reactor (SBR). In addition, the species dynamics, as monitored by qPCR (quantitative real-time PCR), showed a stable coexistence of C. pasteurianum and C. butyricum during this fermentation.

Conclusions

The four pure Clostridium strains and the artificial co-cultures tested in this study were shown to efficiently produce H₂ using glucose and starch as carbon sources. The artificial co-cultures produced H₂ at higher rates than the pure strains, while the H₂ yields were only slightly affected.     

Bioconversion of acid-hydrolyzed poplar hemicellulose to acetic acid byClostridium thermoaceticum

Summary Clostridium thermoaceticum was used to ferment carbohydrate released from pretreated oat splet xylan and hemicellulose isolated from hybrid poplar. Hydrolysis with dilute sulfuric acid (2.5% (v/v) for oat spelt xylan and 4.0% (v/v) for poplar hemicellulose) at 100°C for 60 min was found to release the highest concentration of fermentable substrate.C. thermoaceticum, when grown in non-pH controlled batch culture at 55°C under a headspace of 100% CO₂, typically produced 14gl^–1 acetic acid during a 48 h fermentation in medium containing 2% xylose. In fed-batch fermentations this organism was able to produce 42gl^–1 acetic acid after 116h when the concentration of xylose was maintained at approximately 2% and the pH was controlled at 7.0.     

Acidogenic fermentation of corn stover

Corn stover was fermented by anaerobic acidogenic bacteria to produce volatile (C₂–C₆) organic acids. Mild pretreatment with dilute alkali solutions produced a two-fold increase in fermentability. A mixture of lime and sodium carbonate was found to be a better pretreatment agent than sodium hydroxide. Methane generation was inhibited by low temperature (? 25°C) and high solids [≥ 2.5% (w/v)] fermentation. Volatile acid yields of 0.5–0.55 g acetic acid equiv/g dry ash-free (DAF) stover could be obtained in batch fermentations. Several extractants and extraction solvents for organic acids were found to be nontoxic to acidogenic fermentation. The data show that acidogenic fermentation can produce useful volatile fatty acids in high yields from a complex lignocellulosic feedstock. These fermentations are nonsterile, need no stirring, and are easy to run. Moreover, cellulose, pentosans, and other carbohydrates are directly utilized by acidogenic bacteria. Hence, acidogenic fermentation could be useful in converting biomass to chemical feedstocks and fuel.     

Potential of ochratoxin A production by Aspergillus carbonarius strains isolated from grapes at different ecological factors

Aspergillus carbonarius is known to colonize and produce ochratoxin A (OTA) on grapes and its derived products which is harmful to humans. We screened and tested A. carbonarius strains which isolated from grapes for production of OTA and selected three high OTA producing strains (ACSP1, ACSP2, ACSP3) for this study. These strains were further tested for their ability to produce OTA at different ecological factors [temperature 15, 25, 30, 35°C; water activity (a_w) 0.98, 0.95, 0.90, 0.88; and pH 4.0, 7.0, 9.0, 10.0]. Out of the three strains tested, A. carbonarius ACSP3 produced high levels of OTA than ACSP2 and ACSP1 in all the ecological factors. At 30°C A. carbonarius strains produced the highest OTA compared with other temperature regimes. With reference to water activity, a_w 0.98 favoured mycelial growth and accumulation of more OTA with all the three A. carbonarius strains. Further, pH 4.0 was encouraged the greatest production of OTA in all the strains. No growth was observed at a_w 0.88 and pH 10.0 in all the three strains except the strain ACSP3 at high pH. Our work demonstrated that temperature 30°C, a_w 0.98 and pH 4.0 is optimum for growth and production of OTA by A. carbonarius strains. Maximum amounts of OTA were found at earlier growth stages (7–9 days of incubation) in all the strains of A. carbonarius. The present study revealed that different ecological factors had great impact on OTA production by A. carbonarius which is useful for understanding OTA contamination and to develop proper management practices in future research programmes.     

Improvement of Omega-3 Docosahexaenoic Acid Production by Marine Dinoflagellate Crypthecodinium cohnii Using Rapeseed Meal Hydrolysate and Waste Molasses as Feedstock

Rapeseed meal and waste molasses are two important agro-industrial by-products which are produced in large quantities. In this study, solid state fermentation and fungal autolysis were performed to produce rapeseed meal hydrolysate (RMH) using fungal strains of Aspergillus oryzae, Penicillium oxalicum and Neurospora crassa. The hydrolysate was used as fermentation feedstock for heterotrophic growth of microalga Crypthecodinium cohnii that produce docosahexaenoic acid (DHA). The addition of waste molasses as a supplementary carbon source greatly increased the biomass and DHA yield. In the batch fermentations using media composed of diluted RMH (7%) and 1-9% waste molasses, the highest biomass concentration and DHA yield reached 3.43 g/L and 8.72 mg/L, respectively. The algal biomass produced from RMH and molasses medium also had a high percentage of DHA (22-34%) in total fatty acids similar to that of commercial algal biomass. RMH was shown to be rich in nitrogen supply comparable to the commercial nitrogen feedstock like yeast extract. Using RMH as sole nitrogen source, waste molasses excelled other carbon sources and produced the highest concentration of biomass. This study suggests that DHA production of the marine dinoflagellate C. cohnii could be greatly improved by concomitantly using the cheap by-products rapeseed meal hydrolysate and molasses as alternative feedstock.     

Isolation and utilization of indigenous yeast strain for brewing of millet beer (OYOKPO)

Three yeast strains were isolated from a spontaneously fermented native millet (Pennisetum typhoideum) malt beer (Oyokpo). One of the yeast isolates found to have the most highly fermenting capacity was characterised and identified as a strain of Saccharomyces cerevisiae. The yeast was then utilised as the pitching yeast in a subsequent controlled fermentation of millet wort at 20°C for 120 hours. Bitter leaf (Vernonia amagdalina) extract was used as the bittering and flavouring agent. The Oyokpo beer sample produced under these conditions was found to possess both chemical and organoleptic qualities comparable to some extent, to the conventional barley malt beer. At the end of fermentation, the pH, specific gravity, alcohol content, reducing sugar content and protein content of the beer were 4.11, 1.0308, 2.81% (v/v), 4.00 (mg/ml) and 0.84 (mg/ml) respectively.     

Control of Carbon and Electron Flow in Clostridium acetobutylicum Fermentations: Utilization of Carbon Monoxide to Inhibit Hydrogen Production and to Enhance Butanol Yields

Extracts prepared from non-solvent-producing cells of Clostridium acetobutylicum contained methyl viologen-linked hydrogenase activity (20 U/mg of protein at 37°C) but did not display carbon monoxide dehydrogenase activity. CO addition readily inhibited the hydrogenase activity of cell extracts or of viable metabolizing cells. Increasing the partial pressure of CO (2 to 10%) in unshaken anaerobic culture tube headspaces significantly inhibited (90% inhibition at 10% CO) both growth and hydrogen production by C. acetobutylicum. Growth was not sensitive to low partial pressures of CO (i.e., up to 15%) in pH-controlled fermentors (pH 4.5) that were continuously gassed and mixed. CO addition dramatically altered the glucose fermentation balance of C. acetobutylicum by diverting carbon and electrons away from H₂, CO₂, acetate, and butyrate production and towards production of ethanol and butanol. The butanol concentration was increased from 65 to 106 mM and the butanol productivity (i.e., the ratio of butanol produced/total acids and solvents produced) was increased by 31% when glucose fermentations maintained at pH 4.5 were continuously gassed with 85% N₂-15% CO versus N₂ alone. The results are discussed in terms of metabolic regulation of C. acetobutylicum saccharide fermentations to achieve maximal butanol or solvent yield.     

Biosynthesis of flavour-active furanones by Saccharomyces cerevisiae during fermentation depends on the malt type used in medium preparation

Extracts of a coloured malt contained 4-hydroxy-5-monomethyl-3(2H)-furanone (HMMF), 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) but not 4-hydroxy-5(or 2)-ethyl-2(or 5)-methyl-3(2H)-furanone (HEMF). Extracts of a pale malt did not contain any of the furanones. HMMF and HDMF were produced by Saccharomyces cerevisiae during fermen-tation of both types of malt extract. About 0.09 mg HEMF l ⁻¹ was synthesised during fermentation of the coloured malt extract but none was produced with the pale malt extract. Final concentrations of HDMF (2.0 mgl ⁻¹) and HEMF (0.09 mgl ⁻¹) were in excess of their aroma threshold values in water (0.16 and 0.02 mgl ⁻¹ respectively) after fermen-tation of the coloured malt extract. This revised version was published online in November 2006 with corrections to the Cover Date.     

Evaluation of liquid residues from beer and potato processing for the production of docosahexaenoic acid (C22:6n-3, DHA) by native thraustochytrid strains

Liquid residues from beer (RB) and potato (RP) processing were evaluated as carbon sources for the production of docosahexaenoic acid (C22:6n-3, DHA) by two native Thraustochytriidae sp., M12-X1 and C41, in shaking flask experiments. Results were compared with those obtained in the fermentations of glucose, maltose, soluble starch and ethanol. Both strains produced the highest biomass concentration (2.3 g/L) in the fermentation of RB supplemented with nitrogen sources [yeast extract (YE) and monosodium glutamate (MSG)]. DHA content in the fatty acids produced by the native thraustochytrids was dependent on the fermented carbon source; the fatty acids from biomass grown on carbon sources that permitted a lower growth rate contained more DHA. The highest DHA productivity [55.1 mg/(day L)] was obtained in the fermentation of RB-YE-MSG by M12-X1 strain. In this medium, M12-X1 strain grew at a specific growth rate of 0.014 h^?1 and total fatty acid content in the biomass was 41.3%. Production of DHA by M12-X1 strain followed a non-growth rate associated pattern and DHA content in the biomass decreased significantly after growth ceased.     

A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings

Background

Fermentations using Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST), and Zymomonas mobilis AX101 are compared side-by-side on corn steep liquor (CSL) media and the water extract and enzymatic hydrolysate from ammonia fiber expansion (AFEX)-pretreated corn stover.

Results

The three ethanologens are able produce ethanol from a CSL-supplemented co-fermentation at a metabolic yield, final concentration and rate greater than 0.42 g/g consumed sugars, 40 g/L and 0.7 g/L/h (0-48 h), respectively. Xylose-only fermentation of the tested ethanologenic bacteria are five to eight times faster than 424A(LNH-ST) in the CSL fermentation. All tested strains grow and co-ferment sugars at 15% w/v solids loading equivalent of ammonia fiber explosion (AFEX)-pretreated corn stover water extract. However, both KO11 and 424A(LNH-ST) exhibit higher growth robustness than AX101. In 18% w/w solids loading lignocellulosic hydrolysate from AFEX pretreatment, complete glucose fermentations can be achieved at a rate greater than 0.77 g/L/h. In contrast to results from fermentation in CSL, S. cerevisiae 424A(LNH-ST) consumed xylose at the greatest extent and rate in the hydrolysate compared to the bacteria tested.

Conclusions

Our results confirm that glucose fermentations among the tested strains are effective even at high solids loading (18% by weight). However, xylose consumption in the lignocellulosic hydrolysate is the major bottleneck affecting overall yield, titer or rate of the process. In comparison, Saccharomyces cerevisiae 424A(LNH-ST) is the most relevant strains for industrial production for its ability to ferment both glucose and xylose from undetoxified and unsupplemented hydrolysate from AFEX-pretreated corn stover at high yield.     

Optimization of the fermentation media for sophorolipid production from Candida bombicola ATCC 22214 using a simplex centroid design

This article describes the use of a simplex centroid mixture experimental design to optimize the fermentation medium in the production of sophorolipids (SLs) using Candida bombicola. In the first stage, 16 media ingredients were screened for the ones that have the most positive influence on the SL production. The sixteen ingredients that were chosen are five different carbohydrates (fructose, glucose, glycerol, lactose, and sucrose), five different nitrogen sources (malt extract, peptone extract, soytone, urea, and yeast extract), two lipid sources (mineral oil and oleic acid), two phosphorus sources (K₂HPO₄ and KH₂PO₄), MgSO₄, and CaCl₂. Multiple regression analysis and centroid effect analysis were carried out to find the sugar, lipid, nitrogen source, phosphorus source, and metals having the most positive influence. Sucrose, malt extract, oleic acid, K₂HPO₄, and CaCl₂ were selected for the second stage of experiments. An augmented simplex centroid design for five ingredients requiring 16 experiments was used for the optimization stage. This produced a quadratic model developed to help understand the interaction amongst the ingredients and find the optimal media concentrations. In addition, the top three results from the optimization experiments were used to obtain constraints that identify an optimal region. The model together with the optimal region constraints predicts the maximum production of SLs when the fermentation media is composed of sucrose, 125 g/L; malt extract, 25 g/L; oleic acid, 166.67 g/L; K₂HPO₄, 1.5 g/L; and CaCl₂, 2.5 g/L. The optimal media was validated experimentally and a yield of 177 g/L was obtained. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

In Vitro Kinetic Analysis of Oligofructose Consumption by Bacteroides and Bifidobacterium spp. Indicates Different Degradation Mechanisms

The growth of pure cultures of Bacteroides thetaiotaomicron LMG 11262 and Bacteroides fragilis LMG 10263 on fructose and oligofructose was examined and compared to that of Bifidobacterium longum BB536 through in vitro laboratory fermentations. Gas chromatography (GC) analysis was used to determine the different fractions of oligofructose and their degradation during the fermentation process. Both B. thetaiotaomicron LMG 11262 and B. fragilis LMG 10263 were able to grow on oligofructose as fast as on fructose, succinic acid being the major metabolite produced by both strains. B. longum BB536 grew slower on oligofructose than on fructose. Acetic acid and lactic acid were the main metabolites produced when fructose was used as the sole energy source. Increased amounts of formic acid and ethanol were produced when oligofructose was used as an energy source at the cost of lactic acid. Detailed kinetic analysis revealed a preferential metabolism of the short oligofructose fractions (e.g., F₂ and F₃) for B. longum BB536. After depletion of the short fractions, the larger oligofructose fractions (e.g., F₄, GF₄, F₅, GF₅, and F₆) were metabolized, too. Both Bacteroides strains did not display such a preferential metabolism and degraded all oligofructose fractions simultaneously, transiently increasing the fructose concentration in the medium. This suggests a different mechanism for oligofructose breakdown between the strain of Bifidobacterium and both strains of Bacteroides, which helps to explain the bifidogenic nature of inulin-type fructans.     

Effect of ragi and Lxxx-lactate-producing cultures on enteric pathogens in a rice-based weaning food

The use of the Southeast-Asian starter culture ragi in enhancing the safety of a rice-based model weaning food is described and compared with the use of diastatic malt extract. Ragi was shown to be an effective saccharifying agent convenient for use in weaning-food preparation on a domestic scale. Saccharification and fermentation with ragi alone produced some antimicrobial effect against the three enteric bacterial pathogens tested but this was much improved when ragi was used in conjunction with the Lxxx-lactate-producing Lactococcus lactis or Lactobacillus bavaricus. The latter showed the greatest inhibition of pathogens, reducing viable numbers by more than a factor of 10⁴ within 4 h. The antibacterial effects observed correlated with the total acid produced (ragi alone giving 0.2%; ragi with Lc. lactis giving 0.3% and ragi with Lb. bavaricus giving 0.4%). The proportion of the physiological Lxxx-lactate isomer was highest in Lc. lactis fermentations (>99% compared with 80% with Lb. bavaricus). There was no evidence of any pronounced antimicrobial effect due to the nisin produced during fermentation by Lc. lactis (150 IU/g). Whereas bacteriocin production may play little role in pathogen control, it may be desirable as a way of preventing fermentations conducted non-aseptically from becoming dominated by lactic acid bacteria producing unacceptable amounts of Dxxx-lactate.R.M. Yusof is with the Department of Nutrition and Community Health, Faculty of Human Ecology, University Pertanian Malaysia, 43400 UPM, Serdang, Selangor, Malaysia. T.A. Baker, J.B. Morgan and M.R. Adams are with the School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK.     

Clostridium quinii sp. nov., a new saccharolytic anaerobic bacterium isolated from granular sludge

A new species of sporulating saccharolytic anaerobe, designated as Clostridium quinii sp. nov., is described. A gram-positive strain BS1, was isolated from the granular metanogenic sludge (UASB) from a waste-water treatment plant at a sugar refinery. The strain exhibits a series of morphological stages, developing from a spore to a small rod to a motile rod (peritrichous flagella) in the exponential growth phase, and then swelling to form cigar-shaped cells, exhibiting tumbling movements, in the late exponential growth phase before finally becoming large nonmotile ovoid cells in the stationary phase. Swelling occurs as a result of glucose being taken up and stored as a glycogen-like substance. The main fermentation products when growing on glucose is H₂, CO₂, formate, acetate and ethanol as well as small amounts of butyrate during exponential growth. Lactate is formed during the stationary phase, when glucose is abundant. Optimal conditions for growth is 40–45°C and pH of around 7.4. The type strain BS1 contains 28.0% mol G+C.     

Characterization of a Symbiotic Coculture of Clostridium thermohydrosulfuricum YM3 and Clostridium thermocellum YM4

Clostridium thermohydrosulfuricum YM3 and C. thermocellum YM4 were isolated from a coculture which was obtained from an enrichment culture inoculated with volcanic soil in Izu Peninsula, Japan. Strain YM3 had advantages over reported C. thermohydrosulfuricum strains in that it fermented inulin and could accumulate ethanol up to 1.3% (wt/vol). The highest ethanol yield obtained was 1.96 mol/mol of anhydroglucose unit in cellobiose. Strain YM4 had features different from those reported in C. thermocellum strains: it formed spores rarely (at a frequency of <10^-5), it required CO₂ and Na₂CO₃ for growth, and it fermented sucrose. Strain YM4 completely decomposed 1% Avicel within 25 h when the inoculum constituted 2% of the culture medium volume, and it produced 0.22 U of Avicelase and 2.21 U of carboxymethylcellulase per ml of the medium. The doubling times on Avicel, cellobiose, and glucose were 2.7, 1.1, and 1.6 h, respectively. Reconstructed cocultures of strains YM3 and YM4 were very stable and degraded Avicel more rapidly than did strain YM4 monoculture. Without yeast extract, neither microorganism was able to grow. However, the coculture grew on cellulose without yeast extract and produced ethanol in high yield. Moreover, cell-free spent culture broth of strain YM3 could replace yeast extract in supporting the growth of strain YM4. The symbiotic relationship of the two bacteria in cellulose fermentation is probably a case of mutualism.     

Saccharide production from methanol by transposon 5 mutants derived from the extracellular polysaccharide-producing bacterium Methylobacillus sp. strain 12S

A CH₃OH-utilizing bacterium that has the ability to produce extracellular polysaccharide (EPS) was isolated from a soil sample, and was identified as the obligate methylotroph Methylobacillus sp. strain 12S on the basis of its 16S rDNA sequence and growth-substrate specificity. The EPS produced by strain 12S was purified and the sugar composition was analysed by GC-MS and HPLC to reveal that the EPS was a heteropolymer composed of glucosyl, galactosyl, and mannosyl residues in the molar ratio 3:1:1. In order to produce mono- and/or oligosaccharides by single-step fermentation from CH₃OH, stain 12S was mutagenized by transposon 5. Among eleven EPS-deficient mutants, three strains were found to accumulate significant amounts of reducing sugars in the media. The amounts of the reducing sugars produced by the mutants (>ca. 700 mg glucose equivalent/l) were >11–22 times higher than those produced by the wild-type strain (<ca. 60 mg glucose equivalent/l). The GC-MS analysis showed that all the mutants accumulated glucose, erythrose, threose and a disaccharide-like compound in the media. Received: 25 August 1999 / Received revision: 15 March 2000 / Accepted: 24 March 2000     

Inulinase production and mathematical modeling from carob extract by using Aspergillus niger

The main objectives of the study were to produce inulinase from carob extract by Aspergillus niger A42 (ATCC 204447) and to model the inulinase fermentation in the optimum carob extract-based medium. In the study, carob extract was used as a novel and renewable carbon source in the production of A. niger inulinase. For medium optimization, eight different variables including initial sugar concentration (°Bx), (NH₄)₂HPO₄, MgSO₄.7H₂O, KH₂PO₄, NH₄NO₃, yeast extract, peptone, and ZnSO₄.7H₂O were employed. After fermentations, optimum medium composition contained 1% yeast extract in 5°Bx carob extract. As a result of the fermentation, the maximum inulinase activity, maximum invertase-type activity, I/S ratio, maximum inulinase- and invertase-type activity rates, maximum sugar consumption rate, and sugar utilization yield were 1507.03 U/ml, 1552.86 U/ml, 0.97, 175.82 and 323.76 U/ml/day, 13.26 g/L/day, and 98.52%, respectively. Regarding mathematical modeling, the actual inulinase production and sugar consumption data were successfully predicted by Baranyi and Cone models based on the model evaluation and validation results and the predicted kinetic values, respectively. Consequently, this was the first report in which carob extract was used in the production of inulinase as a carbon source. Additionally, the best-selected models can serve as universal equations in modeling the inulinase production and sugar consumption in shake flask fermentation with carob extract medium.     

Guanacastane-type diterpenoids from Coprinus plicatilis

Four new guanacastane-type diterpenoids (1–4), named plicatilisins E-H, together with the known compound, plicatilisin D (5), were isloated from two fermentation extracts (i.e., solid state fermentation on PDA medium & static fermentation of malt extract broth culture medium) of the fungal strain Coprinus plicatilis 82. Their structures were elucidated on the basis of extensive spectroscopic analysis, including 1D and 2D NMR as well as FT-ICR-MS, UV, and IR, and comparison with literature data. All the compounds showed insignificant cytotoxicities against the cancer cell line HCT116.     

Fermentation of Xylan,Corn Fiber,or Sugars to Acetoin and Butanediol by Bacillus polymyxa Strains

Bacillus polymyxa can produce levo-butanediol, a potential biogradable anti-freeze, and ethanol, a fuel additive, using starch-based fermentations. To explore use of less expensive biomass fermentation substrates, we screened B. polymyxa strains for good growth on xylans. During aerobic growth on glucose, six selected xylanolytic strains produced mainly acetoin and butanediol plus lesser amounts of acetaldehyde and ethanol. Undesirable acetoin formation was eliminated by anaerobic growth on glucose, but substrate usage, butanediol, and other fermentation products were greatly reduced. High xylanase activity occurred with growth on xylans or corn fiber, and about 50–65% of oatspelt xylan and 25–35% of the corn fiber were used during aerobic growth, but unexpectedly no butanediol and only small levels of acetoin were produced. Aerobic growth on arabinose, arabinose plus glucose, or xylose plus glucose resulted in both acetoin and butanediol formation. Little or no butanediol was made from xylose alone. Growth on an acid hydrolysate of corn fiber that contained a mixture of these sugars resulted in the formation of acetoin, acetaldehyde, and ethanol, but very little butanediol. The data suggest B. polymyxa is limited in conversion of xylan-rich biomass sources or their hydrolysates to butanediol. This limitation might be overcome by using better cultivation conditions and/or genetically engineered strains.     

Glycerol Overproduction by Engineered Saccharomyces cerevisiae Wine Yeast Strains Leads to Substantial Changes in By-Product Formation and to a Stimulation of Fermentation Rate in Stationary Phase

Six commercial wine yeast strains and three nonindustrial strains (two laboratory strains and one haploid strain derived from a wine yeast strain) were engineered to produce large amounts of glycerol with a lower ethanol yield. Overexpression of the GPD1 gene, encoding a glycerol-3-phosphate dehydrogenase, resulted in a 1.5- to 2.5-fold increase in glycerol production and a slight decrease in ethanol formation under conditions simulating wine fermentation. All the strains overexpressing GPD1 produced a larger amount of succinate and acetate, with marked differences in the level of these compounds between industrial and nonindustrial engineered strains. Acetoin and 2,3-butanediol formation was enhanced with significant variation between strains and in relation to the level of glycerol produced. Wine strains overproducing glycerol at moderate levels (12 to 18 g/liter) reduced acetoin almost completely to 2,3-butanediol. A lower biomass concentration was attained by GPD1-overexpressing strains, probably due to high acetaldehyde production during the growth phase. Despite the reduction in cell numbers, complete sugar exhaustion was achieved during fermentation in a sugar-rich medium. Surprisingly, the engineered wine yeast strains exhibited a significant increase in the fermentation rate in the stationary phase, which reduced the time of fermentation.