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Use of acetate, propionate and butyrate for reduction of nitrate and sulfate 1 and methanogenesis in microcosms and bioreactors simulating an oil 2 reservoir 3 4 Chuan Chen1,2,*, Yin Shen1, Dongshan An1 and Gerrit Voordouw1 5 1Petroleum Microbiology Research Group, Department of Biological Sciences, 6 University of Calgary, Calgary, Alberta, T2N 1N4, Canada. 7 2State Key laboratory of Urban Water Resource and Environment, Harbin Institute 8 of Technology, Harbin, HeiLongjiang Province, 150090, China. 9 10 11 *Address correspondence to: Chuan Chen, cchen@hit.edu.cn 12

AEM Accepted Manuscript Posted Online 27 January 2017Appl. Environ. Microbiol. doi:10.1128/AEM.02983-16Copyright © 2017 American Society for Microbiology. All Rights Reserved.

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Abstract 13 Acetate, propionate and butyrate (volatile fatty acids, VFA) occur in oil field waters 14 and are frequently used for microbial growth of oil field consortia. We determined 15 the kinetics of use of these VFA components (3 mM of each) by an anaerobic oil 16 field consortium in microcosms containing 2 mM sulfate and either 0, 4, 6, 8 or 13 17 mM of nitrate. Nitrate was reduced first with preference for acetate and 18 propionate. Sulfate reduction then proceeded with propionate (not butyrate) as 19 the electron donor, whereas the fermentation of butyrate (not propionate) was 20 associated with methanogenesis. Microbial community analyses indicated 21 Paracoccus-Thauera, Desulfobulbus and Syntrophomonas-Methanobacterium as the 22 dominant taxa catalyzing these three processes. Most probable number assays 23 showed the presence of up to 107/ml of propionate-oxidizing SRB in waters from 24 the Medicine Hat Glauconitic C field. Bioreactors with the same concentrations of 25 sulfate and VFA responded similarly to increasing concentrations of injected 26 nitrate as observed in the microcosms: sulfide formation was prevented by adding 27 approximately 80% of the nitrate dose needed to completely oxidize VFA to CO2 in 28 both. Thus this work has demonstrated that simple time-dependent observations 29 of the use of acetate, propionate and butyrate for nitrate reduction, sulfate 30 reduction and methanogenesis in microcosms are a good proxy for these 31 processes in bioreactors of which monitoring is more complex. 32

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33 34 35 Importance: 36 Oil field volatile fatty acids acetate, propionate and butyrate were specifically used 37 for nitrate reduction, sulfate reduction and methanogenic fermentation. Time-38 dependent analyses in microcosms served as a good proxy for these processes in a 39 bioreactor, mimicking a sulfide-producing (souring) oil reservoir: 80% of the 40 nitrate dose required to oxidize volatile fatty acids to CO2 was needed to prevent 41 souring in both. Our data also suggest that propionate is a good substrate to 42 enumerate oil field SRB. 43 44 Introduction 45 The injection of water into oil fields to maintain reservoir pressure often 46 leads to increased concentrations of sulfide (souring) through the activity of 47 sulfate-reducing bacteria (SRB), which derive energy for growth from the 48 oxidation of oil organics to reduce sulfate to sulfide (1, 2). The sulfide formed 49 represents a health hazard, decreases the value of produced oil and/or accelerates 50 corrosion of facilities and infrastructure. 51 Nitrate injection is a well-known strategy for control of souring (3, 4). The 52 mechanism of inhibition of sulfate reduction by nitrate injection includes 53

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biocompetitive exclusion of SRB by heterotrophic nitrate-reducing bacteria 54 (hNRB), which assumes that these use the same electron donors for reduction of 55 nitrate as used by SRB for reduction of sulfate (5-9). 56 Volatile fatty acids (VFA), which include acetate, propionate and butyrate, 57 are considered to be major electron donors shared by hNRB and SRB. A survey of 58 21 formation waters from wells accessing oil reservoirs on the Norwegian 59 continental shelf (10) gave average concentrations of 4.7 mM acetate (range 0.32 60 to 16.9 mM), 0.48 mM propionate (range 0.05 to 1.53 mM) and 0.14 mM butyrate 61 (range 0 to 0.82 mM). Produced waters from fields in the Danish sector of the 62 North Sea and from the Kuparuk oil field in Alaska had concentrations in the same 63 range (11, 12). All of these oil fields had a high down-hole temperature of 60 to 64 90oC. Produced waters from a mesothermic Alaskan oil field contained on average 65 3.5 mM acetate, 0.65 mM propionate and 0.12 mM butyrate with some samples 66 having zero VFA (13). Produced waters from the Medicine Hat Glauconitic C 67 (MHGC) field with a downhole temperature of 30oC (3) had lower VFA 68 concentrations with 0.12 mM acetate detected. In view of these results microbial 69 tests have been done with acetate and propionate (14) or with acetate, propionate 70 and butyrate (15). 71 VFA are formed by anaerobic metabolism of oil hydrocarbons. Their 72 concentrations thus result from rates of formation and of use. Transient formation 73 of acetate was demonstrated in a bioreactor, containing heavy MHGC oil, 74 whenever injection of nitrate was terminated and reduction of sulfate re-started 75 (16). VFA may be formed by beta-oxidation of alkanes, with even chain length 76

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alkanes forming acetate (equation i) and uneven chain length alkanes forming 77 acetate and propionate (equation ii). Butyrate may be formed if the final beta-78 oxidation step in the degradation of even chain length alkanes (equation iii) 79 remains incomplete. These reactions are catalyzed by syntrophic Firmicutes or 80 Deltaproteobacteria (17-19) and are driven forward by removal of the H2-formed 81 by hydrogenotrophic methanogens (equation iv) or SRB (equation v) (17, 19, 20, 82 21). High VFA concentrations will result if the rates of these hydrogenotrophic 83 reactions, exceed those for acetotrophic methanogenesis, syntrophic acetate 84 oxidation or the use of VFA for sulfate reduction. 85 VFA are also used by hNRB in biological nitrogen removal in wastewater 86 treatment. In this process, acetate is used first, followed by propionate and 87 butyrate, and finally valerate (22). In addition, VFA are important electron donors 88 in enhanced biological phosphorus removal and are precursors for biosynthesis of 89 polyhydroxyalkanoates (23). They also function as electron donors in microbial 90 fuel cells for electricity generation or hydrogen production (24, 25). 91 Previous work on the use of VFA as electron donors in microcosms has 92 shown that use of propionate and butyrate by SRB to reduce sulfate to sulfide is 93 accompanied by acetate production (26). Nitrate reduction by hNRB occurred 94 with all three VFA components (26). Once nitrate was depleted, methanogenesis 95 was also observed (4, 26-28). In this study, the effects of nitrate addition on sulfate 96 reduction and methanogenesis by an anaerobic oil field consortium in microcosms 97 were compared with those in an up-flow bioreactor operated under similar 98 conditions. Bioreactors mimic souring in reservoir environments subjected to 99

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nitrate injection more accurately, but are more laborious to run. We conclude that, 100 despite differences in kinetics, both approaches give similar results with respect to 101 the nitrate dose needed to control souring. 102 103 Experimental section 104 105 Media and enrichment cultures. CSBA medium (pH 7.2-7.4) contained per L: 7 g 106 NaCl, 0.2 g KH2PO4, 0.4 g MgCl26H2O, 0.5 g KCl, 0.15 g CaCl22H2O and 0.25 g 107 NH4Cl (29). Following dissolution and autoclaving, the medium was cooled under 108 a gas mixture of 90% (v/v) N2 and 10% CO2 (N2-CO2). To cooled anoxic medium, 109 the following were added aseptically (per L): 1 ml trace element solution, 1 ml 110 selenite-tungstate solution and 30 ml of 1 M NaHCO3 (30). CSBA medium was 111 further amended with sulfate to 2 mM (CSBA-S) and with VFA to 3 mM (3 mM each 112 of acetate, propionate and butyrate) for use in serum bottle tests and in a 113 bioreactor. Alternatively, acetate and propionate, propionate only, or butyrate 114 only were added (all to 3 mM). Medium (50 ml) was placed in 122 ml serum 115 bottles, which were flushed with N2-CO2 for 5 min to remove oxygen from the head 116 space. The bottles were then sealed with butyl rubber stoppers and closed with 117 aluminum caps. SRB enrichment culture E2PW was made by inoculating 5 mL of 118 produced water 2PW from the MHGC field (3) into 50 mL of CSBA, containing 5 119 mM sulfate and 3 mM VFA. Enrichments were incubated for 14 days at room 120 temperature (22oC) and transferred at least twice prior to use as inoculum for 121 serum bottles and the bioreactor. 122

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123 Kinetics of use of VFA by MHGC oil field produced waters. A schematic of the 124 MHGC field, indicating make-up water sources, water plants, injection wells and 125 producing wells, sampled monthly, has been presented elsewhere (3). Five ml of 126 produced waters 2PW, 4PW, 7PW or 13PW (3) were inoculated into 122 mL 127 serum bottles containing 50 mL of CSBA or 50 ml of CSBA-S medium and either 3 128 mM VFA, 3 mM propionate or 3 mM butyrate. Before inoculation, the medium was 129 flushed with N2-CO2 for 5 min to remove oxygen. Each experimental condition was 130 set up in duplicate and incubated at 22oC. Aliquots of 0.5 mL liquid sample were 131 removed periodically with a 1 mL syringe flushed with N2-CO2 to determine 132 concentrations of sulfate, sulfide, nitrate, nitrite, or VFA. The sulfide concentration 133 was analyzed immediately after each sampling. The remainders of the samples 134 were centrifuged and the supernatants were frozen at -20oC for further analysis. 135 Aliquots (0.2 ml) of headspace gas were analyzed for methane. 136 137 Effect of nitrate on the kinetics of VFA use by hNRB, SRB and methanogens. 138 CSBA-S medium (50 mL) with 3 mM VFA or its components was amended with 139 nitrate to 0, 4, 8 or 13 mM and inoculated with 5 mL of SRB enrichment E2PW. 140 Incubation and sampling were as described above. 141 142 Comparison of most probable numbers of lactate- and of propionate-143 utilizing SRB. Aliquots (100 μl) of MHGC field samples collected in July 2016 were 144

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serially diluted in triplicate in 48-well microtiter plates containing Postgate 145 medium B (900 μl). Postgate medium B (pH 7.0-7.5) contains (g L-1): KH2PO4 (0.5), 146 NH4Cl (1.0), CaSO4 (1.0), MgSO4·7H2O (2.0), sodium lactate (4.0: 60% w/w), yeast 147 extract (1.0), ascorbic acid (0.1), thioglycolate (0.1), FeSO4·7H2O (0.5). For 148 medium in which propionate served as electron donor for sulfate reduction 149 sodium lactate was replaced by 2.2 g/L of sodium propionate. Microtiter plates 150 were sealed with a Titer-Tops® polyethylene membrane. The samples were 151 incubated anaerobically for 3 weeks at 30oC in an anaerobic hood with an 152 atmosphere of 5% H2, 10% CO2 and 85% N2.. Wells exhibiting a black FeS 153 precipitate were scored positive. Most probable numbers (MPNs) were derived 154 from the data using appropriate statistical tables (31). Because the microtiter 155 plate wells had a headspace of only 0.6 ml, the available H2 would reduce 156 maximally 0.3 mM sulfate, whereas the concentrations of lactate (21.4 mM) and 157 propionate (22.9 mM) would reduce 10.7 and 17.3 mM sulfate, respectively, of the 158 17.3 mM sulfate present in the medium. The MPN method counted, therefore, 159 lactate- or propionate-oxidizing SRB, not H2-oxidizing SRB. 160 161 Bioreactor setup and start-up. A 60-mL syringe column (2.5x16 cm) was fitted 162 with glass wool and polymeric mesh at the bottom, packed with sand having an 163 average grain size of 225 μm (Sigma-Aldrich) and then fitted with one layer of 164

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polymeric mesh at the top (32). All materials including sand, tubing, glass wool 165 and polymeric mesh were autoclaved before use. The pore volume (PV = 28 ml) of 166 the column was determined by flooding with CSBA medium using a multichannel 167 peristaltic pump (Gilson Inc., Minipuls-3, 8-channel head) and determining the 168 difference between the wet and dry weights of the column. The column had a 22% 169 liquid porosity with an effective area of 1.1 cm2 (Aeff=0.22 лR2). Three-way valves 170 were placed at the influent and effluent ports for sampling. The flow rate was 42 171 mL/d with a retention time of 16 h. For start-up, 0.5 PV of SRB enrichment E2PW 172 was pumped into the column via the influent port. The bioreactor was shut down 173 and incubated without flow for 1 week to allow SRB growth. CSBA-S medium with 174 3 mM VFA was then injected at 21 mL/d. When the 2 mM sulfate in this medium 175 was completely reduced, the flow rate was increased to 42 mL/d and kept at this 176 rate through the remaining operation. The bioreactor was run at room 177 temperature (22oC) with the operating conditions being changed when a new 178 steady state was established as indicated by constant concentrations of sulfate, 179 sulfide, nitrate, nitrite and VFA. 180 181 DNA extraction. Samples of enrichments (3 mL) taken at the end of serum bottle 182 incubations and of bioreactor effluents (5 mL) were centrifuged for 5 min at 183 13200 rpm at 4 oC. The cell pellets were collected for DNA isolation with the Fast 184 DNA Spin Kit for Soil and the Fast Prep Instrument (MP Biomedicals, Santa Ana, CA) 185 according to the manufacturer’s instructions. The concentrations of extracted DNA 186 were quantified using a Qubit fluorimeter (Invitrogen). 187

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188 Pyrosequencing. Pyrosequencing of 16S amplicons was done for 15 enrichments 189 and 7 bioreactor effluents. PCR amplification involved 35 cycles using 16S primers 190 926Fw (AAACTYAAAKGAATTGRCGG) and 1392R (ACGGGCGGTGTGTRC), followed 191 by 15 cycles with FLX titanium primers 454T_RA_X and 454T_FwB, as described 192 elsewhere (33). Purified 16S amplicons (~125 ng) were sequenced at the Genome 193 Quebec and McGill University Innovation Centre, Montreal, Quebec with a Genome 194 Sequencer FLX Instrument, using a GS FLX Titanium Series Kit XLR 70 (Roche 195 Diagnostics Corporation). Data analysis was conducted with Phoenix 2, a 16S 196 rRNA data analysis pipeline, developed in house (34). High quality sequences, 197 which remained following quality control and chimeric sequence removal, were 198 clustered into operational taxonomic units at 3% distance by using the average 199 linkage algorithm (35). A taxonomic consensus of all representative sequences 200 from each of these was derived from the recurring species within 5% of the best 201 bitscore from a BLAST search against the SSU Reference data set SILVA102 (36). 202 Amplicon libraries were clustered into a Newick-formatted tree using the UPGMA 203 algorithm with the distance between libraries calculated with the thetaYC 204 coefficient (37) as a measurement of their similarity in the Mothur software 205 package (38, 39). The Newick format of the sample relation tree was visualized 206 using Dendroscope (40). The entire set of the raw reads is available from the 207 Sequence Read Archive at NCBI under accession number SRX684441. 208 209 Chemical analyses. Aqueous sulfide concentrations were determined 210

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colorimetrically with N,N-dimethyl-p-phenylenediamine (41). Ammonium was 211 measured colorimetrically at 635 nm using the indophenol blue method (42). 212 Sulfate, nitrate and nitrite were assayed by high-pressure liquid chromatography 213 (HPLC), using a Waters 600E HPLC instrument equipped with a Waters 423 214 conductivity detector, a Waters 2489 UV/VIS detector (wavelength set at 200 nm) 215 and a Waters IC-PAK Anion HC column (4.6 by 150 mm; Waters, Japan) with a 216 mobile phase flow rate of 2 mL/min. The mobile phase contained per L of water: 217 20 mL butanol, 120 mL acetonitrile and 20 mL borate/gluconate concentrate 218 (boric acid 18 g/L; sodium D-gluconate 16 g/L; sodium tetraborate decahydrate 219 25 g/L; glycerol 250 ml/L). The concentrations of lactate, acetate, propionate, and 220 butyrate were determined using an HPLC instrument equipped with a Waters 221 2489 UV/VIS detector at 210 nm, using a Prevail Organic Acids 5u column (250.0 222 by 4.6 mm; Alltech) with a mobile phase of 25 mM KH2PO4 (pH 2.5) at a flow rate 223 of 1.0 mL/min. Methane production was detected by injection of 0.2 ml of culture 224 headspace into an HP 5890 gas chromatograph equipped with a stainless steel 225 Porapak R 80/100 column (0.049 by 5.49 m). Injector and detector temperatures 226 were 150 and 200°C, respectively (33). 227 228 Chemical reactions considered in the Results and Discussion sections. 229 Sequential beta-oxidation of octadecanoate to acetate and H2: 230

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C17H35COO- + 16H2O→9C2H3O2-+ 8H++16H2 ` (i) 231 Degradation of heptadecanoate to acetate, propionate and H2: 232 C16H33COO- + 14H2O→7C2H3O2-+ C3H5O2-+7H++14H2 (ii) 233 Conversion of butyrate to two acetate and H2 (final step of i) 234 C4H7O2-+2H2O →2C2H3O2-+ H+ +2H2 (iii) 235 Reduction of CO2 with H2: 236 CO2+4H2→CH4+2H2O (iv) 237 Reduction of sulfate with H2: 238 SO42- +4H2+H+ →HS-+4H2O (v) 239 Reduction of sulfate coupled to oxidation of propionate to acetate and CO2: 240 4C3H5O2-+3SO42- +3H+ →4C2H3O2-+3HS-+4CO2+4H2O (vi) 241 Fermentation of butyrate to two acetates coupled to methanogenesis: 242 2C4H7O2-+CO2+2H2O→4C2H3O2-+ CH4 +2H+ (vii) 243 Reduction of sulfate coupled to oxidation of butyrate to two acetates: 244 2C4H7O2-+SO42-→4C2H3O2-+HS-+H+. (viii) 245 Combination of (vii) and (viii): 246 3C4H7O2-+1.2SO42-+0.3CO2+0.6H2O→6C2H3O2-+1.2HS-+0.3CH4+1.8H+ (ix) 247 Redox half reactions for oxidation of butyrate, propionate and acetate to CO2: 248 C4H7O2-+6H2O→4CO2 +19H++20e (x) 249 C3H5O2-+4H2O→3CO2 +13H++14e (xi)250

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C2H3O2-+2H2O→2CO2 +7H++8e (xii) 251 Redox half reaction for reduction of nitrate: 252 NO3-+6H++5e ½N2 +3H2O (xiii) 253 Complete oxidation of 3 each of acetate, propionate and butyrate to CO2: 254 3C4H7O2-+3C3H5O2-+3C2H3O2-+25.2NO3-+34.2H+→27CO2+12.6N2+39.6H2O (xiv) 255 Complete oxidation of 3 each of acetate and propionate to CO2: 256 3C3H5O2-+3C2H3O2-+13.2NO3-+19.2H+→15CO2+6.6N2+21.6H2O (xv) 257 Complete oxidation of 3 propionate to CO2: 258 3C3H5O2+8.4NO3-+11.4H+→9CO2+4.2N2+13.2H2O (xvi) 259 Incomplete oxidation of propionate to acetate and CO2: 260 C3H5O2-+2H2O →C2H3O2-+CO2+3H2 (xvii) 261 In equations (xiii) to (xvii) we have assumed N2 as the sole product of nitrate 262 reduction by MHGC consortia. In the presence of excess electron donor, neither 263 nitrite (NO2-) nor nitrous oxide (N2O) accumulated when tested (results not 264 shown). Nitrate was not reduced to ammonium (43). 265 266 Results 267

268 Kinetics of use of VFA by oil-field microcosms. MHGC-produced waters 269 contained on average 0.117 mM of acetate (N=29; range 0.056-0.315 mM). 270 Propionate and butyrate were not detected. When 10% (v/v) of duplicate samples 271

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of produced waters from producing wells 2PW, 4PW, 7PW or 13PW were 272 inoculated into CSBA-S medium, containing 2 mM sulfate and 3 mM VFA, all 273 showed similar use of VFA components except for one of the replicates of 7PW 274 (7PWb). SRB first reduced sulfate to sulfide (1.7 mM) with a preference for 275 propionate as the electron donor, which was converted to acetate (equation vi). 276 Subsequent methanogenesis was coupled to the conversion of butyrate to two 277 acetates (equation vii). These two processes increased the acetate concentration 278 from 3 to 11 mM (Fig. 1 B, E and Fig. S1 B, F). Sulfate, propionate and butyrate 279 were completely consumed at the end of the incubations. Up to 1.1 mM methane 280 was detected in the headspace of the bottles. Correcting for the difference in 281 volume of the headspace (67 ml) and aqueous phase (55 ml) indicates formation 282 of 1.3 mM methane, which is close to the theoretical maximum of 1.5 mM methane 283 formed from 3 mM butyrate according to equation (vii). 284 However, in microcosm 7PWb butyrate was used preferentially as electron 285 donor for sulfate reduction. Only 1 mM propionate was oxidized with 2 mM 286 propionate remaining (Fig. S1C). The decreased availability of butyrate allowed 287 production of only 0.5 mM methane (corrected for volume) in microcosm 7PWb 288 (Fig.S1D). This was much lower than that in microcosm 7PWa in which propionate 289 was used for sulfate reduction (as in all other microcosms) and in which butyrate 290 was used for methanogenesis with production of 1.5 mM methane (corrected for 291 volume; Fig.S1D). Since the VFA components were used with similar kinetics in all 292 other microcosms, the microcosm inoculated with 2PW was assumed to be 293

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representative of all microcosms and was used throughout in further experiments 294 (Table 2: E2PW) 295 In microcosms with 2 mM sulfate and 3 mM propionate, sulfate reduction 296 started immediately: 1.2 mM sulfide was produced and all propionate was 297 oxidized with the production of 3 mM acetate, as per equation (vi). No methane 298 production was observed (Fig. 2 A, B, C). In microcosms with 2 mM sulfate and 3 299 mM butyrate sulfate reduction started after a lag phase of 150 h (Fig. 2D). No 300 methane formation was observed during this period. Subsequent oxidation of 3 301 mM butyrate to 6 mM acetate was coupled to the reduction of 1.2 mM sulfate to 302 sulfide and the formation of 0.3 mM methane (corrected for volume; Fig. 2 D, E, F), 303 as per equation (ix). Incubations with 3 mM VFA in the absence of sulfate showed 304 that only butyrate was used for methanogenesis. Metabolism of 3 mM butyrate 305 caused the acetate concentration to increase to 9 mM and the corrected methane 306 concentration to increase to 1.3 mM (Fig. 3), somewhat less than the theoretically 307 expected 1.5 mM methane (equation vii). Propionate was not used under these 308 conditions. 309 310 Use of VFA in the presence of nitrate. When 4 mM nitrate was added to CSBA-S 311 medium containing 2 mM sulfate and 3 mM VFA, nitrate was completely reduced 312 within 25 h. No nitrite intermediate was detected. A small decrease in the 313 ammonium concentration in the medium was noted (Fig. S2A). The ammonium 314 was probably used as the nitrogen source in biomass synthesis. These results 315 indicate that nitrate was mostly reduced to N2 and not to ammonium, as shown 316

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elsewhere (43). Nitrate reduction was coupled to preferential oxidation of acetate 317 and propionate (Fig. S2C). Reduction of 2 mM sulfate (Fig. S2B) then proceeded 318 with use of the remaining propionate and butyrate as the electron donor, forming 319 5 mM acetate (Fig. S2C). A little methane was formed in the headspace (Fig. S2D: 320 0.1 mM). 321 When added at 8 mM, nitrate was first reduced to N2 with removal of 3 mM 322 acetate, 3 mM propionate and 1 mM butyrate (Fig. S3A). This stoichiometry shows 323 that not all VFA were oxidized to CO2 as per equations (x) to (xii), which would 324 yield two-fold more electrons (86 mM) than required to reduce 8 mM nitrate to N2 325 (40 mM, as per xiii). Hence, some VFA were used as the carbon source for the 326 formation of biomass and associated storage polymers. Sulfate (1.4 mM) was then 327 reduced to sulfide (Fig. S3B) with the oxidation of the residual 2 mM butyrate and 328 of biomass to form 5 mM acetate (Fig. S3C). The kinetics of acetate formation was 329 considerably slower than that of butyrate use (Fig. S3C), suggesting acetate 330 formation via a more complex route than mere oxidation of butyrate. No methane 331 was formed. Upon addition of 13 mM nitrate, nitrate reduction proceeded with the 332 complete removal of all VFA within 25 h (Fig. S4A). This stoichiometry indicates 333 partial oxidation of VFA to CO2 and use of VFA for formation of biomass and 334 storage polymers. The latter participated in reduction of 0.7 mM sulfate to sulfide 335 from 100 h onwards (Fig. S4B) and in formation of 2 mM acetate at 300 h (Fig. 336 S4C). No methanogenesis was observed. Thus, addition of 0, 4, 8 or 13 mM nitrate 337 resulted in remaining sulfate concentrations of 0, 0, 0.7 and 1.3 mM and 338 production of 2, 2, 1.3 and 0.7 mM sulfide, indicating increased but not complete 339

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souring control. The theoretical nitrate dose at which all of 3 mM VFA are oxidized 340 to CO2 is 25.2 mM (equation xiv). 341 342 Effect of nitrate on souring in bioreactors. To establish microbial activity, the 343 bioreactor was inoculated with 0.5 PV (14 ml) of the SRB enrichment E2PW. The 344 bioreactor was then incubated for one week without flow after which CSBA 345 medium containing 2 mM sulfate and 3 mM VFA was pumped in at a flow rate of 346 42 mL/d. The concentration of injected sulfate (2 mM) and flow rate were 347 constant throughout. The nitrate concentration and the kinds of VFA injected (at 3 348 mM each) are shown in Table 1. 349 When 3 mM VFA and 0 mM nitrate were injected, complete reduction of 350 sulfate to 1.6 mM of aqueous sulfide was obtained after day 10 (Fig. 4: stage I). No 351 propionate or butyrate were detected in the effluent, which contained a low 352 concentration of acetate of 1 to 4 mM, compared to 11 to 12 mM in comparable 353 microcosms (Fig. 1B, E). These lower acetate concentrations suggested increased 354 formation of methane, which was not monitored in the bioreactors. Injection of 4 355 mM nitrate did not limit sulfate reduction because 2 mM sulfide were still detected 356 in the effluent. However, the concentrations of acetate and propionate in the 357 effluent increased to 8.6 and 1 mM, respectively (Fig. 4: stage II). This acetate 358 concentration was similar to that observed in the comparable microcosm (Fig. 359 S2C), indicating inhibition of methanogenesis by the nitrate injection. When 8 mM 360 nitrate was injected some sulfate remained, the average sulfide concentration in 361 the effluent was 1.9 mM and the average acetate concentration dropped to 6 mM. 362

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(Fig. 4: stage III), similar to the 5 mM acetate observed in the microcosm in Fig. 363 S3C. No propionate was observed in the effluent. Souring was partially controlled 364 by injecting 13 mM nitrate, which limited reduction of sulfate to 0.7 mM sulfide, 365 while the acetate concentration decreased further to 3 mM (Fig. 4: stage IV). This 366 can be compared to the microcosm of Fig. S4, in which 0.7 mM sulfide and 2 mM 367 acetate were observed. No propionate, butyrate, nitrate or nitrite was found in the 368 effluent, similar to the findings in the comparable microcosm (Fig. S4). When 369 nitrate injection was stopped the sulfide concentration rose immediately to 2 mM 370 and the acetate and propionate concentrations increased to 9.2 mM and 2 mM, 371 respectively (Fig. 4: stage V). This indicated that the sulfate reduction had been 372 restored to the same level as during startup (Fig. 4: stage I). However, increased 373 concentrations of acetate and propionate in stage V, as compared to stage I, 374 suggest that nitrate injection provided long-term inhibition of methanogenic 375 activity. 376 When 3 mM acetate and 3 mM propionate and 4, 8 or 13 mM nitrate were 377 injected, the sulfide concentration in the effluent decreased to 1, 0.5, and 0 mM, 378 respectively (Fig. 4: stages VI, VII and VIII). The concentrations of acetate in the 379 effluent were 3, 1 and 0 mM, respectively. No propionate was observed. Injection 380 of 13 mM nitrate led on average to breakthrough of 1 mM nitrate and up to 4 mM 381 nitrite in the bioreactor effluent (Fig. 4: stage VIII). As indicated in equation xv, 382 13.2 mM nitrate can completely oxidize 3 mM propionate and 3 mM acetate to CO2. 383 The average eluted concentrations of 1 mM nitrate and 4 mM nitrite indicate that 384 nitrate was only about 80% reduced. Hence, 20% of injected acetate and 385

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propionate most likely served as the carbon source for biomass formation. When 386 nitrate injection was stopped the concentrations of sulfide and acetate in the 387 effluent increased within 10 days to 1.7 and 5.0 mM, respectively (Fig. 4: stage IX), 388 whereas the concentration of propionate was 0.71 mM in the same period. This is 389 consistent with propionate being incompletely oxidized to acetate and CO2 while 390 serving as the electron donor for sulfate reduction. 391 The available electron donor was then further decreased from 3 mM aceate 392 and 3 mM propionate to 3 mM propionate only. In the absence of nitrate, complete 393 reduction of 2 mM sulfate to sulfide was observed with production of 2-3 mM 394 acetate in the effluent (Fig. 4: stage X). Subsequent injection of 4 and 6 mM nitrate 395 resulted in partial and complete control of souring (Fig. 4: stages XI and XII, 396 respectively). No acetate was observed in the effluent when 6 mM nitrate was 397 injected. Although 8.4 mM nitrate is needed to completely oxidize 3 mM 398 propionate to CO2 (equation xvi), the observation that 6 mM nitrate was sufficient 399 to control souring indicates again that part of the propionate was used as carbon 400 source for biomass formation. When the injected nitrate concentration was 401 decreased to 2 mM, the sulfide and acetate concentrations increased to 1.5 and 2 402 mM, respectively (Fig. 4: stage XIII). 403 404 Microbial community analyses. DNA was isolated, subjected to PCR 405 amplification of 16S rRNA genes and pyrosequencing yielding 7 amplicon libraries 406 for bioreactor effluents and 15 amplicon libraries for microcosms (Table 2). 407

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Comparison in a dendrogram (Fig. 5A) indicated clade I for libraries of 408 bioreactor effluents and enrichments, inoculated with E2PW, and clade II for 409 libraries of microcosms directly inoculated with field samples (2PW_S2V3, 410 4PW_S2V3, 7PWa_S2V3, 7PWb_S2V3 and 13PW_S2V3). Because the enrichments 411 were harvested after prolonged incubation, where nitrate and sulfate had already 412 been reduced (see arrows in Figs. 1-3 and S1-S4), the physiological states of these 413 enrichments corresponded to those of the bioreactor effluents, which were also 414 mostly devoid of electron acceptors. Microbial community compositions are 415 presented in Table 3. Microbial communities in clade II for microcosms inoculated 416 with field samples had high fractions of hydrogenotrophic methanogens (44, 45, 417 46), including Methanocalculus (1.8-51.6%), Methanocorpusculum (0-53.3%) and 418 Methanofollis (0.3-18.6%). Interestingly, a high proportion (2.0-48.7%) of the 419 acetotrophic methanogen Methanosaeta was present (Table 3) even though 420 methane production from the metabolism of acetate was not detected in these 421 microcosms. Microbial communities in clade I are discussed below. 422 423 Use of propionate for measuring the MPN of SRB in oil field samples. Our 424 results have shown that VFA components served different functions with 425 propionate being used for sulfate reduction and butyrate being used for syntrophy 426 and methanogenesis. The presence of SRB in oil field samples is often evaluated by 427 MPN assays in which lactate is used as the electron donor for sulfate reduction 428 (31). Because propionate is readily used by SRB in the MHGC field, we compared 429 MPNs in lactate-containing media with those obtained in propionate-containing 430

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media. The data for 13 samples showed that log MPN obtained with propionate 431 was linearly related with log MPN for lactate (Fig. S5: r2=0.86). Although lactate-432 utilizing SRB were more numerous than propionate-utilizing SRB, the difference 433 was less than 10-fold at high MPN (107/ml). 434 435 Discussion 436

437 The effect of nitrate on sulfide-producing oil field consortia has been 438 studied in microcosms (4, 7-9, 11, 26, 43, 49, 50), bioreactors (14, 16, 29, 32) and 439 in the field (3, 4, 5, 11, 15, 43). Despite this wealth of information some 440 misconceptions remain. One of these is that the inhibitory effect of nitrate is due to 441 competitive exclusion (8) in which hNRB outcompete SRB for the same electron 442 donors. This is typically demonstrated in bottle tests (microcosms) with a high 443 concentration of nitrate relative to available electron donor (e.g. VFA). Because 444 nitrate is always reduced first, no sulfate reduction is observed under these 445 conditions. However, in the presence of excess VFA, nitrate reduction will be 446 followed by sulfate reduction giving only transient inhibition as observed here in 447 microcosms and in bioreactors (Fig. 4, Figs. S2-S4), and as shown elsewhere (26, 448 29, 32). Excess electron donor is expected in oil fields and transient inhibition was 449 indeed demonstrated in the MHGC field, when injection water with 1 mM sulfate 450 was continuously amended with 2 mM nitrate (3, 4). Interestingly, competitive 451 exclusion applies in the presence of excess electron donor at high temperature 452 (>50oC), because nitrate reduction stops at nitrite under these conditions (49). 453

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Nitrite is a very strong and specific SRB inhibitor (39), which targets the sulfide-454 producing dissimilatory sulfite reductase (Dsr). 455 A second misconception, related to the first, is that when VFA serve as 456 electron donor for the reduction of nitrate and sulfate in oil fields, the nitrate dose 457 required to prevent souring can be derived from determining the concentrations 458 of VFA in oil field waters. VFA are constantly being formed from syntrophic 459 degradation of oil (16, 19, 20) and their concentrations reflect differences in rates 460 of formation and use. Accumulation of up to 6 mM acetate was demonstrated in a 461 bioreactor in which heavy MHGC oil served as sole electron donor for the 462 reduction of sulfate and nitrate (16). VFA accumulation was credited to 463 preferential use of H2, formed during water-mediated fermentation of 464 hydrocarbons (equations i and ii), by oil field SRB (16). 465 Thus VFA are formed from oil components and are significant electron 466 donors. As shown here, VFA have some specificity for different physiological 467 processes. When present in excess, acetate and propionate were used 468 preferentially for nitrate reduction, propionate for sulfate reduction and butyrate 469 for methanogenic fermentation (Figs. 1-3 and S1-S3). Interestingly, although the 470 kinetics of VFA use and associated nitrate and/or sulfate reduction were much 471 slower in microcosms than in the bioreactor, the effect of nitrate dosing was the 472 same. In the bioreactor, injection of 0, 4, 8 and 13 mM nitrate resulted in 1.7, 2, 1.8 473 and 0.7 mM of aqueous sulfide, whereas in microcosms this was 2, 2, 1.3 and 0.7 474 mM of aqueous sulfide. Plots of the residual concentration of sulfide in the 475 bioreactor effluent and of the injected nitrate concentration (Fig. 6), indicated that 476

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higher nitrate doses were needed to limit souring (the production of sulfide) with 477 increased injection of VFA. The dose needed to eliminate all sulfide was 478 approximately 80% of that required to oxidize all injected VFA to CO2 (Fig. 6, 479 arrows; equations xiv, xv and xvi). These results indicate that 20% of the VFA 480 were used as the carbon source for biomass formation. 481 The microbial community compositions (Table 3) indicated that 482 Desulfobulbus was the prominent SRB with a fraction of 1.8-14.0%, except in the 483 amplicon library from microcosm 7PWb_S2V3, where it was only 0.2%. 484 Desulfobulbus was also the most prominent SRB in a previous bioreactor study 485 (32). Many species of this genus are able to use propionate as electron donor for 486 sulfate reduction (47). This is consistent with the physiology of these enrichments, 487 which showed use of propionate as electron donor for sulfate reduction in 488 microcosms 2PW_S2V3, 4PW_S2V3, 7PWa_S2V3 and 13PW_S2V3, but not in 489 7PWb_S2V3 (Figs. 1 and S1). Microcosm 13PW_S2V3 had a high proportion of 490 Syntrophomonas (Table 3: 14.0%), which is a potential syntrophic butyrate-491 degrader forming hydrogen, formate and acetate in syntrophic coculture with 492 methanogens (17-19, 48). Enrichment E_S2B3 with 2 mM sulfate and 3 mM 493 butyrate had a high fraction of Syntrophomonas (12.1%) and of the methanogen 494 Methanobacterium (28.4%), indicating that this culture supported itself by 495 methanogenic, syntrophic butyrate degradation at the time of sampling. This 496 culture had the highest proportions of the SRB Desulfovibrio and Desulfofustis 497 (Table 3: 0.6%), indicating that a syntrophic coculture of Syntrophomonas and 498

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these SRB may have catalyzed butyrate-dependent sulfate reduction earlier in this 499 culture. 500 Amplicon libraries for enrichments with 2 mM sulfate, 3 mM VFA and 4, 8 501 or 13 mM nitrate had high fractions of the hNRB Paracoccus (49-71.1%) and 502 Thauera (8.4-42.2%). The fraction of Paracoccus increased with increasing 503 concentrations of nitrate. Methanogens were minor components in enrichments 504 with nitrate (Table 3). Desulfobulbus was also only a minor component (0-1%) in 505 these libraries and Syntrophomonas was not found. These results are consistent 506 with the physiological data obtained for these microcosms. 507

VFA concentrations in oil field produced waters do not define the nitrate 508 dose needed to eliminate souring, as VFA are constantly formed from syntrophic 509 degradation of oil and some hNRB can couple reduction of nitrate to oxidation of 510 selected oil components, such as toluene (4, 50). Collectively our data indicate that, 511 when water amended with sulfate and nitrate is injected into the MHGC field, 512 nitrate is reduced first with toluene (4, 50), other oil components and VFA as 513 electron donors. Sulfate is reduced further along the flow path with H2, propionate 514 and other products from syntrophic oil degradation as substrates. Syntrophic 515 degradation substrates, including butyrate and acetate, are used for further 516 syntrophy and methanogenesis, which occurs even deeper in the reservoir. In the 517 MHGC field the overall outcome of this is that produced waters are free of nitrate 518 and sulfate but contain some acetate (this study: 0.12 mM) and sulfide (3, 4). This 519

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is similar to what was observed in microcosms and in bioreactor effluents in this 520 study. The latter had higher acetate concentrations especially following nitrate 521 treatments, which appeared to cause long-term inhibition of acetotrophic 522 methanogenesis. 523 524 Acknowledgements 525 526 This work was supported through a Natural Sciences and Engineering Research 527 Council (NSERC) Industrial Research Chair awarded to GV, which was also 528 supported by Baker Hughes, BP, Computer Modeling Group Ltd., ConocoPhillips, 529 Intertek, Dow Microbial Control, Enbridge, Enerplus Corporation, Oil Search 530 Limited, Shell Global Solutions International, Suncor Energy Inc., Yara Norge and 531 Alberta Innovates Energy and Environment Solutions (AIEES). We are grateful to 532 Rhonda Clark for administrative support and to Baker Hughes and Enerplus 533 Corporation and Suncor for providing the field samples used in this study. 534 535 References 536 1. Sunde E, Torsvik T. 2005. Microbial control of hydrogen sulfide production in 537 oil reservoirs, p 201–213. In Ollivier B, Magot M (ed), Petroleum microbiology. 538 ASM Press, Washington, DC. 539

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2. Vance I, Thrasher DR. 2005. Reservoir souring: mechanisms and prevention, 540 2005, p 123–142. In Ollivier B, Magot M (ed), Petroleum microbiology. ASM 541 Press, Washington, DC. 542 3. Voordouw G, Grigoryan AA, Lambo A, Lin SP, Park HS, Jack TR, Coombe D, 543 Clay B, Zhang F, Ertmoed R, Miner K, Arensdorf JJ. 2009. Sulfide remediation 544 by pulsed injection of nitrate into a low temperature Canadian heavy oil 545 reservoir. Environ Sci Technol 43:9512-9518. 546 4. Agrawal, A.; Park, H.S.; Nathoo, S.; Gieg, L.M.; Jack, T.R.; Miner, K.; Ertmoed, 547 R.; Benko, A. and Voordouw, G. Toluene depletion in produced oil 548 contributes to souring control in a field subjected to nitrate injection. Environ. 549 Sci. Technol. 2012, 46, 1285-1292. 550 5. Telang, A. J.; Ebert, S.; Foght, J. M.; Westlake, D. W. S.; Jenneman,G. E.; 551 Gevertz, D.; Voordouw, G. The effect of nitrate injection on the microbial 552 community in an oil field as monitored by reverse sample genome probing. 553 Appl. Environ.Microbiol. 1997, 63, 1785–1793. 554 6. Greene, E. A.; Hubert, C.; Nemati, M.; Jenneman, G. E.; Voordouw, G. Nitrite 555 reductase activity of sulfate-reducing bacteria prevents their inhibition by 556 nitrate-reducing, sulfide-xidizing bacteria. Environ. Microbiol. 2002, 5, 607–557 617. 558

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45. Imachi, H.; Sakai, S.; Nagai, H.; Yamaguchi, T.; Takai, K. Methanofollis 695 ethanolicus sp. nov., an ethanol-utilizing methanogen isolated from a lotus field. 696 Int.J. Evol. Micr. 2009, 59, 800-805. 697 46. Zhu, J.X.; Liu, X.L.; Dong, X.Z. Methanobacterium movens sp. nov. and 698 Methanobacterium flexile sp. nov., isolated from lake sediment. Int.J. Evol. Micr. 699 2011, 61, 2974-2978. 700 47. Widdel F, Pfennig N. 1982. Studies on dissimilatory sulfate-reducing bacteria 701 that decompose fatty acids. II. Incomplete oxidation of propionate by 702 Desulfobulbus propionicus gen. nov., sp. nov. Arch. Microbiol. 131:360-365. 703 48. Liu, P.F.; Qiu, Q.F.; Lu, Y.H. Syntrophomonadaceae-affiliated species as active 704 butyrate-ultilizing syntrophs in paddy field soil. Appl. Environ. Microbiol. 2011, 705 77, 3884-3887. 706 49. Fida, T.T.; Chen C.; Okpala G.; Voordouw G. Implications of limited 707 thermophilicity of nitrite reduction for control of sulfide production in oil 708 reservoirs. Appl Environ Microbiol. 2016, . pii: AEM.00599-16. 709 50. Lambo, A.J.; Noke, K.; Larter, S.R.; Voordouw, G. Competitive, microbially-710 mediated reduction of nitrate with sulfide and aromatic oil components in a 711 low-temperature, western Canadian oil reservoir. Environ. Sci. Technol. 2008, 712 42, 8941-8946. 713 714

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Table and Figure Captions 715 716 Table 1 Concentrations (mM) of the indicated analytes in the bioreactor effluent 717 during stages I to XIII1). 718 719 Table 2 Sequence accession (SA) numbers, number of quality controlled (QC) 720 reads and derived numbers of operational taxonomic units (OTUs) and taxa for 22 721 amplicon libraries. The Shannon index and the fraction (%) of bacterial and 722 archaeal reads are also indicated. 723 724 Table 3 Taxa in 16S rRNA libraries from microcosms and bioreactor effluents, 725 presented in the same order as in the dendrogram (Fig. 5A)1). The numbers shown 726 are fractions (%) of quality controlled pyrosequencing reads (Table 2). Fractions 727 in excess of 1% are in bold. The table is ranked according to average fraction. 728 Missing entries (e.g. #15-19) could not be assigned at the genus level. 729 730 Figure 1 Sulfate reduction (yellow) and methanogenesis (orange) in CSBA-S 731 medium with 3 mM VFA inoculated with produced water 2PW (A-C; microcosm 732 2PW_S2V3_18) or 4PW (D-F; microcosm 4PW_S2V3_19). The concentrations of 733 sulfate, sulfide and VFA in the aqueous phase and of methane in the headspace are 734

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shown as a function of time. The data are averages of duplicate incubations with 735 standard deviations shown when these exceed the size of the symbols. The 736 numbered arrows indicate sampling for DNA isolation. 737 738 Figure 2 Sulfate reduction (yellow) and methanogenesis (orange) in CSBA-S 739 medium with 3 mM of propionate (A, B, C; microcosm E_S2P3_15) or butyrate (D, 740 E, F; microcosm E_S2B3_16) and inoculated with 2PW enrichment. (D, E, F) Colour 741 overlap indicates that both processes occur simultaneously. The concentrations of 742 sulfate, sulfide, propionate, butyrate and acetate in the aqueous phase and of 743 methane in the headspace are shown as a function of time. The data are averages 744 of duplicate incubations with standard deviations shown when these exceed the 745 size of the symbols. The numbered arrows indicate sampling for DNA isolation. 746 747 Figure 3 Syntrophic butyrate degradation and coupled methanogenesis (orange) 748 in CSBA medium with 3 mM VFA in microcosm E_V3_17 . The concentrations of 749 aqueous acetate, propionate and butyrate and of headspace methane are shown as 750 a function of time. The data are averages of duplicate incubations; standard 751 deviations did not exceed the size of the symbols. 752 753

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Figure 4 Effect of nitrate (either 0, 2, 4, 6, 8 or 13 mM) on souring in an up-flow, 754 packed-bed bioreactor injected with CSBA medium with 2 mM sulfate and electron 755 donors as follows: stage I to V, 3 mM each of acetate, propionate and butyrate; 756 stage VI to IX: 3 mM acetate and 3 mM propionate; stage X to XIII: 3 mM 757 propionate. The concentrations of sulfate, sulfide, nitrate, nitrite, acetate, 758 propionate and butyrate in the effluent are shown as a function of time. The 759 numbered arrows indicate sampling for DNA isolation. 760 761 Figure 5 Analysis of 16S rRNA gene sequences (reads) in 22 amplicon libraries, 762 identified by name and number as in Table 2. (A) Relational tree; the bar 763 represents 0.1 substitutions per nucleotide position. Clades I and II are indicated, 764 as explained in the text. (B) Fractions of phyla (% of total reads) and (C) fractions 765 of classes (% of Proteobacteria reads). 766 767 Figure 6 Effect of increased injection of nitrate on the concentration of produced 768 sulfide in bioreactor effluents. Injected electron donors were 3 mM each of 769 propionate, acetate and butyrate (diamonds), of propionate and acetate (squares) 770 and of propionate only (triangles). The nitrate doses required to oxidize these 771 concentrations of electron donors to CO2 are indicated (↓). 772

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Table 1 Concentrations (mM) of the indicated analytes in the bioreactor effluent during stages I to XIII1).

Stage (days) Input2) Analytes in effluent (mM) DNA Sulfate Sulfide Nitrate Nitrite Acetate Propionate day 3)

I (1-13) 3 VFA 0.03±0.08 1.63±0.08 - - 4.04±0.2 0 II (14-22) 3 VFA, 4 NO3

- 0.01±0.01 2.05±0.02 0 0 8.57±0.11 1.06±0.1 III (23-35) 3 VFA, 8 NO3

- 0.28±0.12 1.87±0.08 0 0 6.09±0.13 0 IV (36-49) 3 VFA, 13 NO3

- 1.40±0.08 0.67±0.1 0 0 3.01±0.09 0 45 V (50-64) 3 VFA 0 2.08±0.09 - - 9.17±0.42 1.9±0.1 60 VI (65-83) 3 ace, 3 prop, 4 NO3

- 1.05±0.08 1.11±0.13 0 0 3.10±0.06 0 83 VII (84-95) 3 ace, 3 prop, 8 NO3

- 1.67±0.07 0.47±0.1 0 0 1.19±0.17 0 95 VIII (96-112) 3 ace, 3 prop, 13 NO3

- 2.2±0.05 0 2.4±0.3 7.2±0.4 0 0 111 IX (113-125) 3 ace, 3 prop 0.49±0.06 1.72±0.05 - - 5.01±0.17 0.72±0.09 X (127-134) 3 prop 0 2.01±0.03 - - 2.48±0.07 0.02±0.02 XI (136-147) 3 prop, 4 NO3

- 1.70±0.10 0.47±0.11 0 0 0.57±0.12 - 145 XII (149-163) 3 prop, 6 NO3

- 2.21±0.14 0.05±0.05 0 0.04±0.06 0 0 161 XIII (164-187) 3 prop, 2 NO3

- 0.7±0.07 1.39±0.07 0 0 1.82±0.05 0 1) Data are shown in Fig. 4; the flow rate was 42 mL/d (1.5 PV/d); 2 mM sulfate was injected throughout; butyrate was not detected. 2) Numbers are concentrations in mM; ace is acetate, prop is propionate. 3) Days on which effluent samples were collected for 454 sequencing.

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Table 2 Sequence accession SA numbers, number of quality controlled QC reads and derived numbers of operational taxonomic units OTUs and taxa for amplicon libraries. The Shannon index and the fraction % of bacterial and archaeal reads are also indicated. Library_name_

number1) Day or

inoculum2) SA number QC reads OTUs Taxa Shannon

index Archaea

(%) Bacteria

(%) BE_S2N13V3_1 45 SRR1555313 8338 31 52 1.19 0.6 99.4

BE_S2V3_2 60 SRR1555314 3743 65 65 2.17 8.6 91.3 BE_S2N4A3P3_3 83 SRR1555315 7876 28 55 1.25 1.3 98.7 BE_S2N8A3P3_4 95 SRR1555331 5371 29 43 1.03 0.0 100.0

BE_S2N13A3P3_5 111 SRR1555323 6551 75 86 2.39 0.1 99.9 BE_S2N4P3_6 145 SRR1555332 7832 27 49 0.57 0.1 99.9 BE_S2N6P3_7 161 SRR1555334 6467 41 53 1.43 0.0 99.9

E_S2V3_8 E2PW SRR1555316 7283 18 25 0.75 77.4 22.6 E_S2N4V3_9 E2PW SRR1555320 5041 27 35 1.07 2.0 98.0 E_S2N8V3_10 E2PW SRR1555321 12006 19 38 0.90 0.1 99.9

E_S2N13V3_11 E2PW SRR1555322 9291 22 36 1.19 0.1 99.9 E_S2N4V3_12 E2PW SRR1555317 8717 33 47 1.10 10.6 89.4 E_S2N8V3_13 E2PW SRR1555318 10728 26 32 0.82 0.3 99.7

E_S2N13V3_14 E2PW SRR1555319 1831 32 28 1.02 1.3 98.7 E_S2P3_15 E2PW SRR1555328 7603 31 49 1.12 5.4 94.6 E_S2B3_16 E2PW SRR1555329 7190 31 30 1.32 30.3 69.7 E_V3_17 E2PW SRR1555330 7129 21 25 1.15 58.1 41.9

2PW_S2V3_18 2PW SRR1555324 7773 55 56 1.62 88.0 12.0 4PW_S2V3_19 4PW SRR1555325 6860 67 67 2.33 62.6 37.5 7PWa_S2V3_20 7PW SRR1555326 9040 70 64 2.08 81.6 18.4 7PWb_S2V3_21 7PW SRR1555333 12261 41 58 1.75 64.5 35.5 13PW_S2V3_22 13PW SRR1555327 6208 55 49 1.77 62.0 38.0 Samples were from bioreactor effluent BE or from microcosms. )nputs were: S, sulfate; N, nitrate; V, VFA; A, acetate, P, propionate, B, butyrate; the numbers are the concentrations in mM. All had mM sulfate S , except , which only had mM VFA. Day on which the BE sample was taken or the inoculum used to start the microcosm, either produced water PW, PW, PW or PW or PW enrichment E PW .

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Table 3 Taxa in S rRNA libraries from microcosms and bioreactor effluents, presented in the same order as in the dendrogram Fig. A . The numbers shown are fractions % of total pyrosequencing reads. Fractions in excess of % are in bold. The table is ranked according to average fraction. Missing entries e.g. # - 9 could not be assigned at the genus level.

1)Library numbers to are as indicated in Figures to and Figure S to S .

Tao

#

Ph lu or lass Ge us BE_S

NP

_

BE_S

NA

P_

E_S

P_

E_V

_

E_S

V_

E_S

B_

BE_S

NA

P_

BE_S

V_

BE_S

NA

P_

E_S

NV

_

E_S

NV

_

E_S

NV

_

BE_S

NV

_

E_S

NV

_

E_S

NV

_

E_S

NV

_

BE_S

NP

_

PW_S

V_

PWa_

SV

_

PW_S

V_

PW_S

V_

PW_S

V_

Average

Betaproteo a teria Thauera . . . . . . . . . . . . . . . . . . . . . .Alphaproteo a teria Para o us . . . . . . . . . . . . . . .Ga aproteo a teria Pseudo o as . . . . . . . . . . . . . . . . . . . . . . .Metha o a teria Metha o a teriu . . . . . . . . . . . . . . . . . . .Metha o i ro ia Metha o al ulus . . . . . . . . . . . . . . . . .Metha o i ro ia Metha osaeta . . . . . . . . . . . . . . . . .Metha o i ro ia Metha o orpus ulu . . . . . .Deltaproteo a teria Desulfo ul us . . . . . . . . . . . . . . . . . . . .Deferri a teres Geovi rio . . . . . . . . . . . . .Fir i utes S tropho o as . . . . . . . . . . .Fir i utes A holeplas a . . . . . . . . . . . . . . . . . . . . . . .Metha o i ro ia Metha ofollis . . . . . . . . . . .Spiro haetes Spiro haeta . . . . . . . . . . . . . . . . . . . . . . .Fir i utes Fusi a ter . . . . . . . . . . . . . . . . .Metha o i ro ia Metha o ulleus . . . . . . . . . . . . . . . . .Ga aproteo a teria Ste otropho o as . . . . . . .:S ergistetes Ther a aerovi rio . . . . . . . . . . . . . . . . . . . . . . .Metha o i ro ia Metha osar i a . . . . . . .Spiro haetes Lepto e a . . . .Betaproteo a teria Der ia . . . . . .Fir i utes A ida i o a ter . . . . . . . . . . . . . . .Alphaproteo a teria Rhizo iu . . . . . . . . .Ther otogae Kos otoga . . . . . . . . . . . . . . . . . . .Metha o i ro ia Metha ospirillu . . . . . .Fir i utes A aerovora . . . . . . . . . . . . . . . . . .Betaproteo a teria Diaphoro a ter . . . . .Metha o i ro ia Metha oli ea . . . . . . . . . . . . . .Alphaproteo a teria Sphi go o as . . . . . . . . . .Fir i utes Sedi e ti a ter . . . . . . . . . . . . . . .Deltaproteo a teria Desulfo i ro iu . . . . . .Deltaproteo a teria Desulfovi rio . . . . . . . . .Alphaproteo a teria Brevu di o as . . . . . .Fir i utes Er sipelothri . . . . . . . . . . .Deltaproteo a teria Desulforegula . . . . . . . . .Deltaproteo a teria Desulfofustis . . . . . .

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