See discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/283426415 Variations in microbial carbon sources and cycling in the deep continental subsurface ARTICLE in GEOCHIMICA ET COSMOCHIMICA ACTA · OCTOBER 2015 Impact Factor: 4.33 · DOI: 10.1016/j.gca.2015.10.003 READS 28 16 AUTHORS, INCLUDING: Gregory F Slater Barbara Sherwood Lollar McMaster University University of Toronto 116 PUBLICATIONS 2,226 CITATIONS 153 PUBLICATIONS 4,227 CITATIONS SEE PROFILE SEE PROFILE Maggie Lau Tullis Onstott Princeton University Princeton University 25 PUBLICATIONS 565 CITATIONS 10 PUBLICATIONS 14 CITATIONS SEE PROFILE SEE PROFILE All in-text references underlined in blue are linked to publications on ResearchGate, Available from: Gregory F Slater letting you access and read them immediately. Retrieved on: 23 November 2015 Available online at www.sciencedirect.com ScienceDirect Geochimica et Cosmochimica Acta 173 (2016) 264–283 www.elsevier.com/locate/gca Variations in microbial carbon sources and cycling in the deep continental subsurface Danielle N. Simkus a, Greg F. Slater a,⇑, Barbara Sherwood Lollar b, Kenna Wilkie b, Thomas L. Kieft c, Cara Magnabosco d, Maggie C.Y. Lau d, Michael J. Pullin e, Sarah B. Hendrickson e, K. Eric Wommack f, Eric G. Sakowski f, Esta van Heerden g, Olukayode Kuloyo g, Borja Linage g, Gaetan Borgonie g,1, Tullis C. Onstott d a School of Geography and Earth Sciences, McMaster University, Hamilton, Ontario, Canada b Department of Earth Sciences, University of Toronto, Toronto, Ontario, Canada c Department of Biology, New Mexico Institute of Mining and Technology, Socorro, NM, USA d Department of Geosciences, Princeton University, Princeton, NJ, USA e Department of Chemistry, New Mexico Institute of Mining and Technology, Socorro, NM, USA f Department of Plant and Soil Sciences, Delaware Biotechnology Institute, Newark, DE, USA g Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa Received 11 December 2014; accepted in revised form 6 October 2015; Available online 19 October 2015 Abstract Deep continental subsurface fracture water systems, ranging from 1.1 to 3.3 km below land surface (kmbls), were inves- tigated to characterize the indigenous microorganisms and elucidate microbial carbon sources and their cycling. Analysis of phospholipid fatty acid (PLFA) abundances and direct cell counts detected varying biomass that was not correlated with depth. Compound-specific carbon isotope analyses (d13C and D14C) of the phospholipid fatty acids (PLFAs) and carbon substrates combined with genomic analyses did identify, however, distinct carbon sources and cycles between the two depth ranges studied. In the shallower boreholes at circa 1 kmbls, isotopic evidence indicated microbial incorporation of biogenic CH4 by the in situ microbial community. At the shallowest site, 1.05 kmbls in Driefontein mine, this process clearly dominated the iso- topic signal. At slightly deeper depths, 1.34 kmbls in Beatrix mine, the isotopic data indicated the incorporation of both bio- genic CH4 and dissolved inorganic carbon (DIC) derived from CH4 oxidation. In both of these cases, molecular genetic analysis indicated that methanogenic and methanotrophic organisms together comprised a small component (<5%) of the microbial community. Thus, it appears that a relatively minor component of the prokaryotic community is supporting a much larger overall bacterial community in these samples. In the samples collected from >3 kmbls in Tau Tona mine (TT107, TT109 Bh2), the CH4 had an isotopic signature sug- gesting a predominantly abiogenic origin with minor inputs from microbial methanogenesis. In these samples, the isotopic 13 14 enrichments (d C and D C) of the PLFAs relative to CH4 were consistent with little incorporation of CH4 into the biomass. 13 The most C-enriched PLFAs were observed in TT107 where the dominant CO2-fixation pathway was the acetyl-CoA ⇑ Corresponding author at: School of Geography and Earth Sciences, Room 306, General Sciences Building, McMaster University, Hamilton, Ontario L8S 4L8, Canada. Tel.: +1 (905) 525 9140x26388; fax: +1 (905) 546 0463. 1 Current address: Extreme Life Isyensya, PB 65, 9050 Gentbrugge, Belgium. http://dx.doi.org/10.1016/j.gca.2015.10.003 0016-7037/Ó 2015 Elsevier Ltd. All rights reserved. D.N. Simkus et al. / Geochimica et Cosmochimica Acta 173 (2016) 264–283 265 pathway by non-acetogenic bacteria. The differences in the d13C of the PLFAs and the DIC and DOC for TT109 Bh2 were À24‰ and 0‰, respectively. The dominant CO2-fixation pathways were 3-HP/4-HB cycle > acetyl-CoA pathway > reduc- tive pentose phosphate cycle. Ó 2015 Elsevier Ltd. All rights reserved. 1. INTRODUCTION microorganism. Oxidation of CH4 under anaerobic condi- tions by bacteria has also been shown to occur by the den- The carbon sources and carbon cycling processes uti- itrifying Candidatus ‘‘Methylomirabilis oxyfera”, which lized by the Earth’s deep continental subsurface biosphere produces intracellular O2 by dismutation of NO (Ettwig are still poorly understood, despite the global significance et al., 2010, 2012). Although aerobic CH4 oxidation has of these microbial systems (Onstott et al., 1998; Whitman been reported in terrestrial deep subsurface habitats et al., 1998; Pfiffner et al., 2006). Constraining these pro- (Bowman et al., 1993; Kotelnikova, 2002; Mills et al., cesses is an integral step in defining the limits of habitability 2010), the occurrence of anaerobic CH4 oxidation in this on Earth, while providing insight into the potential for life setting has not yet been reported in the literature. to exist in the deep subsurface of other planetary bodies. Comparison of the natural abundances of 13C and 14Cin Although photosynthetically derived organic carbon buried membrane phospholipid fatty acids (PLFAs) and in poten- within Earth’s subsurface can be utilized as a carbon source tial carbon sources can elucidate microbial carbon sources by subsurface microbial communities, this organic carbon (Petsch et al., 2001; Slater et al., 2005). Further, such com- source is limiting in many deep terrestrial subsurface envi- parison can identify the putative pathways being used to ronments (Pedersen, 2000). In systems where complex assimilate carbon and, in some cases, deduce the in situ organic carbon is limited, microbial communities must rely metabolic rates. The d13C value of microbial PLFAs on either autotrophic fixation of dissolved inorganic carbon depends on the following: (1) the d13C value of the source (DIC) by chemolithoautotrophs, including methanogens, of the assimilated carbon; (2) kinetic isotope effects (KIEs) acetogens, sulphate reducers and iron reducers (Chivian associated with the carbon assimilation pathway (e.g. et al., 2008; Beal et al., 2009; Lau et al., 2014; Stevens autotrophy vs. heterotrophy vs. CH4 oxidation); and (3) and McKinley, 1995; Pedersen, 2000; Sherwood Lollar KIEs involved in the microbial synthesis of PLFAs et al., 2006; Magnabosco et al., 2015) or oxidation of (Hayes, 2001; Boschker and Middelburg, 2002). Carbon fix- CH4 by methanotrophs (Bowman et al., 1993; ation pathways involved in autotrophic metabolisms gener- Kotelnikova, 2002; Mills et al., 2010). ally produce organic components, and particularly PLFAs, Energy sources for chemolithoautotrophic communities that are depleted in 13C relative to the DIC source, but the (e.g. H2) can be produced via several abiotic processes, extent of this carbon isotope fractionation is highly variable including magmatic gas reactions, cataclasis of silicates, (Boschker and Middelburg, 2002; Berg et al., 2010). For hydrolysis of ferrous minerals, the gas shift reaction and example, autotrophic sulphate-reducing bacteria have been radiolytic decomposition of water (Apps and van de found to produce PLFAs that are up to 58‰ more depleted Kamp, 1993; Pedersen, 1997; Lin et al., 2005; Etiope and in 13C than DIC (Londry et al., 2004); whereas other auto- Sherwood Lollar, 2013; Stevens and McKinley, 1995). trophic bacteria produce PLFAs that are only several ‰ Methanogens and acetogens couple DIC reduction to H2 more depleted than DIC (Boschker and Middelburg, oxidation to produce CH4 and acetate, respectively. Alter- 2002). For heterotrophic metabolisms, PLFAs produced natively, abiogenic hydrocarbon production via Fischer– in aerobic environments generally show relatively small car- 13 Tropsch-type synthesis reactions, whereby CO/CO2 and bon isotope fractionations, producing d CPLFA values that H2 react to produce hydrocarbons of various molecular are typically 4–8‰ more depleted than the dissolved weights including CH4 (Sherwood Lollar et al., 2002), can organic carbon (DOC) source. However, in anaerobic envi- provide CH4 and higher hydrocarbons as potential carbon ronments, heterotrophic bacteria have been shown to pro- substrates for methanotrophs and heterotrophs to support duce PLFAs that are up to 21‰ more depleted than their subsurface microbial communities independent of DOC substrate (Teece et al., 1999; Boschker and photosynthetically-derived organic matter. Middelburg, 2002); and sulphate-reducing bacteria produce Microbial oxidation of CH4 is carried out by both aero- PLFAs that are 9.5‰ more enriched when oxidizing acetate bic bacteria that utilize CH4 monooxygenase (MMO; (Londry et al., 2004). Lastly, microbial aerobic oxidation of 13 Bowman, 2006) and anaerobic archaea that utilize portions CH4 generally results in particularly depleted