Microcalorimetric investigations of the metabolism of yeasts VII. Flow-calorimetry of aerobic batch cultures

Summary The heat evolution of aerobic batch cultures of growing yeast (Saccharomyces cerevisiae) in glucose media was investigated by a combination of a flow-microcalorimeter with a fermentor vessel. The course of heat production, cell production and the rate of oxygen consumption were qualitatively the same for all glucose concentrations between 10 mM and 100 mM. Under optimal aerobic conditions a triphasic growth was observed due to the fermentation of glucose to ethanol, respiration of ethanol to CO₂ and acetate, and respiration of acetate to C0₂. Energy and carbon were found to be in balance for all glucose concentrations.     

Influence of cytochrome composition on the energy conservation of acinetobacter calcoaceticus 69-V

Acinetobacter calcoaceticus 69-V contained cytochromes b, o, d and traces of cytochrome a₁ after growth on ethanol. Cytochromes o, d, and a₁ are known to be potentially capable of functioning as terminal oxidases. After growth on acetate only smaller amounts of cytochromes b and o were detectable. Cytochrome spectra of acetate plus glucose-grown cells resembled more those of ethanolgrown cells. Cytochrome spectra of acetate plus ethanol-grown cells were more similar to those of acetate-grown cells but already showed small amounts of cytochrome d. Ethanol-grown cells were energized only by the oxidation of glucose. But the velocity of ATP synthesis was so low that the P/O quotient amounted to practically zero. The stoichiometries of ATP synthesis of the otherwise-grown cells were generally higher for the oxidation of glucose than for the oxidations of succinate and D-lactate. Similar high P/O quotients were measured for the oxidations of glucose and ethanol in acetate plus ethanol-grown cells. No significant energization could be observed during the oxidations of gluconate and acetate. The findings indicate that electrons delivered by the PQQ-dependent glucose as well as by NAD⁺- dependent ethanol oxidations have probably a common place of entry into the respiratory chain. Glucose and NADH appeared to be energetically equivalent although at least two energetically different pathways might be involved in substrate oxidation.     

Effect of iron and EDTA on ethyl acetate accumulation in Candida utilis

Summary Production of economically-recoverable products from dilute sugar or ethanol is of practical importance. Conversion of glucose to ethyl acetate by Candida utilis was inhibited by FeCl₃ supplementation as low as 10 uM. EDTA added at the onset of growth on glucose relieved such an inhibition and also caused faster and greater amounts of accumulation of the ester. Addition of EDTA during conversion of ethanol to ethyl acetate showed little effect. EDTA may affect cell permeability and/or oxidative metabolism. For continual ethyl acetate production iron limitation must be maintained during as well as before ethanol utilization.EDTA = Ethylenediaminetetraacetic acid.     

Energetics of glucose uptake in Salmonella typhimurium

We have studied the energetics of glucose uptake in Salmonella typhimurium. Strain PP418 transprots glucose via the phosphoenolpyruvate: glucose phosphotransferase system, while strain PP1705 lacks this system and can only use the galactose permease for glucose uptake. These two strains were cultured anaerobically in glucose-limited chemostats. Both strains produced ethanol and acetate in equimolar amounts but a significant difference was observed in the molar growth yield on glucose (Y _Glc). It is suggested that this difference is due to a difference in the energetics of the glucose uptake systems in the two strains.Assuming an equal Y _ATP for both strains, we could calculate that uptake of 1 mole of glucose via the galactose permease consumes the equivalent of 0.5 mole of ATP. With the additional assumption that one proton is transported in symport with one glucose molecule, these results imply a stoichiometry of two protons per ATP hydrolysed.Abbreviations PTS Phosphoenolpyruvate: carbohydrate phosphotransferase system - D dilution rate (h^-1 - DW dry weight - GalP galactose permease - EtOH ethanol - HAc acetate - Lact lactate - Suc succinate - HFo formate - Glc Glucose - Y _Glc, Y _ATP yield of cells per glucose or ATP - q specific production rate     

Metabolism of Acetivibrio cellulolyticus during optimized growth on glucose,cellobiose and cellulose

Summary The growth of Acetivibrio cellulolyticus in 2.5 l batch cultures was optimized by controlling the growth pH at 6.7, the dissolved inorganic sulphide concentration at 0.4–0.6 mM, and by constant removal of hydrogen from the cultures by sparging with N₂/CO₂ or N₂ gas. An initial ethanol concentration of 0.15% (w/v) in cellobiose media resulted in specific growth rates which were reduced by about 75% compared to growth rates of 0.17 h^–1 in control cultures. Acetivibrio cellulolyticus had to be adapted for growth on glucose and ¹⁴C-radiotracer studies indicated that glucose was metabolized by the Embden-Meyerhof pathway. The specific growth rate (=0.03h^–1) and molar growth yield (Y_glucose=21.5) were considerably lower than those obtained (=0.17 h^–1, Y_cellobiose=68.9) in cellobiose media. A Y_ATP of 12.8 was obtained during growth on cellobiose. The mol product formed per mol Avicel cellulose fermented (on anhydroglucose equivalent basis) were 3.70 H₂, 2.64 CO₂, 0.73 acetate, 0.39 ethanol and 0.03 total soluble sugars on glucose basis. Maximum cellulase activity was observed in cellulose-grown cultures.National Research council of Canada No. 20826     

Physiological properties of Cellulomonas fermentans,a mesophilic cellulolytic bacterium

Summary The fermentation of cellobiose, glucose and cellulose MN 300 by Cellulomonas fermentans was studied. The molar growth yields (i.e. grams of cells per mole of hexose equivalent) were similar on cellobiose and cellulose at low sugar consumption levels (47.8 and 46.5 respectively), but was lower on glucose (38.0). The occurrence of cellobiose phosphorylase activity, detected in cellobiose- and cellulose-grown cells, might explain this result. The specific growth rates measured in cultures on cellobiose, glucose and cellulose were 0.055 h^-1, 0.040 h^-1 and 0.013 h^-1 respectively. Growth inhibition was observed, and a drop in Y_H occurred after relatively low but different quantities of hexose were consumed (2.2 mM, 5 mM and 8 mM hexose equivalent with cellulose, glucose and cellobiose respectively), which coincided with a change in the fermentative metabolism from a typical mixed acid metabolism (1 ethanol, 1 acetate and 2 formate synthesized by consumed hexose) to a more ethanolic fermentation. When growth ceased in cellulose cultures, consumption of cellulose continued, as did production of ethanol.Molar growth yields of C. fermentans were similar in anaerobic and aerobic cellobiose cultures (47.8 g/mol and 42.2 g/mol respectively). Specific growth rates were also quite similar under both culture conditions (0.055±0.013 h^-1 and 0.070±0.007 h^-1 respectively). Aerobic metabolism was studied using ¹⁴C glucose. During the exponential growth phase, acetate, succinate and nonidentified compound(s) accumulated in the supernatant, but no ¹⁴CO₂ was produced. During the stationary phase, acetate was oxidized and ¹⁴CO₂ produced, but without any further biomass synthesis. It seems that a blocking of metabolite oxidation may have occurred in C. fermentans except in the case of acetate, but acetate oxidation was apparently not coupled with production of energy utilizable in biosynthesis.     

Exopolysaccharide Production and Peculiarities of C6-Metabolism in Acinetobacter sp. Grown on Carbohydrate Substrates

An Acinetobacter sp. strain grown on carbohydrate substrates (mono- and disaccharides, molasses, starch) was shown to synthesize exopolysaccharides (EPS). Glucose catabolism proved to proceed via the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways. Pyruvate entered the tricarboxylic acid cycle due to pyruvate dehydrogenase activity. Pyruvate carboxylation by pyruvate carboxylase was the anaplerotic reaction providing for the synthesis of intermediates for the constructive metabolism of Acinetobacter sp. grown on C₆-substrates. The C₆-metabolism in Acinetobacter sp. was limited by coenzyme A. Irrespective of the carbohydrate growth substrate (glucose, ethanol), the activities of the key enzymes of both C₂- and C₆-metabolism was high, except for the isocitrate lyase activity in glucose-grown bacteria. Isocitrate lyase activity was induced by C₂-compounds (ethanol or acetate). After their addition to glucose-containing medium, both substrates were utilized simultaneously, and an increase was observed in the EPS synthesis, as well as in the EPS yield relative to biomass. The mechanisms responsible for enhancing the EPS synthesis in Acinetobacter sp. grown on a mixture of C₂- and C₆-substrates are discussed.     

Effect of substrate loading on hydrogen production during anaerobic fermentation by Clostridium thermocellum 27405

We have investigated hydrogen (H₂) production by the cellulose-degrading anaerobic bacterium, Clostridium thermocellum. In the following experiments, batch-fermentations were carried out with cellobiose at three different substrate concentrations to observe the effects of carbon-limited or carbon-excess conditions on the carbon flow, H₂-production, and synthesis of other fermentation end products, such as ethanol and organic acids. Rates of cell growth were unaffected by different substrate concentrations. H₂, carbon dioxide (CO₂), acetate, and ethanol were the main products of fermentation. Other significant end products detected were formate and lactate. In cultures where cell growth was severely limited due to low initial substrate concentrations, hydrogen yields of 1 mol H₂/mol of glucose were obtained. In the cultures where growth ceased due to carbon depletion, lactate and formate represented a small fraction of the total end products produced, which consisted mainly of H₂, CO₂, acetate, and ethanol throughout growth. In cultures with high initial substrate concentrations, cellobiose consumption was incomplete and cell growth was limited by factors other than carbon availability. H₂-production continued even in stationary phase and H₂/CO₂ ratios were consistently greater than 1 with a maximum of 1.2 at the stationary phase. A maximum specific H₂ production rate of 14.6 mmol g dry cell⁻¹ h⁻¹ was observed. As cells entered stationary phase, extracellular pyruvate production was observed in high substrate concentration cultures and lactate became a major end product.     

The Role of Ethylene Diisothiocyanate (EDI) in the Antifungal Action of Disodium Ethylene Bisdithiocarbamate (Nabam). I. The Mode of Action of EDI on the Metabolism of Yeast

With glucose as a substrate, the oxygen consumption in yeast in inhibited by 2· 10^-5M ethylene diisothiocyanate. The degree of inhibition was only to a small extent dependant on pH. Radiorespirometric experiments with uniformely labelled glucose showed that the CO₂-production from glucose increased, probably due to increased glycolytic activity. Conversion of C-1 to CO₂ was unaffected by the inhibitor, while the evolution of CO₂ from C-6 was strongly inhibited. The same was the case with CO₂ from C-1 in acetate. Respiration of ethanol was more strongly inhibited than that of glucose or acetate. Experiments with dual wavelength spectrophotometry showed the inhibition to be located on the Krebs cycle side of the respiratory flavoproteins. It is concluded that the action of ethylene diisothiocyanate on respiration must be located at the mitochondria.     

Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO₂ to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.     

Rhizomorph Formation in Fungi I. Stimulation by Ethanol and Acetate and Inhibition by Disul-firam of Growth and Rhizomorph Formation in Armillaria mellea

Rhizomorph formation in Armillaria mellea (Vahl ex Fr.) Quél. has been studied on a synthetic glucose medium with ethanol and acetate as inducers. Disulfiram (antabuse) inhibited growth and rhizomorph formation when ethanol was the inducer, while the stimulating effect of acetate was unaffected. This indicates that ethanol is metabolised by the normal pathway via acetate. Growth, ethanol- and glucose uptake were measured as a function of time. Ethanol was used up in 12 days; after that, growth and glucose uptake declined. A further supply of ethanol gave a significant increase in the growth rate. The results show that ethanol is a limiting growth factor during the entire growth period.     

Application of acetate buffer in pH adjustment of sorghum mash and its influence on fuel ethanol fermentation

A 2 M sodium acetate buffer at pH 4.2 was tried to simplify the step of pH adjustment in a laboratory dry-grind procedure. Ethanol yields or conversion efficiencies of 18 sorghum hybrids improved significantly with 2.0–5.9% (3.9% on average) of relative increases when the method of pH adjustment changed from traditional HCl to the acetate buffer. Ethanol yields obtained using the two methods were highly correlated (R ² = 0.96, P < 0.0001), indicating that the acetate buffer did not influence resolution of the procedure to differentiate sorghum hybrids varying in fermentation quality. Acetate retarded the growth of Saccharomyces cerevisiae, but did not affect the overall fermentation rate. With 41–47 mM of undissociated acetic acid in mash of a sorghum hybrid at pH 4.7, rates of glucose consumption and ethanol production were inhibited during exponential phase but promoted during stationary phase. The maximum growth rate constants (μ _max) were 0.42 and 0.32 h⁻¹ for cells grown in mashes with pH adjusted by HCl and the acetate buffer, respectively. Viable cell counts of yeast in mashes with pH adjusted by the acetate buffer were 36% lower than those in mashes adjusted by HCl during stationary phase. Coupled to a 5.3% relative increase in ethanol, a 43.6% relative decrease in glycerol was observed, when the acetate buffer was substituted for HCl. Acetate helped to transfer glucose to ethanol more efficiently. The strain tested did not use acetic acid as carbon source. It was suggested that decreased levels of ATP under acetate stress stimulate glycolysis to ethanol formation, increasing its yield at the expense of biomass and glycerol production. Names are necessary to report factually on available data; however, the U.S. Department of Agriculture neither guarantees nor warrants the standard of the product, and use of the name by the U.S. Department of Agriculture implies no approval of the product to the exclusion of others that may also be suitable.     

Regulatory aspects of bakers' yeast metabolism in aerobic fed-batch cultures

A highly instrumented computer-coupled bioreactor is used to investigate metabolic changes of Saccharomyces cerevisiae in aerobic fed-batch systems which are generally applied in bankers' yeast manufacture. The four types of metabolism (oxidation of glucose, aerobic fermentation, oxidation of glucose and ethanol, and oxidation of ethanol) appearing in such systems are characterized by four significant fermentation parameters: Respiratory quotient (RQ), glucose uptake rate (Q_g), ethanol turnover rate (Q_EtOH), and growth yield on glucose (Y_g). Below the critical glucose concentration glucose and ethanol are utilized simultaneously. The shift from aerobic fermentation to nondiauxic growth on glucose and ethanol is not only dependent on glucose concentration. but also on the precultivation on cells. The uptake of ethanol is controlled by the glucose supply except in the case when ethanol is limiting; the oxygen uptake rate (Q_o2), however, is unaffected by the ratio of Q_g and Q_EtOH. Critical glucose concentration is not a constant value for a particular strain, but varies corresponding to the nutritional state of the cells.     

Ethanol production from xylose with a pyruvate-formate-lyase mutant of Klebsiella planticola carrying a pyruvate-decarboxylase gene from Zymomonas mobilis

Summary Grown anaerobically on d-xylose, Klebsiella planticola ATCC 33531 produced acetate, formate, lactate, CO₂ and ethanol as major end-products. A Mu-insertion mutant which lacked pyruvate-formate-lyase showed among its fermentation products more than 70% d-lactate with residual acetate, 2,3-butanediol, and traces of ethanol, formate, and CO₂. After the introduction of a plasmid carrying the gene for the enzyme pyruvate decarboxylase from Zymomonas mobilis, this Klebsiella mutant became an efficient ethanol producer. The recombinant strain produced 387 mM ethanol from 275 mM xylose in 80 h, about 83% of the theoretical maximal yield. Furthermore, this mutant consumed more than double the amount of xylose (41 g/l) compared to the wild-type, due to reduced production of inhibiting acids during growth.Dedicated to Professor Dr. Zähner on the occasion of his 60th birthday     

Metabolic selectivity and growth of<Emphasis Type="Italic"> Clostridium thermocellum</Emphasis> in continuous culture under elevated hydrostatic pressure

The continuous culture of Clostridium thermocellum, a thermophilic bacterium capable of producing ethanol from cellulosic material, is demonstrated at elevated hydrostatic pressure (7.0 MPa, 17.3 MPa) and compared with cultures at atmospheric pressure. A commercial limitation of ethanol production by C. thermocellum is low ethanol yield due to the formation of organic acids (acetate, lactate). At elevated hydrostatic pressure, ethanol:acetate (E/A) ratios increased >10² relative to atmospheric pressure. Cell growth was inhibited by approximately 40% and 60% for incubations at 7.0 MPa and 17.3 MPa, respectively, relative to continuous culture at atmospheric pressure. A decrease in the theoretical maximum growth yield and an increase in the maintenance coefficient indicated that more cellobiose and ATP are channeled towards maintaining cellular function in pressurized cultures. Shifts in product selectivity toward ethanol are consistent with previous observations of hydrostatic pressure effects in batch cultures. The results are partially attributed to the increasing concentration of dissolved product gases (H₂, CO₂) with increasing pressure; and they highlight the utility of continuous culture experiments for the quantification of the complex role of dissolved gas and pressure effects on metabolic activity.     

Anaerobic Elemental Sulfur Reduction by Fungus Fusarium oxysporum

Reduction of inorganic sulfur compounds by the fungus Fusarium oxysporum was examined. When transferred from a normoxic to an anoxic environment, F. oxysporum reduced elemental sulfur to hydrogen sulfide (H₂S). This reaction accompanied fungal growth and oxidation of the carbon source (ethanol) to acetate. Over 2-fold more of H₂S than of acetate was produced, which is the theoretical correlation for the oxidation of ethanol to acetate. NADH-dependent sulfur reductase (SR) activity was detected in cell-free extracts of the H₂S-producing fungus, and was found to be up-regulated under the anaerobic conditions. On the other hands both O₂ consumption by the cells and cytochrome c oxidase activity by the crude mitochondrial fractions decreased. These results indicate that H₂S production involving SR was due to a novel dissimilation mechanism of F. oxysporum, and that the fungus adapts to anaerobic conditions by replacing the energy-producing mechanism of O₂ respiration with sulfur reduction.     

Observer design for differential-algebraic model of an aerobic culture of a recombinant yeast

The mathematical model of an aerobic culture of recombinant yeast presented in work by Zhang et al. (1997) is given by a differential-algebraic system. The classical nonlinear observer algorithms are generally based on ordinary differential equations. In this paper, first we extend the nonlinear observer synthesis to differential-algebraic dynamical systems. Next, we apply this observer theory to the mathematical model proposed in Zhang et al. (1997). More precisely, based on the total cell concentration and the recombinant protein concentration, the observer gives the online estimation of the glucose, the ethanol, the plasmid-bearing cell concentration and a parameter that represents the probability of plasmid loss of plasmid-bearing cells. Numerical simulations are given to show the good performances of the designed observer.Symbols C ₁ activity of pacing enzyme pool for glucose fermentation (dimensionless) - C ₂ activity of pacing enzyme pool for glucose oxidation (dimensionless) - C ₃ activity of pacing enzyme pool for ethanol oxidation (dimensionless) - E ethanol concentration (g/l) - G glucose concentration (g/l) - k _a regulation constant for (g glucose/g cell h^–1) - k _b regulation constant for (dimensionless) - k _c regulation constant for (g glucose/g cell h^–1) - k _d regulation constant for (dimensionless) - K _m1 saturation constant for glucose fermentation (g/l) - K _m2 saturation constant for glucose oxidation (g/l) - K _m3 saturation constant for ethanol oxidation (g/l) - L ( t) time lag function (dimensionless) - p probability of plasmid loss of plasmid-bearing cells (dimensionless) - P recombinant protein concentration (mg/g cell) - q _G total glucose flux culture time (g glucose/g cell h) - t culture time (h) - t _lag lag time (h) - X total cell concentration (g/l) - X ⁺ plasmid-bearing cell concentration (g/l) - Y ^F _{X / G} cell yield for glucose fermentation pathway (g cell/g glucose) - Y ^O _{X / G} cell yield for glucose oxidation pathway (g cell/g glucose) - Y _{X / E} cell yield for ethanol oxidation pathway (g cell/g ethanol) - Y _{E / X} ethanol yield for fermentation pathway based on cell mass (g ethanol·g cell) - ₂ glucoamylase yield for glucose oxidation (units/g cell) - ₃ glucoamylase yield for ethanol oxidation (units/g cell) - µ₁ specific growth rate for glucose fermentation (h^–1) - µ₂ specific growth rate for glucose oxidation (h^–1) - µ₃ specific growth rate for ethanol oxidation (h^–1) - µ_1max maximum specific growth rate for glucose fermentation (h^–1) - µ_2max maximum specific growth rate for glucose oxidation (h^–1) - µ_3max maximum specific growth rate for ethanol oxidation (h^–1)     

Metabolic control of Clostridium thermocellum via inhibition of hydrogenase activity and the glucose transport rate

Clostridium thermocellum has the ability to catabolize cellulosic biomass into ethanol, but acetic acid, lactic acid, carbon dioxide, and hydrogen gas (H₂) are also produced. The effect of hydrogenase inhibitors (H₂, carbon monoxide (CO), and methyl viologen) on product selectivity was investigated. The anticipated effect of these hydrogenase inhibitors was to decrease acetate production. However, shifts to ethanol and lactate production are also observed as a function of cultivation conditions. When the sparge gas of cellobiose-limited chemostat cultures was switched from N₂ to H₂, acetate declined, and ethanol production increased 350%. In resting cell suspensions, lactate increased when H₂ or CO was the inhibitor or when the cells were held at elevated hyperbaric pressure (6.8 atm). In contrast, methyl-viologen-treated resting cells produced twice as much ethanol as the other treatments. The relationship of chemostat physiology to methyl viologen inhibition was revealed by glucose transport experiments, in which methyl viologen decreased the rate of glucose transport by 90%. C. thermocellum produces NAD⁺ from NADH by H₂, lactate, and ethanol production. When the hydrogenases were inhibited, the latter two products increased. However, excess substrate availability causes fructose 1,6-diphosphate, the glycolytic intermediate that triggers lactate production, to increase. Compensatory ethanol production was observed when the chemostat fluid dilution rate or methyl viologen decreased substrate transport. This research highlights the complex effects of high concentrations of dissolved gases in fermentation, which are increasingly envisioned in microbial applications of H₂ production for the conversion of synthetic gases to chemicals.     

Laboratory metabolic evolution improves acetate tolerance and growth on acetate of ethanologenic Escherichia coli under non-aerated conditions in glucose-mineral medium

In this work, Escherichia coli MG1655 was engineered to produce ethanol and evolved in a laboratory process to obtain an acetate tolerant strain called MS04 (E. coli MG1655: ΔpflB, ΔadhE, ΔfrdA, ΔxylFGH, ΔldhA, PpflB::pdc _Zm -adhB _Zm , evolved). The growth and ethanol production kinetics of strain MS04 were determined in mineral medium, mainly under non-aerated conditions, supplemented with glucose in the presence of different concentrations of sodium acetate at pH?7.0 and at different values of acid pH and a constant concentration of sodium acetate (2?g/l). Results revealed an increase in the specific growth rate, cell mass formation, and ethanol volumetric productivity at moderate concentrations of sodium acetate (2–10?g/l), in addition to a high tolerance to acetate because it was able to grow and produce a high yield of ethanol in the presence of up to 40?g/l of sodium acetate. Genomic analysis of the ΔpflB evolved strain identified that a chromosomal deletion of 27.3?kb generates the improved growth and acetate tolerance in MG1655 ΔpflB derivative strains. This deletion comprises genes related to the respiration of nitrate, repair of alkylated DNA and synthesis of the ompC gene coding for porin C, cytochromes C, thiamine, and colonic acid. Strain MS04 is advantageous for the production of ethanol from hemicellulosic hydrolysates that contain acetate.