CHARACTERIZATION OF H2-PRODUCING BACTERIAL COMMUNITIES FROM HEAT-TREATED SOIL AND ISOLATION OF DOMINANT CLOSTRIDIUM SPP.
Open Access
- Author:
- Luo, Yonghua
- Graduate Program:
- Soil Science
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 12, 2007
- Committee Members:
- Maryann Victoria Bruns, Committee Chair/Co-Chair
John Michael Regan, Committee Member
David Robert Huff, Committee Member
Hangsheng Lin, Committee Member
Curtis James Dell, Committee Member - Keywords:
- Biohydrogen production
bacterial community
H2-producing Clostridium spp.
L-cysteine
RISA
16S rRNA - Abstract:
- Hydrogen is an attractive alternative to conventional fossil fuels. Currently most hydrogen is produced from nonrenewable natural gas, oil and coal. However, from the perspectives of economics and environmental quality, hydrogen production from renewable resources is gaining more and more attention. Biohydrogen production from the fermentation of wastewater containing carbohydrates is one approach for exploiting renewable resources. Much literature has been published on the effects of external factors such as substrate concentration, temperature, pH, etc. on hydrogen production by pure cultures or defined mixed cultures. However, only a few studies have been conducted on the effects of these external factors on H2-producing microbial communities from uncharacterized, mixed inocula. The objectives of this research were threefold: to determine the effect of glucose concentration on H2-producing bacterial community composition in the fermentation of synthetic wastewater; to determine the effect of L-cysteine and successive transfer on H2 producing bacterial community composition in batch cultures; and to isolate and characterize a new strain of H2-producing Clostridium spp. from mixed soil inocula using L-cysteine as reducing agent. In the first study, a continuous flow bioreactor was continuously fed with synthetic wastewater for periods of 7-14 days. Heat-treated soil was introduced into the reactor as a mixed inoculum and L-cysteine was added to a final concentration of 0.5g/L as reducing agent. PCR-based ribosomal RNA intergenic spacer analysis (RISA) was used to characterize the bacterial community composition in the bioreactor. It was revealed that RISA profiles of bacterial communities grown in media with 5.0, 7.5 or 10g/L glucose yielded partial 16S rRNA sequences most related to Clostridium spp. In contrast, RISA profiles of cultures grown in 2.5 g/L glucose yielded more diverse DNA sequences. The lowest glucose concentration gave rise to sequences representing two bacterial families, Clostridiaceae and Acidaminococcaeae, in the low G+C Gram-positive Firmicutes division, and one family, Enterobacteraceae in the gamma Proteobacteria division. Using an oligonucleotide probe ¡°LYHI¡±, complementary to rRNA of an isolate obtained from the reactor containing 10 g/L glucose, it was shown that about 90% percent of the bacterial cells in microscope fields from bioreactor samples at 10 g/L glucose hybridized with the LYH1 probe. In samples from the bioreactor fed with 2.5 g/L glucose, only 26% of bacterial cells hybridized to LYH1 probe. These results further confirmed the effect of glucose on the composition of bacterial communities as judged by RISA. In the second study, batch experiments were carried out with the same nutrient levels used in the continuous flow bioreactor. Regardless of the glucose concentration and number of serial transfers, RISA profiles of bacterial communities grown without L-cysteine yielded partial 16S rRNA sequences closely related to Enterobacteriaceae. In contrast, RISA profiles of batch cultures with L-cysteine yielded results similar to those observed with the continuous flow bioreactor, which indicated that 5g/L appeared to be the lowest favorable glucose concentration for the successful competition of Clostridium spp. over other bacterial species. L-cysteine could have affected the composition of bacterial communities in batch experiments either due to its function as a quick oxygen scavenger or its role as a spore germinant. In the third study, a new Clostridium sp. strain LYH2 was isolated from a silty clay loam soil using L-cysteine as a reducing agent in the growth medium. Closest cultured relatives of strain LYH2 were Clostridium acidisoli, Clostridium akagii and Clostridium pasteurianum. This dissertation showed that heat-treated soil provided a useful source of Clostridium spp. for producing biohydrogen from renewable wastes, and that glucose concentrations greater than 5g/L and L-cysteine enhanced their growth and H2 production.