Discussion
Patterns of microbial community assembly  and interaction in river sediments are far from clear, evidenced by the high  proportion of uncultured and unclassified organisms present in subsurface  environments together with the overwhelming discovery of new microbial lineages  involved in major geochemical cycles (Anantharaman et al. 2016). In this study, we showed that nitrate amendment to sulfide-rich river sediments overwrote the redox conditions and thus simplified the microbial community composition and interaction by enriching sulfur-oxidizing denitrifiers.
 
Most denitrifiers are heterotrophs and a minor group are chemoautotrophs that utilize inorganic energy sources such as reduced sulfur and Fe(II) (Hayakawa et al. 2013). After nitrate amendment, we identified (i) a consistent nitrate reduction and the coupled AVS oxidation, (ii) an accumulation of nitrite and sulfate, and (iii) a drastic Fe(II) oxidation and the coupled Fe(III) accumulation in the final stage (Figure S6). Notably, more nitrate in stoichiometry was consumed than the sum consumption  of AVS and Fe(II) if N2 was the end product (by denitrification), so  there must have been cryptic electron donors fueling nitrate reduction (see  supplementary information: the cryptic electron donor).
 Among the identified genera, the control and early nitrate communities were dominated by methanogens (Patel & Sprott 1990; Ma et al. 2005; Imachi et al. 2008; Braeuer et al. 2011; Kroeninger et al. 2017) and syntrophic bacteria (de Bok et al. 2001; McInerney et al. 2007; Qiu et al. 2008). When exposed to elevated nitrate, most genera were gradually outcompeted including some denitrifiers. Members of Thiobacillus were characterized as autotrophic sulfide oxidizers and denitrifiers (Bosch et al. 2012; Dolejs et al. 2015); T. ferrooxidans might even feed on Fe(II) (Nemati et al. 1998). The Thiobacillus detected from late nitrate communities shared the highest 16S rRNA gene similarity with T. thiophilus strain D24TN (99%) and T. denitrificans strain NCIMB 9548 (98%), both were obligate chemolithotrophs and facultative anaerobes, using thiosulfate or AVS as the electron donor, nitrate or oxygen as the electron acceptor, and CO2 as the sole carbon source (Kelly & Wood 2000; Kellermann & Griebler 2009).
The dominance of Thiobacillus (in abundance) reappeared in the community enrichments (Figure 2E, 5A). Moreover, pure culture of isolated T. thiophilus was performing simultaneous nitrate-reduction, AVS-oxidation and thiosulfate-oxidation (Table 1) so that it dominated NS enrichments (in abundance) because of its high metabolic fitness. Consistently, several studies identified an enhanced sulfide oxidation alongside the rise of Thiobacillus after nitrate amendment (Xu et al. 2014; He et al. 2017; He et al. 2018). The other winner Luteimonas was barely detectable at first but significantly enriched later, in agreement with a recent discovery in which Luteimonas dominated communities of sediment samples with an increasing proportion of recalcitrant carbon (Wu et al. 2018). Luteimonas spp. might be a potential Fe(II) oxidizer, as Zhang et al. identified a predicted ferric reductase gene and two subunits of ferredoxin nitrite reductase from the genome of L. abyssi XH301T (Zhang et al. 2015).
There are studies pointing out that community functionality is a direct reflection of physiochemical condition (Nelson et al. 2016; Gibbons 2017; Louca et al. 2017), and the rise of Luteimonas and Thiobacillus well substantiated that. Among so many denitrifiers, why lithotrophic Luteimonas and Thiobacillus rather than those organotrophs gained dominance remained unclear, under conditions of so abundant TOC.
Keystone taxa are believed important in maintaining the functionality and integrity of an ecosystem, whose extinction often leads to community fragmentation or even collapse (Wu et al. 2016). In this study, microbial interactive networks under nitrate amendment were getting smaller and simpler along with the loss of keystone taxa which predominantly comprised of members of Class Proteobacteria and Chloroflexi, as we hypothesized. We identified no strong correlation between OTU abundances and their interactive significance, although keystone nodes turned to be sparse OTUs. Instead, the network connectivity was speculated highly associated with microbial cross-feedings or metabolic interdependencies that were predominantly sustained by native auxotrophs (Mee et al. 2014; Embree et al. 2015; Ponomarova & Patil 2015; Hubalek et al. 2017). For example, syntrophic bacteria (e.g. Smithella and Syntrophorhabdus) and methanogens (e.g. Methanolinea) were auxotrophs, performing extensive interspecies cross-feeding, and were keystone taxa in the microbial interactive networks.
 Nitrate amendment might have other effects on microbial communities than simplification. We identified several rapid microbial responses to elevated nitrate, whereas the community richness, α-diversity and evenness were little affected (Figure 2, Day 0 - 4). Several genera such as Methanomassiliicoccus, Vulcanibacillus, Thermomonas and Bacillus were significantly enriched on Day 4 but were eventually suppressed or eliminated (Figure 1E, 1F). So elevated nitrate introduced both days- and weeks- effects upon microbial communities of sulfide-rich river sediment. For the first few days it boosted biodiversity possibly by niche creation (e.g. bridging metabolic interdependencies between nitrate reducers and AVS oxidizers), in agreement with our previous work (Xu et al. 2014).
In summary, this study provides insights about the microbial assemblage and interactions in river sediments in the presence of elevated nitrate. Microbial communities were eventually simplified and specialized in nitrate reduction coupled with sulfide and Fe(II) oxidation. The significant loss of biodiversity and other functions were considered the negative ecological impacts of nitrate amendments because biodiversity increases community resistance to environmental perturbations (Griffiths & Philippot 2013; Isbell et al. 2015; Evans et al. 2017; Xun et al. 2019). Thiobacillus and Luteimonas gained dominance while keystone taxa appeared to be diminished or extinct, leading to fragmentated interactive networks.