Hydrogen sulfide production reduced or suppressed by nitrate and molybdate treatments
To assess if these supplements resulted in metabolic changes that reduce or inhibit H2S production, we measured H2S in the headspace of the in vitro microcosms from Well D. Untreated microcosms from Well D generated, on average, approximately 220 ppm (+/- 35) of headspace H2S after 10 days of cultivation (Figure 3B ). Nitrate-treated microcosms reduced the amount of H2S to ~150 ppm on average, while the molybdate and combined treatments limited the H2S to approximately 2 ppm or less. Taken together with the sequencing data, this suggest that the nitrate treatment here likely allowed for competitive metabolism of nitrate over sulfate as has been reported to decrease H2S production.11,41,43 Biostimulation with nitrate alone, however, was not able to completely inhibit H2S production and enriched for specific organisms that can utilize both nitrate and sulfate,35 likeSulfurospirillium, 11,46. We hypothesize that these taxa may have either simultaneously utilized both electron acceptors or switched to sulfate metabolism once the nitrate was consumed, resulting in modest decreases in H2S production. Additionally, nitrate supplementation generally allows other species an advantage over the sulfate-reducing bacteria likeDesulfovibrio and Dethiosulfovibrio species, which were present only in our controls, but does not directly inhibit these organisms that contribute to the production of H2S. On the other hand, molybdate proved very effective at inhibiting H2S generation both on its own and in combination with nitrate. Molybdate has been proposed to inhibit H2S generation by binding to the sulfate adenylyltransferase (Sat, ATP sulfurylase) complex and blocking the generation of ammonium persulfate precursor to sulfide while also depleting the ATP pools of the cell.43 We believe that this form of selection may explain why the molybdate effectively inhibits H2S formation while also limiting the number of H2S generating organisms in the community, which is also consistent with our decreased alpha diversity finding (Figure 2 ). Based on this understanding, molybdate strongly inhibits the sulfate-reducers, which allows other microbes to ferment the molasses to organic acids and gases for MEOR, whereas nitrate only limits H2S production through competition until it is all consumed, after which the presence of microbes that can consume both nitrate and sulfate may dominate.