Heterotrophic diazotrophs in a eutrophic temperate bay (Jiaozhou Bay) broadens the domain of N₂ fixation in China's coastal waters

Highlights

• The occurrence of N₂ fixation was demonstrated under N-replete conditions in the temperate Jiaozhou bay, Northern China.

• Organic carbon may be an important regulation of N₂ fixation in coastal areas like Jiaozhou Bay.

• Phytoplankton-derived organic carbon may stimulate N₂ fixation in Jiaozhou Bay.

• This study may provide new insights into the regulation of marine N₂ fixation and its relationship with carbon fixation.

Abstract

N₂ fixation in coastal waters represents an overlooked process in the global marine nitrogen cycle, yet the mechanisms and controls are still poorly understood. This study first conducted simultaneous measurements of N₂ fixation rates, primary production, chlorophyll a (Chl a) concentration, organic carbon concentration, and the composition of nitrogen-fixing genes (nifH) in a eutrophic temperate bay (Jiaozhou Bay, the western Yellow Sea) from 2015 to 2017. The activity of N₂ fixation (not detected–1.53 nmol N L ⁻¹ h ⁻¹) were observed at most sampling stations, suggesting that the N-replete conditions in Jiaozhou Bay did not prevent N₂ fixation. The major N₂ fixers in JB were heterotrophic, while N₂ fixation were mainly contributed by > 10 μm fraction, indicating the possible active N₂ fixation of heterotrophic diazotrophs on large particles or planktonic aggregates. Positive correlations between N₂ fixation and organic carbon concentration were observed in spring. Further estimates of the level of phytoplankton-derived organic carbon and its potential relationship with N₂ fixation suggest that phytoplankton processes may stimulate N₂ fixation in Jiaozhou Bay by increasing the bioavailability of organic carbon or/and providing suitable environmental conditions for N₂ fixation. Our results first infer the co-regulation of physical factors and biological processes on N₂ fixation. As Jiaozhou Bay has been subject to anthropogenic pollution of inorganic nutrients, our results provide insights into the dominant controls on N₂ fixation and relevant biogeochemical processes in coastal waters under anthropogenic disturbance.

Introduction

N₂ fixation is an essential source of bio-available fixed-N to the ocean, which plays a key role in marine biogeochemical cycles and impacts climate on a global scale (Capone et al., 2005; Karl et al., 2002). In recent years, relevant researches have extended the investigation of N₂ fixation from oligotrophic open oceans (Ahmed et al., 2017; Capone et al., 2005; Karl et al., 2002) to dark, colder ecosystems, and coastal regions (Balis et al., 2012; Farnelid, 2013; Jiang et al., 2018; Mulholland et al., 2019; Rees et al., 2009; Shiozaki et al., 2017), where N₂ fixation was originally thought to be insignificant. The view that N₂ fixation contributes minimally to the N loading in coastal/estuarine and other N-replete ecosystems has been increasingly challenged (Damashek and Francis, 2017; Li et al., 2019a,b; Mulholland et al., 2019). The nitrogenase nifH genes from non-cyanobacterial diazotrophs (e.g. heterotrophic bacteria) had been suggested to be widespread, and may be more abundant in coastal and estuarine regions, even under N-replete conditions (Affourtit et al., 2001; Bombar et al., 2016; Jenkins et al., 2004), inferring the greater potential for N₂ fixation in coastal waters relative to the open ocean (Farnelid, 2011, 2013) and the possible regulation of organic carbon on N₂ fixation. Organic carbon is a major growth-limiting factor for oceanic heterotrophic and mixotrophic bacteria (Wambeke et al., 2008). The influence of dissolved organic carbon (DOC) on heterotrophic N₂ fixation has been reported in both the open ocean (Moisander et al., 2014; Zehr and Karl, 2007) and coastal areas (Li et al., 2019a,b; Rahav et al., 2013). The enhancement of heterotrophic N₂ fixation by particulate organic carbon (POC) has also been reported in the North Pacific subtropical gyre (Farnelid et al., 2018). Hence, the absence of N₂ fixation in some circumstances is generally attributed to physical and chemical constraints in the marine environment, rather than the lack of genetic potential (Farnelid, 2013).

Jiaozhou Bay (JB, ~120°E, ~36°N), an inlet of the Yellow Sea (a marginal sea of the China seas), may be an ideal location to assess the occurrence and the dominant controls on N₂ fixation in coastal waters subject to anthropogenic impact. There are several rivers flowing into the bay, with an average freshwater discharge of ~6.7 × 10⁸ m⁻³ yr⁻¹ (Sheng et al., 2014). Over the past 30 years, anthropogenic development (urbanization, industrialization, reclamation, etc.) has decreased the bay area from 535 km² in 1863 to 343 km² in 2012 (Ma et al., 2014). Further, human activity has caused an increase in nutrient content and the change of nutrient construction (Shen, 2001, 2006; Yuan et al., 2018; Zhao et al., 2010), and thus altered the annual variability of chlorophyll a (Chl a) concentrations and primary productivity (PP) (Sun et al., 2011). Though N₂ fixation rates have not yet been reported to date, there are several evidences suggesting the occurrence of N₂ fixation here: primarily, heterotrophic diazotrophic bacteria (mainly Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria and Epsilonproteobacteria) were detected in the coastal waters of JB in the summer of 2011 (Zhang et al., 2015a,b), thus confirming their diversity and wide distribution in marine and estuarine waters (Farnelid, 2013). Further, the occurrence of N₂ fixation has been reported in the adjacent temperate Yellow Sea (Zhang et al., 2012). It is likely that diazotrophs may frequently enter JB from the Yellow sea, due to the relatively rapid mixing rate between the two water bodies (renewal time ~10 d; Yang et al., 2013). Finally, geochemical mass balance calculation, though of relatively large uncertainties, indicated a net new N input besides other known N sources (i.e., riverine input and aeolian deposition) in such eutrophic coastal waters (Yuan et al., 2018). N₂ fixation may therefore be a potential candidate for this N source in JB.

As an interface between terrestrial and marine ecosystems, JB may also be suitable to assess the regulation of organic carbon on N₂ fixation. Terrestrial inputs, including river inputs and wastewater discharge, bring large amount of organic carbon into JB (Li et al., 2010; Yang et al., 2010). In addition, phytoplankton is also an important source of organic carbon in JB (Yuan et al., 2018). Phytoplankton is a major source of ‘fresh’ organic carbon (Biddanda and Benner, 1997), playing a major role in shaping local nutrient fields and distributing available organic matter by releasing photosynthetic carbon (Kirchman et al., 2001). The bioavailability of organic matter in eutrophic coastal waters may provide significant benefit for non-cyanobacterial diazotrophs to be active (Zhang et al., 2015a,b). The effect of macroalgal blooms on diazotrophic abundance and community composition have been reported in the coastal surface waters in the Yellow Sea (Zhang et al., 2015a,b). These findings infer the possible correlation between phytoplankton-derived organic carbon and N₂ fixation in coastal waters, which have been hypothesized in our previous study in the subtropical Daya Bay located in the northern South China sea (Li et al., 2019a,b).

The main goals of this study were as follows: (1) to measure the possible N₂ fixation rates and determine the composition of nifH in JB, (2) to determine the influence of organic carbon on N₂ fixation in JB, and (3) to identify the regulation of phytoplankon-derived organic carbon on N₂ fixation. Our results will build upon previous knowledge of N₂ fixation in coastal areas and provide new insights into the impacts of anthropogenic activities on coastal biochemical dynamics.