Micronutrient supplements control metabolism and shape microbiome composition in vitro
To investigate if micronutrients can modulate the microbiome composition of the microcosms for top-down engineering in MEOR, our in vitroscreening system was used to track metabolism and community composition under various micronutrient supplements. In vitro screening focused on Well D because it was one of the two most responsive wells. Moreover, Well G was heavily dominated by one genus (Malaciobacter , class Campylobacteria) (Supplemental Figure 1 ), with less microbial diversity to adapt to the micronutrient conditions. Again, Well D was stimulated with molasses and supplemented with a variety of micronutrients including chloride (NaCl and KCl), phosphate (K2HPO3 and KH2PO3), nitrate (KNO3), molybdate (Na2MoO4), and a combination of both nitrate and molybdate. Chloride salts were used as a negative control while phosphate salts were tested as a phosphate source, which have been added in other MEOR studies.9,31,36 To reduce sulfate reduction, nitrate and molybdate salts were tested. Nitrate is a competing electron acceptor in place of sulfate in several sulfur-reducing bacteria,11,39–41 while molybdate has been shown to inhibit H2S generation in different aqueous and marine contexts.40–43
Gas and organic acid production varied strongly as a function of micronutrient supplementation (Figure 2A, Supplemental Figure 3 ). Nitrate- and molybdate-supplemented communities from Well D produced significant amounts of pressure. The cumulative effects of combination treatments were additive, resulting in the most observed pressure generation. Similarly, nitrate dropped the pH of the culture the most (to a pH ~4) where the molybdate treatment had a slightly smaller effect (Supplemental Figure 3B). These results suggest that chloride and phosphorous do not alter microbial metabolism in the microcosms and that nitrate best stimulated the production of gases and acids for MEOR. There was some additional benefit to adding molybdate in combination with nitrate as it generated the most gas and one of the larger pH changes. Because only select microbes can convert nitrate into N2 and/or ammonia, or use it as a nitrogen source for synthesis of DNA and amino acids, supplemented nitrate will confer a competitive advantage to specific microbes in an otherwise nitrogen-limited environment such as an oil well. In turn, we expect that the differences in the metabolic outputs (Figure 2A ) are reflective of the differences in the microbial community composition as a result of the added compounds.