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

Nov 10, 2014 - Over the past few decades, human activities have accelerated the rates and extents ... Denitrification by Affecting Transcriptional Reg...
0 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/est

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 State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China

Downloaded via TULANE UNIV on January 10, 2019 at 18:15:34 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: Over the past few decades, human activities have accelerated the rates and extents of water eutrophication and global warming through increasing delivery of biologically available nitrogen such as nitrate and large emissions of anthropogenic greenhouse gases. In particular, nitrous oxide (N2O) is one of the most important greenhouse gases, because it has a 300-fold higher global warming potential than carbon dioxide. Microbial denitrification is a major pathway responsible for nitrate removal, and also a dominant source of N2O emissions from terrestrial or aquatic environments. However, whether the release of zinc oxide nanoparticles (ZnO NPs) into the environment affects microbial denitrification is largely unknown. Here we show that the presence of ZnO NPs lead to great increases in nitrate delivery (9.8-fold higher) and N2O emissions (350- and 174-fold higher in the gas and liquid phases, respectively). Our data further reveal that ZnO NPs significantly change the transcriptional regulations of glycolysis and polyhydroxybutyrate synthesis, which causes the decrease in reducing powers available for the reduction of nitrate and N2O. Moreover, ZnO NPs substantially inhibit the gene expressions and catalytic activities of key denitrifying enzymes. These negative effects of ZnO NPs on microbial denitrification finally cause lower nitrate removal and higher N2O emissions, which is likely to exacerbate water eutrophication and global warming.

■

range of industrial applications and consumer products.10,11 The increasing manufacture and utilization of nanomaterials will accelerate their releases into terrestrial and aquatic environments,12−14 which might cause potential risks to humans and the environment.15,16 For example, the pervasive use of engineered nanomaterials has recently been regarded as a substantial threat to human health.10,11,17 Nevertheless, few studies have been concerned with the possible impacts of nanomaterials on environmental processes, especially microbial denitrification.18 To the best of our knowledge, it remains unclear at present whether the release of ZnO NPs into the environment causes negative effects on nitrate removal and N2O emissions. Here we report the potential influences of ZnO NPs on nitrate reduction and N2O emissions under denitrification conditions. During the exposure period, we identify a large set of differentially expressed genes (DEGs) involved in the intracellular metabolism and also measure the catalytic activities of the key enzymes related to glycolysis, polyhydroxybutyrate

INTRODUCTION Since nitrogen is an important limiting factor for plant growth, the wide use of artificial nitrogen fertilizer has made a significant contribution to global food production.1 Unfortunately, this behavior has led to some global environmental problems, such as water eutrophication and climate change.2−4 Over the past few decades, human activities have accelerated the rates and extents of these environmental changes through increasing delivery of biologically available nitrogen such as nitrate and large emissions of anthropogenic greenhouse gases.5,6 In particular, nitrous oxide (N2O) is one of the most important greenhouse gas because it has a 300-fold higher global warming potential than carbon dioxide4 and can cause stratospheric ozone destruction.7 It is already known that microbial denitrification is the primary pathway by which fixed nitrogen such as nitrate returns to the atmosphere from terrestrial and aquatic environments.8 Nevertheless, the incomplete denitrification can cause large N2O emissions, which might result in global warming and stratospheric ozone destruction.6 As a result, microbial denitrification is mainly responsible for nitrate removal8 and is also a dominant source of N2O emissions from the environments.9 Over the past decade, man-made nanomaterials, such as zinc oxide nanoparticles (ZnO NPs), have been used in a wide © 2014 American Chemical Society

Received: Revised: Accepted: Published: 13800

August 29, 2014 November 5, 2014 November 10, 2014 November 10, 2014 dx.doi.org/10.1021/es504251v | Environ. Sci. Technol. 2014, 48, 13800−13807

Environmental Science & Technology

Article

control) and presence of 50 mg/L ZnO NPs. Briefly, the cells were harvested at 16 h after exposure to ZnO NPs, followed by centrifugation at 10 000 rpm for 10 min at 4 °C, and then lysed in TRIzol reagent (Invitrogen) for extraction of total RNA. To avoid genomic DNA contamination, the extracted RNA was treated with DNase I (Ambion) according to the manufacturer’s protocol. Thereafter, mRNA was isolated from the DNA-free total RNA using the MICROBExpress Bacterial mRNA Enrichment Kit (Ambion), and prepared for Illumina sequencing using the mRNA-Seq Sample Preparation Kit (Illumina) according to manufacturer’s instructions. The RNASeq libraries were finally sequenced using an Illumina HiSeq 2000. All sequencing data have been submitted to the National Center for Biotechnology Information (NCBI) short-read archive (SRA) under accession numbers SRR953150 and SRR953225. The next generation sequencing (NGS) quality control (QC) toolkit v2.2.1 was used to filter the raw reads23 by removing the reads with (1) sequence adapters, (2) more than 5% “N” bases, and (3) more than 50% QA ≤ 15 bases. The obtained clean reads were aligned to the reference genome using SOAP2,24 and no more than 3 mismatches were allowed in the alignment for each read. The gene expression level was calculated using the RPKM (reads per kilobase of exon region per million mappable reads) method,25 and the differentially expressed genes were identified based on the criteria: absolute fold change > 2 and false discovery rate (FDR) < 0.05. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using Blast2GO using default annotation parameters.26 Interactive Pathways (ipath) analysis was carried out via interactive pathways explorer v2 (http://pathways.embl.de/). Enzyme Assays. The measurements of key enzymes activities can be used to examine the NPs-induced potential influences according to the literature.27,28 Briefly, after 24 h of exposure, cells were harvested by centrifugation (5000 rpm for 10 min), washed thrice with 0.1 M phosphate-buffered saline (PBS) (pH 7.4), and resuspended in the same buffer at 4 °C. Thereafter, crude cell extracts were prepared by disrupting cells for 1 min by sonication (4 °C, 20 kHz) followed by centrifugation at 16000 rpm for 10 min at 4 °C and were immediately used for determination of enzyme activities. The protein concentrations of cell extracts were measured using Protein Assay Kit (Bio-Rad) with bovine serum albumin (BSA) as a standard. Glucokinase (GK) activity was determined by measuring the formation of NADPH at 340 nm as described previously.29 The reaction mixture (a total volume of 1 mL) contained 100 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 0.5 mM NADP, 1 mM ATP, 10 mM glucose, 2 U glucose-6-phosphate dehydrogenase, and 50 μL of cell extract. Phosphofructokinase (PFK) activity was assayed by monitoring the decrease of NADH in absorbance at 340 nm as described in ref 30. The reaction mixture (1 mL) contained 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.15 mM NADH, 0.5 mM ATP, 5 mM fructose-6phosphate, 2 U each of aldolase, triosephosphate isomerase and glycerol-3-phosphate dehydrogenase, and 50 μL of cell extract. Pyruvate kinase (PK) activity was measured spectrophotometrically at 340 nm through the oxidation of NADH to NAD+.31 The reaction mixture (1 mL) contained 50 mM TrisHCl (pH 7.5), 10 mM MgCl2, 0.2 mM NADH, 5 mM ADP, 10 mM dithiothreitol, 1 mM fructose 1,6-diphosphate, 10 U lactate dehydrogenase, and 50 μL of cell extract.

(PHB) synthesis, and denitrification. The data from this work help elucidate the possible reasons for these negative effects of ZnO NPs on denitrifying bacteria. This mechanism will make it possible to find a proper way to mitigate the risks of engineered nanomaterials to water eutrophication and global warming.

■

MATERIALS AND METHODS Engineered ZnO NPs. In this study, ZnO NPs were purchased from Sigma-Aldrich (St. Louis, MO, USA). To characterize the NPs, X-ray diffraction (XRD) analysis was conducted using a Rigaku D/Max-RB diffractometer equipped with a rotating anode and a Cu Kα radiation source (Figure S1, Supporting Information), and a transmission electron microscope (TEM) image was taken by a Philips Tecnai F20 microscope at an accelerating voltage of 200 kV (Figure S2, Supporting Information). To prepare a stock suspension, 500 mg of ZnO NPs were dispersed in 1 L of Milli-Q water followed by ultrasonication (40 kHz and 250 W) for 1 h at room temperature as described previously.19 Dynamic light scattering (DLS) analysis via a Malvern Autosizer 4700 (Malvern Instruments, UK) showed that the typical sizes of particles in the stock suspension were in the range 80−100 nm. Cell Culture. Paracoccus denitrificans was obtained from ATCC (Manassas, VA, USA) and grown in Difco nutrient broth at 30 °C and 200 rpm to an optical density at 600 nm (OD600) of 0.8−1.0. P. denitrificans Exposed to ZnO NPs. To examine the possible effects of ZnO NPs on microbial denitrification, P. denitrificans was exposed to 0, 1, 10, and 50 mg/L ZnO NPs in a mineral medium modified from the literature.20 The mineral medium contained (per liter) 7.0 g K2HPO4, 3.0 g KH2PO4, 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 element feed was documented in our previous publication.19 Bacterial cultures were inoculated at an initial OD600 of 0.05 and anaerobically cultured in the dark at 30 °C and 200 rpm with different concentrations of ZnO NPs. The concentrations of NO3−, NO2−, N2O, glucose, and PHB were estimated at intervals of 4 h for a total time of 24 h. The measurements of NO3−, NO2−, glucose, and PHB were performed as described previously19,21 and detailed in the Supporting Information. The gaseous and dissolved N2O was analyzed using gas chromatography with an electron capture detector (ECD) according to the literature.22 Dissolution of ZnO NPs and P. denitrificans Exposed to Released Zinc Ions. The dissolution of ZnO NPs in the medium was determined according to our previous study.19 Briefly, a series of serum bottles that respectively contained 50 mL of the mineral medium with 0, 1, 10, and 50 mg/L ZnO NPs were shaken at 30 °C and 200 rpm. At different time points, ZnO NPs were removed by high speed centrifugation (16000 rpm) for 10 min, and 0.5 mL of the supernatant was added to 4.5 mL of Milli-Q water (containing 2% ultrahigh purity HNO3). The concentration of zinc ions in the resulting solution was measured by inductively coupled plasma mass spectrometry (ICP-MS, Agilent Technologies, USA). Then, P. denitrificans was exposed to the measured concentration of released zinc ions according to the method described in the P. denitrificans exposed to ZnO NPs section, and the final concentrations of NO3− and N2O were measured to determine the potential effects of released zinc ions on the denitrification process. RNA-Seq. The RNA-Seq analysis was used to determine the transcriptional profiling of P. denitrificans in the absence (the 13801

dx.doi.org/10.1021/es504251v | Environ. Sci. Technol. 2014, 48, 13800−13807

Environmental Science & Technology

Article

Figure 1. Effect of ZnO NPs on the denitrifying bacterium P. denitrificans. (a) TEM image and EDX measurement of P. denitrificans exposed to ZnO NPs. (b and c) Relative LDH release (an indicator of membrane damage) and bacterial growth curve in response to ZnO NPs, respectively. For b and c, data are shown as mean value ± standard deviation from three independent measurements.

Figure 2. Effect of ZnO NPs on the denitrification process of P. denitrificans. NO3− reduction (a), NO2− variation (b), and N2O accumulation (c) in response to ZnO NPs. The data are shown as mean value ± standard deviation from three independent measurements.

the reaction. After 30 min of incubation, the concentration of NO3−, NO2−, NO, or N2O was determined, and the specific enzyme activity was calculated. The detailed procedures of the measurements of NO 3 − , NO 2 − , NO, and N 2 O were documented in the Supporting Information. Analytical Method. Transmission electron microscopy analysis, lactate dehydrogenase release assay, and measurements of key trace metal ions in the absence and presence of ZnO NPs were documented in the Supporting Information. Statistical Analysis. All tests in this study were performed in triplicate. An analysis of variance was used to test the significance of results, and p < 0.05 was considered to be statistically significant.

Pyruvate dehydrogenase (PDH) activity was estimated spectrophotometrically at 340 nm by the pyruvate-dependent reduction of NAD+ according to the literature.32 The reaction mixture (1 mL) contained 100 mM M Tris-HCl (pH 7.5), 10 mM MgCl2, 1.2 mM NAD+, 6 mM dithiothreitol, 10 mM pyruvate, 0.12 mM CoA, and 50 μL of cell extract. Acetyl-CoA acetyltransferase (ACAT) activity was determined spectrophotometrically at 303 nm by measuring the decrease of acetoacetyl-CoA.33 The reaction mixture (1 mL) contained 0.1 M Tris-HCl (pH 8.3), 25 mM MgCl2, 50 mM KCl, 20 nM acetoacetyl-CoA, 20 nM CoA, and 50 μL of cell extract. The 3hydroxybutyryl-CoA dehydrogenase (Hbd) activity was assayed by determining the decrease in absorbance at 263 nm.34 The reaction mixture (1 mL) contained 100 mM Tris-HCl (pH 7.5), 30 μM crotonyl-CoA, and 50 μL of cell extract. PHA synthase activity (Pha) was measured spectrophotometrically at 412 nm by reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) as described previously.35 The reaction mixture (1 mL) contained 25 mM Tris-HCl (pH 7.5), 1 mM DTNB, 30 mM D-(−)-3-hydroxybutyryl-CoA, and 50 μL of cell extract. The measurements of nitrate reductase (NAR), nitrite reductase (NIR), nitric oxide reductase (NOR), and nitrous oxide reductase (N2OR) activities were performed through the consumption of electron acceptor (NO3−, NO2−, NO, or N2O) with methyl viologen as electron donor as described previously.36,37 Briefly, these denitrifying enzymes were assayed at 22 °C in anaerobic cuvettes with rubber stoppers. The cuvettes contained 2 mL of the reaction mixture (10 mM potassium phosphate buffer (pH 7.1), 10 mM methyl viologen, 5 mM Na2S2O4, 1 mM electron acceptor (NO3−, NO2−, NO, or N2O)). Then, 100 μL of the cell extract was injected to initiate

■

RESULTS AND DISCUSSION Bacterial Structure and Denitrification in the Presence of ZnO NPs. To determine the possible effects of ZnO NPs on microbial denitrification, we first examined the growth and metabolism of P. denitrificans (a model denitrifying bacterium) in the absence and presence of ZnO NPs. Transmission electron microscopy (TEM) images showed that ZnO NPs in the medium adhered to the cell membrane of P. denitrificans (Figure 1a). Using lactate dehydrogenase (LDH) release as an indicator of membrane damage, we found that the LDH releases in the absence and presence of ZnO NPs had no significant differences (P > 0.05, Student’s t-test) (Figure 1b), suggesting that these NPs did not change the membrane integrity of P. denitrificans. It can be also observed that the presence of ZnO NPs caused no negative effects on the bacterial population density during 24 h of exposure (P > 0.05, Student’s t-test) (Figure 1c). 13802

dx.doi.org/10.1021/es504251v | Environ. Sci. Technol. 2014, 48, 13800−13807

Environmental Science & Technology

Article

Figure 3. Effects of released zinc ions on the denitrification process due to the dissolution of ZnO NPs. (a) Dissolution curves of ZnO NPs in the culture medium during 24 h of exposure. (b) Concentrations of NO3−−N and total N2O in the absence and presence of ZnO NPs and its released zinc ions at the end of exposure. The data are shown as mean value ± standard deviation from three independent measurements.

Figure 4. GO functional enrichment analysis of the DEGs in response to 50 mg/L ZnO NPs. The genes that are up-regulated and down-regulated are shown in green and red, respectively.

Further studies indicated that increasing ZnO NPs concentrations caused the inhibitory effects on the denitrification process. After 24 h of exposure, the final concentration of NO3−−N was remarkably greater in the presence of 50 mg/L ZnO NPs than its absence (42.1 versus 3.9 mg/L) (Figure 2a), although little NO2−−N was measured in effluent no matter whether ZnO NPs were present or not (Figure 2b). Meanwhile, we observed large changes in the N2O production during exposure to different concentrations of ZnO NPs. The average N2O concentrations in both gas and liquid phases were very low (0.04% and 0.02% of total nitrogen (TN), respectively) in the absence of ZnO NPs (the control). In contrast, those concentrations were 13.96% and 3.48% of TN in the presence

of 50 mg/L ZnO NPs, which were approximately 350- and 174fold higher than those of the control, respectively (Figure 2c). Clearly, our data indicated that increasing release of ZnO NPs into the environment not only inhibited the nitrate reduction but also led to the significant increases in N2O emissions. Generally, nanomaterials can easily lead to the reactive oxygen species (ROS) production, because of their extremely small sizes and high catalytic activities.10 This behavior might induce the changes in membrane integrity and cell morphology of living organisms.38,39 However, the presence of ZnO NPs in the culture medium did not affect the surface integrity and viability of P. denitrificans (Figure 1). This phenomenon might be due to the absence of oxygen in this study (anoxic 13803

dx.doi.org/10.1021/es504251v | Environ. Sci. Technol. 2014, 48, 13800−13807

Environmental Science & Technology

Article

Figure 5. Effects of ZnO NPs on the glucose degradation and polyhydroxybutyrate (PHB) synthesis of P. denitrificans. (a) Schematic metabolic pathways of intracellular substances (mainly glucose degradation and PHB synthesis) in P. denitrificans. Key gene expressions involved in glycolysis (b), relative glucose degradation efficiency (c), relative activities of GK, PFK, and PK (d), key gene expressions involved in PHB synthesis (e), relative PHB content (f), and relative activities of PDH, ACAT, Hbd, and PhaC (g) in response to ZnO NPs. For c, d, f, and g, data are shown as mean value ± standard deviation from three independent measurements. Student’s t-test, compared with the control, *P < 0.05.

presence of 50 mg/L ZnO NPs, because bacterial growth and metabolism are closely related to the changes in transcriptional regulation. It is well-known that the high-throughput RNA sequencing (RNA-Seq) is a highly accurate technology for quantifying expression levels of transcripts under different conditions.41,42 Using this method, we obtained a total number of 26 774 234 high-quality reads after raw sequences processing and filtering (Table S1, Supporting Information). After mapping these clean reads to the reference genome of P. denitrificans, we found that 95.4% of the total reads matched to the reference genome and 89.8% matched to unique genomic locations (Table S1, Supporting Information). By comparing with the control, we identified 1714 genes that displayed transcriptional fluctuation in response to 50 mg/L ZnO NPs (considering absolute fold change > 2 and FDR < 0.05). These differentially expressed genes (DGEs) were subsequently used for functional annotation and enrichment analysis, and were further divided into three primary categories of Gene Ontology (GO) classification: biological process, cellular component, and molecular function. Although no obvious changes in metabolic pathways were found in the presence of ZnO NPs (Figure S3, Supporting Information), the gene expressions involved in 29 functional subcategory

conditions), since ROS is generated primarily by the reduction of molecular oxygen.11 On the other hand, ZnO NPs are slightly soluble in water, and thus can release zinc ions under neutral condition. Figure 3a showed that the average stable concentrations of released zinc ions in the presence of 1, 10, and 50 mg/L ZnO NPs were 0.65, 3.81, and 6.47 mg/L, respectively. It was further found that the presence of 3.81 and 6.47 mg/L zinc ions caused the increases in the final concentrations of NO3−−N and total N2O (Figure 3b), suggesting that the release of zinc ions was one of the important reasons for the negative effects of ZnO NPs on microbial denitrification. Many investigations indicate that the released zinc ions are mainly responsible for the toxicity of ZnO NPs to model organisms.38,40 Our previous study also found that the dissolution of ZnO NPs could inhibit biological nitrogen and phosphorus removal in wastewater treatment plants.19 Nevertheless, to date, the mechanism of ZnO NPsinduced inhibition to microbial denitrification is poorly understood, and needs to be investigated. Transcriptional Profiling of P. denitrificans in Response to ZnO NPs. To understand how ZnO NPs affect microbial denitrification, we investigated the genome-wide transcriptional profiling of P. denitrificans in the absence and 13804

dx.doi.org/10.1021/es504251v | Environ. Sci. Technol. 2014, 48, 13800−13807

Environmental Science & Technology

Article

Figure 6. Effects of ZnO NPs on nitrate reduction and key denitrifying enzymes in P. denitrificans. (a) Schematic nitrate reduction pathway and gene clusters of the key denitrifying enzymes.8 Key gene expressions involved in nitrate reduction (b), relative concentrations of Fe, Mo, and Cu ions in the medium (c), and relative activities of NAR, NIR, NOR, and N2OR (d) in response to ZnO NPs. For b, c, and d, data are shown as mean value ± standard deviation from three independent measurements. Student’s t-test, compared with the control, *P < 0.05.

ZnO NPs concentrations significantly enhanced the genes expressions (Figure 5e) and relative activities of PDH, ACAT, Hbd, and PhaC (Figure 5g), which was mainly responsible for the higher PHB contents in P. denitrificans exposed to ZnO NPs (Figure 5f). Hence, these results revealed that the enhanced PHB accumulation could compete for the electrons used for microbial denitrification, which resulted in the negative effects on the reduction of nitrate and N2O. Microbial denitrification is very much dependent on the expressions and activities of four essential denitrifying enzymes: nitrate reductase (NAR), nitrite reductase (NIR), nitric oxide reductase (NOR), and nitrous oxide reductase (N2OR), which are encoded by the gene clusters of narGHJI (encoding respiratory nitrate reductase), napABC (encoding periplasmic nitrate reductase), nirSECF, norCBQDEF, and nosRZD, respectively (Figure 6a). Figure 6b displays that the gene expressions of these denitrifying enzymes are substantially down-regulated during exposure to 50 mg/L ZnO NPs, which was adverse to the reduction of nitrate and N2O. It can be found that the down-regulated folds for narG, napA, nirS, norB, and nosZ were 2.8, 0.3, 4.2, 2.3, and 23.7, respectively. Clearly, these results showed that the down-regulated fold for nos operon was much greater than the other operons, which might be an important reason for the observed N2O accumulation. Moreover, it has been reported in the literature that these denitrifying enzymes are metalloenzymes containing Fe, Mo, or Cu ions,45−47 and the decreases of Fe and Cu ions in pure culture systems were observed to decline the activities of NAR and NIR in model bacteria, such as Pseudomonas sp. and P. denitrificans.48 Thus, these results suggested that the activities of key denitrifying enzymes are closely related to the concentrations of these important metal ions.49,50 It is well-known that nanomaterial usually has an excellent adsorption capability due to its higher specific surface area. Hence, we examined the concentrations of Fe, Mo, and Cu ions in the medium after exposure to ZnO NPs. It was found that the presence of 50 mg/L ZnO NPs significantly decreased the concentrations of these trace metal ions in the medium (Figure 6c), which might

annotations showed large differences, especially the annotations of catalytic activity, binding, antioxidant activity, molecular transducer activity, nucleic acid binding, transcription factor activity, and transporter activity (Figure 4). Hence, these results suggested that the presence of ZnO NPs was able to cause substantial influences on cellular regulation and metabolism of P. denitrificans. Gene Expressions and Catalytic Activities of Key Enzymes Involved in Denitrification. Previous studies have confirmed that microbial denitrification needs to degrade organic matters to supply electrons, which are essential for the complete reduction of nitrate to N2.8 Hence, in this study, the glucose metabolism plays an important role in the reduction of nitrate and N2O involved in microbial denitrification (Figure 5a). We analyzed the transcriptional levels of key genes related to glycolysis in the absence and presence of 50 mg/L ZnO NPs (Figure 5b). It can be found that the gene expressions of these vital enzymes, such as glucokinase (GK), phosphofructokinase (PFK), and pyruvate kinase (PK), were significantly downregulated in the presence of ZnO NPs. This might be an important reason for the decreased glucose degradation efficiency after 24 h of exposure to ZnO NPs (Figure 5c). In particular, GK, PFK, and PK catalyze the irreversible reactions in glycolysis, and thus are regarded as the most important enzymes relevant to glucose degradation.43 We found that the activities of GK, PFK, and PK decreased dramatically with increasing ZnO NPs concentrations (Figure 5d). Our data showed that the presence of ZnO NPs suppressed glucose degradation due to significant decreases in the gene expressions and catalytic activities of key enzymes involved in glycolysis. It should be noted that P. denitrificans can accumulate the intracellular polyhydroxybutyrate (PHB), and this process consumes the electrons and energy produced by glucose degradation.44 Figure 5a shows that pyruvate dehydrogenase (PDH), acetyl-CoA acetyltransferase (ACAT), 3-hydroxybutyryl-CoA dehydrogenase (Hbd), 3-hydroxybutyryl-CoA epimerase, and polyhydroxyalkanoate synthase (PhaC) play essential roles in the PHB formation. We found that increasing 13805

dx.doi.org/10.1021/es504251v | Environ. Sci. Technol. 2014, 48, 13800−13807

Environmental Science & Technology

Article

L. W.; Dahm, C. N.; Dodds, W. K.; Grimm, N. B.; Johnson, S. L.; McDowell, W. H.; Poole, G. C.; Valett, H. M.; Arango, C. P.; Bernot, M. J.; Burgin, A. J.; Crenshaw, C. L.; Helton, A. M.; Johnson, L. T.; O’Brien, J. M.; Potter, J. D.; Sheibley, R. W.; Sobota, D. J.; Thomas, S. M. Nitrous oxide emission from denitrification in stream and river networks. Proc. Natl. Acad. Sci. USA 2011, 108, 214−219. (10) Colvin, V. L. The potential environmental impact of engineered nanomaterials. Nat. Biotechnol. 2003, 21, 1166−1170. (11) Nel, A.; Xia, T.; Madler, L.; Li, N. Toxic potential of materials at the nanolevel. Science 2006, 311, 622−627. (12) Wiesner, M. R.; Lowry, G. V.; Alvarez, P.; Dionysiou, D.; Biswas, P. Assessing the risks of manufactured nanomaterials. Environ. Sci. Technol. 2006, 40, 4336−4345. (13) Klaine, S. J.; Alvarez, P. J. J.; Batley, G. E.; Fernandes, T. F.; Handy, R. D.; Lyon, D. Y.; Mahendra, S.; McLaughlin, M. J.; Lead, J. R. Nanomaterials in the environment: Behavior, fate, bioavailability, and effects. Environ. Toxicol. Chem. 2008, 27, 1825−1851. (14) Gottschalk, F.; Sonderer, T.; Scholz, R. W.; Nowack, B. Modeled environmental concentrations of engineered nanomaterials (TiO2, ZnO, Ag, CNT, Fullerenes) for different regions. Environ. Sci. Technol. 2009, 43, 9216−9222. (15) Setyawati, M. I.; Tay, C. Y.; Chia, S. L.; Goh, S. L.; Fang, W.; Neo, M. J.; Chong, H. C.; Tan, S. M.; Loo, S. C. J.; Ng, K. W.; Xie, J. P.; Ong, C. N.; Tan, N. S.; Leong, D. T. Titanium dioxide nanomaterials cause endothelial cell leakiness by disrupting the homophilic interaction of VE-cadherin. Nat. Commun. 2013, 4, 1673. (16) Brayner, R.; Ferrari-Iliou, R.; Brivois, N.; Djediat, S.; Benedetti, M. F.; Fievet, F. Toxicological impact studies based on Escherichia coli bacteria in ultrafine ZnO nanoparticles colloidal medium. Nano Lett. 2006, 6, 866−870. (17) Oberdorster, G.; Oberdorster, E.; Oberdorster, J. Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles. Environ. Health Perspect. 2005, 113, 823−839. (18) Yang, Y.; Zhu, H.; Colvin, V. L.; Alvarez, P. J. Cellular and transcriptional response of Pseudomonas stutzeri to quantum dots under aerobic and denitrifying conditions. Environ. Sci. Technol. 2011, 45, 4988−4994. (19) Zheng, X.; Wu, R.; Chen, Y. Effects of ZnO nanoparticles on wastewater biological nitrogen and phosphorus removal. Environ. Sci. Technol. 2011, 45, 2826−2832. (20) Blaszczyk, M. Effect of medium composition on the denitrification of nitrate by Paracoccus denitrif icans. Appl. Environ. Microbiol. 1993, 59, 3951−3953. (21) Jiang, Y.; Chen, Y.; Zheng, X. Efficient polyhydroxyalkanoates production from a waste-activated sludge alkaline fermentation liquid by activated sludge submitted to the aerobic feeding and discharge process. Environ. Sci. Technol. 2009, 43, 7734−7741. (22) Rosamond, M. S.; Thuss, S. J.; Schiff, S. L. Dependence of riverine nitrous oxide emissions on dissolved oxygen levels. Nat. Geosci. 2012, 5, 715−718. (23) Patel, R. K.; Jain, M. NGS QC toolkit: A toolkit for quality control of next generation sequencing data. PLoS One 2012, 7, e30619. (24) Li, R.; Yu, C.; Li, Y.; Lam, T. W.; Yiu, S.; Kristiansen, K.; Wang, J. SOAP2: An improved ultrafast tool for short read alignment. Bioinformatics 2009, 25, 1966−1967. (25) Mortazavi, A.; Williams, B. A.; Mccue, K.; Schaeffer, L.; Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 2008, 5, 621−628. (26) Conesa, A.; Gotz, S.; Garcia-Gomez, J. M.; Terol, J.; Talon, M.; Robles, M. Blast2GO: A universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 2005, 21, 3674−3676. (27) Wigginton, N. S.; De Titta, A.; Piccapietra, F.; Dobias, J.; Nesatty, V. J.; Suter, M. J. F.; Bernier-Latmani, R. Binding of silver nanoparticles to bacterial proteins depends on surface modifications and inhibits enzymatic activity. Environ. Sci. Technol. 2010, 44, 2163− 2168. (28) Schug, H.; Isaacson, C. W.; Sigg, L.; Ammann, A. A.; Schirmer, K. Effect of TiO2 nanoparticles and UV radiation on extracellular

be one reason for the inhibited activities of NAR, NIR, NOR, and N2OR in P. denitrificans (Figure 6d). Therefore, the presence of ZnO NPs could cause the decreases in the gene expressions and catalytic activities of the key denitrifying enzymes, which finally induced the declined nitrate reduction and large N2O emissions. In summary, we have described the potential risks of ZnO NPs to nitrate removal and N2O emissions during the microbial denitrification. Increasing release of ZnO NPs into the environment leads to lower nitrate removal and higher N2O production, which might accelerate human-induced water eutrophication and global warming. Our data further indicate that the presence of ZnO NPs affects the metabolism of intracellular substances (such as glucose and polyhydroxybutyrate) and inhibits the gene expressions and catalytic activities of key denitrifying enzymes, which results in the negative effects on the reduction of nitrate and N2O. These results offer an opportunity to develop approaches for the mitigation of ZnO NPs-induced effects on microbial denitrification.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional analytical methods, Tables S1 and S2, and Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org/.

■

AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 21 65981263. Fax: +86 21 65986313. E-mail address: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the National Hi-Tech Research and Development Program of China (Grant no. 2011AA060903), National Natural Science Fund for Distinguished Young Scholar, National Natural Science Foundation of China (Grant no. 41301558 and 51278354), and Collaborative Innovation Center for Regional Environmental Quality.

■

REFERENCES

(1) Erisman, J. W.; Sutton, M. A.; Galloway, J.; Klimont, Z.; Winiwarter, W. How a century of ammonia synthesis changed the world. Nat. Geosci. 2008, 1, 636−639. (2) Gruber, N.; Galloway, J. N. An Earth-system perspective of the global nitrogen cycle. Nature 2008, 451, 293−296. (3) Davidson, E. A. The contribution of manure and fertilizer nitrogen to atmospheric nitrous oxide since 1860. Nat. Geosci. 2009, 2, 659−662. (4) Canfield, D. E.; Glazer, A. N.; Falkowski, P. G. The evolution and future of Earth’s nitrogen cycle. Science 2010, 330, 192−196. (5) McIsaac, G. F.; David, M. B.; Gertner, G. Z.; Goolsby, D. A. Nitrate flux in the Mississippi river. Nature 2001, 414, 166−167. (6) Wuebbles, D. J. Nitrous oxide: No laughing matter. Science 2009, 326, 56−57. (7) Ravishankara, A. R.; Daniel, J. S.; Portmann, R. W. Nitrous oxide (N2O): The dominant ozone-depleting substance emitted in the 21st century. Science 2009, 326, 123−125. (8) Zumft, W. G. Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev. 1997, 61, 533−616. (9) Beaulieu, J. J.; Tank, J. L.; Hamilton, S. K.; Wollheim, W. M.; Hall, R. O.; Mulholland, P. J.; Peterson, B. J.; Ashkenas, L. R.; Cooper, 13806

dx.doi.org/10.1021/es504251v | Environ. Sci. Technol. 2014, 48, 13800−13807

Environmental Science & Technology

Article

generation by bacterial nitric oxide reductase. Science 2010, 330, 1666−1670. (47) Brown, K.; Tegoni, M.; Prudencio, M.; Pereira, A. S.; Besson, S.; Moura, J. J.; Moura, I.; Cambillau, C. A novel type of catalytic copper cluster in nitrous oxide reductase. Nat. Struct. Biol. 2000, 7, 191−195. (48) Granger, J.; Ward, B. B. Accumulation of nitrogen oxides in copper-limited cultures of denitrifying bacteria. Limnol. Oceanogr. 2003, 48, 313−318. (49) Moura, I.; Moura, J. J. G. Structural aspects of denitrifying enzymes. Curr. Opin. Chem. Biol. 2001, 5, 168−175. (50) Labbe, N.; Parent, S.; Villemur, R. Addition of trace metals increases denitrification rate in closed marine systems. Water Res. 2003, 37, 914−920.

enzyme activity of intact heterotrophic biofilms. Environ. Sci. Technol. 2014, 48, 11620−11628. (29) Dols, M.; Chraibi, W.; RemaudSimeon, M.; Lindley, N. D.; Monsan, P. F. Growth and energetics of Leuconostoc mesenteroides NRRL B-1299 during metabolism of various sugars and their consequences for dextransucrase production. Appl. Environ. Microbiol. 1997, 63, 2159−2165. (30) Alice, A. F.; Perez-Martinez, G.; Sanchez-Rivas, C. Existence of a true phosphofructokinase in Bacillus sphaericus: Cloning and sequencing of the pf k gene. Appl. Environ. Microbiol. 2002, 68, 6410−6415. (31) Sridhar, J.; Eiteman, M. A.; Wiegel, J. W. Elucidation of enzymes in fermentation pathways used by Clostridium thermosuccinogenes growing on inulin. Appl. Environ. Microbiol. 2000, 66, 246−251. (32) Pauling, D. C.; Lapointe, J. P.; Paris, C. M.; Ludwig, R. A. Azorhizobium caulinodans pyruvate dehydrogenase activity is dispensable for aerobic but required for microaerobic growth. Microbiology 2001, 147, 2233−2245. (33) Tanaka, T.; Shima, Y.; Ogawa, N.; Nagayama, K.; Yoshida, T.; Ohmachi, T. Expression, identification and purification of dictyostelium acetoacetyl-CoA thiolase expressed in Escherichia coli. Int. J. Biol. Sci. 2011, 7, 9−17. (34) Bond-Watts, B. B.; Bellerose, R. J.; Chang, M. C. Y. Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways. Nat. Chem. Biol. 2011, 7, 222−227. (35) Valentin, H. E.; Steinbuchel, A. Application of enzymatically synthesized short-chain-length hydroxy fatty-acid coenzyme-A thioesters for assay of polyhydroxyalkanoic acid synthases. Appl. Microbiol. Biotechnol. 1994, 40, 699−709. (36) Kristjansson, J. K.; Hollocher, T. C. First practical assay for soluble nitrous oxide reductase of denitrifying bacteria and a partial kinetic characterization. J. Biol. Chem. 1980, 255, 704−707. (37) Zhu, X.; Chen, Y. Reduction of N2O and NO generation in anaerobic-aerobic (low dissolved oxygen) biological wastewater treatment process by using sludge alkaline fermentation liquid. Environ. Sci. Technol. 2011, 45, 2137−2143. (38) Xia, T.; Kovochich, M.; Liong, M.; Madler, L.; Gilbert, B.; Shi, H. B.; Yeh, J. I.; Zink, J. I.; Nel, A. E. Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. ACS Nano 2008, 2, 2121− 2134. (39) Kocbek, P.; Teskac, K.; Kreft, M. E.; Kristl, J. Toxicological aspects of long-term treatment of keratinocytes with ZnO and TiO2 nanoparticles. Small 2010, 6, 1908−1917. (40) Franklin, N. M.; Rogers, N. J.; Apte, S. C.; Batley, G. E.; Gadd, G. E.; Casey, P. S. Comparative toxicity of nanoparticulate ZnO, bulk ZnO, and ZnCl2 to a freshwater microalga (Pseudokirchneriella subcapitata): The importance of particle solubility. Environ. Sci. Technol. 2007, 41, 8484−8490. (41) Nagalakshmi, U.; Wang, Z.; Waern, K.; Shou, C.; Raha, D.; Gerstein, M.; Snyder, M. The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 2008, 320, 1344−1349. (42) Wang, Z.; Gerstein, M.; Snyder, M. RNA-Seq: A revolutionary tool for transcriptomics. Nat. Rev. Genet. 2009, 10, 57−63. (43) Saltiel, A. R.; Kahn, C. R. Insulin signalling and the regulation of glucose and lipid metabolism. Nature 2001, 414, 799−806. (44) Ueda, S.; Yabutani, T.; Maehara, A.; Yamane, S. Molecular analysis of the poly(3-hydroxyalkanoate) synthase gene from a methylotrophic bacterium Paracoccus denitrif icans. J. Bacteriol. 1996, 178, 774−779. (45) Butler, C. S.; Charnock, J. M.; Bennett, B.; Sears, H. J.; Reilly, A. J.; Ferguson, S. J.; Garner, C. D.; Lowe, D. J.; Thomson, A. J.; Berks, B. C.; Richardson, D. J. Models for molybdenum coordination during the catalytic cycle of periplasmic nitrate reductase from Paracoccus denitrif icans derived from EPR and EXAFS spectroscopy. Biochemistry 1999, 38, 9000−9012. (46) Hino, T.; Matsumoto, Y.; Nagano, S.; Sugimoto, H.; Fukumori, Y.; Murata, T.; Iwata, S.; Shiro, Y. Structural basis of biological N2O 13807

dx.doi.org/10.1021/es504251v | Environ. Sci. Technol. 2014, 48, 13800−13807