Figure 3 – Top-down nutrient treatments inhibit and enrich
specific microbes and their metabolism. A) Microbiome composition of
microcosms with and without treatment across days 3 – 14. B)
H2S headspace measurements of microcosms after 14 days
of treatment, T-tests performed with respect to None except where
indicated. Error bars represent standard deviation, n=4 (except for
“None”, n=3); * = p < 0.05, ** = p < 0.005, and
*** = p < 0.001. Enterobacteriaceae * andLachnospiraceae * indicate unspecified genera within these two
Families.
To evaluate how persistent the impacts of these treatments are, we
passaged the communities into media that contained the same, different,
or no supplements. After three days of growth, microcosms were passaged
three separate times into fresh media with and without supplements.
Subcultures of untreated communities with either no supplement or only
nitrate produced moderate amounts of H2S although lower
than the untreated parent culture, while molybdate treatment of
untreated subcultures matched that of the initial molybdate-treated
cultures with little to no H2S formation after the
passages (≤7 ppm H2S) (Figure 4, Supplemental
Figure 5A ). When cultures were passaged from nitrate treatments and
selection pressure was removed or kept the same (Figure 4B ; no
supplement or NO3-1 only,
respectively), the H2S production resumed and remained a
little lower than the level of the parent culture (~100
ppm). This suggests that the nitrate treatment may have enriched for
H2S-reducing bacteria or at best suppressed their
metabolism initially. However, if nitrate selection is applied to
molybdate cultures, H2S levels remained below that of
nitrate treatment of the native microbiome. Therefore, we suspect that
the reduced alpha diversity of molybdate treatment (Figure 2 )
reflects a direct inhibition or loss of many of the sulfur-reducing
species, which are either temporarily limited or possibly enriched by
the nitrate treatment. As before, molybdate was able to limit the
H2S production of the sulfate-reducing communities to
very low levels (Figure 4B ), further demonstrating that it was
effective even against actively H2S-generating
communities like those from the nitrate-treated group that otherwise
produce H2S (Figure 4C ). Molybdate-treated
subcultures, regardless of parent culture, generated less
H2S (Supplemental Figures 5A and D ).
Interestingly, when molybdate microcosms were subcultured and had their
selection pressure removed (Figure 4C , Supplemental
Figure 5B and C ), H2S was produced although at low
levels (<25 ppm) with each sequential passage producing more
H2S demonstrating that continuous application of this
selection pressure is needed to permanently suppress
H2S. This suggest that H2S-generation is
inhibited by molybdate, but the associated microbes may still be present
at low levels. Lastly, subcultures of microcosms generated from
combination treatment (Figure 4D ) behaved like molybdate
subcultures for combined supplement and molybdate passages. However,
after three passages, H2S generation of these
communities treated with nitrate began to return to higher levels. This
suggests that the supplemented nitrate substrate in the combination
treatment may have continued enriching for nitrate consumers,
specifically some that harbor both nitrate and sulfate reducing
pathways, so that sulfate reduction resumed once the nitrate was
consumed. Throughout the subculture cultivations, the microbial
community compositions were similar to their respective nutrient
screening microcosms which were dominated by Enterobacteriaceae,
Bacteroides, and Lachnoclostridium (Figure 3, Supplemental
Figure 6 ). Ultimately, the combination of molybdate and nitrate best
limits H2S (Supplemental Figure 5E ) and
provides some redundancy of inhibitors in the case one of the selection
pressures is resisted or lost; however, molybdate is the most important
for effectively limiting H2S production from these oil
well communities (Supplemental Figure 5D ). From this in
vitro screening system, we showed that oil well microbial communities
can be engineered and controlled from the top down using nitrate and
molybdate, structural analogs of sulfate, to inhibit sulfate-reducing
bacteria and H2S generation.