78 Chapter 3 cultivations enable us to observe the results of specific stressors on the microbial community composition without the confounding factors of a highly dynamic natural ecosystem, leading to the discovery of novel taxa potentially involved in driving nutrient cycling under eutrophic conditions in the environment (Arshad et al., 2017; Dalcin Martins et al., 2022, Chapter 4; Delgado Vela et al., 2021) or observing the effect of salinization on ammonium and methane oxidizing microbial communities in a controlled environment (Frank et al., 2023). Moreover, there is an untapped potential for novel metabolism associated with chemolithotrophic coastal microbiomes as many microbial taxa of such microbiomes cannot be assigned to known references (Dalcin Martins et al., 2024, Chapter 2). In this study, we investigated the effects of high and low nitrogen (nitrate and ammonium) and sulfide loading under high methane concentrations on the microbial community structure and function of a coastal anoxic sediment microbiome. Moreover, to assess the effect of salt intrusion on nutrient cycling, we analyzed the effect of salinity increase on a long-term brackish-acclimated culture. We conducted a long-term continuous bioreactor study by monitoring physicochemical parameters, 16S rRNA gene amplicon, and metagenomic sequencing of the two differing bioreactors with a focus on the methanotrophic and sulfide-oxidizing community performing denitrification or dissimilatory nitrate reduction (DNRA). In the process, we co-enriched a member of the betaproteobacterial MBAE14-like family (Pseudomonadales IMCC2047), and betaproteobacterial Rugosibacter species (order Nitrosomonadales) with genomes containing putative novel copper-dependent membrane-bound monooxygenases (CuMMOs). EXPERIMENTAL PROCEDURES Inoculum and bioreactors operation Two 6.8 L bioreactors (Applikon, Delft, The Netherlands) were operated as sequencing batch reactors (SBR) for 15 months (schematic overview of experimental procedures in Supplementary Figure 1). From month 3-4 onwards, a wallassociated biofilm developed (Supplementary Figure 2). Both bioreactors were inoculated anoxically with sediment: each received 25 g from a mixture of all
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