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The Pennsylvania State University The Graduate School Department of Civil and Environmental Engineering ANALYSIS OF MICROBIAL COMMUNITIES AND DESIGN OF BIOREACTORS USED FOR PERCHLORATE REMEDIATION AND BIOHYDROGEN PRODUCTION A Thesis in Environmental Engineering by Husen Zhang © 2005 Husen Zhang Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2005 The thesis of Husen Zhang has been reviewed and approved* by the following: Bruce E. Logan Kappe Professor of Environmental Engineering Thesis Advisor Chair of Committee Mary Ann Bruns Assistant Professor of Crop and Soil Sciences John M. Regan Assistant Professor of Environmental Engineering William D. Burgos Associate Professor of Environmental Engineering Andrew Scanlon Professor of Civil Engineering Head of the Department of Civil and Environmental Engineering *Signatures are on file in the Graduate School. ABSTRACT A chemolithoautotrophic bacterium (strain HZ) was isolated from biofilm samples in an unsaturated-flow, packed-bed reactor treating perchlorate-contaminated groundwater. Dilution- to-extinction method was used for isolation. Purity was initially examined microscopically, and confirmed by identical intergenic ribosomal RNA spacer sequences from multiple clones. Strain HZ is a Gram-negative, rod-shaped facultative anaerobe that can use oxygen, perchlorate, chlorate, or nitrate as an electron acceptor and hydrogen gas or acetate as an electron donor. Growth on hydrogen gas was coupled with complete perchlorate (10 mM) reduction to chloride with a maximum doubling time of 8.9 hours. Autotrophic growth with carbon dioxide as the sole carbon source was confirmed by demonstrating that biomass carbon (100.9%) was derived from 14 CO2. Phylogenetic analysis based upon the 16S rRNA sequence indicated that strain HZ belongs to the genus Dechloromonas within the β subgroup of the Proteobacteria. Biofilm samples from a pilot-scale, fixed-bed, perchlorate-reducing reactor were analyzed for microbial species from inoculated and indigenous populations. The bioreactor was inoculated with Dechlorosoma sp. strain KJ and fed groundwater containing indigenous microorganisms. The reactor was flushed weekly to remove accumulated biomass. Perchlorate was reduced to non-detectable levels (< 4 µg L-1) after 26 days of operation and remained so during proper reactor operation in a six-month long test. Plastic media in the reactor were collected from top, middle, and bottom locations. Genomic DNA was extracted from successive washes of thawed biofilm material for PCR-based community fingerprinting by 16S-23S ribosomal intergenic spacer analysis (RISA). No DNA sequences closely related to that of strain KJ were recovered. The most intense bands yielded DNA sequences with high similarities (> 98%) to Dechloromonas spp. Other sequences from RISA fingerprints indicated presence of the iii low G+C Gram-positive bacteria and the Cytophaga-Flavobacterium-Bacteroides (CFB) group. Fluorescence in situ hybridization (FISH) was also used to examine biofilms using genus- specific 16S ribosomal RNA probes. Numbers of bacteria hybridizing to the Dechloromonas probe were most abundant at the biofilm surface (23% of all cells), and decreased as biofilm depth increased. The bacteria hybridizing to Dechlorosoma probes constituted less than 1 % of all cells in the biofilms examined, except in the deepest portions where they represented 3-5%. Biological hydrogen production was investigated using a laboratory-scale, trickle-bed biofilm reactor. The reactor was inoculated with Clostridium acetobutylicum and fed mineral medium with glucose as the sole carbon and energy source. The flowrate was 1.6 mL/min (0.096 L/h), producing a hydraulic retention time of 2.1 min. Continuous hydrogen production rates were 14, 20, 25, and 27 mL/h at influent glucose concentrations of 1.0, 3.3, 4.5, and 10.5 g/L, respectively. Gas-phase hydrogen concentrations of 70-79% were obtained at influent glucose concentrations of 1.0, 3.3, 4.5, and 10.5 g/L. The major fermentation byproducts were acetate and butyrate. The measured hydrogen yields indicated an overall conversion efficiency of 15-26% based on a theoretical stoichiometry of 4 moles hydrogen from 1 mole of glucose. -1 -1 The normalized hydrogen production rate was 676 to 1265 (mL-H2)(g-glucose) (L-reactor) , which is within the range of values reported using continuously stirred tank reactors. Another H2-producing bioreactor was operated at four hydraulic retention times (HRT) (10, 5, 2.5 and 1 h) and four glucose concentrations (10, 7.5, 5, and 2.5 g/L). Biomass suspensions were analyzed by Ribosomal Intergenic Spacer Analysis (RISA) to obtain qualitative information on bacterial populations at each condition. Results showed that microbial species composition responded more to influent glucose concentration than to HRT. Populations detected at 2.5 g/L glucose concentration were taxonomically more diverse than at 5, 7.5, and 10 iv g/L glucose concentrations. The most intense band in RISA profiles at 10, 7.5, and 5 g/L glucose yielded sequences with high similarity (> 97%) to Clostridium acidisoli. At 2.5 g/L glucose, a Selenomonadaceae spp. was identified as being present in the reactor. v Table of Contents Page List of Tables……………………………………………………………………….......... viii List of Figures……………………………………………………………………… …… ix Acknowledgments………………………………………………………………………. x Chapter 1 Introduction……………………………………………………………………. 1 1.1 Problem Statements………………………………………………………………. 1 1.1.1 Perchlorate bioremediation………………………………………………… 1 1.1.2 Biological hydrogen generation ……………………………………………. 2 1.2 Organization of Dissertation……………………………………………………….. 3 1.3 References ……………………………………………………………………. 5 Chapter 2 Perchlorate reduction by a novel chemolithoautotrophic,hydrogen-oxidizing bacterium……………………………………………………………………………………10 2.1 Introduction………………………………………………………………………… 11 2.2 Results……………………………………………………………………………… 13 2.2.1 Enrichment and isolation…………………………………………………….. 13 2.2.2 Phenotypic characteristics…………………………………………………… 13 2.2.3 Autotrophic CO2 fixation by strain HZ……………………………………… 14 2.2.4 Phylogenetic analysis………………………………………………………… 14 2.3 Discussion………………………………………………………………………… 15 2.4 Experimental Procedures…………………………………………………………. 17 2.4.1 Medium and cultivation……………………………………………………… 17 2.4.2 Enrichment and isolation…………………………………………………….. 18 2.4.3 Electron microscopy……………………………………………..…………. 18 14 2.4.4 Determination of CO2 incorporation……………………………………… 19 2.4.5 Analytical techniques……………………………………………………….. 20 2.4.6 Phylogenetic analysis………………………………………………………… 20 2.5 References………………………………………………………………………… 22 Chapter 3 Molecular assessment of inoculated and indigenous bacteria in biofilms from a pilot- scale perchlorate-reducing bioreactor……………………………………………………… 30 3.1 Introduction………………………………………………………………………. 31 3.2 Materials and Methods……………………………………………………………. 34 3.3 Results…………………………………………………………………………….. 40 3.4 Discussion ……………………………………………………………………... 43 3.5 References………………………………………………………………………… 52 Chapter 4 Biological hydrogen production in a trickle-bed bioreactor……………………. 63 4.1 Introduction……………………………………………………………………….. 64 4.2 Materials and Methods…………………………………………………………… 65 4.2.1 Medium and culture conditions……………………………………… …….. 65 4.2.2 Reactor design and operation………………………………………… …… 66 4.2.3 Calculation…………………………………………………… …….……… 67 4.2.4 Determination of the reactor’s hydraulic retention time……....……………. 68 vi 4.2.5 Analytical procedures………………………………………………………. 68 4.3 Results and Discussion…………………………………………………………… 69 4.4 References………………………………………………………………………… 78 Chapter 5 Microbial community shifting as a function of hydraulic retention time (HRT) and substrate concentration in a chemostat H2-producing reactor……………………………... 81 5.1 Introduction……………………………………………………………………….. 81 5.2 Materials and Methods……………………………………………………………. 82 5.3 Results and Discussion…………………………………………………………… 84 5.4 References ………………………………………………………………………89 Appendix Data used to generate figures in chapter 2 to chapter 4………………………… 92 vii List of Tables Table Page Table 3.1 FISH probe sequences, target sites, formamide concentrations in the hybridization buffer and sodium chloride concentrations in the washing buffer……………………….. 51 Table 3.2 Phylogenetic summary of perchlorate-reducing community from cloning and sequencing results………………………………………………………………………… 52 Table 4.1 Summary of H2 production rates and conversion efficiency……………… 71 Table 4.2 Summary of volatile fatty acids production………………………………. 72 Table 4.3 Comparison of hydrogen production rates in the trickle-bed reactor with those reported using a CSTR……………………………………………………………………. 73 Table 5.1 Phylogenetic summary of hydrogen-producing community from cloning and sequencing results…………………………………………………………………………. 87 viii List of Figures Figure Page Figure 2.1 Perchlorate reduction by the chemolithoautotrophic hydrogen-oxidizing enrichment culture…………………………………………………………………………. 27 Figure 2.2 Scanning electron micrograph of anaerobically grown cells of strain HZ… 27 Figure 2.3 Growth of strain HZ with hydrogen as the electron donor and perchlorate (10 mM) as the electron acceptor with carbon dioxide as the only carbon source………… 28 Figure 2.4 Neighbor-joining phylogenetic tree of 16S rDNA sequences of strain HZ and others…………………………………………………………………………………... 29 Figure 3.1 Perchlorate concentration profile in the reactor with two flow rates……… 58 Figure 3.2 Chemical profiles in the reactor of acetate, oxygen, nitrate, and perchlorate at 0.34 L

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