241 Synthesis and discussion In Chapter 2, we emphasize the importance of improving biogeochemical models via the incorporation of AOM inhibitory thresholds, particularly given the dynamic and anthropogenically impacted nature of coastal ecosystems. Here, we tested the effect of different sulfide concentrations (0.5, 1, 2, and 4 mM) on S-AOM in the anoxic sediment of the Stockholm Archipelago site that showed highest methanotrophic abundance, belonging to the hypoxic bottom water redox condition site (Lilla Värtan). We were able to estimate a half inhibitory constant for S-AOM at around ~ 1 mM, contributing to valuable data for gene-based model refinement. In the case of understanding the specific growth of methanotrophs that couple methane oxidation to sulfate, Fe and Mn oxides (Lenstra et al., 2023) presents challenges. Improving models that use specific Michaelis-Menten kinetics to calculate microbial growth and efficiency (Figure 1) is complicated given the inherent difficulties of cultivating specific ANME microorganisms and characterizing their specific growth and turnover rates. ANME form complex syntrophic partnerships with marine SRB (Murali et al., 2023b), putative freshwater SRB (Lake Cadagno in Chapter 7) or rely on nitrite scavengers (Chapters 5 and 6) to avoid build-up of toxic compounds. Thereby, they are part of complex microbial consortia, being hard to isolate and capture specific cell methane oxidation rates independently. Furthermore, they are slow growing microorganisms (order of weeks) with low activity rates (in ‘t Zandt et al., 2018). In fact, as seen in Chapter 2, S-AOM required two months to become active before sulfide toxicity experiments could be started, and sensitive 13C-CH4 activity assays were required to monitor activity. These incubation approaches make it difficult to obtain maximum biomass growth rates, or even cell division estimation compared to classical OD600 measurements (Figure 1). Continuous culture systems pose additional challenges. Methane, being poorly soluble, must be provided at saturation, and electron acceptors (such as sulfate or nitrate) must be carefully controlled to avoid substrate limitation. Furthermore, headspace gas exchange is critical to mitigate sulfide toxicity, while also considering that sulfide can form FeS precipitates or be re-oxidized to sulfate by manganese oxides, both of which need to be considered when preparing the medium, as in Chapter 6. ANME exhibit significant metabolic plasticity, being capable of using multiple electron acceptors - sulfate, iron, and manganese - depending on environmental conditions. This versatility highlights 8
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