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CO2 Conversion to Methane and Biomass in Obligate Methylotrophic Methanogens In bioRxiv preprint doi: https://doi.org/10.1101/528562; this version posted January 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-NC-ND 4.0 International license. 1 CO2 conversion to methane and biomass in obligate methylotrophic methanogens in 2 marine sediments 3 Xiuran Yin1,2,3*, Weichao Wu2,4*‡, Mara Maeke1,3, Tim Richter-Heitmann1, Ajinkya C. 4 Kulkarni1,2,3, Oluwatobi E. Oni1,2, Jenny Wendt2,4, Marcus Elvert2,4 and Michael W. Friedrich1,2 5 1Microbial Ecophysiology Group, Faculty of Biology/Chemistry, University of Bremen, Bremen, 6 Germany 7 2MARUM - Center for Marine Environmental Sciences, Bremen, Germany 8 3International Max-Planck Research School for Marine Microbiology, Max Planck Institute for Marine 9 Microbiology, Bremen, Germany 10 4Department of Geosciences, University of Bremen, Bremen, Germany 11 Running title: Methane formation from CO2 in Methanococcoides 12 Correspondence: 13 Michael W. Friedrich, 14 Microbial Ecophysiology Group, Faculty of Biology/Chemistry, University of Bremen, PO 15 Box 33 04 40, D-28334 Bremen, Germany 16 Email: [email protected] 17 * These authors contributed equally to this work. 18 ‡ Current address: Department of Biogeochemistry of Agroecosystems, University of Goettingen, 19 Goettingen, Germany 1 bioRxiv preprint doi: https://doi.org/10.1101/528562; this version posted January 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-NC-ND 4.0 International license. 20 Abstract 21 Methyl substrates are important compounds for methanogenesis in marine sediments but 22 diversity and carbon utilization by methylotrophic methanogenic archaea have not been clarified. 23 Here, we demonstrate that RNA-stable isotope probing (SIP) requires 13C-labeled bicarbonate as 24 co-substrate for identification of methylotrophic methanogens in sediment samples of the 25 Helgoland mud area, North Sea. Using lipid-SIP, we found that methylotrophic methanogens 26 incorporate 60 to 86% of dissolved inorganic carbon (DIC) into lipids, and thus considerably 27 more than what can be predicted from known metabolic pathways (~40% contribution). In slurry 28 experiments amended with the marine methylotroph Methanococcoides methylutens, up to 12% 29 of methane was produced from CO2, indicating that CO2-dependent methanogenesis is an 30 alternative methanogenic pathway and suggesting that obligate methylotrophic methanogens 31 grow in fact mixotrophically on methyl compounds and DIC. Thus, the observed high DIC 32 incorporation into lipds is likely linked to CO2-dependent methanogenesis, which was triggered 33 when methane production rates were low. Since methylotrophic methanogenesis rates are much 34 lower in marine sediments than under optimal conditions in pure culture, CO2 conversion to 35 methane is an important but previously overlooked methanogenic process in sediments for 36 methylotrophic methanogens. 2 bioRxiv preprint doi: https://doi.org/10.1101/528562; this version posted January 30, 2019. 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-NC-ND 4.0 International license. 37 Introduction 38 Methanogenesis is the terminal step of organic matter mineralization in marine sediments [1]. 39 There are three main pathways producing methane, i.e., hydrogenotrophic (H2/CO2), acetoclastic 40 (acetate) and methylotrophic (e.g., methanol, methylamine) methanogenesis [2] with the former 41 two pathways considered dominant [3]. However, the importance of methylated compounds for 42 methanogenesis in marine sediments has been acknowledged in recent years. Geochemical 43 profiles and molecular analysis have shown that methylotrophic methanogenesis is the most 44 significant pathway for methane formation in hypersaline sediments [4,5] and in the sulfate 45 reduction zone (SRZ) in marine environments [6,7] where methanol concentration of up to 69 46 µM had been measured [8,9]. Especially in the SRZ, methylated compounds are regarded as non- 47 competitive substrates for methanogenesis, since sulfate reducing microorganisms apparently do 48 not compete with methanogens for these compounds [10]. In sediments of the Helgoland mud 49 area, specifically, high relative abundances of potential methylotrophic methanogens were 50 observed [11] of which many are unknown. The potential for methylotrophic methanogenesis 51 was recently even predicted from two metagenome-assembled genomes of uncultivated 52 Bathyarchaeota, assembled from a shotgun metagenome [12]. 53 The formation of methane via the three main pathways in methanogenic archaea has been studied 54 intensively [13,14,15]. Much less is known regarding assimilation of carbon into biomass under 55 in situ conditions and to which extend different carbon sources in the environment are utilized. 56 Discrepancies between predicted pathways known and the actual carbon metabolism measured 57 appear to be based on different cellular functions of carbon dissimilation and assimilation, 58 originate from reaction equilibria operative, intermediate carbon cross utilization as well as 59 interplay between different microbial communities [14, 16,17,18,19]. For example, 3 bioRxiv preprint doi: https://doi.org/10.1101/528562; this version posted January 30, 2019. 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-NC-ND 4.0 International license. 60 mixotrophically growing cultures of Methanosarcina barkeri form their biomass equally from 61 methanol and CO2, however, almost all the methane is formed from methanol rather than from 62 CO2 [20] since methanol is disproportionated to methane and CO2 according to the following 63 reaction: 64 4 CH3OH 3 CH4 + CO2 + 2 H2O (1) 65 But apart from such culture studies using the nutritionally versatile Methanosarcina barkeri, 66 which is capable of hydrogenotrophic, acetoclastic and methylotrophic methanogenesis, the 67 respective contribution of CO2 and methylated carbon substrates to biomass formation during 68 methylotrophic methanogenesis, especially for “obligate” methylotrophic methanogens, in 69 natural sediments has not been studied to date. 70 Nucleic acids (RNA ~20%, DNA ~3%, of dry biomass, respectively), lipids (7–9%) and proteins 71 (50–55%) are crucial cell components in living microorganisms [21], and thus, are suitable 72 markers of carbon assimilation. In order to characterize carbon assimilation capabilities, stable 73 isotope probing (SIP) techniques exist, among which RNA-SIP is very powerful for identifying 74 active microorganisms based on separating 13C-labeled from unlabeled RNA using isopycnic 75 centrifugation [22,23]. In combination with downstream sequencing analysis, RNA-SIP provides 76 high phylogenetic resolution in detecting transcriptionally active microbes [24,25] but is limited 77 in its sensitivity by requiring more than 10% of 13C incorporation into RNA molecules for 78 sufficient density gradient separation [26]. To date, a number of SIP studies successfully 79 detected methylotrophic bacteria [27,28,29] but the detection of methylotrophic methanogens by 80 RNA-SIP with 13C labeled methyl compounds might be hampered by mixotrophic growth [20], 81 i.e., simultaneous assimilation of carbon from methylated compounds and CO2. 4 bioRxiv preprint doi: https://doi.org/10.1101/528562; this version posted January 30, 2019. 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-NC-ND 4.0 International license. 82 In contrast to RNA-SIP, lipid-SIP has a lower phylogenetic resolution, but can detect very 83 sensitively δ13C-values in lipid derivatives by gas chromatography combustion isotope ratio mass 84 spectrometry (GC-IRMS), thereby facilitating quantitative determination of small amounts of 85 assimilated carbon [30,31]. 86 In this study, we aimed to identify methylotrophic methanogens by RNA-SIP and elucidate 87 carbon assimilation patterns in marine sediments. We hypothesized that the large pool of ambient 88 dissolved inorganic carbon (DIC) in sediments [6] alters carbon utilization patterns in 89 methylotrophic methanogens compared to pure cultures. To address these questions, we tracked 90 carbon dissimilation into methane and quantified assimilation into lipids by lipid-SIP in slurry 91 incubations and pure cultures. We found an unexpectedly high degree of methanogenesis from 92 DIC by previously considered obligate methylotrophic methanogens, i.e., using only methyl 93 groups for methane formation. This mixotrophic methanogenesis might be the basis for our 94 observation that more inorganic carbon was assimilated into biomass than could be expected 95 from known pathways. 96 Materials and Methods 97 Sediment incubation setup for SIP 98 Sediment was collected from the Helgoland mud area (54°05.23'N, 007°58.04'E) by gravity 99 coring in 2015 during the RV HEINCKE cruise HE443. The geochemical profiles
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