bioRxiv preprint doi: https://doi.org/10.1101/2020.07.28.225276; this version posted February 24, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-NC-ND 4.0 International license. 1 Biogeochemical and omic evidence for paradoxical methane production via multiple 2 co-occurring mechanisms in aquatic ecosystems 3 4 Authors 5 Elisabet Perez-Coronel1,2*, J. Michael Beman1 6 1Environmental Systems Graduate Group, and Sierra Nevada Research Institute, University of 7 California Merced, Merced, USA. 8 2Ecology, Behavior and Evolution Section, University of California San Diego, La Jolla, USA. 9 10 11 *Corresponding author: Elisabet Perez-Coronel. University of California San Diego. 9500 12 Gilman Dr, La Jolla, CA, 92093 USA 13 e-mail for correspondence: [email protected] 14 Phone: +12093558131 15 16 17 Abstract 18 Aquatic ecosystems are globally significant sources of the greenhouse gas methane (CH4) to the 19 atmosphere. However, CH4 is produced ‘paradoxically’ in oxygenated water via poorly 20 understood mechanisms, fundamentally limiting our understanding of overall CH4 production. 13 21 Here we resolve paradoxical CH4 production mechanisms through CH4 measurements, δ CH4 22 analyses, 16S rRNA sequencing, and metagenomics/metatranscriptomics applied to freshwater 23 incubation experiments with multiple time points and treatments (addition of a methanogenesis 24 inhibitor, dark, high-light). We captured significant paradoxical CH4 production, as well as 25 consistent metabolism of methylphosphonate by abundant bacteria—resembling observations 13 26 from marine ecosystems. Metatranscriptomics and δ CH4 analyses applied to experimental 27 treatments identified an additional CH4 production mechanism associated with 28 (bacterio)chlorophyll metabolism and photosynthesis by Cyanobacteria, and especially by 29 Proteobacteria. Both mechanisms occured together within metagenome-assembled genomes, 30 and appear widespread in freshwater. Our results indicate that multiple, co-occurring, and 31 broadly-distributed bacterial groups and metabolic pathways produce CH4 in aquatic ecosystems. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.28.225276; this version posted February 24, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-NC-ND 4.0 International license. 32 Introduction 33 Atmospheric concentrations of the potent greenhouse gas methane (CH4) have increased 34 significantly due to anthropogenic activity, representing an important component of climate 35 change (1). However, these increases are superimposed on substantial spatial and temporal 36 variability in natural sources of CH4 to the atmosphere. Of all natural CH4 sources, freshwater 37 lakes are particularly important but poorly understood, with their estimated contribution ranging 38 from 6 to 16% of all natural CH4 emissions—despite accounting for only ~0.9% of the Earth’s 39 surface area (2). CH4 emissions from lakes are conventionally viewed to be regulated by CH4 40 production (occurring predominantly in anoxic sediments) and subsequent CH4 oxidation in 41 surface sediments and the water column (3). However, oversaturation of CH4 has been 42 consistently observed in oxygenated waters of aquatic systems (4). This observation indicates 43 that CH4 is produced under oxic conditions, and that the rate of CH4 production exceeds CH4 44 oxidation. Since archaeal methanogenesis is an obligate anaerobic process (5), oxic CH4 45 production is typically referred to as the “methane paradox,” and has been observed in oceans (6, 46 7), lakes (8, 9, 10), and aerobic wetland soils (11). Notably, paradoxical CH4 production occurs 47 near the surface, and so any produced CH4 may readily flux to the atmosphere. Identifying which 48 mechanisms produce CH4 in oxygenated waters is therefore essential for our understanding of 49 CH4 fluxes and their contribution to climate change. 50 Although multiple mechanisms for paradoxical aerobic CH4 production have been 51 proposed, the degree to which these are active in freshwater lakes remains unknown. Initial 52 studies suggested that CH4 production under oxygenated conditions could be occurring in anoxic 53 microsites in the water column—such as fecal pellets, detritus, and the gastrointestinal tracts of 54 larger organisms such as fish or zooplankton (12, 13, 14). Several studies have also demonstrated 55 a correlation between phytoplankton or primary production and CH4 production (8, 9, 10). 56 However, the underlying reason(s) for this relationship is unknown. One possibility is that 57 methanogens reside on the surface of phytoplankton cells and produce CH4 in presumably anoxic 58 microsites (8). Bogard et al. (9) and Donis et al. (15) also hypothesized that several groups of 59 methanogens have oxygen-tolerant or detoxifying pathways that could aid in CH4 production in 60 the presence of oxygen. For example, Angle et al. (11) characterized a methanogen candidate 61 that possesses the enzymes to detoxify oxygen and produce CH4 under aerobic conditions. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.28.225276; this version posted February 24, 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-NC-ND 4.0 International license. 62 In contrast, the current prevailing view of marine ecosystems is that methylphosphonate 63 (MPn) is the main precursor of CH4 production under oxic conditions—particularly in 64 phosphorus (P)-stressed ecosystems such as the open ocean (6, 16). MPn is the simplest form of 65 organic carbon (C)-P bonded compounds in aquatic ecosystems; microbial utilization of MPn, 66 and the consequent breakdown of the C-P bond, releases CH4 as a by-product (6, 16, 17). A 67 broad range of marine and freshwater bacteria have the genomic potential to metabolize MPn 68 and produce CH4, based on the presence of the multi-gene C-P lyase pathway in their 69 genomes. This includes multiple groups of Proteobacteria, Firmicutes, Bacteroidetes, 70 Chloroflexi, and Cyanobacteria (17, 18, 19). While expression of this pathway is thought to be 71 regulated by phosphate availability (17, 18, 19), the degree to which this occurs in freshwater 72 ecosystems is not well known (20, 21, 22). Finally, recent work indicates that cultures of marine 73 and freshwater Cyanobacteria can directly produce CH4 (23). However, outside of a single 74 experiment (24), this has not been examined in aquatic ecosystems. More significantly, the exact 75 mechanism by which this occurs remains unknown. Given the widespread distribution of 76 cyanobacteria in the ocean and freshwater, identifying the potential mechanism(s) by which 77 cyanobacteria produce methane—and whether this capability may be present in other 78 photosynthetic organisms—is of broad relevance. 79 These proposed mechanisms for CH4 production—(1) methanogenesis aided by 80 detoxifying genes or in anoxic microsites, (2) CH4 production by breakdown of methylated 81 compounds, and (3) CH4 production by Cyanobacteria—point to multiple mechanisms by which 82 CH4 can be produced under oxygenated conditions. Many of these are recently discovered and 83 therefore poorly understood, and the degree to which they occur within different aquatic 84 ecosystems is largely unknown. We developed an experimental approach to disentangle these 85 mechanisms and determine which may produce CH4 in freshwater lakes. We conducted 86 incubation experiments using surface waters from high-elevation lakes, in order to rule out 87 physical transport and focus on potential biological mechanisms of oxic CH4 production. We 88 investigated specific mechanisms using a combination of CH4 measurements over time, 89 experimental treatments and inhibitors, and 16S rRNA gene and transcript sequencing, while 90 also applying stable isotope analyses and metagenome and metatranscriptome sequencing to 91 selected experiments. Paradoxical CH4 production was evident in multiple experiments and 92 experimental treatments, and could be attributed to MPn breakdown via widely-distributed 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.28.225276; this version posted February 24, 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-NC-ND 4.0 International license. 93 members of the Comamonadaceae family. However, experimental treatments, stable isotope 13 94 δ C signatures of CH4, and metatranscriptomic data also point to a new potential mechanism of 95 aerobic CH4 production carried out by photosynthetic bacteria. 96 97 Results and Discussion 98 Our experiments provide multiple lines of evidence for paradoxical CH4 production in freshwater 99 lakes. Out of 19 total experiments conducted in five lakes in Yosemite National Park, 26% of 100 experiments showed unequivocal, monotonic CH4 production in unamended controls; 16% 101 showed net oxidation in controls; 21% exhibited significant nonlinear patterns; and at least one 102 experimental treatment in 37% of experiments also