Published: October 21, 2011 r2011 American Chemical Society 10155 dx.doi.org/10.1021/es202569b | Environ. Sci. Technol. 2011, 45, 10155–10162 ARTICLE pubs.acs.org/est Interactions between Perchlorate and Nitrate Reductions in the Biofilm of a Hydrogen-Based Membrane Biofilm Reactor He-Ping Zhao,* ,† Steve Van Ginkel, † Youneng Tang, † Dae-Wook Kang, † Bruce Rittmann, † and Rosa Krajmalnik-Brown † † Swette Center for Environmental Biotechnology, Biodesign Institute at Arizona State University, P.O. Box 875701, Tempe, Arizona 85287-5701, United States b S Supporting Information ’ INTRODUCTION Perchlorate (ClO 4 À ) is a strong oxidizing agent that has been widely used in rocket propellants, explosives, and fireworks. 1 ClO 4 À contamination in water is a health problem because it interferes with the production of thyroid hormones that are needed for pre- and postnatal growth and development as well as for normal metabolism in adults. 2 Although the US EPA has not yet established a maximum contaminant level (MCL) for ClO 4 À , some states have established cleanup levels ranging from 2 to 18 μg/L for ClO 4 À in drinking water. 3 Strategies for removing ClO 4 À from water include activated carbon, membrane filtration, ion exchange (IX), chemical or electrochemical reduction, and biological reduction. 1,4,5 Biologi- cal processes have gained interest in the past decade due to their ability to destroy ClO 4 À and their lower cost. 3 Among the biological-reduction methods is the H 2 -based membrane biofilm reactor (MBfR), in which H 2 gas is delivered to bacteria by diffusion through the wall of a gas-transfer fiber. H 2 is an electron donor that drives the respiratory reduction of one or a mixture of oxidized contaminants, e.g., nitrate (NO 3 À ), ClO 4 À , selenate, chromate, and chlorinated solvents. 6À9 The biological reduction of ClO 4 À involves ClO 4 À reductase (pcrABCD), which reduces ClO 4 À to chlorite (ClO 2 À ), 10 and chlorite dismutase (cld), which catalyzes the reduction of chlorite (ClO 2 À ) to chloride (Cl À ) and oxygen (O 2 ), 11 which is subse- quently reduced to H 2 O. Because the cld gene is not specific to perchlorate-reducing bacteria (PRB), targeting a pcr gene is most appropriate for detecting PRB. 12 A frequent cocontaminant with ClO 4 À is ammonium nit- rate (NH 4 NO 3 ), which is a main component in rocket fuel and explosives. In these situations, the concentration of NO 3 À typi- cally is 2 to 5 orders of magnitude higher than ClO 4 À . 13 NO 3 À and its immediate reduction product nitrite (NO 2 À ) also are serious water contaminants. The USA drinking-water standards for NO 3 À and NO 2 ‑ are 10 and 1 mg N/L, respectively. 14 Bacteria also readily reduce NO 3 À and NO 2 ‑ to harmless N 2 gas, a process called denitrification. NO 3 À reductases (Nar or Nap) reduce NO 3 À to NO 2 À ; NO 2 À is reduced to nitric oxide (NO) by either a copper (Cu-Nir) (nirK) or a cytochrome cd1 NO 2 ‑ reductase (Cd-Nir) (nirS); NO reductase (Nor) reduces NO to nitrous oxide (N 2 O); and N 2 O reductase (Nos) reduces N 2 O to N 2 . 15,16 Figure S1 in the Supporting Information summarizes Received: July 25, 2011 Accepted: October 21, 2011 Revised: October 19, 2011 ABSTRACT: We studied the microbial functional and struc- tural interactions between nitrate (NO 3 À ) and perchlorate (ClO 4 À ) reductions in the hydrogen (H 2 )-based membrane biofilm reactor (MBfR). When H 2 was not limiting, ClO 4 À and NO 3 À reductions were complete, and the MBfR’s biofilm was composed mainly of bacteria from the ε- and β-proteobacteria classes, with autotrophic genera Sulfuricurvum, Hydrogenophaga, and Dechloromonas dominating the biofilm. Based on func- tional-gene and pyrosequencing assays, Dechloromonas played the most important role in ClO 4 À reduction, while Sulfuricur- vum and Hydrogenophaga were responsible for NO 3 À reduction. When H 2 delivery was insufficient to completely reduce both electron acceptors, NO 3 À reduction out-competed ClO 4 À reduction for electrons from H 2 , and mixotrophs become important in the MBfR biofilm. β-Proteobacteria became the dominant class, and Azonexus replaced Sulfuricurvum as a main genus. The changes suggest that facultative, NO 3 À -reducing bacteria had advantages over strict autotrophs when H 2 was limiting, because organic microbial products became important electron donors when H 2 was severely limiting.