245 Synthesis and discussion In this Chapter 8, we investigated whether the Methylomonadaceae described in Chapter 3 possessed an extensive repertoire of multiheme c-type cytochromes (MHC), similar to the findings for “Ca. Methanoperedens” in Chapter 7, which could indicate potential for extracellular electron transfer (EET). For that, we analyzed the 11 high-quality Methylomonadaceae metagenome-assembled genomes (MAGs) described in Chapter 3 and screened for genes encoding for three or more CXXCH motifs in their genomes, similar to our approach in Chapter 7. This allowed us to identify a variety of MHC-type proteins with three or more CXXCH motifs. The counts of MHCs (with more than three CXXCH motifs) for each MAG were as follows: “Methylobacter 1” (6), “Methylocystis” (3), “Methylomonas 1” (15), “Methylomonas 3” (5), “Methylomonas 5” (2), “Methylovulum” (6), “Methylobacter whittenburyi” (4), “Methylomonadaceae KS41 1” (3), “Methylomonas 2” (6), “Methylomonas 4” (1), and “Methylotetracoccus 1” (1). Notably, “Methylobacter 1” and “Methylomonas_1” each harbored MHC proteins with 10 or more CXXCH motifs, highlighting their significant potential for EET. Additionally, all these MAGs contained electroconductive pilA and Type IV pilus assembly proteins, further supporting their potential for EET. Most MAGs also possessed riboflavin synthase, a key enzyme associated with producing electron shuttles, enhancing potential EET capabilities. The MAGs containing riboflavin synthase were: “Methylobacter_1”, “Methylobacter whittenburyi”, “Methylocystis”, “Methylomonadaceae KS41 1”, “Methylomonas 1”, “Methylomonas 2”, and “Methylomonas 3”. Similarly, Li et al. (2023) studied iron-rich freshwater anoxic sediments from Lake Fuxian and identified MAGs from the genus Methylomonas as key methanotrophs involved in methane-dependent iron reduction, with potential for EET via large extracellular MHCs, PilA, and riboflavin metabolism. Another study highlighted the family Methylomonadaceae as the most active and abundant in Tibet Lake sediments, with potential for nitrate/nitrite reduction and iron reduction (Deng et al., 2024). Oxidation of short chain hydrocarbons by divergent Cu-MMO harboring microorganisms In Chapter 3, we explored a methane-saturated (micro)anoxic bioreactor operating under low sulfide, ammonium, and nitrate conditions, simulating an oligotrophic environment. Using this untargeted enrichment approach, we successfully coenriched novel chemolitho(auto)trophic microorganisms. These included members 8
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