The Pennsylvania State University The Graduate School Department of Crop and Soil Sciences CHARACTERIZATION OF H2-PRODUCING BACTERIAL COMMUNITIES FROM HEAT-TREATED SOIL AND ISOLATION OF DOMINANT CLOSTRIDIUM SPP. A Thesis in Soil Science by Yonghua Luo © 2007 Yonghua Luo Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2007 The thesis of Yonghua Luo was reviewed and approved* by the following: Mary Ann Bruns Associate Professor of Soil Science Thesis Advisor Chair of Committee John M.Regan Assistant Professor of Civil and Environmental Engineering David R.Huff Associate Professor of Turfgrass Breeding and Genetics Hery Lin Assistant Professor of Hydropedology/Soil Hydrology Curtis J.Dell Soil Scientist and Adjunct Professor of Soil Science David M.Sylvia Professor of Soil Microbiology Head of the Department of Crop and Soil Sciences *Signatures are on file in the Graduate School iii 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 iv 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, v 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. vi TABLE OF CONTENTS ABSTRACT…………………………………………………………………………....iii TABLE OF CONTENTS……………………………………………………………......vi LIST OF FIGURES…………………………………... ...............................................vviiii LIST OF TABLES........................................................................................................xiii ACKNOWLEDGEMENTS..........................................................................................xiv Chapter 1 Introduction…………………………………………………………………...1 REFERENCES…………………………………………………………………………..9 Chapter 2 The effect of glucose concentration on composition of H2- producing bacterial communities in a continuous flow bioreactor………………….………...…....14 ABSTRACT…………………………………………………………………………… 15 INTRODUCTION…………………………………………………………………….. ...17 MATERIALS AND METHODS……………………………………………………….19 RESULTS……………………………………………………………………………..... .26 DISCUSSION………………………………………………………………………......30 REFERENCES………………………………………………………………………......37 Chapter 3 The effect of L-cysteine on bacterial community composition and H2 production in batch cultures of heat-treated soil inocula……………………………….57 ABSTRACT………………………………………………………………………........58 INTRODUCTION…………………………………………………………….……. …...60 MATERIALS AND METHODS……………………………………………………......61 RESULTS…………………………………………………………………………….....66 DISCUSSION…………………………………………………………………………..69 vii REFERENCE………………………………………………………………………….74 Chapter 4 Strain LYH2, a N2-fixing, ferric-iron reducing Clostridium sp. isolated from a silty clay loam soil………………………………………………………….………….85 ABSTRACT……………………………………………………………………………...86 INTRODUCTION……………………………………………………………………….87 MATERIALS AND METHODS……………………………………………………….88 RESULTS……………………………………………………………………………...93 DISCUSSION……………………………………………………………………….....96 REFERENCE…………………………………………………………………………..99 Chapter 5 GENERAL CONCLUSIONS……………………………………………..110 Conclusion Appendix A…………………………………………………………………………..114 Appendix B…………………………………………………………………………... 116 viii LIST OF FIGURES Figure 1-1: H2 production pathways…………………………………………………..3 Figure 1-2: Pathway to hydrogen production by fermentation………………………...4 Figure 1-3 Structural formula of L-cysteine. The thiol group can effectively bind oxygen in liquid medium.………………………...................................................7 Figure 2-1: RISA profiles showing the bacterial community fingerprints of bioreactor samples with different glucose concentrations and hydraulic retention times. The glucose concentrations and hydraulic retention times are indicated at the top. The sizes of DNA marker bands are indicated by the arrows. The different DNA bands in bioreactor samples are indicated with arrows in their respective lanes ..………………………………………………………………………………....44 Figure 2-2: Petri plate after 6 days of incubation on anaerobic medium containing L-cysteine and 10g/L glucose, showing colonies of H2 producing bacteria...…...47 Figure 2-3: RISA fingerprint of genomic DNA of isolate from 10g/L glucose culture. The sizes of DNA bands are indicated by the arrows………………………….. 48 Figure 2-4: Neighbor-joining dendrogram depicting phylogenetic relationships of strain LYH1 with other Clostridium spp. based on nearly complete 16S rRNA sequences, using Bacillus subtilis as outgroup. GenBank accession numbers are in parentheses. The numbers at the nodes represent bootstrap values of 1,000 replicates. The scale bar represents the calculated number of changes per nucleotide position………………………………………………………………49 Figure 2-5: Comparison of biogas production (A) and H2 percentage in biogas (B) in ix samples grown with 0.5g/L L-cysteine and without L-cysteine. Column heights represent means and error bars indicate standard deviations. …………………....50 Figure 2-6: Accumulated gas production during growth of strain LYH1in batch cultures with and without L-cysteine (squares) and without L-cysteine (diamonds) …………………………………………………………………………………...51 Figure 2-7: Alignments of the LYH1 probe sequence, its target site, and sequences of corresponding sites in reference organisms. 17 related clostridia, Selenomonas strain SB90, Enterobacter cloacae strain CP1 and Bacillus subtilis were selected as reference organisms. The target sequence is displayed in the upper row; mismatches between target and sequences of corresponding sites in reference organisms are shadowed……………………………………………...52 Figure 2-8: Confocal Scanning Laser Microscope images of sample culture at 10g/L glucose concentration with 10-h HRT: (A) The hybridization of sample cells with universal EUB probe labeled Cy-3 and LYH1 probe labeled by Cy-5. (B) Morphology of sample cells examined by differential interference