126 Chapter 4 stressor than toxicity. MAG 32 had all genes encoding substrate oxidation and electron transport chain proteins in the anammox pathway (de Almeida et al., 2016), as well as carbon fixation via the Wood–Ljungdahl pathway, which were transcribed at all time points (Figure 4). The second microorganism that persisted throughout all regular and experimental conditions was represented by MAG 44, 93.1% complete with 3.4% contamination, a divergent Planctomycetes genome. Based on metagenomic and transcriptomic analyses, the most likely metabolism performed by this organism was heterotrophic denitrification via narGHI and nirS (Supplementary Table 2). While normalized genome coverage of this MAG varied only between 16-50x across regular and experimental conditions, summed TPM values for all genes transcribed in each time point were highest in the preparatory phase for the sulfide and NO toxicity experiment, as well as before and after the experiment (T3-5; TPM = ~ 168-361) in comparison to previous time points (T0-2; TPM = ~ 33-43; Supplementary Table 2). Microbial community transcriptional responses to substrate removal and toxicity stresses provide insights into community dynamics and resiliency Gene-centric analyses aimed to elucidate the transcriptional responses of the reactor community to two experimental conditions. The first, ammonium removal from the medium while methane, sulfide and nitrate were still provided to the reactor, tested the strength of microbial interactions for the supply of ammonium via DNRA to community members - in particular, to anammox bacteria. The second, sulfide and NO addition while no other substrates were provided to the reactor, aimed to enrich sulfide-oxidizing nitric oxide reducers and to test the limits of microbial community resiliency to these stresses. A major response to ammonium removal was a general inhibition of methane, ammonium and sulfide oxidizers, as indicated by downregulation of their key genes mcrA, pmoA, hzsA, sqr, soxB, sorA, and dsrABC (Figure 5). RT-qPCR of mcrA, pmoA, and hzsA confirmed these trends (Supplementary Table 3). Not surprisingly, anammox bacteria were particularly affected, with an, approximately, 18-fold decrease in hzsA transcription. On the other hand, nitrogen fixation nifDHK genes, ammonium transporter amtB genes, and the ammonium-forming nitrite reductase nrfA genes
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