Microbial diversity and methanogenic activity of Antrim Shale formation waters from recently fractured wells

Abstract

The Antrim Shale in the Michigan Basin is one of the most productive shale gas formations in the U.S., but optimal resource recovery strategies must rely on a thorough understanding of the complex biogeochemical, microbial, and physical interdependencies in this and similar systems. We used Illumina MiSeq 16S rDNA sequencing to analyze the diversity and relative abundance of prokaryotic communities present in Antrim shale formation water of three closely spaced recently fractured gas-producing wells. In addition, the well waters were incubated with a suite of fermentative and methanogenic substrates in an effort to stimulate microbial methane generation. The three wells exhibited substantial differences in their community structure that may arise from their different drilling and fracturing histories. Bacterial sequences greatly outnumbered those of archaea and shared highest similarity to previously described cultures of mesophiles and moderate halophiles within the Firmicutes, Bacteroidetes, and δ-and ε-Proteobacteria. The majority of archaeal sequences shared highest sequence similarity to uncultured euryarchaeotal environmental clones. Some sequences closely related to cultured methylotrophic and hydrogenotrophic methanogens were also present in the initial well water. Incubation with methanol and trimethylamine stimulated methylotrophic methanogens and resulted in the largest increase in methane production in the formation waters, while fermentation triggered by the addition of yeast extract and formate indirectly stimulated hydrogenotrophic methanogens. The addition of sterile powdered shale as a complex natural substrate stimulated the rate of methane production without affecting total methane yields. Depletion of methane indicative of anaerobic methane oxidation (AMO) was observed over the course of incubation with some substrates. This process could constitute a substantial loss of methane in the shale formation.

Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales

“…It is possible that these compounds are also assimilated as a nitrogen source (MMA) or are oxidized by Pseudomonas and Marinobacter (methanol) at earlier time points 17 . Although Methanohalophilus 16S rRNA genes have been reported from Antrim 8,9 and Burket/Geneseo 4 fractured shales, our genomic and metabolite findings identify the endogenous and exogenous sources of methyltrophic substrates, show their co-occurrence with methanogens, and confirm the metabolic pathways for methanogenesis.…”

“…Hydraulic fracturing (input, noted as T0) and shale-produced fluids were collected from well heads (days 3-14) and gas-fluid separators (49, 82 and 328 days), with fluids from well 1 used for more detailed metagenomics and NMR metabolite analyses here. For our Utica samples, injected fluids and produced fluids from gasfluid separators 4,8,9 were collected between July 2014 and May 2015 from an oil-gas well in Ohio, USA. The gas-fluid separators at the Marcellus and Utica sites had a capacity of ∼5,560 l, approximately half gas and half produced fluids (2,780 l).…”

“…Halanaerobium are the dominant members in produced fluids from Barnett, Marcellus, Burket/Geneseo and Antrim fractured shales 4,6,7,9 . Our shale-hosted Halanaerobium genomes also have the capacity to ferment amino acids (for example, alanine; Supplementary Discussion), sucrose, fructose, glucose and maltose (Supplementary Table 7).…”

“…Initial work by our group and others [4][5][6][7][8][9] used single marker gene analyses to identify microorganisms from several geographically distinct shale formations. These analyses showed similar halotolerant taxa in produced fluids several months after hydraulic fracturing.…”

Comparative analysis of metagenomes from three methanogenic hydrocarbon-degrading enrichment cultures with 41 environmental samples

“…Many previous studies have shown that microbial communities in methanogenic hydrocarbonassociated environments (for example, Chang et al, 2005;Kasai et al, 2005;Sun and Cupples, 2012;Wawrik et al, 2012;An et al, 2013) or enrichment cultures (Ficker et al, 1999;Fowler et al, 2012) can be highly diverse. Literature-wide compilations examining such communities (Gray et al, 2011;Strapoc et al, 2011;Kleinsteuber et al, 2012) have revealed that they frequently contain hydrogenotrophic (for example, Methanoculleus, Methanolinea, Candidatus Methanoregula, Methanospirillum), acetotrophic (for example, Methanosaeta), and/or methylotrophic methanogens (for example, Methanolobus; Wuchter et al, 2013). Deltaproteobacteria (for example, Syntrophus/ Smithella, Desulfovibrio, Geobacter) and Firmicutes (for example, Desulfotomaculum, Desulfosporosinus, Pelotomaculum) are commonly abundant bacteria, and are often identified as the putative hydrocarbonactivating organisms (Gray et al, 2011;Kleinsteuber et al, 2012).…”