J Soils Sediments (2012) 12:1435–1444 DOI 10.1007/s11368-012-0542-9
SEDIMENTS, SEC 2 • PHYSICAL AND BIOGEOCHEMICAL PROCESSES • RESEARCH ARTICLE
Nitrate reduction coupled with microbial oxidation of sulfide in river sediment Xunan Yang & Shan Huang & Qunhe Wu & Renduo Zhang
Received: 24 December 2011 / Accepted: 19 May 2012 / Published online: 26 June 2012 # Springer-Verlag 2012
Abstract Purpose Nitrate (NO3−) is often considered to be removed mainly through microbial respiratory denitrification coupled with carbon oxidation. Alternatively, NO3− may be reduced by chemolithoautotrophic bacteria using sulfide as an electron donor. The aim of this study was to quantify the NO3− reduction process with sulfide oxidation under different NO3− input concentrations in river sediment. Materials and methods Under NO3− input concentrations of 0.2 to 30 mM, flow-through reactors filled with river sediment from the Pearl River, China, were used to measure the processes of potential NO3− reduction and sulfate (SO42−) production. Molecular biology analyses were conducted to study the microbial mechanisms involved. Results and discussion Simultaneous NO3− removal and SO42− production were observed with the different NO3− concentrations in the sediment samples collected at different depths. Potentially, NO3− removal reached 72 to 91 % and SO 4 2− production rates ranged from 0.196 to 0.903 mM h−1. The potential NO3− removal rates were linearly correlated to the NO3− input concentrations. While the SO42− production process became stable, the NO3− reduction process was still a first-order reaction within the range of NO3− input concentrations. With low NO3− input concentrations, the NO3− removal was mainly through the pathway of dissimilatory NO3− reduction to NH4+, while with higher NO3− concentrations the NO3− removal was through the denitrification pathway. Responsible editor: Marcel van der Perk X. Yang : S. Huang : Q. Wu (*) : R. Zhang (*) School of Environmental Science and Engineering, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Environmental Pollution Control and Remediation Technology, Guangzhou, Guangdong 510275, People’s Republic of China e-mail:
[email protected] e-mail:
[email protected]
Conclusions While most of NO3− in the sediment was reduced by denitrifying heterotrophs, sulfide-driven NO3− reduction accounted for up to 26 % of the total NO3− removal under lower NO3− concentrations. The vertical distributions of NO3− reduction and SO42− production processes were different because of the variable bacterial communities with depth. Keywords Flow-through reactor . Nitrate reduction . Sediment . Sulfide oxidation
1 Introduction Anthropogenic activities have dramatically increased nitrogen (N) loading to aquatic ecosystems, particularly in the highly developing and densely populated regions (Galloway et al. 2004; Boyer et al. 2006). The N loading has resulted in considerable increase in nitrate (NO3−) concentrations in rivers, contributing to coastal eutrophication and hypoxia (Qiu et al. 2010). Nevertheless, most of the N loading to terrestrial soils and freshwater disappears before reaching coastal waters (Forshay and Stanley 2005; Seitzinger et al. 2006). These changes in N cycling lead to the question: what processes are involved in N removal from rivers? Most attention has been devoted to study NO3− removal by respiratory denitrification with organic carbon as an electron donor, and dissimilatory NO3− reduction to ammonium (DNRA) by fermentative bacteria (Burgin and Hamilton 2007). Alternatively, autotrophic denitrifiers can use NO3− to oxidize sulfide and elemental S to SO42− and their biogeochemical importance has been recognized (Jørgensen and Gallardo 1999; Shao et al. 2010). Nevertheless, most research activities have been focused on sulfide-driven autotrophic denitrifiers in marine sediments (Fossing et al.
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1995; Sayama et al. 2005; Zhang et al. 2009). Several taxa, including the members of the genera Thiobacillus, Thiocapsa, and Beggiatoa, have been established with diverse metabolic characteristics (Brettar and Rheinheimer 1991; Jørgensen and Gallardo 1999; Kojima and Fukui 2003). More recently, the importance of sulfide-driven autotrophic denitrifiers in freshwater ecosystems has been investigated. For example, Kamp et al. (2006) enriched the Beggiatoa from a NO3−-rich stream and observed its ability to oxidize sulfide with NO3−. Burgin and Hamilton (2008) studied the pathways of sulfide-driven NO3− reduction in freshwater bodies. Payne et al. (2009) revealed the coupling respond of N and S in wetlands. There are two pathways for NO3− removal through sulfide oxidation: reducing NO3− to N2 in the form of denitrification; and reducing NO3− to NH4+ in the form of DNRA (Burgin and Hamilton 2008). Environmental factors in river sediment, such as sediment depth, NO3− concentrations, and bacterial population and activities, should affect the NO3− removal process with S oxidation and the associated pathways. However, the potential and mechanisms of sulfide-driven NO3− reduction in river sediment are still poorly understood. Therefore, the objectives of this study were: (1) to investigate the potential rates of sulfide-driven NO3− reduction with different NO3− input concentrations in different depths of river sediment; and (2) to explore the dominate pathways of the sulfidedriven NO3− reduction process.
J Soils Sediments (2012) 12:1435–1444
2.2 Flow-through reactor experiment Six FTRs in this study were constructed following Pallud et al. (2007). The inner diameter of the reactor cell was the same as that of the sediment core (7.0 cm). A buffer room and a sintered disk were included to homogenize the influent. The 30 cm sediment core was cut into slices at depths of 0–2, 5–7, 10–12, 15–17, 20–22 and 25–27 cm. Each sediment slice was put into the polyvinyl chloride cell of the FTR with nitrocellulose filter (0.22 μm pore size) on each side. The FTR experiments were conducted in an incubator, in which the temperature was set at 28 °C based on the temperatures of bottom water in the river (range from 12 to 28 °C). The influent of FTR was controlled at a constant flow rate (15 cm3 h−1) using a peristaltic pump. Input solutions included five NO3− concentrations (0.2, 1, 5, 15 and 30 mM) and one Br− solution (1 mM). The NO3− input concentrations were selected based on the literature (e.g., Laverman et al. 2007; Pallud et al. 2007). To ensure an anaerobic condition in the cell, each reactor was wrapped with airproof material and the input solution was vigorously purged with argon gas before entering the cell. For each input solution, the experiment was carried out until reaching a steady-state outflow concentration (about 20 h). The Br− breakthrough curves were used to obtain the transport parameters (Pallud et al. 2007). The steady-state rate of NO3− reduction or SO42− production was calculated as follows:
2 Materials and methods R¼ 2.1 Sample collection Sediment samples were collected from a drain outlet (113° 17′1.66″E, 23°6′50.41″N) of the Pearl River in Guangzhou, China. The section Pearl River crossing Guangzhou City is close to the Pearl River Estuary, which links the highly developing urban area and the South China Sea, and represents an important ecosystem. In recent years, the Pearl River has been subjected to a high load of anthropogenic contaminants from wastewater runoff because of the increasing population and economic development in the Pearl River Delta (Jiang et al. 2009; Lu et al. 2009). The river is also affected by tides of the South China Sea. On July 14th, 2010, two sediment cores were collected using a core sampler with a diameter of 7.0 cm and length of 50 cm. Water samples above the sediment–water interface, and the top 30 cm of the sediment cores were retained for the following experiments: (1) one of the sediment cores was used for flow-through reactor (FTR) experiments (described below); and (2) the other core was used to analyze chemical and microbial conditions of the sediment before the FTR experiments.
ðCi Co ÞQ V
ð1Þ
where: Ci is the input concentration; Co is the steady-state concentration in the outflow; Q is the volumetric flow rate; and V is the volume of the sediment slice in the reactor. 2.3 Analysis of environmental parameters Water content of the sediment samples was measured using the gravimetric method. Concentrations of Br−, NO2−, NO3− and SO42− of the water samples, interstitial water samples from the sediment sub-samples, and the outflow samples of the FTR experiments were measured using ion chromatography (Metrohm 882, Metrohm AG, Herisau, Switzerland), and NH4+ using spectrophotometric detection with Nessleri’s reagent. Total organic carbon content (TOC) was determined with the potassium dichromate dilution heat colorimetric method and the total nitrogen (TN) content was determined using a Foss Kjeltec 2300 Analyzer Unit (Foss Tecator AB, Höganäs, Sweden). Analyses of acid-volatile sulfur (AVS) concentrations in the sediment were based on the cold-acid purge-and-trap method (Chen et al. 2006).
J Soils Sediments (2012) 12:1435–1444
2.4 Molecular biology analysis 2.4.1 DNA extraction and PCR amplification Total DNA was extracted from each sediment slice with Fast DNA spin kit (Bio 101, Qbiogene Inc., CA, USA) following manufacturer’s instruction manual. The following primer sets were used for PCR amplification of the genes encoding 16S rRNA: the forward primer 27F (5′-AGAGTTT GATCMTGGCTCAG-3′) labeled at the 5′ end with the dye carboxyfluorescein (FAM; synthesized within the primer by Genolab Co., Ltd., China.) and the reverse primer 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR mixture contained 50 ng of extracted DNA, 2 μl of 5 mM concentrations of each primer, 3.2 μl of 2.5 mM concentrations of dNTP, 0.4 μl of 5 U/μl TaKaRa Taq DNA polymerase (TaKaRa Bio Inc., Shiga, Japan), and 5 μl of 10× PCR buffer for TaKaRa Taq, then replenish with ddH2O to 50 μl. The PCR amplifications were performed in a total volume of 50 μl in 0.2 ml reaction tubes using the PCR reactor (T-gradient, Biometra, USA) with the following procedure, 94 °C for 5 min and then 30 cycles each consisting of 30 s at 94 °C, 30 s at 55 °C; and 45 s at 72 °C; and finally 10 min at 72 °C to complete the primer extension. The presence of PCR products was shown using 1 % agarose gel electrophoresis. 2.4.2 Terminal-restriction fragment length polymorphism analysis After the processes of purification and concentration of the PCR products with a Wizard SV gel and a PCR clean-up system (AxyPrep PCR clean kit, China), 5 μl of the amplicons were digested with 10 U of the restriction enzyme HaeIII, HhaI, and MspI (TaKaRa, Japan), respectively, in the manufacturer’s recommended reaction buffers for 4 h at 37 °C. Enzymes were subsequently inactivated by incubation at 65 °C for 20 min. Cleaved PCR products were purified by ethanol precipitation and dissolved in 20 μl of sterile ddH2O. Ten μl of the purified products together with 0.5 μl of internal standard (ROX-500) were denatured at 95 °C for 2 min, cooled on ice, and subject to electrophoresis on a ABI 3730XL DNA analyzer (Applied Biosystems, USA). After electrophoresis, the size of the fluorescently labeled terminal-restriction fragments (T-RFs) was determined by comparing with the size standard using the software GeneMarker 1.97. To avoid detecting primers and uncertainties in size determination, terminal-restriction fragment lengths smaller than 50 bp were excluded. The percentage of detected T-RF was calculated from the peak height to the total peak height (>1 %). T-RFs were identified by the T-RFLP analysis package built by the Center for Limnology, University of Wisconsin–Madison, USA
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(https://secure.limnology.wisc.edu/trflp), then the taxonomy information was searched from the U.S. National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). 2.4.3 Real-time PCR analysis The primers selected for amplification of the different genes encoding narG, nirS, nirK, nosZ and nrfA are narG328f– narG497r (Reyna et al. 2010), nirS3f–nirS5r (Braker et al. 1998), nirK2f–nirK3r (Thräback et al. 2004), nos1527f– nos1773r (Scala and Kerkhof 1998), and nrfA2F–nrfA2R (Smith et al. 2007). Each assay contained a standard using a serial dilution of known copies of PCR fragments of the respective functional genes, independent triplicate sediment DNA templates for each sediment slice, and triplicate no template controls. Experimental Q-PCR triplicates for each DNA sample were then averaged to obtain a single gene copy number. Real-time PCRs were carried out in LightCycler480 with Sequence Detection Software v1.4 (Applied Biosystems, USA). Each PCR mixture (10 μl) was composed of 5 μl of SYBR Premix Ex TaqTM II (2×), 0.4 μl 10 nM of each forward and reverse primers, 0.2 μl ROX Reference Dye II (50×)×3, 3.2 μl ddH2O and 2.0 μl of template DNA (TaKaRa Biotechnology, Japan). PCR amplification and detection were performed in LightCycler480 Multiwell (384-well) reaction plates with optical cap (Applied Biosystems, USA). The PCR procedure was as follows, 30 s at 94 °C, 40 cycles of 5 s at 94 °C; 30 s at the specific annealing temperature (61, 57, 57, 57, and 60 °C for narG, nirS, nirK, nosZ, and nrfA, respectively); and 30 s at 70 °C. A melting curve analysis for SYBR Green assay was conducted after the amplification to distinguish the targeted from the non-targeted PCR product.
3 Results 3.1 Physical and chemical characteristics of the sediment core Physical and chemical properties of the sediment core are listed in Table 1, including water content, porosities, concentrations of NO3−, NH4+, TN, TOC and AVS of the sediment, and concentrations of NO3− and NH4+ in the interstitial water. The water contents and porosities decreased from 67 % to 29 % and from 82 % to 54 %, respectively, with increasing core depths. The NO3− concentrations in the sediment and interstitial water reached the maximum values (0.056 mmol kg−1 and 0.0802 mM, respectively) at a depth of 10-12 cm, then decreased with the depth. The NH4+ concentrations were much larger than the NO3− concentrations and the maximum NH4+ values in the sediment and interstitial water (39.0 mmol kg−1 and 5.48 mM,
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J Soils Sediments (2012) 12:1435–1444
Table 1 Physical and chemical characteristics of the sediment core Depth
Water content
Porosity
Sediment
(cm)
%
%
NO3− (mmol kg−1)
NH4+ (mmol kg−1)
TN (g kg−1)
TOC (g kg−1)
AVS (mmol kg−1)
NO3− (mM)
NH4+ (mM)
0.038 0.038 0.056 0.054 0.039 0.037
14.1 16.2 39.0 24.1 22.3 29.5
1.11 1.71 2.80 1.45 1.40 1.44
15.2 36.2 57.1 27.4 20.3 18.9
12.0 26.9 25.0 33.7 27.1 27.6
0.0310 0.0273 0.0802 0.0255 0.0283 0.0291
1.069 1.547 5.484 1.211 2.671 2.109
0–2 5–7 10–12 15–17 20–22 25–27
67.6 60.0 54.0 40.7 36.9 28.6
81.8 77.9 74.2 56.8 63.2 53.6
Interstitial water
respectively) also occurred at a depth of 10-12 cm. The TOC and TN values were highly correlated with a coefficient of determination (R2) of 0.934 (p
