A mutation in the AdhE alcohol dehydrogenase of Clostridium thermocellum increases tolerance to several primary alcohols, including isobutanol, n-butanol and ethanol.

Clostridium thermocellum is a good candidate organism for producing cellulosic biofuels due to its native ability to ferment cellulose, however its maximum biofuel titer is limited by tolerance. Wild type C. thermocellum is inhibited by 5 g/L n-butanol. Using growth adaptation in a chemostat, we increased n-butanol tolerance to 15 g/L. We discovered that several tolerant strains had acquired a D494G mutation in the adhE gene. Re-introducing this mutation recapitulated the n-butanol tolerance phenotype. In addition, it increased tolerance to several other primary alcohols including isobutanol and ethanol. To confirm that adhE is the cause of inhibition by primary alcohols, we showed that deleting adhE also increases tolerance to several primary alcohols.

Growth and expression of relevant metabolic genes of Clostridium thermocellum cultured on lignocellulosic residues.

The plant cell wall is a source of fermentable sugars in second-generation bioethanol production. However, cellulosic biomass hydrolysis remains an obstacle to bioethanol production in an efficient and low-cost process. Clostridium thermocellum has been studied as a model organism able to produce enzymatic blends that efficiently degrade lignocellulosic biomass, and also as a fermentative microorganism in a consolidated process for the conversion of lignocellulose to bioethanol. In this study, a C. thermocellum strain (designated B8) isolated from goat rumen was characterized for its ability to grow on sugarcane straw and cotton waste, and to produce cellulosomes. We also evaluated C. thermocellum gene expression control in the presence of complex lignocellulosic biomasses. This isolate is capable of growing in the presence of microcrystalline cellulose, sugarcane straw and cotton waste as carbon sources, producing free enzymes and residual substrate-bound proteins (RSBP). The highest growth rate and cellulase/xylanase production were detected at pH 7.0 and 60 °C, after 48 h. Moreover, this strain showed different expression levels of transcripts encoding cellulosomal proteins and proteins with a role in fermentation and catabolic repression.

Differences in biomass degradation between newly isolated environmental strains of Clostridium thermocellum and heterogeneity in the size of the cellulosomal scaffoldin.

Cellulolytic bacterial strains with high activity were isolated from cellulose degrading enrichment cultures derived from thermophilic biogas plants and environmental samples. The 16S rRNA gene sequences of the strains revealed >99.8% sequence identity and affiliation with the species Clostridium thermocellum. The strains differed in their ability to degrade crystalline cellulose, especially at an elevated temperature of up to 67 °C and at relatively low pH values (pH 6.5). To evaluate the influence of amino acid sequences on the discrepancies in cellulose degradation efficacy, the gene for the major cellulosomal component CelR was sequenced for all strains. The sequences were found to be almost identical (>99%). In contrast, the cellulosomal scaffoldin gene cipA showed more differences in the amino acid sequence and contained 8 or 9 cohesin modules, which indicated a different size of the cellulosome depending on the isolate. Based on MALDI-TOF MS analysis the relative abundance of important cellulosomal enzyme classes was determined. The strains with better biomass degradation properties (BC1 and NB2) had a significantly higher fraction of xylanases.

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.

Anaerobic high-throughput cultivation method for isolation of thermophiles using biomass-derived substrates.

Flow cytometry (FCM) techniques have been developed for sorting mesophilic organisms, but the difficulty increases if the target microbes are thermophilic anaerobes. We demonstrate a reliable, high-throughput method of screening thermophilic anaerobic organisms using FCM and 96-well plates for growth on biomass-relevant substrates. The method was tested using the cellulolytic thermophiles Clostridium thermocellum (T(opt) = 55 °C), Caldicellulosiruptor obsidiansis (T(opt) = 78 °C) and the fermentative hyperthermophiles, Pyrococcus furiosus (T(opt) = 100 °C) and Thermotoga maritima (T(opt) = 80 °C). Multi-well plates were incubated at various temperatures for approximately 72-120 h and then tested for growth. Positive growth resulting from single cells sorted into individual wells containing an anaerobic medium was verified by OD(600). Depending on the growth substrate, up to 80 % of the wells contained viable cultures, which could be transferred to fresh media. This method was used to isolate thermophilic microbes from Rabbit Creek, Yellowstone National Park (YNP), Wyoming. Substrates for enrichment cultures including crystalline cellulose (Avicel), xylan (from Birchwood), pretreated switchgrass and Populus were used to cultivate organisms that may be of interest to lignocellulosic biofuel production.

Influences of pH and hydraulic retention time on anaerobes converting beer processing wastes into hydrogen.

To convert high-solids organic wastes (3% w./w.) to high-value hydrogen, a full factorial experimental design was employed in planning the experiments for learning the effects of pH and hydraulic retention time (HRT) on the hydrogen production in a chemostat reactor using waste yeast obtained from beer processing wastes. For determining which experimental variable settings affect hydrogen production, predictive polynomial quadratic equation and response surface methodology were employed to determine and explain the conditions required for high-value hydrogen production. Experimental results indicate that a maximum hydrogen production rate of 460 mL/gVSS/d was obtained at pH = 5.8 and HRT = 32 hours. Moreover, hydrogenase targeted RT-PCR results indicate that Clostridium thermocellum and Klebsiella pneumoniae predominated.