Introduction
Microorganisms in nature co-exit as communities, and species interactions are essential to stabilize their composition and functionality (Donaldson et al. 2016). Members of a community may interact bidirectionally or unidirectionally, and microscale communities frequently assemble and disassemble by migration, attachment, and detachment from surfaces or cells, leading to mutualism, parasitism or competition among them (Cordero & Datta 2016).
Environmental fluctuations are altering the biodiversity, which in turn is a key driver of biogeochemical processes. Reactive nitrogen (Nr, all nitrogen species other than N2) is widely utilized by plants and algae (Chen et al. 2011) but may cause ecological issues if too abundant. In the past few decades, vast quantities of Nr have been produced to meet the increasing demand for food and energy (Lutz et al. 2001). Many of them can be nitrified to highly mobile nitrate and are leached into rivers, leading to eutrophication and seasonal hypoxia (McIsaac et al. 2001; Fowler et al.2013). Rivers were reported to convert (50 ± 20)% of Nr inputs from adjacent ecosystems into N2 while transporting the other half to coastal water (Xia et al. 2018). River sediments contributed a significant part of total nitrogen transformation.
In subsurface or deeper sediments where oxygen cannot penetrate, nitrate ranks the best alternative electron acceptor in association with anaerobic oxidation of organic matters, sulfide and ferrous iron (Burgin & Hamilton 2007). It was reported that nitrate reduction accounted for 10 to 40% nitrate removal in the sediments of Michigan wetlands, lakes and creeks (Brunet & GarciaGil 1996; Burgin & Hamilton 2008). A number of studies have obtained positive effects of nitrate input into the sulfide-rich river sediments by stimulating denitrifiers, autotrophic sulfide oxidizers and Fe(II) oxidizers (He et al. 2017; Heet al. 2018). Our previous study also identified an enrichment of functional genes involved in nitrogen (e.g. nir , nif ,nor and nas ) and sulfur (e.g. sox , dsr ,apr and sir ) cycles when native microorganisms were exposed to elevated nitrate for 6 days (Xu et al. 2014).
In an ecological view however, external nitrate injection is considered an environmental perturbation, and how it shapes microbial communities, especially in a long run, has not yet been studied. Next-generation sequencing (NGS) provides extensive inferences about microbial community structure and functional profile, which are high-throughput, culture-independent, and reproducible, but are far less convincing than solid experimental data. In 2012, an approach for identifying new strains, microbial interactions and functions by culturing not a single isolate but the entire community with serial dilution was proposed (Lagier et al. 2012; Lagier et al. 2016). This approach has been so far explored in human gut and animal rumen microbiomes (Zehavi et al. 2018). Considering the key role of nitrate in biosphere and the complexity of microbial network involving in nitrogen cycling, the combination of NGS and community enrichment could be the promising strategy.
In this study, we hypothesized (i) that elevated nitrate would enrich native denitrifiers in association with a suppression of methanogens and sulfate reducers, resulting in much specialized sediment communities; (ii) that the overall community functionality would gradually converge at nitrate reduction coupled with sulfide oxidation; and (iii) that the microbial interactive networks would be remodeled due to the rise of denitrifiers and their metabolic specializations. Excess nitrate was injected to river sediments in the Pearl River Delta, which was rich in organic carbon and sulfide, to simulate a natural nitrate surge. Community successions and physio-chemical conditions were monitored for 32 days. In addition to 16S rRNA gene amplicon sequencing, community enrichments were cultured by serial dilution to isolate strains of sulfur-oxidizing denitrifiers, and to match the results to those of NGS. Our work describes new entry points for the undefined microbial mechanisms of nitrate-induced biogeochemical cycling in river sediments.