bioRxiv preprint doi: https://doi.org/10.1101/2021.08.26.457739; this version posted August 26, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 Subsurface Hydrocarbon Degradation Strategies in Low- and High-Sulfate Coal Seam 2 Communities Identified with Activity-Based Metagenomics 3 4 Authors: Hannah Schweitzer1,2,†§**, Heidi Smith1,2,§, Elliott P. Barnhart1,3, Luke McKay1,4, 5 Robin Gerlach1,5,6, Alfred B. Cunningham1,5,7, Rex R. Malmstrom8, Danielle Goudeau8, and 6 Matthew W. Fields1,2,5** 7 8 Affiliations: 9 1Center for Biofilm Engineering, Montana State University, Bozeman, MT 59717, USA 10 2Department of Microbiology & Cell Biology, Montana State University, Bozeman, MT 11 59717, USA 12 3US Geological Survey, Wyoming-Montana Water Science Center, Helena, MT 59601,USA 13 4Department of Land Resources and Environmental Sciences, Montana State University, 14 Bozeman, MT 59717, USA 15 5Energy Research Institute, Montana State University, Bozeman, MT 59717, USA 16 6Department of Biological and Chemical Engineering, Montana State University, Bozeman, 17 MT 59717, USA 18 7Department of Civil Engineering, Montana State University, Bozeman, MT 59717, USA 19 8DOE Joint Genome Institute, Berkeley, CA 94720, USA 20 21 §Indicates both authors contributed equally to this work 22 †Now at UiT - The Arctic University of Norway, 9019 Tromsø, Norway 23 24 **Corresponding authors 25 H.D. Schweitzer, Post Doctoral Researcher 26 UiT - The Arctic University of Norway 27 The Norweigian College of Fishery Science 28 Muninbakken 21 29 9019 Tromsø, Norway 30 [email protected] 31 32 M.W. Fields, Professor 33 Montana State University 34 Center for Biofilm Engineering 35 366 EPS Building 36 Bozeman, MT 59717, USA 37 [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.26.457739; this version posted August 26, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 38 One Sentence Summary: 39 Identifying hydrocarbon degradation strategies across redox gradients via metagenomic 40 analysis of environmental and translationally active (BONCAT-FACS) samples from 41 subsurface coal beds. 42 43 44 45 46 Abstract 47 Environmentally relevant metagenomes and BONCAT-FACS derived translationally active 48 metagenomes from Powder River Basin coal seams were investigated to elucidate potential 49 genes and functional groups involved in hydrocarbon degradation to methane in coal seams 50 with high- and low-sulfate levels. An advanced subsurface environmental sampler allowed the 51 establishment of coal-associated microbial communities under in situ conditions for 52 metagenomic analyses from environmental and translationally active populations. 53 Metagenomic sequencing demonstrated that biosurfactants, aerobic dioxygenases, and 54 anaerobic phenol degradation pathways were present in active populations across the sampled 55 redox gradient. In particular, results suggested the importance of anaerobic degradation 56 pathways under high-sulfate conditions with an emphasis on fumarate addition. Under low- 57 sulfate conditions, a mixture of both aerobic and anaerobic pathways were observed but with 58 a predominance of aerobic dioxygenases. The putative low-molecular weight biosurfactant, 59 lichysein, appeared to play a more important role compared to rhamnolipids. The novel 60 methods used in this study—subsurface environmental samplers in combination with 61 metagenomic sequencing of both translationally active metagenomes and environmental 62 genomes—offer a deeper and environmentally relevant perspective on community genetic 63 potential from coal seams poised at different redox potentials broadening the understanding of 64 degradation strategies for subsurface carbon. 65 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.26.457739; this version posted August 26, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 66 Introduction 67 The terrestrial subsurface contains the majority of Earth’s organic carbon 68 (~90%)1, and much of the carbon can be converted to methane under anaerobic 69 conditions through biogasification (i.e., biological decomposition of organic matter 70 into methane and secondary gases). Biogasification can take place in coal, black shale, 71 and petroleum reservoirs and is estimated to account for over 20% of the world’s 72 natural gas resources2. Factors influencing biogasification include coal rank, redox 73 conditions (e.g., presence or absence of oxygen and oxyanions), and the genetic 74 potential and activity of the microbial community. Coal is a heterogeneous and highly 75 complex hydrocarbon consisting of polycyclic aromatic hydrocarbons, alkylated 76 benzenes, and long and short chain n-alkanes3, and despite the recalcitrant nature of 77 coal, degradation by microbial consortia has been shown in a variety of coal 78 formations4. It is generally accepted that shallower coal beds that contain sulfate do 79 not produce methane because sulfate-reducing bacteria (SRB) outcompete 5,6 80 methanogens for substrates (e.g., acetate, CO2 and hydrogen) . In methanogenic coal 81 beds6,7, hydrogenotrophic and acetoclastic methanogens are commonly identified, 82 including different types of acetoclastic methanogens (e.g., Methanothrix, 83 Methanosarcina), which have distinct pathways for acetate utilization. It remains 84 unknown what type of methanogenesis predominates in situ for different coal seams 85 under different physicochemical conditions8,9. 86 New coal degradation pathways are still being discovered and the involvement 87 of different pathways in the turnover of refractory carbon under various redox 88 conditions remains largely unresolved10–12. The majority of coal degradation research 89 has focused on fumarate addition, while less is known about alternate coal degradation 90 strategies such as phenol degradation by carboxylation and hydroxylation of alkanes, 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.26.457739; this version posted August 26, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 91 benzene and ethylbenzene13,14. The fumarate addition pathway involves the activation 92 of n-alkanes by the addition of fumarate via the double bonds at the terminal or sub 93 terminal carbon13,15–19. Several fumarate addition genes (e.g., ass-alkylsuccinate 94 synthase for alkanes, bss-benzylsuccinate synthase for alkylbenzenes, and nms- 95 naphylmethylsuccinate synthase) are often used as catabolic biomarkers for anaerobic 96 hydrocarbon degradation15–17. These genes have been characterized from many 97 subsurface hydrocarbon-containing environments,13,16,20–25 but the importance under 98 different redox conditions is still unclear. Carboxylation and hydroxylation strategies 99 are less well documented mechanisms of anaerobic degradation, although, in recent 100 years work has begun to suggest importance in anaerobic hydrocarbon 101 degradation14,18,26,27, yet how these strategies vary across redox transition zones in situ 102 and detecting organisms responsible for degradation warrants further investigation. 103 While biosurfactants have not been identified in situ in coal seams and are not 104 considered a necessary hydrocarbon degradation gene, previous laboratory-based 105 research demonstrates a potentially important role of these compounds in decreasing 106 the hydrophobicity of the solid coal surface, allowing for cellular and/or protein 107 interactions at the coal surface28. Biosurfactant-producing microorganisms likely play 108 direct and indirect roles in hydrocarbon degradation28–31. The accumulation of the 109 esterase hydrolase enzyme has been correlated to biosurfactant production and is often 110 used as a biomarker for biosurfactant production32. Biosurfactants are routinely 111 observed in environments that consist of complex hydrocarbons and are therefore 112 hypothesized to be an interdependent, complex, and coordinated means of increasing 113 coal bioavailability. However, studies that have demonstrated active biosurfactant- 114 producing microorganisms in coal environments are lacking. 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.26.457739; this version posted August 26, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 115 Aerobic hydrocarbon degradation is the most documented form of hydrocarbon 116 degradation via the aerobic activation of alkanes with dioxygenase enzymes that use 117 oxygen as an electron acceptor and as a reactant in hydroxylation33. Aerobic 118 hydrocarbon degradation in coal environments is often disputed due to the uncertainty 119 of the presence of oxygen and more research