Thermophilic degradation of cellulose by a triculture of Clostridium thermocellum, Methanobacterium sp. and Methanosarcina MP

Abstract The fermentation of cellulose at 55°C by different associations of the 3 bacteria Clostridium thermocellum, Methanobacterium sp. and Methanosarcina MP, was studied. C. thermocellum alone produced acetate, lactate, ethanol, H₂ and CO₂. The co-culture C. thermocellum-Methanobacterium sp. produced more acetate and less ethanol than the monoculture of Clostridium .
Methanosarcina MP used acetate only in the triculture including Methanobacterium sp. When methanol was added (5 mM) to the triculture, Methanosarcina MP had a shorter lag phase on acetate and degraded much more acetate. maximum methane production was 8.5 mmol CH₄/g cellulose degraded.     

Influence of the presence of Zymomonas anaerobia on the conversion of cellobiose, glucose, and xylose to ethanol by Clostridium saccharolyticum

To convert sugar mixtures containing cellobiose, glucose, and xylose to ethanol in a single step, the possibility of using a coculture consisting of Clostridium saccharolyticum and Zymomonas anaerobia was studied. In monoculture, C. saccharolyticum utilized all three sugars; however, it preferentially utilized glucose and produced acetic acid in addition to ethanol. The formation of acetic acid from the metabolism of glucose inhibited the growth of C. saccharolyticum and, consequently, the utilization of cellobiose and xylose. In monoculture, Z. anaerobia utilized glucose at a rate of 50 g/L day, but it did not ferment cellobiose or xylose. In coculture, Z. anaerobia converted most of the glucose to ethanol during the lag phase of growth of C. saccharolyticum, which then converted cellobiose and xylose to ethanol. The use of this coculture increased both the rate and the efficiency of the conversion of these three sugars to ethanol, and produced relatively small amounts of acetic acid.     

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.     

嗜热厌氧纤维素降解细菌的分离、鉴定及其系统发育分析

利用纤维素降解细菌和纤维素粘附的方法分别从新鲜牛粪、高温堆肥和本实验室保存的纤维素降解富集物中分离得到4株嗜热厌氧纤维素降解细菌。分离菌株为革兰氏染色阴性,直的或稍弯曲杆菌,菌体大小为0.4μm～0.6μm×3μm～15μm,严格厌氧,不还原硫酸盐,形成芽孢。多数芽孢着生于菌体顶端。分离菌株能利用纤维素滤纸、纤维素粉Whatman CFII、微晶纤维素、纤维素粉MN300和未经处理的玉米秆芯、甘蔗渣、水稻秸杆。分离菌株在pH6.2～8.9、温度45℃～65℃范围内利用纤维素,最适pH为7.0～7.5,最适温度为55℃～60℃,发酵纤维素产生乙醇、乙酸、H2和CO2。分离菌株还可利用纤维二糖、葡萄糖、果糖、麦芽糖、山梨醇作为碳源。部分长度的16S rDNA序列分析表明,分离菌株EVAI与Clostridium thermocellum具有99.8%相似性。     

Conversion of Cellulose to Methane and Carbon Dioxide by Triculture of Acetivibrio cellulolyticus, Desulfovibrio sp., and Methanosarcina barkeri

The fermentation of cellulose by monocultures of Acetivibrio cellulolyticus and cocultures of A. cellulolyticus-Methanosarcina barkeri, A. cellulolyticus-Desulfovibrio sp., and A. cellulolyticus-M. barkeri-Desulfovibrio sp. was studied. The monoculture produced ethanol, acetate, H₂, and CO₂. More acetate and less ethanol was formed by the cocultures than by the monoculture. Acetate was utilized by M. barkeri in coculture with A. cellulolyticus after a lag period, whereas ethanol was metabolized by the sulfate reducer only under conditions of low H₂ partial pressure, i.e., when cocultured with A. celluloyticus-M. barkeri or when grown together with the methanogen. Only the three-component culture carried out the rapid conversion of cellulose to CO₂ and methane. Furthermore, this culture hydrolyzed the most cellulose—85% of that initially present. This amount was increased to 90% by increasing the population of M. barkeri in the triculture. Methane production was also increased, and a quicker fermentation rate was achieved.     

Ethanol production from cellobiose, amorphous cellulose, and crystalline cellulose by recombinant Klebsiella oxytoca containing chromosomally integrated Zymomonas mobilis genes for ethanol production and plasmids expressing thermostable cellulase genes from Clostridium thermocellum.

The Zymomonas mobilis genes for ethanol production have been integrated into the chromosome of Klebsiella oxytoca M5A1. The best of these constructs, strain P2, produced ethanol efficiently from cellobiose in addition to monomeric sugars. Utilization of cellobiose and cellotriose by this strain eliminated the requirement for external beta-glucosidase and reduced the amount of commercial cellulase needed to ferment Solka Floc SW40 (primarily crystalline cellulose). The addition of plasmids encoding endoglucanases from Clostridium thermocellum resulted in the intracellular accumulation of thermostable enzymes as coproducts with ethanol during fermentation. The best of these, strain P2(pCT603T) containing celD, was used to hydrolyze amorphous cellulose to cellobiose and produce ethanol in a two-stage process. Strain P2(pCT603T) was also tested in combination with commercial cellulases. Pretreatment of Solka Floc SW40 at 60 degrees C with endoglucanase D substantially reduced the amount of commercial cellulase required to ferment Solka Floc. The stimulatory effect of the endoglucanase D pretreatment may result from the hydrolysis of amorphous regions, exposing additional sites for attack by fungal cellulases. Since endoglucanase D functions as part of a complex in C. thermocellum, it is possible that this enzyme may complex with fungal enzymes or bind cellulose to produce a more open structure for hydrolysis.     

Ethanol production from cellobiose, amorphous cellulose, and crystalline cellulose by recombinant Klebsiella oxytoca containing chromosomally integrated Zymomonas mobilis genes for ethanol production and plasmids expressing thermostable cellulase genes from Clostridium thermocellum.

The Zymomonas mobilis genes for ethanol production have been integrated into the chromosome of Klebsiella oxytoca M5A1. The best of these constructs, strain P2, produced ethanol efficiently from cellobiose in addition to monomeric sugars. Utilization of cellobiose and cellotriose by this strain eliminated the requirement for external beta-glucosidase and reduced the amount of commercial cellulase needed to ferment Solka Floc SW40 (primarily crystalline cellulose). The addition of plasmids encoding endoglucanases from Clostridium thermocellum resulted in the intracellular accumulation of thermostable enzymes as coproducts with ethanol during fermentation. The best of these, strain P2(pCT603T) containing celD, was used to hydrolyze amorphous cellulose to cellobiose and produce ethanol in a two-stage process. Strain P2(pCT603T) was also tested in combination with commercial cellulases. Pretreatment of Solka Floc SW40 at 60 degrees C with endoglucanase D substantially reduced the amount of commercial cellulase required to ferment Solka Floc. The stimulatory effect of the endoglucanase D pretreatment may result from the hydrolysis of amorphous regions, exposing additional sites for attack by fungal cellulases. Since endoglucanase D functions as part of a complex in C. thermocellum, it is possible that this enzyme may complex with fungal enzymes or bind cellulose to produce a more open structure for hydrolysis.     

Acetic acid production from lactose by an anaerobic thermophilic coculture immobilized in a fibrous-bed bioreactor

An anaerobic thermophilic coculture consisting of a heterofermentative bacterium (Clostridium thermolacticum) and a homoacetogen (Moorella thermoautotrophica) was developed for acetic acid production from lactose and milk permeate. The fermentation kinetics with free cells in conventional fermentors and immobilized cells in a recycle batch fibrous-bed bioreactor were studied. The optimal conditions for the cocultured fermentation were found to be 58 degrees C and pH 6.4. In the free-cell fermentation, C. thermolacticum converted lactose to acetate, ethanol, lactate, H(2) and CO(2), and the homoacetogen then converted lactate, H(2), and CO(2) to acetate. The overall acetate yield from lactose ranged from 0.46 to 0.65 g/g lactose fermented, depending on the fermentation conditions. In contrast, no ethanol was produced in the immobilized-cell fermentation, and the overall acetate yield from lactose increased to 0.8-0.96 g/g lactose fermented. The fibrous-bed bioreactor also gave a higher final acetate concentration (up to 25. 5 g/L) and reactor productivity (0.18-0.54 g/L/h) as compared to those from the free-cell fermentation (final acetate concentration, 15 g/L; productivity, 0.06-0.08 g/L/h). The superior performance of the fibrous-bed bioreactor was attributed to the high cell density (20 g/L) immobilized in the fibrous-bed and adaptation of C. thermolacticum cells to tolerate a higher acetate concentration. The effects of yeast extract and trypticase as nutrient supplements on the fermentation were also studied. For the free-cell fermentation, nutrient supplementation was necessary for the bacteria to grow in milk permeate. For the immobilized-cell fermentation, plain milk permeate gave a high acetate yield (0.96 g/g), although the reactor productivity was lower than those with nutrient supplementation. Balanced growth and fermentation activities between the two bacteria in the coculture are important to the quantitative conversion of lactose to acetic acid. Lactate and hydrogen produced by C. thermolacticum must be timely converted to acetic acid by the homoacetogen to avoid inhibition by these metabolites.     

Clostridium thermocellum ATCC27405 transcriptomic, metabolomic and proteomic profiles after ethanol stress

ABSTRACT: BACKGROUND: Clostridium thermocellum is a candidate consolidated bioprocessing biocatalyst, which is a microorganism that expresses enzymes for both cellulose hydrolysis and its fermentation to produce fuels such as lignocellulosic ethanol. However, C. thermocellum is relatively sensitive to ethanol compared to ethanologenic microorganisms such as yeast and Zymomonas mobilis that are used in industrial fermentations but do not possess native enzymes for industrial cellulose hydrolysis. RESULTS: In this study, C. thermocellum was grown to mid-exponential phase and then treated with ethanol to a final concentration of 3.9 g/L to investigate its physiological and regulatory responses to ethanol stress. Samples were taken pre-shock and 2, 12, 30, 60, 120, and 240 min post-shock, and from untreated control fermentations for systems biology analyses. Cell growth was arrested by ethanol supplementation with intracellular accumulation of carbon sources such as cellobiose, and sugar phosphates, including fructose-6-phosphate and glucose-6-phosphate. The largest response of C. thermocellum to ethanol shock treatment was in genes and proteins related to nitrogen uptake and metabolism, which is likely important for redirecting the cells physiology to overcome inhibition and allow growth to resume. CONCLUSION: This study suggests possible avenues for metabolic engineering and provides comprehensive, integrated systems biology datasets that will be useful for future metabolic modeling and strain development endeavors.     

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

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

Cellobiose: A true inducer of cellulosome in different strains of Clostridium thermocellum

Abstract Growth and production of cellulosome by three strains (YS, LQRI and NCIB 10682) of Clostridium thermocellum were compared using Avicel (microcrystalline cellulose) and cellobiose as carbon sources. All three strains grew faster on cellobiose than on Avicel and produced 0.71–0.74 IU of endoglucanase/ml compared to 0.88–1.18 IU/ml on Avicel. Also, the cellulase produced by these strains in the presence of 0.2–1% cellobiose and Avicel, when compared on the basis of equal units of endoglucanase (0.5 IU), degraded cotton almost completely. SDS-PAGE further confirmed the production of cellulosome by all three strains when grown on cellobiose and Avicel. Thus, the cellobiose, like Avicel, acts as a true inducer of cellulosome in C. thermocellum .     

Bioconversion of Cellulose to Acetate with Pure Cultures of Ruminococcus albus and a Hydrogen-Using Acetogen

Bioconversion of cellulose to acetate was accomplished with cocultures of two organisms. One was the cellulolytic species Ruminococcus albus. It ferments crystalline cellulose (Avicel) to acetate, ethanol, CO(inf2), and H(inf2). The other organism (HA) obtains energy for growth by using H(inf2) to reduce CO(inf2) to acetate. HA is a gram-negative coccobacillus that was isolated from horse feces. Coculture of R. albus with HA in batch or continuous culture alters the fermentation products formed from crystalline cellulose by the ruminococcus via interspecies H(inf2) transfer. The major product of the fermentation by R. albus and HA coculture is acetate. High concentrations of acetate (333 mM) were obtained when batch cocultures grown on 5% cellulose were neutralized with Ca(OH)(inf2). Continuous cocultures grown at retention times of 2 and 3.1 days produced 109 and 102 mM acetate, respectively, when fed 1% cellulose with utilization of 84% of the substrate.     

High ethanol titers from cellulose by using metabolically engineered thermophilic, anaerobic microbes

This work describes novel genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employed counter-selections developed from the native C. thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene and was used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta). The Δldh Δpta mutant was evolved for 2,000 h, resulting in a stable strain with 40:1 ethanol selectivity and a 4.2-fold increase in ethanol yield over the wild-type strain. Ethanol production from cellulose was investigated with an engineered coculture of organic acid-deficient engineered strains of both C. thermocellum and T. saccharolyticum. Fermentation of 92 g/liter Avicel by this coculture resulted in 38 g/liter ethanol, with acetic and lactic acids below detection limits, in 146 h. These results demonstrate that ethanol production by thermophilic, cellulolytic microbes is amenable to substantial improvement by metabolic engineering.     

Simultaneous and Enhanced Production of Thermostable Amylases and Ethanol from Starch by Cocultures of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum

Clostridium thermohydrosulfuricum and Clostridium thermosulfurogenes produced ethanol and amylases with different components as primary metabolites of starch fermentation. Starch fermentation parameters were compared in mono- and cocultures of these two thermoanaerobes to show that the fermentation was dramatically improved as a consequence of coordinate action of amylolytic enzymes and synergistic metabolic interactions between the two species. Under given monoculture fermentation conditions, neither species completely degraded starch during the time course of the study, whereas in coculture, starch was completely degraded. In monoculture starch fermentation, C. thermohydrosulfuricum produced lower levels of pullulanase and glucoamylase, whereas C. thermosulfurogenes produced lower levels of β-amylase and glucoamylase. In coculture fermentation, improvement of starch metabolism by each species was noted in terms of increased amounts and rates of increased starch consumption, amylase production, and ethanol formation. The single-step coculture fermentation completely degraded 2.5% starch in 30 h at 60°C and produced 9 U of β-amylase per ml, 1.3 U of pullulanase per ml, 0.3 U of glucoamylase per ml, and >120 mM ethanol with a yield of 1.7 mol/mol of glucose in starch. The potential industrial applications of the coculture fermentation and the physiological basis for the interspecies metabolic interactions are discussed.     

Fermentation of cellulose to acetic acid by Clostridium lentocellum SG6: induction of sporulation and effect of buffering agent on acetic acid production

AIMS: To determine the growth, correlation between sporulation and acetic acid production and effect of buffering agent at high substrate cellulose concentrations of the strain Clostiridium lentocellum SG6. METHODS AND RESULTS: The strain SG6 was grown in cellulose mineral salt medium containing cellulose (Whatman No. 1 filter paper, Whatmore International Ltd., Maidstone, UK) or cellobiose. The strain fermented cellulose even after several transfers on cellobiose medium. The formation of endospores on third day onwards indicated the lowering of pH in the medium because of the formation of acetic acid. Maintaining the pH 7.2 at higher substrate concentrations resulted in increase of biomass, cellulose fermentation, acetic acid production, etc. CONCLUSIONS: The strain SG6, with its high fermentation yields and sporulating character can become a potential strain for acetic acid production and also as a probiotic strain in animal nutrition. SIGNIFICANCE AND IMPACT OF THE STUDY: The direct conversion of cellulosic biomass to acetic acid can eliminate expensive three-step saccharification, fermentation processes. The strain SG6 can ferment cellulose at high substrate concentrations.     

Cellulose Fermentation by a Rumen Anaerobic Fungus in Both the Absence and the Presence of Rumen Methanogens

The fermentation of cellulose by an ovine rumen anaerobic fungus in the absence and presence of rumen methanogens is described. In the monoculture, moles of product as a percentage of the moles of hexose fermented were: acetate, 72.7; carbon dioxide, 37.6; formate, 83.1; ethanol, 37.4; lactate, 67.0; and hydrogen, 35.3. In the coculture, acetate was the major product (134.7%), and carbon dioxide increased (88.7%). Lactate and ethanol production decreased to 2.9 and 19%, respectively, little formate was detected (1%), and hydrogen did not accumulate. Substantial amounts of methane were produced in the coculture (58.7%). Studies with [2-¹⁴C]acetate indicated that acetate was not a precursor of methane. The demonstration of cellulose fermentation by a fungus extends the range of known rumen organisms capable of participating in cellulose digestion and provides further support for a role of anaerobic fungi in rumen fiber digestion. The effect of the methanogens on the pattern of fermentation is interpreted as a shift in flow of electrons away from electron sink products to methane via hydrogen. The study provides a new example of intermicrobial hydrogen transfer and the first demonstration of hydrogen formation by a fungus.     

Clostridium lentocellum SG6--a potential organism for fermentation of cellulose to acetic acid

A cellulolytic, acetic acid producing anaerobic bacterial isolate, Gram negative, rod-shaped, motile, terminal oval shaped endospore forming bacterium identified as Clostridium lentocellum SG6 based on physiological and biochemical characteristics. It produced acetic acid as a major end product from cellulose fermentation at 37°C and pH 7.2. Acetic acid production was 0.67 g/g cellulose substrate utilized in cellulose mineral salt (CMS) medium. Yeast extract (0.4%) was the best nitrogen source among the various nitrogenous nutrients tested in production medium containing 0.8% cellulose as substrate. No additional vitamins or trace elemental solution were required for acetic acid fermentation. This is the highest acetic acid fermentation yield in monoculture fermentation for direct conversion of cellulose to acetic acid.     

Ethanol production from cellulose by a coculture ofZymomonas mobilis and a clostridium

Zymomonas mobilis and a mesophilic cellulolytic clostridium (strain C7) were grown in coculture in a medium containing cellulose as fermentable substrate. The coculture was stable through at least ten serial transfers and produced markedly higher amounts of ethanol than monocultures of the cellulolytic clostridium. Glucose and cellobiose, derived from the breakdown of cellulose, accumulated in strain C7 monocultures, but not in cocultures. The molar ratio of ethanol to acetate was higher in cocultures than in monocultures of strain C7. The cellulolytic clostridium was relatively ethanol-tolerant, inasmuch as it grew and fermented cellulose in media containing up to 7 g of ethanol/100 ml. Cellulase (Avicelase) activity of strain C7 was inhibited by cellobiose, but not by glucose.