(533w) Methanotrophic Activity in the Deep Environment: Enhancement of Methane Catalysis Rates

Methane is a powerful greenhouse gas, second to carbon dioxide in abundance and yet over 25-times as effective in trapping heat. The impact of methanefluxes from the deep biosphere of non-coal mines on microorganisms and the associated microbiome involved in methane oxidation has not been previously studied. There are still several intriguing questions about methanotrophs and their key methane monooxygenase (MMO) enzymes. MMO in methanotrophs are uniquely responsible for the oxidative catalysis of an important biochemical process that is the conversion of methane to methanol. There are two forms of MMO that exists in methanotrophs- cytoplasmic or soluble methane monooxygenase (sMMO) and transmembrane or particulate methane monooxygenase (pMMO). However, there is a controversy regarding the active site(s) in pMMO, specifically whether the active site(s) are in the pmoB (α subunit), pmoA (β subunit), or pmoC (γ subunit). Also, MMOs have lower affinity towards methane.

With our years of exploration in the deep biosphere (300 to 5,000 ft. levels) of the Sanford Underground Research Facility (SURF) at Homestake Gold Mine (Lead, SD, USA), we have confirmed the synergy among more than 44 different bacterial and archaea phyla in water, soil, sediments, and rock samples. Our results indicated the domination of Rhodobacteraceae, Alcaligenaceae, Bradyrhizobiaceae, Mycobacteriaceae, and Pseudonocardiaceae families in deep biosphere of SURF. Certain microbial members of these families had sMMO. The relative importance of Rhodobacteraceae in SURF and our results with Rhodobacter sp. showing their ability to carry out methanotrophy suggest that evolutionary groups other than conventional methanotrophs are involved in methane oxidation. These findings suggest that there may be more novel methanotrophs that are unidentified and virtually unstudied.

Using systems biology approaches, we unveiled whether the active site(s) of pMMO is located within the copper center or within the vicinity of the copper center using an obligatory aerobic methanotroph Methylosinus trichosporium OB3b as a model organism. To increase the catalysis rates of methane in pMMO of OB3b, selected amino acid residues interacting at the binding site of ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene were mutated. Based on screening the strain energy, docking energy, and physiochemical properties, five mutants were down selected, B:Leu31Ser, B:Phe96Gly, B:Phe92Thr, B:Trp106Ala and B:Tyr110Phe, which showed docking energies of -6.3, -6.7, -6.3, -6.5 and -6.5 kcal/mol, respectively as compared to the wild type (-5.2 kcal/mol) with ethylbenzene. These results suggest that these five mutants would likely increase methane oxidation rates compared to the wild type pMMO.

Furthermore, in our wet lab experiment, we did take into consideration the physiochemical method of enhancement of methane catalysis rate in Methylosinus trichosporium OB3b using supplementation of copper. A decade of methanotrophic research witnessed the significance of copper as a crucial metal in the differential expression of sMMO and particulate pMMO forms of MMOs. In certain methanotrophs, expressing both forms of MMOs, where the expression of either form is usually controlled by the concentration of Cu, following a mechanism referred as the “Cu switch”. The switch has an enthralling role play during the bioremediation of effluents as sMMO and pMMO degrades broad range organic substrates but diverges in specificity. In our study, Methylosinus trichosporium OB3b was used as a model bacterium to investigate the range of Cu concentration that triggers the expression of sMMO to pMMO, the ensuing effect of altering Cu concentration on the growth, methane oxidation capabilities, and the expression of individual genes within the methanobactin operon of OB3b. The Cu switch was observed to be regulated within 3 µM to 5 µM of Cu concentration with a strict increase in the methane consumption rates per day from 49 to 78 mg/L (at the mid exponential phase). The consumption of oxygen was observed to be proportional to the methane utilization rate and was highly controlled by the di-heme enzyme within the methanobactin operon. The ICP-MS data showed that Cu has been utilized at a greater rate when more Cu was supplemented in the media, and simultaneously the upregulation of methanobactin synthesis genes (mbnABC, and mbnT) signified that more Cu was harvested when pMMO genes were upregulated (≥ 5µM). A newly developed quantitative assay based on Naphthalene-Molisch principle were performed to distinguish between the sMMO and pMMO expressing cells. The gene expression results coincided with the assay where naphthol concentration was detected to be higher at low Cu concentration, and gradually decreased to no naphthol production where pMMO was expressed and acted as the sole methane oxidizer. However, the trend of increase in methane oxidation became inconsistent when the cells reached its toxicity level (40 µM). Therefore, to elucidate the mechanism and the involvement of crucial genes towards copper toxicity and copper homeostasis, more proteins were considered using physical protein-protein interaction. This resulted in 7 transporter protein, 3 cell wall biosynthesis/degradation protein, a Cu resistance operon (cop), and 20 hypothetical proteins may also be involved in the Cu response to the environment and Cu regulation within the organism.