Lakes and reservoirs contribute substantially to atmospheric concentrations of the potent greenhouse gas methane. Lake sediments produce large amounts of methane, which accumulate in the oxygen-depleted bottom waters of stratified lakes. Climate change and eutrophication may increase the number of lakes with methane storage in the future. Whether stored methane escapes to the atmosphere during annual lake overturn is a matter of controversy and depends critically on the response of the methanotroph assemblage. Here we show, by combining 16S rRNA gene and pmoA mRNA amplicon sequencing, qPCR, CARD-FISH and potential methane-oxidation rate measurements, that the methanotroph assemblage in a mixing lake underwent both a substantial bloom and ecological succession. As a result, methane oxidation kept pace with the methane supplied from methane-rich bottom water and most methane was oxidized. This aspect of freshwater methanotroph ecology represents an effective mechanism limiting methane transfer from lakes to the atmosphere.

16Lakes and reservoirs contribute substantially to atmospheric concentrations of the potent greenhouse 17 gas methane. Lacustrine sediments produce large amounts of methane, which accumulate in oxygen-18 depleted hypolimnia of stratified lakes. Due to climate change and progressing eutrophication, the 19 number of lakes with hypolimnetic methane storage may increase in the future. However, whether 20 stored methane eventually reaches the atmosphere during lake overturn is a matter of controversy 21 and depends critically on the response of the methanotroph assemblage. We show that the 22 methanotroph assemblage in a mixing lake underwent both a substantial bloom and ecological 23 succession. As a result, the methane oxidation capacity of the mixed layer kept pace with the methane 24 supplied from the hypolimnion and most of the stored methane was oxidized. This previously 25 unknown aspect of freshwater methanotroph ecology represents an effective mechanism limiting 26 methane transfer from lakes to the atmosphere. 27Here we present a field study covering the entire three months of the autumn lake overturn of shallow 53 eutrophic Lake Rotsee, Switzerland. We asked (i) if the MOB assemblage grows fast enough to oxidize 54 the methane mobilized from the bottom water before outgassing and (ii) whether the standing MOB 55 assemblage is activated, or a new assemblage successively takes over in the changing lake. We used 56 16S rRNA gene amplicon sequencing, pmoA mRNA sequencing and qPCR, CARD-FISH and potential 57 methane oxidation rate measurements to investigate succession, growth and methane oxidation 58 capacity of the MOB assemblage during lake overturn. The rates of physical mixing and the transfer 59 and transformation of methane have been analyzed with a process-based model in a parallel study 14 . 60The present study provides detailed insights into the dynamics of freshwater lake MOB during the 61 critical overturn period. We show that a new and highly abundant MOB assemblage, representing up 62 to 28% of 16S rRNA gene sequences, thrived in the expanding mixed layer. In parallel with MOB 63 abundance, the methane oxidation capacity of the lake increased substantially, thereby limiting 64 methane emissions to a small percentage of the stored methane. 65 From October to December, a total of 4.2 Mg C (ref. 14) of stored methane gradually entered the 80 expanding mixed layer ( Supplementary Fig. 2). Nevertheless, median methane concentrations in the 81 surface layer stayed low, ranging from 0.1 -1.1 µM. During overturn, oxygen levels in the mixed layer 82 dropped down to approx. 175 µM (Dec 12, Fig. 1C), likely due to methane and other reduced 83 substances from the hypolimnion triggering abiotic and biotic oxygen consumption. From December 84 to January, oxygen concentrations increased again. 85The methane oxidizing assemblage prior to lake overturn 86 We analyzed the MOB assemblage with a set of independent and mutually supportive methods. We 87 used 16S rRNA gene sequencing to phylogenetically identify know...

Abstract. In lakes, large amounts of methane are produced in anoxic sediments. Methane-oxidizing bacteria effectively convert this potent greenhouse gas into biomass and carbon dioxide. These bacteria are present throughout the water column, where methane concentrations can range from nanomolar to millimolar. In this study, we tested the hypothesis that methanotroph assemblages in a seasonally stratified freshwater lake are adapted to the contrasting methane concentrations in the epi- and hypolimnion. We further hypothesized that lake overturn would change the apparent methane oxidation kinetics as more methane becomes available in the epilimnion. In addition to the change in the methane oxidation kinetics, we investigated changes in the transcription of genes encoding methane monooxygenase, the enzyme responsible for the first step of methane oxidation, with metatranscriptomics. Using laboratory incubations of the natural microbial communities, we show that the half-saturation constant (Km) for methane – the methane concentration at which half the maximum methane oxidation rate is reached – was 20 times higher in the hypolimnion than in the epilimnion during stable stratification. During lake overturn, however, the kinetic constants in the epi- and hypolimnion converged along with a change in the transcriptionally active methanotroph assemblage. Conventional particulate methane monooxygenase appeared to be responsible for methane oxidation under different methane concentrations. Our results suggest that methane availability is one important factor for creating niches for methanotroph assemblages with well-adapted methane oxidation kinetics. This rapid selection and succession of adapted lacustrine methanotroph assemblages allowed the previously reported high removal efficiency of methane transported to the epilimnion to be maintained – even under rapidly changing conditions during lake overturn. Consequently, only a small fraction of methane stored in the anoxic hypolimnion is emitted to the atmosphere.

<p><strong>Abstract.</strong> In freshwater lakes, large amounts of methane are produced in anoxic sediments. Methane-oxidizing bacteria effectively convert this potent greenhouse gas into biomass and carbon dioxide. These bacteria are present throughout the water column where methane concentrations can range from nanomolar to millimolar concentrations. In this study, we tested the hypothesis that methanotroph assemblages in seasonally stratified lakes are adapted to the contrasting methane concentrations in the epi- and hypolimnion. We further hypothesized that lake overturn would change the methane oxidation kinetics as more methane becomes available in the epilimnion. Together with the change of methane oxidation kinetics, we investigated changes in the transcription of genes encoding methane monooxygenase, the enzyme responsible for the first step of methane oxidation, with metatranscriptomics. We show that the half-saturation constant (K<sub>m</sub>) for methane, obtained from laboratory experiments with the natural microbial community, differed by two orders of magnitude between epi- and hypolimnion during stable stratification. During lake overturn, however, the kinetic constants in the epi- and hypolimnion converged along with a change of the transcriptionally active methanotroph assemblage. Conventional particulate methane monooxygenase appeared to be responsible for methane oxidation under different methane concentrations. Our results suggest that methane availability is important for creating niches for methanotroph assemblages with well-adapted methane-oxidation kinetics. This rapid selection and succession of adapted lacustrine methanotroph assemblages allows high methane removal efficiency of more than 90 % to be maintained even under rapidly changing conditions during lake overturn. Consequently, only a small fraction of methane stored in the anoxic hypolimnion is emitted to the atmosphere.</p>

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