Physiological and biochemical characteristics of the carbon ion beam irradiation-generated mutant strain Clostridium butyricum FZM 240 in vitro and in vivo.

Clostridium butyricum (C. butyricum) represents a new generation of probiotics, which is beneficial because of its good tolerance and ability to produce beneficial metabolites, such as short-chain fatty acids and enzymes; however, its low enzyme activity limits its probiotic efficacy. In this study, a mutant strain, C. butyricum FZM 240 was obtained using carbon ion beam irradiation, which exhibited greatly improved enzyme production and tolerance. The highest filter paper, endoglucanase, and amylase activities produced by C. butyricum FZM 240 were 125.69 U/mL, 225.82 U/ mL, and 252.28 U/mL, which were 2.58, 1.95, and 2.21-fold higher, respectively, than those of the original strain. The survival rate of the strain increased by 11.40 % and 5.60 % after incubation at 90 °C for 5 min and with simulated gastric fluid at pH 2.5 for 2 h, respectively, compared with that of the original strain. Whole-genome resequencing and quantitative real-time PCR(qRT-PCR) analysis showed that the expression of genes related to enzyme synthesis (GE000348, GE001963 and GE003123) and tolerance (GE001114) was significantly up-regulated, while that of genes related to acid metabolism (GE003450) was significantly down-regulated. On this basis, homology modeling and functional prediction of the proteins encoded by the mutated genes were performed. According to the results, the properties related to the efficacy of C. butyricum as a probiotic were significantly enhanced by carbon ion beam irradiation, which is a novel strategy for the application of Clostridium spp. as feed additives.

Long-term effect of inoculum pretreatment on fermentative hydrogen production by repeated batch cultivations: homoacetogenesis and methanogenesis as competitors to hydrogen production.

Long-term effects of inoculum pretreatments (heat, acid, loading-shock) on hydrogen production from glucose under different temperatures (37 °C, 55 °C) and initial pH (7 and 5.5) were studied by repeated batch cultivations. Results obtained showed that it was necessary to investigate the long-term effect of inoculum pretreatment on hydrogen production since pretreatments may just temporarily inhibit the hydrogen consuming processes. After long-term cultivation, pretreated inocula did not enhance hydrogen production compared to untreated inocula under mesophilic conditions (initial pH 7 and pH 5.5) and thermophilic conditions (initial pH 7). However, pretreatment could inhibit lactate production and lead to higher hydrogen yield under thermophilic conditions at initial pH 5.5. The results further demonstrated that inoculum pretreatment could not permanently inhibit either methanogenesis or homoacetogenesis, and methanogenesis and homoacetogenesis could only be inhibited by proper control of fermentation pH and temperature. Methanogenic activity could be inhibited at pH lower than 6, both under mesophilic and thermophilic conditions, while homoacetogenic activity could only be inhibited under thermophilic condition at initial pH 5.5. Microbial community analysis showed that pretreatment did not affect the dominant bacteria. The dominant bacteria were Clostridium butyricum related organisms under mesophilic condition (initial pH 7 and 5.5), Thermoanaerobacterium sp. related organisms under thermophilic condition (initial pH 7), and Thermoanaerobacterium thermosaccharolyticum related organisms under thermophilic condition (initial pH 5.5). Results from this study clearly indicated that the long-term effects of inoculum pretreatments on hydrogen production, methanogenesis, homoacetogenesis and dominant bacteria were dependent on fermentation temperature and pH.