Closing the carbon balance for fermentation by Clostridium thermocellum (ATCC 27405)

Our lab and most others have not been able to close a carbon balance for fermentation by the thermophilic, cellulolytic anaerobe, Clostridium thermocellum. We undertook a detailed accounting of product formation in C. thermocellum ATCC 27405. Elemental analysis revealed that for both cellulose (Avicel) and cellobiose, >92% of the substrate carbon utilized could be accounted for in the pellet, supernatant and off-gas when including sampling. However, 11.1% of the original substrate carbon was found in the liquid phase and not in the form of commonly-measured fermentation products - ethanol, acetate, lactate, and formate. Further detailed analysis revealed all the products to be <720 da and have not usually been associated with C. thermocellum fermentation, including malate, pyruvate, uracil, soluble glucans, and extracellular free amino acids. By accounting for these products, 92.9% and 93.2% of the final product carbon was identified during growth on cellobiose and Avicel, respectively.     

Identification of the cellulose-binding domain of the cellulosome subunit S1 from Clostridium thermocellum YS

The 3' region of a gene designated cipB, which shows strong homology with cipA that encodes the cellulosome SL subunit of Clostridium thermocellum ATCC 27405, was isolated from a gene library of C. thermocellum strain YS. The truncated S1 protein encoded by the cipB derivative bound tightly to cellulose. The cellulose-binding domain in this polypeptide consisted of a C-terminal proximal 167 residue sequence which showed complete identity with residues 337-503 of mature SL from C. thermocellum strain ATCC 27405. The cellulose-binding domain interacted with both crystalline and amorphous cellulose, but not with xylan.     

Molecular characterization and expression of pyruvate formate-lyase-activating enzyme in a ruminal bacterium,Streptococcus bovis

To clarify the significance of the activation of pyruvate formate-lyase (PFL) by PFL-activating enzyme (PFL-AE) in Streptococcus bovis, the molecular properties and gene expression of PFL-AE were investigated. S. bovis PFL-AE was deduced to consist of 261 amino acids with a molecular mass of 29.9 kDa and appeared to be a monomer protein. Similar to Escherichia coli PFL-AE, S. bovis PFL-AE required Fe(2+) for activity. The gene encoding PFL-AE (act) was found to be polycistronic, and the PFL gene (pfl) was not included. However, the act mRNA level changed in parallel with the pfl mRNA level, responding to growth conditions, and the change was contrary to the change in the lactate dehydrogenase (LDH) mRNA level. PFL-AE synthesis appeared to change in parallel with PFL synthesis. Introduction of a recombinant plasmid containing S. bovis pfl and the pfl promoter into S. bovis did not affect formate and lactate production, which suggests that the activity of the pfl promoter is low. When the pfl promoter was replaced by the S. bovis ldh promoter, PFL was overexpressed, which caused an increase in the formate-to-lactate ratio. However, when PFL-AE was overexpressed, the formate-to-lactate ratio did not change, suggesting that PFL-AE was present at a level that was high enough to activate PFL. When both PFL-AE and PFL were overexpressed, the formate-to-lactate ratio further increased. It is conceivable that LDH activity is much higher than PFL activity, which may explain why the formate-to-lactate ratio is usually low.     

Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence of Methanobacterium thermoautotrophicum.

The fermentation of cellulose and cellobiose by Clostridium thermocellum monocultures and C. thermocellum/Methanobacterium thermoautotrophicum cocultures was studied. All cultures were grown under anaerobic conditions in batch culture at 60 degrees C. When grown on cellulose, the coculture exhibited a shorter lag before initiation and growth and celluloysis than did the monoculture. Cellulase activity appeared earlier in the coculture than in the monoculture; however, after growth had ceased, cellulase activity was greater in the monoculture. Monocultures produced primarily ethanol, acetic acid, H2 and CO2. Cocultures produced more H2 and acetic acid and less ethanol than did the monoculture. In the coculture, conversion of H2 to methane was usually complete, and most of the methane produced was derived from CO2 reduction rather than from acetate conversion. Agents of fermentation stoppage were found to be low pH and high concentrations of ethanol in the monoculture and low pH in the coculture. Fermentation of cellobiose was more rapid than that of cellulose. In cellobiose medium, the methanogen caused only slight changes in the fermentation balance of the Clostridium, and free H2 was produced.     

Effect of Yeast Extract and Vitamin B(12) on Ethanol Production from Cellulose by Clostridium thermocellum I-1-B

Addition to media of yeast extract, a vitamin mixture containing vitamin B(12), biotin, pyridoxamine, and p-aminobenzoic acid, or vitamin B(12) alone enhanced formation of ethanol but decreased lactate production in the fermentation of cellulose by Clostridium thermocellum I-1-B. A similar effect was not observed with C. thermocellum ATCC 27405 and JW20.     

Isolation and characterization of a hydrogen- and ethanol-producing Clostridium sp. strain URNW

Identification, characterization, and end-product synthesis patterns were analyzed in a newly identified mesophilic, anaerobic Clostridium sp. strain URNW, capable of producing hydrogen (H?) and ethanol. Metabolic profiling was used to characterize putative end-product synthesis pathways of the Clostridium sp. strain URNW, which was found to grow on cellobiose; on hexose sugars, such as glucose, sucrose, and mannose; and on sugar alcohols, like mannitol and sorbitol. When grown in batch cultures on 2 g cellobiose·L?1, Clostridium sp. strain URNW showed a cell generation time of 1.5 h, and the major end-products were H2, formate, carbon dioxide (CO?), lactate, butyrate, acetate, pyruvate, and ethanol. The total volumetric H? production was 14.2 mmol·(L culture)?1 and the total production of ethanol was 0.4 mmol·(L culture)?1. The maximum yield of H? was 1.3 mol·(mol glucose equivalent)?1 at a carbon recovery of 94%. The specific production rates of H?, CO?, and ethanol were 0.45, 0.13, and 0.003 mol·h?1·(g dry cell mass)-1, respectively. BLAST analyses of 16S rDNA and chaperonin 60 (cpn60) sequences from Clostridium sp. strain URNW revealed a 98% nucleotide sequence identity with the 16S rDNA and cpn60 sequences from Clostridium intestinale ATCC 49213. Phylogenetic analyses placed Clostridium sp. strain URNW within the butyrate-synthesizing clostridia.     

Continuous hydrogen production during fermentation of alpha-cellulose by the thermophillic bacterium Clostridium thermocellum

Continuous hydrogen (H2) production during fermentation of alpha-cellulose was established using the thermophillic, anaerobic bacterium Clostridium thermocellum ATCC 27405. The objectives of this work were to characterize growth of C. thermocellum, quantify H2 production and determine soluble end-product synthesis patterns during fermentation of a cellulosic substrate under continuous culture conditions. A 5 L working volume fermentor was established and growth experiments were maintained for over 3,000 h. Substrate concentrations were varied from 1 to 4 g/L and the feed was introduced with continuous nitrogen gas sparging to prevent clogging of the feed-line. The pH and temperature of the reactor were maintained at 7.0 and 600 degrees C, respectively, throughout the study. At concentrations above 4 g/L, the delivery of alpha-cellulose was impaired due to feed-line clogging and it became difficult to maintain a homogenous suspension. The highest total gas (H2 plus CO2) production rate, 56.6 mL L(-1) h(-1), was observed at a dilution rate of 0.042 h(-1) and substrate concentration of 4 g/L. Under these conditions, the H2 production rate was 5.06 mmol h(-1). Acetate and ethanol were the major soluble end-products, while lactate and formate were greatly reduced compared to production in batch cultures. Concentrations of all metabolites increased with increasing substrate concentration, with the exception of lactate. Despite a number of short-term electrical and mechanical failures during the testing period, the system recovered quickly, exhibiting substantial robustness. A carbon balance was completed to ensure that all end-products were accounted for, with final results indicating near 100% carbon recovery. This study shows that long-term, stable H2 production can be achieved during direct fermentation of an insoluble cellulosic substrate under continuous culture conditions.     

Molecular Characterization and Expression of Pyruvate Formate-Lyase-Activating Enzyme in a Ruminal Bacterium, Streptococcus bovis

To clarify the significance of the activation of pyruvate formate-lyase (PFL) by PFL-activating enzyme (PFL-AE) in Streptococcus bovis, the molecular properties and gene expression of PFL-AE were investigated. S. bovis PFL-AE was deduced to consist of 261 amino acids with a molecular mass of 29.9 kDa and appeared to be a monomer protein. Similar to Escherichia coli PFL-AE, S. bovis PFL-AE required Fe²⁺ for activity. The gene encoding PFL-AE (act) was found to be polycistronic, and the PFL gene (pfl) was not included. However, the act mRNA level changed in parallel with the pfl mRNA level, responding to growth conditions, and the change was contrary to the change in the lactate dehydrogenase (LDH) mRNA level. PFL-AE synthesis appeared to change in parallel with PFL synthesis. Introduction of a recombinant plasmid containing S. bovis pfl and the pfl promoter into S. bovis did not affect formate and lactate production, which suggests that the activity of the pfl promoter is low. When the pfl promoter was replaced by the S. bovis ldh promoter, PFL was overexpressed, which caused an increase in the formate-to-lactate ratio. However, when PFL-AE was overexpressed, the formate-to-lactate ratio did not change, suggesting that PFL-AE was present at a level that was high enough to activate PFL. When both PFL-AE and PFL were overexpressed, the formate-to-lactate ratio further increased. It is conceivable that LDH activity is much higher than PFL activity, which may explain why the formate-to-lactate ratio is usually low.     

Complete genome sequence of the cellulolytic thermophile Clostridium thermocellum DSM1313

Clostridium thermocellum DSM1313 is a thermophilic, anaerobic bacterium with some of the highest rates of cellulose hydrolysis reported. The complete genome sequence reveals a suite of carbohydrate-active enzymes and demonstrates a level of diversity at the species level distinguishing it from the type strain ATCC 27405.     

Utilization of cellobiose and D-glucose by Clostridium thermocellum ATCC-27405

Cultures of Clostridium thermocellum ATCC-27405, maintained on cellulose and not adapted to grow on glucose utilize cellobiose preferentially over D-glucose, and are only able to initiate growth on D-glucose when the cellobiose has been exhausted from the growth medium. However, D-glucose is the carbon source preferentially utilized when cultures of this microorganism, previously adapted for growth on glucose, are transferred to a medium with equivalent concentrations of both sugars. One reason for the preferential utilization of glucose over that of cellobiose might be the competitive inhibition of cellobiose phosphorylase by intracellular glucose accumulation. When in the glucose-adapted cultures the pressure to grow on glucose as the sole carbon source is again released, both sugars can be simultaneously utilized.     

Clostridium alkalicellum sp. nov., an obligately alkaliphilic cellulolytic bacterium from a soda lake in the Baikal region

The first anaerobic alkaliphilic cellulolytic microorganism has been isolated from the Verkhnee Beloe soda lake (Buryatiya, Russia) with pH 10.2 and a salt content of up to 24 g/l. Five strains were characterized. Strain Z-7026 was chosen as the type strain. The cells of the isolate are gram-positive spore-forming rods. A mucous external capsule is produced. The microorganism is obligately alkaliphilic, growing in a pH range of 8.0-10.2, with an optimum at pH 9.0. Sodium ions and, in carbonate-buffered media, sodium chloride are obligately required. The microorganism is slightly halophilic; it grows at 0.017-0.4 M Na+ with an optimum at 0.15-0.3 M Na+. The metabolism is fermentative and strictly anaerobic. Cellulose, cellobiose, and xylan can be used as growth substrates. Plant and algal debris can be fermented. Lactate, ethanol, acetate, hydrogen, and traces of formate are produced during cellulose or cellobiose fermentation. Yeast extract or vitamins are required for anabolic purposes. The microorganism fixes dinitrogen and is nitrogenase-positive. It is tolerant to up to 48 mM Na2S. Growth is not inhibited by kanamycin or neomycin. Chloramphenicol, streptomycin, penicillin, ampicillin, ampiox, bacillin, novobiocin, and bacitracin suppress growth. The DNA G+C content is 29.9 mol %. According to the nucleotide sequence of its 16S rRNA gene, strain Z-7026 is phylogenetically close to the neutrophilic cellulolytic bacteria Clostridium thermocellum (95.5%), C. aldrichii (94.9%), and Acetivibrio cellulolyticus (94.8%). It is proposed as a new species: Clostridium alkalicellum sp. nov.     

Pyruvate degradation via pyruvate formate-lyase (EC 2.3.1.54) and the enzymes of formate fermentation in the green alga Chlorogonium elongatum

Formate was formed in extracts of Chlorogonium elongatum via direct cleavage of pyruvate by a pyruvate formate-lyase (PFL, EC 2.3.1.54). The conversion of PFL to the catalytically active form required S-adenosylmethionine, ferric (2+), photoreduced deazariboflavin as reductant, pyruvate as allosteric effector and strict anaerobic conditions. At the optimum pH (pH 8.0), PFL catalyzed formate formation, pyruvate synthesis and the isotope exchange from [¹⁴C]formate into pyruvate with rates of 30.0, 1.5 and 1.2 nmol min^-1 mg^-1 protein, respectively. Treatment of the active enzyme with O₂ irreversibly inactivated PFL activity (half-time 2 min). In addition to PFL, the activities of phosphotransacetylase (EC 2.3.1.8), acetate kinase (EC 2.7.2.1), aldehyde dehydrogenase (CoA acetylating, EC 1.2.1.10) and alcohol dehydrogenase (EC 1.1.1.1) were also detected in extracts of C. elongatum. The occurrence of these enzymes indicates pyruvate degradation via a formate-fermentation pathway during anaerobiosis of algal cells in the dark.Abbreviations DTT dithiothreitol - Hepes 4-(2-hydroxyethyl)-1-piperazine+ethane sulfonic acid - PFL pyruvate formate-lyase     

Growth phase-dependant enzyme profile of pyruvate catabolism and end-product formation in Clostridium thermocellum ATCC 27405

End-product synthesis and enzyme activities involved in pyruvate catabolism, H₂ synthesis, and ethanol production in mid-log (OD₆₀₀  0.25), early stationary (OD₆₀₀  0.5), and stationary phase (OD₆₀₀  0.7) cell extracts were determined in Clostridium thermocellum ATCC 27405 grown in batch cultures on cellobiose. Carbon dioxide, hydrogen, ethanol, acetate and formate were major end-products and their production paralleled growth and cellobiose consumption. Lactate dehydrogenase, pyruvate:formate lyase, pyruvate:ferredoxin oxidoreductase, methyl viologen-dependant hydrogenase, ferredoxin-dependant hydrogenase, NADH-dependant hydrogenase, NADPH-dependant hydrogenase, NADH-dependant acetaldehyde dehydrogenase, NADH-dependant alcohol dehydogenase, and NADPH-dependant alcohol dehydrogenase activities were detected in all extracts, while pyruate dehydrogenase and formate dehydrogenase activities were not detected. All hydrogenase activities decreased (2–12-fold) as growth progressed from early exponential to stationary phase. Alcohol dehydrogenase activities fluctuated only marginally (<45%), while lactate dehydrogenase, pyruvate:formate lyase, and pyruvate:ferredoxin oxidoreductase remained constant in all cell extracts. We have proposed a pathway involved in pyruvate catabolism and end-product formation based on enzyme activity profiles in conjunction with bioinformatics analysis.     

Regulation of cellulase synthesis in batch and continuous cultures of Clostridium thermocellum

Regulation of cell-specific cellulase synthesis (expressed in milligrams of cellulase per gram [dry weight] of cells) by Clostridium thermocellum was investigated using an enzyme-linked immunosorbent assay protocol based on antibody raised against a peptide sequence from the scaffoldin protein of the cellulosome (Zhang and Lynd, Anal. Chem. 75:219-227, 2003). The cellulase synthesis in Avicel-grown batch cultures was ninefold greater than that in cellobiose-grown batch cultures. In substrate-limited continuous cultures, however, the cellulase synthesis with Avicel-grown cultures was 1.3- to 2.4-fold greater than that in cellobiose-grown cultures, depending on the dilution rate. The differences between the cellulase yields observed during carbon-limited growth on cellulose and the cellulase yields observed during carbon-limited growth on cellobiose at the same dilution rate suggest that hydrolysis products other than cellobiose affect cellulase synthesis during growth on cellulose and/or that the presence of insoluble cellulose triggers an increase in cellulase synthesis. Continuous cellobiose-grown cultures maintained either at high dilution rates or with a high feed substrate concentration exhibited decreased cellulase synthesis; there was a large (sevenfold) decrease between 0 and 0.2 g of cellobiose per liter, and there was a much more gradual further decrease for cellobiose concentrations >0.2 g/liter. Several factors suggest that cellulase synthesis in C. thermocellum is regulated by catabolite repression. These factors include: (i) substantially higher cellulase yields observed during batch growth on Avicel than during batch growth on cellobiose, (ii) a strong negative correlation between the cellobiose concentration and the cellulase yield in continuous cultures with varied dilution rates at a constant feed substrate concentration and also with varied feed substrate concentrations at a constant dilution rate, and (iii) the presence of sequences corresponding to key elements of catabolite repression systems in the C. thermocellum genome.     

Kinetics and relative importance of phosphorolytic and hydrolytic cleavage of cellodextrins and cellobiose in cell extracts of Clostridium thermocellum

Rates of phosphorolytic cleavage of beta-glucan substrates were determined for cell extracts from Clostridium thermocellum ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)- and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temperatures, an assay protocol was developed featuring discrete sampling at 60 degrees C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35 degrees C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by > or = 20-fold for both cellobiose and cellopentaose over a 10-fold range of beta-glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM). Rates of phosphorolytic cleavage of beta-glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures. Comparisons of V(max) values indicated that cellobiose- and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control. The apparent K(m) for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel- and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM).