Overflow metabolism and growth cessation in Clostridium thermocellum DSM1313 during high cellulose loading fermentations.

As a model thermophilic bacterium for the production of second-generation biofuels, the metabolism of Clostridium thermocellum has been widely studied. However, most studies have characterized C. thermocellum metabolism for growth at relatively low substrate concentrations. This outlook is not industrially relevant, however, as commercial viability requires substrate loadings of at least 100 g/L cellulosic materials. Recently, a wild-type C. thermocellum DSM1313 was cultured on high cellulose loading batch fermentations and reported to produce a wide range of fermentative products not seen at lower substrate concentrations, opening the door for a more in-depth analysis of how this organism will behave in industrially relevant conditions. In this work, we elucidated the interconnectedness of overflow metabolism and growth cessation in C. thermocellum during high cellulose loading batch fermentations (100 g/L). Metabolic flux and thermodynamic analyses suggested that hydrogen and formate accumulation perturbed the complex redox metabolism and limited conversion of pyruvate to acetyl-CoA conversion, likely leading to overflow metabolism and growth cessation in C. thermocellum. Pyruvate formate lyase (PFL) acts as an important redox valve and its flux is inhibited by formate accumulation. Finally, we demonstrated that manipulation of fermentation conditions to alleviate hydrogen accumulation could dramatically alter the fate of pyruvate, providing valuable insight into process design for enhanced C. thermocellum production of chemicals and biofuels. Biotechnol. Bioeng. 2017;114: 2592-2604. © 2017 Wiley Periodicals, Inc.

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.

Facultative Anaerobe Caldibacillus debilis GB1: Characterization and Use in a Designed Aerotolerant, Cellulose-Degrading Coculture with Clostridium thermocellum.

Development of a designed coculture that can achieve aerotolerant ethanogenic biofuel production from cellulose can reduce the costs of maintaining anaerobic conditions during industrial consolidated bioprocessing (CBP). To this end, a strain of Caldibacillus debilis isolated from an air-tolerant cellulolytic consortium which included a Clostridium thermocellum strain was characterized and compared with the C. debilis type strain. Characterization of isolate C. debilis GB1 and comparisons with the type strain of C. debilis revealed significant physiological differences, including (i) the absence of anaerobic metabolism in the type strain and (ii) different end product synthesis profiles under the experimental conditions used. The designed cocultures displayed unique responses to oxidative conditions, including an increase in lactate production. We show here that when the two species were cultured together, the noncellulolytic facultative anaerobe C. debilis GB1 provided respiratory protection for C. thermocellum, allowing the synergistic utilization of cellulose even under an aerobic atmosphere.

Comparative genotyping of Clostridium thermocellum strains isolated from biogas plants: genetic markers and characterization of cellulolytic potential.

Clostridium thermocellum is among the most prevalent of known anaerobic cellulolytic bacteria. In this study, genetic and phenotypic variations among C. thermocellum strains isolated from different biogas plants were determined and different genotyping methods were evaluated on these isolates. At least two C. thermocellum strains were isolated independently from each of nine different biogas plants via enrichment on cellulose. Various DNA-based genotyping methods such as ribotyping, RAPD (Random Amplified Polymorphic DNA) and VNTR (Variable Number of Tandem Repeats) were applied to these isolates. One novel approach - the amplification of unknown target sequences between copies of a previously discovered Random Inserted Mobile Element (RIME) - was also tested. The genotyping method with the highest discriminatory power was found to be the amplification of the sequences between the insertion elements, where isolates from each biogas plant yielded a different band pattern. Cellulolytic potentials, optimal growth conditions and substrate spectra of all isolates were characterized to help identify phenotypic variations. Irrespective of the genotyping method used, the isolates from each individual biogas plant always exhibited identical patterns. This is suggestive of a single C. thermocellum strain exhibiting dominance in each biogas plant. The genotypic groups reflect the results of the physiological characterization of the isolates like substrate diversity and cellulase activity. Conversely, strains isolated across a range of biogas plants differed in their genotyping results and physiological properties. Both strains isolated from one biogas plant had the best specific cellulose-degrading properties and might therefore achieve superior substrate utilization yields in biogas fermenters.

Isolation and characterization of two thermophilic cellulolytic strains of Clostridium thermocellum from a compost sample.

AIMS: To isolate, identify and characterize new thermophilic cellulolytic bacterial strains from a compost sample. METHODS AND RESULTS: Two thermophilic and cellulolytic bacterial strains were isolated via enrichment on cellulose (milled filter paper) and characterized. Both strains, CS7 and CS8, were rod-shaped, Gram-positive and spore-forming bacteria, sharing the same optimal temperature (60°C) and pH (7.0) for growth. Both were highly cellulolytic and produced ethanol and acetate as the major fermentation products, but lacked xylanase activity. They only grew on cellulose (both filter paper and crystalline cellulose) and cellobiose and produced yellow pigment, without growing on other substrates including glucose. Based on 16S rRNA gene sequence analysis, CS7 and CS8 are closely related (99% sequence identity) to Clostridium thermocellum ATCC 27405. However, they had significantly higher specific cellulase activities and ethanol/acetate ratios than Cl. thermocellum ATCC 27405. CONCLUSIONS: CS7 and CS8 are two new highly cellulolytic and ethanologenic Cl. thermocellum strains. SIGNIFICANCE AND IMPACT OF THE STUDY: First report of applying the cloning-RFLP-sequencing approach for purity confirmation of the isolates beside conventional methods. Strains CS7 and CS8 might be of potential application in research and development of cellulosic bioconversion.

[Cellulose degradation and ethanol production of different Clostridium strain].

Cellulose degradation and ethanol production of two types of cellulosic materials with different concentration were evaluated in batch system of mono-cultures of cellulolytic ethanol producing strains (Clostridium thermocellum strain LQRI and Clostridium thermocellum strain VPI), and co-cultures of LQRI or VPI in combination with one of the non-cellulolytic ethanol producing strains (Thermoanaerobacter ethanolicus strains X514 or Thermoanaerobacter ethanolicus 39E). Results demonstrated that higher cellulose degradation abilities about 1.2 times were detected in LQRI mono-culture than in VPI mono-culture, while no significant difference of ethanol yields was found between the two mono-cultures. Abilities of cellulose degradation and ethanol production decreased significantly with the increasing of substrate cellulose concentration (1%, 2%, 5%). In the co-culture system, cellulose degradation abilities of LQRI were also significantly higher than VPI, the former is 1.28-1.58 times of the latter. Cellulose degradation rate of LQRI + Thermoanaerobacter and VPI + Thermoanaerobacter decreased gradually with the increasing of substrate cellulose concentration, while the absolute value of cellulose degradation was also affected by the partner Thermoanaerobacter strain. Additionally, the ethanol yields in the co-cultures of LQRI + Thermoanaerobacter were significantly higher than that in the co-cultures of VPI + Thermoanaerobacter with same Thermoanaerobaeter partner, the former is 1.27-1.77 times of the latter. However, ethanol yields in the co-cultures have not significantly declined with the increasing of substrate cellulose concentration.

Community analysis of hydrogen-producing extreme thermophilic anaerobic microflora enriched from cow manure with five substrates.

The present study analyzed the community structures of anaerobic microflora producing hydrogen under extreme thermophilic conditions by two culture-independent methods: denaturing gradient gel electrophoresis (DGGE) and clone library analyses. Extreme thermophilic microflora (ETM) was enriched from cow manure by repeated batch cultures at 75 degrees C, using a substrate of xylose, glucose, lactose, cellobiose, or soluble starch, and produced hydrogen at yields of 0.56, 2.65, 2.17, 2.68, and 1.73 mol/mol-monosaccharide degraded, respectively. The results from the DGGE and clone library analyses were consistent and demonstrated that the community structures of ETM enriched with the four hexose-based substrates (glucose, lactose, cellobiose, and soluble starch) consisted of a single species, closely related to a hydrogen-producing extreme thermophile, Caldoanaerobacter subterraneus, with diversity at subspecies levels. The ETM enriched with xylose was more diverse than those enriched with the other substrates, and contained the bacterium related to C. subterraneus and an unclassified bacterium, distantly related to a xylan-degrading and hydrogen-producing extreme thermophile, Caloramator fervidus.

Effect of fermentation temperature on hydrogen production from cow waste slurry by using anaerobic microflora within the slurry.

We examined hydrogen production from a dairy cow waste slurry (13.4 g of volatile solids per liter) by batch cultures in a temperature range from 37 to 85 degrees C, using microflora naturally present within the slurry. Without the addition of seed bacteria, hydrogen was produced by simply incubating the slurry, using the microflora within the slurry. Interestingly, two peaks of fermentation temperatures for hydrogen production from the slurry were observed at 60 and 75 degrees C (392 and 248 ml H2 per liter of slurry, respectively). After the termination of the hydrogen evolution, the microflora cultured at 60 degrees C displayed hydrogen-consuming activity, but hydrogen-consuming activity of the microflora cultured at 75 degrees C was not detected, at least for 24 days. At both 60 and 75 degrees C, the main by-product was acetate, and the optimum pH of the slurry for hydrogen production was around neutral. Bacteria related to hydrogen-producing moderate and extreme thermophiles, Clostridium thermocellum and Caldanaerobacter subterraneus, were detected in the slurries cultured at 60 and 75 degrees C, respectively, by denaturing gradient gel electrophoresis analyses, using the V3 region of 16S rDNA.