PUBLICATIONS Journal of Geophysical Research: Biogeosciences RESEARCH ARTICLE 10.1002/2017JG003766 Key Points: • Fermentation reaction is the dominant pathway of DNRA in the Yangtze Estuary • Organic matter and NO2 stimulated the metabolism of DNRA microorganisms 2+ • Oxidation of Fe and sulfide can enhance fermentative DNRA by providing extra free energy
Supporting Information: • Supporting Information S1 Correspondence to: L. Hou,
[email protected]
Citation: Yin G., L. Hou, M. Liu, X. Li, Y. Zheng, J. Gao, X. Jiang, R. Wang, C. Yu, and X. Lin (2017), DNRA in intertidal sediments of the Yangtze Estuary, J. Geophys. Res. Biogeosci., 122, doi:10.1002/ 2017JG003766. Received 25 MAR 2017 Accepted 20 JUL 2017 Accepted article online 24 JUL 2017
DNRA in intertidal sediments of the Yangtze Estuary Guoyu Yin1,2 , Lijun Hou3 , Min Liu1,2 , Xiaofei Li1,2 , Yanling Zheng1,2 Xiaofen Jiang3 , Rong Wang3 , Chendi Yu3, and Xianbiao Lin1,2
, Juan Gao3
,
1
Key Laboratory of Geographic Information Science (Ministry of Education), East China Normal University, Shanghai, China, School of Geographic Sciences, East China Normal University, Shanghai, China, 3State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai, China
2
Abstract Dissimilatory nitrate reduction to ammonium (DNRA) plays an important role in regulating the fate of reactive nitrogen in estuarine and coastal ecosystems. In this work, intertidal sediments of the Yangtze Estuary were collected in January and August 2015. Potential rates of DNRA and associated functional gene were investigated with nitrogen isotope-tracing and molecular techniques. The measured DNRA rates ranged from 0.14 to 5.57 μmol 15N kg1 h1 in the intertidal sediments. DNRA rates were tightly related to abundance of nrfA gene (p < 0.001), demonstrating that fermentation reaction may be the dominant pathway of DNRA in the study area. Redundancy analysis (RDA) showed a relationship between DNRA and organic matter and NO2, suggesting that these substrates stimulated the metabolism of DNRA microorganisms. On the other hand, the correlation between abundances of nrfA gene and Fe2+ and sulfide in the RDA analysis implied that oxidation of both Fe2+ and sulfide can enhance fermentative DNRA by providing extra free energy. DNRA converted approximately 2.29 × 105 t of nitrate to ammonia annually in the sampling area of the Yangtze Estuary, and most of the converted ammonium was retained in the estuarine ecosystem. DNRA may further contribute to eutrophication in the Yangtze Estuary and also in the other hypereutrophic estuaries.
1. Introduction Nitrate overloading in estuarine and coastal regions can lead to various environmental problems, such as eutrophication, seasonal hypoxia, and harmful algal blooms [Burgin and Hamilton, 2007; Conley et al., 2009; Canfield et al., 2010; Deegan et al., 2012; Yin et al., 2014a]. Excess nitrate may be transformed via different nitrate reduction pathways in estuarine and coastal areas before reaching the open oceans, which makes estuaries important sites for nitrogen recycling [Howarth et al., 2011; Robertson et al., 2016]. Of the nitrate reduction processes, only denitrification and anaerobic ammonium oxidation (anammox) can remove bioavailable nitrogen permanently from estuarine ecosystems in the form of dinitrogen gas. Although the above two processes are the major nitrate reduction pathways removing approximately 30% to 60% of total anthropogenic reactive nitrogen in estuarine and coastal regions [Dong et al., 2009; Crowe et al., 2012; Fernandes et al., 2012; Hou et al., 2013; Gomez-Velez et al., 2015], dissimilatory nitrate reduction to ammonium (DNRA) is also a competing nitrate reduction process converting nitrate into ammonium [Giblin et al., 2013; Cheng et al., 2016; Robertson et al., 2016; Shan et al., 2016]. Therefore, DNRA plays a different role from denitrification and anammox by retaining nitrogen as a more bioavailable form in aquatic ecosystems, further contributing to eutrophication [Deng et al., 2015; Murphy et al., 2016; Yin et al., 2016]. DNRA can be performed by fermentative and chemolithotrophic microorganisms. Organic carbon is used as the electron donor by heterotrophic microbes in the fermentative DNRA (equation (1)), and chemolithoautotrophic microbes can also conduct DNRA via oxidizing reduced inorganic substrates (e.g., sulfide or ferrous iron) with nitrate (equations (2) and (3)) [Giblin et al., 2013; Song et al., 2014; Robertson et al., 2016]:
©2017. American Geophysical Union. All Rights Reserved.
YIN ET AL.
2CH2 O þ NO3 þ H2 O→NH4 þ þ 2HCO3
(1)
HS þ NO3 þ Hþ þ H2 O→NH4 þ þ SO4 2
(2)
6Fe2þ þ NO2 þ 16H2 O→NH4 þ þ 6FeðOHÞ3 þ 10Hþ
(3)
The enzymes and functional genes used in the fermentative DNRA pathway have been well studied [Simon, 2002; Song et al., 2014]. A pentaheme cytochrome C nitrite reductase (NrfA) is the major enzyme for conducting DNRA, so the nrfA gene is frequently targeted as a marker gene encoding the conversion of nitrite to
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ammonia in the DNRA process [Smith et al., 2007; Decleyre et al., 2015]. NrfA mainly mediates nitrite reduction to ammonium in the fermentative DNRA [Einsle et al., 1999], and octaheme tetrathionate reductase (Otr) and octaheme cytochrome C nitrite reductase (Onr) are primary enzymes catalyzing chemolithoautotrophic DNRA [Giblin et al., 2013]. However, NrfA can also use inorganic substrates as alternative to reduce nitrite to ammonium in a few DNRA bacteria [Simon, 2002; Simon et al., 2011]. While studies have examined the nrfA gene in sediments, research on the occurrence and abundance of nrfA gene in hypereutrophic estuaries is limited [Takeuchi, 2006; Smith et al., 2007; Song et al., 2014; Smith et al., 2015]. DNRA is closely related to various physicochemical factors of sediments, such as salinity, temperature, organic matter, metal oxides, nutrients, and sulfide [Yin et al., 2002; Gardner et al., 2006; Lu et al., 2013; Bernard et al., 2015; Deng et al., 2015; Cheng et al., 2016; Robertson et al., 2016; Shan et al., 2016]. However, it still remains unclear about the relative importance of factors controlling DNRA and specific relationships between physicochemical characteristics and DNRA in hypereutrophic estuarine and coastal areas. The Yangtze Estuary is a subtropical estuary and located in one of the most industrialized regions in China, with a large population of over 25 million [Zhao et al., 2015; Cheng et al., 2016]. Huge amounts of anthropogenic reactive nitrogen have been transported into the Yangtze Estuary over the past several decades, and nitrogen pollution is a dominant environmental issue in this area [Hou et al., 2013; Deng et al., 2015; Chen et al., 2016]. Although nitrate reduction processes in the Yangtze Estuary have received more attention in recent years, few studies have focused on DNRA [Hou et al., 2013; Zheng et al., 2016]. In this work, potential rates of DNRA in the intertidal sediments from the Yangtze Estuary were measured via slurry experiments combined with nitrogen isotope-tracing technique [Yin et al., 2014b]. The abundances of nrfA gene encoding the conversion of nitrite to ammonia in the DNRA process were also quantified. The main purposes of this work are (1) to elucidate the potential rates of DNRA in the intertidal zone of the Yangtze Estuary and associated relationships with environmental factors, (2) to investigate the occurrence and abundance of DNRA functional gene in the Yangtze Estuary, and (3) to evaluate the role of DNRA in retaining reactive nitrogen in the hypereutrophic estuarine environment.
2. Materials and Methods 2.1. Study Area The Yangtze Estuary, covering an area of about 8500 km2, is the largest estuary in China. The average tidal amplitudes ranges from 2.4 to 4.6 m in this estuary, which is irregular and semidiurnal [Deng et al., 2015]. The river runoff carries approximately 4.18 × 108 t of suspended sediment into the Yangtze Estuary annually, which contributes to the development of extensive intertidal zones [Chen et al., 2001]. The intertidal sediment is dominated by mud and fine sand, with about 67% and 95% of the sediment grain size finer than 50 μm and 100 μm, respectively [Yang et al., 2008]. Tidal current and river runoff cause a distinct salinity gradient in the estuary, with a range from 0 to 30 [Li et al., 2009]. Due to the anthropogenic activities, the Yangtze Estuary is the most polluted estuarine ecosystem in China [Yin et al., 2016]. Over the past few decades, nitrate concentration in the estuarine ecosystem has risen from 10 μmol L1 to over 130 μmol L1 [Deng et al., 2015]. Excessive inputs of nitrate lead to serious environmental issues in this area, such as harmful algal blooms and hypereutrophication [Hou et al., 2013]. 2.2. Sampling and Pretreatment Along the intertidal zone of the Yangtze Estuary, eight sites were selected to conduct the field surveys in January and August 2015 (Figure 1). Surface sediments (0–5 cm) were collected in triplicate using box corers at each sampling site. In situ tidal water was also sampled at each site for the incubation experiments. The sediment cores were preserved in sterile plastic bags and transported to the laboratory within 2 h. Subsequently, each sediment core was thoroughly mixed under helium atmosphere to obtain one homogenized sample. One portion of the mixed sample was immediately incubated to measure the DNRA rates via slurry experiments, and the other part was used to determine the abundances of nrfA gene and physicochemical characteristics. 2.3. Slurry Experiments Potential rates of DNRA were measured using slurry incubations incorporated with 15N isotope-tracing technique [Yin et al., 2014b]. Briefly, sediment samples were mixed with site water at a ratio of 1:7 to make
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homogenized slurries [Hou et al., 2014]. The slurries were purged with helium and stirred vigorously for about 30 min [Hou et al., 2013]. The mixed slurries were distributed into 16 respective 12 mL vials (Labco Exetainer, High Wycombe, UK) under a helium atmosphere and then sealed with butylrubber septa and screw caps to make it gas tight. Preincubation was conducted at near in situ temperature (Table 1) on a shaker table (200 rpm) for over 24 h to deplete the background nitrate, nitrite, and oxygen [Hou et al., 2014]. After the preincubation, 15NO3 was spiked into the slurry vials (final concentration approximately 100 μmol L1 and final Figure 1. Map of the Yangtze Estuary showing the sampling sites. % 15N approximately 90–99%). Four of the slurry vials were immediately preserved as initial samples by adding 100 μL of saturated HgCl2 solution [Hou et al., 2012]. The remaining vials were incubated and shaken (200 rpm) at near in situ temperature (Table 1). After being incubated for 2 h, 4 h, and 8 h, four of the remaining slurry vials were preserved with HgCl2 as described above, respectively. The produced 15NH4+ during the incubation was quantified using membrane inlet mass spectrometry (HPR-40, Hiden Analytical, UK) after being oxidized into 29N2 and/or 30 N2 with 0.2 mL hypobromite iodine oxidant [Yin et al., 2014b]. Potential rates of DNRA were calculated based on the linear regression of the process-specific 15NH4+ concentrations versus incubation time, according to the following equation: R ¼ KVW 1
(4)
where R (μmol 15N kg1 h1) is the potential DNRA rate, K is the slope calculated from 15NH4+ concentrations versus incubation time (R2 ≥ 0.96), V (L) is the volume of the incubation vial, and W (kg) is the dry weight of the sediment. 2.4. Geochemical Analysis A known amount of wet sediments were dried at 80°C to a constant value, and the contents of sediment water were estimated from the weight loss [Deng et al., 2015]. Sediment salinity was measured with an YSI-30 portable salinity meter, after sediments were mixed with deionized water at the ratio of 1:2.5 [Zheng et al., 2014]. Nutrients including nitrate, ammonium, and nitrite in the fresh sediments were extracted by 2 mol L1 KCl solution [Hou et al., 2013], and their concentrations were determined using a continuous-flow analyzer (SAN plus, Skalar Analytical B.V., Breda, the Netherlands) with a detection limit of 0.1 μmol L1 for a
Table 1. Physicochemical Characteristics of Sediment Samples at Each Site XP
Water content (%) Temperature (°C) Salinity 1 TOC (mg g ) 2+ 1 Fe (mg g ) 3+ 1 Fe (mg g ) 1 NO3 (μmol g ) 1 NO2 (μmol g ) + 1 NH4 (μmol g ) 1 Sulfide (μmol g ) a
QPH
LHK
WSK
BLG
CY
LCG
FX
Jan
Aug
Jan
Aug
Jan
Aug
Jan
Aug
Jan
Aug
Jan
Aug
Jan
Aug
Jan
Aug
33.3 3.7 0.2 14.1 8.2 7.9 2.0 0.04 0.47 1.03
24.3 28.9 0.2 15.6 9.6 5.7 2.2 0.06 0.77 1.65
37.1 4.0 0.2 12.9 16.0 2.4 1.2 0.05 1.86 1.72
38.2 29.5 0.2 15.0 16.2 5.0 3.3 0.07 2.21 1.36
39.1 4.2 0.2 11.6 13.4 4.6 1.2 0.04 1.38 1.54
32.5 31.8 0.2 13.1 16.4 3.3 2.8 0.05 0.76 0.56
43.5 4.9 0.2 14.6 20.3 4.9 3.2 0.12 2.17 1.42
32.4 31.4 0.2 15.7 18.6 1.8 1.5 0.14 2.61 1.92
44.3 3.4 0.2 16.2 24.3 4.5 1.1 0.08 0.84 1.78
47.9 32.6 0.2 18.2 20.9 8.8 1.9 0.10 0.91 5.44
60.0 4.6 4.5 12.0 13.0 17.8 1.5 0.04 0.92 2.70
56.5 32.7 3.8 14.2 12.6 12.9 1.6 0.04 1.09 1.38
44.6 2.8 9.4 12.6 12.1 7.0 0.5 0.03 1.02 1.42
49.3 34.3 8.3 13.6 10.0 11.6 1.3 0.03 2.36 4.30
32.6 5.7 8.1 12.3 14.5 3.8 0.5 0.05 0.42 0.74
26.6 33.1 7.5 13.3 7.9 6.1 1.1 0.04 0.39 0.85
TOC: total organic carbon.
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nitrate and nitrite and 0.5 μmol L1 for ammonium [Yin et al., 2016]. Contents of sulfide in the sediment were analyzed via an Orion Sure-flow® combination silver-sulfide electrode (Thermo Scientific Orion) with a detection limit of 0.09 μmol L1 [Hou et al., 2012]. Total organic carbon (TOC) in sediments was measured using a CHNOS (carbon-hydrogen-nitrogen) Elementary Analyzer (Vario EL III), after sedimentary carbonate was removed by leaching with 0.1 mol L1 HCl [Hou et al., 2012]. Concentrations of amorphous Fe oxides were quantified by extracting with 30 mL of 0.5 mol L1 HCl from 0.5 g of fresh sediment, followed by photometric analysis using Ferrozine assay [Yin et al., 2014a]. All parameters of physicochemical characteristics in the sediment samples were determined in triplicate. 2.5. Real-Time Quantitative PCR Abundances of DNRA functional gene in sediment samples of each site were analyzed. The nrfA gene was quantified as a marker gene, as it encodes a periplasmic nitrite reductase catalyzing the conversion of nitrite to ammonia [Smith et al., 2007]. PowersoilTM DNA Isolation Kits (MO BIO, USA) were used to extract total DNA in sediment samples, following the instructions of the manufacturer. The abundances of nrfA gene in the extracted DNA were measured by conducting real-time quantitative polymerase chain reaction (PCR) assays. Primers nrfA-2F (50 -CAC GAC AGC AAG ACT GCC G-30 ) and nrfA-2R (50 -CCG GCA CTT TCG AGC CC-30 ) were used to amplify the nrfA gene fragments in the extracted DNA [Smith et al., 2007]. The copy numbers of nrfA gene were quantified in triplicate using the SYBR green qPCR method with an ABI 7500 Sequence Detection System (Applied Biosystems, Canada). The qPCR systems contained 12.5 μL of Maxima SYBR Green/Rox qPCR Master Mix (Fermentas, Lithuania), 1 μL of template DNA, 1 μL of each primer (10 μmol L1, Sangon, China), and 10.5 μL of sterile ddH2O. The concentrations of template DNA ranged from 3.64 to 12.95 ng μL1 in all samples (Figure S1 and Table S2 in the supporting information). The qPCR reactions were performed with the thermal cycling conditions of 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 30 s at 95°C, 1 min at 60°C, and 1 min at 72°C. A known amount of plasmid DNA including the target fragment was diluted to build the standard curves. Consistency of real-time qPCR was validated by strong linear correlations of the standard curves between the log10 values of gene copy numbers and the threshold cycle (CT) (R2 = 0.9963) and the high amplification efficiency of 98.5%. The specificity of the qPCR reaction was demonstrated by the single peak at 80.0°C of the melt curve for the samples and standards (Figure S2 in the supporting information). Agarose gel electrophoresis was also conducted to ensure that the amplicon sizes (67 bp) of the PCR products were correct (Figure S3 in the supporting information). Three negative controls without template DNA were prepared for all assays in order to eliminate potential contamination. The abundances of nrfA gene were calculated according to the constructed standard curve and then converted through the following equation and expressed as copies g1 dry sediment (detection limit
