BackgroundThe 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.ResultsThe 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.ConclusionsPyruvate 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.

Consolidated bioprocess assures an efficient lignocellulosic conversion to fermentable sugars and subsequently to bioethanol. Such a single-step hydrolysis and anaerobic fermentation was achieved with acclimated Clostridium thermocellum DSM 1313 on different mildly pre-treated agricultural lignocellulosic residues without any additional enzymes/and strains. Acclimation was achieved by serially sub-culturing in increasing concentration of individual substrates, such as rice husk, sugarcane bagasse, and banana pseudostem in the standard media, with cellobiose as an adjunct. The acclimated cellulolytic thermophile exhibited an early log phase entry with enhanced growth compared to the direct inoculation experiments with unacclimated culture. Around 672 mg/g of reducing sugar was produced from sugarcane bagasse media and 636 mg/g from rice husk media and 513 mg/g from banana pseudostem media with the acclimated organism. Bioethanol production also doubled in experiments with serially acclimated cultures, with a maximum of 1.21 and 1.0 g/L ethanol titre from sugarcane bagasse and rice husk, respectively. The serial acclimation experiments have increased the saccharification potentials of the organism towards the respective lignocellulosic substrates and also enhanced the bioethanol production.

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