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.