70 Chapter 2 in surface sediments could not account for the 7-fold higher benthic methane efflux in Site 7 relative to Site 5, given that these two sites had nearly identical depth and thickness of the SMTZ (0-10 cm) and similar potential methane production rates in top sediments (Figure 4). Instead, our experimental results in combination with the virtual disappearance of ANME-2 sequences at Site 7, but their persistence at Site 5, suggests that the disruption of the methane biofilter likely accounted for this 7-fold difference. We infer that the putative distribution of ANME-2 at our study sites is most likely linked to high sulfide concentrations (0.5-1.2 mmol L-1) and especially the higher annual exposure to sulfide (0.88 mmol year-1) at Site 7 compared with Site 5 (0.39 mmol year-1), which could have directly caused sulfide toxicity in ANME-2 cells. We note that the relationship between sulfide and AOM-rates is non-linear (Figure 6), which could explain why the difference in sulfide exposure between the two sites (~ 2-fold) does not mathematically account for the difference in benthic methane efflux (7-fold) between the two sites. In addition, we cannot rule out an effect of other (abiotic) factors, such as differences in the type of organic matter and sediment composition in general between the two sites. These results agree with an early study on environmental controls on ANME abundances, which found that sulfide concentrations negatively correlated with 16S-based ANME-2 abundances, while positively correlated with ANME-1 abundances, potentially due to sulfide-oxidizing bacteria co-occurring with ANME-1 but not with ANME-2 (Biddle et al., 2012). Additionally, sulfide could hamper the enzymatic activity of the F420-dependent sulfite reductase (Fsr) via product inhibition, leading to sulfite toxicity as well. Sulfite is known to inhibit methanogenesis (Balderston & Payne, 1976), and the Fsr enzyme, first described in Methanocaldococcus jannaschii, detoxifies sulfite by reducing it to sulfide, which can then be assimilated. While sulfite toxicity generally occurs due to its intracellular reaction with proteins and sulfhydryl groups (Johnson & Mukhopadhyay, 2005; Wedzicha, 1992), in methanogens specifically sulfite reacts in vitro with and inactivates the key methanogenic enzyme, methyl-coenzyme M reductase (Becker & Ragsdale, 1998; Mahlert et al., 2002). The recent crystal structure of Fsr from Methanothermococcus thermolithotrophicus revealed Fsr as the simplest sulfite reductase crystallized so far, with similar traits to assimilatory sulfite reductases, having, interestingly, a
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