Figure 2 – Non-essential nutrients stimulate metabolism and
manipulate the microbiome composition. A) Pressure generated in
microcosms from Well D & G when treated with various nutrient
supplements; T-tests performed with respect to None. B) Diversity score
(Pielou’s evenness) of microcosms after treatment with micronutrients
(days 3, 4, 5 and 7). C) Unweighted Unifrac PCoA plot of Well D
microcosms after micronutrient supplementation; shapes represent days,
colors indicate treatment group: spheres = day 1, diamonds = day 3, star
= day 4, rings = day 5, cones = day
7. Error bars represent standard
deviation, n=3; * = p < 0.05, ** = p < 0.005, and
*** = p < 0.001.
While the fate of nitrate is ultimately determined by the microbial
community, we found that nitrate and molybdate supplements shifted the
microbial community composition in different ways. Nitrate, molybdate,
and the nitrate-molybdate combination treatments provide a selective
pressure that selects for or enriches specific species reducing the
alpha diversity (Shannon index) of the cultures from Well D
(Figure 2B ). However, the treatments do not enrich for the same
species - the beta diversity of the communities as estimated by
principal coordinate analysis (PCoA) analysis of the 16S rRNA gene
composition (Figure 2C ) reveal distinct groups of data points.
Moreover, the time evolution of these communities diverge over the first
three days (spheres to diamonds) as a result of their different
selective pressures. However, the combined molybdate and nitrate
treatment converges on that of molybdate alone (Figure 3C )
suggesting that the selection pressure applied by molybdate is stronger
than that of nitrate and, ultimately, determines microbial metabolism
and community structure. Hydrogen sulfide inhibition in the literature
is more sensitive to molybdate than nitrate42,43,
adding further support for the strong selective pressure of molybdate.
To determine which taxa were specifically selected against and enriched
in these microcosms, we evaluated
the 16S rRNA gene composition of the microbial community. Interestingly,
we found that after Day 1 of cultivation, there was little effect across
the treatments with only a slight increase in theEnterobacteriaceae in the molybdate and combined treatments
(Supplemental Figure 4 ). After Day 3, however, the treatments
varied noticeably from the untreated microcosms (Figure 3A ).
For example, nitrate microcosms saw a bloom of Sulfospirillium in
days 3 and 4, while the molybdate and combined treatments each saw a
bloom in Enterobacteriaceae sp. and Lachnoclostridiumsp., which are MEOR-relevant bacteria. ManySulfospirillium species contain both nitrate- and sulfur-reducing
pathways, so it is reasonable to expect that the nitrate substrate
allowed these species to outgrow organisms that could not use this
nutrient. Similarly, a few Enterobacteriaceae have been found to
reduce molybdate44,45 which may have provided a
similar advantage to these microbes. At the same time, molybdate is
structurally similar to sulfate and has been shown to inhibit the growth
of H2S producers43 which may account
for the absence of Desulfovibrio , Dethiosulfovibrio ,Sulfospirillium , and other sulfate reducing bacteria in the
molybdate and combined treatment microcosms. After 7 days, the cultures
primarily consisted of members of Bacteroides andLachnoclostridium regardless of treatment suggesting that the
community may have shifted to scavenge or use other nutrient sources to
survive. Similar progressions of microcosms toward primarily
Bacteroidetes and Clostridia have been seen in previous
studies36 suggesting these groups of bacteria have the
advantage over other organisms in anaerobic consortia when the initial
nutrients have been depleted at the end of the cultivation.
Additionally, no known H2S reducing bacteria were
detected with molybdate and combination treatments suggesting that the
effects of the molybdate were effective over this whole time-course of
the cultivation. In contrast, Sulfurospirillium species were
present at several timepoints in the nitrate microcosms. In a previous
H2S inhibition study, molybdate was also found to be the
strongest inhibitor of H2S reducing
bacteria43 from marine enrichment cultures. Where
previous findings suggested that the addition of these inhibitors had no
effect on the composition of the microbial
community,42 we find that they can drastically shape
the community composition and metabolism. However, our findings that
Well D and G (Supplemental Figure 4) respond differently to the
nitrate and molybdate inhibitors further support previous assertations
that the efficiency of H2S inhibitors depends on the
composition of the microbiome.42,43 While changes in
microbial community composition indicate a response to our top-down
engineering, they are not definitive of metabolic output, which must be
characterized directly.