Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1–2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed.     

Production of ethanol from straw and bamboo pulp by primary isolates of Clostridium thermocellum

Clostridium thermocellum strains SS8 and GS1 grew poorly on crude blopolymers but termented them easily after alkall treatment. With 1% alkall-extracted rice straw (AERS) and dellgnified bamboo pulp (DBP), the ethanol-to-substrate (E/S) ratios were almost the same as those obtained when using fillter paper. Increasing the substrate concentrations decreased the percentage substrate degraded and the E/S ratio and concomitantly increased the amount of reducing sugars accumulated. A maximum amount of 8.6 g ethanol/l was produced by strain SS8 out of 37.5 g DBP degraded. Strain GS1 accumulated reducing sugars at substrate concentrations >50 g/l, thereby accounting for about 70% of AERS degraded. This strain produced cellulase on both cellulose and cellobiose. Both the strains grew in the presence of 1.5% (v/v) ethanol. Strain SS8 fermented starch, but the ethanol yield was low compared to that from cellulose. About 75% of starch degraded accumulated as reducing sugars at a substrate concentration of 40 g/l. The Inhibitory effects of ethanol (2 to 4%) were less drastic when growing cultures were challenged than when they were formed in situ. The effect of ethanol depended upon the phase of the culture.The authors are with the Department of Microbiology, Osmania University, Hyderabad-500007, India.     

Ethanol production byClostridium thermocellum SS8, a newly isolated thermophilic bacterium

Summary Clostridium thermocellum SS8, has a broad substrate spectrum. It produced 0.25–0.29 g. of ethanol per g. of cellulose consumed. Cellulose fermentation was repressed by both glucose and cellobiose. pH had an effect on ethanol productivity at high substrate concentration. Best results were obtained at 30 g/l with an E/S and E/A ratios of 0.29 and 2.4 respectively.     

Enhancement of ethanol production in the presence of metabolic inhibitors inClostridium thermocellum strain SS8

Summary Clostridium thermocellum strain SS8 produced 0.25g of ethanol and 0.24g of acetic acid per g cellulose consumed. Enhancement in ethanol production upto 0.39g/g substrate was observed in the presence of 0.15mM concentration of sodium azide and 7% polyethyleneglycol along with significant repression in acetic acid formation.     

Characterization of and Ethanol Hyper-production by Clostridium thermocellum I-1-B

An ethanol hyper-producing clostridial strain, I-1-B, was isolated from Shibi hot spring, Kagoshima prefecture and identified as Clostridium thermocellum based on morphological and physiological proper­ ties. The carbohydrates used as energy sources were glucose, fructose, cellobiose, cellulose and esculin. Fermentation products were ethanol, lactate, acetate, formate, carbon dioxide, and hydrogen. The optimum, maximum, and minimum temperature for growth are about 60, 70, and 47°C, respectively. Optimum pH for growth is about 7.5, and growth occurs at starting pH between 6.0 and 9.0. I-1-B strain has strong tolerance for ethanol and hyper ethanol-productivity. Ethanol concentrations causing 50%. decrease of growth yield are 27 and 16g/liter for I-1-B and ATCC27405 of C. thermocellum, respectively. The organism was cultured on a medium containing 80 g/liter cellulose at 60°C for 156 h. The culture was fed with a vitamin mixture containing vitamin B₁₂ and mineral salts solution at intervals. In this culture the organism produced 23.6 g/liter (512mM) ethanol, 8.5 g/liter (94mM) lactate, 2.9 g/liter (48mM) acetate, and 0.9 g/liter (20mM) formate. The molar ratio of ethanol to total acidic products was 3.2. The ethanol productivity of the strain I-1-B is superior to any of the wild and mutant strains of C. thermocellum so far reported.     

Improved ethanol tolerance and production in strains of Clostridium thermocellum

Two Clostridium thermocellum strains were improved for ethanol tolerance, to 5% (v/v), by gradual adaptation and mutation. The best mutant gave an ethanol yield of 0.37 g/g substrate, with a growth yield 1.5 times more than its parent. Accumulation of acids and reducing sugars by the mutant strain with 5% (v/v) ethanol was lower than that of the parent strain with 1.5% (v/v) ethanol.     

Optimization of Clostridium thermocellum growth on cellulose and pretreated wood substrates

Summary Two strains of Clostridium thermocellum ATCC 27405 and NRCC 2688 demonstrated similar product yields and cellulase activities when grown on solka floc. A sequential culture of C. thermocellum and Zymomonas anaerobia supplemented with cellobiase could produce 1.8 mg/ml of ethanol when grwon on 1% solka floc. Different media were evaluated for their ability to enhance the product and cellulase yields of C. thermocellum grown on cellulose substrates. Ethanol and reducing sugar values of 1.5 and 3.8 mg/ml respectively and an endoglucanase activity of 3 IU/ml were obtained after growth of Clostridium thermocellum in a modified medium containing 1% solka floc. Three different pretreated wood fractions were assessed as substrates for growth. A steam exploded wood fraction gave comparable values to those obtained after growth on solka floc. Sequential cultures of C. thermocellum and Zymomonas anaerobia grown on a 1% steam exploded wood fraction could produce 1.6 mg/ml ethanol after 3 days growth.     

Variation in amount of enzyme protein in natural populations

Among strains of Drosophila melanogaster each derived from a single fertilized female taken from natural populations, there is variation in both alcohol dehydrogenase (ADH) activity and the amount of ADH protein. The correlation between ADH activity and number of molecules over all strains examined is 0.87 or 0.96 in late third instar larvae depending on whether the substrate is 2-propanol or ethanol. With respect to the two common electrophoretic allozymic forms, F and S, segregating in these populations, the FF strains on the whole have higher ADH activities and numbers of ADH molecules than the SS strains. Over all strains examined, enzyme extracts from FF strains have a mean catalytic efficiency per enzyme molecule higher than that of enzyme extracts from SS strains when ethanol is the substrate, and much higher when 2-propanol is the substrate. One FF strain had an ADH activity/ADH protein ratio characteristic of SS strains.     

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.

     

Analysis of composition and structure of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> membranes from wild-type and ethanol-adapted strains

Clostridium thermocellum is a candidate organism for consolidated bioprocessing of lignocellulosic biomass into ethanol. However, commercial use is limited due to growth inhibition at modest ethanol concentrations. Recently, an ethanol-adapted strain of C. thermocellum was produced. Since ethanol adaptation in microorganisms has been linked to modification of membrane lipids, we tested the hypothesis that ethanol adaptation in C. thermocellum involves lipid modification by comparing the fatty acid composition and membrane anisotropy of wild-type and ethanol-adapted strains. Derivatization to fatty acid methyl esters provided quantitative lipid analysis. Compared to wild-type, the ethanol-adapted strain had a larger percentage of fatty acids with chain lengths >16:0 and showed a significant increase in the percentage of 16:0 plasmalogens. Structural identification of fatty acids was confirmed through mass spectral fragmentation patterns of picolinyl esters. Ethanol adaptation did not involve modification at sites of methyl branching or the unsaturation index. Comparison of steady-state fluorescence anisotropy experiments, in the absence and presence of ethanol, provided evidence for the effects of ethanol on membrane fluidity. In the presence of ethanol, both strains displayed increased fluidity by approximately 12%. These data support the model that ethanol adaptation was the result of fatty acid changes that increased membrane rigidity that counter-acted the fluidizing effect of ethanol.     

Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria

Characteristics of 13 newly isolated thermophilic, anaerobic, and cellulolytic strains were compared with previously described strains of Clostridium thermocellum: ATCC 27405 and JW20 (ATCC 31549). Colony morphology, antibiotic sensitivity, fermentation end-products, and cellulose degradation were documented. All 13 strains were sensitive to erythromycin (5 μg/ml) and chloramphenicol (25 μg/ml), and all strains but one were sensitive to kanamycin (20 μg/ml). Polymerase chain reaction (PCR) amplification using primers based on gene sequences from C. thermocellum ATCC 27405 was successful for all 13 strains in the case of the hydrogenase gene and 11 strains in the case of phosphotransacetylase/acetate kinase genes. Ten strains amplified a product of the expected size with primers developed to be specific for C. thermocellum 16SrRNA primers. Two of the 13 strains did not amplify any product with the PCR primers designed for the phosphotransacetylase/acetate kinase and 16SrRNA primers. A MboI-like GATC- recognizing restriction activity was present in all of the five strains examined. The results of this study have several positive implications with respect to future development of a transformation system for cellulolytic thermophiles. Journal of Industrial Microbiology & Biotechnology (2001) 27, 275–280. Received 12 September 2000/ Accepted in revised form 20 November 2000     

Ethanol Production by Thermophilic Bacteria: Fermentation of Cellulosic Substrates by Cocultures of Clostridium thermocellum and Clostridium thermohydrosulfuricum

The fermentation of various saccharides derived from cellulosic biomass to ethanol was examined in mono- and cocultures of Clostridium thermocellum strain LQRI and C. thermohydrosulfuricum strain 39E. C. thermohydrosulfuricum fermented glucose, cellobiose, and xylose, but not cellulose or xylan, and yielded ethanol/acetate ratios of >7.0. C. thermocellum fermented a variety of cellulosic substrates, glucose, and cellobiose, but not xylan or xylose, and yielded ethanol/acetate ratios of ~1.0. At nonlimiting cellulosic substrate concentrations (~1%), C. thermocellum cellulase hydrolysis products accumulated during monoculture fermentation of Solka Floc cellulose and included glucose, cellobiose, xylose, and xylobiose. A stable coculture that contained nearly equal numbers of C. thermocellum and C. thermohydrosulfuricum was established that fermented a variety of cellulosic substrates, and the ethanol yield observed was twofold higher than in C. thermocellum monoculture fermentations. The metabolic basis for the enhanced fermentation effectiveness of the coculture on Solka Floc cellulose included: the ability of C. thermocellum cellulase to hydrolyze α-cellulose and hemicellulose; the enhanced utilization of mono- and disaccharides by C. thermohydrosulfuricum; increased cellulose consumption; threefold increase in the ethanol production rate; and twofold decrease in the acetate production rate. The coculture actively fermented MN300 cellulose, Avicel, Solka Floc, SO₂-treated wood, and steam-exploded wood. The highest ethanol yield obtained was 1.8 mol of ethanol per mol of anhydroglucose unit in MN300 cellulose.     

Evaluation of ethanol productivity from cellulose by clostridium thermocellum

Clostridium thermocellum, a thermophillic anaerobe, directly converts cellulose to ethanol. To estimate its ethanol production from cellulose, we used a new method based on material balance by which the efficiencies of the enzymes that convert cellulose to ethanol were calculated. Using this method, the maximum efficiency of ethanol production of two strains of C. thermocellum was estimated to be 0.05, with 0.67 as the theoretical maximum.     

The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum

Clostridium thermocellum ferments cellulose, is a promising candidate for ethanol production from cellulosic biomass, and has been the focus of studies aimed at improving ethanol yield. Thermoanaerobacterium saccharolyticum ferments hemicellulose, but not cellulose, and has been engineered to produce ethanol at high yield and titer. Recent research has led to the identification of four genes in T. saccharolyticum involved in ethanol production: adhE, nfnA, nfnB and adhA. We introduced these genes into C. thermocellum and observed significant improvements to ethanol yield, titer, and productivity. The four genes alone, however, were insufficient to achieve in C. thermocellum the ethanol yields and titers observed in engineered T. saccharolyticum strains, even when combined with gene deletions targeting hydrogen production. This suggests that other parts of T. saccharolyticum metabolism may also be necessary to reproduce the high ethanol yield and titer phenotype in C. thermocellum.     

Proteomic profile changes in membranes of ethanol-tolerant <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, has potential for commercial exploitation in converting fibrous biomass to ethanol. However, ethanol concentrations above 1% (w/v) are inhibitory to growth and fermentation, and this limits industrial application of the organism. Recent work with ethanol-adapted strains suggested that protein changes occurred during ethanol adaptation, particularly in the membrane proteome. A two-stage Bicine-doubled sodium dodecyl sulfate-polyacrylamide gel electrophoresis protocol was designed to separate membrane proteins and circumvent problems associated with membrane protein analysis using traditional gel-based proteomics approaches. Wild-type and ethanol-adapted C. thermocellum membranes displayed similar spot diversity and approximately 60% of proteins identified from purified membrane fractions were observed to be differentially expressed in the two strains. A majority (73%) of differentially expressed proteins were down-regulated in the ethanol-adapted strain. Based on putative identifications, a significant proportion of these down-regulated proteins were involved with carbohydrate transport and metabolism. Approximately one-third of the up-regulated proteins in the ethanol-adapted species were associated with chemotaxis and signal transduction. Overall, the results suggested that membrane-associated proteins in the ethanol-adapted strain are either being synthesized in lower quantities or not properly incorporated into the cell membrane.     

Prospects for ethanol production from cellulose withClostridium thermocellum-Bacillus stearothermophilus co-cultures

Summary A lactate dehydrogenase deficient, high ethanol yielding mutant ofB. stearothermophilus was successfully co-cultured withC. thermocellum on cellulose. The co-culture produced 50% more ethanol and a 75% higher level of CMCase activity than theC. thermocellum mono-culture.     

Thermophilic methanogenesis from sugar beet pulp by a defined mixed bacterial culture

Summary Thermophilic degradation of sugar beet pulp was studied in batch cultures at 55°C by different associations of bacteria, includingClostridium thermocellum,Methanobacterium sp. andMethanosarcina MP.C. thermocellum produced acetate, succinate, methanol, ethanol, H₂ and CO₂. The coculture ofC. thermocellum andMethanobacterium sp. produced trace amounts of ethanol and succinate; acetate concentration was about three times higher than in theC. thermocellum monoculture. The association of this coculture withMethanosarcina MP produced 5.5 mmol CH₄/g dry weight sugar beet pulp.     

Isolation and characterization of a new cellulosome-producing Clostridium thermocellum strain

The anaerobic thermophilic bacterium, Clostridium thermocellum, is a potent cellulolytic microorganism that produces large extracellular multienzyme complexes called cellulosomes. To isolate C. thermocellum organisms that possess effective cellulose-degrading ability, new thermophilic cellulolytic strains were screened from more than 800 samples obtained mainly from agriculture residues in Thailand using microcrystalline cellulose as a carbon source. A new strain, C. thermocellum S14, having high cellulose-degrading ability was isolated from bagasse paper sludge. Cellulosomes prepared from S14 demonstrated faster degradation of microcrystalline cellulose, and 3.4- and 5.6-fold greater Avicelase activity than those from C. thermocellum ATCC27405 and JW20 (ATCC31449), respectively. Scanning electron microscopic analysis showed that S14 had unique cell surface features with few protuberances in contrast to the type strains. In addition, the cellulosome of S14 was resistant to inhibition by cellobiose that is a major end product of cellulose hydrolysis. Saccharification tests conducted using rice straw soaked with sodium hydroxide indicated the cellulosome of S14 released approximately 1.5-fold more total sugars compared to that of ATCC27405. This newly isolated S14 strain has the potential as an enzyme resource for effective lignocellulose degradation.     

A blue native-PAGE analysis of membrane protein complexes in Clostridium thermocellum

Background  

Clostridium thermocellum is a Gram-positive thermophilic anaerobic bacterium with the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. Identification and characterization of protein complexes in C. thermocellum are important toward understanding its metabolism and physiology.     

Nutritional interdependence betweenThermoanaerobacter thermohydrosulfuricus andClostridium thermocellum

Thermoanaerobacter thermohydrosulfuricus strain YM3 and Clostridium thermocellum strain YM4, obtained originally as a stable coculture, required yeast extract to grow separately. Cell-free broths of T. thermohydrosulfuricus strain YM3 and C. thermocellum strain YM4 monocultures replaced yeast extract in supporting the growth of strains YM4 and YM3, respectively. T. thermohydrosulfuricus strain YM3 produced vitamin B₆, B₁₂ analog(s), p-aminobenzoic acid and folic acid, which were required by C. thermocellum strain YM4. Likewise, strain YM4 produced niacin-active compound(s), thiamine, and methionine required by strain YM3. Received: 17 March 1995 / Accepted: 27 March 1995