High ethanol tolerance of new isolates of Clostridium thermocellum strains SS21 and SS22

Clostridium thermocellum strains SS21 and SS22, producing high yields of ethanol, were tolerant to 4.0 and 5.0% (v/v) ethanol, respectively. This is the highest ethanol tolerance so far reported by wild type strains of C. thermocellum. In the presence of added ethanol, both the strains had extended period of growth arrest. On addition of ethanol at different culture ages increase in ethanol tolerance upto 7.0 and 8.0% (v/v) by strains SS21 and SS22, respectively was observed. The optimum growth temperature for strain SS21 decreased as the concentration of ethanol in the medium increased and remained constant for strain SS22. Both the strains were tolerant to various solvents and acetic acid indicating that high ethanol tolerance of the strains is due to the general solvent tolerance of the organisms.     

High-throughput screening and characterization of xylose-utilizing, ethanol-tolerant thermophilic bacteria for bioethanol production

Aims: To develop a high‐throughput assay for screening xylose‐utilizing and ethanol‐tolerant thermophilic bacteria owing to their abilities to be the promising ethanologens. Methods and Results: Based on alcohol oxidase and peroxidase‐coupled enzymatic reaction, an assay was developed by the formation of the coloured quinonimine to monitor the oxidation of ethanol in the reaction and calculate the concentration of ethanol. This assay was performed in 96‐well microtitre plate in a high‐throughput and had a well‐linear detection range of ethanol from 0 up to 2·5 g l^?1 with high accuracy. The assay was then verified by screening soil samples from hot spring for xylose‐utilizing and ethanol production at 60°C. Three isolates LM14‐1, LM14‐5 and LM18‐4 with 3–5% (v/v) ethanol tolerance and around 0·29–0·38 g g^?1 ethanol yield from xylose were obtained. Phylogenetic and phenotypic analysis showed that the isolates clustered with members of the genus Bacillus or Geobacillus subgroup. Conclusions: The developed double enzyme‐coupled, high‐throughput screening system is effective to screen and isolate xylose‐utilizing, ethanol‐producing thermophilic bacteria for bioethanol production at the elevated temperature. Significance and Impact of the Study: Our research presented a novel high‐throughput method to screen thermophilic bacteria for producing ethanol from xylose. This screening method is also very useful to screen all kinds of ethanologens either from natural habitats or from mutant libraries, to improve bioethanol production from lignocellulosic feedstocks.     

High ethanol tolerance of the thermophilic anaerobic ethanol producer Thermoanaerobacter BG1L1

The low ethanol tolerance of thermophilic anaerobic bacteria, generally less than 2% (v/v) ethanol, is one of the main limiting factors for their potential use for second generation fuel ethanol production. In this work, the tolerance of thermophilic anaerobic bacterium Thermoanaerobacter BG1L1 to exogenously added ethanol was studied in a continuous immobilized reactor system at a growth temperature of 70°C. Ethanol tolerance was evaluated based on inhibition of fermentative performance e.g. inhibition of substrate conversion. At the highest ethanol concentration tested (8.3% v/v), the strain was able to convert 42% of the xylose initially present, indicating that this ethanol concentration is not the upper limit tolerated by the strain. Long-term strain adaptation to high ethanol concentrations (6–8.3%) resulted in an improvement of xylose conversion by 25% at an ethanol concentration of 5% v/v, which is the concentration required in practice for economically efficient product recovery. For all ethanol concentrations tested, relatively high and stable ethanol yields (0.40–0.42 g/g) were seen. The strain demonstrated a remarkable ethanol tolerance, which is the second highest displayed by thermophilic anaerobic bacteria known to the authors. This appears to be the first study of the ethanol tolerance of these microorganisms in a continuous immobilized reactor system.     

Saccharomyces cerevisiae strains with different degrees of ethanol tolerance exhibit different adaptive responses to produced ethanol

Two Saccharomyces cerevisiae strains with different degrees of ethanol tolerance adapted differently to produced ethanol. Adaptation in the less ethanol-tolerant strain was high and resulted in a reduced formation of ethanol-induced respiratory deficient mutants and an increased ergosterol content of the cells. Adaptation in the more ethanol-tolerant strain was less pronounced. Journal of Industrial Microbiology & Biotechnology (2000) 24, 75–78. Received 22 June 1999/ Accepted in revised form 06 October 1999     

典型产纤维小体梭菌的遗传改造及其在纤维素乙醇中的应用研究进展简

以解纤维梭菌（ Clostridium cellulolyticum）和热纤梭菌（ Clostridium thermocellum）为代表的产纤维小体梭菌可以直接完成从木质纤维素原料到乙醇的生物转化,是用于通过整合生物加工技术生产纤维素乙醇的优良候选菌株。然而,这些产纤维小体梭菌的纤维素降解效率及乙醇产量尚不能满足工业化生产的要求,其遗传改造技术的不成熟严重制约了通过定向代谢工程改造提高生产性能的进程。针对这些典型的产纤维小体菌株,各国科学家近年来在基于二类内含子的嗜中温及嗜高温遗传改造平台建立方面取得了较大突破,并通过靶向代谢工程改造,显著提高纤维素乙醇的产量。笔者对这些前期研究工作以及国内外相关研究成果进行系统的总结,并对构建的遗传改造工具的应用前景进行展望。     

High ethanol production by new isolates of Clostridium thermocellum

Summary Among twelve strains of Clostridium thermocellum isolated from faecal droppings of various herbivorous animals and birds, three of the strains, SS21, SS22 and SS19, produced 0.37, 0.33 and 0.32 g of ethanol per g of the substrate consumed and had ethanol to acetate ratios of 2.21, 2.45 and 1.72 respectively. These are the highest substrate conversion yields of ethanol amongst the wild strains of C. thermocellum reported so far. The optimum temperature and pH for growth and ethanol production were 60 °C and 7.5, respectively.     

Lactose/whey utilization and ethanol production by transformed Saccharomyces cerevisiae cells

Strains of Saccharomyces cerevisiae transformed with a multicopy expression vector bearing both the Escherichia coli beta-galactosidase gene under the control of the upstream activating sequence of the GAL1-10 genes and the GAL4 activator gene release part of beta-galactosidase in the growth medium. This release is due to cell lysis of the older mother cells; the enzyme maintains its activity in buffered growth media. Fermentation studies with transformed yeast strains showed that the release of beta-galactosidase allowed an efficient growth on buffered media containing lactose as carbon source as well as on whey-based media. The transformed strains utilized up to 95% of the lactose and a high growth yield was obtained in rich media. High productions of ethanol were also observed in stationary phase after growth in lactose minimal media.     

Genetic engineering of Clostridium thermocellum DSM1313 for enhanced ethanol production

Background

The twin problem of shortage in fossil fuel and increase in environmental pollution can be partly addressed by blending of ethanol with transport fuel. Increasing the ethanol production for this purpose without affecting the food security of the countries would require the use of cellulosic plant materials as substrate. Clostridium thermocellum is an anaerobic thermophilic bacterium with cellulolytic property and the ability to produce ethanol. But its application as biocatalyst for ethanol production is limited because pyruvate ferredoxin oxidoreductase, which diverts pyruvate to ethanol production pathway, has low affinity to the substrate. Therefore, the present study was undertaken to genetically modify C. thermocellum for enhancing its ethanol production capacity by transferring pyruvate carboxylase (pdc) and alcohol dehydrogenase (adh) genes of the homoethanol pathway from Zymomonas mobilis.

Results

The pdc and adh genes from Z. mobilis were cloned in pNW33N, and transformed to Clostridium thermocellum DSM 1313 by electroporation to generate recombinant CTH-pdc, CTH-adh and CTH-pdc-adh strains that carried heterologous pdc, adh, and both genes, respectively. The plasmids were stably maintained in the recombinant strains. Though both pdc and adh were functional in C. thermocellum, the presence of adh severely limited the growth of the recombinant strains, irrespective of the presence or absence of the pdc gene. The recombinant CTH-pdc strain showed two-fold increase in pyruvate carboxylase activity and ethanol production when compared with the wild type strain.

Conclusions

Pyruvate decarboxylase gene of the homoethanol pathway from Z mobilis was functional in recombinant C. thermocellum strain and enhanced its ability to produced ethanol. Strain improvement and bioprocess optimizations may further increase the ethanol production from this recombinant strain.

     

Development of ethanol tolerance in Clostridium thermocellum: effect of growth temperature.

The growth of Clostridium thermocellum ATCC 27405 and of C9, an ethanol-resistant mutant of this strain, at different ethanol concentrations and temperatures was characterized. After ethanol addition, cultures continued to grow for 1 to 2 h at rates similar to those observed before ethanol was added and then entered a period of growth arrest, the duration of which was a function of the age of inocula. After this period, cultures grew at an exponential rate that was a function of ethanol concentration. The wild-type strain showed a higher energy of activation for growth than the ethanol-tolerant derivative. The optimum growth temperature of the wild type decreased as the concentration of the ethanol challenge increased, whereas the optimum growth temperature for C9 remained constant. The results are discussed in terms of what is known about the effects of ethanol and temperature on membrane composition and fluidity.     

Increased ethanol production by metabolic modulation of cellulose fermentation in Clostridium thermocellum

Significant quantitative differences in ethanol yields along with repression in acetic acid production were observed in Clostridium thermocellum strains SS21 and SS22 in the presence of H 2 , acetone and sodium azide. Exogenous H 2 addition (1.0 atm) increased the ethanol yields to 0.40 g/g and ethanol to acetate ratio to 5.75 in strain SS21 but was inhibitory in strain SS22. Addition of acetone reversed the inhibition caused by H 2 and increased the ethanol yields and ethanol to acetate ratio of strain SS22 up to 0.40 g/g and 7.9, respectively. Enhancement in ethanol yields up to 0.40 g/g and 0.41 g/g and ethanol to acetate ratio up to 3.63 and 8.1 were observed in the presence of 0.2 mM and 0.15 mM concentration of sodium azide by strains SS21 and SS22, respectively.     

Superiority of the PCR-based approach for cloning the acetate kinase gene of Clostridium thermocellum

Cloning of Clostridium thermocellum acetate kinase (ack) and/or phosphotransacetylase (pta) genes in Escherichia coli by functional complementation of ack and/or pta mutants was complicated by an alternative acetate assimilation pathway involving acetyl-CoA synthetase (ACS). In addition to the problems encountered with the complementation approach, cloning of these genes was not readily achieved using heterologous probing with corresponding genes from Escherichia coli and Methanosarcina thermophila due to the lack of sufficient homology. The use of a PCR-based approach, on the other hand, yielded a specific C. thermocellum gene fragment which showed significant sequence identity to the ack gene for which primers were designed. The subcloned ack fragment was then successfully used as a probe for the isolation of the corresponding gene and restriction analysis of that region. Received 22 January 1998/ Accepted in revised form 31 August 1998     

Whole cell immobilization as a means of enhancing ethanol tolerance

Cell immobilization by covalent linkage to an epoxide derivative of hydroxyalkyl methacrylate gel via glutaraldehyde-diamine spacers improves the tolerance of Saccharomyces cerevisiae cells to ethanol. This was attributed to membrane compositional changes accompanying this mode of cell attachment. The stability of the membrane alterations was tested under salt stress, and the character of stimuli inducing the phenotype changes of attached cells is discussed. Received 12 May 1998/ Accepted in revised form 08 February 1999     

Methane production from organic acid-rich wastewaters by immobilized thermophilic methane-producing bacteria

Thermophilic methane-producing bacteria isolated from a wastewater treatment facility have been immobilized in acetylcellulose filter with agar. The immobilized cells produced methane from wastewaters in rich organic acid (acetic, propionic and butyric acids) at the rate of 1.4 μmol mg protein⁻¹ h⁻¹. The optimum conditions for methane production by immobilized whole cells were 52–55°C and pH 7.0–8.0. The immobilized cells retained 80% of the initial activity after exposure to air. The immobilized thermophilic bacteria produced methane continuously over 10 days at 52°C.     

重组肠道细菌作为产乙醇生物催化剂的研究进展

以木质纤维素水解液发酵生产的燃料酒精作为一种清洁的可再生能源引起人们极大的关注。本文介绍了燃料酒精工业发展的最新动态,并对多种产乙醇重组肠道细菌的研究进展作了综述。     

Inferring ethanol tolerance of Saccharomyces and non-Saccharomyces yeasts by progressive inactivation

The kinetics of cell inactivation in the presence of ethanol at 20, 22.5% and 25% (v/v), was measured by progressive sampling and viable counting, and used as an inference of the ethanol resistance status of five non-Saccharomyces strains and one strain of Saccharomyces cerevisiae. The capacity of standard inocula of the same strains to establish growth at increasing initial ethanol concentrations was employed as a comparison. The effect of various different pre-culture conditions on the ethanol resistance of the 6 strains was analysed by the cell inactivation method and by the cell growth method. Exposing cells to 25% (v/v) ethanol for 4 min enabled the differentiation of the yeasts in terms of their resistance to ethanol. The results suggest that the two methods are generally concordant and that the cell inactivation method can, thus, be used to infer ethanol resistance of yeast strains.     

A parametric study on ethanol production from xylose byPichia stipitis

Characteristics of ethanol production by a xylose-fermenting yeast,Pichia stipitis Y-7124, were studied. The sugar consumption rate and specific growth rate were higher in the glucose-containing medium than in the xylose-containing medium. Specific activities of xylose reductase and xylitol dehydrogenase were higher in the medium with xylose than glucose, suggesting their induction by xylose. Maximum specific growth rate and ethanol yield were achieved at 30 g xylose/L concentration without formation of by-products such as xylitol and acetic acid whereas a maximum ethanol concentration was obtained at 130 g/L xylose. Adding a respiratory inhibitor, rotenone, increased a maximum ethanol concentration by 10% compared with the control experiment. In order to evaluate the pattern of ethanol inhibition on specific growth rate, a kinetic model based on Luong’s equations was applied. The relationship between ethanol concentration and specific growth rate was hyperbolic for glucose and parabolic for xylose. A maximum ethanol concentration at which cells did not grow was 33.6 g/L for glucose and 44.7 g/L for xylose.     

Onsite bio-detoxification of steam-exploded corn stover for cellulosic ethanol production

In the process of ethanol production from steam-exploded corn stover (SECS), a cellulose-degradation strain of Aspergillus nidulans (FLZ10) was investigated whether it could remove the inhibitors released from steam exploded pretreatment , and thereby be used for biological detoxification on Saccharomycescerevisiae. The results showed that FLZ10 removed 75.2% formic acid, 53.6% acetic acid, and 100% hydroxymethyl furfural (5-HMF) and furfural from the hydrolysate washed from SECS after 72 h cultivation. A cellulase activity of 0.49 IU/ml was simultaneously produced while the biological detoxification occurred. An ethanol yield of 0.45 g/g on glucose was obtained in the hydrolysate biodetoxified by FLZ10. The glucose consumption rate of FLZ10 was much lower than that of S. cerevisiae, thereby it had little competition with S. cerevisiae on glucose consumption. Based on SECS to ethanol mass balance analysis, with the onsite bio-detoxification, fermentation using S. cerevisiae effectively converted monomeric glucose with 94.4% ethanol yield.     

Abundance and distribution of anaerobic protozoa and their contribution to methane production in Lake Cadagno (Switzerland)

Abstract In the uupermost layers of the anoxic sediment in Lake Cadagno, 9 different species of anaerobic protozoa were identified. The total number of these organisms was about 580 cells·ml⁻¹ sediment. Most pf these protozoa contained endosymbiotic methanogenic bacteria which in total amounted to 10⁶ methanogens·ml⁻¹ sediment. In addition to the methanogenic endosymbionts, cells of Metopus setosus and Caenomorpha lata also contained a non-fluorescent bacterial rod inside the cytoplasm. In some individual cells of C. lata this second type of endosymbiotic bacterium was sometimes the only endosymbiont observed. Contrary to earlier suggestions, anaerobic protozoa do not seem to play a major role in methane production at least in Lake Cadagno. No significant methane production due to the anaerobic protozoa and their methanogenic endosymbionts was found in situ. Isolated ciliates and amoebae produced methane at 12°C, but not at 6°C, probably as a result of temperature limitation. In the sediment of Lake Cadagno sulfate reduction seemed to be the dominant terminal degradation process.     

Enhancing effect of albumin hydrolysate on ethanol production employing Saccharomyces sake

The enhancing effect of albumin hydrolysate on ethanol production was investigated in ethanol fermentations using Saccharomyces sake. In batchwise ethanol production, addition of supplemental albumin hydrolysate and phosphatidylcholine, or albumin hydrolysate alone, brought about a more than 60% increase in final ethanol concentration (148 or 144 g/L compared with 88 g/L with no supplementation [control] after 72 h). The effect of the supplements is believed to be due to an enhanced alcohol tolerance of cells grown in media containing the supplements. Cells grown in media containing albumin hydrolysate were enriched in phenyalanine, tyrosine, and methionine in their plasma membranes. All three amino acids were also present in considerable amounts in the albumin hydrolysate. This fact suggests that the three amino acids, which are present in albumin hydrolysate, are incorporated into the plasma membranes of cells. Under ethanol production conditions in which only one amino acid among the components of albumin hydrolysate was excluded, namely phenlalanine, tyrosine, or methionine, significant reductions in ethanol production resulted. (c) 1995 John Wiley & Sons, Inc.