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.