1 Complete perchlorate reduction using methane as the sole 2 electron donor and carbon source 3 Yi-Hao Luo1, †, Ran Chen1, †, Li-Lian Wen1, Fan Meng1, Yin Zhang1, Chun-Yu Lai1, 4 Bruce E. Rittmann2, He-Ping Zhao1, *, Ping Zheng1 5 6 1. MOE Key Lab of Environmental Remediation and Ecosystem Health, College of 7 Environmental and Resource Science, Zhejiang University, Hangzhou, China. 8 2. 9 State University, P.O. Box 875701, Tempe, Arizona 85287-5701, U.S.A. Swette Center for Environmental Biotechnology, Biodesign Institute at Arizona 10 11 * Correspondence to Dr. He-Ping Zhao. Tel: 0086-571-88982739, Fax: 0086-571-88982739, 12 E-mail: zhaohp@zju.edu.cn 13 † Contribute equally. 1 14 Abstract 15 Using a CH4-based membrane biofilm reactor (MBfR), we studied perchlorate (ClO4-) 16 reduction by a biofilm performing anaerobic methane oxidation coupled to 17 denitrification (ANMO-D). 18 (NO2-) surface loadings on ClO4- reduction and on the biofilm community’s 19 mechanism for ClO4- reduction. 20 ClO4- to a non-detectable level using CH4 as the only electron donor and carbon 21 source when CH4 delivery was not limiting; NO3- was completely reduced as well 22 when its surface loading was ≤ 0.32 g N/m2-d. 23 NO3- inhibited ClO4- reduction by competing for the scarce electron donor. 24 inhibited ClO4- reduction when its surface loading was ≥ 0.10 g N/m2-d, probably due 25 to cellular toxicity. 26 dominated the ClO4--reducing ANMO-D biofilm, and gene copies of the particulate 27 methane mono-oxygenase (pMMO) correlated to the increase of respiratory gene 28 copies. 29 involved chlorite (ClO2-) dismutation to generate the O2 needed as a co-substrate for 30 the mono-oxygenation of CH4. 31 Key Words: methane, perchlorate, oxidation, reduction, membrane-biofilm reactor We focused on the effects of nitrate (NO3-) and nitrite The ANMO-D biofilm reduced up to 5 mg/L of When CH4 delivery was limiting, NO2- Although Archaea were present through all stages, Bacteria These pieces of evidence support that ClO4- reduction by the MBfR biofilm 32 2 33 TOC Art 34 35 3 36 Introduction 37 Perchlorate (ClO4-) is a strong oxidizing agent that has been widely used in rocket fuel, 38 munitions, and explosives (EPA, 2005).1 39 interfering with the production of thyroid hormones needed for growth and 40 development (Coates & Achenbach, 2004).2 41 in groundwater is lower than 100 µg/L, but in some cases it can reach concentrations 42 of 20 mg/L or more. 3, 4 Although a nationwide maximum contaminant level (MCL) 43 for ClO4- has not yet established by the US EPA, some states have established cleanup 44 levels ranging from 2 to 18 µg/L for ClO4- in drinking water (Gu & Coates, 2006).5 45 Nitrate (NO3-) is an oxyanion commonly co-occurring with ClO4- in groundwater, for 46 example, at military bases that house rockets (USEPA, 2001).6 47 methemoglobinemia in infants, the MCL for NO3- in drinking water is regulated at 10 48 mg N/L (USEPA, 2009).7 49 common electron donor when the electron donor is insufficient. 8, 9 50 Different electron donors have been applied to achieve complete ClO4- and NO3- 51 removal by microbiological reduction.10-12 52 reduction using methane as the sole electron donor and carbon source,13, 14 since 53 methane is inexpensive and widely available.15-17 54 Methane oxidation coupled to denitrification (MO-D) has been extensively studied 55 during the past decade.14, 15, 18-21 56 out MO-D. 57 23 58 methane oxidizers (methanotrophs) and denitrifiers.22, 24, 25 It causes serious health problems by The typical perchlorate concentration Because NO3- causes NO3- inhibits ClO4- reduction due to competition for the An interesting example is nitrogen Two microbial processes are capable of carrying One is aerobic methane oxidation coupled to denitrification (AMO-D),22, which is performed by the combined actions of two distinct bacterial groups: The second is anaerobic 4 59 methane oxidation coupled to denitrification (ANMO-D).21, 26 60 microorganisms include a bacterial group affiliated with the candidate division NC10 61 and an archaeal group distantly related to anaerobic methanotrophic archaea.19, 27-29 62 AMO-D occurs in the presence of O2, because methanotrophs require O2 for the initial 63 mono-oxygenation step. 64 their catabolism and anabolism, for example, methanol, acetate, and citrate, and the 65 intermediates can be further utilized by denitrifiers as electron donors.15, 22, 30 66 steps are illustrated schematically in panel A of Figure S1 in Supplemental 67 Information. 68 amount of O2 is necessary to promote AMO-D.30, 31 69 ANMO-D can follow two pathways, illustrated schematically in panels B and C of 70 Figure S1. 71 which involves the combined action of Archaea and denitrifying bacteria.19 The 72 Archaea carry out reverse methanogenesis to generate H2 that is shuttled to the 73 denitrifying bacteria that respire NO2- to N2. 74 population ANME-2d (Methanoperedens nitroreducens) catalyzed CH4 oxidation by 75 methylcoenzyme M reductase (mcrABCDG) through a reverse-methanogenesis 76 pathway using NO3- as their terminal electron acceptor and generating NO2-; the 77 NC10 bacteria then reduced NO2- to N2.14 78 The second ANMO-D pathway is called the “Intra-Aerobic” pathway,21, 32 and only 79 one denitrifying microorganism (Candidatus Methylomirabilis oxyfera) was involved. ANMO-D The methanotrophs can release organic intermediates from These Although a high concentration of O2 inhibits denitrification, a certain Raghoebarsing et al. hypothesized the “Reverse Methanogesis” pathway, Haroon et al. reported that Archaea 5 80 Denitrification was carried out by stepwise reduction of NO2- to NO using nitrate 81 reductase (narGHJI) and nitrite reductase (nirSJFD/GH/L); then, NO was 82 disproportionated to produce O2 intracellularly and N2 using an unknown dismutase 83 enzyme. 84 methane mono-oxygenation by a membrane-bound particulate methane 85 mono-oxygenase (pMMO). 86 While the true mechanism of ANMO-D is unresolved, it is thermodynamically 87 feasible, as shown by redox equations 1 and 2 for NO3- or NO2- as the terminal 88 electron acceptor:19 89 5CH4 + 8NO3- + 8H+ = 5CO2 + 4N2 + 14H2O ΔG0’= -765 KJmol-1CH4 (1) 90 3CH4 + 8NO2- + 8H+ = 3CO2 + 4N2 + 10H2O ΔG0’= -928 KJmol-1CH4 (2) 91 When perchlorate (ClO4-) is the electron acceptor, a similar reaction between ClO4- 92 and CH4 also is thermodynamically feasible: 33 93 CH4 + ClO4- = HCO3- + Cl- + H2O 94 The pathway for dissimilatory perchlorate reduction begins with reduction of ClO4- to 95 chlorite (ClO2-, catalyzed by perchlorate reductase, pcrA) and ends with dismutation 96 of ClO2- to yield chloride (Cl-, catalyzed by chlorite dismutase, cld) and molecular 97 oxygen (O2), which is essential for the methane oxidation.2 98 complete CH4 removal coupled with ClO2- dismutation by a mixture of methanotrophs 99 and the perchlorate-reducing bacterium Dechloromonas agitate CKB.34 The 100 methanotrophs used extracellular O2 derived from disproportion of ClO2- by D. 101 agitate CKB to oxidize CH4 aerobically; thus, it was an extracellular-aerobic pathway The O2 was then used by the same microorganism as a co-substrate for ΔG0’= -792 KJmol-1CH4 (3) Miller et al. confirmed 6 102 that required that the substrate for dismutation, ClO2-, be supplied. 103 culture did not oxidize CH4 when supplied with ClO4- or ClO3-; thus, they concluded 104 that O2 produced via ClO4- reduction was unavailable for the aerobic methanotrophs. 105 So far, no study has successfully reduced ClO4- using CH4 as the sole electron donor 106 and carbon source. 107 are able to reduce ClO4- (using either nitrate or perchlorate reductase) means that a 108 ANMO-D or AMO-D culture has the possibility to reduce ClO4- using CH4 as electron 109 donor and carbon source. 110 The H2-based membrane biofilm reactor (MBfR) has been applied successfully for 111 microbial removal of oxidized contaminants, including NO3- and ClO4-.11, 35-37 112 non-porous walls of hollow-fiber membranes transfer H2 directly to a biofilm of 113 H2-oxidizing bacteria that reduce one or more electron acceptors.35 114 “bubbleless” membranes and the rapid oxidation of H2 in the biofilm allow nearly 100% 115 utilization of H2, preventing H2 losses to the atmosphere or effluent liquid.36, 38 116 The MBfR also could provide a means for the safe and efficient supply of CH4 to 117 drive ANMO-D, AMO-D, and ClO4- reduction. Supporting the concept, Sun et al 118 reported that an aerobic methane-based MBfR removed up to 97% of NO3- applied at 119 a concentration of 30 mg N/L,39 and Shi et al achieved 86 mg N/m2-d NO3- removal in 120 an anaerobic MBfR provided with CH4 as carbon source and electron donor.40 121 The objective of this study was to evaluate ClO4- reduction in a CH4-based MBfR. The mixed However, thermodynamics and the reality that most denitrifiers The The use of 7 122 Specifically, we studied the reduction patterns of NO2-, NO3-, and ClO4- when we 123 exposed the biofilm to different relative loadings. 124 CH4-permeation coefficient through the membrane wall to determine if the delivery 125 rate of CH4 was limiting, and we used quantitative real-time PCR (qPCR) to monitor 126 how the abundances of functional genes key to respiration reactions were affected by 127 the acceptor loadings. 128 mechanistic interpretation about what controls ClO4- reduction by the biofilm and the 129 likely pathways by which NO3- and ClO4- reductions occur when CH4 is the sole 130 electron donor. We quantified the Based on several types of evidence, we are able to provide 8 131 Materials and Methods 132 CH4 permeation 133 Steady-state CH4-permeation experiments were carried out in the same system (shown 134 schematically in Supporting Information (SI) Figure S2) Tang et al. used for 135 quantifying H2 permeation.41 136 (total volume of 1.6 × 10-4 m3, liquid volume of 0.6 × 10-4 m3) at a flow rate of 7.2 × 137 10-4 m3/d. 138 purity, Shanghai Gas Company, China) at a pressure of 1.0 bar (14.5 psi). 139 diffused through the hollow-fiber wall, dissolved in the water, and partitioned into the 140 headspace. 141 CH4 was rapidly partitioned to the gas phase. 142 measure its CH4 partial pressure. 143 pressure was stable for at least 40 hydraulic retention times (HRTs).41 We then 144 calculated the CH4 permeability of the membrane fiber based on the method in Tang 145 et al.41 146 summarized in the Supplementary Information (Table S1 & S2). 147 MBfR Setup 148 We used a two-column MBfR system similar to Zhao et al.8 149 composite hollow fibers (hydrophobic microporous polyethylene fiber, 280-µm o.d., 150 and a 180-µm i.d., pore size 0.1-0.15 µm) manufactured by Mitsubishi Rayon (Model Deionized water was pumped through the serum bottle The hollow fibers in the serum bottle were pressurized with CH4 (99.99% CH4 A magnetic stirring bar ensured complete mixing of the liquid and that We took the headspace gas samples to Steady state was achieved when the CH4 partial The equations and experimental parameters for the CH4-permeation test are The MBfR had 9 151 MHF-200TL, Mitsubishi, Ltd., Japan). The fibers were glued into a CH4-supply 152 manifold at the bottom of the MBfR column, and the top of each fiber was sealed. 153 The total volume of the MBfR was 65 mL, and the total membrane surface area was 154 7.0 cm2. 155 (Longer Pump, model 1515X, Longer Precision Pump Co, Ltd, China) at 100 156 mL/min. 157 Start up and continuous operation of the MBfR 158 We inoculated the MBfR with 10 mL of ANMO-D culture (original maintained 159 anaerobic) donated by Dr. Wei Xiang Wu at Zhejiang University (China) and enriched 160 the community by recirculating a mineral salt medium (described below) containing 2 161 mg N/L NO2- for 2 days. 162 mg N/L of NO2- continuously for 40 days, when complete NO2- reduction was 163 achieved. 164 with ClO4- and NO2- at influent concentrations of 1 mg/L and 2 mg N/L, respectively, 165 in Stage 1. 166 N/m2-d) or improve ClO4- removal at medium (0.1-0.6 g N/m2-d) or small (<0.1g 167 N/m2-d) loadings in a H2-based MBfR,9 we then systematically changed the influent 168 concentrations of NO3- and ClO4- in Stages 2 through 6. 169 reach steady state, which was defined as effluent concentrations stable (<10% 170 variation) for a minimum of three HRTs. 171 2: The MBfR was completely mixed by recirculation with a peristaltic pump To accumulate enough biomass, we fed the MBfR with 2 To investigate ClO4- reduction in the presence of NO2-, we fed the MBfR Since NO3- may inhibit ClO4- reduction at high surface loadings (> 0.6 g 1 mg/L ClO4-; Stage 3: We allowed each stage to The influent concentrations were: 1 mg/L ClO4- and 1.1 mg N/L of NO3-; Stage 4: Stage 1 10 172 mg/L ClO4- and 11.3 mg N/L of NO3-; Stage 5: 173 NO3-; and Stage 6: 174 NO2- after high ClO4--removal efficiency had been achieved, we fed the MBfR 5 175 mg/L of ClO4- and 5 mg N/L of NO2- in Stage 7. 176 varied slightly from the targets and are presented in Table S3 and Figure 1. 177 The influent feeding rate was 0.5 mL/min (HRT of 130 min), the CH4 pressure was 10 178 psi (0.69 bar) for Stage 1-3 and 15 psi (1.03 bar) for the latter stages, and the 179 temperature was 29±1oC for all experiments. The medium pH was adjusted to 180 7.0±0.2 with hydrochloric acid and contained the following mineral salts (analytical 181 grade or purer) per L of demineralized water: 182 MgSO4•7H2O 5 mg, KaH2PO4 0.2 g, Na2HPO4•12H2O 0.4 g, 1 mL acid trace element 183 solution (HCl 100 mM, 2.085 g of FeSO4•7H2O, 68 mg of ZnSO4•7H2O, 14 mg of 184 H3BO3, 120 mg of CoCl2•6H2O, 500 mg of MnCl2•4H2O, 320 mg of CuSO4, 95 mg of 185 NiCl2•6H2O per liter), and 1 mL alkaline trace element solution (NaOH 10 mM, 67 186 mg of SeO2, 50 mg of Na2WO4•2H2O, 242 mg of Na2MoO4•2H2O per liter). 187 medium was de-gassed with N2 to maintain an anaerobic condition. 188 During Stage 4, the CH4 supply was accidently lost for 48 hours, and we immediately 189 substituted N2 gas to keep the fibers pressurized. 190 in Stage 4, we intentionally stopped the CH4 supply for 30 hours in Stage 5. 191 CH4 supply was reinstated when the removal percentages were zero for NO3- and 192 ClO4-. 5 mg/L ClO4-. 1 mg/L ClO4- and 4.5 mg N/L of To investigate ClO4- reduction in the presence of Actual influent concentrations CaCl2 1 mg, NaHCO3 0.3 g, The To re-evaluate the response found The 11 193 Analyses 194 We measured the CH4 partial pressure (PCH4) of gas samples using a gas 195 chromatograph (Agilent Technologies GC system, model 7890A, Agilent 196 Technologies Inc., U.S.A) equipped with a flame ionization detector and a packed 197 column (30 m long, 0.32 mm i.d., 0.5 µm thickness, cross-linked polydimethysiloxane 198 film, J&W scientific, U.S.A.). 199 0.96 bar and a constant flow rate of 0.065 m3/d, and the temperature conditions for 200 injection and detector were 200 and 260oC, respectively. 201 used for standard calibration curves and for the experiments. 202 We took liquid samples from the MBfR with 5-mL syringes and filtered them 203 immediately through a 0.2-µm membrane filter (LC+PVDF membrane, Shanghai 204 Xinya, China). 205 833 Basic IC plus, Switzerland) with an A-Supp-5 column, an eluent containing 3.2 206 mM NaHCO3, 1.0 mM Na2CO3, and 5% Acetone in a flow rate of 1 mL/min. ClO4- 207 was measured using ion chromatography (Metrohm 833 Basic IC plus, Switzerland) 208 with an AS 16 column and AG 16 pre-column, eluent concentration of 35 mM KOH, 209 and a 1.5 mL/min flow rate. Dissolved O2 was measured with a dissolved oxygen 210 probe (Starter, model 300D, Ohaus Instruments Company, Germany), and the 211 concentrations for ~0.2 mg/L for the influent and ≤0.1 mg/L for the effluent. 212 values of the influent and effluent were measured by a pH meter (Seven Easy, Mettler 213 Toledo, Switzerland) and were between 7.4 and 7.7 for all stages. N2 was the carrier gas fed at a constant pressure of Analytical grade CH4 was We assayed for NO3- and NO2- using ion chromatography (Metrohm The pH 12 214 Flux Calculations 215 We calculated the NO3- and ClO4- removal fluxes (g/m2-d) according to: 216 J = (So-S)Q/A (4) 217 in which S° and S are the influent and effluent NO3- or ClO4- concentration (g/L), Q is 218 the influent flow rate to the MBfR system (L/d), and A is the membrane surface area 219 (m2). 220 stoichiometries shown in equations 5 through 7.42 The CH4 flux was calculated from the removal fluxes and reaction NO2- + 0.828CH4 + H+ = 0.04CO2 + 0.42N2 + 0.158C5H7O2N + 1.6H2O (5) NO3- + 1.2CH4 + H+ = 0.2CO2 + 0.4N2 + 0.2C5H7O2N + 2.2H2O (6) ClO4- + 1.613CH4 + 0.175NO3- +0.175H+ = Cl- + 0.737CO2+ 2.7H2O + 0.175C5H7O2N (7) 221 We compared the actual CH4 flux to the maximum CH4 flux that can be delivered 222 through the composite hollow fiber at the applied CH4 pressure to indicate if CH4 223 delivery was limiting.41 224 Biofilm Sampling and DNA extraction 225 We collected biofilm samples when the reactor reached a steady state for all stages 226 except Stage 2. 227 we cut off one ~10-cm-long section from the coupon fiber and then sealed the 228 remaining by tying the end into a knot. 229 Blood & Tissue Kit (Qiagen, USA) as previously described by Zhao et al (2011).38 Sparging with N2 gas at the sampling point to preclude O2 exposure, We then extracted DNA using the DNeasy 230 13 231 Quantification of 16S rRNA genes for Bacterial, Archaea and other functional genes 232 We used plasmids containing target fragments as positive controls and to produce 233 calibration curves.8 234 described for pcrA – reductase for ClO4-,43 narG – reductase for NO3-,44 nirS – 235 reductase for NO2-,45 mcrA – formation of methane from most of methanogens,46 236 pMMO – CH4 mono-oxygenase,47 the16S rRNA gene for bacteria,48 and the 16S rRNA 237 for archaea.49 238 performed qPCR as previously described by Zhao et al. (2011).8 The copy numbers 239 of each gene were calculated by comparison to standard curves. Negative controls 240 included water instead of template DNA in the PCR reaction mix. We performed 241 triplicate PCR reactions for all samples and negative controls. 242 plasmid standard curves and efficiency values for quantification by qPCR are shown 243 in Table S4. The primers and qPCR conditions were the same as previously We used the SYBR Premix Ex Taq Kit (Takara Bio Inc, Japan) and The slopes of the 244 14 245 Results and Discussion 246 Methane permeability 247 Figure 1 shows the headspace pressures during the CH4-permeation experiment. 248 Steady state was achieved at ~15 hours for the composite fiber. 249 permeability for the composite fiber was 1.03×10−7 m3 CH4 at standard temperature 250 and pressure - m membrane thickness/m2 hollow fiber surface area - d - bar. 251 permeability is about 10-fold smaller than the H2 permeability for the same composite 252 fiber and temperature.41 Although the Henry’s law constant of CH4 is only slightly 253 smaller than for H2, its mass-to-mole ratio (16 g/mol) is about 8 times greater than for 254 H2 (2 g/mol), making the CH4 molecule bulkier and more slowly diffusing through the 255 membrane wall. 256 Perchlorate reduction in the presence of nitrate and nitrite 257 Figure 2-A shows the influent and effluent concentrations of NO2-, NO3-, and ClO4- 258 for the entire set of experiments, and Figure 2-B shows the corresponding removal 259 percentages. ClO4- reduction could be achieved when CH4 was the sole electron 260 donor and carbon source. 261 major partial reduction was achieved in Stages 1 and 7. Though Miller et al 262 established a link between ClO2- and CH4 consumption in soils and mixed cultures by 263 D. agitate CKB and methanotrophs (Methylococcus capsulatus Bath or 264 Methylomicrobium album BG8) using acetate as the electron donor and carbon source, The CH4 This 100% reductions occurred in Stages 2, 3, 5, and 6, and 15 265 they did not find any upstream connection between ClO4- or ClO3- reduction and 266 methane oxidation.34 267 reduction was negligible or unavailable for aerobic methanotrophs. 268 results clearly show that the MBfR biofilm was able to reduce ClO4- using CH4 as the 269 sole electron donor. 270 methane oxidation suggests that reduction of other anions might also be coupled to 271 anaerobic methane oxidation. 272 bromate, selenate, chromate and other anion contaminants could be reduced in similar 273 CH4-based systems. 274 Comparison among Stages 1, 2, 6, and 7 shows that NO2- inhibited ClO4- reduction: 275 ClO4- reduction was complete when NO2- was absent in the influent in Stages 2 and 6, 276 but it decreased to < 50% when NO2- was present in the influent at a surface loadings 277 of 0.1-0.4 g N/m2-d (1.69±0.006 mg N/L for Stages 1, 5.22±0.13 mg N/L for Stage 7 278 in the influent, respectively). 279 Comparison among Stages 2, 3, 4, 5, and 6 shows that NO3- also inhibited ClO4- 280 reduction, but only at high NO3- surface loadings. 281 were <0.32±0.003 g N/m2-d (Stages 2, 3, 5, and 6), ClO4- and NO3- reductions were 282 complete. 283 in Stage 4, ClO4- reduction dropped to ≤5%, with NO3- reduction declining to ≤85%. 284 This trend is consistent with Tang et al,9 who used biofilm modeling to quantify the 285 impact of NO3- loading on perchlorate reduction when H2 was the electron donor. They concluded that oxygen generation during perchlorate In contrast, our This success of coupling perchlorate reduction with anaerobic Hence, it would be interesting to explore whether When the NO3- surface loadings However, when the NO3- surface loading increased to 0.78±0.09 g N/m2-d 16 286 High NO3- loading slowed ClO4- reduction by competing for the common donor (H2 287 for Tang et al.9 and CH4 here). 288 The MBfR accidently lost its CH4 supply for 48 hours (days 75-77), and we provided 289 N2 gas to keep the fibers pressurized (Figure S3-A). 290 to 2% before the CH4 supply was recovered, but it returned to 70% within 12 hours. 291 However, ClO4- removal remained low (2%) after recovery of the CH4 supply, 292 although it recovered to 100% in Stage 5, when the nitrate loading was smaller. 293 reinforce that methane was the electron donor responsible for perchlorate and nitrate 294 reduction, we repeated the CH4-loss experience during Stage 5 by intentionally 295 replacing the CH4 supply with N2 gas for 30 hours beginning on day 94 (Figure S3-B). 296 NO3- and ClO4- removals dropped to 0 within 12 hours for ClO4- and 24 hours for 297 NO3-, but both returned to 100% after the CH4 supply was recovered. 298 We calculated the consumption fluxes of NO2-, NO3-, and ClO4-, along with the 299 stoichiometric fluxes of CH4 (from equations 5 – 7). 300 Table 1 for each steady state. 301 consumption and the maximum possible CH4 flux. 302 mmol CH4/m2-d for Stages 1 – 4 and 86.8 mmol CH4/m2-d for Stages 5-7, both 303 calculated from the Km of CH4 of the composite fiber for the experimental conditions. 304 The maximum CH4 delivery flux for Stages 1, 2, 3, 5, 6, and 7 was substantially 305 greater than the observed CH4 fluxes, and CH4 delivery should not have been limiting. 306 Stage 4 may have been limited by CH4 delivery, because the actual CH4 flux NO3- removal dropped sharply To The fluxes are summarized in One important comparison is between the actual CH4 The maximum fluxes were 57.9 17 307 (47.5±7.20 mmol/m2-d) was close to the maximum CH4 flux (57.9 mmol CH4/m2-d). 308 In Stage 4, the effluent concentration of nitrate was stable at 1.39±0.21 mg N/L for 2 309 days before the methane supply was lost (Figure S3). 310 maximum methane-delivery rate could remove NO3- at a maximum flux of 0.67 g 311 N/m2-d, which corresponds to 100% removal of an influent concentration at 10.2 mg 312 N/L. The actual influent concentration was 11.3±0.40 mg N/L in Stage 4, which 313 explains the partial NO3- removal and that competition of CH4 is why ClO4- reduction 314 remained very low throughout Stage 4. 315 Because donor limitation was not an issue for Stages 1 and 7, the negative impact of 316 NO2- on ClO4- reduction probably was due to toxicity of NO2-, not to competition for 317 CH4. 318 inhibited by adding NO2-.50 319 methane oxidation by methanotrophs, and the inhibition was inversely proportional to 320 headspace methane concentrations.51 321 Functional Community Structure through Functional Gene Analysis 322 Figure 3 shows the 16S rRNA gene copies for Bacteria and Archaea, functional-gene 323 copy numbers, and fluxes of the tested electron acceptors through all stages. 324 copy number of the pcrA gene gradually increased from Stage 1 to Stage 5, and this 325 was parallel to overall increasing flux of ClO4- + NO3- and accumulation of more 326 bacteria, illustrated by the increasing gene copies for the 16S rRNA gene. 327 number of the pcrA gene decreased when the flux of all electron acceptors Based on stoichiometry, the Kluber & Conrad reported that methanogenesis activity could be significantly King & Schnell reported that NO2- could inhibit the The The copy 18 328 significantly decreased in Stage 6 (due to the absence of NO3- and NO2-), but 329 increased again when NO2- was re-introduced at a flux of 0.39±0.01 g N/m2-d in 330 Stage 7. 331 harbor the pcrA gene,52, 53 it is not surprising that the abundance of the pcrA gene was 332 significantly related with the NO3- flux in our study (Table S5), as well as in previous 333 MBfR studies with H2.8, 54, 55 334 Similar to the pcrA gene, nirS and narG genes gradually increased from Stage 1 to 335 Stage 5, though the fluxes of NO3-+NO2- decreased from Stage 4 to 5; again, the 336 increases likely were due to accumulating bacteria overall. 337 were absent in Stage 6, the nirS and narG abundances dropped by 0.5 to 1 order of 338 magnitude. 339 Stage 7 when NO2- was re-introduced into the MBfR system. However, the narG 340 abundance was similar to nirS in Stage 1, when NO2- was fed at a low loading, and 341 became much lower than nirS in Stage 7 when NO2- was fed at a higher loading. 342 Because the NarG gene is not selective for all DB,56 nirS is mostly used to quantify 343 the DB.57 344 Overall, Bacteria (16S rRNA gene) were ~2 orders of magnitudes higher than Archaea 345 (Archaeal 16S rRNA gene) through all stages, suggesting that Bacteria dominated 346 Archaea. 347 Stages 1 and 3, the pMMO gene increased much more by Stage 5 and in parallel to the 348 large increase in the flux of CH4. Since most denitrifying bacteria (DB) are able to reduce ClO4- and may When NO3- and NO2- Also similar with the pcrA trend, the nirS and narG genes increased in While the abundances of mcrA and pMMO genes were about the same in The mcrA and pMMO genes decreased in Stage 6, 19 349 when NO3- and NO2- were absent in the system, resulting in a much lower CH4 flux. 350 The pMMO gene abundance returned to its Stage 5 level with the increase of CH4 flux 351 Stage 7, but the mcrA gene remained low in Stage 7. 352 Archaea are necessary for the “Reverse Methanogesis” ANMO-D pathway, as they 353 produce electrons for denitrification. 354 supports the “Reverse Methanogenesis” was not important in the ClO4--reducing, 355 CH4-oxidizing biofilm. 356 gene copies of mcrA and respiration genes had no correlation. 357 Intracellularly generated O2 is essential for the “Intra-Aerobic Type” ANMO-D 358 pathway, in which Candidatus M. oxyfera (or a similar methanotroph) oxidizes CH4 359 via an initial mono-oxygenation reaction. 360 pMMO correlated to the gene copies for narG+nirS+pcrA, which supports an essential 361 role of O2 generation associated with ClO4- reduction. 362 the key bacteria reducing ClO4- used a chlorite dismutase in a manner similar to NO 363 disproportionation in denitrification.21 364 intracellularly, ClO4- reduction occurred via an “Intra-Aerobic Type” ANMO-PR 365 pathway, which is illustrated in Figure 5-A. 366 Rikken et al found that O2 was released extracellularly during complete ClO4- 367 reduction.58 368 reduction by a mixture of methanotrophs and perchlorate-reducing bacteria using 369 pMMO and pcrA separately. The low abundance of Archaea (Fig. 3) Further support is given in Figure 4, which shows that the Figure 4 shows that the gene copies of This association is logical if If O2 were produced and consumed Thus, another possibility is that CH4 oxidation was coupled to ClO4- Miller et al. reported that a variety of 20 370 methane-oxidizing bacteria, e.g., M. capsulatus Bath, M. album BG8, and M. 371 trichlsporium OB3b, were able to utilize O2 released from the disproportion of ClO2- 372 by dissmilatory perchlorate-reducing bacteria.34 Sun et al. reported that,39 in an 373 AMO-D process, co-existing methanotrophs consumed O2 preferentially, creating a 374 micro-aerobic environment conducive for denitrification. 375 methanotrophs released organic intermediates that served as electron donors for 376 denitrification.15, 22, 30 We name this potential mechanism “micro-Aerobic Methane 377 Oxidation coupled to Perchlorate Reduction,” or “mAMO-PR”. 378 Figure 5-B. 379 In summary, we found that the biofilm in an MBfR was able to reduce up to 5 mg/L of 380 ClO4- to non-detectable levels using CH4 as the only electron donor and carbon source 381 in the presence of NO3- at a surface loading of ≤ 0.32 g N/m2-d. 382 surface loadings (e.g., 0.78 g N/m2-d) inhibited ClO4- reduction due to electron-donor 383 competition, NO2- inhibited ClO4- reduction at low surface loadings (e.g., 0.1 g 384 N/m2-d), probably due to toxicity of NO2- to the ClO4--reducing cells. 385 much more abundant than Archaea in the biofilm, and pMMO gene copies correlated 386 to the increase of respiratory gene copies, while mcrA did not; thus, the CH4-oxidizing 387 biofilm likely respired ClO4- by a pathway that involved generating O2 using ClO2- 388 dismutation, with the O2 utilized as a co-substrate for the mono-oxygenation of CH4. 389 Two options are possible: 390 intracellular O2, and (2) mAMO-PR, in which ClO4--reducing bacteria produce 391 extracellular O2 by ClO2- dismutation, while methanotrophs uses O2 as a co-substrate In addition, the It is illustrated in While NO3- at high Bacteria were (1) ANMO-PR via a single strain producing and utilizing 21 392 to initiate oxidation of CH4. This study shows that it is feasible to use methane as an 393 electron donor to biologically remove perchlorate, which is a new option for 394 perchlorate reduction and a new application for the MBfR. 395 demonstrate that methane is a versatile electron donor, like hydrogen, for reducing 396 oxidized contaminants in water and wastewater treatment, then methane could be used 397 as an inexpensive electron donor. Should further study 22 398 Acknowledgments 399 Authors greatly thank “The National Key Technology R&D Program 400 (2014ZX07101-012)”, “National Natural Science Foundation of China (Grant No. 401 21107091, Grant No. 21377109)”, and “National High Technology Research and 402 Development Program of China (2013AA06A205)” for their financial support. 403 Supporting Information Available 404 Table S1-5 and Figure S1-3. 405 at http://pubs.asc.org. This material is available free of charge via the Internet 406 23 407 References 408 (1) USEPA IRIS, 2005. 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