Ethanol production from xylose by Thermoanaerobacter ethanolicus in batch and continuous culture

The fermentation of xylose by Thermoanaerobacter ethanolicus ATCC 31938 was studied in pH-controlled batch and continuous cultures. In batch culture, a dependency of growth rate, product yield, and product distribution upon xylose concentration was observed. With 27 mM xylose media, an ethanol yield of 1.3 mol ethanol/mol xylose (78% of maximum theoretical yield) was typically obtained. With the same media, xylose-limited growth in continuous culture could be achieved with a volumetric productivity of 0.50 g ethanol/liter h and a yield of 0.42 g ethanol/g xylose (1.37 mol ethanol/mol xylose). With extended operation of the chemostat, variation in xylose uptake and a decline in ethanol yield was seen. Instability with respect to fermentation performance was attributed to a selection for mutant populations with different metabolic characteristics. Ethanol production in these T. ethanolicus systems was compared with xylose-to-ethanol conversions of other organisms. Relative to the other systems, T. ethanolicus offers the advantages of a high ethanol yield at low xylose concentrations in batch culture and of a rapid growth rate. Its disadvantages include a lower ethanol yield at higher xylose concentrations in batch culture and an instability of fermentation characteristics in continuous culture.     

Growth and Fermentation Characteristics of a Strain of the Wine Yeast Kluyveromyces thermotolerans Isolated in Greece

Summary During the single culture fermentation of grape must K. thermotolerans, strain TH941, isolated in a wine-producing region in northern Greece, reached a very high cell concentration of 8.4 log (c.f.u ml⁻¹), followed by a rapid decline of the viable cells. The yeast produced 9.6 g L-lactic acid l⁻¹ during the growth phase, 7.58% v/v of ethanol and showed a limited degradation of L-malic acid as well as a low production of volatile acidity. In the presence of 3% v/v and 6% v/v of ethanol the K. thermotolerans isolate was able to grow. At 9% v/v of ethanol it could not grow but showed no loss of viability for 10 days.     

Fermentation of xylose and hemicellulose hydrolysates by an ethanol-adapted culture ofBacteroides polypragmatus

Bacteroides polypragmatus type strain GP4 was adapted to grow in the presence of 3.5% (w/v) ethanol by successive transfers into 1% (w/v)d-xylose media supplemented with increasing concentrations of ethanol. The maximum specific growth rate of the ethanol-adapted culture (=0.30 h^-1) was not affected by up to 2% (w/v) ethanol but that of the non-adapted strain declined by about 50%. The growth rate of both cultures was limited by nutrient(s) contained in yeast extract. The ethanol yield of the adapted culture (1.01 mol/mol xylose) was higher than that (0.80 mol/mol xylose) of the non-adapted strain. The adapted culture retained the ability to simultaneously ferment pentose and hexose sugars, and moreover it was not inhibited by xylose concentrations of 7–9% (w/v). This culture also readily fermented hemicellulose hydrolysates obtained by mild acid hydrolysis of either hydrogen fluoride treated or steam exploded Aspen wood. The ethanol yield from the fermentation of the hydrolysates was comparable to that obtained from xylose.This paper is issued as NRCC No. 26338     

Ethanol production during batch fermentation with Saccharomyces cerevisiae: changes in glycolytic enzymes and internal pH

During batch fermentation, the rate of ethanol production per milligram of cell protein is maximal for a brief period early in this process and declines progressively as ethanol accumulates in the surrounding broth. Our studies demonstrate that the removal of this accumulated ethanol does not immediately restore fermentative activity, and they provide evidence that the decline in metabolic rate is due to physiological changes (including possible ethanol damage) rather than to the presence of ethanol. Several potential causes for the decline in fermentative activity have been investigated. Viability remained at or above 90%, internal pH remained near neutrality, and the specific activities of the glycolytic and alcohologenic enzymes (measured in vitro) remained high throughout batch fermentation. None of these factors appears to be causally related to the fall in fermentative activity during batch fermentation.     

Ethanol production during batch fermentation with Saccharomyces cerevisiae: changes in glycolytic enzymes and internal pH.

During batch fermentation, the rate of ethanol production per milligram of cell protein is maximal for a brief period early in this process and declines progressively as ethanol accumulates in the surrounding broth. Our studies demonstrate that the removal of this accumulated ethanol does not immediately restore fermentative activity, and they provide evidence that the decline in metabolic rate is due to physiological changes (including possible ethanol damage) rather than to the presence of ethanol. Several potential causes for the decline in fermentative activity have been investigated. Viability remained at or above 90%, internal pH remained near neutrality, and the specific activities of the glycolytic and alcohologenic enzymes (measured in vitro) remained high throughout batch fermentation. None of these factors appears to be causally related to the fall in fermentative activity during batch fermentation.     

Sugar fermentation by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> to produce ethanol

As a first step in the research on ethanol production from lignocellulose residues, sugar fermentation by Fusarium oxysporum in oxygen-limited conditions is studied in this work. As a substrate, solutions of arabinose, glucose, xylose and glucose/xylose mixtures are employed. The main kinetic and yield parameters of the process are determined according to a time-dependent model. The microorganism growth is characterized by the maximum specific growth rate and biomass productivity, the substrate consumption is studied through the specific consumption rate and biomass yield, and the product formation via the specific production rate and product yields. In conclusion, F. oxysporum can convert glucose and xylose into ethanol with product yields of 0.38 and 0.25, respectively; when using a glucose/xylose mixture as carbon source, the sugars are utilized sequentially and a maximum value of 0.28 g/g ethanol yield is determined from a 50% glucose/50% xylose mixture. Although fermentation performance by F.␣oxysporum is somewhat lower than that of other fermenting microorganisms, its ability for simultaneous lignocellulose-residue saccharification and fermentation is considered as a potential advantage.     

Effect of pretreatment chemicals on xylose fermentation by Pichia stipitis

Pretreatment of biomass with dilute H₂SO₄ results in residual acid which is neutralized with alkalis such as Ca(OH)₂, NaOH and NH₄OH. The salt produced after neutralization has an effect on the fermentation of Pichia stipitis. Synthetic media of xylose (60 g total sugar/l) was fermented to ethanol in the presence and absence of the salts using P. stipitis CBS 6054. CaSO₄ enhanced growth and xylitol production, but produced the lowest ethanol concentration and yield after 140 h. Na₂SO₄ inhibited xylitol production, slightly enhanced growth towards the end of fermentation but had no significant effect on xylose consumption and ethanol concentration. (NH₄)₂SO₄ inhibited growth, had no effect on xylitol production, and enhanced xylose consumption and ethanol production.     

Study of sugarcane pieces as yeast supports for ethanol production from sugarcane juice and molasses

Due to the environmental concerns and the increasing price of oil, bioethanol was already produced in large amount in Brazil and China from sugarcane juice and molasses. In order to make this process competitive, we have investigated the suitability of immobilized Saccharomyces cerevisiae strain AS2.1190 on sugarcane pieces for production of ethanol. Electron microscopy clearly showed that cell immobilization resulted in firm adsorption of the yeast cells within subsurface cavities, capillary flow through the vessels of the vascular bundle structure, and attachment of the yeast to the surface of the sugarcane pieces. Repeated batch fermentations using sugarcane supported-biocatalyst were successfully carried out for at least ten times without any significant loss in ethanol production from sugarcane juice and molasses. The number of cells attached to the support increased during the fermentation process, and fewer yeast cells leaked into fermentation broth. Ethanol concentrations (about 89.73–77.13 g/l in average value), and ethanol productivities (about 59.53–62.79 g/l d in average value) were high and stable, and residual sugar concentrations were low in all fermentations (0.34–3.60 g/l) with conversions ranging from 97.67–99.80%, showing efficiency (90.11–94.28%) and operational stability of the biocatalyst for ethanol fermentation. The results of this study concerning the use of sugarcane as yeast supports could be promising for industrial fermentations. L. Liang and Y. Zhang have contributed equally to this work.     

Clostridium neopropionicum sp. nov., a strict anaerobic bacterium fermenting ethanol to propionate through acrylate pathway

Strain X4 was isolated several years ago from an anaerobic mesophilic plant treating vegetable cannery waste waters. It was the first example of propionic fermentation from ethanol. Morphologic and physiologic characterizations of the strain are presented here. This strain is described as type strain of a new species, Clostridium neopropionicum sp. nov. Whole cells of strain X4 ferment [1-¹³C]ethanol and CO₂ to [2-¹³C]propionate, [1-¹³C]acetate and [2-¹³C]propanol, suggesting the absence of a randomizing pathway during the propionate formation. Enzymes involved in this fermentation were assayed in cell-free extracts of cells grown with ethanol as sole substrate. Alcohol dehydrogenase, aldehyde dehydrogenase, phosphate acetyl transferase, acetate kinase, pyruvate synthase, lactate dehydrogenases, and the enzymes of the acrylate pathway were detected at activities sufficient to be involved in ethanol fermentation. The same pathway may be used for the degradation of lactate or acrylate to acetate.     

Synergism among lactic acid, sulfite, pH and ethanol in alcoholic fermentation of Saccharomyces cerevisiae (PE-2 and M-26)

Summary The industrial production of ethanol is affected mainly by contamination by lactic acid bacteria besides others factors that act synergistically like increased sulfite content, extremely low pH, high acidity, high alcoholic content, high temperature and osmotic pressure. In this research two strains of Saccharomyces cerevisiae PE-2 and M-26 were tested regarding the alcoholic fermentation potential in highly stressed conditions. These strains were subjected to values up to 200 mg NaHSO₃ l⁻¹, 6 g lactic acid l⁻¹, 9.5% (w/v) ethanol and pH 3.6 during fermentative processes. The low pH (3.6) was the major stressing factor on yeasts during the fermentation. The M-26 strain produced higher acidity than the other, with higher production of succinic acid, an important inhibitor of lactic bacteria. Both strains of yeasts showed similar performance during the fermentation, with no significant difference in cell viability.     

Pyruvate metabolism in rice coleoptiles under anaerobiosis

Relative importance of ethanolic, lactate and alanine fermentation pathways was estimated in coleoptiles of rice seedlings (Oryza sativa L.) subjected to anoxic stress. The in vitro activities of alcohol dehydrogenase (ADH, EC 1.1.1.1), pyruvate decarboxylase (PDC, EC 4.1.1.1) and alanine aminotransferase (AlaAT, EC 2.6.1.2) in the coleoptiles increased in anoxia, whereas no significant increase was measured in lactate dehydrogenase (LDH, EC 1.1.1.27) activity. At 48 h, the ADH, PDC and AlaAT activities in anoxic coleoptiles were 62-, 15- and 7.6-fold greater, respectively, than those in the presence of oxygen. Ethanol and alanine in the coleoptiles accumulated rapidly under anoxia, increasing by 48 h, 57- and 5.6-fold compared with those in the presence of oxygen, respectively. However, lactate concentration did not increase and no initial burst of lactate production was detected. The relative ratio of carbon flux from pyruvate to ethanol, lactate and alanine in anoxic coleoptiles was estimated to be 92, 1 and 7% of the total carbon flux, respectively. These results suggest that the potential carbon flux from pyruvate to ethanol may be much greater than the potential flux from pyruvate to lactate and alanine in rice coleoptiles during anoxia.     

Intracellular accumulation of AMP as a cause for the decline in rate of ethanol production by Saccharomyces cerevisiae during batch fermentation

A general hypothesis is presented for the decline in the rate of ethanol production (per unit of cell protein) during batch fermentation. Inhibition of ethanol production is proposed to result from the intracellular accumulation of AMP during the transition from growth to the stationary phase. AMP acts as a competitive inhibitor of hexokinase with respect to ATP. When assayed in vitro in the presence of ATP and AMP concentrations equivalent to those within cells at different stages of fermentation, hexokinase activity declined in parallel with the in vivo decline in the rate of ethanol production. The coupling of glycolytic flux and fermentation to cell growth via degradation products of RNA may be of evolutionary advantage for Saccharomyces cerevisiae. Such a coupling would reduce the exposure of nongrowing cells to potentially harmful concentrations of waste products from metabolism and would conserve nutrients for future growth under more favorable conditions.     

Intracellular accumulation of AMP as a cause for the decline in rate of ethanol production by Saccharomyces cerevisiae during batch fermentation.

A general hypothesis is presented for the decline in the rate of ethanol production (per unit of cell protein) during batch fermentation. Inhibition of ethanol production is proposed to result from the intracellular accumulation of AMP during the transition from growth to the stationary phase. AMP acts as a competitive inhibitor of hexokinase with respect to ATP. When assayed in vitro in the presence of ATP and AMP concentrations equivalent to those within cells at different stages of fermentation, hexokinase activity declined in parallel with the in vivo decline in the rate of ethanol production. The coupling of glycolytic flux and fermentation to cell growth via degradation products of RNA may be of evolutionary advantage for Saccharomyces cerevisiae. Such a coupling would reduce the exposure of nongrowing cells to potentially harmful concentrations of waste products from metabolism and would conserve nutrients for future growth under more favorable conditions.     

Enhanced ethanol fermentation of brewery wastewater using the genetically modified strain <Emphasis Type="Italic">E. coli </Emphasis>KO11

We have used liquid waste obtained from a beer brewery process to produce ethanol. To increase the productivity, genetically modified organism, Escherichia coli KO11, was used for ethanol fermentation. Yeast was also used to produce ethanol from the same feed stock, and the ethanol production rates and resulting concentrations of sugars and ethanol were compared with those of KO11. In the experiments, first the raw wastewater was directly fermented using two strains with no saccharification enzymes added. Then, commercial enzymes, α-amylase, pectinase, or a combination of both, were used for simultaneous saccharification and fermentation, and the results were compared with those of the no-enzyme experiments for KO11 and yeast. Under the given conditions with or without the enzymes, yeast produced ethanol more rapidly than E. coli KO11, but the final ethanol concentrations were almost the same. For both yeast and KO11, the enzymes were observed to enhance the ethanol yields by 61–84% as compared to the fermentation without enzymes. The combination of the two enzymes increased ethanol production the most for the both strains. The advantages of using KO11 were not demonstrated clearly as compared to the yeast fermentation results.     

Inhibition of <Emphasis Type="Italic">Pichia stipitis</Emphasis> fermentation of hydrolysates from olive tree cuttings

The ethanolic fermentation of liquid fractions (hydrolysates) issued from dilute acid pre-treatment of olive tree biomass by Pichia stipitis is reported for the first time. On the one side, P. stipitis has been reported as the most promising naturally occurring C5 fermenting microorganism; on the other side, olive tree biomass is a renewable, low cost, and lacking of alternatives agricultural residue especially abundant in Mediterranean countries. The study was performed in two steps. First, the fermentation performance of P. stipitis was evaluated on a fermentation medium also containing the main inhibitors found in these hydrolysates (acetic acid, formic acid, and furfural), as well as glucose and xylose as carbon sources. The effect of inhibitors, individually or in a mixture, on kinetic and yield parameters was calculated. In a second step, hydrolysates obtained from 1% (w/w) sulfuric acid pre-treatment of olive tree biomass at 190°C for 10 min were used as a real fermentation medium with the same microorganism. Due to inhibition, effective fermentation required dilution of the hydrolysate and either overliming or activated charcoal treatment. Results show that ethanol yields obtained from hydrolysates, ranging from 0.35 to 0.42 g/g, are similar to those from synthetic medium, although the process proceeds at lower rates. Inhibiting compounds affect the fermentation performance in a synergistic way. Furfural is rapidly assimilated by the yeast; acetic acid and formic acid concentrations decrease slowly during the process. Activated charcoal or overliming detoxification improve the fermentability of diluted hydrolysates.     

Metabolic engineering of a Lactobacillus plantarum double ldh knockout strain for enhanced ethanol production

Lactobacillus plantarum ferments glucose through the Embden–Meyerhof–Parnas pathway: the central metabolite pyruvate is converted into lactate via lactate dehydrogenase (LDH). By substituting LDH with pyruvate decarboxylase (PDC) activity, pyruvate may be redirected toward ethanol production instead of lactic acid fermentation. A PDC gene from the Gram-positive bacterium Sarcina ventriculi (Spdc) was introduced into an LDH-deficient strain, L. plantarum TF103, in which both the ldhL and ldhD genes were inactivated. Four different fusion genes between Spdc and either the S. ventriculi promoter or three Lactococcus lactis promoters in pTRKH2 were introduced into TF103. PDC activity was detected in all four recombinant strains. The engineered strains were examined for production of ethanol and other metabolites in flask fermentations. The recombinant strains grew slightly faster than the parent TF103 and produced 90–130 mM ethanol. Although slightly more ethanol was observed, carbon flow was not significantly improved toward ethanol, suggesting that a further understanding of this organism’s metabolism is necessary.     

Alcohol conversion by Desulfobulbus propionicus Lindhorst in the presence and absence of sulfate and hydrogen

Ethanol was rapidly degraded to mainly acetate in anaerobic freshwater sediment slurries. Propionate was produced in small amounts. Desulfovibrio species were the dominant bacteria among the ethanol-degrading organisms. The propionate-producing Desulfobulbus propionicus came to the fore under iron-limited conditions in an ethanol-limited chemostat with excess sulfate inoculated with anaerobic intertidal freshwater sediment. In the absence of sulfate, ethanol was fermented by D. propionicus Lindhorst to propionate and acetate in a molar ratio of 2.0.l-Propanol was intermediately produced during the fermentation of ethanol. In the presence of H₂ and CO₂, ethanol was quantitatively converted to propionate. H₂-plus sulfate-grown cells of D. propionicus Lindhorst were able to oxidize l-propanol and l-butanol to propionate and butyrate respectively with the concomitant reduction of acetate plus CO₂ to propionate. Growth was also observed on acetate alone in the presence of H₂ and CO₂ D. propionicus was able to grow mixotrophically on H₂ plus an organic compound. Finally, a brief discussion has been given of the ecological niche of D. propionicus in anaerobic freshwater sediments.     

Ethanol vapour pressure as a control factor during alcoholic fermentation

Aqueous solutions of glucose/fructose mixtures with varying concentrations of ethanol were used to study the effects on fermentation of ethanol vapour pressure and water activity. Water vapour pressure was found to increase significantly with temperature in the range 15 to 30‡C. The effects on glucose fermentation bySaccharomyces cerevisiae Bg7FL of the variables glucose concentration, Tween 80 concentration, temperature and ammonium and ethanol concentrations were examined using central composite design. A best fit equation describing the main, quadratic and interactive effects of the five variables on yeast growth rate was produced. Further model systems were analysed in which the effects of ethanol vapour pressure, water vapour pressure and ethanol concentration on maximal growth rate of the yeast strain were studied. Above 18‡C, neither ethanol concentration nor ethanol vapour pressure controlled the fermentation rate. Ethanol toxicity was shown to be associated with its vapour pressure rather than its concentration.     

Ethanol and acetate production by <Emphasis Type="Italic">Clostridium ljungdahlii</Emphasis> and <Emphasis Type="Italic">Clostridium autoethanogenum</Emphasis> using resting cells

Combined gasification and fermentation technologies can potentially produce biofuels from renewable biomass. Gasification generates synthesis gas consisting primarily of CO, CO₂, H₂, N₂, with smaller amounts of CH₄, NO_x, O₂, C₂ compounds, ash and tars. Several anaerobic bacteria species can ferment bottled mixtures of pure synthesis gas constituents. However, there are challenges to maintaining culture viability of synthesis gas exposed cells. This study was designed to enhance culture stability and improve ethanol-to-acetate ratios using resting (non-growing) cells in synthesis gas fermentation. Resting cell states were induced in autotrophic Clostridium ljungdahlii cultures with minimal ethanol and acetate production due to low metabolic activity compared to growing cell production levels of 5.2 and 40.1 mM of ethanol and acetate. Clostridium autoethanogenum cultures were not induced into true resting states but did show improvement in total ethanol production (from 5.1 mM in growing cultures to 9.4 in one nitrogen-limited medium) as well as increased shifts in ethanol-to-acetate production ratios.     

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.