Anaerobic Methanotrophic Archaea of the ANME-2d Cluster Are Active in a Low-sulfate, Iron-rich Freshwater Sediment

ANaerobic MEthanotrophic (ANME) archaea remove the greenhouse gas methane from anoxic environments and diminish its flux to the atmosphere. High methane removal efficiencies are well documented in marine environments, whereas anaerobic oxidation of methane (AOM) was only recently indicated as an important methane sink in freshwater systems. Freshwater AOM-mediating microorganisms lack taxonomic identification and only little is known about metabolic adaptions to prevailing biogeochemical conditions. One of the first study sites providing information about AOM activity in freshwater sediment is Lake Ørn, a low-sulfate, iron-rich Danish lake. With the aim to identify freshwater AOM-mediating archaea, we incubated AOM-active anoxic, nitrate-free freshwater sediment from Lake Ørn with 13C-labeled methane (13CCH4) and 13C-labeled bicarbonate (13CDIC) and followed the assimilation of 13C into RNA by stable isotope probing. While AOM was active, 13CCH4 and probably also 13CDIC were incorporated into uncultured archaea of the Methanosarcinales-related cluster ANME-2d, whereas other known ANME lineages were not detected. This finding strongly suggests that ANME-2d archaea perform AOM coupled to sulfate and/or iron reduction and may have the capability of mixed assimilation of CH4 and DIC. ANME-2d archaea may thus play an important role in controlling methane emissions from nitrate-depleted and low-sulfate freshwater systems.


SR and AOM rates
In both control and 13 CH 4 -amended core incubations, sulfate reduction rates ranged from ~6 ± 0.1 to 32 ± 2 nmol cm -3 day -1 without showing a trend over time ( Figure   2D). In the same cores, AOM rates were in the range of 1 ± 0.1 to 9 ± 8 nmol cm -3 day -1 , whereas highest rates were measured in the first two time points and thus, AOM activity apparently decreased with time ( Figure 2E). In slurry incubations, SR rates decreased over time both in control and 13 CH 4 -amended incubations from 19 ± 0.6 nmol cm -3 day -1 to 1 ± 0 nmol cm -3 day -1 ( Figure 3D). AOM rates increased over time from the detection limit, reaching up to 33 ± 21 nmol cm -3 day -1 at day 72 ( Figure   3E), and clearly exceeded SR rates at this last time point of the incubation.
Lake Ørn sediment is characterized by rapid iron-driven reoxidation processes and cryptic sulfur cycling involving microbial disproportionation of intermediate sulfur species (Norði et al., 2013, Weber et al., 2016. Therefore, microbially produced 35 S sulfides were most likely partially reoxidized and SR rates may be underestimated several fold, thus reflecting net instead of gross rates (Moeslund et al., 1994). At the same time, a previous Lake Ørn study (Norði et al., 2013) proposed that methanogenesis was co-occurring with AOM in the AOM zone, which was also indicated in our incubations by relatively constant methane concentrations despite AOM activity and a slight net methane accumulation in the control incubations.
Therefore, even though 13 C DIC produced by anaerobic methane oxidizers would have been diluted in a large DIC pool and consequently, the error should be relatively small, some of it could have been microbially re-converted to 13 CH 4 , causing an underestimation of AOM rates.
Therefore, SR and AOM rates presented here serve to confirm the activity of microbial sulfate reducers and anaerobic methane oxidizers. However, the magnitude of the rates as well as the corresponding SR:AOM ratio may be interpreted with caution. In the 13 C DIC slurry incubation, the 13  The results of the T-RFLP analysis suggested that more taxonomical units may potentially assimilate methane and/or bicarbonate than indicated by the 16S rRNA clone libraries or that cross-feeding of 13 C-labeled metabolites occurred.

Identified archaea in AOM zone
The 16S rRNA clone libraries and the T-RFLP analysis provided further information about diversity and identities of archaea that were -besides members of ANME-2dactive in the AOM zone of Lake Ørn sediment. The identified groups were (1) The linage Thermoplasmatales showed a high contribution to the RNA pool at incubation start in the AOM zone of Lake Ørn sediment. Previously, Thermoplasmatales were found in numerous freshwater and marine habitats (e.g.,     Table S1. Taxonomic affiliation, numbers and operational taxonomical units (OTUs) of sequences obtained from 16S rRNA clone libraries at incubation start (T 0 ) for one representative sample of 13 C CH4 and 13 C DIC incubations, each from the 13 C fraction and the 12 C fraction of the same CsTFA gradients.  Table S2. Relative abundances (%) of Operational Taxonomical Units (OTUs) obtained from T-RFLP analysis at incubation start (T 0 core and slurry) and for one representative sample of the 13 C fraction and the 12 C fraction of the same CsTFA gradient of the 13 C CH4 and 13 C DIC incubations, respectively.