South Dakota School of Mines & Technology (SDSMT) collaborated with Montana State University (MSU) and University of Oklahoma (OU) to fill a large knowledge gap in the field of methane regulation in extreme environments prevalent in our jurisdictions as well as in many others in the US. To fill this knowledge gap, the BuG ReMeDEE consortium was formed, and Genome to Phenome (GtP) expertise were combined from three institutions into an integrated research and education effort, focusing on biogeochemical mechanisms of methane oxidation in extreme environments. This consortium provided a collaborative infrastructure that enabled the three institutions to use the Sanford Underground Research Facility (SURF) in SD and Yellowstone National Park (YNP) as testbeds for extreme environments in the deep biosphere and thermal systems, respectively. The consortium goals were to i) Establish multidisciplinary, inter-jurisdictional infrastructure to investigate complex biogeochemical mechanisms involved during methane cycling in extreme environments; ii) Facilitate collaboration among investigators across disciplines and across three jurisdictions to coalesce GtP expertise; iii) Edit genomes of previously unexplored and novel methanotrophs for phenotypic improvement in methane uptake and oxidation and its conversion into useful products; iv) Recruit eminent researchers to increase competitiveness in GtP research at three institutions, and v) Develop career pathways for junior faculty, and research scientists, graduate students, and under-represented Native American students.

Products and consortium sustainability: The BuG ReMeDEE consortium participants have been awarded by 39 proposals ($76,300,412) during the project. The participants published 72 peer-reviewed papers, 21 book chapters, edited 8 books, made 290 conference presentations, and 18 keynote and invited talks.

Intellectual Merit: This RII T2 project provided insights on methane regulation in previously unexplored deep and extreme environments (SURF, YNP and various local sites in Norman, OU), as well as developed new innovative biological routes for converting methane into biofuels and value-added products (e.g., methanol, biopolymers, and bioelectricity). Methanotrophs were isolated from methane oxidizing communities existed in SURF and YNP as well as Zodletone and other Oklahoma sites. We obtained six novel microbial isolates: four strains of Methylomonas, one strain of Methylocystis, and one strain of Methylovulum. These strains are being characterized with respect to several phylogenomic and physiological characteristics and will formally be described and named in the International Journal of Systematic and Evolutionary Microbiology. A microbial diversity and geochemistry census of geothermal features in Yellowstone National Park was performed. A benchmark study targeted at revealing microbial physiology by combining bioorthogonal non-canonical amino acid tagging (BONCAT) and fluorescence activated cell sorting (FACS) was provided. Methanotrophic communities in Yellowstone hot springs and metagenomic predictions in mesocosms were studied. Metabolic models for both Type I and Type II methanotroph metabolism were constructed. These in silico investigations are being used to predict methanotroph acclimation to oxygen limitation and analysis of effect of copper limitation on energy metabolism in methanotrophs.

We verified that the α-subunit of pMMO of Methylosinus trichosporium OB3b is a catalytic center for methane oxidation as opposed to β- or γ-subunit reported in the literature. We also identified 5 mutants of pMMO that will likely increase the methane oxidation rate in OB3b. One mutation could enhance methane oxidation 2-folds, and we also found that overexpression of mbnT could reduce the copper toxicity. This will likely increase growth rates thus increase methane oxidation rates. We tested biopolymer producing capability of a new isolate, Methylocystis sp. NLS7, and biopolymer characterization, and determined four candidate genes to knockout for overproduction of biopolymer. We demonstrated exoelectrogenic properties of three model methylotrophs and mixed microbial methanotrophs enriched from SURF. The efficiency of the extracellular electron transfer in R. sphaeroides biocatalyst was increased using plasma exfoliated graphene coated nickel electrodes. We also demonstrated exoelectrogenic properties of mixed microbial methylotrophs enriched from hydraulic fracturing flowback water.

Broader Impacts: The RII T2 has provided the education and training for 55 undergraduates, 31 graduates, 4 interns, 5 postdocs, and 10 early-career faculty. The BuG ReMeDEE team took deliberate steps to ensure effective collaboration and integration among research groups through active training and exercises. For example, senior faculty mentored junior faculty on mentoring graduate students. The graduate students hosted workshops in an REU-like programs (e.g., Summer Undergraduate Research Education as SDSMT and Native American Summer Science Program at OU) for undergraduates and Native American high school students. At all levels, the participants learned effective communication, teamwork and collaboration, large dataset analysis, and mentoring skills. The research and training methods were shared through unique means like symposia dedicated to methanotrophs, genome to phenome, and greenhouse gases. Metabolic models were developed for methanotrophs which could be used to predict the impact of methanotroph metabolism on climate change.

Last Modified: 11/28/2022
Modified by: Rajesh K Sani