Contrasting impact of elevated atmospheric CO2 on nitrogen cycle in eutrophic water with or without Eichhornia crassipes (Mart.) Solms

Science of the Total Environment 666 (2019) 285–297

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Contrasting impact of elevated atmospheric CO2 on nitrogen cycle in eutrophic water with or without Eichhornia crassipes (Mart.) Solms Man Shi a,b,1, Jiangye Li b,1, Weiguo Zhang b, Qi Zhou a, Yuhan Niu a,b, Zhenhua Zhang b,c, Yan Gao b,⁎, Shaohua Yan b a b c

College of Forestry, Nanjing Forestry University, Nanjing 210037, China Institute of Agricultural Resources and Environment, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China School of Agriculture and Environment, The University of Western Australia, Perth, WA 6009, Australia

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Elevated atmospheric CO2 (e[CO2]) promotes denitrification and N2O emissions from eutrophic water without plant. • Algal activity is the main driving factor leading to inhibited nitrification but enhanced N2O emissions under e[CO2]. • Floating aquatic plant cultivation attenuate the negative effect on enhanced N2O emissions caused by e[CO2]

a r t i c l e

i n f o

Article history: Received 24 October 2018 Received in revised form 14 February 2019 Accepted 14 February 2019 Available online 15 February 2019 Editor: Charlotte Poschenrieder Keywords: CO2 concentration Eutrophication N-transformation Regulation by plant Microorganism

a b s t r a c t The elevation of atmospheric CO2 is an inevitable trend that would lead to significant impact on the interrelated carbon and nitrogen cycles through microbial activities in the aquatic ecosystem. Eutrophication has become a common trophic state of inland waters throughout the world, but how the elevated CO2 affects N cycles in such eutrophic water with algal bloom, and how vegetative restoration helps to mitigate N2O emission remains unknown. We conducted the experiments to investigate the effects of ambient and elevated atmospheric CO2 (a [CO2], e[CO2]; 400, 800 μmol﹒mol−1) with and without the floating aquatic plant, Eichhornia crassipes (Mart.) Solms, on N-transformation in eutrophic water using the 15N tracer method. The nitrification could be slightly inhibited by e[CO2], due mainly to the competition for dissolved inorganic carbon between algae and nitrifiers. The e[CO2] promoted denitrification and N2O emissions from eutrophic water without growth of plants, leading to aggravation of greenhouse effect and forming a vicious cycle. However, growth of the aquatic plant, Eichhornia crassipes, slightly promoted nitrification, but reduced N2O emissions from eutrophic water under e[CO2] conditions, thereby attenuating the negative effect of e[CO2] on N2O emissions. In the experiment, the N transformation was influenced by many factors such as pH, DO and algae density, except e[CO2] and plant presence. The pH could be regulated through diurnal photosynthesis and respiration of algae and mitigated the acidification of water caused by e[CO2], leading to an appropriate pH range for both nitrifying and denitrifying microbes. Algal respiration at night could consume DO and enhance abundance of denitrifying functional genes (nirK,

⁎ Corresponding author at: Institute of Agricultural Resources and Environment, Jiangsu Academy of Agricultural Sciences, 50 Zhongling Street, Nanjing 210014, China. E-mail address: [email protected] (Y. Gao). 1 These authors contributed equally to this study.

https://doi.org/10.1016/j.scitotenv.2019.02.224 0048-9697/© 2019 Elsevier B.V. All rights reserved.

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nosZ) in water, which was also supposed to be a critical factor affecting denitrification and N2O emissions. This study clarifies how the greenhouse effect caused by e[CO2] mediates N biogeochemical cycle in the aquatic ecosystem, and how vegetative restoration mitigates greenhouse gas emission. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Concentration of atmospheric CO2 has been rising since the Industrial Revolution and it is believed that the current trend will continue (Feely et al., 2009). It was predicted that the average atmospheric CO2 concentration will increase from current 400 μmol﹒mol−1 to about 800 μmol﹒ mol−1 by 2100 (IPCC, 2013). Elevation in atmospheric CO2 will have a significant impact on carbon (C) and nitrogen (N) biogeochemical cycles in various ecosystems, such as forests, farmlands, grasslands, oceans, lakes and other ecosystems (Kammann et al., 2008; Balmer and Downing, 2011; Zong and Shangguan, 2016; Paschalis et al., 2017). It has been estimated that 25–33% of anthropogenic CO2 emissions could have been absorbed by the water bodies that occupy 3/4 of the global surface area (Richier et al., 2014). Therefore, the impact of the dissolution and exchange of CO2 at the water-air interface on water pH and the balance of carbonate system, also known as the C cycles in aquatic ecosystems, should not be underestimated as the atmospheric CO2 concentration continues to rise. The NH3 could be oxidized to NO3− through nitrification, and most nitrifiers take inorganic carbon as carbon source. Meanwhile, NO3− could be eventually converted back to N2 through denitrification and various denitrifiers take organic carbon as carbon source. Therefore, C and N are closely interrelated through microbial activities in various ecosystems, which calls for our attention to the impact of atmospheric CO2 concentration on the N biogeochemical cycle of water bodies. Elevated atmospheric CO2 (e[CO2]) can affect N biogeochemical cycles in water by changing the physical and chemical environment of water and affecting the propagation and growth of microorganisms (Feely et al., 2009; Joint et al., 2011). Thus far, related studies have mainly focused on the influence of e[CO2] on the transformation of N in seawater (Beman and Karl, 2011). The results showed that e[CO2] would lower the pH of seawater and cause the chemical equilibrium “NH3+H+ ⇌ NH4+” in waters to shift towards the production of more NH4+, and thus NH3/NH4+ will drop, resulting in the decrease of ammoxidation efficiency of ammonia-oxidizing bacteria (AOB) (Beman and Karl, 2011) and leading to the inhibition of microbial nitrification. However, in inland waters, especially in eutrophic waters, N and P are abundant, resulting in the mass propagation of algae which is able to consume CO2 in the waters. Hence, these waters have a pH above 7 in general, even up to 10.6 during the algae bloom (Green et al., 1996). In this case, the elevation of CO2 concentration will have a minor effect on water pH. Therefore, whether e[CO2] will inhibit nitrification occurred in eutrophic waters still remains unknown. In addition, e[CO2] may directly or indirectly affect denitrification. Earlier research had shown that, elevated CO2 damaged the bacterial membrane and directly inhibited the transport and consumption of intracellular electrons by suppressing the activity and synthesis of key denitrifying enzymes, thus inhibiting denitrification (Wan et al., 2016). The indirect impact on denitrification mainly depended on the changes of environmental parameters, such as pH, dissolved oxygen (DO) and carbon sources (Barnard et al., 2004). Previous studies showed that the suboptimal pH of 6.8 inhibited denitrification activity in Pseudomonas denitrificans (Baumann et al., 1997), and a pH of 5 negatively impacted denitrification gene expression. However, levels of nirS and cnorB gene expression were not affected by pH treatment in Pseudomonas mandelii cultures grown at pH 6, 7 and 8 (Salehlakha et al., 2009). In addition, anoxic conditions favored the denitrification process and DO concentrations above 10 mg· L−1 in the water inhibited this process (Luo et al., 2016). Beyond that, when there is abundant NO3−, sufficient organic carbon would act as electron donors to promote denitrification by reducing oxygen

concentration and expanding the anaerobic habitat through aerobic decomposition (Arango et al., 2010). Thus far, most of the earlier studies were concerned with the impact of environmental factors on denitrification activity and were based on the investigation of each single factor in seawater or liquid media. However, the environment in eutrophic water bodies are more complex and changeable due to the massive growth of algae, with large diurnal variation of DO and pH, large quantity of easily degradable organic matter and interactions between algae and bacteria (Balmer and Downing, 2011; Verspagen et al., 2014; Gao et al., 2016). Moreover, algal mucus can hinder the diffusion of O2 within the algal aggregates thus algal aggregates are able to provide an anaerobic microenvironment that may favor the denitrification process (Ploug et al., 1997). Aquatic plants could have a direct impact on the N cycle in waters (Veraart et al., 2011). Floating aquatic plants, such as Eichhornia crassipes (Mart.) Solms, Pistia stratiotes L. and Ipomoea aquatica Forsk., are often used for the bioremediation of eutrophic waters (Lu et al., 2018). The well-developed root systems of floating aquatic plants, which are suspended in water, have an excellent ability to exude O2 and organic carbon (Ma et al., 2014; Kosten et al., 2016), hence providing an alternating anaerobic-aerobic root microenvironment for colonization and propagation of nitrifying and denitrifying microbes. This allows them to regulate the process of nitrification and denitrification in waters (Snooknah, 2000; Gao et al., 2012). Previous studies implied that e[CO2] can promote the exudation of O2 and organic carbon from plant roots (Phillips et al., 2011). Therefore, whether the floating aquatic plants, under the elevated atmospheric CO2 concentration, wield a positive or negative impact on microbial transformation of N, e.g. nitrification or denitrification, in eutrophic waters is worthy to be further discussed. To fill up these research gaps, we have used the N-15 stable isotope tracing method in this paper to study the effect of e[CO2] on the microbial transformation of N, with an emphasis on nitrification and/or denitrification, in eutrophic waters with or without the growth of Eichhornia crassipes. Based on the literature review, we hypothesize that: (1) Elevated CO2 may stimulate algal propagation in eutrophic waters, change the physical and chemical environment of waters, e.g. pH, dissolved oxygen and inorganic carbon, and lead to resource competition between autotrophic nitrifying bacteria and algae for inorganic carbon, O2, etc. By contrast, algal metabolism can provide more organic carbon for heterotrophic denitrifying bacteria, which may eventually inhibit nitrification but promote denitrification. (2) Elevated CO2 may promote the release of N2O from eutrophic waters without growth of aquatic plants and aggravate the greenhouse effect. (3) With growth of aquatic plants, elevated CO2 may promote the assimilation of N by the plants and the release of O2 by the aquatic plants, change the water environment, and eventually promote nitrification in eutrophic waters but reduce the release of N2O. This study will clarify how the nitrogen cycle in eutrophic waters would be mediated by elevated CO2, in the direction to contribute as a valuable supplementary material for scientists to further investigate the impact of the elevated atmospheric CO2. 2. Material and methods 2.1. Preparation of eutrophic water and aquatic plants Seedlings of Eichhornia crassipes were cultivated in a eutrophic pond (32° 02′ 21“ N, 118° 52’ 37” E) located in Jiangsu Academy of Agricultural Sciences for 30 days before the experiment. Plants of similar sizes and biomass were selected for the microcosm experiment.

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Eutrophic water was collected from the cultivating eutrophic pond to be used in the experiment. Subsequently, 5.175 g of 15NH4Cl (30.15%, Shanghai Engineering Research Center of Stable Isotope, China) and 0.052 g of KH2PO4 were added to the collected water to achieve the desired N concentrations (~15 mg L−1). The physical and chemical properties of the experimental eutrophic water were shown in Table 1.

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concentration in the chamber was adjusted using 0.02 mol ·L−1 Na2CO3 solution and 0.1 mol ·L−1 NaOH solution. Environmental conditions within the device were as follows: temperature of 26–27 °C, humidity of 80–85%, illumination with LED incandescent lamp (14 W, 8:00 am-20:00 pm, 12 h ·d−1). The second experiment started in October 2017, conducting the same process as the first experiment without growth of plants.

2.2. Experimental device 2.4. Sampling and measurement of water and gas samples The device used in the experiment was a plexiglass chamber with the dimensions of 1.0 m × 0.4 m × 0.8 m (Fig. 1). In the chamber, four 5.3-L cylindrical plexiglass hydroponic tanks (3) were placed for loading of experimental eutrophic water and growth of Eichhornia crassipes. The chamber was externally connected to a steel cylinder of high-purity air (79% N2, 21% O2, 2 μmol﹒mol−1 CO2) (14) in order to replace the air and reduce the background concentration of CO2 in the chamber. Two storage bags (11) were externally connected to the top of the chamber, for HCl and Na2CO3 solutions respectively. Elevated atmospheric CO2 was produced by the reaction of HCl and Na2CO3 solutions in the reaction tank (4) within the chamber. The CO2 concentration in the chamber was measured in real time using a CO2 detector (17) to ensure a constant CO2 concentration within the chamber. Four silicone hoses (9) with inner diameter of 0.2 cm, were connected to the four hydroponic tanks and immersed in the water, with the parts beyond the chamber being clamped by pinchcock to avoid air leakage. Water samples in the tank were collected by an injector connected to the silicone hose. The front and back of the chamber body were inlaid with two rubber plugs (SHIMADZU, 201–35,584). Gas samples were collected using an air-tight glass syringe. A three-way valve was installed between the glass syringe and the needle. Before gas sampling, fans installed on the inner wall of the device were turned on for 10 min to mix the gas within the chamber. 2.3. Experimental design The first experiment consisted of four treatments with four replicates for each treatment, including normal or elevated CO2 concentrations, and with or without growth of the floating aquatic plant, Eichhornia crassipes. Details of the treatments were shown in Table 2. The second experiment consisted of two treatments including normal or elevated CO2 concentrations, with four replicates for each treatment. No floating aquatic plant was grown in the second experiment. The first experiment started in September 2017 in a plant cultivation room. Each hydroponic tank in the chamber was filled with 5 L of the prepared eutrophic water containing 15N tracer, and three Eichhornia crassipes seedlings of similar sizes (total fresh biomass of 140 ± 2 g) were placed in each tank. Thereafter, the sample inlets for water sample collection were closed. Fans within the chamber and the steel cylinder containing high-purity air were turned on to minimize the background concentration of CO2 within the chamber. All the valves connected to the chamber were closed when the CO2 concentration within the chamber fell lower than 50 μmol ·mol−1. Subsequently, 500 mL of HCl solution (0.02 mol · L−1) was added into the reaction tank using an infusion hose while 150 mL of Na2CO3 solution (0.02 mol · L−1) was dripped into the reaction tank slowly to produce CO2 through the reaction of HCl and Na2CO3. After the initial concentration of CO2 reached 400 ± 20 μmol ·mol−1 or 800 ± 50 μmol ·mol−1 in the corresponding treatments, the fans were turned off. During the experiment, the CO2

2.4.1. Sampling The first experiment lasted for 21 days. Water samples (100 mL) were collected from each hydroponic tank on day 0, 2, 4, 6, 8, 14 and 21. Concentrations of NH4+, NO3− and NO2− were determined immediately. On day 0, 7, 14 and 21, an additional water sample of 550 mL was collected from each hydroponic tank for extraction of the total genomic DNA of microorganisms in the water. On day 1, 2, 3, 4, 5, 6, 7, 8, 14 and 21, 15 mL of gas samples were collected from the headspace of each chamber and injected into vacuum septum-capped glass vials (Labco Exetainer, Labco Limited, UK) for the immediate assessment of concentrations. At the end of the experiment, plants were collected with their leaves and roots kept separately. Part of fresh roots were sampled for DNA extraction of root-attached microorganisms, leaves and remaining roots were separately dried to measure the biomass of plants and the abundance of 15N in leaves and roots. The algae in the water was centrifuged followed by freeze-drying while the algae attached to the wall of hydroponic tanks was collected and freeze-dried. The combined samples were weighted as the biomass of algae and used to determine the abundance of 15N in algae. A portion of each water samples was filtered using a 0.45 μm filtration membrane, and 80 mL filtered water was mixed with two drops of saturated HgCl2 solution and stored at −20 °C to determine the concentrations of 15NH4+, 15NO3− and 15 NO2−. The second experiment lasted for 14 days, and water samples (100 mL) for determining the NH4+, NO3− and NO2− concentrations and air samples (15 mL) for N2O concentration were collected on day 0, 3, 5, 7, 9 and 14. The methods of sampling are the same as the first experiment. 2.4.2. Determination of chemical indexes Total nitrogen (TN) in the water samples was digested using potassium persulfate and determined through ultraviolet spectrophotometry (Lu, 2000). Concentration of NH4+ was determined using the Nessler's reagent method (SEPA, 2002). Concentration of NO3− was determined using double-band ultraviolet spectrophotometry (Lu, 2000). Concentration of NO2− was determined by N-(1-naphthyl)-ethylenediamine spectrophotometry (SEPA, 2002). Concentration of N2O was determined by gas chromatograph GC-2010 Plus (SHIMADZU, Japan). 15N isotopic abundance of 15NH4+, 15NO3−, 15NO2− and 15N2O was determined using a MAT253 stable isotope ratio mass spectrometer (Cao et al., 2013). 2.4.3. DNA extraction and fluorescent quantitative polymerase chain reaction (qPCR) analysis The 550 mL of water sample was filtered using a 0.22 μm filtermembrane. DNA of the microorganisms presented in the filtermembrane was extracted using DNeasy Power Water Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The 10 g of fresh fibrous roots evenly sampled from the whole roots were

Table 1 Initial physical and chemical properties of eutrophic water. Indexes

−1 NH+ 4 -N mg·L

−1 NO− 2 -N mg·L

−1 NO− 3 -N mg·L

TP mg·L−1

pH

Chlorophyll a mg·m−3)

The first experiment The second experiment

15.18 14.29

0.18 0.33

0.91 2.35

1.82 1.73

7.65 7.66

106.66 85.39

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Fig. 1. Experimental device. 1. Chamber, 2. Sample Inlet, 3. Hydroponic Tank, 4. Reaction Tank, 5. Fan, 6. Condenser, 7. Outlet of Condenser Pipe, 8. Inlet of Condenser Pipe, 9. Silicone Hose (inner diameter of 0.2 cm), 10. Discharge Outlet, 11. Storage Bag of CO2 Reaction Solution, 12. Speed Control Valve, 13. Conduit Outlet, 14. Steel Cylinder of High-purity Air, 15. Reducing Valve, 16. Thermometer and Hygrometer, 17. CO2 detector, 18. Silicone Hose (inner diameter of 0.8 cm), 19. Hollow Stud, 20. Light supplement unit, 21. Pinchcock, 22. Air Inlet, 23. Platform, 24. Rubber Plug.

transferred into 200 mL of sterile water. Bacteria attached to roots were detached by ultrosonication for 10 min (Shumei KQ-500E ultrasonic bath，Kunshan ultrosonic instrument), followed by vigorous shaking for 30 min (18.3 Hz, Thermomixer Eppendorf) (Gao et al., 2014), and filtered through a 5 μm sterile filter to remove the impurities. The resultant filtrates were filtered through 0.22 μm filter-membrane and DNA of root-attached microorganisms presented in the filter-membrane was extracted using the same kit and method as the water samples. Bacterial 16S rRNA gene, ammonia-oxidizing bacteria (amoA-AOB) and bacterial denitrifying functional genes (nirK, nosZ) were quantified using ABI 7500 real-time system (Life Technology, USA). Quantification was repeated for 3 times. Amplification was performed in 20 μL reaction mixtures using SYBR® Premix Ex Taq™ (Tli RNaseH Plus) qPCR kit (Takara bio, Dalian, China) as described. Details of primer and thermal profiles are listed in Table S1. 2.5. Statistical analyses and calculation The date used for statistical analysis fitted normal distribution or fitted normal distribution after log10 transformation. Multivariate analysis of variance (MANOVA) for repeated measuring was conducted to examine the effects of treatments over time on changes of N-related physicochemical indexes (NH4+, NO3−, NO2−, N2O,15NH4+, 15NO3−, 15 NO2−, 15N-N2O) and functional gene (16S rRNA, amoA, nirK, nosZ). One-way ANOVA was carried out to compare the effects of treatments on the amount 15N in water, N2O and unrecovered N. Independentsample t-test was used to compare the effects of treatments on the amount 15N in leaf, roots and algae. These above statistical analyses were carried out by SPSS 20.0. Redundancy analysis (RDA) of environmental factors and functional genes was constructed by Canoco 4.5.

Other graphs and the rising slope of line obtained by linear regression were constructed using SigmaPlot 12.5. 15 N-related indexes were calculated as below: 15−C ¼ ð15AP−NAPÞ  C n

ð1Þ

where 15-C is the 15NH4+, 15NO3−, 15NO2− concentration (mg · L−1) or 15 N-N2O concentration (mg · m−3); 15AP is the corresponding 15 NH4+, 15NO3−, 15NO2− atom percentage in the water (%) or 15N-N2O atom percentage in the gas (%); NAP is the 15N natural abundance atom percentage (0.3663%); C is corresponding NH4+, NO3−, NO2− concentration (mg · L−1) or N2O concentration (mg · m−3); n is the number of N atoms. M ¼ C V

ð2Þ

where M is the 15N amount in water (mg) or 15N-N2O amount in gas; C is the combined concentration of 15NH4+, 15NO3− and 15NO2− in water (mg · L−1) or 15N-N2O concentration in gas; V is the volume of water used to calculate the 15N amount in water or is the chamber volume water volume for calculating 15N-N2O amount in gas. 15−M ¼ ð15AP−NAPÞ m

ð3Þ

where 15-M is 15N amount in leaves, roots or algae (mg); 15AP is 15N atom percentage in leaves, roots or algae (%); NAP is the 15N natural abundance atom percentage (0.3663%); m is the dry weight of leaves, roots or algae (mg). 15

15

Unrecovered N ðmgÞ ¼ Total Added N− 15 N in Water Sample − 15 N in Leaves; Roots and Algae− 15 N−N2 O: ð4Þ

Table 2 Factorial experimental design. Treatment

Abbreviation

15

N Tracer in the water

−1

a[CO2] 400 μmol·mol CO2 800 μmol·mol−1 CO2 e[CO2] −1 400 μmol·mol CO2 + Eichhornia crassipes a[CO2] + plant 800 μmol·mol−1 CO2 + Eichhornia crassipes e[CO2] + plant

C(NH+ 4 ) = 15.18 mg·L−1

15

  15 N Recovery ð%Þ ¼ amount15 N in Sample=Total N added to water 100:

ð5Þ

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N2O emission rate was calculated using the equation modified from Wang et al. (2018): F ¼ ðdc=dtÞ  ðM=VÞ ð273=273 þ TÞ ½ðVc −Vw Þ=Aw   1000

ð6Þ

where F is N2O emission rate (μg N2O-N m−2 h−1); dc/dt is the slope of the linear regression for the N2O concentration gradient within the initial eight days of the experiment (μmol mol−1 h−1); M is the molar mass of N (14.0 g mol−1);V is the N2O molar volume (L mol−1) under standard conditions; T is the air temperature in the chamber (°C); Vc is the volume of the chamber (m3), Vw is the volume of water (m3); Aw is the area of water (m2). 3. Results 3.1. Effect of e[CO2] on N-transformation in eutrophic water Concentrations of NH4+ and 15NH4+ constantly decreased during the experiment (Figs. 2 and 3) and transformed into NO2− and 15NO2−, NO3− and 15NO3− as well as N2O and 15N-N2O due to assimilation, nitrification, denitrification and coupled nitrification-denitrification in the eutrophic water. Greater levels of NH4+ and 15NH4+ (concentrations were decreased by 32.12 ± 6.41% and 42.96 ± 2.50% respectively) in a[CO2] treatment were transformed to other forms of N, as compared to that in e[CO2] treatment (concentrations were decreased by 20.46 ± 3.86%

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and 35.89 ± 1.96% respectively) (p b 0.05, p b 0.05). Moreover, at the end of the experiment, NO3− concentration in e[CO2] treatment was 65.01% of that in a[CO2] treatment (p b 0.05), and 15NO3− concentration in e[CO2] treatment was 13.86% of that in a[CO2] treatment. This suggested that nitrification might have been inhibited by e[CO2], but implied enhanced transformation of 15NO3− to 15NO2− and 15N-N2O via denitrification under e[CO2] conditions at the same time. Concentrations of NO2− and 15NO2− increased rapidly during the first 4 days. The increases of NO2− and 15NO2− production were obviously lower in e [CO2] treatment (slope = 0.0919 mg·L−1·d−1, R2 = 0.86; slope = 0.0119 mg·L−1·d−1, R2 = 1) than a[CO2] treatment (slope = 0.3577 mg·L−1·d−1, R2 = 0.96; slope = 0.0503 mg·L−1·d−1, R2 = 1), indicating the lower generation rate of NO2− and 15NO2− in e[CO2] treatment enhanced transformation of NO2− to N2O and 15NO2− to 15NN2O during the early stage of the experiment. The emissions of N2O in e[CO2] treatment was about 9 times higher than that in a[CO2] treatment (p b 0.05), which was a direct demonstration of promoted N2O emission under e[CO2] conditions. As N2O may result from both 15NH4+ added to the water and NO3− background in water, the produced 15N-N2O would have been diluted by the enhanced emission of N2O in e[CO2] treatment, leading to lower concentration (0.014 ± 0.001 mg·m−3) of 15N-N2O in e[CO2] treatment than that (0.040 ± 0.006 mg·m−3) in a[CO2] treatment. During the initial four days, the rising slope (0.0173 mg·m−3·d−1, R2 = 0.94) of N2O was about 12 times greater than that (0.0014 mg·m−3·d−1, R2 = 1) of 15N-

− − Fig. 2. Concentration variation of NH+ 4 (a), NO2 (b), NO3 (c) in water, and total N2O generation (d) over time. Data was shown as mean value ±standard deviation from four replicates.

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15 15 15 Fig. 3. Concentration variation of 15NH+ NO− NO− 4 (a), 2 (b), 3 (c) in water and N-N2O generation (d) over time. Data was shown as mean value ±standard deviation from four replicates.

N2O in a[CO2] treatment; while the rising slope (0.2065 mg·m−3·d−1, R2 = 0.87) of N2O was about 295 times greater than that (slope = 0.0007 mg·m−3·d−1, R2 = 1) of 15N-N2O in e[CO2] treatment. Although the generation rate of N2O and 15N-N2O slowed down during the later stage of the experiment, they continued displaying the same changing pattern as observed during the earlier stage. All of

these implied that enhanced emission of N2O under e[CO2] conditions might have been mainly due to NO3− background in eutrophic water. In second experimental, we observed elevated reduction of NO3− concentrations when the initial NO3− concentration was higher in e [CO2] treatment, as compared to that in the first experiment (p b

− 15 15 Fig. 4. Effect of elevated atmospheric CO2 on the transformation of N in eutrophic waters with different initial concentrations of NO− NO− N3 . NO3 reduction (a), 3 variation (b), N2O and −1 N2O accumulation (c) in response to elevated atmospheric CO2. The first experiment lasted 21 days with NO− concentration of 0.91 mg·L and the second experiment lasted 14 days with 3 −1 NO− . Data was shown as mean value ±standard deviation from four replicates. 3 concentration of 2.35 mg·L

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0.05). On the contrary, 15NO3− concentration derived from 15NH4+ with higher initial NO3− concentration was less than that with lower initial NO3− concentration (p b 0.05) (Fig. 4). Although the initial concentration of NO3− in the second experiment was 2.58 times of that in the first experiment, N2O emission in the second experiment was 9.22 times of that in the first experiment under e[CO2] conditions (p b 0.05), it was notable that the productions of 15N-N2O were almost similar between the two experiments. This further confirmed that N2O was mainly derived from the initial NO3− background in eutrophic water. The higher the initial NO3− concentration in the background, the more pronounced the promotion effect would be. 3.2. Effect of Eichhornia crassipes on N-transformation in eutrophic water under e[CO2] conditions 15

NH4+

In the presence of Eichhornia crassipes, the concentration of decreased dramatically (Figs. 5 and 6) due to plant assimilation and microbial biotransformation which had occurred in the water and rhizosphere. In e[CO2] + plant treatment, the concentrations of NH4+ and 15 NH4+ were decreased by 46.53 ± 3.36% and 76.63 ± 2.86% respectively. The drop were 36.79% and 14.58% higher than that in a[CO2] + plant treatment (p b 0.05, p b 0.05). We observed greater accumulation of NO2− in e[CO2] + plant treatment during the earlier stage of the experiment (from D-0 to D-2), and greater accumulation of NO3− and 15NO3− in

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e[CO2] + plant treatment than that in a[CO2] + plant treatment during the first 4 days of the experiment. The differences in concentrations of different N forms between e[CO2] + plant and a[CO2] + plant treatments indicated that nitrification in eutrophic water might have been slightly promoted by the presence of the plant. During the later stage of the experiment, NO 3− concentration remained almost unchanged in e [CO 2 ] + plant treatment, while greater reduction of NO 3− and 15 NO 3− was occurred in a[CO 2 ] + plant treatment. In addition, a [CO 2 ] + plant treatment generated higher levels of N 2 O and 15 NN2O (2.01 ± 0.09, 0.30 ± 0.02, mg·m−3) than e[CO2] + plant treatment (1.36 ± 0.18,0.26 ± 0.03, mg·m−3) (p b 0.05, p b 0.05). These indicated that plant growth under e[CO 2 ] conditions may reduce N2O generation in eutrophic water. 3.3. Effect of e[CO2] on the fate of 15N in eutrophic water with or without aquatic plants In e[CO2] treatment, the proportion of the remaining 15N in water and unrecovered 15N were 64.19% and 25.42% respectively, which was not much different from that in a[CO2] treatment (66.01%, 28.30%). However, there was a significant difference with regards to the 15N in algae and 15N-N2O between a[CO2] treatment and e[CO2] treatment (p b 0.05). In e[CO2] treatment, algae contained 10.37% of the total 15N added to the water, about twice as much as that (5.63%) in a[CO2]

− − Fig. 5. Concentration variation of NH+ 4 (a), NO2 (b), NO3 (c) in water, and total N2O generation (d) in the Presence of Eichhornia Crassipes. Data was shown as mean value ±standard deviation from four replicates.

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15 15 15 Fig. 6. Concentration variation of 15NH+ NO− NO− N-N2O generation (d) in the Presence of Eichhornia Crassipes. Data was shown as mean value ± 4 (a), 2 (b), 3 (c) in water, and total standard deviation from four replicates.

treatment, while 15N-N2O contained about 0.02% of the total 15N, which was significantly lower than that (0.06%) in a[CO2] treatment (Fig. 7). In the presence of plants, 15NH4+ added to water was mainly assimilated by the plants and metabolized by microorganisms, but algae were

almost non-existent due to the growth of the plants. The proportion of N remaining in water accounted for about 1/3 of the total 15N added to the water. In e[CO2] + plant treatment, the proportions of the 15N remaining in water (35.66%), assimilated by the plant roots (11.65%) and 15

Fig. 7. Fate of 15N. Data was shown as mean value ±standard deviation from four replicates. Bars marked with different letters were significantly different (p b 0.05) in different treatments from each part of 15N recovered.

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unrecovered (21.10%) were not significantly different from these (37.39%, 10.82%, 28.56%) in a[CO2] + plant treatment, but the proportions of the 15N assimilated by the plant leaves (31.22%) and generated 15 N-N2O (0.37%) were significantly different as compared with these (22.81%, 0.42%) in a[CO2] + plant treatment (p b 0.05).

3.4. Effect of e[CO2] on functional genes related to N-transformation The abundance of 16S rRNA gene and amoA gene of free-living bacteria in eutrophic water without the plant had decreased during the experiment, with no significant difference between a[CO2] and e [CO2] treatments. On the contrary, the abundance of denitrificationrelated nirK and nosZ genes increased during incubation, and there was a significant difference between a[CO2] and e[CO2] treatments (p b 0.05) during the later stage of the experiment, with nirK and nosZ gene abundance in e[CO 2 ] treatment at 178.93% and 58.20% higher than that in a[CO2] treatment respectively. This implied that elevated CO 2 may have promoted the propagation of denitrifying bacteria (Fig. 8). In the presence of the plants, more bacteria were attached to the roots of the plants, and the abundance of their functional genes was 2–4 orders of magnitude different from that in the treatment without plants. For root-attached bacteria, the abundance of 16S rRNA gene, amoA, nirK and nosZ gene had no significant difference between a[CO 2 ] + plant and e[CO 2] + plant treatments. For freeliving bacteria in the water, the abundance of each functional gene was significantly lower with the presence of plants than that in the treatment without plants after 14 days of the experiment (p b 0.05). The abundance of each functional gene in e[CO2] + plant treatment was lower than that in a[CO2] + plant treatment on the 14-day (p b 0.05). Since then, the differences in abundance of all functional genes between a[CO2] + plant and e[CO2] + plant treatment were gradually diminished.

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3.5. Relationships between functional gene and environmental variables Redundancy analysis (RDA) of environmental factors and functional genes with Monte Carlo permutation (Fig. 9) showed that 74.9% of the total variances of all functional genes could have been accounted for by all canonical axes, with the first axis accounting for 71.73% of the total variances (Table S2). The concentration of NH4+ showed significant positive correlation with bacterial 16S rRNA, bacterial amoA and amoA/ nirK (p b 0.01), and also showed positive correlation with nirK gene and nosZ gene, which indicated that it regulated nitrification and coupled nitrification-denitrification. The concentration of NO3− showed significant negative correlation with bacterial 16S rRNA, bacterial amoA, nirK, nosZ and amoA/nirK (p b 0.01), and significant positive correlation with nirK/nosZ and N2O (p b 0.01), which indicated that the reduction of NO3− concentration was closely related to the changes of all functional genes abundance associated with N-transformation, and that NO3− was involved in the regulation of NO reduction, N2 production and N2O generation. The concentration of N2O was negatively correlated with all functional genes, but positively correlated with nirK/nosZ and amoA/nosZ (a greater extent of correlation with nirK/nosZ), and negatively correlated with amoA/nirK, which indicated that the generation of N2O was mainly due to both nitrification and denitrification but denitrification might have played a major role. The correlation between pH and nirK as well as nosZ was much more significant than that between pH and bacterial amoA gene abundance, which suggested that pH might have a greater effect on denitrification than nitrification in this experiment. 4. Discussion The results implied that e[CO2] could slightly inhibit nitrification but promote denitrification and N2O emissions in eutrophic water without the growth of plants, however, with the growth of plant, e[CO2] could slightly promote nitrification in eutrophic water and alleviate the

Fig. 8. The abundance variation of 16S rRNA gene (a), amoA gene(b), nirK gene(c) and nosZ gene(d) in eutrophic water and attached to roots over time. The unit of copys g−1 refers to dry weight. Data was shown as mean value ±standard deviation from four replicates. Bars marked with different letters were significantly different (p b 0.05) in different treatments.

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Fig. 9. RDA of functional genes and environmental factors in different treatments. All functional genes refer to bacteria in eutrophic water. Note: N in the figure represents (14+15) N.

negative effects of e[CO2] on N2O emission. Photosynthesis by algae in eutrophic water during day-light releases oxygen and removes CO2 from the water column, resulting in higher pH (pH was 7.6–8.8 (Fig. S1) in this experiment) and higher buffer capacity for the changes in pH caused by e[CO2]. The pH between 7 and 9 would favor the growth of nitrifying bacteria in water (Jiménez et al., 2011; Le et al., 2018). Therefore, pH levels might not be the main environmental factor influencing nitrification in eutrophic water. The reason could have been that algae and nitrifying bacteria may compete directly for inorganic carbon and other resources (Risgaard-Petersen et al., 2004; Choi et al., 2010), because algae are generally more capable of enriching HCO3– (Kaplan and Reinhold, 1999) than bacteria, while most of the nitrifying bacteria in water are autotrophic and utilize CO2 or HCO3– as carbon sources. In addition, as O2 is required as an electron acceptor for nitration, competition for O2 consumption among nitrifying bacteria, algae and its attached heterotrophic bacteria, is especially pronounced during night time, when the respiratory processes of algae and heterotrophic bacteria consume the produced O2 and release CO2 into water. In our experiment, the continuous decrease of DIC content in eutrophic water by 7.75%–12.23% was observed during the initial 7-day incubation (Fig.S2). This was accompanied by an increase in algal density but no significant changes in AOB abundance, hence indicating that nitrifying bacteria were at a disadvantage in competing for inorganic carbon. In addition, the direct competition between microalgae and AOB for O2 in darkness, and AOB reached b20% of its potential activity (Risgaard-Petersen et al., 2004). In our study, when the concentration of CO2 was elevated, algae density in e[CO2] treatment increased by 25.86 ± 10.49% but bacterial amoA gene abundance showed no significant difference between a[CO2] and e[CO2] treatments. However, analysis of the N-15 isotope data demonstrated the slight inhibition of nitrification process, which implied that the activity of AOB might have been inhibited, and thus nitrification in water was slightly inhibited. Few studies have paid attention to the effect of e[CO2] on the denitrification process in eutrophic water, while studies on soil and liquid medium have shown that elevated CO2 can promote or inhibit denitrification for the changing of environment (Robinson and Conroy, 1998; Wan et al., 2016). Taking specificity of the eutrophic water into consideration, we must not neglect the impact of changes in pH and algae density caused by e[CO 2] in eutrophic water (Verspagen et al., 2014; Eshel and Singer, 2016). Some studies have shown an increase in denitrification rates when pH is increased to the range of 7–9 (Cuhel et al., 2010). The pH of our experimental eutrophic water was kept within the optimum pH range for the growth of denitrifying bacteria. RDA also showed that the correlation between pH and denitrification-functional genes (nirK, nosZ) was significantly positive. Therefore, pH of eutrophic water under e[CO2]

conditions may favor the denitrification process in eutrophic water. As most of denitrifying bacteria are heterotrophic microorganisms, they developed a mutually beneficial relationship with algae (Ramanan et al., 2016) that provided sufficient dissolved organic carbon (DOC) and favorable alternating aerobic-anaerobic microenvironments for denitrifying bacteria to propagate under the conditions of e[CO2 ] (Zhao et al., 2013; Ramanan et al., 2016). Ishida et al. (2008) suggested higher diatom abundance was accompanied by higher denitrification rates. Our results demonstrated that e [CO2] had increased algae biomass, and accompanied by greater enhanced the gene abundances of denitrification-related functional genes nirK and nosZ. Therefore, algae density may be another major factor that determines the potential of denitrification in eutrophic waters. In addition, NO3− could also be a major factor affecting denitrification. Previously published results showed that NO3− concentration was one of the main environmental factors affecting the abundance and diversity of denitrifying bacteria (Santoro et al., 2006), and constituting the functional structure of microbial community (Xu et al., 2014). RDA analysis in our study clearly showed that NO3− concentration was the main environmental factor affecting the abundance of microbial functional genes (Table S3), which was in significant negative correlation with nirK, nosZ abundance, but in significant positive correlation with nirK/nosZ and N2 O (p b 0.01). This was consistent with the results that NO3− concentration continually decreased under e[CO2] conditions but more N2O was generated and denitrification was promoted. These indicated the critical role of NO 3− in affecting the abundance of each denitrifying functional gene and in regulating the reduction of substrate NO to N2O as well as the transformation of N2O to N2. Hence, the enhanced denitrification potential in eutrophic water under e[CO 2] conditions were caused by the combination of factors such as pH, NO3− concentration and algae density. The regulating effect of aquatic plant Eichhornia crassipes on N transformation under e[CO2] condition may be related to the growth status of Eichhornia crassipes and its root microenvironment. The cultivation of Eichhornia crassipes would affect N transformation by assimilating or releasing the water nutrients during growth or senescence (Fig. 5-a), and by inhibiting the growth of algae (Jin et al., 2003), moreover, by decreasing DO (Ma et al., 2014), pH (Fig. S1) and dissolved organic carbon (Fig. S3) in the eutrophic water. In addition, the roots of Eichhornia crassipes are well-developed and they facilitate the attachment of microorganisms in water (Snooknah, 2000). Therefore, the abundance of bacteria and functional genes related to N-transformation decreased rapidly in the water while was highly attached to roots during the experiment. In general, the O2 released by the roots is quickly captured by the root-attached bacteria, and thus aerobic and anaerobic zones are formed near the roots. During the earlier stage of the experiment, Eichhornia crassipes grew well, e[CO2] promoted the root secretion of O2 and organic carbon that helped root-attached bacteria propagate and provided a better O2 microenvironment for nitrification. As the experiment progressed, nutrients in the water were depleted and Eichhornia crassipes grew worse than its initial state at the beginning under normal CO2 concentration conditions. Thus the O2 secreted by the roots might have been insufficient to meet the respiration needs of the root-attached bacteria under a[CO2] conditions, which resulted in a greater anaerobic microdomain (Hamersley and Howes, 2002). Such conditions were not conducive to the growth of nitrifying bacteria, but might have been beneficial to denitrification. By contrast, e[CO2] promoted the growth of Eichhornia crassipes, leading to better Eichhornia crassipes growth and greater O2 production by the plant roots, providing a more favorable water environment for nitrification. Furthermore, there was a competition for N between plants and microbes, and Eichhornia crassipes was well-known for its high nitrogen assimilation capacity (Kaye and Hart, 1997; Inselsbacher et al., 2010). Thus, less N substrates were left for microbial metabolism. Enhanced inputs of labile carbon under elevated CO2 via root exudation and

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295

Table 3 Comparison of N2O emission rates from various types of water bodies. Type of water body

River

Eutrophic river

Eutrophic lake Pond

Eutrophic water

N2O emission rate (μg N2O-N m−2 h−1) Mean

Minimum

Maximum

25.20 ± 10.08

–

–

54.13

−0.06

182.7

56.1

41.1

87.7

– –

1.33 1.87

45.08 40.8

5.00

−56.51

51.90

21.58 ± 8.75

2.91

427.58

154.9

1.30

1164.38

135 – 8.17

9.5 7.00 −0.99

372 19.73 32.03

3.42 ± 0.71

−0.33

10.34

2.39 35.34 26.15 24.75

–

–

trophic status (mg N L−1)

Notes

References

NO− 3 —N: 0.95–2.00 NH+ 4 —N: 0.03–3.45 NO− 3 —N: 0.02–4.00 + NH4 —N: 0.39–0.90 NO− 3 —N: 0.67–1.63 DIN: 2.2–50 NO− 3 —N: 0.74–2.08 TN: 6.7 NO− 3 —N: 1.6 NH+ 4 —N: 0–6.58 − NO3 —N: 0–3.22 NH+ 4 —N: 0.1–32.1 NO− 3 —N: 0.01–17.9 NO− 3 —N: 0.05–3.50 TN: 2.56–5.60 TN: 0.77–3.52 NH+ 4 —N: 0.95–4.91 NO− 3 —N: 0.03–0.39

Mississippi River

Turner et al., 2016

The Guadalete River Estuary

Burgos et al., 2015

A sewage-enriched river in the Taihu region a subtropical Brisbane River estuary, Australia The Changjiang river

Musenze et al., 2014 Yan et al., 2012

Meiliang Bay, in the north part of Taihu Lake

Wang et al., 2006

The Pearl River Estuary

Lin et al., 2016

Nitrogen-enriched rivers, Chao Lake basin, China

Yang and Lei, 2018

San Joaquin River, California Reed-dominated zones in Baiyangdian Lake,China Taihu Lake

Hinshaw and Dahlgren, 2013 Yang et al., 2012 Wang et al., 2009

Aquaculture pond

Yang et al., 2015

a[CO2] a[CO2] + plant e[CO2] e[CO2] + plant

This study

NH+ 4 —N: 15.18 NO− 3 —N: 0.91

increased fine root turnover may increase the microbial N demand, resulting in increased competition between plants and microorganisms for available N (Müller et al., 2009). In our study, in the presence of Eichhornia crassipes, the atom percentage of 15N in the plant leaves in e[CO2] treatment was 4.2 ± 0.2%, significantly higher than that (3.6 ± 0.2%) in a[CO2] treatment (p = 0.011). By contrast, N2O generation was significantly lower in e[CO2] treatment than that in a[CO2] treatment, which indicated that e[CO2] promoted the assimilation of N through plants, thus reducing N availability for microbes and lowering the potential for N2O production. Elevated atmospheric CO2 would promote N2O emission in eutrophic water, which was related to the trophic state of water. When the initial concentration of NO3− in eutrophic water was increased from 0.91 to 2.31 mg L−1, N2O emission rate under e[CO2] conditions (152.08 μg N2O-N m−2 h−1) had increased by a factor of 13. To the best of our knowledge, it is the first time that relevant studies have considered the interaction between CO2 and N2O emissions from eutrophic waterbodies, although there were some previous studies concerning CO2 and N2O emissions or their interaction in lake or river (Wang et al., 2009; Yang and Lei, 2018). The related researches mostly occurred in soil. Applying high rates of N fertilizer in Lolium perenne sward (Baggs and Blum, 2004), wheat field (Lam et al., 2011), semi-arid grasslands (Dijkstra et al., 2010), tropical flooded rice soil (Bhattacharyya et al., 2013) enhanced N2O emission under e[CO2] condition. It was attributed to the effects of elevated CO2 on soil moisture or root-derived available C or both (Dijkstra et al., 2012). Elevated CO2 increased soil moisture by improving the efficiency of plant water-use (Morgan et al., 2011), thus creating anaerobic conditions which were conductive to denitrification (Arnone Iii and Bohlen, 1998). Moreover, it enhanced root-derived available C, which acted as an energy source for denitrification (Moser et al., 2018). In eutrophic water, similar mechanisms have been found to demonstrate that e[CO 2] promoted algal growth and decomposition through its life cycle and provided abundant labile organic carbon as energy for denitrification. Moreover, algal diurnal photosynthesis and respiration dynamics created alternating aerobic-anaerobic microenvironments which are favorable for denitrification. Eutrophic rivers generally had higher N2O emission rates as compared to normal rivers with lower available N (Table 3). Several earlier studies suggested that N2O emissions were strongly correlated with inorganic nitrogen (NH4+ + NO3−) concentrations, but perhaps not significantly

Xia et al., 2013

correlated with NO3− concentration (Wang et al., 2006; Wang et al., 2009). This was consistent with our results that similar N2O emission rates were generated between different initial concentrations of NO3− in eutrophic water (same DIN concentration) under normal CO2 concentration. Under a[CO2] conditions, N2O emission rates in our study were 2.39 and 35.34 μg N2O-N m−2 h−1 without and with the growth of aquatic plants respectively. These rates were comparable with Wang's, Lin's and Yang's research (Table 3), but lower than the emission rates observed in other eutrophic rivers. The low estimated N2O emission rates may be attributed to the depletion of NH4+ and NO3− in the experimental water. In natural eutrophic waters, there will be continuous excessive N input that may contribute to higher N2O emission rates, especially under e[CO2] conditions in the future. Hence, taking the increasing eutrophication of rivers and lakes in many developing countries into account, the elevation of atmospheric CO2 concentrations would boost N2O emissions from eutrophic rivers, resulting in aggravation of greenhouse effect and forming a vicious cycle. However, vegetative restoration of aquatic ecosystems would attenuate the negative effects of e [CO2] on global warming. 5. Conclusion The e[CO2] has contrasting impact on nitrification and denitrification in eutrophic water with or without the growth of the floating aquatic plant, Eichhornia crassipes. e[CO2] promotes algal growth, resulting in the competition between algae and nitrifying bacteria for dissolved inorganic carbon, and inhibits nitrification slightly, but promotes denitrification and N2O emissions in the absence of plants. Vegetative restoration by floating aquatic plant cultivation could attenuate the negative effects of e[CO2] on N2O emissions by promoting nitrification and reducing denitrification in eutrophic water under e[CO2] conditions. The pH, DIC, algae density and DO, which could be changed by elevated atmospheric CO2, are the essential environmental factors influencing N transformation in eutrophic water, and the N2O emissions from eutrophic water under elevated CO2 and the regulation of plants deserve more attention. Acknowledgements The authors are grateful for the financial support from the State Natural Science Foundation of China (No. 41571458, 31600419,

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