Methane oxidation in anoxic lake water stimulated by nitrate and sulfate addition

Summary Methanotrophic bacteria play a key role in limiting methane emissions from lakes. It is generally assumed that methanotrophic bacteria are mostly active at the oxic‐anoxic transition zone in stratified lakes, where they use oxygen to oxidize methane. Here, we describe a methanotroph of the genera Methylobacter that is performing high‐rate (up to 72 μM day−1) methane oxidation in the anoxic hypolimnion of the temperate Lacamas Lake (Washington, USA), stimulated by both nitrate and sulfate addition. Oxic and anoxic incubations both showed active methane oxidation by a Methylobacter species, with anoxic rates being threefold higher. In anoxic incubations, Methylobacter cell numbers increased almost two orders of magnitude within 3 days, suggesting that this specific Methylobacter species is a facultative anaerobe with a rapid response capability. Genomic analysis revealed adaptations to oxygen‐limitation as well as pathways for mixed‐acid fermentation and H2 production. The denitrification pathway was incomplete, lacking the genes narG/napA and nosZ, allowing only for methane oxidation coupled to nitrite‐reduction. Our data suggest that Methylobacter can be an important driver of the conversion of methane in oxygen‐limited lake systems and potentially use alternative electron acceptors or fermentation to remain active under oxygen‐depleted conditions.

. Methane concentration over time in the incubation experiment with 12 m depth summer samples, with oxygen (grey triangles) or humic substance (orange circles) added. No significant change in methane concentration over time was observed, the R 2 of the linear regression analysis was 0.04 for the oxygen addition experiment and 0.03 for the experiment with the addition of humic substances. Table S3.   S4. Genome-inferred metabolic pathways of MAG bin-63. Pathways indicated in green were detected, sequences of grey pathways were lacking. Genes were indicated where possible; numbers refer to EC database numbers. For details, see Supplementary File S1.

Fig. S5.
Maximum likelihood phylogenetic tree based on 34 concatenated single-copy, proteincoding genes (following the method of Dombrowski et al., 2018) of the three highest average abundance  detected in the winter incubation experiment under anoxic conditions and amended with methane. Fig. S6. GC coverage plots including the contigs of the 10 most abundant bins obtained in the sequenced sample with (A) the taxonomic classification of GTDB-Tk at the level of family, and (B) with the taxonomic classification of CheckM, indicating that the MAG bin-63, indicated in dark blue in panel B, was affiliated to the family Methylomonadaceae, indicated in teal in panel A. Table S1. Methane oxidation rates (MOR) and additional information regarding the rate measurements. The R 2 given is the R 2 of the linear regression analysis used to determine the methane oxidation rate. The oxidizing equivalents surplus/deficiency indicates the µM of methane that could have been oxidized by the electron acceptor, after the amount that was oxidized within the 24 h incubation experiment was deducted. .08 a the methane oxidation rate is based on the linear regression analysis, whilst the actual methane oxidation consumption in the incubation vials was lower due to methane limitation. b a subset of the data points was used (t6, t12 and t24) c Using a 8:3 ratio of NO3:CH4 and a 1:1 ratio of SO4:CH4 (Segarra et al. 2013) d Low R 2 of regression analysis Table S2. Relative abundances (%) of known methanotrophs detected in the Lacamas Lake water column and incubation experiments (C denotes control) as determined by 16S rRNA gene amplicon sequencing. Any group with a percentage <0.1% is considered not significant and displayed as zero. Total reads obtained are indicated. Total 16S rRNA gene copies per liter were determined using quantitative PCR of the total prokaryotic community, with SD being the standard deviation of 3 experimental replicates. The taxonomic assignment of each of the mentioned methanotrophs is shown in Fig. 3.

Natural conditions (depth in m)
Incubations (12 m Methylobacter spp. Reads per sample (x10 6 ) 1.7 1.4 1.9 1.8 0.9 1.5 1.7 1.5  Table S3. pmoA relative abundance (%) in samples of the summer and winter water column and incubations (C denotes control). The tentative taxonomic assignment of the denovo sequences can be observed in Figure 5.

Natural conditions (depth in m)
Incubations (12 m  Classification was inferred by GTDB-Tk. An unassigned species (i.e., s__) indicates that the query genome is either i) placed outside a named genus or ii) the ANI to the closest intra-genus reference genome with an AF >=0.65 is not within the species-specific ANI circumscription radius. Classification was performed by placement of the genome in the reference tree and by using the relative evolutionary divergence (RED). Red value indicates the relative evolutionary divergence for a query genome. aa_percent: indicates the percentage of the multiple sequence alignment spanned by the genome (i.e. percentage of columns with an amino acid).
X. single sampling (for DNA purposes) #. sampling in biological duplicate +. sampling in biological quadruplicate Table S7. List of 34 single-copy marker genes used for phylogenetic analysis of MAGs. Table S8. Relative abundance of methanotroph OTUs as percentage of all reads in the sample (C denotes control). The taxonomic affiliation of the OTUs is shown in Figure 3.