Copper oxide nanoparticles inhibited denitrifying enzymes and electron transport system activities to influence soil denitrification and N2O emission

Journal Pre-proof Copper oxide nanoparticles inhibited denitrifying enzymes and electron transport system activities to influence soil denitrification and N2O emission Shuyuan Zhao, Xiaoxuan Su, Yiyu Wang, Xiangyu Yang, Mohan Bi, Qiang He, Yi Chen PII:

S0045-6535(19)32634-7

DOI:

https://doi.org/10.1016/j.chemosphere.2019.125394

Reference:

CHEM 125394

To appear in:

ECSN

Received Date: 3 July 2019 Revised Date:

9 November 2019

Accepted Date: 16 November 2019

Please cite this article as: Zhao, S., Su, X., Wang, Y., Yang, X., Bi, M., He, Q., Chen, Y., Copper oxide nanoparticles inhibited denitrifying enzymes and electron transport system activities to influence soil denitrification and N2O emission, Chemosphere (2019), doi: https://doi.org/10.1016/ j.chemosphere.2019.125394. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Graphical abstract

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Copper oxide nanoparticles inhibited denitrifying enzymes

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and electron transport system activities to influence soil

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denitrification and N2O emission

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Shuyuan Zhao a, b, Xiaoxuan Su a, b, Yiyu Wang a, b, Xiangyu Yang a, b, Mohan Bi a, b,

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Qiang He a, b, Yi Chen a, b *

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a

Key Laboratory of the Three Gorges Reservoir Region's Eco-Environment, Ministry of

Education, Chongqing University, Chongqing 400045, PR China. b

College of Environment and Ecology, Chongqing University, Chongqing 400045,

China.

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*

Corresponding author address: 174 Shazhengjie Street,

Shapingba District,

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Chongqing 400044, P.R. China. Tel.: 86-23-65120750; fax: 86-23-65120750.

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E-mail address: [email protected] (Y. Chen).

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Abstract

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Nanopesticides are widely applied in modern agricultural systems to replace

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traditional pesticides, which inevitably leads to their accumulation in soils.

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Nanopesticides based on copper oxide nanoparticles (CuO NPs) may affect the soil

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nitrogen cycle, such as the denitrification process; however, the mechanism remains

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unclear. Here, acute exposure experiments for 60 h were conducted to explore the

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effects of CuO NPs (10, 100, 500 mg kg-1) on denitrification. In this study, Cu

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speciation, activities of denitrifying enzymes, electron transport system activity (ETSA),

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expression of denitrifying functional genes, composition of bacterial communities and

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reactive oxygen species (ROS) were determined. In all treatments, Cu ions was the

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dominant form and responsible for the toxicity of CuO NPs. The results indicated that

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CuO NPs treatments at 500 mg kg-1 remarkably inhibited denitrification, led to an

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11-fold increase in NO3− accumulation and N2O emission rates decrease by 10.2~24.1%.

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In the denitrification process, the activities of nitrate reductase and nitric oxide

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reductase reduced by 21.1~42.1% and 10.3~16.3%, respectively, which may be a reason

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for the negative effect of CuO NPs. In addition, ETSA was significantly inhibited with

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CuO NPs applications, which reflects the ability of denitrification to accept electrons.

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Denitrifying functional genes and bacterial communities composition were changed,

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thus further influencing the denitrification process. ROS analysis showed that there

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were no significant differences among NPs treatments. This research improves the

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understanding of CuO NPs impact on soil denitrification. Further attention should be

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paid to the nitrogen transformation in agricultural soils in the presence of

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nanopesticides.

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Keywords: Denitrification; Nanoparticles; Denitrifying enzyme activity; Nitrous oxide

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emission; Copper ions; Nanopesticides

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1. Introduction

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Nanopesticides based on copper oxide nanoparticles (CuO NPs) are applied widely

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in agricultural systems to replace traditional pesticides. Due to their unique antifungal

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and antimicrobial properties (Ray et al., 2015; Ingle and Rai, 2017; Keller et al., 2017),

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CuO NPs are used on fungicides, insecticides, herbicides, fertilizers, soil remediation

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solutions, and growth regulators to increase crop yields (Gogos et al., 2012; Zhu et al.,

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2012; Weitz et al., 2015; Hong et al., 2016; Du et al., 2017). CuO NPs have also been

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applied as electrical-nanosensors and bionanosensors for use in the identification of

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plant pathogens (Dubey and Mailapalli, 2016). Repeated applications of CuO NPs can,

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however, lead to elevated concentrations of these metal oxides in surface soils. In

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addition to direct application, the accumulation of CuO NPs in soils could indirectly be

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caused by their use in catalysts, coatings, and cosmetics (Blinova et al., 2010; Lee, 2010;

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Zhang et al., 2017). Previous reports have suggested that, once they have been released

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into the environment, CuO NPs may pose risks to human health and ecosystems (Colvin,

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2003; Andre et al., 2006) by causing cell death (Kumar et al., 2011), inhibited the

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removal of nitrogen and phosphorus (Zheng et al., 2017) and reduced microbial

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abundance (Xu et al., 2015). Therefore, increased attention should be paid to

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investigating the harm caused by CuO NPs.

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Currently, the extensive use of artificial nitrogen fertilizers introduces nitrogen to

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the soil and causes environmental problems such as greenhouse gas emissions and water

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eutrophication (Hu et al., 2018). Denitrification is a sequential bioreduction process that

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maintains the balance of environmental nitrogen by converting nitrate (NO3−) to nitrite

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(NO2−), nitric oxide (NO), nitrous oxide (N2O) and nitrogen gas (N2) (Zumft, 1997;

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Beaulieu et al., 2011). N2O is a key atmospheric greenhouse gas that contributes to

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global warming and the destruction of stratospheric ozone (Ravishankara et al., 2009).

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Studies have shown that the greenhouse effect has seriously jeopardized the

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environment and led global temperatures to rise by 0.74°C over the past 100 years.

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Because of their special properties, nanoparticles entry into the environments may affect

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the emission of N2O. Cu NPs have been recorded to influence the N2O production by

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decreased nitrite accumulation in the activated sludge process (Chen et al., 2012).

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Zheng et al. (2017) also showed that the response of nitrifier N2O production to Ag NPs

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exhibited low-dose stimulation and high-dose inhibition in aquatic environments.

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Although the effects of different nanoparticles on N2O emission in aquatic environments

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have been investigated, their effects on microbial denitrification process and N2O

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emission in soil are not fully understood.

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Denitrification is catalyzed by four key enzymes including nitrate reductase (NAR),

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nitrite reductase (NIR), nitric oxide reductase (NOR) and nitrous oxide reductase (NOS)

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(Ravishankara et al., 2009). Electrons are controlled by the electron transport system

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and consumed by enzymes during the denitrification process (Zumft, 1997; Wan et al.,

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2016). Denitrification relies on electron transport and consumption; thus, factors

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disturbing these processes affect denitrification performance. Previous studies have

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shown that increasing the concentration of CuO NPs changes soil-based bacterial

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communities and structures, which potentially affects the process of denitrification (Xu

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et al., 2015). Some studies have also shown that CuO NPs release copper ions (Cu2+)

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and produce reactive oxygen species (ROS) (Karlsson et al., 2008). ROS are a major

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cause of the oxidative stress (OS) response, which can lead to disorders of cellular

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oxidation and antioxidant systems, potentially influencing microbial activity. Therefore,

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whether CuO NPs have an effect on denitrification and its molecular mechanism are

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worth exploring.

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We hypothesized that CuO NPs disturb electron transport systems, denitrifying

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enzymes and denitrifying genes to impact soil denitrification and the emission of N2O.

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To test this hypothesis, we aimed to: (1) investigate the effects of CuO NPs on the

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emission rates of NO3−, NO2− and N2O emission rates; (2) explore whether the toxicity

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associated with CuO NPs is due to Cu2+ or the nanoparticles themselves; (3) reveal the

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effects of CuO NPs on electron transport system activity (ETSA) and enzyme activities

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during denitrification; (4) analyze changes in functional gene abundances and microbial

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communities during denitrification.

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2. Materials and methods

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2.1 Preparation of CuO NPs and collection of soil samples

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Spherical CuO NPs (purity > 99.9%) were purchased from Sigma-Aldrich (St.

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Louis, MO, USA). The average particle size was <50 nm and the specific surface area

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was 29 m2g-1. Surface soil samples (0~20 cm deep) were collected from a paddy field

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located at the Institute of Jiangxi Red Soil (28°15′ N, 116°55′ E), Chinese Academy of

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Sciences, China. This station is located in the typical agricultural area and red soil cover

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2.04 million square km2 in China which accounting for 30% of the country's arable land

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(Xu et al., 2003). To minimize spatial heterogeneity, five samples were collected at

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randomly chosen points and combined. The soil samples were air-dried at 20~25°C,

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passed through a 2 mm sieve. The physicochemical properties of the soil samples are

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presented in Table S1.

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2.2 Experimental design

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2.2.1 Experiment 1

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In this study, three concentrations (10, 100 and 500 mg kg-1) of CuO NPs in soil

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were used; soil samples containing no CuO NPs were used as controls. Three doses of

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CuO NPs were established in this study according to previous research (Heinlaan et al.,

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2008; Xu et al., 2015). To investigate the effects of CuO NPs on soil denitrification,

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microcosms consisting of 80 g dry soil and 56 mL Milli-Q water containing glucose

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(7.14 mg mL-1) and NO3−-N (0.16 mg mL−1) were created in 150 mL-serum bottles.

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CuO NPs were added to each bottle to achieve the target concentrations of 10, 100 and

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500 mg kg-1. Then, N2 was injected to maintain soil anaerobic conditions. All bottles

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were incubated in the dark at 25°C for 60 h in an anaerobic glove box. The

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concentrations of NO3−-N, NO2−-N, NH4+-N and N2O were detected at 0, 4, 8, 12, 24,

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36, 48 and 60 h. After this period, the N2O emission rates was calculated and different

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forms of Cu, ETSA, denitrifying enzyme activities, denitrifying functional gene

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abundances, bacterial community composition and production of ROS were measured.

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The schematic diagram of experimental design concept of this study was added to the

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Supplementary Materials (Figure S1).

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2.2.2 Experiment 2

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At the end of experiment 1, the different forms of Cu released from CuO NPs were

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determined. To clearly differentiate toxic effects of CuO NPs themselves and released

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Cu ions, we took fresh soil again for culture, and all treatment and cultural conditions

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were the same as experiment 1. Briefly, microcosms consisting of 80 g dry soil and 56

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mL Milli-Q water containing glucose (7.14 mg mL-1) and NO3−-N (0.16 mg mL−1).

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Subsequently, different concentrations of CuCl2 were added to the fresh soil to replace

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the determined concentrations of Cu ions. Then, N2 was injected to maintain soil

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anaerobic conditions. To investigate the effects of Cu ions on soil denitrification, we

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also conducted a 60 h experiment to determine the effects of Cu ions on NO3−-N and

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N2O emission rates in the denitrification process.

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2.3 Determination of denitrification intermediates and different forms of Cu released

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from CuO NPs

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The NO3−-N, NO2−-N and NH4+-N were detected following the method reported by

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(Miller et al., 2008). The detailed methods of extraction and measurement were

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described in our previous study (Hu et al., 2018). Briefly, 2 g dry soil was added 20 mL

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of 2 M KCL and incubated at 200 rpm for 30 min. The mixture was centrifuged for 10

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min and the supernatant was passed through a 0.45 µm filter. The resulting sample was

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analyzed using ion chromatography ICS-600 (Thermo Fisher Scientific, USA). The

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N2O sample was analyzed using gas chromatography (2010 plus, SHIMADZU Japan)

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fitted with an electron capture detector (Liu et al., 2017). The N2O emission rates were

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calculated according to a reported method (Kampschreur, 2008).

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Different forms of Cu were extracted from the soil using the single extraction

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method (Quevauviller, 1998). Air-dried soil samples (5 g) were added to 50 mL

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centrifuge tubes. Aqua regia was used to extract total Cu. The water-soluble forms of

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Cu were extracted with 25 mL of 0.01 M calcium chloride (CaCL2), while shaking the

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tubes at 200 rpm for 2 h (Xu et al., 2015). Two identical samples to extract

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exchangeable forms of Cu. The first sample was extracted using 25 mL of 0.05 M

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ethylenediaminetetraacetic acid (EDTA) for 1 h (200 rpm). The second sample was

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extracted with a mixture solution consist of 0.005 M diethylenetriaminepentaacetic acid

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(DTPA), 0.1 M triethanolamine (TEA) and 0.01 M CaCL2 for 2 h (200 rpm). (Lindsay.

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and Norvell, 1978; Quevauviller, 1998). The microbial available Cu was extracted by

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CHCL3 using the same procedure used for the fumigation-extraction method, with 1 M

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of NH4NO3 as the extractant (Khan et al., 2009). Microbial available metal = the metal

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extracted from fumigated soil − metal extracted from non-fumigated soil. The detailed

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method was described in previous study (Khan et al., 2009). All extractions were

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centrifuged for 10 min at 1200 rpm and the supernatants were passed through a 0.22 µm

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filter. The resulting samples were analyzed using an Inductively Coupled Plasma

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Chromatograph (iCAP 6300 Duo, Thermo, America).

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2.4 Determination of electron transport system activity

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The ETSA was measured according to previous studies (Broberg, 1985; Su et al.,

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2019). Briefly, 5 g of air-dried soils were washed twice with 10 mL 0.1 M phosphate

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buffered saline (PBS) and centrifuged (4000 g for 10 min, 4°C). Then 1 mL of

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2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) and 1 mg of

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nicotinamide adenine dinucleotide (NADH) were injected after soils were resuspended

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in 0.1 M PBS. All samples were incubated in darkness for 30 min and shaken (200 rpm)

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to oxidize INT to formazan (INF); then 1 mL formaldehyde (HCHO) was added to stop

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the reaction. Afterwards, the samples were centrifuged and supernatants discarded.

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Methanol (CH3OH; 5 mL) was added to extract the INF. The samples were centrifuged

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again and this time the supernatants were investigated using a UV-spectrophotometer at

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490 nm.

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2.5 Determination of denitrifying enzyme activity

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Four soil denitrifying enzymes were extracted and measured according to our

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previous study (Chen et al., 2019). Briefly, a reaction mixture was created to include 5

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mM sodium hyposulfite (Na2S2O4), 10 mM methyl viologen, 10 mM PBS and 1mM

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electron acceptor (NO3−, NO2−, NO or N2O). Next, 1 mL of enzyme solution was

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injected into 2 mL of the reaction mixture to start the reaction. The mixtures were

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incubated in darkness for 30 min under anaerobic conditions (28°C). The amount of

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NO2− production or reduction was used to represent NAR or NIR activity and the

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increase or decrease in N2O concentration was used to represent NOR or NOS activity.

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2.6 Denitrifying functional genes and microbial community structure

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Denitrifying genes (narG, nirK, nirS and nosZ) were investigated to determine

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how microbial mechanisms associated with CuO NPs affect denitrification (Ellen et al.,

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2006; Henry et al., 2006; Jr et al., 2014). At the end of the period of exposure to CuO

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NPs, 5 g soil samples were collected from each treatment to explore the presence of

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denitrification functional genes and microbial communities. Soil DNA was extracted

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using a Fast DNA SPIN KIT according to the manufacturer’s instructions (Yang et al.,

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2019). Real-time quantitative Polymerase Chain Reaction (RT-qPCR) was used to

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quantify the expression of denitrifying functional genes. The primers are listed in Table

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S2. Soil microbial communities were determined by high-throughput Illumina MiSeq

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sequencing of the 16S rRNA gene. The details are provided in previous studies

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(Caporaso, 2012; Wang et al., 2019).

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2.7 Determination of ROS

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Intracellular ROS were detected using a Cellular ROS Assay Kit (Jiancheng

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Technology Company, Nanjing, China), according to the manufacturer’s instructions. In

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detail, soil samples were washed with PBS (0.1 M, pH 7.4) three times to remove

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impurities. The precipitates were resuspended in the same PBS with 50 µM

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2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, Molecular Probes, Invitrogen)

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and then cultured at 28°C in darkness for 40 min. Following incubation, the supernatants

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were discarded by centrifugation in order to remove extracellular DCFH-DA. The

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samples were resuspended in nanoparticle solutions containing 10, 100 or 500 mg kg-1

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CuO NPs. Fluorescein dichloroflurescein (DCF) production was measured after 4.5 h

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using a fluorescein microplate reader (Multiskan Mk3, Thermo Fisher Scientific

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Instrument Co, Ltd, Shanghai, China) at 530 nm emission and 502 nm excitation. The

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calculation of ROS was: ROS (%) = F test / F control × 100 %.

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2.8 Statistical analysis

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All experimental parameters were tested in triplicate and the data were expressed

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as mean ± standard deviation. A one-way analysis of variance (ANOVA) was used to

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compare the effects of CuO NPs concentrations on the different forms of Cu, ETSA

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values, enzyme activities, abundances of denitrifying functional genes and bacterial

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community structures. Data analyses were conducted using SPSS software (version

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19.0). Probability values (P) of <0.05 were considered to be significant.

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3. Results and discussion

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3.1 Effect of CuO NPs on the process of soil denitrification

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To investigate the effects of CuO NPs on denitrification, the concentrations of

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NO3−, NO2−, and N2O were measured over the course of 60 h. As shown in Figure 1a,

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the final concentrations of NO3− ranged from 0.71 to 7.84 mg kg-1. Compared with the

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control, CuO NPs treatments significantly inhibited NO3− reduction and the final

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concentration of NO3− was increased 11-fold under Cu500 treatment (P<0.05). Figure

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1b indicates that the NO2− accumulation increased significantly (P<0.05) with

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increasing concentrations of CuO NPs. After 60 h, the NO2− accumulation in the Cu500

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treatment was 24-fold higher than that in the control, indicating that high concentrations

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of CuO NPs could lead to significant accumulation of NO2− in soil. Similar trends have

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also been observed in previous studies. Su et al. (2015) also demonstrated that the

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presence of CuO NPs leads to less efficient NO3− reduction and causes NO2−

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accumulation (Su et al., 2015).

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Figure 1c demonstrates that the application of CuO NPs resulted in the significant

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decrease in N2O emission rates. The N2O emission rates was reduced by 15.31, 10.18

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and 24.05% in Cu10, Cu100 and Cu500 treatments, respectively (P<0.05). Van den

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Heuveo et al. (2011) reported that N2O emission consists of two processes: production

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(NO to N2O) and consumption (N2O to N2); the study also denotes that the latter process

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may be more important (Van den Heuvel et al., 2011). Our previous studies have also

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documented that pollutants created by human activity could effect N2O production and

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consumption processes (Hu et al., 2018). We speculated that the inhibition of CuO NPs

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during N2O production was higher than during the consumption process, which may be

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the reason for the decreased N2O emission rates.

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3.2 Extraction of different forms Cu and their toxic effects

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The toxicity mechanisms of CuO NPs are complex and related to their

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physicochemical properties and soil composition (Gunawan et al., 2011). The

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application of CuO NPs to soil releases Cu ions, which can precipitate and complex

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with soil organic matter, Fe and Mn oxides and clay minerals to produce different forms

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of Cu (Misra et al., 2012). Therefore, we measured not only the total amount of Cu, but

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also its ion concentrations in water soluble form, exchangeable form, and

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microbial-available form. The extraction of water soluble Cu was carried out by CaCl2,

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which has a weaker binding force. EDTA and DTPA have strong chelation properties

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and were used to extract exchangeable form Cu ions. Table S3 indicates that the

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concentrations of released Cu ions in the three treatments (10, 100, 500 mg kg-1 CuO

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NPs) were 8.88, 83.79, 437.74 mg Cu kg-1 soil, respectively. The recovery rate ranged

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between 83.8~88.8%, which may be due to the inhomogeneity of CuO NPs in the soil.

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The level of Cu ions released under all treatments rose with increasing concentrations of

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CuO NPs. The water-soluble content of Cu was found to be very low, most likely due to

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the high content of organic matter in the soil. Cu ions rapidly combine with organic

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matter to form an exchangeable form of the metal. The content of exchangeable forms

278

of Cu was significantly higher than that of the water soluble forms and this is consistent

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with a previous study (Gunawan et al., 2011). The microbially available form of Cu was

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rare, may due to the toxicity of CuO NPs.

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The effects of Cu ions on denitrification were investigated using 8.88, 83.79, and

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437.74 mg kg-1 CuCl2. The data indicated that the NO3− concentration was raised and

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N2O emission rates suppressed by increasing the concentration of Cu ions from 8.88 to

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437.74 mg kg-1 (Figure 2). The high concentration promoted the highest NO3−

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concentration (5.7 mg N kg-1 dry soil) and lowest N2O emission rates (8.8 µg kg-1 h-1).

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The NO3− concentrations in Cu ions treatments (8.88, 83.79, 437.74 mg kg-1) were

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40.0%, 106.2% and 72.7% of different CuO NPs treatments (10, 100, 500 mg kg-1 CuO

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NPs). The N2O emission rates in three Cu ions treatments were 79.6%, 80.6% and 90.6%

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of different CuO NPs treatments. The effects of Cu ions on NO3− and N2O is basically

290

the same as that of CuO NPs, suggesting that Cu ion release is, in the main, responsible

291

for the toxic effects of CuO NPs on microbial denitrification.

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3.3 Effect of CuO NPs on ETSA

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Denitrification requires the acceptance of electrons to complete a series of

294

bioreduction reactions (Berks et al., 1995; J and M, 2013). Exposure to CuO NPs might

295

disturb microbial electron transport efficiency during denitrification. These process can

296

be assessed by detecting ETSA. As shown in Figure 3, the ETSA value was 0.021 µg O2

297

mg−1 protein min−1 in control, which was similar to previous published results (Colvin,

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2003). However, ETSA values decreased to 0.016 and 0.013 µg O2 mg−1 protein·min−1

299

under Cu10 and Cu100 treatments, respectively (P<0.05). The ETSA was just 0.011 µg

300

O2 mg−1 protein min−1 when the CuO NPs concentration was further increased to 500

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mg kg-1, indicating that the CuO NPs significantly affected the value. A previous report

302

suggested that the reduction in ETSA may inhibit the denitrification process (Wan et al.,

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2016). In our study, the lower ETSA values under CuO NPs treatments suggested the

304

presence of an inhibitory effect on the electron transport process, which may hinder

305

NO3−, NO2−, NO and N2O from obtaining electrons, thus affecting the denitrification

306

process.

307

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3.4 Effect of CuO NPs on denitrifying enzymes

309

Denitrifying enzymes have been reported to be responsible for the bioreductions of

310

NO3−, NO2−, NO and N2O during denitrification. Thus, we analyzed the activities of

311

denitrifying reductases in the absence and presence of CuO NPs. NAR is crucial to

312

denitrification and is responsible for the reduction of NO3− to NO2−. Figure 4a indicates

313

that the activity of NAR was reduced by 21.1%, 26.3% and 42.7% in Cu10, Cu100 and

314

Cu500 treatments, respectively, when compared with the control. This suggests that

315

CuO NPs have a significant inhibitory effect on NAR activity. Hou et al. (2016) studied

316

the impacts of CuO NPs on total nitrogen removal and the results showed that NAR

317

activity was obviously reduced, resulting in the accumulation of NO3− (Hou et al., 2016).

318

Figure 4b shows that there were no significant differences in NIR activity among the

319

control, Cu10 and Cu500 treatments (P>0.05). However, its activity under Cu100

320

treatment was raised. As Cu is a component of NIR, Cu ions released by the CuO NPs at

321

100 mg kg-1 may have promoted NIR activity.

322

As shown in Figure 4c and d, the activity of NOR in the control was significantly

323

higher than that in other treatments (P<0.01). This indicated that increasing CuO NPs

324

exposure inhibited NOR activity thus inhibited the production process of N2O. There

325

were no significant differences between the control and CuO NPs treatments in terms of

326

NOS activity (P>0.05), indicating that the reduction process of N2O was not

327

significantly affected. The N2O emission was a combination of the production process

328

and reduction process of N2O. When N2O production decreases, N2O reduction

329

increases or remains unchanged, N2O emission decreases. In this experiment, the

15

330

decrease of NOR activity was the major cause of the N2O emission rates decreased.

331

N2O emission was related to the inhibition of denitrification reductase activity and the

332

application of NPs had an inhibitory effect on NOR activity (Zheng et al., 2014). Chen

333

et al. (2012) also demonstrated that N2O generation was inhibited by the application of

334

Cu NPs (Chen et al., 2012). As mentioned above, the significant inhibition of NAR and

335

NOR activity by CuO NPs could be a reason for the effect of CuO NPs on the

336

denitrification process.

337 338

3.5 Effect of CuO NPs on gene abundances and soil microbial community

339

Denitrifying reductases are products of the denitrification genes expression, which

340

is likely to be affected by CuO NPs. As shown in Figure 5, copy numbers of the

341

denitrification genes narG, nirS, nirK, norB and nosZ ranged from 104 to 106 copies g-1

342

dry soil. The abundance of narG in all treatments was lower than that for the other

343

genes, which is inconsistent with a study by Li et al. (2017), who reported that the copy

344

numbers of narG were highest in most natural soils. Our results suggested that the

345

up-regulation of narG may be affected by red soil (Li et al., 2017). In this study, the

346

copy numbers of nirK were higher than those of nirS. These differences might depend

347

on soil properties such as pH and nutrient content (Enwall et al., 2010). It should be

348

noted that the significant reduction of nirS (P<0.001) abundance with increasing

349

concentrations of CuO NPs, resulted in reduced NIR activity, which may be one of the

350

causes of NO2− accumulation. Compared with the control, norB abundance in the CuO

351

NPs treatments was reduced by 13.2%, resulting in decreases NOR activity. The

16

352

abundance of nosZ rose by 5.7% and 4.1% in Cu100 and Cu500 treatments,

353

respectively, resulting in a slight increase in NOS activity, which is related to the

354

reduction process (N2O to N2).

355

Communities and structures of soil bacteria in different treatments at the phylum,

356

class and genus levels are shown in Figure 6. The dominant bacteria were Firmicutes,

357

Chloroflexi, Proteobacteria and Actinobacteria, which accounted for more than 94% of

358

the bacteria present in all treatments. Firmicutes was the most sensitive to CuO NPs and

359

its levels decreased from 91.6% to 91.5%, 76.8% and 60.0% under all treatments,

360

respectively. However, Chloroflexi, Proteobacteria and Actinobacteria populations

361

increased from 2.5, 1.8 and 2.4% to 11.1, 13.1 and 9.7%, respectively, following the

362

application of CuO NPs at 500mg kg-1. Classes with relative abundances >0.2% were

363

selected for analysis in this study. The most abundant taxa were Bacillus, Clostridia,

364

Actinobacteria,

365

relatively abundances of Bacillus and Clostridia reduced from 69.9% to 59.7%, and

366

from 22.0% to 0.4%, respectively, when exposed to CuO NPs. Actinobacteria levels

367

increased from 2.4% (control) to 9.8% (Cu500 treatment), while the β-Proteobacteria

368

and α-Proteobacteria levels increased from 0.6% to 9.6%, and from 1.0% to 3.3%,

369

respectively. The relative abundances of Proteobacteria, including α- and

370

β-Proteobacteria are closely related to the denitrification process, were significantly

371

increased by the application of CuO NPs (Srinandan et al., 2011). At the genus level,

372

the taxonomic groups displayed > 0.1% abundance genera were chosen to compare the

373

microbial richness at different CuO NPs concentrations (Figure 6c). Bacillus and

Ktedonobacteria,

β-Proteobacteria

17

and

α-Proteobacteria.

The

374

Clostridium were the dominant taxa and the relative abundances had an obvious

375

decrease after CuO NPs application, from 59.2% to 55.9% and 21.9% to 0.1% in control

376

and Cu500 treatments, respectively. Previous studies have documented that Bacillus and

377

Clostridium potentially contribute to denitrification in natural ecosystems, which are

378

inhibited by CuO NPs stress (Verbaendert et al., 2011; Libing Chu, 2016). The present

379

results demonstrated that the presence of CuO NPs shift abundances and community

380

compositions of bacteria associated with denitrification, thus affecting denitrification

381

process.

382 383

3.6 Effects of CuO NPs on ROS

384

Nanoparticles can enter cells and implement the production of ROS due to their

385

small sizes and high catalytic activities (Andre et al., 2006). Cell membrane integrity

386

can be damaged due to lipid peroxidation, which is induced by ROS. Previous studies

387

have shown that TiO2 NPs inhibit denitrification through the production of ROS. In our

388

study, ROS production was used to indicate the level of oxidative stress. As seen as

389

Figure S2, the production of ROS showed no significant differences under the four

390

treatments (P>0.05), indicating that high concentrations of CuO NPs do not promote

391

ROS production. The reason for this may be that ROS are produced though oxygen

392

reduction, whereas denitrification is an anaerobic process and there was a lack of

393

oxygen in this experiment (Andre et al., 2006). Based on a survey of ZnO NPs, Zheng

394

et al. (2014) found that ROS were not formed during the denitrification process. Their

395

experimental results showed that oxygen is a precursor to generating ROS. Therefore,

18

396

ROS may not be responsible for the toxicity of CuO NPs to soil denitrification

397

processes in our experiment.

398 399

3.7 Relationships among denitrifying enzymes, ETSA and soil denitrification

400

Regression analyses were used to investigate the relationships among NO3− and

401

N2O emission rates with denitrifying enzymes and ETSA. Figure S3a shows that the

402

final NO3− concentration was negatively correlated with NAR activity (R2=0.48). CuO

403

NPs led to NO3− accumulation by inhibiting NAR activity, because NAR can reduce

404

NO3− to NO2−. Similarly, NO2− accumulation was negatively correlated with NAR and

405

NIR activities (Figure S3b). The slope of the fitted line for NO2− accumulation and NAR

406

was greater than the fitted line for NO2− accumulation and NIR, indicating that NAR

407

activity is more sensitive to CuO NPs than NIR activity. As shown in Figure S3c and d,

408

the N2O emission rates was significantly positively correlated with NAR (R2=0.42) and

409

NOR activities (R2=0.27). Decreased NOR activity leads to inhibition of N2O

410

production, because NOR reduces NO to N2O. In this experiment, CuO NPs had a much

411

greater inhibitory effect on NOR than NOS, resulting in a significant decrease in N2O

412

emission rates. There were significant positive correlations between ETSA values and

413

NAR or NOR activities (Figure S3e and f). These results demonstrate that reduced

414

electron transport may be responsible for the effect of CuO NPs on denitrification.

415 416 417

3.8 Environmental significance Applications of CuO NPs have increased 2.81-fold from 2014 (570 tons) to 2025

19

418

(1600 tons) . In general, CuO NPs have favorable impacts on crop growth. Qiang et al.

419

(2012) indicated that CuO NPs used in tomato fertilizer can increase yields compared

420

with the use of conventional fertilizer (urea) (Qiang et al., 2012). However, the

421

influences of CuO NPs on the soil nitrogen cycle are not clear. Our research illustrated

422

that increasing CuO NPs in the soil not only increase NO3− concentrations, but also

423

decrease N2O emission rates, which helps to maintain soil fertility and reduce

424

greenhouse gas emissions. In the future, the production and use of NPs should be

425

conducted from the source, and researchers should develop new soil biological

426

treatments similar to NPs-based nanopesticides, which are beneficial to crop growth and

427

less harmful to the environment. In addition, the impact of new nanopesticides and other

428

nanoparticles on the nitrogen cycle in soil should be investigated further.

429

4. Conclusion

430

In this study, CuO NPs at concentrations of 10 and 100 mg kg-1 did not

431

significantly inhibit denitrification; whereas 500 mg kg-1 caused an 11-fold increase in

432

NO3− accumulation, but decreased the N2O emission rates by 24.1%. The released Cu

433

ions was found to be a major reason for the toxic effects of CuO NPs on

434

microorganisms, due to the high bioavailability and dissolution of CuO NPs in soil.

435

Compared with the control, ETSA values were strongly reduced by 23.8~47.6% under

436

CuO NPs treatments. Additionally, NAR and NOR activities were reduced by 21.1~42.1%

437

and 10.3~16.3%, respectively. Moreover, CuO NPs altered the abundances of

438

denitrification functional genes and the structures of microbial communities. These may

439

be reasons for the negative effects of CuO NPs on the denitrification process. ROS

20

440

analysis showed that there were no significant differences among the treatments.

441

Overall, the impacts of NPs on agricultural land and the development of environmental

442

friendly pesticides should be the focus of further research.

443 444

Acknowledgments

445

This work was financially supported by the National Natural Science Foundation

446

of China (NO.51708056) and the Fundamental Research Funds for the Central

447

Universities of Chongqing University (Grant No. 2019CDXYCH0027). In addition, we

448

would like to thank Analytical and Testing Center of Chongqing University for

449

Inductively Coupled Plasma Chromatograph.

450 451

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Figure captions

578

Figure 1. Effects of copper oxide nanoparticles (CuO NPs) on the variations of NO3−

579

concentrations (a), NO2− concentration (b), N2O emission rates (c) and NH4+

580

concentration (d) during denitrification. Error bars represent the standard deviations of

581

triplicate tests.

582

Figure 2. Effects of absence and presence of CuO NPs and released copper ions on the

25

583

final NO3− concentration and N2O emission rates after 60 h. Error bars represent the

584

standard deviations of triplicate tests.

585

Figure 3. The values of ETSA in the control and CuO NPs treatments after 60 h. Error

586

bars represent the standard deviations of triplicate tests. Statistically significant

587

differences from the control: *P < 0.05, moderately significant; **P < 0.01, highly

588

significant; ***P < 0.001, noticeably significant.

589

Figure 4. Effects of CuO NPs on the activity of NAR (a), NIR (b), NOR (c) and NOS

590

(d) after 60 h. Error bars represent the standard deviations of triplicate tests. Statistically

591

significant difference from the control: *P < 0.05, moderately significant; **P < 0.01,

592

highly significant; ***P < 0.001, noticeably significant.

593

Figure 5. Effects of CuO NPs on the relative gene abundances of narG, nirS, norK,

594

norB and nosZ after 60 h. Error bars represent the standard deviations of triplicate tests.

595

Statistically significant difference from the control: *P < 0.05, moderately significant;

596

**P < 0.01, highly significant; ***P < 0.001, noticeably significant.

597

Figure 6. Effects of CuO NPs on bacterial composition at the Phylum level (a) Class

598

level (b) and genus level (c).

599

26

10mg kg-1 CuO NPs 100mg kg-1CuO NPs

80

500mg kg-1CuO NPs

60 40 20 0

0

8

16

24

32

40

48

56

NO2--N (mgN kg-1dry soil)

a

Control

64

35

b

30 25 20 15 10 5 0

0

8

16

Time (h)

c

100

**

* ***

80 60 40 20 0

Control

10

24

32

40

48

56

64

Time (h)

120

100

500

CuO NPs concentration (mg kg-1)

NH4+-N (mgN kg-1dry soil)

NO3--N (mgN kg-1dry soil) N2O emission rates (% control)

100

7.0

d 6.5 6.0 5.5 5.0 4.5 4.0

0

8

16

24

32

40

48

56

64

Time (h)

Figure 1. Effects of copper oxide nanoparticles (CuO NPs) on the variations of NO3− concentrations (a), NO2− concentration (b), N2O emission rates (c) and NH4+ concentration (d) during denitrification. Error bars represent the standard deviations of triplicate tests.

N2O emission rates 8

14 13

7

12

6 11 5 10 4 9

3 2

8

1

7

0

N2O emission rates (µg kg-1h-1)

NO3 -N concentration (mg kg-1)

NO3--N concentration

9

6 control

10

100

500

CuO NPs (mg kg-1)

10

100

500

Released Cu2+ (mg kg-1)

Figure 2. Effects of absence and presence of CuO NPs and released copper ions on the final NO3− concentration and N2O emission rates after 60 h. Error bars represent the standard deviations of triplicate tests.

ETSA (µg O2 g-1 pro min-1)

0.025

0.020 ** 0.015

**

**

0.010

0.005

0.000

control

1

100

500

CuO NPs concentration (mg kg-1)

Figure 3. The values of ETSA in the control and CuO NPs treatments after 60 h. Error bars represent the standard deviations of triplicate tests. Statistically significant differences from the control: *P < 0.05, moderately significant; **P < 0.01, highly significant; ***P < 0.001, noticeably significant.

*

***

0.15 0.10 0.05

NIR ( µm ol NO 2 - -N g -1 soil*h -1 )

a 0.20

*

0.00

3.0

b

***

2.4 1.8 1.2 0.6 0.0

c 0.40

**

0.32

***

***

0.24 0.16 0.08 0.00

Control

10

100

500 -1

CuO NPs concentration (mg kg )

NO S ( µm ol N 2 O -N g -1 soil*h -1 )

NOR ( µm ol N 2 O -N g -1 soil*h -1 ) NAR ( µm ol NO 2 - -N g -1 soil*h -1 )

0.25

d 0.16 0.12 0.08 0.04 0.00

Control

10

100

500 -1

CuO NPs concentration (mg kg )

Figure 4. Effects of CuO NPs on the activity of NAR (a), NIR (b), NOR (c) and NOS (d) after 60 h. Error bars represent the standard deviations of triplicate tests. Statistically significant difference from the control: *P < 0.05, moderately significant; **P < 0.01, highly significant; ***P < 0.001, noticeably significant.

Relative abundance (% of control)

1.6

Control CuO10 CuO100 CuO500

*

1.4 1.2 1.0

*

* **

0.8

* * ***

0.6

***

0.4 0.2 0.0

narG

nirK

nirS

norB

nosZ

Figure 5. Effects of CuO NPs on the relative gene abundances of narG, nirS, norK, norB and nosZ after 60 h. Error bars represent the standard deviations of triplicate tests. Statistically significant difference from the control: *P < 0.05, moderately significant; **P < 0.01, highly significant; ***P < 0.001, noticeably significant.

-1 CuO NPs concentration (mg kg )

a

other Bacteroidetes Verrucomicrobia Nitrospirae Gemmatimonadetes Planctomycetes Acidobacteria Actinobacteria Proteobacteria Chloroflexi Firmicutes

500

100

10

control

-1 CuO NPs concentration (mg kg )

0.0

0.2

0.4 0.6 0.8 Relative abundance of Phylum level

1.0

b

0ther Thermomicrobia TK10 Gemmatimonadetes Planctomycetacia Acidobacteria Alphaproteobacteria Betaproteobacteria Ktedonobacteria Actinobacteria Clostridia Bacilli

500

100

10

control 0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Relative abundance of Class level Bacillus

59.40

c

Clostridium Ammoniphilus 55.00

Gaiellales Acidobacteriaceae Acidothermus

30.00

Sphingomonas Planctomycetaceae Gemmatimonadaceae

6.00

Acidimicrobiales Massilia Ammoniphilus

0.80

Burkholderia Bryobacter 0.00

Other

control

10

100

500

CuO NPs concentration (mg kg-1)

Figure 6. Effects of CuO NPs on bacterial composition at the Phylum level (a) Class level (b) and genus level (c).

Highlights:
CuO NPs inhibited soil denitrification and decreased N2O emission rates.
The released Cu ions was major reason for the toxicity effects of CuO NPs.
CuO NPs have significantly affected on electron transport system activity.
CuO NPs decreased the nitrate reductase and nitric oxide reductase activities.
Communities and structures of bacteria were altered by CuO NPs.

Authors: Shuyuan Zhao

a, b

, Xiaoxuan Su

a, b

, Yiyu Wang

a, b

Qiang He a, b, Yi Chen a, b * 1. Shuyuan Zhao: Did the experiment and wrote the paper. 2. Xiaoxuan Su: Data analyse 3. Yiyu Wang: Analyse the electron transport system activity 4. Xiangyu Yang: Analyse the denitrifying enzyme activity 5. Mohan Bi: Did the experiment 6. Qiang He: Design the experiment and revise the paper. 7. Yi Chen: Design the experiment, wrote and revise the paper.

, Xiangyu Yang

a, b

, Mohan Bi

a, b

,

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: