Transformation and fate of nitrate near the sediment–water interface of Copano Bay

Continental Shelf Research 35 (2012) 86–94

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Transformation and fate of nitrate near the sediment–water interface of Copano Bay Lijun Hou a,n, Min Liu b, Stephen A. Carini c,1, Wayne S. Gardner c a

State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai 200062, People’s Republic of China Department of Geography, East China Normal University, Shanghai 200062, People’s Republic of China c The University of Texas at Austin Marine Science Institute, 750 Channel View Drive, Port Aransas, TX 78373, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 June 2011 Received in revised form 26 December 2011 Accepted 3 January 2012 Available online 11 January 2012

This study investigated potential transformation processes and fates of nitrate at the sediment–water interface of Copano Bay during a period of drought by conducting continuous-flow and slurry experiments combined with a 15NO3 addition technique. Rates of 15NO3 -based denitrification, anaerobic ammonium oxidation (ANAMMOX) and potential dissimilatory nitrate reduction to ammonium (DNRA) were in the range of 27.7–40.1, 0.26–1.6 and 1.4–3.8 mmol 15N m–2 h–1, respectively. Compared with the total 15NO3 fluxes into sediments, dissimilatory processes contributed 29–49% to loss of the spiked 15NO3 . Based on the mass balance of 15NO3 , microbial assimilation was estimated to consume about 50–70% of the added 15NO3 , indicating that most of nitrate was incorporated by microorganisms in this N-limiting system. In addition, significant correlations of nitrate transformation rates with sediment characteristics reflect that the depth related behaviors of nitrate transformations in core sediments were coupled strongly to organic matter, iron (Fe) and sulfur (S) cycles. & 2012 Elsevier Ltd. All rights reserved.

Keywords: Nitrogen Denitrification ANAMMOX DNRA Assimilation Copano Bay

1. Introduction Over the past several decades, reactive nitrogen production has increased by 120% (Galloway et al., 2008), and global nitrogen overload was identified as a main emerging environmental issue in this century (Munn et al., 1999; Yan et al., 2003; Seitzinger, 2008). The reactive nitrogen, mainly nitrate, has exerted a serious threat to the environmental quality of estuarine and coastal ecosystems (Burgin and Hamilton, 2007; Diaz and Rosenberg, 2008). To some extent, increasing nitrogen input is an important driver of water pollution (e.g. coastal eutrophication, the development of hypoxic zones and harmful algae blooms) (Vitousek et al., 1997; Rabalais, 2002; Burgin and Hamilton, 2007; Booth and Campbell, 2007). Therefore, an improved understanding of nitrogen (N) transformations and fates on local and global scales is required to develop strategies to protect the water quality and health of coastal environments and other aquatic ecosystems (Gardner and McCarthy, 2009; Dong et al., 2011). Nitrogen is the most limiting nutrient for primary production in Texas coastal ecosystems (Gardner et al., 2006 and references therein). Furthermore, oxidized inorganic N forms (mainly

n

Corresponding author. Tel.: þ86 21 62233931; fax: þ86 21 62546441. E-mail address: [email protected] (L. Hou). 1 Present address: Department of Biology and Marine Biology, University of North Carolina Wilmington, Wilmington, NC 28403-5915, USA 0278-4343/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.csr.2012.01.004

nitrate) are often more abundant than ammonium in overlying water. This pattern implies that nitrate may help regulate the occurrence of algal blooms, subsequently leading to losses of sea grass beds. Therefore, the transformations and fates of nitrate in the coastal systems are needed to understand eutrophication issues in coastal ecosystems. Denitrification, dissimilatory nitrate reduction to ammonium (DNRA), anaerobic ammonium oxidization (ANAMMOX) and assimilation by marine organisms are important N transformation processes (Koike and Hattori, 1978; Seitzinger, 1988; Sørensen, 1978; Cornwell et al., 1999; Kelso et al., 1999; Veuger et al., 2007), which may influence nitrate dynamics in these shallow ecosystems. However, detailed information on transformations of oxidized inorganic N remains poor at the study area. The objectives of the present work were (1) to investigate nitrate reduction rates, (2) to elucidate the potential fates of nitrate and (3) to measure profile patterns of potential nitrate transformation rates in the sediments of this bay.

2. Material and methods 2.1. Study area Copano Bay is located in the Texas coastal zone, which is a semi-enclosed and shallow bay with an average water depth of ˜ ez-James et al., 2009). The bay has a southwest– 3 m (Nan northeast orientation and is bordered by Port Bay to the south,

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system consisted of a 10-L gas-tight bag containing the site water, a multi-channel peristaltic pump, Peek transmission tubing and an acetol plunger with Viton o-ring (McCarthy et al., 2008; Lin et al., 2011). The plunger was positioned  5 cm above the sediment surface to give  230 ml of overlying water volume. This system was designed to be gas-tight. The pump transferred water from the gas-tight bag and continuously displaced overlying water above the core at a flow rate of 1.5 ml min  1. Inflow and outflow water samples for each core were collected for dissolved nitrogen gas and nutrient analyses after incubation of the cores for about 24 h at near in situ temperature to establish steady-state exchange conditions (Gardner and McCarthy, 2009). Subsequently, the inflow water was enriched with 15NO3 (final concentration ca. 100 mmol L  1, final% 15N ca. 90–99%, depending on the background nitrate concentration). The flow was continued overnight to allow the chambers to approach steady-state and the inflow and outflow waters were sampled again for gas and nutrient analyses. Samples for dissolved gas analysis were preserved with 200 mL ZnCl2 solution (50% of saturation concentration) and analyzed within 6 h. Nutrient samples were filtered through 0.2 mm pore-size nylon membrane syringe filters and frozen at  20 1C. The rates of N-dynamic processes at the sediment–water interface were calculated from concentration differences between inflow and outflow samples, flow rates and cross-sectional area of the cores (Lavrentyev et al., 2000). Nitrate transformation rates for the continuous-flow experiments were expressed as mmol 15N m–2 h–1. Fig. 1. Sketch map of Copano Bay on the Texas coast showing the sampling sites. A and B represent the west and east sampling sites, respectively.

Mission Bay to the north and Aransas Bay to the east (Fig. 1). Primary freshwater sources for Copano Bay are the Mission River, which flows into Mission Bay before entering Copano Bay, and the Aransas River. Seawater exchanges with the Gulf of Mexico through Aransas Bay. Inputs of freshwater are episodic in this region where rainfall is sporadic and salinities can range from fresh to hypersaline levels (Solis and Powell, 1999). This study was conducted during a drought period when salinities of the Bay were about 30 ppt. 2.2. Collection and pretreatment of samples Twelve intact sediment cores (10–20 cm deep) were collected at each site (Fig. 1) in March and July of 2009 with a coring device equipped with a core cylinder, a PVC pipe handle and a one-way valve (Gardner et al., 2006). Water from about 1 m above the sediment surface was also collected for nutrient analysis and water column incubation experiments. After collection, all samples were transported to the laboratory within 4 h and nine of twelve sediment cores from each station were used for the continuous-flow and slurry experiments. The remaining three cores were sectioned at 1–2 cm intervals inside a N2-filled chamber within a few hours after returning to the laboratory. A portion of each section was placed in a 60-ml centrifuge tube, centrifuged for 40 min and supernatant water was filtered through 0.2 mm pore-size nylon membrane syringe filters to provide porewater samples for dissolved inorganic nitrogen and sulfide analyses. Centrifuge tubes were filled completely with N2 to prevent oxidation of the sulfide during centrifugation. The remaining sediment of each section was air-dried at room temperature for later determination of grain size, amorphous Fe oxide, organic carbon and nitrogen. 2.3. Continuous-flow experiments Six cores from each site were wrapped with aluminum foil, and installed into a continuous-flow system. The continuous-flow

2.4. Slurry experiments Three cores from each site were segmented immediately at 1–2 cm depth intervals inside an anaerobic chamber, and the subsamples were homogenized by hand mixing in a glove box. About 30 g of homogenized sediment was transferred into respective individual 160 ml serum bottles. The remaining headspace was displaced by anaerobic near-bottom seawater, which was first purged by helium for 40 min, and the serum bottles were sealed with black butyl stoppers and crimped with aluminum caps. After the slurries were mixed vigorously and settled, 20 ml of initial water samples were taken from the serum bottles using syringes with needles for dissolved nitrogen gas and nutrient analyses. Simultaneously, equivalent anaerobic seawater was supplemented to the serum bottles with a second syringe using a needle to keep the ratio of sediment to incubation water relatively constant. Subsequently, these slurry bottles were spiked with 15NO3 (final concentration ca. 100 mmol L  1, final% 15N ca. 90–99%, depending on the background nitrate concentration). The slurry bottles were placed onto a shaker table (200 rpm) and incubated for about 8 h at near in situ temperature. The incubation duration period was based on a pre-incubation experiment, which showed that nitrate transformation processes in the slurry experiments changed linearly within 8 h. After the slurry bottles were removed from the shaker table and allowed to settle for 30 min, the aqueous phases of the slurries were sampled directly from the slurry bottles via syringes. All samples collected at the slurry experiments were processed as described above. Potential denitrification, DNRA, ANAMMOX and assimilation rates in slurries were estimated by quantifying the process-specific 15N-labeled products and expressed as mmol 15N kg  1 h  1. 2.5. Calculation of nitrate transformation rates Prior to this study, a preliminary 15NH4þ tracer experiment confirmed the occurrence of the ANAMMOX process at the study area. Thus, the 15NO3 -based ANAMMOX rates, together with denitrification rates in the continuous-flow and slurry experiments could

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be calculated using the methods developed by Thamdrup and Dalsgaard (2002) and Trimmer et al (2003). In brief, the production of 29N2 during the incubation is derived from denitrification and ANAMMOX. The contribution of each respective process to 29N2 production was quantified by the following equation: P 29 ¼ A29 þ D29

ð1Þ

where P29 is the total, measured 29N2 production rates during the incubation, A29 represents the production rates of 29N2 due to ANAMMOX and D29 denotes the production rates of 29N2 due to denitrification. Here, D29 can be obtained using Eq. (2), given random paring of 14N and 15N from 14NO3 or 15NO3 (Nielsen, 1992; RisgaardPetersen et al., 2003): D29 ¼ P 30  2ð1F N ÞF 1 N

Dt ¼ D29 þ2P 30

ð3Þ

A29 ¼ P 29 D29

ð4Þ 15

NO3 -based

denitrification where Dt and A29 represent the rates of and ANAMMOX, respectively. Potential DNRA rates were estimated in the continuous-flow experiments according to the following equation (Lavrentyev et al., 2000): RDNRA ¼ ð½15 NH4þ Outflow ½15 NH4þ Inflow ÞVA

ð5Þ 15

where RDNRA means the rates of DNRA, [ NH4þ ]Outflow [15NH4þ ]Inflow represent the concentrations of 15NH4þ in the

and outflow and inflow waters, V denotes the flow rate, A represents the cross-sectional area of the cores. The following equation is used to quantify DNRA rates in the slurry experiments (Porubsky et al., 2008): RDNRA ¼ ð½15 NH4þ Final ½15 NH4þ Initial ÞW 1 T 1 NH4þ ]Final

3. Results

ð2Þ

where P30 denotes the total, measured production rates of 30N2 during the incubation, FN is the fraction of 15N in NO3 . Hence, the rates of 15NO3 -based denitrification and ANAMMOX can be quantified by the following equations:

15

2002). The respective contents of organic carbon and nitrogen were determined by a CHN elementary analyzer (VVario ELIII) after removing carbonate by leaching with 0.1 mol L  1 HCl (Hou et al., 2009). Sulfide was measured using an Orion Sure-flows combination silver–sulfide electrode (Thermo Scientific Orion) with a detection limit of 0.09 mmol L  1. Concentration of amorphous Fe oxides was quantified by extracting 0.5 g of sediment with 30 ml of 0.5 mol L  1 HCl, followed by colorimetric (Ferrozine) determination (Roden and Lovley, 1993). Sediment grain size was measured using a LS 13 320 Laser grain sizer.

15

ð6Þ

NH4þ ]Initial

where [ and [ are the concentrations of 15 NH4þ at the end and start of the slurry experiments, W is the sediment weight, T is the incubation time. Note that these rates are conservative because they do not account for possible cation exchange reactions between 14NH4þ and 15NH4þ in the sediments. The rates of 15NO3 -based assimilation within the continuousflow and slurry experiments were obtained by the following 15 NO3 mass balance equation: RAssimi ¼ F 15 NO3 Dt A29 RDNRA

ð7Þ

where RAssimi denotes the rates of nitrate assimilation, F 15 NO3 means the total removal fluxes (or amounts) of 15NO3 over the incubation experiments, Dt, A29 and RDNRA represent respective 15 NO3 -based denitrification, ANAMMOX and DNRA rates. 2.6. Geochemical analysis Water temperature, salinity and dissolved oxygen (DO) were measured in situ using a YSI-600 XLM, multi-parameter water quality monitor. The concentration and atom% 15N of NH4þ in water samples were measured by high performance liquid chromatography (Gardner et al., 1995). After NO3 (plus nitrite) in water was reduced to NH4þ by zinc dust, the analyses of its concentration and atom% 15N followed the procedure of ammonium (Carini et al., 2010). Dissolved nitrogen gases were measured by membrane inlet mass spectrometry (Kana et al., 1994; An et al., 2001) and 30-ppt artificial seawater held at different temperatures (21 and 30 1C) were used as external standards to calculate the concentrations of 29N2 and 30N2 (An and Gardner,

3.1. Station characteristics Site water depths on the different sampling occasions ranged between 2.4 and 3.2 m, and salinities varied between 30.3 and 34.5 ppt (Table 1) during this period of drought conditions. Temperatures ranged from 17.2–19.3 1C in March and 29.5–31.4 1C in July. Dissolved oxygen was near saturation, with the ranges of 7.1–7.3 mg L  1 and 6.3–6.7 mg L  1 in March and July, respectively. Ammonium concentrations were below the detection limit (ca. 0.1 mmol L  1), whereas NO3 (plus nitrite) was the dominant form of inorganic nitrogen, with values of 8.2–14.5 mmol L  1 in March and 3.7–4.6 mmol L  1 in July. Site sediments are characterized by silt–clay, with respective clay ( o4 um) and silt (4–63 mm) contents accounting for about 40.4–56.3% and 30.1–46.3% of total sediments (Table 2). Organic carbon and nitrogen in the core sediments of the Copano west site varied from 594.2 to 734.2 mmol g  1 and from 31.4 to 70.2 mmol g  1, respectively. In comparison, relatively high organic carbon and nitrogen contents were detected at the Copano east site, with values of 603.3–785.7 mmol g  1 and 45.6–81.4 mmol g  1, respectively. Also, molar ratios of organic carbon to nitrogen were lower at the Copano east site (9.9–20.9) than at the Copano west site (9.4–13.3), indicating a potential higher organic matter availability at the Copano east site. Reactive Fe oxide concentrations generally decreased with depth, in the ranges of 10.7–78.6 mmol g  1 and 50.0–144.6 mmol g  1 at the Copano west and east sites, respectively. No significant differences in inorganic nitrogen concentrations were observed between the west and east sites of the Copano Bay (one-way ANOVA, p40.5). Ammonium concentrations in porewater of the core sediments varied between 40.4 and 113.5 mmol L  1 in March. Higher ammonium concentrations of 60.3–151.2 mmol L  1 were detected in July. In contrast, nitrate concentrations were significantly higher in March (5.8–15.9 mmol L  1) than in July (2.8–7.5 mmol L  1). Sulfide concentrations in porewater were not significantly different between the two sampling sites (one-way ANOVA, p40.4). However, seasonal sulfide values varied significantly (one-way Table 1 Physiochemical characteristics of overlying water at the sampling stations. T, S and DO are the abbreviations for temperature, salinity and dissolved oxygen, respectively, and nd denotes that ammonium concentrations are below the detection limit (0.1 mmol L  1). DO (mg L  1)

NH4þ (mmol L  1)

NO3 (mmol L  1)

17.2 30.3 29.5 33.2

7.1 6.3

nd nd

14.5 4.6

19.3 31.4 31.4 34.5

7.3 6.7

nd nd

8.2 3.7

Season Water depth (m)

T (1C)

Site A March July

2.4 2.7

Site B March July

3.0 3.2

S (ppt)

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Table 2 Physiochemical characteristics of core sediments at the sampling stations. OC and ON represent organic carbon and nitrogen, respectively. Layer (cm)

OC (mmol g  1)

ON (mmol g  1)

Fe oxides (mmol g  1)

NH4þ (mmol L  1)

NO3 (mmol L  1)

Sulfide (mmol L  1)

Jul.

Mar.

Jul.

Mar.

Jul.

Mar.

Mar.

Jul.

Mar.

Jul.

Mar.

Jul.

30.1 34.5 35.3 36.2 32.4

35.6 36.3 36.2 34.4 33.3

695.0 602.5 609.2 605.1 619.3

734.2 570.0 619.1 594.2 660.1

70.2 42.1 41.4 35.0 32.8

68.6 36.4 39.3 33.6 31.4

78.6 50.0 51.8 26.7 16.1

64.3 30.4 37.5 19.6 10.7

40.4 56.3 74.8 87.1 99.3

60.3 66.1 75.9 100.9 148.2

10.0 14.9 8.0 7.2 5.8

3.2 6.8 4.9 4.0 2.8

0.1 0.1 1.5 3.2 2.1

1.8 2.4 2.3 4.5 4.2

40.6 43.2 42.5 45.7 42.3

43.4 42.5 46.3 42.1 41.4

767.5 762.4 693.3 669.2 603.3

760.8 785.7 700.4 676.6 613.3

81.4 76.3 57.8 59.3 48.6

79.3 72.1 61.4 55.7 45.6

144.6 82.1 83.9 58.8 51.7

133.9 92.8 76.7 62.5 50.0

49.1 66.8 76.4 87.6 113.5

54.2 87.3 99.2 123.8 151.2

8.6 14.1 15.9 11.7 6.3

3.6 7.5 5.4 3.6 3.3

0.1 0.4 1.5 2.7 3.0

1.8 2.4 4.4 5.3 5.6

Clay (%)

Silt (%)

Mar.

Jul.

Mar.

Site A 0–1 1–2 2–3 3–5 5–7

53.2 54.1 55.4 52.5 50.3

56.3 52.7 51.2 55.1 54.4

Site B 0–1 1–2 2–3 3–5 5–7

42.2 45.3 43.6 40.4 43.7

44.5 43.7 42.2 41.3 41.4

ANOVA, po0.05), from 0.1–3.2 mmol L  1 in March to 1.8– 5.6 mmol L  1 in July. 3.2. Continuous-flow experiments Before the 15NO3 additions, 15NO3 fluxes were not measurable at either station (Fig. 2). However, considerable 15NO3 diffused into sediments after 15NO3 additions. In March, 15NO3 fluxes were  79.2 and 86.3 mmol 15N m–2 h–1 at the west and east sites of the Copano Bay, respectively. An obvious increase in 15 NO3 flux was detected in July, with the values of  111.8 mmol 15 N m–2 h–1 at the west site and  104.9 mmol 15N m–2 h–1 at the east site. Fig. 2 also shows 15NO3 -based potential denitrification, DNRA, ANAMMOX and assimilation rates. As expected, no 15NO3 transformation processes were measurable before the 15NO3 addition. After the 15NO3 addition, the transformation rates of 15 NO3 in March were 34.8–40.1 mmol 15N m–2 h–1 for denitrification, 1.4–1.9 mmol 15N m–2 h–1 for DNRA and 0.3–0.4 mmol 15N m– 2 –1 h for ANAMMOX. In July, 15NO3 -based denitrification, DNRA and ANAMMOX rates were in the range of 27.7–28.2 mmol 15N m– 2 –1 h , 2.9–3.8 mmol 15N m–2 h–1 and 0.4–1.6 mmol 15N m–2 h–1, respectively. The calculated rates of 15NO3 assimilation were 42.6–44.0 mmol 15N m–2 h–1 in March. In contrast, higher 15 NO3 assimilation rates were detected in July, with values of 73.7–78.7 mmol 15N m–2 h–1. 3.3. Slurry experiments The down-core 15NO–3 removal and transformation rates measured in slurries of core sediments are shown in Fig. 3. In March, the reduction fluxes of added 15NO–3 during incubations ranged from 2.5 to 12.5 mmol 15N kg  1 h  1 and from 3.2 to 17.0 mmol 15N kg  1 h  1 in the core sediments of both the west and east sites, respectively. In contrast, relatively higher potential 15NO–3 reduction rates were observed in July, with fluxes of 3.2–18.0 mmol 15N kg  1 h  1 at the west site and 5.5–26.0 mmol 15N kg  1 h  1 at the east site. In general, potential denitrification rates decreased with depth and the most rapid denitrification rates occurred during a cool season (March). At the west site of the Copano Bay, potential denitrification rates in core sediments varied from 0.7 to 4.8 mmol 15N kg  1 h  1 in March and from 0.2 to 7.9 mmol 15N kg  1 h  1 in July. More active denitrification was observed at the east site than at the west site, with rates of 0.2–6.8 mmol 15N kg  1 h  1 and 0.2–11.7 mmol 15 N kg  1 h  1 in March and July, respectively. The vertical distributions of potential ANAMMOX rates were characterized by increasing rates with depth. In March, the rates of ANAMMOX ranged from 0.5 to 0.9 mmol 15N kg  1 h  1 and from 0.1 to 1.4 mmol 15N kg  1 h  1, respectively, at the west and east sites of the Copano Bay. In this

Jul.

study, relatively higher ANAMMOX rates were measured in July, with the values of 0.5–1.2 mmol 15N kg  1 h  1 and 0.6–1.6 mmol 15 N kg  1 h  1 at the west and east sites, respectively. During the slurry experiments, 15NH4þ production in core sediments had a depth profile similar to denitrification rates. In March, potential DNRA rates varied from 0.1 to 1.1 mmol 15N kg  1 h  1 and from 0.2 to 0.9 mmol 15N kg  1 h  1, respectively, at the west and east sites. In contrast, relatively higher 15NH4þ production rates were measured in July, in the range of 0.3–1.6 mmol 15N kg  1 h  1 and 0.2–1.1 mmol 15 N kg  1 h  1 at the west and east sites, respectively. The rates of 15 NO–3 assimilation in March were estimated at 0.8–6.3 mmol 15 N kg  1 h  1 and 0.2–9.2 mmol 15N kg  1 h  1 at the west and east sites, respectively. In comparison with March, July assimilation rates of 15NO–3 were relatively higher, with the values of 1.2–12.1 mmol 15 N kg  1 h  1at the west site and 2.1–8.5 mmol 15N kg  1 h  1 at the east site. In addition, the changes of the transformation processes of 15 NO–3 related closely to the physio-chemical characteristics of core sediments (Table 3).

4. Discussion 4.1. Dissimilatory reduction of nitrate across the sediment–water interface Denitrification to N2, anaerobic ammonium oxidation and DNRA are important dissimilatory pathways of nitrate removal in aquatic environments (Seitzinger, 1988; Binnerup et al., 1992; An and Gardner, 2002; Dagg et al., 2007; Veuger et al., 2007). However, they play diverse roles in the fates of nitrate. Denitrification and ANAMMOX are considered mechanisms of permanently removing nitrate from ecosystems (Seitzinger, 1988). In contrast, DNRA reduces nitrate to ammonium, thus retaining the transformed inorganic nitrogen in ecosystems as a bioavailable form (An and Gardner, 2002; Burgin and Hamilton, 2007; Veuger et al., 2007; Gardner and McCarthy, 2009). In the continuous-flow experiments, denitrification was the major dissimilatory process reducing the added nitrate from overlying waters and removed 43.0–46.5% and 25.2–26.3% of the added 15NO3 in March and July, respectively. Compared with July, relatively rapid denitrification rates in March may have resulted from a relatively optimal temperature for metabolisms of denitrifying bacteria at the study area. A diagenetic model (Kelly-Gerreyn et al., 2001) predicted that denitrification is the favored pathway of nitrate removal at temperatures between 14 and 17 1C. Also, relatively lower potential denitrification rates in July are attributed partly to enhanced sulfide concentrations (Table 2). Increased sulfide production can inhibit the activities

-120 -180 60

1 2 Days of incubation

40 20 0 1 2 Days of incubation

15NO3

-60

DNF (umol m-2 h-1)

July

0

-20 6 DNRA (umol m-2 h-1)

March

4

2

60 0 -60 -120 -180 60

20 0

4

2

1 2 Days of incubation ANAMMOX (umol m-2 h-1)

1 2 Days of incubation ANAMMOX (umol m-2 h-1)

1 2 Days of incubation

0

0

2.0 1.5 1.0 0.5 0.0

2.0 1.5 1.0 0.5 0.0

1 2 Days of incubation

1 2 Days of incubation

100

100 Assimi (umol m-2 h-1)

Assimi (umol m-2 h-1)

1 2 Days of incubation

40

-20 6 DNRA (umol m-2 h-1)

15NO3

DNF (umol m-2 h-1)

60

flux (umol m-2 h-1)

L. Hou et al. / Continental Shelf Research 35 (2012) 86–94

flux (umol m-2 h-1)

90

75 50 25 0

75 50 25 0

1 2 Days of incubation

1 2 Days of incubation

Fig. 2. 15NO3 fluxes and the rates of 15NO3 -based potential denitrification (DNF), DNRA and ANAMMOX before and after 15NO3 addition. Arrows show the time of addition. Bars represent standard error of triplicate cores. A and B denote the west and east sites of the Copano Bay, respectively.

of denitrifying bacteria (Brunet and Garcia-Gil, 1996). The decrease in the activities of denitrifying bacteria in summer may have related to sulfide inhibition of NO- and N2O-reductases and/or competition for organic electron donors with sulfate reducing bacteria. In addition, slightly higher denitrification rates observed at the Copano east site could have resulted from increased availability and concentrations of organic matter ¨ (Thamdrup and Dalsgaard, 2002; Engstrom et al., 2005; Dodla et al., 2008).

DNRA consumed about 1.8–2.2% and 2.5–3.6% of the added 15NO3 in March and July, respectively. Enhancement of DNRA rates in summer (July) may be related to sulfide- or sulfate-reducing bacteria (Rysgaard et al., 1996) and/or increased temperature (Gardner and McCarthy, 2009). DNRA is favored by the presence of sulfate reducing bacteria because DNRA can be a byproduct of denitrification, whereas sulfide inhibits the final step of denitrification but enhances DNRA (Brunet and Garcia-Gil, 1996). This conclusion is supported by relatively high sulfide concentrations in this season (Table 2).

L. Hou et al. / Continental Shelf Research 35 (2012) 86–94

15

-

NO 3 (umol kg-1 h-1)

6

12

18

24

30

0

0

0

2

2 Depth (cm)

Depth (cm)

0

15

4 March July

6

6

24

30

DNF (umol kg-1 h-1)

4

8

12

16

0

0

0

2

2

Depth (cm)

Depth (cm)

18

8 0

4 6

4

8

12

16

4 6

8

8 ANAMMOX (umol kg-1 h-1)

0.0 0

0.5

1.0

1.5

ANAMMOX (umol kg-1 h-1) 0.0 0

2.0

0.5

1.0

1.5

2.0

2 Depth (cm)

2 Depth (cm)

12

4

DNF (umol kg-1 h-1)

4

4 6

6

8

8 DNRA (umol kg-1 h-1) 0.0 0

0.6

1.2

DNRA (umol kg-1 h-1) 1.8

0.0

0.6

1.2

1.8

0

2

2 Depth (cm)

Depth (cm)

-

NO 3 (umol kg-1 h-1)

6

8

4 6

4 6

8

8 Assimi (umol kg-1 h-1) 0

3

6

9

12

Assimi (umol kg-1 h-1) 0

15

0

0

2

2 Depth (cm)

Depth (cm)

91

4 6

6

9

12

15

4 6

8 Fig. 3. Depth distributions of

3

8 15

NO3 -based

denitrification (DNF), DNRA and ANAMMOX at the Copano west (A) and east (B) sites.

However, potential DNRA rates may have been underestimated in this study because the 15NH4þ produced from 15NO3 via DNRA may have exchanged with 14NH4þ adsorbed on sediments as it was released from the sediments into overlying water (Rosenfeld, 1979; Gardner et al., 1991, 2006). If such exchange occurred, a portion of

15

NH4þ would have displaced 14NH4þ on sediment exchange sites. This process was evidenced by the determination of the adsorbed 15 NH4þ in sediments (data not shown). This exchange would decrease the release of 15NH4þ into the overflowing water and cause an underestimation of potential DNRA rates.

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L. Hou et al. / Continental Shelf Research 35 (2012) 86–94

Table 3 Linear regression analyses between nitrate transformation rates and characteristics of core sediments (n¼20). DNF, ANAMMOX, DNRA, Assimi, OC and ON are the abbreviations for denitrification, anaerobic ammonium oxidation, dissimilatory nitrate reduction to ammonium, assimilation of nitrate, organic carbon and nitrogen, respectively. Transformations (mmol 15 N kg  1 h  1)

Clay (%) Silt (%)

OC (mmol g  1)

ON (mmol g  1)

C:N

Correlation DNF ANAMMOX DNRA Assimi

 0.011  0.304 0.029  0.025

0.099 0.309 0.140 0.031

0.651  0.380 0.516 0.637

0.696  0.308 0.572 0.685

 0.572 0.709 0.408  0.364  0.477 0.560  0.536 0.691

0.484 0.096 0.279 0.459

0.339 0.062 0.451 0.448

0.001 0.049 0.010 0.001

0.000 0.094 0.004 0.000

Sig. (1-tailed) DNF ANAMMOX DNRA Assimi

In comparison with denitrification and DNRA, ANAMMOX was of minor importance to nitrate removal, accounting for 0.3–0.52% and 0.4–1.45% of nitrate loss in March and July, respectively. In the warm season (July), relatively higher ANAMMOX rates may be attributed to higher temperature. Although ANAMMOX bacteria can grow over a wide range of temperatures, they have an optimum temperature of about 35 1C (Strous et al., 1999). Temperature effect may be the reason for the relatively high ANAMMOX rates appearing in July. Additionally, the difference in ANAMMOX rates between the two sites may relate to the ¨ availability and concentrations of organic matter (Engstrom et al., 2009). The slurry experiments also support this conclusion. 4.2. Depth-related behavior of dissimilatory nitrate transformations The transformations of nitrate in core sediments may be strongly coupled to organic matter, Fe and/or S cycles (Burgin and Hamilton, 2007). Denitrification rates in the core sediments related positively to the concentrations of organic carbon, nitrogen and Fe oxides, whereas they correlated negatively with C:N ratios of sedimentary organic matter and sulfide in porewater (Table 3). The correlations of denitrification rates with organic carbon and nitrogen contents imply that denitrification in sediment cores at this Bay was driven by microbial decomposition of organic matter (Burgin and Hamilton, 2007). Furthermore, the relationship between denitrification rates and C:N ratios suggests that the activities of denitrifying bacteria were affected by organic matter availability. Both quantity and quality could be important in regulating denitrification (Dodla et al., 2008 and references therein). However, the denitrification activity in sediments may be inhibited by sulfide. This inhibitation may be associated with the effect of sulfide on NO- and N2O-reductases (Brunet and Garcia-Gil, 1996 and references therein). This conclusion is supported by the negative correlation of denitrification rates with sulfide concentrations in porewater (Table 3). In addition, the correlation of the denitrification rates with the Fe-oxide contents may implicate the existence of autotrophic denitrification coupled to ferrous Fe oxidation in sediments. At this pathway, NO3 can be converted to NO2 by ferrous Fe as an electron donor, followed by the rapid reduction of NO2 to N2 (Burgin and Hamilton, 2007). ANAMMOX rates related negatively to organic carbon content in the core sediments (Table 3). However, they correlated positively with C:N ratios of organic matter. The correlations of ANAMMOX rates with organic carbon concentration and ANAMMOX rates may be regulated not only by the quantity of organic matter but also by its availability. Increasing rates of ANAMMOX correlated inversely with availability of reactive organic matter and relatively low concentrations of organic

0.004 0.21 0.038 0.007

Fe oxides (%)

0.000 0.057 0.005 0.000

NH4þ (mmol L  1)

NO3 (mmol L  1)

Sulfide (mmol L  1)

 0.248 0.290  0.359  0.528

 0.110 0.025  0.063  0.228

 0.458 0.570  0.409  0.376

0.181 0.014 0.061 0.008

0.323 0.458 0.396 0.166

0.021 0.004 0.037 0.051

carbon favored ANAMMOX activity (Thamdrup and Dalsgaard, ¨ et al., 2005). In addition, the positive correlation 2002; Engstrom of ANAMMOX rates with sulfide concentrations implies that the existence of sulfide can accelerate the activities of ANAMMOX bacteria by inhibiting the competition of denitrifiers with ANAMMOX bacteria for nitrite and/or nitrate (Brunet and Garcia-Gil, 1996). Also, it may be associated with sulfate reduction coupled to organic matter decomposition. Ammonium was produced during the mineralization process and may have supported the metabolic activities of ANAMMOX bacteria (Canfield et al., 2010). The depth profiles of DNRA rates indicate that the process of nitrate reduction to ammonium occurs mainly at the surface sediments. Generally, ammonium production via DNRA is considered to be coupled to the microbial reduction of sulfate (Rysgaard et al., 1996; Brunet and Garcia-Gil, 1996). However, the negative relationship between DNRA rates and sulfide concentrations in the core sediments shows that the reduction of sulfate was not the unique mechanism regulating microbial reduction of nitrate to ammonium. Interestingly, a significant correlation existed between DNRA rates and reactive Fe oxide concentrations in the core sediments. Such a relationship implies that ammonium production via DNRA may be driven by oxidation of ferrous Fe, consequently controlling the profile distributions of DNRA process. Addition of nitrate can cause immediate oxidation of ferrous Fe coupled to reduction of nitrate to ammonium (Weber et al., 2006). To some extent, the inter-station difference in DNRA rates obtained at the continuous-flow experiments also supports the existence of Fe-driven DNRA. In addition, the positive correlations of DNRA rates with organic carbon and nitrogen also reflect that the microbial fermentation makes a significant contribution to ammonium production during the process of nitrate reduction to ammonium (Burgin and Hamilton, 2007). 4.3. Contributions of assimilation to nitrate fate Assimilation of 15NO3 within the continuous-flow systems, as measured by the difference between 15NO3 removal from solution and that accounted for by the sum of the measured dissimilatory processes, consumed about 50–53.7% and 69–70.7% of the added 15NO3 in March and July, respectively. The results reflect that assimilation is the major fate of nitrate in the N-limiting shallow bay. Likewise, 50–100% of the added 15NO3 was incorporated or retained by bacterial, algae and fauna in a N-limiting intertidal system (Veuger et al., 2007). However, the contribution of assimilation to nitrate removal may be overestimated, considering that some 15NO3 may have been retained in the sediment porewater. Unfortunately, the remaining 15NO3 in sediment porewater was not measured at the end of the continuous-flow

L. Hou et al. / Continental Shelf Research 35 (2012) 86–94

experiments. In addition, the profile distributions of assimilation rates reflect that the assimilation of nitrate occurred mainly at the top of sediment cores, probably because there is abundant bacterial (and/or algal) biomass in surface sediments. In conclusion, although nitrate assimilation was the main transformation pathway, the dissimilatory processes reducing nitrate in sediments competed with assimilation processes in controlling the fate of nitrate at the sediment–water interface. Also, the relative importance of the different nitrate transformation processes varied seasonally and with depth in the sediments, depending on organic matter, Fe and sulfide contents.

Acknowledgments The field research was conducted while Hou was a visiting scientist at Gardner’s laboratory at the University of Texas Marine Science Institute, via support from the National Natural Science Foundations (nos. 41071135, 41130525 and 40701167) and the State Key Laboratory of Estuarine and Coastal Research (no. 2010RCDW07). The U.S. National Oceanic and Atmospheric Administration (NOAA) National Estuarine Research Reserve (NERR) program provided field research support in the Mission Aransas NERR site. The investigation was also supported by the Fundamental Research Funds for the Central Universities and the Marine Scientific Research Project for Public Interest (no. 200905007). We thank Dr. Edward J. Buskey, Director of Research at the Mission Aransas NERR, for field support and analytical assistance; Mark J. McCarthy for sharing his analytical expertise on N-dynamics measurements; the CSR Editor, Denise Bruesewitz, and an anonymous reviewer for constructive comments on the manuscript.

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