(23a) Improved Butanol Production from Clostridium Pasteurianum from Crude Glycerol Feedstock

Biodiesel production results in crude glycerol waste from the transesterification of fatty acids (10% w/w). The solventogenic Clostridium pasteurianum, an anaerobic Firmicute, can produce butanol from glycerol as the sole carbon source. Coupling butanol fermentation with biodiesel production can improve the overall economic viability of biofuels. However, crude glycerol contains growth inhibiting byproducts which reduce feedstock consumption and solvent production. To obtain a strain with improved characteristics, a random mutagenesis and directed evolution selection technique was used. A wild type C. pasteurianum (ATCC 6013) culture was chemically mutagenized and the resulting population underwent 10 days of selection in increasing concentrations of crude glycerol (80 to 150 g/L). The best performing mutant (M150B) showed a 72% increase in butanol production in 100 g/L crude glycerol compared to the wild type strain, as well as increased growth rate, a higher final optical density, and less production of the side product PDO (1,3-propanediol). Wild type and M150B strains were sequenced via Single Molecule Real-Time (SMRT) sequencing. Mutations introduced to the M150B genome were identified by sequence comparison to the wild type and published closed sequences. A major mutation in key transcriptional regulator Spo0A was identified. Complementation studies were done with both wild type and mutated spo0A genes in a constructed Spo0A-deficient strain. RNAseq identifies differences to the transcriptomes of the strains during growth on crude glycerol. Methylation patterns on genomic DNA identified with SMRT sequencing were used to aid in optimization of transformation efficiency of plasmid DNA to elude C. pasteurianum endogenous restriction endonucleases.