Butanol production under microaerobic conditions with a symbiotic system of Clostridium acetobutylicum and Bacillus cereus.

BACKGROUND: One major problem of ABE (acetone, butanol and ethanol) fermentation is high oxygen sensitivity of Clostridium acetobutylicum. Currently, no single strain has been isolated or genetically engineered to produce butanol effectively under aerobic conditions. In our previous work, a symbiotic system TSH06 has been developed successfully by our group, and two strains, C. acetobutylicum TSH1 and Bacillus cereus TSH2, were isolated from TSH06. RESULTS: Compared with single culture, TSH06 showed promotion on cell growth and solvent accumulation under microaerobic conditions. To simulate TSH06, a new symbiotic system was successfully re-constructed by adding living cells of B. cereus TSH2 into C. acetobutylicum TSH1 cultures. During the fermentation process, the function of B. cereus TSH2 was found to deplete oxygen and provide anaerobic environment for C. acetobutylicum TSH1. Furthermore, inoculation ratio of C. acetobutylicum TSH1 and B. cereus TSH2 affected butanol production. In a batch fermentation with optimized inoculation ratio of 5 % C. acetobutylicum TSH1 and 0.5 % B. cereus TSH2, 11.0 g/L butanol and 18.1 g/L ABE were produced under microaerobic static condition. In contrast to the single culture of C. acetobutylicum TSH1, the symbiotic system became more aerotolerant and was able to produce 11.2 g/L butanol in a 5 L bioreactor even with continuous 0.15 L/min air sparging. In addition, qPCR assay demonstrated that the abundance of B. cereus TSH2 increased quickly at first and then decreased sharply to lower than 1 %, whereas C. acetobutylicum TSH1 accounted for more than 99 % of the whole population in solventogenic phase. CONCLUSIONS: The characterization of a novel symbiotic system on butanol fermentation was studied. The new symbiotic system re-constructed by co-culture of C. acetobutylicum TSH1 and B. cereus TSH2 showed excellent performance on butanol production under microaerobic conditions. B. cereus TSH2 was a good partner for C. acetobutylicum TSH1 by providing an anaerobic environment. During fermentation process, the high ratio of Clostridium and low ratio of Bacillus composition indicated that this symbiotic system was an effective and easily controlled cultivation model for ABE fermentation under microaerobic conditions.

Isolation and characterisation of non-anaerobic butanol-producing symbiotic system TSH06.

Butanol-producing microorganisms are all obligate anaerobes. In this study, a unique symbiotic system TSH06 was isolated to be capable of producing butanol under non-anaerobic condition. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal RNA (rRNA) revealed that two strains coexist in TSH06. The two strains were identical to Clostridium acetobutylicum and Bacillus cereus, respectively. They were isolated individually and named as C. acetobutylicum TSH1 and B. cereus TSH2. C. acetobutylicum TSH1 is a butanol-producing, obligate anaerobic strain. Facultative anaerobic B. cereus TSH2 did not possess the ability of butanol production; however, it offered C. acetobutylicum TSH1 the viability under non-anaerobic condition. Moreover, B. cereus TSH2 enhanced butanol yield and speed of fermentation. TSH06 produced 12.97 g/L butanol and 15.39 g/L total solvent under non-anaerobic condition, which is 25 and 24 %, respectively, higher than those of C. acetobutylicum TSH1. In addition, TSH06 produced butanol faster under non-anaerobic condition than under anaerobic condition. Butanol accounted for more than 80 % of total solvent, which is higher than the known report. TSH06 was stable during passage. In all, TSH06 is a promising candidate for industrialisation of biobutanol with high yield, high butanol proportion, easy-handling and time-saving system. These results demonstrated the potential advantage of symbiosis. This study also provides a promising strategy for butanol fermentation.