Environ Sci Pollut Res (2014) 21:14138–14145 DOI 10.1007/s11356-014-3328-3
RESEARCH ARTICLE
Nitrate removal under different ecological remediation measures in Taihu Lake: a 15 N mass-balance approach Dandan Liu & Zhengkui Li & Wanguang Zhang
Received: 26 February 2014 / Accepted: 11 July 2014 / Published online: 23 July 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Ecological remediation is an important measure for the protection of lake water quality in removing nutrients, such as nitrate (NO3−). In this study, four bioremediation processes (bare sediment, immobilized nitrogen cycling bacteria (INCB) added, Elodea nuttallii added, E. nuttallii-INCB assemblage) were operated at a lab to elucidate the effect of macrophyte appearance and INCB addition on NO3− removal and achieve the optimal processes for biomediation. 15 N-NO3 solution was added to microcosms to identify the key nitrogen transformation processes responsible for NO3− removal. Results showed that nitrate removal was significantly enhanced after the addition of INCB and E. nuttallii. In the treatments with INCB added, E. nuttallii added, and INCB and E. nuttallii-INCB assemblage, nitrate removal ratio achieved 94.74, 98.76, and 99.15 %, respectively. In contrast, only 23.47 % added nitrate was removed in the control. Plant uptake and denitrification played an important role in nitrogen removal. The water quality was substantially improved by the addition of INCB and macrophyte that can accelerate denitrification and promote nitrogen assimilation of plants. The results indicated that plant uptake and microbial denitrification were key processes for nitrate removal.
Keywords Nitrogen transformation . Plant assimilation . N2O emission . Elodea nuttallii-INCB assemblage . Taihu Lake
Responsible editor: Hailong Wang D. Liu : Z. Li (*) : W. Zhang State Key Laboratory of Pollutant Control and Resources Reuse, School of the Environment, Institute of Oceanographic Research, Nanjing University, 163 Xianlin Avenue, 210023 Nanjing, People’s Republic of China e-mail: [email protected]
Introduction In the past decades, nitrogen inputs to aquatic ecosystems have increased dramatically. Excess nutrient loading from agricultural runoff, untreated industrial and urban discharges, leads to eutrophication, which has received growing concerns in many countries (Picek et al. 2008; Scheffer et al. 2003; Smith et al. 1999). Therefore, suitable steps should be taken to protect natural water resource from eutrophication by reducing nutrient inputs (Hamilton and Landman 2011; Sollie and Verhoeven 2008). Lake Taihu is a large (2,338 km2), shallow (1.9 m mean depth), and well-mixed lake in China. The annual water inflow is approximately 8×109 m3, and the residence time of the lake is approximately 5 months (Zeng et al. 2009). Meiliang Bay is located in the northern part of Lake Taihu with a water surface area of 135 km2 and a mean water depth of 2.1 m. It provides drinking water, irrigation, and industrial waters for Wuxi City. It receives the municipal and industrial wastewater from the surrounding river, resulting in the deterioration of water quality in this area. Serious cyanobacterial blooms in Meiliang Bay have increased in frequency and intensity in recent years and have seriously affected the function of the water body (Qiao et al. 2006; Zeng et al. 2012). Phytoremediation is an important measure to remove nutrients from polluted lakes (Sollie and Verhoeven 2008). It can remove nutrients through plant uptake and rhizosphere denitrification. Plant uptake represents a temporary store of nutrients, and harvesting biomass may completely remove nutrients from the system. Denitrification, the stepwise reduction of nitrate to N2 under low-oxygen and anaerobic conditions, represents a permanent nitrogen removal from aquatic systems (Lee et al. 2009; Seitzinger et al. 2006). However, N removal rates of lakes are different in many studies (Epstein et al. 2012; Schubert et al. 2006; Soana and Bartoli 2014), which are affected by background nitrogen concentration,
Environ Sci Pollut Res (2014) 21:14138–14145
presence of macrophyte, abundance of nitrogen cycle cycling microorganisms, and climatic condition (Pina-Ochoa and Alvarez-Cobelas 2006; Schubert et al. 2006). More studies should be conducted to determine the key limiting factors for nitrate removal of different ecological remediation treatments. Numerous studies have reported that macrophyte revegetation and the addition of immobilized nitrogen cycling bacteria (INCB) can stimulate N removal in sediments. Rooted submerged macrophytes may create favorable conditions for coupled nitrification-denitrification in the sediment by creating heterogeneous oxygen conditions in the root zone and by excreting organic carbon from their roots (Caffrey and Kemp 1992; Reddy et al. 1989; Ullah et al. 2014). The INCB technology can increase the quantities of nitrogen cycling bacteria through screening, immobilizing, and releasing them to the water column. INCB have been applied for nitrogen removal from domestic water, seawater, and freshwater (Chen et al. 2012; Joerdening et al. 2006). The integrated restoration method with macrophytes and INCB technology can enhance the activity of aquatic nitrogen removal. The integrated Elodea nuttallii-INCB technology could increase sediment denitrification rate by 162 % in the sediments (Wang et al. 2013). However, information about the nitrogen removal processes is limited for the different treatment methods. Thus, investigating the effects of various treatments (bare sediment, INCB added, E. nuttallii added, E. nuttallii-INCB assemblage) on benthic nitrogen cycling and quantifying the contribution of different removal pathways are theoretically and practically essential for optimal application of the processes in eutrophic lake restoration. The main objectives of this study were as follows: (1) to investigate the effect of different ecological remediation treatments (bare sediment, INCB added, E. nuttallii added, E. nuttallii-INCB assemblage) on nitrate removal and transformations in Taihu Lake and (2) to quantify the contribution of different removal pathways to nitrate removal under different ecological remediation treatments.
Materials and methods Study site and sampling A bare zone without macrophyte in the central position of Meiliang Bay (31° 12′ 14″ N, 119° 55′ 12″ E) was chosen as the sampling site (Fig. 1). Sampling of bare sediments, water, and E. nuttallii was conducted in this site in January 2013. Clear Plexiglas tubes (length 60 cm and inside diameter 9 cm) were pressed ~20 cm into the sediment, and a total of 24 intact sediment cores were collected for the following incubation experiments in the laboratory. The overlying water was simultaneously collected with plastic barrels. E. nuttallii was
14139
collected and put into a tank filled with lake water. All the sediment, water samples, and E. nuttallii were transported to the laboratory within 4 h at 5±1 °C. Preparation of INCB INCB were prepared as described previously (Wang et al. 2013). Indigenous nitrogen cycling bacteria were isolated from Lake Taihu water using the method of plate streaking and identified by 16S rRNA sequences. To get porous carriers for immobilization, 2-hydroxyethyl acrylate (HEA), 2-hydroxyethyl methylacrylate (HEMA), and distilled water were mixed at a volume ratio of 3:3:14. The mixture was irradiated at −78 °C with 60 Co gamma ray source at 10 kGy for 24 h. Then, solid polymer carriers were synthesized. The resultant polymer carriers were cut into small pieces, approximately 5×5×5 mm in dimension, and then the carriers were washed in excess deionized water to remove the unreacted components. The prepared carriers were immersed in deionized water for 3 days in order to be fully swollen. The swollen carriers with a pore size of about 20–100 μm were added to the mixture of precultured nitrogen cycle bacteria and nutrient medium. After alternate aerobic (8 h) and anaerobic incubation (4 h) for six times (total 72 h), the polymer carrier of immobilized nitrogen cycling bacteria was cultivated in an incubator at 28±1 °C under gentle rotary shaking (80 rpm). The porous carriers proportioned a suitable 3-D microhabitat for nitrogen cycling bacteria to colonize and reproduce (Cao et al. 2002; Zhang et al. 2007). After being incubated, nitrogen cycling bacteria were immobilized in the carriers and the INCB were prepared. The total quantity of nitrogen cycling bacteria immobilized was about 2.1×1012 cells/g (lipid-P biomass) on average. Experimental design Twenty-four cores from Meiliang Bay were divided into four restoration treatments. Each treatment had six replicates: treatment A, bare sediment core, as control group without any restoration treatments; treatment B, sediment + INCB; treatment C, sediment + E. nuttallii, five shoots of E. nuttallii (10 cm in length) cleaned with ultrapure water were planted into sediment; treatment D, sediment + E. nuttallii + INCB, prepared carrier with INCB (50±1 g tube−1) was added to the core to allow bacterial movement into the upper sediment layer. After restoration treatment, all the cores were filled immediately with lake water using a plastic syringe. For each treatment, three replicates were used to determine N2O fluxes, nitrate removal process analyses, and the other three were used to investigate the O2 profile. Lake water and INCB were renewed once a week. All cores after different restoration treatments were incubated at room temperature (25±1 °C) under natural light from the window in the lab throughout
14140
Environ Sci Pollut Res (2014) 21:14138–14145
Fig. 1 Geographical location of sampling points in Meiliang Bay, Taihu Lake
the whole experiment. The influence of different restoration treatments on N cycling process was evaluated after incubation for about 6 weeks.
N2O fluxes For measurement of N2O, a static closed chamber method was used. On the top of the core, a gas collection chamber (10 cm in height and 9 cm in inner diameter) was used to quantify N2O fluxes. At the beginning, the chamber was closed by lids which were made of rubber and coated with Apiezon high vacuum grease (type M). The incubation lasted for 40 h at room temperature under natural light condition. Gas samples were transferred from the chamber to previously evacuated glass vials by using double-sided needles. Each vial was allowed to fill for 30 s. The concentration of N2O was determined by a gas chromatograph (Agilent 4890) equipped with a flame ionization detector (FID) and an electron capture detector (ECD). The N2O portion was separated using a 1-m stainless steel column with an inner diameter 2-mm Porapak Q (80/100 mesh) and was measured using the ECD, which was set at 330 °C. The ECD used high-pure nitrogen as a carrier gas, at a flow rate of 35 mL/min. The column temperatures were maintained at 55 °C (Song et al. 2006). The gas flux was calculated according to the following equation: J¼
dc M P T 0 ⋅ ⋅ ⋅ ⋅H dt V 0 P0 T
where dc/dt is the slope of the gas concentration curve variation along with time; M is the mole mass of each gas; P is the atmospheric pressure in the sampling site; T is the absolute temperature during sampling; V0, T0, and P0 are the gas mole volume, air absolute temperate, and atmospheric pressure under standard conditions, respectively; and H is the height of the chamber above the water surface.
Sediment oxygen profile and characteristics At the end of the restoration incubation, sediment oxygen profiles were determined by a O2 microprobe with a tip diameter less than 0.1 mm (PreSens, Germany). The insertion of the O2 microprobe was controlled by a 3-D motorized micromanipulator (PreSens, Germany). The microprobe was inserted to the center of the core to make sure that no plant roots were touched. Six values were taken from each treatment, and the mean of these six values represented the O2 concentration of the treatment at the measured time point. 15
N chamber experiments
On the day of the experiment, the dosing solution (99 at.% 15 N-NO3− as NaNO3− was prepared based on chamber volume (L) and ambient NO3− concentrations to enrich background concentrations in the entire core by 10 %. Each chamber was dosed using a backpack sprayer. 15 N-NO3− was added to each chamber as shown in Table 1. The 15 N-NO3− balance experiment lasted for 40 h.
Environ Sci Pollut Res (2014) 21:14138–14145
14141
Table 1 Fate of transformed 15 N-NO3− during the experiment 15
N balance component
A Mass of 15 N (μg)
B Transformed 15 N- Mass of 15 N (μg) NaNO3− (%)
Amount of 15 N added 459 Detected in macrophytes Detected in sediment Detected in the water column (nitrate) Detected in N2O Unaccounted for [1 −(2+3+4+5)]
C Transformed 15 N- Mass of 15 N (μg) NaNO3− (%)
1,268
D Transformed 15 N- Mass of 15 N (μg) NaNO3− (%)
485
Transformed 15 NNaNO3− (%)
932
0
0
0
0
119±3.2
24.54
50±1.6
5.36
20±1.3 373±4.1
4.36 76.53
6±0.2 67±2.4
0.47 5.28
11±0.9 6±0.4
2.27 1.24
5±0.2 8±0.3
0.53 0.85
0.1±0.03
0.02
4±0.24
0.32
0.006
2±0.20
0.21
65.9±7.7
19.09
1,191± 3.7
93.93
0.03± 0.01 348.97± 1.4
71.94
867±0.5
93.05
Sample processing and analysis Water samples were collected manually in 250 mL HDPE bottles. Sediment was sampled using a cutting ring. Sediment samples were sieved (6 mm mesh) to remove large rocks and debris and dried at 60 °C (24–48 h) before preparation for 15 N analysis. Samples of macrophytes were collected from 12 chambers and clipped at the ground surface. A subset of each sample was immediately dried at 60 °C (24–48 h) and weighed to determine biomass (g/m2). After drying, samples were ground to a uniform texture (fine powder) by using a ball grinder. A small amount of each solid sample was weighed; macrophytes and sediment were transferred into encapsulated tins and stored in microtiter plates with individually sealed wells. Samples (macrophytes and sediment) were sent to the Stable Isotope Laboratory at the Nanjing Normal University (Nanjing, China) for analysis of N-isotope composition and percentage of element N by mass spectrometry using a Europa Integra continuous flow isotope ratio mass spectrometer (Europa Scientific, Seron, Cheshire, UK) coupled to an in-line elemental analyzer. Isotopic composition of the NH4+ and NO3− was measured using an automated C/N analyzer isotope ratio mass spectrometer (Europa Scientific Integra, Crewe, UK). NH4+ and NO3− were separated for 15 N measurements by distillation with magnesium oxide and Devarda’s alloy (Feast and Dennis 1996; Zhang et al. 2012). In detail, a portion of the extract was steam-distilled with MgO to separate NH4+; thereafter, the sample in the flask was distilled again after the addition of Devarda’s alloy to separate NO3−. The liberated NH3 was trapped with boric acid solution in a conical flask. The trapped N was acidified and converted to (NH4)2SO4 using 0.02 mol L−1 H2SO4 solution. The H2SO4 solution containing NH4+ was then evaporated to dryness at 65 °C in an oven and analyzed for 15 N abundance. Before separating NH4+ and NO3− in the extract
using the steam distillation system, the recovery of NH4+ and NO3− in a standard solution (1 g ammonia (NH4+-N) L−1 and 1 g nitrate (NO3−-N) L−1) was determined. The results showed that almost all (>99 %) of the NH4+-N in the solution could be recovered and the recovery of NO3− was >95 %. The amount of excess 15 N in sediment, overlying water, macrophytes, and N2O at the end of the experiment was calculated by subtracting the amount of background 15 N from the values of the final samples. N content of the macrophytes and sediment was calculated directly from the dry mass of the samples (g), the N concentration (g N/g). N-NO3− content in the water column was calculated from the mean N concentration in the water (mg/L) by the volume of water in each core. N2O content was calculated from the N2O fluxes, the volume, and experiment time in each core. NH 4+-N, nitrite (NO2−-N), NO3−-N, and total nitrogen (TN) were determined by Nessler’s reagent spectrophotometry, N-(1naphthyl)-ethylenediamine spectrophotometry, UV spectrophotometric method with reading absorbance at 220 and 275 mm (UV-2450, Shimadzu, Kyoto, Japan), and alkaline potassium persulfate UV spectrophotometry, respectively. Data analysis Statistical analysis was performed using the SPSS statistical package 13.0. One- or two-way ANOVA was run to determine significant differences between different treatments. If differences were determined, Tukey’s post hoc analysis was also conducted. Pearson correlation analysis was further used to evaluate correlation between variables. Untransformed data in all cases satisfied assumptions of normality. Significant differences were accepted for different treatments at the level of P
RESEARCH ARTICLE
Nitrate removal under different ecological remediation measures in Taihu Lake: a 15 N mass-balance approach Dandan Liu & Zhengkui Li & Wanguang Zhang
Received: 26 February 2014 / Accepted: 11 July 2014 / Published online: 23 July 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Ecological remediation is an important measure for the protection of lake water quality in removing nutrients, such as nitrate (NO3−). In this study, four bioremediation processes (bare sediment, immobilized nitrogen cycling bacteria (INCB) added, Elodea nuttallii added, E. nuttallii-INCB assemblage) were operated at a lab to elucidate the effect of macrophyte appearance and INCB addition on NO3− removal and achieve the optimal processes for biomediation. 15 N-NO3 solution was added to microcosms to identify the key nitrogen transformation processes responsible for NO3− removal. Results showed that nitrate removal was significantly enhanced after the addition of INCB and E. nuttallii. In the treatments with INCB added, E. nuttallii added, and INCB and E. nuttallii-INCB assemblage, nitrate removal ratio achieved 94.74, 98.76, and 99.15 %, respectively. In contrast, only 23.47 % added nitrate was removed in the control. Plant uptake and denitrification played an important role in nitrogen removal. The water quality was substantially improved by the addition of INCB and macrophyte that can accelerate denitrification and promote nitrogen assimilation of plants. The results indicated that plant uptake and microbial denitrification were key processes for nitrate removal.
Keywords Nitrogen transformation . Plant assimilation . N2O emission . Elodea nuttallii-INCB assemblage . Taihu Lake
Responsible editor: Hailong Wang D. Liu : Z. Li (*) : W. Zhang State Key Laboratory of Pollutant Control and Resources Reuse, School of the Environment, Institute of Oceanographic Research, Nanjing University, 163 Xianlin Avenue, 210023 Nanjing, People’s Republic of China e-mail: [email protected]
Introduction In the past decades, nitrogen inputs to aquatic ecosystems have increased dramatically. Excess nutrient loading from agricultural runoff, untreated industrial and urban discharges, leads to eutrophication, which has received growing concerns in many countries (Picek et al. 2008; Scheffer et al. 2003; Smith et al. 1999). Therefore, suitable steps should be taken to protect natural water resource from eutrophication by reducing nutrient inputs (Hamilton and Landman 2011; Sollie and Verhoeven 2008). Lake Taihu is a large (2,338 km2), shallow (1.9 m mean depth), and well-mixed lake in China. The annual water inflow is approximately 8×109 m3, and the residence time of the lake is approximately 5 months (Zeng et al. 2009). Meiliang Bay is located in the northern part of Lake Taihu with a water surface area of 135 km2 and a mean water depth of 2.1 m. It provides drinking water, irrigation, and industrial waters for Wuxi City. It receives the municipal and industrial wastewater from the surrounding river, resulting in the deterioration of water quality in this area. Serious cyanobacterial blooms in Meiliang Bay have increased in frequency and intensity in recent years and have seriously affected the function of the water body (Qiao et al. 2006; Zeng et al. 2012). Phytoremediation is an important measure to remove nutrients from polluted lakes (Sollie and Verhoeven 2008). It can remove nutrients through plant uptake and rhizosphere denitrification. Plant uptake represents a temporary store of nutrients, and harvesting biomass may completely remove nutrients from the system. Denitrification, the stepwise reduction of nitrate to N2 under low-oxygen and anaerobic conditions, represents a permanent nitrogen removal from aquatic systems (Lee et al. 2009; Seitzinger et al. 2006). However, N removal rates of lakes are different in many studies (Epstein et al. 2012; Schubert et al. 2006; Soana and Bartoli 2014), which are affected by background nitrogen concentration,
Environ Sci Pollut Res (2014) 21:14138–14145
presence of macrophyte, abundance of nitrogen cycle cycling microorganisms, and climatic condition (Pina-Ochoa and Alvarez-Cobelas 2006; Schubert et al. 2006). More studies should be conducted to determine the key limiting factors for nitrate removal of different ecological remediation treatments. Numerous studies have reported that macrophyte revegetation and the addition of immobilized nitrogen cycling bacteria (INCB) can stimulate N removal in sediments. Rooted submerged macrophytes may create favorable conditions for coupled nitrification-denitrification in the sediment by creating heterogeneous oxygen conditions in the root zone and by excreting organic carbon from their roots (Caffrey and Kemp 1992; Reddy et al. 1989; Ullah et al. 2014). The INCB technology can increase the quantities of nitrogen cycling bacteria through screening, immobilizing, and releasing them to the water column. INCB have been applied for nitrogen removal from domestic water, seawater, and freshwater (Chen et al. 2012; Joerdening et al. 2006). The integrated restoration method with macrophytes and INCB technology can enhance the activity of aquatic nitrogen removal. The integrated Elodea nuttallii-INCB technology could increase sediment denitrification rate by 162 % in the sediments (Wang et al. 2013). However, information about the nitrogen removal processes is limited for the different treatment methods. Thus, investigating the effects of various treatments (bare sediment, INCB added, E. nuttallii added, E. nuttallii-INCB assemblage) on benthic nitrogen cycling and quantifying the contribution of different removal pathways are theoretically and practically essential for optimal application of the processes in eutrophic lake restoration. The main objectives of this study were as follows: (1) to investigate the effect of different ecological remediation treatments (bare sediment, INCB added, E. nuttallii added, E. nuttallii-INCB assemblage) on nitrate removal and transformations in Taihu Lake and (2) to quantify the contribution of different removal pathways to nitrate removal under different ecological remediation treatments.
Materials and methods Study site and sampling A bare zone without macrophyte in the central position of Meiliang Bay (31° 12′ 14″ N, 119° 55′ 12″ E) was chosen as the sampling site (Fig. 1). Sampling of bare sediments, water, and E. nuttallii was conducted in this site in January 2013. Clear Plexiglas tubes (length 60 cm and inside diameter 9 cm) were pressed ~20 cm into the sediment, and a total of 24 intact sediment cores were collected for the following incubation experiments in the laboratory. The overlying water was simultaneously collected with plastic barrels. E. nuttallii was
14139
collected and put into a tank filled with lake water. All the sediment, water samples, and E. nuttallii were transported to the laboratory within 4 h at 5±1 °C. Preparation of INCB INCB were prepared as described previously (Wang et al. 2013). Indigenous nitrogen cycling bacteria were isolated from Lake Taihu water using the method of plate streaking and identified by 16S rRNA sequences. To get porous carriers for immobilization, 2-hydroxyethyl acrylate (HEA), 2-hydroxyethyl methylacrylate (HEMA), and distilled water were mixed at a volume ratio of 3:3:14. The mixture was irradiated at −78 °C with 60 Co gamma ray source at 10 kGy for 24 h. Then, solid polymer carriers were synthesized. The resultant polymer carriers were cut into small pieces, approximately 5×5×5 mm in dimension, and then the carriers were washed in excess deionized water to remove the unreacted components. The prepared carriers were immersed in deionized water for 3 days in order to be fully swollen. The swollen carriers with a pore size of about 20–100 μm were added to the mixture of precultured nitrogen cycle bacteria and nutrient medium. After alternate aerobic (8 h) and anaerobic incubation (4 h) for six times (total 72 h), the polymer carrier of immobilized nitrogen cycling bacteria was cultivated in an incubator at 28±1 °C under gentle rotary shaking (80 rpm). The porous carriers proportioned a suitable 3-D microhabitat for nitrogen cycling bacteria to colonize and reproduce (Cao et al. 2002; Zhang et al. 2007). After being incubated, nitrogen cycling bacteria were immobilized in the carriers and the INCB were prepared. The total quantity of nitrogen cycling bacteria immobilized was about 2.1×1012 cells/g (lipid-P biomass) on average. Experimental design Twenty-four cores from Meiliang Bay were divided into four restoration treatments. Each treatment had six replicates: treatment A, bare sediment core, as control group without any restoration treatments; treatment B, sediment + INCB; treatment C, sediment + E. nuttallii, five shoots of E. nuttallii (10 cm in length) cleaned with ultrapure water were planted into sediment; treatment D, sediment + E. nuttallii + INCB, prepared carrier with INCB (50±1 g tube−1) was added to the core to allow bacterial movement into the upper sediment layer. After restoration treatment, all the cores were filled immediately with lake water using a plastic syringe. For each treatment, three replicates were used to determine N2O fluxes, nitrate removal process analyses, and the other three were used to investigate the O2 profile. Lake water and INCB were renewed once a week. All cores after different restoration treatments were incubated at room temperature (25±1 °C) under natural light from the window in the lab throughout
14140
Environ Sci Pollut Res (2014) 21:14138–14145
Fig. 1 Geographical location of sampling points in Meiliang Bay, Taihu Lake
the whole experiment. The influence of different restoration treatments on N cycling process was evaluated after incubation for about 6 weeks.
N2O fluxes For measurement of N2O, a static closed chamber method was used. On the top of the core, a gas collection chamber (10 cm in height and 9 cm in inner diameter) was used to quantify N2O fluxes. At the beginning, the chamber was closed by lids which were made of rubber and coated with Apiezon high vacuum grease (type M). The incubation lasted for 40 h at room temperature under natural light condition. Gas samples were transferred from the chamber to previously evacuated glass vials by using double-sided needles. Each vial was allowed to fill for 30 s. The concentration of N2O was determined by a gas chromatograph (Agilent 4890) equipped with a flame ionization detector (FID) and an electron capture detector (ECD). The N2O portion was separated using a 1-m stainless steel column with an inner diameter 2-mm Porapak Q (80/100 mesh) and was measured using the ECD, which was set at 330 °C. The ECD used high-pure nitrogen as a carrier gas, at a flow rate of 35 mL/min. The column temperatures were maintained at 55 °C (Song et al. 2006). The gas flux was calculated according to the following equation: J¼
dc M P T 0 ⋅ ⋅ ⋅ ⋅H dt V 0 P0 T
where dc/dt is the slope of the gas concentration curve variation along with time; M is the mole mass of each gas; P is the atmospheric pressure in the sampling site; T is the absolute temperature during sampling; V0, T0, and P0 are the gas mole volume, air absolute temperate, and atmospheric pressure under standard conditions, respectively; and H is the height of the chamber above the water surface.
Sediment oxygen profile and characteristics At the end of the restoration incubation, sediment oxygen profiles were determined by a O2 microprobe with a tip diameter less than 0.1 mm (PreSens, Germany). The insertion of the O2 microprobe was controlled by a 3-D motorized micromanipulator (PreSens, Germany). The microprobe was inserted to the center of the core to make sure that no plant roots were touched. Six values were taken from each treatment, and the mean of these six values represented the O2 concentration of the treatment at the measured time point. 15
N chamber experiments
On the day of the experiment, the dosing solution (99 at.% 15 N-NO3− as NaNO3− was prepared based on chamber volume (L) and ambient NO3− concentrations to enrich background concentrations in the entire core by 10 %. Each chamber was dosed using a backpack sprayer. 15 N-NO3− was added to each chamber as shown in Table 1. The 15 N-NO3− balance experiment lasted for 40 h.
Environ Sci Pollut Res (2014) 21:14138–14145
14141
Table 1 Fate of transformed 15 N-NO3− during the experiment 15
N balance component
A Mass of 15 N (μg)
B Transformed 15 N- Mass of 15 N (μg) NaNO3− (%)
Amount of 15 N added 459 Detected in macrophytes Detected in sediment Detected in the water column (nitrate) Detected in N2O Unaccounted for [1 −(2+3+4+5)]
C Transformed 15 N- Mass of 15 N (μg) NaNO3− (%)
1,268
D Transformed 15 N- Mass of 15 N (μg) NaNO3− (%)
485
Transformed 15 NNaNO3− (%)
932
0
0
0
0
119±3.2
24.54
50±1.6
5.36
20±1.3 373±4.1
4.36 76.53
6±0.2 67±2.4
0.47 5.28
11±0.9 6±0.4
2.27 1.24
5±0.2 8±0.3
0.53 0.85
0.1±0.03
0.02
4±0.24
0.32
0.006
2±0.20
0.21
65.9±7.7
19.09
1,191± 3.7
93.93
0.03± 0.01 348.97± 1.4
71.94
867±0.5
93.05
Sample processing and analysis Water samples were collected manually in 250 mL HDPE bottles. Sediment was sampled using a cutting ring. Sediment samples were sieved (6 mm mesh) to remove large rocks and debris and dried at 60 °C (24–48 h) before preparation for 15 N analysis. Samples of macrophytes were collected from 12 chambers and clipped at the ground surface. A subset of each sample was immediately dried at 60 °C (24–48 h) and weighed to determine biomass (g/m2). After drying, samples were ground to a uniform texture (fine powder) by using a ball grinder. A small amount of each solid sample was weighed; macrophytes and sediment were transferred into encapsulated tins and stored in microtiter plates with individually sealed wells. Samples (macrophytes and sediment) were sent to the Stable Isotope Laboratory at the Nanjing Normal University (Nanjing, China) for analysis of N-isotope composition and percentage of element N by mass spectrometry using a Europa Integra continuous flow isotope ratio mass spectrometer (Europa Scientific, Seron, Cheshire, UK) coupled to an in-line elemental analyzer. Isotopic composition of the NH4+ and NO3− was measured using an automated C/N analyzer isotope ratio mass spectrometer (Europa Scientific Integra, Crewe, UK). NH4+ and NO3− were separated for 15 N measurements by distillation with magnesium oxide and Devarda’s alloy (Feast and Dennis 1996; Zhang et al. 2012). In detail, a portion of the extract was steam-distilled with MgO to separate NH4+; thereafter, the sample in the flask was distilled again after the addition of Devarda’s alloy to separate NO3−. The liberated NH3 was trapped with boric acid solution in a conical flask. The trapped N was acidified and converted to (NH4)2SO4 using 0.02 mol L−1 H2SO4 solution. The H2SO4 solution containing NH4+ was then evaporated to dryness at 65 °C in an oven and analyzed for 15 N abundance. Before separating NH4+ and NO3− in the extract
using the steam distillation system, the recovery of NH4+ and NO3− in a standard solution (1 g ammonia (NH4+-N) L−1 and 1 g nitrate (NO3−-N) L−1) was determined. The results showed that almost all (>99 %) of the NH4+-N in the solution could be recovered and the recovery of NO3− was >95 %. The amount of excess 15 N in sediment, overlying water, macrophytes, and N2O at the end of the experiment was calculated by subtracting the amount of background 15 N from the values of the final samples. N content of the macrophytes and sediment was calculated directly from the dry mass of the samples (g), the N concentration (g N/g). N-NO3− content in the water column was calculated from the mean N concentration in the water (mg/L) by the volume of water in each core. N2O content was calculated from the N2O fluxes, the volume, and experiment time in each core. NH 4+-N, nitrite (NO2−-N), NO3−-N, and total nitrogen (TN) were determined by Nessler’s reagent spectrophotometry, N-(1naphthyl)-ethylenediamine spectrophotometry, UV spectrophotometric method with reading absorbance at 220 and 275 mm (UV-2450, Shimadzu, Kyoto, Japan), and alkaline potassium persulfate UV spectrophotometry, respectively. Data analysis Statistical analysis was performed using the SPSS statistical package 13.0. One- or two-way ANOVA was run to determine significant differences between different treatments. If differences were determined, Tukey’s post hoc analysis was also conducted. Pearson correlation analysis was further used to evaluate correlation between variables. Untransformed data in all cases satisfied assumptions of normality. Significant differences were accepted for different treatments at the level of P
