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Supplemental tests of gas trapping device for N2 flux measurement
Ecological Engineering 93 (2016) 9–12
Contents lists available at ScienceDirect
Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng
Short communication
Supplemental tests of gas trapping device for N2 flux measurement Xinhong Liu a,1 , Yan Gao a,1 , Yongqiang Zhao b , Yan Wang a , Neng Yi a , Zhenhua Zhang a , Shaohua Yan a,∗ a b
Institute of Agricultural Resources and Environment, Jiangsu Academy of Agricultural Sciences, 50 Zhong Ling Street, Nanjng 210014, China State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, 71 Beijing road, Nanjing 210008, China
a r t i c l e
i n f o
Article history: Received 11 August 2015 Received in revised form 2 March 2016 Accepted 1 May 2016 Keyword: Gas trapping device N2 flux measurement Non-equilibrium diffusive error Membrane inlet mass spectrometry method
a b s t r a c t The gas trapping device method (GTD) is a relatively new method to measure N2 flux from waters. However, the non-equilibrium diffusion error and the reliability of GTD method compared to other previously established N2 flux measurement methods has not been evaluated. In this study, the diffusive error of GTD, coming from non-equilibrium N2 partial pressure between the headspace inside the gas sample bottle and the air, was estimated using a sterilization experiment. Moreover, the GTD and MIMS methods were compared for measuring N2 flux from water under similar conditions. The results showed that there were maximum diffusion errors of 2.99% in the sample bottles prefilled with pure Helium, while only 1.09–1.76% diffusion errors in bottles prefilled with other N2 standard gas (15% or 75%), indicating minor non-equilibrium diffusion errors. N2 fluxes from water measured by GTD and MIMS methods are quite similar under all three concentrations of nitrate (5.30, 10.55 and 17.25 mg L−1 ) and two levels of temperature (20 and 30 ◦ C). Therefore, the GTD method offers a reliable alternative method to estimate N2 flux rate in aquatic ecosystem. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The N2 flux rate is the major index to evaluate the denitrification rate and self-purification capability in aquatic ecosystems (Canfield et al., 2010; Collins et al., 2010). Various methods have been developed for measuring N2 and N2 O flux (Groffman et al., 2006). Recently, a gas trapping device (GTD) method was developed by Gao et al. (2013), using an floating inverted dome device to continuously collect N2 released from water (Gao et al., 2013; Gao et al., 2016). When using the GTD method, N2 recoveries of standard gas with known concentrations of N2 has been proved as high as 99.1%. Moreover, a zero partial pressure experiment indicated that release of the gas from the sterilized water was minor, suggesting the N2 flux measured by the GTD method should be derived from biological activities. However, difference (non-equilibrium) of N2 partial pressure between the headspace inside gas sample bottle and air occurred
Abbreviation: MIMS, membrane inlet mass spectrometry; GTD, gas trapping device method; GC, gas chromatograph; ECD, Ni63 electron capture detector; TCD, thermal-conductivity detector; DO, dissolved oxygen; ANOVA, analysis of variance; ORP, oxidation reduction potential; Anammox, anaerobic ammonium oxidation. ∗ Corresponding author. E-mail addresses: [email protected] (X. Liu), [email protected] (S. Yan). 1 The authors contribute equally to this article. http://dx.doi.org/10.1016/j.ecoleng.2016.05.009 0925-8574/© 2016 Elsevier B.V. All rights reserved.
after gas samples with different N2 partial pressure has entered into the gas sample bottle. Therefore it is possible that the nonequilibrium diffusion of N2 flux occurs (Smith and Lewis, 1992; Butterbach-Bahl et al., 2002). According to Fick’s law (Nowicki, 1994), there is a concern that the N2 in the collected gas samples may partially come from the air diffusion (Chanson, 1996; Cole and Caraco, 1998). The GTD method assumed that the non-equilibrium diffusion error was minor and could not affect the estimation of N2 flux from water body. However, this assumption has not been tested yet. For direct measurement of N2 flux from water ecosystem, a welldeveloped method is membrane inlet mass spectrometry (MIMS) method (Kana et al., 1994; Cornwell et al., 1999; Zhao et al., 2013). Both GTD method and MIMS method could be applied to estimate N2 flux from microbial activities in aquatic system. The MIMS method estimates the diffusive flux by analyzing the increase of dissolved N2 in the overlaying water (Mccutchan et al., 2003; Groffman et al., 2006), while the GTD investigates the bubble gas from the aquatic system (Gao et al., 2013). Both GTD and MIMS methods calculate N2 flux according to difference value rather than absolute value. The weight difference of a gas sample bottle before and after the experiment is used in the N2 flux calculation formula of GTD method, while slope value of line regression between dissolved N2 concentration in overlying water of the incubation tube and incubation time is used in MIMS method (An et al., 2001).
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X. Liu et al. / Ecological Engineering 93 (2016) 9–12
Table 1 Main properties of sediment and eutrophic water used in the experiment.
2.66 ± 0.05 6.28 ± 0.33
Water Sediment a b
NO3 − (mg L−1 )
NH4 + (mg L−1 )
Media
1.07 ± 0.03 2.80 ± 0.50
TN (N mg L−1 )
TOC/Organic matter 8.40 ± 0.76 7.97 ± 0.22b
4.55 ± 0.09 4.92 ± 0.11
a
TP (P mg L−1 ) 0.52 ± 0.03 1.39 ± 0.05
pH 7.4 ± 0.06 6.75 ± 0.13
Means the TOC concentration (mg L−1 ) in the water. Means the organic matter concentration (%) in sediment.
Thus, it is possible that the N2 flux measured by the two methods is similar under still water body. Therefore, the reliability of GTD method for N2 flux measurement could be compared to MIMS method under similar experimental conditions, which is critical for extensive application of the GTD method. The major objectives of the present study were to (1) test the diffusive error of GTD derived from non-equilibrium of N2 partial pressure between the headspace inside the gas sample bottle and the air, and to (2) test the reliability of the GTD for N2 flux measurement by comparing with MIMS methods under still water body conditions in lab.
2. Material and methods 2.1. Sterilization experiment for non-equilibrium diffusive error check 200L containers, with the open top, were filled with water for the error check. Before the experiment, the water was sterilized with ClO2 (20 mg kg−1 ) to minimize the gas production from microbial activities in water, and then was kept at 15 ◦ C in lab for ten days to achieve the gas equilibrium of water with atmosphere. After that, the GTDs were placed in the water as described in Gao et al. (2013). Then, about 100 ml standard gas was piped into the collecting dome of GTD, which would enter into the sample bottle subsequently. Three standard gases were selected in the test, with the gas composition of 75.32% N2 , 0.50% CO2 , 4.99% CH4 and 19.19% H2 in standard gas I, 15.50% N2 , 2.01% CO2 , 43.30% CH4 and 39.19% H2 in the standard gas II, and 99.99% He2 in standard gas III. All standard gases were purchased from the 55th Institute of China Light and Power Group Corporation (Nanjing, China). The N2 concentration variation of standard gas in the sample bottles was used as the index of non-equilibrium diffusive errors from the GTD method. Gas samples were collected at 0, 20, 40, 60, 80,100, 120, 140, 160 and 180hr after the standard gas was piped into the GTD. All treatments had four replications.
2.2. Comparison of GTD method with MIMS method 2.2.1. Sediment and water preparation Sediment and eutrophic water used in this experiment were collected from a eutrophic pond located in Jiangsu Academy of Agricultural Sciences, Nanjing, China. The sediment collected from the pond was mixed fully and sieved with 4 mm mesh to remove the impurities. The main properties of the sediment and overlying water were listed in Table 1.
2.2.2. Comparison experiment The whole experiment was carried out in the laboratory of the Institute of Soil Science, Chinese Academy of Sciences (ISSAS) at carefully maintained temperatures (20–30 ◦ C). Three treatments (i.e. T1, T2 and T3) with different temperature and nutrients conditions were listed in Table 2. All treatments had three replications. Before the experiment, the same sediment, with 20 cm thickness, was fully mixed and placed evenly at the bottom of water container of GTD method and core tubes of MIMS devices. Subsequently, the same eutrophic water was carefully placed on the sediment. Then, the two methods were started simultaneously after ten days to minimize the man-made interference, and ended at the same time after the experiment. The sampling arrangement of the two methods was listed in Table 2. Sample bottle volume of GTD method was enough for storing bubble gas collected from water. Rubber stoppers in the incubation core tubes of MIMS were sealed tightly during the whole experiment. All the overlying water samples were collected and analyzed immediately.
2.2.3. Net N2 flux determination by GTD method and MIMS method The detailed description of the GTD method and calculation of N2 flux was described in Gao et al., (2013). The gas samples were analyzed using a Gas Chromatography (GC-2010, Shimadzu Corp., Japan) (Liu et al., 2015). Net N2 flux by MIMS method was determined as described in Li et al. (2013). The detailed information of GTD and MIMS methods was shown in the supporting information.
2.3. Methods for analysis of water and sediment samples Water samples were analyzed for the concentrations of NH4 + , NO3 − , TN, TP using a flow injection analyzer (Skalar Analytical, Breda, The Netherlands). The water temperature (t ◦ C), dissolved oxygen (DO), pH and redox potential were measured using a portable meter (YSI Pro Plus, USA). The nutrients background in sediment samples were analyzed according to standard method (APHA, 2005).
2.4. Statistical analysis Analysis of variance (ANOVA) was performed using Statistical software (SPSS18.0), and the graphs were created by Sigmaplot12.0.
Table 2 The sampling arrange of the MIMS method during comparison experiments and condition parameters of water. Test No.
Temperatures
NO3 −
NH4 +
TN
TP
MIMS sampling timea
T1 T2 T3
20 30 30
5.30 ± 1.07 10.55 ± 0.51 17.25 ± 1.70
10.86 ± 0.08 4.28 ± 0.02 6.23 ± 0.04
18.33 ± 1.00 15.36 ± 0.10 25.71 ± 1.68
0.65 ± 0.15 0.1 ± 0.00 0.16 ± 0.00
0,2,4,6,8,24,48,96,120,144,168,192,216,240,264,288 0,2,4,6,8,24,48,96 0,2,4,6,8,26
a Means that MIMS sampling time was set at different time points during the whole treatment process. The sampling time of GTD method was set at the end of each treatment process.
X. Liu et al. / Ecological Engineering 93 (2016) 9–12
11
700
80 -2 -1 N flux of different method (umol.m .h ) 2
N concent ration in sample bot t le (%) 2
600
60
40
20
GTD method MIMS method
500
400 300
200
100
0
0 T1
0
20
40
60
80
10 0
12 0
140
160
180
Sampling period (hours) St andard sample 1
Standard sample 2
T2
T3
Test numbers Fig. 2. N2 flux of gas trapping device method (GTD) and membrane inlet mass spectrometry method (MIMS). The values mean average and 1 standard error.
St andard sample 3
Fig. 1. Variations of N2 concentration in different standard gas samples. The gas composition in standard gas I was 75.32% N2 , 0.50% CO2 , 4.99% CH4 and 19.19% H2 ; the gas composition in standard gas II was 15.50% N2 , 2.01% CO2 , 43.30% CH4 and 39.19% H2 ; the gas composition in standard gas III was pure Helium (99.99% He2 ); n = 4.
3. Results and discussion 3.1. Non-equilibrium diffusive errors of GTD method According to Fick’s diffusion law, the concentration gradient is the key factor to affect diffusive rate (Chanson, 1996). In this study, standard gases containing different concentrations of N2 (0–75.32%) were used to create different gradient difference of N2 partial pressure in order to estimate the non-equilibrium diffusive error in N2 determination using GTD method. The results showed that the maximum value of diffusive error, using GTD method, was 2.99% under the maximum gradient difference of N2 partial pressure when pure Helium (He2 99.999%) was used. Under other difference of N2 partial pressure, when standard gas contains 15% or 75% N2 , only 1.09–1.76% of diffusion errors were detected (Fig. 1). The errors, which were greatly lower than those obtained from the traditional N2 flux method (Hamersley and Howes, 2005; Smith and Lewis, 1992), were minor and acceptable for collecting and analyzing high concentrations of biological N2 production (Chanton et al., 1989). It is possible that the diffusive error was minor because the connecting channel (heavy-wall silicon tube with 6 mm diameter) between the gas sample bottles and water body below the trapping device was narrow, which may have limited the diffusion flux from water to the sample bottles. For the GTD method, therefore, it is necessary to develop a model to rectify the non-equilibrium diffusive error, especially for the samples with low N2 partial pressure (e.g. < 1%) (Cole and Caraco, 1998; Conen and Smith, 2000; Hamersley and Howes, 2005). 3.2. N2 flux estimation using GTD and MIMS methods Using the GTD method, 85.51 ± 25.69 ml, 29.00 ± 1.50 ml and 68.83 ± 3.74 ml of gas was collected in T1, T2 and T3 treatment, respectively. The corresponding gas samples contained 39.08 ± 3.82%, 88.99 ± 2.68% and 46.03 ± 1.09% N2 ,
respectively. The dissolved N2 concentrations during the experiment, using MIMS method, ranged from 264.17 ± 1.50 to 311.55 ± 2.96 mol ml−1 , from 242.10 ± 4.38 to 305.12 ± 8.44 mol ml−1 , and from 239.91 ± 0.68 to 320.28 ± 3.04 mol ml−1 in T1, T2 and T3 treatment, respectively. The N2 fluxes using GTD and MIMS method were 51.97 ± 13.95 and 62.89 ± 3.53 mol m−2 h−1 in T1 treatment, 126.06 ± 3.77 and 127.03 ± 12.64 mol m−2 h−1 in T2 treatment, 610.00 ± 28.79 and 642.20 ± 14.15 mol m−2 h−1 in T3 treatment, respectively (Fig. 2). There were no significant differences in the estimation of N2 flux between using GTD and MIMS methods, with the P values of 0.490 (T1), 0.95 (T2) and 0.37 (T3) respectively. The correlation coefficient of the two methods was 1.04 (r2 = 0.9927). The results suggested that the N2 fluxes estimated by the two methods were at the same level in all three tests (Fig. 2). In this study, sediment was completely homogenized by meshing with a 4 mm mesh and thoroughgoing mixing (See Section 2.2.1). Therefore, we assumed the gas reserved in the sediment was minor, and source of the detected N2 flux using two methods were from the diffusive process and gas bubble release due to the microbial activities rather than external disturbance. According to the gas dissolution equilibrium theory, gas bubble could be formed when the dissolved gas was beyond the saturation of gas solubility at the specific temperature and pressure, in addition, higher pressure could increase the gas solubility in water (Keiski et al., 1993; Colt, 2012; Vogelpohl et al., 2013). Using the GTD method, bubble gas was collected into the gas sample bottle when the diffusive N2 molecules exceeded the upper limit of solubility in the water. Using the MIMS method, the completely sealed core tube prevented gas emission from water and the water pressure in the tube probably increased with the rising gas production. This may lead to the increase of gas dissolution in the water within the sealed core tube and decrease of the bubble gas formation rate. Hence, there was no bubble observed throughout the experiment period, except that about 1 ml bubble gas was found at the end of T3 treatment with relatively higher N2 flux rate (Fig. 2), which was minor and negligible. In this study, the average N2 flux ranged from 51.97 ± 13.95 to 642.20 ± 14.15 mol m−2 h−1 at 20 and 30 ◦ C, which were in the middle range of the reported values (Li et al., 2013; Chanton et al., 1989) and lower than the previous reported values by GTD method (Gao et al., 2013). That was possible because the sediment
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in the natural pond was thicker. However, in order to compare GTD method with MIMS method under similar conditions, simulative experiment was operated in the laboratory using sediment cores and overlying water with consistent properties. At the end of the experiments, there was slight difference of nutrient concentration and physical parameters in the device between GTD and MIMS methods (See supporting Table 1 and 2). The reason was likely that the devices of GTD and MIMS methods existed difference. However, this didn’t lead to the differences in the N2 fluxes at the end of experiment. Therefore, the GTD method offers a reliable alternative method to estimate N2 flux rate in water body. Acknowledgements This work was supported by State Natural Science Foundation of China (No. 41301575, 41471415), Independent Innovation Fund of Agricultural Science and Technology of Jiangsu Province (No. CX (14)5050) and Natural Science Foundation of Jiangsu province (No. BK20140737). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ecoleng.2016.05. 009. References APHA, 2005. Standard Methods for the Examination of Water and Wastewater, 18th ed. American Public Health Association, American Water Works Association, Water Environment Federation, Washington. An, S., Gardner, W.S., Kana, T., 2001. Simultaneous measurement of denitrification and nitrogen fixation using isotope pairing with membrane inlet mass spectrometry analysis. Appl. Environ. Microb. 67, 1171–1178. Butterbach-Bahl, K., Willibald, G., Papen, H., 2002. Soil core method for direct simultaneous determination of N2 and N2O emissions from forest soils. Plant Soil 240, 105–116. Canfield, D.E., Glazer, A.N., Falkowski, P.G., 2010. The evolution and future of earth’s nitrogen cycle. Science 330, 192–196. Chanson, H., 1996. Air-water gas transfer. In: Air Bubble Entrainment in Free-surface Turbulent Shear Flows. Charpt. 4. Academic Press, Burlington, pp. 36–40. Chanton, J.P., Martens, C.S., Kelley, C.A., 1989. Gas transport from methane-saturated: tidal freshwater and wetland sediment. Limnol. Oceanogr. 34, 807–819.
Cole, J.J., Caraco, N.F., 1998. Atmospheric exchange of carbon dioxide in a low-wind oligotrophic lake measured by the addition of SF6 . Limnol. Oceanogr 43, 647–656. Collins, K.A., Lawrence, T.J., Stander, E.K., Jontos, R.J., Kaushal, S.S., Newcomer, T.A., Grimm, N.B., Cole Ekberg, M.L., 2010. Opportunities and challenges for managing nitrogen in urban stormwater. A Rev. Synth. Ecol. Eng. 36, 1507–1519. Colt, J., 2012. Dissolved Gas Concentrations in Water, 2nd ed. Elsevier. Conen, F., Smith, K.A., 2000. An explanation of linear increases in gas concentration under closed chambers used to measure gas exchange between soil and the atmosphere. Eur. J. Soil Sci. 51, 111–117. Cornwell, J., Kemp, W.M., Kana, T., 1999. Denitrification in coastal ecosystems: methods, environmental controls, and ecosystem level controls a review. Aquat. Ecol. 33, 41–54. Gao, Y., Liu, X.H., Yi, N., Wang, Y., Guo, J.Y., Zhang, Z.H., Yan, S.H., 2013. Estimation of N2 and N2 O ebullition from eutrophic water using an improved bubble trap device. Ecol. Eng. 57, 403–412. Gao, Y., Zhang, Z.H., Liu, X.H., Yi, N., Zhang, L., Song, W., Wang, Y., 2016. Seasonal and diurnal dynamics of physicochemical parameters and gas production in vertical water column of a eutrophic pond. Ecol. Eng. 87, 313–323. Groffman, P.M., Alterabet, A.M., Bohlke, J.K., Butterbach-Bahl, K., David, M.B., Firestone, M.K., Giblin, A.E., Kana, T.M., Nielsen, L.P., Voytek, M.A., 2006. Methods for measuring denitrification: diverse approaches to a difficult problem. Ecol. Appl. 16, 2091–2122. Hamersley, M.R., Howes, B.L., 2005. Evaluation of the N2 flux approach for measuring sediment denitrification. Estuar. Coast. Shelf Sci. 62, 711–723. Kana, T.M., Darkangelo, C., Hunt, M.D., Oldham, J.B., Bennett, G.E., Cornwell, J.C., 1994. Membrane inlet mass spectrometer for rapid high-Precision determination of N2 O2 , and Ar in environmental water samples. Anal. Chem. 66, 4166–4170. Keiski, R.L., Desponds, O., Chang, Y.F., Somorjai, G.A., 1993. Kinetics of the water-gas shift reaction over several alkane activation and water-gas shift catalysts. Appl. Catal. A-Gen. 101, 317–338. Li, X., Xia, Y., Li, Y., Kana, T., Kimura, S., Saito, M., Yan, X., 2013. Sediment denitrification in waterways in a rice-paddy-dominated watershed in eastern China. J. Soil Sediments 13, 783–792. Liu, X.H., Gao, Y., Wang, H.L., Guo, J.Y., Yan, S.H., 2015. Applying a new method for direct collection, volume quantification and determination of N2 emission from water. J. Environ. Sci. 27, 217–224. Mccutchan, J.H., Saunders, J.F., Pribyl, A.L., Lewis, W.M., 2003. Open-channel estimation of denitrification. Limnol. Oceanogra-Methods 1, 74–81. Nowicki, B.L., 1994. The effect of temperature oxygen, salinity, and nutrient enrichment on estuarine denitrification rates measured with a modified nitrogen gas flux technique. Estuar. Coast. Shelf. Sci. 38, 137–156. Smith, L.K., Lewis, W.M., 1992. Seasonality of methane emissions from five lakes and associated wetlands of the coloeado rockies. Global Biogeochem. Cycles 6, 323–338. Vogelpohl, C., Brandenbusch, C., Sadowski, G., 2013. High-pressure gas solubility in multicomponent solvent systems for hydroformylation. Part I: carbon monoxide solubility. J. Supercrit. Fluid 81, 23–32. Zhao, Y., Xia, Y., Kana, T.M., Wu, Y., Li, X., Yan, X., 2013. Seasonal variation and controlling factors of anaerobic ammonium oxidation in freshwater river sediments in the Taihu Lake region of China. Chemosphere 93, 2124–2131.
Contents lists available at ScienceDirect
Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng
Short communication
Supplemental tests of gas trapping device for N2 flux measurement Xinhong Liu a,1 , Yan Gao a,1 , Yongqiang Zhao b , Yan Wang a , Neng Yi a , Zhenhua Zhang a , Shaohua Yan a,∗ a b
Institute of Agricultural Resources and Environment, Jiangsu Academy of Agricultural Sciences, 50 Zhong Ling Street, Nanjng 210014, China State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, 71 Beijing road, Nanjing 210008, China
a r t i c l e
i n f o
Article history: Received 11 August 2015 Received in revised form 2 March 2016 Accepted 1 May 2016 Keyword: Gas trapping device N2 flux measurement Non-equilibrium diffusive error Membrane inlet mass spectrometry method
a b s t r a c t The gas trapping device method (GTD) is a relatively new method to measure N2 flux from waters. However, the non-equilibrium diffusion error and the reliability of GTD method compared to other previously established N2 flux measurement methods has not been evaluated. In this study, the diffusive error of GTD, coming from non-equilibrium N2 partial pressure between the headspace inside the gas sample bottle and the air, was estimated using a sterilization experiment. Moreover, the GTD and MIMS methods were compared for measuring N2 flux from water under similar conditions. The results showed that there were maximum diffusion errors of 2.99% in the sample bottles prefilled with pure Helium, while only 1.09–1.76% diffusion errors in bottles prefilled with other N2 standard gas (15% or 75%), indicating minor non-equilibrium diffusion errors. N2 fluxes from water measured by GTD and MIMS methods are quite similar under all three concentrations of nitrate (5.30, 10.55 and 17.25 mg L−1 ) and two levels of temperature (20 and 30 ◦ C). Therefore, the GTD method offers a reliable alternative method to estimate N2 flux rate in aquatic ecosystem. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The N2 flux rate is the major index to evaluate the denitrification rate and self-purification capability in aquatic ecosystems (Canfield et al., 2010; Collins et al., 2010). Various methods have been developed for measuring N2 and N2 O flux (Groffman et al., 2006). Recently, a gas trapping device (GTD) method was developed by Gao et al. (2013), using an floating inverted dome device to continuously collect N2 released from water (Gao et al., 2013; Gao et al., 2016). When using the GTD method, N2 recoveries of standard gas with known concentrations of N2 has been proved as high as 99.1%. Moreover, a zero partial pressure experiment indicated that release of the gas from the sterilized water was minor, suggesting the N2 flux measured by the GTD method should be derived from biological activities. However, difference (non-equilibrium) of N2 partial pressure between the headspace inside gas sample bottle and air occurred
Abbreviation: MIMS, membrane inlet mass spectrometry; GTD, gas trapping device method; GC, gas chromatograph; ECD, Ni63 electron capture detector; TCD, thermal-conductivity detector; DO, dissolved oxygen; ANOVA, analysis of variance; ORP, oxidation reduction potential; Anammox, anaerobic ammonium oxidation. ∗ Corresponding author. E-mail addresses: [email protected] (X. Liu), [email protected] (S. Yan). 1 The authors contribute equally to this article. http://dx.doi.org/10.1016/j.ecoleng.2016.05.009 0925-8574/© 2016 Elsevier B.V. All rights reserved.
after gas samples with different N2 partial pressure has entered into the gas sample bottle. Therefore it is possible that the nonequilibrium diffusion of N2 flux occurs (Smith and Lewis, 1992; Butterbach-Bahl et al., 2002). According to Fick’s law (Nowicki, 1994), there is a concern that the N2 in the collected gas samples may partially come from the air diffusion (Chanson, 1996; Cole and Caraco, 1998). The GTD method assumed that the non-equilibrium diffusion error was minor and could not affect the estimation of N2 flux from water body. However, this assumption has not been tested yet. For direct measurement of N2 flux from water ecosystem, a welldeveloped method is membrane inlet mass spectrometry (MIMS) method (Kana et al., 1994; Cornwell et al., 1999; Zhao et al., 2013). Both GTD method and MIMS method could be applied to estimate N2 flux from microbial activities in aquatic system. The MIMS method estimates the diffusive flux by analyzing the increase of dissolved N2 in the overlaying water (Mccutchan et al., 2003; Groffman et al., 2006), while the GTD investigates the bubble gas from the aquatic system (Gao et al., 2013). Both GTD and MIMS methods calculate N2 flux according to difference value rather than absolute value. The weight difference of a gas sample bottle before and after the experiment is used in the N2 flux calculation formula of GTD method, while slope value of line regression between dissolved N2 concentration in overlying water of the incubation tube and incubation time is used in MIMS method (An et al., 2001).
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X. Liu et al. / Ecological Engineering 93 (2016) 9–12
Table 1 Main properties of sediment and eutrophic water used in the experiment.
2.66 ± 0.05 6.28 ± 0.33
Water Sediment a b
NO3 − (mg L−1 )
NH4 + (mg L−1 )
Media
1.07 ± 0.03 2.80 ± 0.50
TN (N mg L−1 )
TOC/Organic matter 8.40 ± 0.76 7.97 ± 0.22b
4.55 ± 0.09 4.92 ± 0.11
a
TP (P mg L−1 ) 0.52 ± 0.03 1.39 ± 0.05
pH 7.4 ± 0.06 6.75 ± 0.13
Means the TOC concentration (mg L−1 ) in the water. Means the organic matter concentration (%) in sediment.
Thus, it is possible that the N2 flux measured by the two methods is similar under still water body. Therefore, the reliability of GTD method for N2 flux measurement could be compared to MIMS method under similar experimental conditions, which is critical for extensive application of the GTD method. The major objectives of the present study were to (1) test the diffusive error of GTD derived from non-equilibrium of N2 partial pressure between the headspace inside the gas sample bottle and the air, and to (2) test the reliability of the GTD for N2 flux measurement by comparing with MIMS methods under still water body conditions in lab.
2. Material and methods 2.1. Sterilization experiment for non-equilibrium diffusive error check 200L containers, with the open top, were filled with water for the error check. Before the experiment, the water was sterilized with ClO2 (20 mg kg−1 ) to minimize the gas production from microbial activities in water, and then was kept at 15 ◦ C in lab for ten days to achieve the gas equilibrium of water with atmosphere. After that, the GTDs were placed in the water as described in Gao et al. (2013). Then, about 100 ml standard gas was piped into the collecting dome of GTD, which would enter into the sample bottle subsequently. Three standard gases were selected in the test, with the gas composition of 75.32% N2 , 0.50% CO2 , 4.99% CH4 and 19.19% H2 in standard gas I, 15.50% N2 , 2.01% CO2 , 43.30% CH4 and 39.19% H2 in the standard gas II, and 99.99% He2 in standard gas III. All standard gases were purchased from the 55th Institute of China Light and Power Group Corporation (Nanjing, China). The N2 concentration variation of standard gas in the sample bottles was used as the index of non-equilibrium diffusive errors from the GTD method. Gas samples were collected at 0, 20, 40, 60, 80,100, 120, 140, 160 and 180hr after the standard gas was piped into the GTD. All treatments had four replications.
2.2. Comparison of GTD method with MIMS method 2.2.1. Sediment and water preparation Sediment and eutrophic water used in this experiment were collected from a eutrophic pond located in Jiangsu Academy of Agricultural Sciences, Nanjing, China. The sediment collected from the pond was mixed fully and sieved with 4 mm mesh to remove the impurities. The main properties of the sediment and overlying water were listed in Table 1.
2.2.2. Comparison experiment The whole experiment was carried out in the laboratory of the Institute of Soil Science, Chinese Academy of Sciences (ISSAS) at carefully maintained temperatures (20–30 ◦ C). Three treatments (i.e. T1, T2 and T3) with different temperature and nutrients conditions were listed in Table 2. All treatments had three replications. Before the experiment, the same sediment, with 20 cm thickness, was fully mixed and placed evenly at the bottom of water container of GTD method and core tubes of MIMS devices. Subsequently, the same eutrophic water was carefully placed on the sediment. Then, the two methods were started simultaneously after ten days to minimize the man-made interference, and ended at the same time after the experiment. The sampling arrangement of the two methods was listed in Table 2. Sample bottle volume of GTD method was enough for storing bubble gas collected from water. Rubber stoppers in the incubation core tubes of MIMS were sealed tightly during the whole experiment. All the overlying water samples were collected and analyzed immediately.
2.2.3. Net N2 flux determination by GTD method and MIMS method The detailed description of the GTD method and calculation of N2 flux was described in Gao et al., (2013). The gas samples were analyzed using a Gas Chromatography (GC-2010, Shimadzu Corp., Japan) (Liu et al., 2015). Net N2 flux by MIMS method was determined as described in Li et al. (2013). The detailed information of GTD and MIMS methods was shown in the supporting information.
2.3. Methods for analysis of water and sediment samples Water samples were analyzed for the concentrations of NH4 + , NO3 − , TN, TP using a flow injection analyzer (Skalar Analytical, Breda, The Netherlands). The water temperature (t ◦ C), dissolved oxygen (DO), pH and redox potential were measured using a portable meter (YSI Pro Plus, USA). The nutrients background in sediment samples were analyzed according to standard method (APHA, 2005).
2.4. Statistical analysis Analysis of variance (ANOVA) was performed using Statistical software (SPSS18.0), and the graphs were created by Sigmaplot12.0.
Table 2 The sampling arrange of the MIMS method during comparison experiments and condition parameters of water. Test No.
Temperatures
NO3 −
NH4 +
TN
TP
MIMS sampling timea
T1 T2 T3
20 30 30
5.30 ± 1.07 10.55 ± 0.51 17.25 ± 1.70
10.86 ± 0.08 4.28 ± 0.02 6.23 ± 0.04
18.33 ± 1.00 15.36 ± 0.10 25.71 ± 1.68
0.65 ± 0.15 0.1 ± 0.00 0.16 ± 0.00
0,2,4,6,8,24,48,96,120,144,168,192,216,240,264,288 0,2,4,6,8,24,48,96 0,2,4,6,8,26
a Means that MIMS sampling time was set at different time points during the whole treatment process. The sampling time of GTD method was set at the end of each treatment process.
X. Liu et al. / Ecological Engineering 93 (2016) 9–12
11
700
80 -2 -1 N flux of different method (umol.m .h ) 2
N concent ration in sample bot t le (%) 2
600
60
40
20
GTD method MIMS method
500
400 300
200
100
0
0 T1
0
20
40
60
80
10 0
12 0
140
160
180
Sampling period (hours) St andard sample 1
Standard sample 2
T2
T3
Test numbers Fig. 2. N2 flux of gas trapping device method (GTD) and membrane inlet mass spectrometry method (MIMS). The values mean average and 1 standard error.
St andard sample 3
Fig. 1. Variations of N2 concentration in different standard gas samples. The gas composition in standard gas I was 75.32% N2 , 0.50% CO2 , 4.99% CH4 and 19.19% H2 ; the gas composition in standard gas II was 15.50% N2 , 2.01% CO2 , 43.30% CH4 and 39.19% H2 ; the gas composition in standard gas III was pure Helium (99.99% He2 ); n = 4.
3. Results and discussion 3.1. Non-equilibrium diffusive errors of GTD method According to Fick’s diffusion law, the concentration gradient is the key factor to affect diffusive rate (Chanson, 1996). In this study, standard gases containing different concentrations of N2 (0–75.32%) were used to create different gradient difference of N2 partial pressure in order to estimate the non-equilibrium diffusive error in N2 determination using GTD method. The results showed that the maximum value of diffusive error, using GTD method, was 2.99% under the maximum gradient difference of N2 partial pressure when pure Helium (He2 99.999%) was used. Under other difference of N2 partial pressure, when standard gas contains 15% or 75% N2 , only 1.09–1.76% of diffusion errors were detected (Fig. 1). The errors, which were greatly lower than those obtained from the traditional N2 flux method (Hamersley and Howes, 2005; Smith and Lewis, 1992), were minor and acceptable for collecting and analyzing high concentrations of biological N2 production (Chanton et al., 1989). It is possible that the diffusive error was minor because the connecting channel (heavy-wall silicon tube with 6 mm diameter) between the gas sample bottles and water body below the trapping device was narrow, which may have limited the diffusion flux from water to the sample bottles. For the GTD method, therefore, it is necessary to develop a model to rectify the non-equilibrium diffusive error, especially for the samples with low N2 partial pressure (e.g. < 1%) (Cole and Caraco, 1998; Conen and Smith, 2000; Hamersley and Howes, 2005). 3.2. N2 flux estimation using GTD and MIMS methods Using the GTD method, 85.51 ± 25.69 ml, 29.00 ± 1.50 ml and 68.83 ± 3.74 ml of gas was collected in T1, T2 and T3 treatment, respectively. The corresponding gas samples contained 39.08 ± 3.82%, 88.99 ± 2.68% and 46.03 ± 1.09% N2 ,
respectively. The dissolved N2 concentrations during the experiment, using MIMS method, ranged from 264.17 ± 1.50 to 311.55 ± 2.96 mol ml−1 , from 242.10 ± 4.38 to 305.12 ± 8.44 mol ml−1 , and from 239.91 ± 0.68 to 320.28 ± 3.04 mol ml−1 in T1, T2 and T3 treatment, respectively. The N2 fluxes using GTD and MIMS method were 51.97 ± 13.95 and 62.89 ± 3.53 mol m−2 h−1 in T1 treatment, 126.06 ± 3.77 and 127.03 ± 12.64 mol m−2 h−1 in T2 treatment, 610.00 ± 28.79 and 642.20 ± 14.15 mol m−2 h−1 in T3 treatment, respectively (Fig. 2). There were no significant differences in the estimation of N2 flux between using GTD and MIMS methods, with the P values of 0.490 (T1), 0.95 (T2) and 0.37 (T3) respectively. The correlation coefficient of the two methods was 1.04 (r2 = 0.9927). The results suggested that the N2 fluxes estimated by the two methods were at the same level in all three tests (Fig. 2). In this study, sediment was completely homogenized by meshing with a 4 mm mesh and thoroughgoing mixing (See Section 2.2.1). Therefore, we assumed the gas reserved in the sediment was minor, and source of the detected N2 flux using two methods were from the diffusive process and gas bubble release due to the microbial activities rather than external disturbance. According to the gas dissolution equilibrium theory, gas bubble could be formed when the dissolved gas was beyond the saturation of gas solubility at the specific temperature and pressure, in addition, higher pressure could increase the gas solubility in water (Keiski et al., 1993; Colt, 2012; Vogelpohl et al., 2013). Using the GTD method, bubble gas was collected into the gas sample bottle when the diffusive N2 molecules exceeded the upper limit of solubility in the water. Using the MIMS method, the completely sealed core tube prevented gas emission from water and the water pressure in the tube probably increased with the rising gas production. This may lead to the increase of gas dissolution in the water within the sealed core tube and decrease of the bubble gas formation rate. Hence, there was no bubble observed throughout the experiment period, except that about 1 ml bubble gas was found at the end of T3 treatment with relatively higher N2 flux rate (Fig. 2), which was minor and negligible. In this study, the average N2 flux ranged from 51.97 ± 13.95 to 642.20 ± 14.15 mol m−2 h−1 at 20 and 30 ◦ C, which were in the middle range of the reported values (Li et al., 2013; Chanton et al., 1989) and lower than the previous reported values by GTD method (Gao et al., 2013). That was possible because the sediment
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in the natural pond was thicker. However, in order to compare GTD method with MIMS method under similar conditions, simulative experiment was operated in the laboratory using sediment cores and overlying water with consistent properties. At the end of the experiments, there was slight difference of nutrient concentration and physical parameters in the device between GTD and MIMS methods (See supporting Table 1 and 2). The reason was likely that the devices of GTD and MIMS methods existed difference. However, this didn’t lead to the differences in the N2 fluxes at the end of experiment. Therefore, the GTD method offers a reliable alternative method to estimate N2 flux rate in water body. Acknowledgements This work was supported by State Natural Science Foundation of China (No. 41301575, 41471415), Independent Innovation Fund of Agricultural Science and Technology of Jiangsu Province (No. CX (14)5050) and Natural Science Foundation of Jiangsu province (No. BK20140737). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ecoleng.2016.05. 009. References APHA, 2005. Standard Methods for the Examination of Water and Wastewater, 18th ed. American Public Health Association, American Water Works Association, Water Environment Federation, Washington. An, S., Gardner, W.S., Kana, T., 2001. Simultaneous measurement of denitrification and nitrogen fixation using isotope pairing with membrane inlet mass spectrometry analysis. Appl. Environ. Microb. 67, 1171–1178. Butterbach-Bahl, K., Willibald, G., Papen, H., 2002. Soil core method for direct simultaneous determination of N2 and N2O emissions from forest soils. Plant Soil 240, 105–116. Canfield, D.E., Glazer, A.N., Falkowski, P.G., 2010. The evolution and future of earth’s nitrogen cycle. Science 330, 192–196. Chanson, H., 1996. Air-water gas transfer. In: Air Bubble Entrainment in Free-surface Turbulent Shear Flows. Charpt. 4. Academic Press, Burlington, pp. 36–40. Chanton, J.P., Martens, C.S., Kelley, C.A., 1989. Gas transport from methane-saturated: tidal freshwater and wetland sediment. Limnol. Oceanogr. 34, 807–819.
Cole, J.J., Caraco, N.F., 1998. Atmospheric exchange of carbon dioxide in a low-wind oligotrophic lake measured by the addition of SF6 . Limnol. Oceanogr 43, 647–656. Collins, K.A., Lawrence, T.J., Stander, E.K., Jontos, R.J., Kaushal, S.S., Newcomer, T.A., Grimm, N.B., Cole Ekberg, M.L., 2010. Opportunities and challenges for managing nitrogen in urban stormwater. A Rev. Synth. Ecol. Eng. 36, 1507–1519. Colt, J., 2012. Dissolved Gas Concentrations in Water, 2nd ed. Elsevier. Conen, F., Smith, K.A., 2000. An explanation of linear increases in gas concentration under closed chambers used to measure gas exchange between soil and the atmosphere. Eur. J. Soil Sci. 51, 111–117. Cornwell, J., Kemp, W.M., Kana, T., 1999. Denitrification in coastal ecosystems: methods, environmental controls, and ecosystem level controls a review. Aquat. Ecol. 33, 41–54. Gao, Y., Liu, X.H., Yi, N., Wang, Y., Guo, J.Y., Zhang, Z.H., Yan, S.H., 2013. Estimation of N2 and N2 O ebullition from eutrophic water using an improved bubble trap device. Ecol. Eng. 57, 403–412. Gao, Y., Zhang, Z.H., Liu, X.H., Yi, N., Zhang, L., Song, W., Wang, Y., 2016. Seasonal and diurnal dynamics of physicochemical parameters and gas production in vertical water column of a eutrophic pond. Ecol. Eng. 87, 313–323. Groffman, P.M., Alterabet, A.M., Bohlke, J.K., Butterbach-Bahl, K., David, M.B., Firestone, M.K., Giblin, A.E., Kana, T.M., Nielsen, L.P., Voytek, M.A., 2006. Methods for measuring denitrification: diverse approaches to a difficult problem. Ecol. Appl. 16, 2091–2122. Hamersley, M.R., Howes, B.L., 2005. Evaluation of the N2 flux approach for measuring sediment denitrification. Estuar. Coast. Shelf Sci. 62, 711–723. Kana, T.M., Darkangelo, C., Hunt, M.D., Oldham, J.B., Bennett, G.E., Cornwell, J.C., 1994. Membrane inlet mass spectrometer for rapid high-Precision determination of N2 O2 , and Ar in environmental water samples. Anal. Chem. 66, 4166–4170. Keiski, R.L., Desponds, O., Chang, Y.F., Somorjai, G.A., 1993. Kinetics of the water-gas shift reaction over several alkane activation and water-gas shift catalysts. Appl. Catal. A-Gen. 101, 317–338. Li, X., Xia, Y., Li, Y., Kana, T., Kimura, S., Saito, M., Yan, X., 2013. Sediment denitrification in waterways in a rice-paddy-dominated watershed in eastern China. J. Soil Sediments 13, 783–792. Liu, X.H., Gao, Y., Wang, H.L., Guo, J.Y., Yan, S.H., 2015. Applying a new method for direct collection, volume quantification and determination of N2 emission from water. J. Environ. Sci. 27, 217–224. Mccutchan, J.H., Saunders, J.F., Pribyl, A.L., Lewis, W.M., 2003. Open-channel estimation of denitrification. Limnol. Oceanogra-Methods 1, 74–81. Nowicki, B.L., 1994. The effect of temperature oxygen, salinity, and nutrient enrichment on estuarine denitrification rates measured with a modified nitrogen gas flux technique. Estuar. Coast. Shelf. Sci. 38, 137–156. Smith, L.K., Lewis, W.M., 1992. Seasonality of methane emissions from five lakes and associated wetlands of the coloeado rockies. Global Biogeochem. Cycles 6, 323–338. Vogelpohl, C., Brandenbusch, C., Sadowski, G., 2013. High-pressure gas solubility in multicomponent solvent systems for hydroformylation. Part I: carbon monoxide solubility. J. Supercrit. Fluid 81, 23–32. Zhao, Y., Xia, Y., Kana, T.M., Wu, Y., Li, X., Yan, X., 2013. Seasonal variation and controlling factors of anaerobic ammonium oxidation in freshwater river sediments in the Taihu Lake region of China. Chemosphere 93, 2124–2131.