Identification and description of Clostridium sp. and metabolic activities in fermentative hydrogen production

Abstract

Hydrogen is an environmentally friendly and highly efficient energy source. Fermentative hydrogen production is an exciting R&D area that offers a means to produce hydrogen from a variety of renewable resources or even wastewaters. However, the development of fermentative hydrogen production processes has been hampered due to their low yield and relatively high costs. The aim of this thesis was to improve fundamental knowledge of hydrogen-producing bacteria, provide genetic information associated with the hydrogen evolution, and to optimise operating conditions to enhance hydrogen yield. Isolation and identification of hydrogen producing bacteria from activated sludge were conducted using 16S rRNA gene-directed PCR-denaturing gradient gel electrophoresis (DGGE), clone library and heterotrophic plate isolation. The results showed that Clostridium sp. were dominant and active hydrogen producers. For the first time, three hydrogen producers, which harboured the [FeFe] hydrogenase gene, were characterised by 16S rDNA sequencing, and further physiologically identified as Clostridium sp. (W1), Clostridium butyricum (W4) and Clostridium butyricum (W5). The structure of the putative [FeFe] hydrogenase gene cluster of C. butyricum W5 was also described. The changes in [FeFe] hydrogenase mRNA expression of C. butyricum W5 during fermentation were monitored. Statistical analysis showed that both the [FeFe] hydrogenase mRNA expression level and cell growth have positive relationships with hydrogen production. The newly isolated C. butyricum W5 demonstrated highly promising hydrogen fermentation performance and was therefore used as the working strain. Optimization of operating conditions in terms of carbon and nitrogen sources, pH, temperature and inoculum size was carried out in a laboratory scale batch system. Use of molasses and NH₄NO₃ resulted in a high hydrogen production yield. Under the optimized fermentation conditions, 100g/L molasses, 1.2g/L NH₄NO₃, and 9×10⁴ cell/ml initial cell number at 39°C and pH 6.5, a maximum hydrogen yield of 1.85 mol H₂/ mol hexose was achieved. This corresponded to a hydrogen production rate of 17.38 mmol/h/L. Acetic, lactic and butyric acids were found to be the main by-products of the fermentation. The interrelations between the hydrogen yield and other yields of metabolites were statistically analysed corresponding to the variation in operating conditions. The dry cell weight was found to have a power relationship with hydrogen production. The results from this study have provided a better understanding of metabolic processes and gene expression involved in fermentative hydrogen production, and an improved bioengineering process for hydrogen production.