Nitrogen-cycling gene pool shrunk by species interactions among denser bacterial and archaeal community stimulated by excess organic matter and total nitrogen in a eutrophic bay

Highlights

• In a eutrophic bay, microbial density, amount of organic matter (OM) and total nitrogen (TN) strongly positively correlate.

• Eutrophication increases the bacterial and archaeal density and enhances the negative microbial interactions.

• These interactions suppress the relative abundances of density suppressed genes (e.g. amoA, Arch amoA, nirK, and nrfA).

• nirS as a density stable gene independent with microbial densities, OM and TN; while nosZ and hzo were density irrelevant genes.

Abstract

Microbial densities, functional genes, and their responses to environment factors have been studied for years, but still a lot remains unknown about their interactions with each other. In this study, the abundances of 7 nitrogen cycling genes in the sediments from Hangzhou Bay were analyzed along with bacterial and archaeal 16S rRNA abundances as the biomarkers of their densities. The amount of organic matter (OM) and total nitrogen (TN) strongly positively correlated with each other and microbial densities, while total phosphate (TP) and ammonia-nitrogen (NH₃–N) did not. Most studied genes were density suppressed, while nirS was density stable, and nosZ and hzo were density irrelevant. This suggests eutrophication could limit inorganic nitrogen cycle pathways and the removal of nitrogen in the sediment and emit more greenhouse gases. This study provides a new insight of microbial community structures, functions and their interactions in the sediments of eutrophic bays.

Introduction

Estuaries, the linkage of continents and the sea, are special aquatic ecosystems where matter and energy are exchanged along the strong environmental and ecological gradients (Pérez-Ruzafa et al., 2013). Estuaries are more likely to be located by big cities, so not only natural hydrodynamic flows (Pérez-Ruzafa et al., 2013; Yeh et al., 2015), but also anthropogenic disturbances (Nogales et al., 2011) make these areas “melting pots” of environmental stress and foster variable habitats for estuarine life and biodiversity.

Coastal sediments provide ideal habitats for numerous microbes, including bacteria and archaea, since nutrients are accumulated here. Environmental stress may influence these microbes in species abundance, community structure, and function (Awasthi et al., 2014; Reed and Martiny, 2013; Staley et al., 2015). The relationships between species with particular functions and their substrates or products were well studied, for example, our group have learned how ammonia-nitrogen (NH₃–N) influences the ammonia-oxidizing archaea (AOA) and bacteria (AOB) (Zhang et al., 2015b), some other groups focused on how antibiotics increase the abundances of antibiotic resistance genes (Binh et al., 2007; Schmitt et al., 2006). Anthropogenic activities cause a lot of environmental stress like these and change the environmental capacities for all kinds of microbes. An interesting question would be which factors in human's discharge into the estuary has a bigger impact on bacterial and archaeal environmental capacities, nutrients or toxicants. Furthermore, do they alter the microbial community functions and structures directly or through altering the interactions among microbes, which have been overlooked for a long time? How do the functional species and genes respond to the population density in a eutrophic environment? The answers to these questions would be valuable for the control of land-based discharge.

Hangzhou Bay is an ideal area to explore these questions. It is located between Zhejiang Province and Shanghai, China. It is a typical large funnel-shape estuary, which covers an area about 4800 m². It is characterized by a flat bay floor, strong tidal current, and high turbidity (He et al., 2016b). With the booming economy and heavy population of the Yangtze River Delta, Hangzhou Bay became a eutrophic bay. Studies have monitored the high nutrient level (Gao et al., 2011; Qin et al., 2009), heavy metals (Fang and Wang, 2006; Zhang et al., 2008), and polycyclic aromatic hydrocarbons (Chen et al., 2006) in the bay. Eutrophication is a serious problem here, inorganic nitrogen and phosphate were reported to be the main pollutants (Zhejiang Province Ocean and Fisheries Bureau, 2016). Therefore, these pollutants might fuel the extremely diverse nitrogen cycling microbial communities in the sediments and intensify their competition and cooperation.

There are four major microbial inorganic nitrogen cycle processes: nitrification, denitrification, anaerobic ammonia oxidation (ANAMMOX), and dissimilatory nitrate reduction to ammonium (DNRA):

Nitrification is the only known aerobic pathway for oxidizing ammonia to nitrite and nitrate, so it links the reduced N produced by remineralization to the oxidized substrates, nitrite and nitrate required for the N-loss process (Damashek and Francis, 2017). Bacterial amoA and archaeal amoA are responsible for the first and rate-limiting step of nitrification, oxidization of ammonia to hydroxzlamine. Bacteria with amoA gene, i.e. AOB, were long believed to be the only ammonium oxidizer, until a group of archaea with different amoA gene, i.e. AOA, were identified about a decade ago (Konneke et al., 2005). Both populations can dominate in estuaries (Abell et al., 2010; Li et al., 2015; Smith et al., 2015; Wankel et al., 2011). In 2015, some Nitrospira were shown to completely oxidize ammonia to nitrate in one step, a COMAMMOX (complete ammonia oxidation) metabolism (Daims et al., 2015; van Kessel et al., 2015). Their amoA are phylogenetically distinct from the canonical ones (Liu et al., 2020). However, to date, the presence of COMAMMOX bacteria has been rarely demonstrated in coastal areas (Jiang et al., 2019; Liu et al., 2020; Sun et al., 2020), and no studies showed they can outcompete the canonical ammonia oxidizers in estuaries. According to studies from multiple estuaries, nitrification rates can be stimulated by additional relatively small organic matter (OM), but only to a point; past this threshold, further eutrophication causes nitrification to plummet due to oxygen depletion (Caffrey et al., 1993, 2003; Magalhaes et al., 2005; Wankel et al., 2011).

Denitrification and ANAMMOX are two principal microbial processes that remove nitrogen from water ecosystems. Denitrification includes 4 steps to reduce nitrate to nitrogen gas. The rate-limiting one is the reduction of nitrite to nitric oxide, catalyzed by either one from two different nitrite reductases (Nir), the cytochrome cd1 enzyme encoded by nirS or the Cu-containing enzyme encoded by nirK. These two genes don't coexist in most species (Graf et al., 2014; Jung et al., 2012). In general, nirS genes are higher both in diversity which is also somewhat more reflective of 16S diversity and in abundance in estuaries, compared to nirK genes (Abell et al., 2010; Graf et al., 2014; Heylen et al., 2006; Mosier and Francis, 2010; Smith et al., 2015). The last step of denitrification, the reduction of nitrous oxide to dinitrogen, is catalyzed by nitrous oxide reductase encoded by nosZ gene. Denitrifiers with nirS are more likely to have this gene and enzyme, hence, a complete denitrification pathways (Graf et al., 2014), while denitrifiers without this enzyme may release greenhouse gas, nitrous oxide, directly into the atmosphere which may accelerate global warming. Therefore, denitrification in the estuaries has gained much attention not only as a sole N loss process, but also as a process with a substantial impact on the global N₂O budget and global warming (Damashek and Francis, 2017; Kroeze and Seitzinger, 1998; Seitzinger and Kroeze, 1998). In the last few decades, our understanding of N loss from aquatic ecosystems has been complicated by the discovery of ANAMMOX in which ammonia and nitrite are combined into N₂. hzo gene is a common biomarker for it. This gene encodes hydrazine oxidoreductase, which is in charge of the last step of ANAMMOX. Though in some estuarine studies, ANAMMOX did account for the bulk of N loss from estuaries (Engstrom et al., 2005; Teixeira et al., 2012), denitrification still is the main contributor in most studies estuaries (Lisa et al., 2015; Teixeira et al., 2012). Because eutrophication in these areas typically stimulates it and suppresses ANAMMOX (due to competition for nitrite) or simply lowers the percent ANAMMOX due to constant ANAMMOX but massive denitrification rates (Damashek and Francis, 2017).

DNRA is a process competing with denitrification and ANAMMOX for nitrite and nitrate, which turn them into ammonia, so it can also cooperate with ANAMMOX by providing ammonia. Numerous recent studies have measured high benthic DNRA rates compared to denitrification or ANAMMOX (An and Gardner, 2002; Gardner et al., 2006; Song et al., 2014), which brought it to prominence in estuaries. nrfA is its common biomarker, which encodes a nitrite reductase to turn nitrite to ammonia directly. Studying DNRA is critical for understanding the fate of nitrogen in estuaries, since different from denitrification and ANAMMOX, it retain ammonium within in the system.

Due to these processes and genes may exist in all kinds of combinations, in this study, the water and sediment quality, and the abundances of 7 nitrogen cycling genes in the sediments from Hangzhou Bay were analyzed along with bacterial and archaeal 16S rRNA abundances as the biomarkers of their densities. Therefore, we can get a better understanding of the N cycling microbial community's structures and behavior in a eutrophic condition, and fit a piece of puzzle into the whole mysterious map of aquatic microbiome.