150 Chapter 5 0 25 50 DNA RNA DNA RNA DNA RNA DNA RNA Reads or transcripts Others (< 4%) Phycisphaerae g JABWBH01 'Ca. Methanoperedens BLZ2' 'Ca. Methylomirabilis oxyfera' Unbinned Desulfobacillus 2 Thermoanaerobaculia g JACTMI01 2 Aquamicrobium A Aggregatilineales g SB15 Brocadia Aggregatilineales g CFX10 0 25 50 75 100 DNA RNA DNA RNA DNA RNA DNA RNA Reads or transcripts mapped (%) T0 T1 T2 T3 0 25 75 100 DNA RNA DNA RNA DNA RNA DNA RNA Reads or transcripts mapped (%) 17 45 16 26 12 44 11 38 Figure 3. Top MAGs (> 4% metagenomics reads assigned for all time points) community and activity indicated as TPM (> 4%). Additional categories include “Others (<4%) for MAGs that were below the 4% thresholds and unbinned fraction. Categories are ordered from highest to lowest on DNA-based % of T0 values. “Ca. Methanoperedens” percentages are indicated with numbers in the pink box in addition. Despite the shifts in methane oxidation potential, “Ca. Methanoperedens” demonstrated high resilience, as previously reported for oxygen or salt stress (Echeveste Medrano et al., 2024b, Chapter 6; Guerrero Cruz et al., 2018). Notably, “Ca. Methanoperedens” remained the most active community member (RNAbased) for all conditions except for T2, where “Phycisphaerae g JACWBH01” represented almost 50% of the transcripts recovered (Figure 3). MAG “Phycisphaerae g JACWBH01” has previously been hypothesized to act as a nitrate reducing fermenter in the same culture (Legierse et al., 2023). Using cell debris or excreted metabolites, Phycisphaerae could be efficient nitrate reducers outcompeting “Ca. Methanoperedens” under stress. The main nitrite-scavenging microbial community member, “Ca. Methylomirabilis oxyfera”, was previously reported to contribute about 20% to the overall methane oxidation in this culture (Wissink et al., 2024). In our study, “Ca. Methylomirabilis oxyfera” showed a decrease in relative abundance and activity in the DNA and the RNA data throughout the experiment, indicating a gradual inhibition of this methanotroph due to sulfide exposure and toxicity. Consequently, the inhibition of methane oxidation activity observed for “Ca. Methanoperedens”, especially upon long-term sulfide exposure, should be viewed in conjunction with the transient loss of “Ca. Methylomirabilis oxyfera” from the enrichment culture (Figure 3)
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