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Zinc oxide nanoparticles cause inhibition of microbial denitrification by affecting transcriptional regulation and enzyme activity Xiong Zheng, Yinglong Su, Yinguang Chen, Rui Wan, Kun Liu, Mu Li, and Daqiang Yin Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es504251v • Publication Date (Web): 10 Nov 2014 Downloaded from http://pubs.acs.org on November 16, 2014

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Environmental Science & Technology

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Zinc oxide nanoparticles cause inhibition of microbial denitrification by

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affecting transcriptional regulation and enzyme activity

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Xiong Zheng, Yinglong Su, Yinguang Chen*, Rui Wan, Kun Liu, Mu Li, and Daqiang Yin

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State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental Science and

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Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China

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* Corresponding author

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Tel.: +86 21 65981263; fax: +86 21 65986313

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E-mail address: [email protected]

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1

ACS Paragon Plus Environment


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ABSTRACT: Over the past few decades, human activities have accelerated the rates and extents of

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water eutrophication and global warming through increasing delivery of biologically available nitrogen

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such as nitrate and large emissions of anthropogenic greenhouse gases. In particular, nitrous oxide (N2O)

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is one of the most important greenhouse gases, because it has a 300-fold higher global warming potential

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than carbon dioxide. Microbial denitrification is a major pathway responsible for nitrate removal, and

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also a dominant source of N2O emissions from terrestrial or aquatic environments. However, whether

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the release of zinc oxide nanoparticles (ZnO NPs) into the environment affects microbial denitrification is

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largely unknown. Here we show that the presence of ZnO NPs lead to great increases in nitrate delivery

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(9.8-fold higher) and N2O emissions (350- and 174-fold higher in the gas and liquid phases, respectively).

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Our data further reveal that ZnO NPs significantly change the transcriptional regulations of glycolysis and

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polyhydroxybutyrate synthesis, which causes the decrease in reducing powers available for the reduction

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of nitrate and N2O. Moreover, ZnO NPs substantially inhibit the gene expressions and catalytic activities

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of key denitrifying enzymes. These negative effects of ZnO NPs on microbial denitrification finally

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cause lower nitrate removal and higher N2O emissions, which is likely to exacerbate water eutrophication

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and global warming.

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INTRODUCTION

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Since nitrogen is an important limiting factor for plant growth, the wide use of artificial nitrogen

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fertilizer has made a significant contribution to global food production.1

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led to some global environmental problems, such as water eutrophication and climate change.2-4 Over

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the past few decades, human activities have accelerated the rates and extents of these environmental

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changes through increasing delivery of biologically available nitrogen such as nitrate and large emissions

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of anthropogenic greenhouse gases.5, 6

Unfortunately, this behavior has

In particular, nitrous oxide (N2O) is one of the most important

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greenhouse gas, because it has a 300-fold higher global warming potential than carbon dioxide,4 and can

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cause stratospheric ozone destruction.7

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pathway by which fixed nitrogen such as nitrate returns to the atmosphere from terrestrial and aquatic

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environments.8

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result in global warming and stratospheric ozone destruction.6

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mainly responsible for nitrate removal,8 and is also a dominant source of N2O emissions from the

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environments.9

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It is already known that microbial denitrification is the primary

Nevertheless, the incomplete denitrification can cause large N2O emissions, which might As a result, microbial denitrification is

Over the last decade, man-made nanomaterials, such as zinc oxide nanoparticles (ZnO NPs), have

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been used in a wide range of industrial applications and consumer products.10,

11

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manufacture and utilization of nanomaterials will accelerate their releases into terrestrial and aquatic

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environments,12-14 which might cause the potential risks to humans and the environment.15,

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example, the pervasive use of engineered nanomaterials has recently been regarded as a substantial threat

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to human health.10, 11, 17

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on the environmental processes, especially microbial denitrification.18

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remains unclear at present whether the release of ZnO NPs into the environment causes the negative

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effects on nitrate removal and N2O emissions.

The increasing

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For

Nevertheless, few studies have concerned the possible impacts of nanomaterials To the best of our knowledge, it

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Here we report the potential influences of ZnO NPs on nitrate reduction and N2O emissions under

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denitrification conditions. During the exposure period, we identify a large set of differentially expressed

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genes (DEGs) involved in the intracellular metabolism, and also measure the catalytic activities of the key

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enzymes related to glycolysis, polyhydroxybutyrate (PHB) synthesis, and denitrification. The data from

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this work help elucidate the possible reasons for these negative effects of ZnO NPs on denitrifying

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bacteria. This mechanism will make it possible to find a proper way to mitigate the risks of engineered

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nanomaterials to water eutrophication and global warming.

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MATERIALS AND METHODS

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Engineered ZnO NPs. In this study, ZnO NPs were purchased from Sigma-Aldrich (St. Louis, MO,

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USA).

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D/Max-RB diffractometer equipped with a rotating anode and a Cu Kα radiation source (Figure S1,

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Supporting Information), and transmission electron microscope (TEM) image was taken by a Philips

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Tecnai F20 microscope at an accelerating voltage of 200 kV (Figure S2, Supporting Information). To

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prepare a stock suspension, 500 mg of ZnO NPs were dispersed in 1 L of Milli-Q water followed by

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ultrasonication (40 kHz and 250 W) for 1 h at room temperature as described previously.19

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light scattering (DLS) analysis via a Malvern Autosizer 4700 (Malvern Instruments, UK) showed that the

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typical sizes of particles in the stock suspension were in the range 80-100 nm.

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Cell culture. Paracoccus denitrificans was obtained from ATCC (Manassas, VA, USA) and grown in

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Difco nutrient broth at 30 oC and 200 rpm to an optical density at 600 nm (OD600) of 0.8-1.0.

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P. denitrificans exposed to ZnO NPs. To examine the possible effects of ZnO NPs on microbial

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denitrification, P. denitrificans was exposed to 0, 1, 10, and 50 mg/L ZnO NPs in a mineral medium

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modified from the literature.20

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0.5 g MgSO4, 1.0 g (NH4)2SO4, 2.16 g KNO3, 5.0 g glucose, and 50 µL trace element feed. The trace

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element feed was documented in our previous publication.19 Bacterial cultures were inoculated at an

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initial OD600 of 0.05, and anaerobically cultured in dark at 30 °C and 200 rpm with different

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concentrations of ZnO NPs. The concentrations of NO3-, NO2-, N2O, glucose, and PHB were estimated

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at intervals of 4 h for a total time of 24 h. The measurements of NO3-, NO2-, glucose, and PHB were

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performed as described previously,19, 21 and detailed in the Supporting Information. The gaseous and

To characterize the NPs, X-ray diffraction (XRD) analysis was conducted using a Rigaku

Dynamic

The mineral medium contained (per liter) 7.0 g K2HPO4, 3.0 g KH2PO4,

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dissolved N2O was analyzed using a gas chromatography with an electron capture detector (ECD)

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according to the literature.22

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Dissolution of ZnO NPs and P. denitrificans exposed to released zinc ions. The dissolution of ZnO

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NPs in the medium was determined according to our previous study.19 Briefly, a series of serum bottles

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that respectively contained 50 mL of the mineral medium with 0, 1, 10 and 50 mg/L ZnO NPs were

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shaken at 30 oC and 200 rpm.

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centrifugation (16000 rpm) for 10 min, and 0.5 mL of the supernatant was added to 4.5 mL of Milli-Q

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water (containing 2% ultrahigh purity HNO3). The concentration of zinc ions in the resulting solution

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was measured by inductively coupled plasma mass spectrometry (ICP-MS, Agilent Technologies, USA).

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Then, P. denitrificans was exposed to the measured concentration of released zinc ions according to the

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method described in the P. denitrificans exposed to ZnO NPs section, and the final concentrations of NO3-

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and N2O were measured to determine the potential effects of released zinc ions on the denitrification

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process.

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RNA-Seq. The RNA-Seq analysis was used to determine the transcriptional profiling of P. denitrificans

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in the absence (the control) and presence of 50 mg/L ZnO NPs. Briefly, the cells were harvested at 16 h

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after exposure to ZnO NPs, followed by centrifugation at 10000 rpm for 10 min at 4 oC, and then lysed in

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TRIzol reagent (Invitrogen) for extraction of total RNA. To avoid genomic DNA contamination, the

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extracted RNA was treated with DNase I (Ambion) according to the manufacturer’s protocol. Thereafter,

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mRNA was isolated from the DNA-free total RNA using the MICROBExpress Bacterial mRNA

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Enrichment Kit (Ambion), and prepared for Illumina sequencing using the mRNA-Seq Sample

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Preparation Kit (Illumina) according to manufacturer’s instructions. The RNA-Seq libraries were finally

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sequenced using an Illumina HiSeq 2000. All sequencing data have been submitted to the National

At different time points, ZnO NPs were removed by high speed

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Center for Biotechnology Information (NCBI) short-read archive (SRA) under accession numbers

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SRR953150 and SRR953225.

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The next generation sequencing (NGS) quality control (QC) toolkit v2.2.1 was used to filter the raw

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reads23 by removing the reads with (1) sequence adapters, (2) more than 5% ‘N’ bases, and (3) more than

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50% QA ≤ 15 bases. The obtained clean reads were aligned to the reference genome using SOAP2,24

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and no more than 3 mismatches were allowed in the alignment for each read. The gene expression level

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was calculated using the RPKM (reads per kilobase of exon region per million mappable reads) method,25

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and the differentially expressed genes were identified based on the criteria: absolute fold change > 2 and

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false discovery rate (FDR) < 0.05.

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Genomes (KEGG) analyses were performed using Blast2GO using default annotation parameters.26

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Interactive Pathways (ipath) analysis was carried out via interactive pathways explorer v2 (http://

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pathways.embl.de/).

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Enzyme assays. The measurements of key enzymes activities can be used to examine the NPs-induced

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potential influences according to the literature.27, 28

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by centrifugation (5000 rpm for 10 min), washed thrice with 0.1 M phosphate-buffered saline (PBS) (pH

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7.4), and resuspended in the same buffer at 4 oC. Thereafter, crude cell extracts were prepared by

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disrupting cells for 1 min by sonication (4 oC, 20 kHz) followed by centrifugation at 16000 rpm for 10

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min at 4 oC, and were immediately used for determination of enzyme activities.

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concentrations of cell extracts were measured using Protein Assay Kit (Bio-Rad) with bovine serum

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albumin (BSA) as a standard.

Gene ontology (GO) and Kyoto Encyclopedia of Genes and

Briefly, after 24 h of exposure, cells were harvested

The protein

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Glucokinase (GK) activity was determined by measuring the formation of NADPH at 340 nm as

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described previously.29 The reaction mixture (a total volume of 1 mL) contained 100 mM Tris-HCl (pH

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7.5), 10 mM MgCl2, 0.5 mM NADP, 1 mM ATP, 10 mM glucose, 2 U glucose-6-phosphate

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dehydrogenase, and 50 µL of cell extract.

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monitoring the decrease of NADH in absorbance at 340 nm as described in reference.30 The reaction

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mixture (1 mL) contained 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.15 mM NADH, 0.5 mM ATP, 5 mM

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fructose-6-phosphate, 2 U each of aldolase, triosephosphate isomerase and glycerol-3-phosphate

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dehydrogenase, and 50 µL of cell extract.

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spectrophotometrically at 340 nm through the oxidation of NADH to NAD+.31

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mL) contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 0.2 mM NADH, 5 mM ADP, 10 mM

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dithiothreitol, 1 mM fructose 1,6-diphosphate, 10 U lactate dehydrogenase, and 50 µL of cell extract.

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Phosphofructokinase (PFK) activity was assayed by

Pyruvate kinase (PK) activity was measured The reaction mixture (1

Pyruvate dehydrogenase (PDH) activity was estimated spectrophotometrically at 340 nm by the

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pyruvate-dependent reduction of NAD+ according to the literature.32

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contained 100 mM M Tris-HCl (pH 7.5), 10 mM MgCl2, 1.2 mM NAD+, 6 mM dithiothreitol, 10 mM

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pyruvate, 0.12 mM CoA, and 50 µL of cell extract. Acetyl-CoA acetyltransferase (ACAT) activity was

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determined spectrophotometrically at 303 nm by measuring the decrease of acetoacetyl-CoA.33 The

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reaction mixture (1 mL) contained 0.1 M Tris-HCl (pH 8.3), 25 mM MgCl2, 50 mM KCl, 20 nM

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acetoacetyl-CoA, 20 nM CoA, and 50 µL of cell extract. The 3-hydroxybutyryl-CoA dehydrogenase

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(Hbd) activity was assayed by determining the decrease in absorbance at 263 nm.34

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(1 mL) contained 100 mM Tris-HCl (pH 7.5), 30 µM crotonyl-CoA, and 50 µL of cell extract. PHA

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synthase activity (Pha) was measured spectrophotometrically at 412 nm by reduction of

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5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) as described previously.35 The reaction mixture (1 mL)

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contained 25 mM Tris-HCl (pH 7.5), 1 mM DTNB, 30 mM D-(-)-3-hydroxybutyryl-CoA, and 50 µL of

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cell extract.

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The reaction mixture (1 mL)

The reaction mixture


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The measurements of nitrate reductase (NAR), nitrite reductase (NIR), nitric oxide reductase (NOR),

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and nitrous oxide reductase (N2OR) activities were performed through the consumption of electron

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acceptor (NO3-, NO2-, NO, or N2O) with methyl viologen as electron donor as described previously.36, 37

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Briefly, these denitrifying enzymes were assayed at 22 oC in anaerobic cuvettes with rubber stoppers.

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The cuvettes contained 2 mL of the reaction mixture (10 mM potassium phosphate buffer (pH 7.1), 10

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mM methyl viologen, 5 mM Na2S2O4, 1 mM electron acceptor (NO3-, NO2-, NO, or N2O)). Then, 100

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µL of the cell extract was injected to initiate the reaction.

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of NO3-, NO2-, NO, or N2O was determined, and the specific enzyme activity was calculated. The

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detailed procedures of the measurements of NO3-, NO2-, NO, and N2O were documented in the Supporting

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Information.

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Analytical method. Transmission electron microscopy analysis, lactate dehydrogenase release assay,

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and measurements of key trace metal ions in the absence and presence of ZnO NPs were documented in

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the Supporting Information.

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Statistical Analysis. All tests in this study were performed in triplicate.

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used to test the significance of results, and p < 0.05 was considered to be statistically significant.

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RESULTS AND DISCUSSION

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Bacterial structure and denitrification in the presence of ZnO NPs. To determine the possible effects

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of ZnO NPs on microbial denitrification, we first examined the growth and metabolism of P. denitrificans

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(a model denitrifying bacterium) in the absence and presence of ZnO NPs. Transmission electron

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microscopy (TEM) images showed that ZnO NPs in the medium adhered to the cell membrane of P.

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denitrificans (Fig. 1a). Using lactate dehydrogenase (LDH) release as an indicator of membrane damage,

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we found that the LDH releases in the absence and presence of ZnO NPs had no significant differences

After 30 min of incubation, the concentration

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An analysis of variance was

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(P>0.05, Student’s t-test) (Fig. 1b), suggesting that these NPs did not change the membrane integrity of P.

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denitrificans. It can be also observed that the presence of ZnO NPs caused no negative effects on the

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bacterial population density during 24 h of exposure (P>0.05, Student’s t-test) (Fig. 1c).

0.2 μm ZnO NPs

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c

125

0.8

100

0.6

75

OD600

b

Relative LDH release (%)

a

50

0.2

25 0

0.4

Control

1

10

50

ZnO NPs (mg/L)

0.0

0

4

8

12

16

20

24

Time (h)

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Figure 1. Effect of ZnO NPs on the denitrifying bacterium P. denitrificans. a, TEM image and

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EDX measurement of P. denitrificans exposed to ZnO NPs. b and c show the relative LDH release (an

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indicator of membrane damage) and bacterial growth curve in response to ZnO NPs, respectively. For b

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and c, data are shown as mean value ± standard deviation from three independent measurements.

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Further studies indicated that increasing ZnO NPs concentrations caused the inhibitory effects on the

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denitrification process. After 24 h of exposure, the final concentration of NO3--N was remarkably

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greater in the presence of 50 mg/L ZnO NPs than its absence (42.1 mg/L versus 3.9 mg/L) (Fig. 2a),

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although little NO2--N was measured in effluent no matter whether ZnO NPs were present or not (Fig. 2b).

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Meanwhile, we observed large changes in the N2O production during exposure to different concentrations

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of ZnO NPs. The average N2O concentrations in both gas and liquid phases were very low (0.04% and

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0.02% of total nitrogen (TN), respectively) in the absence of ZnO NPs (the control). In contrast, those

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concentrations were 13.96% and 3.48% of TN in the presence of 50 mg/L ZnO NPs, which were

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approximately 350- and 174-fold higher than those of the control, respectively (Fig. 2c). Clearly, our

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data indicated that increasing release of ZnO NPs into the environment not only inhibited the nitrate

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reduction, but also led to the significant increases in N2O emissions.

9


a

b

320

c

15 12

240

9 160 6 80 3 0

183

0

4

8

12

16

20

24

0

0

Time (h)

4

8

12

16

20

24

Time (h)

N2O accumulation (% of TN)


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20 Liquid phase Gas phase

15

10

5

0 Control

1

10

50

ZnO NPs (mg/L)

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Figure 2. Effect of ZnO NPs on the denitrification process of P. denitrificans. a, b, and c show

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respectively the NO3- reduction (a), NO2- variation (b), and N2O accumulation (c) in response to ZnO NPs.

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The data are shown as mean value ± standard deviation from three independent measurements.

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Generally, nanomaterials can easily lead to the reactive oxygen species (ROS) production, because of

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their extremely small sizes and high catalytic activities.10

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membrane integrity and cell morphology of living organisms.38, 39

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the culture medium did not affect the surface integrity and viability of P. denitrificans (Fig. 1). This

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phenomenon might be due to the absence of oxygen in this study (anoxic conditions), since ROS is

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generated primarily by the reduction of molecular oxygen.11 On the other hand, ZnO NPs are slightly

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soluble in water, and thus can release zinc ions under neutral condition. Fig. 3a showed that the average

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stable concentrations of released zinc ions in the presence of 1, 10, and 50 mg/L ZnO NPs were 0.65, 3.81,

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and 6.47 mg/L, respectively.

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caused the increases in the final concentrations of NO3--N and total N2O (Fig. 3b), suggesting that the

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release of zinc ions was one of the important reasons for the negative effects of ZnO NPs on microbial

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denitrification. Many investigations indicate that the released zinc ions are mainly responsible for the

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toxicity of ZnO NPs to model organisms.38, 40

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NPs could inhibit biological nitrogen and phosphorus removal in wastewater treatment plants.19

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Nevertheless, to date, the mechanism of ZnO NPs-induced inhibition to microbial denitrification is poorly

This behavior might induce the changes in However, the presence of ZnO NPs in

It was further found that the presence of 3.81 and 6.47 mg/L zinc ions

Our previous study also found that the dissolution of ZnO

10


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understood, and needs to be investigated.

a

b

Control

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Figure 3. Effects of released zinc ions on the denitrification process due to the dissolution of ZnO

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NPs.

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Concentrations of NO3--N and total N2O in the absence and presence of ZnO NPs and its released zinc

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ions at the end of exposure. The data are shown as mean value ± standard deviation from three

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independent measurements.

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Transcriptional profiling of P. denitrificans in response to ZnO NPs. To understand how ZnO NPs

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affect microbial denitrification, we investigated the genome-wide transcriptional profiling of P.

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denitrificans in the absence and presence of 50 mg/L ZnO NPs, because bacterial growth and metabolism

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are closely related to the changes in transcriptional regulation. It is well-known that the high-throughput

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RNA sequencing (RNA-Seq) is a highly accurate technology for quantifying expression levels of

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transcripts under different conditions.41, 42

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high-quality reads after raw sequences processing and filtering (Table S1, Supporting Information).

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After mapping these clean reads to the reference genome of P. denitrificans, we found that 95.4% of the

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total reads matched to the reference genome, and 89.8% matched to unique genomic locations (Table S1,

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Supporting Information).

a, Dissolution curves of ZnO NPs in the culture medium during 24 h of exposure.

b,

Using this method, we obtained a total number of 26774234

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By comparing with the control, we identified 1714 genes that displayed transcriptional fluctuation in

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response to 50 mg/L ZnO NPs (considering absolute fold change > 2 and FDR < 0.05).

These

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differentially expressed genes (DGEs) were subsequently used for functional annotation and enrichment

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analysis, and were further divided into three primary categories of Gene Ontology (GO) classification:

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biological process, cellular component, and molecular function.

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metabolic pathways were found in the presence of ZnO NPs (Fig. S3, Supporting Information), the gene

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expressions involved in 29 functional subcategory annotations showed large differences, especially the

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annotations of catalytic activity, binding, antioxidant activity, molecular transducer activity, nucleic acid

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binding, transcription factor activity, and transporter activity (Fig. 4). Hence, these results suggested that

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the presence of ZnO NPs was able to cause substantial influences on cellular regulation and metabolism of

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P. denitrificans.

Although no obvious changes in

600 Up Down

Number of genes

500

400

300

200

100

230

Biological process

Cellular component

protein binding transcription factor activity structural molecule activity transporter activity

organelle organelle part antioxidant activity binding catalytic activity enzyme regulator activity molecular transducer activity nucleic acid binding transcription factor activit

response to stimulus signaling cell cell part macromolecular complex membrane membrane part

developmental process establishment of localization growth localization locomotion metabolic process multi-organism process regulation of biological process

biological regulation cellular component organization or biogenesis cellular process

0

Molecular function

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Figure 4. GO functional enrichment analysis of the DEGs in response to 50 mg/L ZnO NPs. The

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genes that are up-regulated and down-regulated are shown in green and red, respectively. 12


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Gene expressions and catalytic activities of key enzymes involved in denitrification.

234

studies have confirmed that microbial denitrification needs to degrade organic matters to supply electrons,

235

which are essential for the complete reduction of nitrate to N2.8

236

metabolism plays an important role in the reduction of nitrate and N2O involved in microbial

237

denitrification (Fig. 5a). We analyzed the transcriptional levels of key genes related to glycolysis in the

238

absence and presence of 50 mg/L ZnO NPs (Fig. 5b). It can be found that the gene expressions of these

239

vital enzymes, such as glucokinase (GK), phosphofructokinase (PFK), and pyruvate kinase (PK), were

240

significantly down-regulated in the presence of ZnO NPs. This might be an important reason for the

241

decreased glucose degradation efficiency after 24 h of exposure to ZnO NPs (Fig. 5c). In particular, GK,

242

PFK, and PK catalyze the irreversible reactions in glycolysis, and thus are regarded as the most important

243

enzymes relevant to glucose degradation.43 We found that the activities of GK, PFK, and PK decreased

244

dramatically with increasing ZnO NPs concentrations (Fig. 5d). Our data showed that the presence of

245

ZnO NPs suppressed glucose degradation due to significant decreases in the gene expressions and

246

catalytic activities of key enzymes involved in glycolysis.

247

Previous

Hence, in this study, the glucose

It should be noted that P. denitrificans can accumulate the intracellular polyhydroxybutyrate (PHB),

248

and this process consumes the electrons and energy produced by glucose degradation.44

249

that pyruvate dehydrogenase (PDH), acetyl-CoA acetyltransferase (ACAT), 3-hydroxybutyryl-CoA

250

dehydrogenase (Hbd), 3-hydroxybutyryl-CoA epimerase, and polyhydroxyalkanoate synthase (PhaC) play

251

essential roles in the PHB formation. We found that increasing ZnO NPs concentrations significantly

252

enhanced the genes expressions (Fig. 5e) and relative activities of PDH, ACAT, Hbd, and PhaC (Fig. 5g),

253

which was mainly responsible for the higher PHB contents in P. denitrificans exposed to ZnO NPs (Fig.

254

5f). Hence, these results revealed that the enhanced PHB accumulation could compete for the electrons

13


Fig. 5a shows


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used for microbial denitrification, which resulted in the negative effects on the reduction of nitrate and

256

N2O.

a Glyceraldehyde phosphate dehydrogenase

Glucose

Glyceraldehyde-3P

NAD+ NADH Phosphoglycerate kinase

bisphosphate ATP Fructose aldolase Glucokinase

ADP

Glucose-6P

pyruvate dehydrogenase E2

Glycerate-1,3P2

Acetyl-dihydrolipoamide

Acetyl-CoA

NAD+ NADH

ADP

NADPH NADP+

acetyl-CoA acetyltransferase


ATP

Glycerate-3P Fructose-1,6P2 Triosephosphate isomerase

Phosphoglycerate mutase

Pyruvate

Acetoacetyl-CoA

Glycerate-2P Glucose-6P isomerase

Phosphofructokinase

Fructose-6P

ATP

Glycerone-P

ATP ADP

Phosphoenolpyruvate

polyhydroxyalkanoate synthase

Polyhydroxybutyrate

Glucose degradation

c

Fructose bisphosphate aldolase Glyceraldehyde phosphate dehydrogenase Phosphoglycerate kinase Phosphoglycerate mutase Enolase


GK

f

pyruvate dehydrogenase E2 acetyl-CoA acetyltransferase 3-hydroxybutyryl-CoA dehydrogenase polyhydroxyalkanoate synthase

g

200

150

100

50

0

257

PFK

ZnO NPs (mg/L) Control ZnO NPs

0

1 mg/L ZnO NPs 10 mg/L ZnO NPs 50 mg/L ZnO NPs

2000

500 1000 1500 2000 Gene expression (RPKM)

Control

2500

1

10

50

PK 1 mg/L ZnO NPs 10 mg/L ZnO NPs 50 mg/L ZnO NPs

200

Relative activity (% of control)

e

500 1000 1500 Gene expression (RPKM)

d Relative activity (% of control)

Control ZnO NPs

0

3-hydroxybutyryl-CoA

Glucose degradation efficiency (%)

Glucokinase Phosphofructokinase

NADPH

NADP+

Polyhydroxybutyrate synthesis

Relative PHB content (%)

b

3-hydroxybutyryl-CoA dehydrogenase

Pyruvate kinase Enolase

ADP

150

100

50

0

PDH

ACAT

Hbd

PhaC

ZnO NPs (mg/L)

258

Figure 5.

259

synthesis of P. denitrificans.

260

glucose degradation and PHB synthesis) in P. denitrificans. b, c, d, e, f, and g show respectively the key

261

gene expressions involved in glycolysis (b), relative glucose degradation efficiency (c), relative activities

262

of GK, PFK, and PK (d), key gene expressions involved in PHB synthesis (e), relative PHB content (f),

263

and relative activities of PDH, ACAT, Hbd, and PhaC (g) in response to ZnO NPs. For c, d, f, and g,

264

data are shown as mean value ± standard deviation from three independent measurements.

265

t-test, compared with the control, *P < 0.05.

266

Effects of ZnO NPs on the glucose degradation and polyhydroxybutyrate (PHB) a, Schematic metabolic pathways of intracellular substances (mainly

Student’s

Microbial denitrification is very much dependent on the expressions and activities of four essential

14


Page 15 of 21


267

denitrifying enzymes: nitrate reductase (NAR), nitrite reductase (NIR), nitric oxide reductase (NOR), and

268

nitrous oxide reductase (N2OR), which are encoded by the gene clusters of narGHJI (encoding respiratory

269

nitrate reductase), napABC (encoding periplasmic nitrate reductase), nirSECF, norCBQDEF, and nosRZD,

270

respectively (Fig. 6a). Fig. 6b displays that the gene expressions of these denitrifying enzymes are

271

substantially down-regulated during exposure to 50 mg/L ZnO NPs, which was adverse to the reduction of

272

nitrate and N2O. It can be found that the down-regulated folds for narG, napA, nirS, norB and nosZ

273

were 2.8, 0.3, 4.2, 2.3, and 23.7, respectively.

274

for nos operon was much greater than the other operons, which might be an important reason for the

275

observed N2O accumulation.

Clearly, these results showed that the down-regulated fold

a

narI narJ narH

Periplasm

NO2-

NO2- NO3-

H+

NAR

e-

Q/QH2

NADH dehydrogenase [H]

[H]

NADH

NAD+

H+

N 2O

NO NIR

Q/QH2

e-

N2OR e-

NO2-

napA

nirS nirE nirC nirF

NOR

norF norE norD norQ norB norC

nosZ

nosD 1 kb

Cytoplasm

Control ZnO NPs

0

276

1000


120

d

100 80 60 40 20 0

Fe

Mo

2000 10000 Gene expression (RPKM)

Cu


120

Relative activity (% of control)

c

nitrate reductase narG nitrate reductase narH nitrate reductase narJ nitrate reductase narI nitrate reductase napA nitrite reductase nirS nitric oxide reductase norC nitric oxide reductase norB nitric oxide reductase norQ nitric oxide reductase norD nitric oxide reductase norE nitrous oxide reductase nosZ

Relative concentration (% of control)

b

napB napC

enosR

NO3-

narG

N2

100 80 60 40 20 0

NAR

NIR

NOR

N2OR

277

Figure 6. Effects of ZnO NPs on nitrate reduction and key denitrifying enzymes in P. denitrificans.

278

a, Schematic nitrate reduction pathway and gene clusters of the key denitrifying enzymes.8

279

show respectively the key gene expressions involved in nitrate reduction (b), relative concentrations of Fe,

280

Mo, and Cu ions in the medium (c), and relative activities of NAR, NIR, NOR, and N2OR (d) in response

281

to ZnO NPs. For b, c and d, data are shown as mean value ± standard deviation from three independent

282

measurements.

Student’s t-test, compared with the control, *P < 0.05.

15


b, c, and d


Page 16 of 21

283

Moreover, it has been reported in the literature that these denitrifying enzymes are metalloenzymes

284

containing Fe, Mo, or Cu ions,45-47 and the decreases of Fe and Cu ions in pure culture systems were

285

observed to decline the activities of NAR and NIR in model bacteria, such as Pseudomonas sp. and P.

286

denitrificans.48 Thus, these results suggested that the activities of key denitrifying enzymes are closely

287

related to the concentrations of these important metal ions.49, 50

288

usually has an excellent adsorption capability due to its higher specific surface area.

289

examined the concentrations of Fe, Mo, and Cu ions in the medium after exposure to ZnO NPs. It was

290

found that the presence of 50 mg/L ZnO NPs significantly decreased the concentrations of these trace

291

metal ions in the medium (Fig. 6c), which might be one reason for the inhibited activities of NAR, NIR,

292

NOR, and N2OR in P. denitrificans (Fig. 6d). Therefore, the presence of ZnO NPs could cause the

293

decreases in the gene expressions and catalytic activities of the key denitrifying enzymes, which finally

294

induced the declined nitrate reduction and large N2O emissions.

It is well-known that nanomaterial Hence, we

295

In summary, we have described the potential risks of ZnO NPs to nitrate removal and N2O emissions

296

during the microbial denitrification. Increasing release of ZnO NPs into the environment leads to lower

297

nitrate removal and higher N2O production, which might accelerate human-induced water eutrophication

298

and global warming.

299

intracellular substances (such as glucose and polyhydroxybutyrate) and inhibits the gene expressions and

300

catalytic activities of key denitrifying enzymes, which results in the negative effects on the reduction of

301

nitrate and N2O. These results offer an opportunity to develop approaches for the mitigation of ZnO

302

NPs-induced effects on microbial denitrification.

303

ACKNOWLEDGEMENTS

304

This work was supported by the National Hi-Tech Research and Development Program of China (Grant

Our data further indicate that the presence of ZnO NPs affects the metabolism of

16


Page 17 of 21


305

no. 2011AA060903), National Natural Science Fund for Distinguished Young Scholar (Grant no.

306

51425252), National Natural Science Foundation of China (Grant no. 41301558 and 51278354), and

307

Collaborative Innovation Center for Regional Environmental Quality.

308

SUPPORTING INFORMATION AVAILABLE

309

This file contains additional analytical methods, Tables S1 and S2, and Figures S1-S3. This information

310

is available free of charge via the Internet at http://pubs.acs.org/.

311

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TOC Art

437 Microbial denitrification:

Effects on transcriptional regulation Number of genes 600

NADH

NAD+

500

NO2-

[H]

N2OR

cellular component organization or biogenesis cellular process

e-

Q/QH2

[H]

400

Q/QH2

biological regulation

NIR e-

e-

NOR

developmental process establishment of localization

Biological process

NO3-

e-

H

N2

300

NAR

NADH dehydrogenase

N 2O

NO

+

200

H

+

0

NO2-

NO2- NO3-

100

Periplasm

growth localization locomotion metabolic process

Cytoplasm

multi-organism process regulation of biological process

Inhibition to nitrate reduction

240

160

4

8

12

Time (h)

16

20

24

signaling cell

Liquid phase Gas phase

15

cell part macromolecular complex membrane membrane part organelle organelle part

10

antioxidant activity binding catalytic activity

5

0

enzyme regulator activity molecular transducer activity nucleic acid binding transcription factor activit protein binding transcription factor activity

Control

1

10

50

ZnO NPs (mg/L)

438

21


structural molecule activity transporter activity

Up

0

response to stimulus

Down

0

Increase in N2O accumulation

Molecular function

80

20

Cellular component

N2O accumulation (% of TN)

320

Zinc Oxide Nanoparticles Cause Inhibition of ... - ACS Publications

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