bioRxiv preprint doi: https://doi.org/10.1101/2020.06.25.170886; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Subsurface carbon monoxide oxidation capacity revealed through genome- 2 resolved metagenomics of a carboxydotroph 3 4 Andre Mu1,2, Brian C. Thomas3, Jillian F. Banfield3,4, and John W. Moreau5,6 * 5 1Department of Microbiology and Immunology, at the Peter Doherty Institute for 6 Infection and Immunity, University of Melbourne, Australia 7 2Doherty Applied Microbial Genomics, Department of Microbiology and Immunology, 8 at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, 9 Australia 10 3Department of Earth and Planetary Science, University of California, Berkeley, United 11 States 12 4Innovative Genomics Institute, University of California, Berkeley, United States 13 5School of Geographical and Earth Sciences, University of Glasgow, United Kingdom 14 6School of Earth Sciences, University of Melbourne, Australia 15 16 Running title: Deep aquifer carboxydotrophy during geosequestration 17 18 Keywords: Metagenomics, CO2 sequestration, geosequestration, CODH, CooS, CO 19 oxidation, carboxydotrophy, Carboxydocella, deep subsurface 20 #Correspondence: 21 Dr. John W. Moreau 22 School of Geographical & Earth Sciences 23 University of Glasgow 24 Email: [email protected] 25 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.25.170886; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 26 Originality-Significance Statement 27 The research conducted in this study is associated with one of the world’s largest 28 demonstrations of carbon geosequestration – The Cooperative Research Centre for 29 Greenhouse Gas Technologies Otway Project (Victoria, Australia). Our results expand 30 the ecology of CO-utilising microbes to include the terrestrial deep subsurface through 31 genome-resolved metagenomics of an aquifer-native carboxydotroph. 32 33 34 Summary 35 Microbial communities play important roles in the biogeochemical cycling of carbon 36 in the Earth’s deep subsurface. Previously, we demonstrated changes to the microbial 37 community structure of a deep aquifer (1.4 km) receiving 150 tons of injected 38 supercritical CO2 (scCO2) in a geosequestration experiment. The observed changes 39 support a key role in the aquifer microbiome for the thermophilic CO-utilising anaerobe 40 Carboxydocella, which decreased in relative abundance post-scCO2 injection. Here, 41 we present results from more detailed metagenomic profiling of this experiment, with 42 genome resolution of the native carboxydotrophic Carboxydocella. We demonstrate a 43 switch in CO-oxidation potential by Carboxydocella through analysis of its carbon 44 monoxide dehydrogenase (CODH) gene before and after the geosequestration 45 experiment. We discuss the potential impacts of scCO2 on subsurface flow of carbon 46 and electrons from oxidation of the metabolic intermediate carbon monoxide (CO). 47 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.25.170886; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 48 Introduction 49 Carbon monoxide (CO) provides a carbon and/or energy source for a wide range of 50 anaerobic microorganisms (Oelgeschläger and Rother, 2008) as its oxidation can be 51 coupled to multiple terminal electron accepting processes (TEAPs), e.g., iron(III)- 52 reduction (Slobodkin et al., 1999), sulfate-reduction (Parshina et al., 2005, 2010) 53 hydrogenogenesis, acetogenesis or methanogenesis (Diender et al., 2015). The 54 electrons produced from CO oxidation can be coupled to the reduction of H2O and 55 metals (Techtmann et al., 2009). The substrate versatility of CO results from the low 56 redox potential (E°; -524 to -558 mV) of the half-reaction for carbon dioxide (CO2) 57 reduction to CO (Ragsdale, 2004; Techtmann et al., 2009). This potential is lower + 58 than that of the H /H2 redox couple, meaning that CO can replace H2 as an electron 59 donor for microorganisms carrying genes encoding for carbon monoxide 60 dehydrogenase (CODH). The CODH gene cluster in the genus, Carboxydocella, 61 consists of 13 genes encoding for: redox- and CO-sensitive transcriptional regulator 62 (cooA), [Ni, Fe]-CODH (cooS), [Ni, Fe]-CODH accessory nickel-insertion protein 63 (cooC), CO-induced hydrogenase membrane anchor (cooM), energy-converging 64 hydrogenase (cooKLXUH), hydrogenase maturation protein (hypA), 4Fe-4S di-cluster 65 domain containing electron transfer protein (cooF), energy-converging hydrogenase 66 catalytic subunit (cooH), and the upstream enigmatic cooSC operon of unknown 67 function (Toshchakov et al., 2018). The upstream cooS gene is notably missing in the 68 closely related Carboxydothermus hydrogenoformans (Toshchakov et al., 2018). The 69 catalytic subunit of CODH is composed of the CooS protein, while the CooF electron- 70 transfer protein is known to associate with redox reactions such as the production of H2 71 or reduced metals (Soboh et al., 2002). Bi-functional CODH enzyme complexes 72 contain domains that catalyse the oxidation of CO (CODH), and formation of acetyl- 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.25.170886; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 73 CoA (Acetyl-CoA synthase; ACS); Ragsdale and Kumar, 1996). Biochemically, the 74 electrons produced by CODH flow on to the ACS module for downstream production 75 of acetyl-CoA. Bi-functional CODH/ACS complexes are therefore fundamental to 76 acetoclastic methanogens and acetogenesis in prokaryotes using the Wood-Ljungdahl 77 pathway to form acetyl-CoA from either CO2 or CO (Ragsdale, 2004). The 78 CODH/ACS complex is also responsible for the majority of autotrophic utilization of 79 CO (Techtmann et al., 2009). An increase in CO2 partial pressure may therefore 80 inhibit microbial carboxydotrophy, resulting in increasing and decreasing levels of CO 81 and H2, respectively. Such changes potentially hold consequences for both carbon and 82 electron flow through various microbial metabolisms (Ragsdale, 2004; Techtmann et 83 al., 2009). 84 85 Using phenotypic and physiological assays, Sokolova et al. (2002) demonstrated that a 86 40% decrease in CO in cultures of the anaerobic Firmicute, Carboxydocella, lead to a 87 30% increase in the gas phase concentrations of H2 and CO2 (Sokolova et al., 2002). 88 Furthermore, pure cultures of Desulfotomaculum kuznetsovii and D. thermobenzoicum 89 subsp. thermosynthrophicum were shown to grow on CO as the sole electron donor, 90 and at concentrations as high as 50-70%, in the presence of hydrogen/CO2. However, 91 co-culture of Desulfotomaculum kuznetsovii and D. thermobenzoicum subsp. 92 thermosynthrophicum with a carboxydotroph (i.e, Carboxydothermus 93 hydrogenoformans) supported sulfate-reducing bacteria (SRB) growth in 100% CO, 2- 94 with CO oxidation coupled to SO4 reduction and acetogenesis (pCO = 120 kPa) 95 (Parshina et al., 2005). 96 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.25.170886; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 97 Injection of massive volumes of supercritical CO2 (scCO2) into deep aquifers forms a 98 principal current strategy for geological carbon storage (Benson and Surles, 2006; 99 Gibbins and Chalmers, 2008). These scCO2 “plumes” will enrich groundwaters 100 locally with respect to dissolved CO2, with implications for both microbial metabolic 101 activity and water-rock chemical reactions (Phillips et al., 2012). Understanding the 102 in situ impacts of increased CO2 on microbial CODH activity will yield insights into 103 the long-term and large-scale potential responses of the subsurface microbial biosphere 104 to geological CO2 sequestration. 105 106 Here we report results from a field-scale geological scCO2 injection project in the 107 Paaratte Formation of the Otway Basin (1.4 km below ground, Southeastern Australia) 108 that revealed a steep decline in the relative abundance of a dominant native 109 carboxydotroph representing >96% of the aquifer microbial community before 110 injection (Mu et al., 2014). Previous studies of subsurface microbial responses to 111 quickly-elevated CO2 levels have inferred their findings on the basis of 16S rRNA gene 112 data (Bordenave et al., 2013; Lavalleur and Colwell, 2013; Mu et al., 2014). Here 113 we used time-series relative abundance in situ metagenomic sampling to resolve a near- 114 complete genome from the carboxydotrophic genus Carboxydocella and established the 115 microbial host of the CODH gene cluster. 116 117 118 2. Results 119 2.1. Metagenomic analysis of the Paaratte Formation 120 A total of 13 samples collected from the Paaratte Formation over the course of a field- 121 scale demonstration of supercritical carbon dioxide (scCO2) geosequestration were 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.25.170886; this version posted June 25, 2020.