Isolation and Characterization of Solventogenic Clostridia for Biobutanol Production

Bhutada V, Shrivastava S

Screening of solventogenic Clostridia was carried out by enrichment of different samples such as compost, soil and mangroves in DRCM broth and thioglycollate broth anaerobically. On Subsequent isolation, 38 different catalase negative and weakly positive isolates were obtained on DRCM agar. Two isolates BS1 obtained from soil of rhizosphere near cactus plant produced maximum of 7.5 g/L of butanol in AAM medium. Butanol, butyric acid and sugar tolerance BS1 was found to be 18 g/L, 4 g/L and 200 g/L respectively. The ability of organism to produce butanol in medium such as RCM, ANS and TYE was tested, RCM was found to be equally effective like AAM, but was found to be uneconomical then AAM.  An isolate CD1 obtained from cow dung was also able to produced 6.1 g/L of butanol did not give reproducible butanol thus BS1 was selected for further study. Growth curve of BS1 was studied in AAM medium to determine the onset of acidogenic and solventogenic phases for optimum butanol production. Growth of BS1 was observed after 16 hours of incubation and it was in the log phase of growth till 36 hours. Maximum pH drop was observed and reached to a steady pH. When the smears of endospore staining were observed under microscope it was observed that cells of BS1 entered sporulation stage after 36hours. At the same time gas chromatography of withdrawn fermentation revealed onset of solvent production and maximum 7.54 g/L butanol was produced by metabolizing 42 g/L of glucose on 68hours of incubation. Biochemical and molecular characterization of BS1 was carried out. It was identified as Clostridium beijerinckii and it shared 99% homology with strain E092 accession no JX267108.