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The effects of graphene oxide on nitrification and N2O emission: Dose and exposure time dependent
Environmental Pollution 252 (2019) 960e966
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Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
The effects of graphene oxide on nitrification and N2O emission: Dose and exposure time dependent Nan Zhou a, Zhirong Zhao a, Huihui Wang a, Xiangyu Chen a, Mingyuan Wang a, Shishi He a, Wen Liu b, Maosheng Zheng a, * a
College of Environmental Science and Engineering, North China Electric Power University, The Key Laboratory of Resources and Environmental Systems Optimization, Ministry of Education, Beijing, 102206, China College of Environmental Sciences and Engineering, Peking University, The Key Laboratory of Water and Sediment Sciences, Ministry of Education, Beijing, 100871, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 February 2019 Received in revised form 9 May 2019 Accepted 3 June 2019 Available online 10 June 2019
With the extensive application of graphene oxide (GO), its leakage and release into wastewater treatment plants become inevitable. However, the toxicity of graphene oxide (GO) on nitrification process and the underlying mechanisms still remain unclear. In this study, the toxic effects of GO at concentration of 10 and 100 mg/L in 4 h and 10 days were evaluated with sealed reactors operated in sequencing batch mode. In the initial 4 h, both GO concentrations showed no negative effect on nitrogen conversion. However, the exposure to 100 mg/L GO significantly weakened the NHþ 4-N and NO- 2-N conversion capabilities and intensified the nitrous oxide (N2O) generation after 10 days. Extracellular polymeric substance (EPS) analysis suggested that 100 mg/L GO decreased the protein content of the nitrifying activated sludge. Moreover, reactive oxygen species (ROS) level was promoted by 100 mg/L GO owing to the impaired endogenous antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT), which caused oxidative stress to bacteria. Finally, quantitative PCR results confirmed that nitriteoxidizing bacteria (NOB) and complete ammonia oxidizing bacteria (CAOB) were more sensitive to GO, which was the primary cause for the significant promotion of N2O generation in the high GO concentration. This study offered new insights in the toxicity of GO on nitrification and N2O generation in the terms of dose and exposure time. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Graphene oxide Nitrification Nitrous oxide Comammox
1. Introduction Graphene oxide (GO) has received wide attention owing to its distinctive physiochemical properties including outstanding compressibility (Chen and Chen, 2018), ideal compatibility in biological behaviors (Chung et al., 2013), superior adsorption capacity (Alqadami et al., 2017) and high thermal conductivities (Chen et al., 2018). Graphene productions market is projected to be worth approximately $1.3 billion by 2023 (Mcwilliams, 2011). Ubiquitous commercial or industrial applications would inevitably result in the exposure and release of these nanomaterials, and eventually accumulate in wastewater treatment plant (Kuhlbusch et al., 2011). Thus, stringent exposure assessments of wastewater disposal
* Corresponding author. Main Building G619, North China Electric Power University, Beijing, 102206, China. E-mail address: [email protected] (M. Zheng). https://doi.org/10.1016/j.envpol.2019.06.009 0269-7491/© 2019 Elsevier Ltd. All rights reserved.
process to GO must be taken into account. Nitrification is the central and rate-limited process in biological nitrogen removal (BNR) in wastewater treatment plant (WWTP), which is mainly contributed by diverse groups of microorganisms including ammonia-oxidizing microbes (AOM), nitrite-oxidizing bacteria (NOB) and complete ammonia oxidizing bacteria (CAOB) (Daims et al., 2015; Zheng et al., 2017). Nitrous oxide (N2O) is one of the most significant greenhouse gases with 298 times of global warming potential than CO2 (Mannina et al., 2018) and BNR is the primary source of N2O generation in WWTP (Mampaey et al., 2016). Toxic substances in receiving water can weaken the activity of these microorganisms and reduce the nitrogen removal efficiency (Zheng et al., 2019; Liu et al., 2019) and even accelerate the generation of nitrous oxide (N2O) which can result in the aggravation of greenhouse effect and depletion of ozone layer (Ahn et al., 2010). The antibacterial activity of GO could raise the oxidative stress and membrane stress by direct contact with bacterial cultures (Liu et al., 2011). Apart from this, low concentration of GO exposure
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could also lead to the differential expression of functionalized proteins and eventually result in cell death (Guo and Mei, 2014; Lv et al., 2018). Acute exposure to GO concentration at 50 ~ 300 mg/L could be accumulated inside the activated sludge and deteriorate the effluent quality through impacting the water turbidity and sludge dewaterability (Ahmed and Rodrigues, 2013). However, existing reports also argued that nano-GOs functionalized with polyethylene glycol was a positive regulator to stimulate the growth of E. coli by enhancing the production of recombinant proteins (Luo et al., 2016). Moreover, 0.06 g/L of GO could instantly improve the bioactivity of both ammonia-oxidizing bacteria (AOB) and NOB in 4 h, posing negligible negative effect on microbial functions of activated sludge (Guo et al., 2018). According to all, the discrepancy of GO toxicity mainly depends on exposure time and GO dose. However, the potential toxicity of GO to nitrifying bacteria under long-term exposure time still remains unclear. Considering the easy combination and coexistence of GO and activated sludge, the risk of long exposure of activated sludge to GO is essential to be evaluated. In this study, the effect of 10 mg/L and 100 mg/L GO on nitrification process and N2O generation was successively evaluated. Moreover, the underlying mechanisms of GO toxicity were explained by analyzing the reactive oxygen species (ROS), extracellular polymeric substance (EPS) and metabolic activity of the nitrifying activated sludge. Finally, the total and the nitrifying bacteria (AOB, NOB and CAOB) was quantified to reveal the variation of the bacterial abundance under the exposure of GO. 2. Materials and methods 2.1. Preparation and characterization of GO GO was made from the flake graphite based on modified Hummers' method (Chen et al., 2015). The concentration of dispersed GO solution was measured at 3.75 g/L. The typical morphology of GO was observed by Scanning Electron Microscopy (Hitachi, Japan) and Transmission Electron Microscope (JEOL-2100, Japan). Moreover, the molecular structure and functional groups were also identified by X-ray Diffraction (Rikagu-XDS-2000, Japan) and Fourier Transform Infrared (Nicolet 8700, USA). The characterization results can be found in Supplementary Material. 2.2. Batch experiments setting Nitrifying activated sludge was taken from a membrane bioreactor (MBR) which has been stably operated in laboratory for more than one year and equally divided into nine sealed shaking bottles with working volume of 300 ml. 10 mL nutrient medium was supplemented into each bottle to start the reaction containing 1.00 g/L K2HPO4, 0.10 g/L MgCl2$6H2O, 0.02 g/L CaCl2$H2O, 0.005 g/ L FeSO4$7H2O, 0.50 g/L KCl, 0.80 g/L NaHCO3 and 1.21 g/L NH4Cl. The mixed liquid suspended solids (MLSS) was about 1700 mg/L and the initial NHþ 4-N concentration was approximately 20 mg/L. Three GO concentrations (0 mg/L, 10 mg/L and 100 mg/L) were triply set in a total of nine bottles. NHþ 4-N, NO- 2-N and NO- 3-N concentrations were measured each hour in the first cycle until NHþ 4-N and NO- 2-N were completely oxidized. Following cycles were manually operated in sequencing batch reactor (SBR) mode with each cycle of 8 h including 6 h of shaking and 2 h of idleness. 10 mL of liquid supernatant was replaced by new nutrient medium before the new cycle to keep the total volume at 160 mL in the shaking bottles. The experiment was operated for ten days and the gas samples were retained in the end of each cycle every two days. N2O concentration was detected using a gas chromatography equipped with an electron capture detector (GC-6890N, Agilent, USA). The
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N2O emission factors were calculated by the percentage of N2O emission accounting for the removed NHþ 4-N amount in one cycle (Zheng et al., 2019). Sludge from the 1 st cycle in the 1 st day and the 30 th cycle in the 10 th day was sampled and stored for further analysis. 2.3. EPS extraction and analysis EPS was extracted by the modified heating method (Adav and Lee, 2008; C.F.Forster, 1971). 5 mL of activated sludge in 1 st cycle and 30 th cycle were taken from the shaking bottles. Sludge samples were centrifuged at 10000g for 20 min, the supernatant was filtered through 0.22 mm to achieve soluble EPS (SEPS). The precipitate was re-suspended with 5 mL of 0.85% NaCl solution and heated in water bath at 80 C for 30 min, then repeating the above procedure to gain bound EPS (BEPS). Total EPS was the sum of SEPS and BEPS (Nielsen et al., 1997). As the two main compositions of EPS, polysaccharides (PS) content in both SEPS and BEPS was measured by the Anthrone method and the protein (PN) content was detected by TGem Spectrophotometer (Tiangen OSE-260, China) using glucose and bovine serum albumin (BSA) as the respective standards. 2.4. Measurement of ROS production and antioxidant activities Overproduction of ROS was regarded as the main cause of oxidative stress, leading to the disruption of the homeostasis inside the cells (Fu et al., 2014). In this study, intracellular ROS measurement was carried out by chemical fluorescence method with 2, 7dichlorofuorescin diacetate (DCFH-DA) as the fluorescence probe (Nanjing Jiancheng Bioengineering Institute, China). DCFH-DA can be hydrolyzed by intracellular esterase to dichlorofluorescin (DCFH), and then be oxidized to dichlorofluorescein (DCF) inside the cells, producing strong green fluorescence. 10 mm of DCFH-DA was added to 2 mL of activated sludge samples, incubating in water bath at 37 C for 30 min. The mixture was washed three times by phosphate buffer saline (PBS) before detection. Fluorescence intensity was measured using a fluorescence spectrometer (PerkinElmer, LS 55, USA) at emission wavelength of 527 nm and excitation wavelength of 488 nm. The enzymatic activities of superoxide dismutase (SOD) and catalase (CAT) from activated sludge under different GO exposure time were determined by assay kit purchased from Nanjing Jiancheng Bioengineering Institute. The enzymatic activities were normalized against the protein content according to a previous study (Wu et al., 2018). 2.5. DNA extraction and real-time quantitative PCR (qPCR) Activated sludge samples were extracted from the bottles in 4 h and 10 d and stored at 20 C before DNA extraction. A total of 2 mL samples were used for DNA extraction using FastDNA™ Spin Kit for Soil (MPbio, USA) according to the instruction manual. The abundances of AOB, CAOB, NOB and 16S rRNA were quantified with StepOne™ Real-time PCR System (Applied Biosystems, USA). Primer pairs and amplifying conditions for qPCR referred to previous studies (Pester et al., 2012; Wang et al., 2018a). 2.6. Statistical analysis All the results were expressed as mean ± SD values. The statistical analyses were conducted by Statistical Product and Service Solution (SPSS) software (IBM Inc. USA). Values of P < 0.05 were considered to be statistically significant.
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3. Results and discussion 3.1. Effect of GO on nitrogen variations and N2O generation The variations of NHþ 4-N, NO- 2-N and NO- 3-N concentrations in the 1 st and 30 th operation cycle were detected every 1 h until NHþ 4-N and NO- 2-N were completely consumed. Compared with the control group in the 1st cycle, 10 mg/L GO showed no obvious impacts in terms of NHþ 4-N and NO- 2-N conversion rates. Intriguingly, 100 mg/L GO significantly promoted NHþ 4-N removal rate and NO- 2-N degradation efficiency. The acceleration occurred in the short-term interaction was probably attributed to the closer interaction between the nitrifying bacteria and the positive ammonia ion through surface chemistry and nano-bio interfaces (Luo et al., 2016). Similar results were also found in previous study that nitrifying bacteria could be positively stimulated by 60 mg/L of GO and thereby facilitated the conversion of NHþ 4-N and NO- 2-N (Guo et al., 2018). Moreover, within the short exposure time the maximal GO concentration used in this study (100 mg/L) could rise trivial ROS generation and cause little oxidative stress to bacteria (discussed in Section 3.3). Nevertheless, when the operation reached the 30 th cycle, the facilitation of nitrification by 100 mg/L GO turned to be inhibited with decreased NHþ 4-N and NO- 2-N conversion rates compared with the control group (Fig. 1 b, d), suggesting that the chronic exposure to GO would probably weaken the ammonia and nitrite oxidizing capabilities of nitrifying bacteria in the activated sludge. N2O was an unwanted by-product generated in biological nitrogen removal (BNR) process which could not be ignored due to its significant greenhouse effect (Xie et al., 2018). In the 1 st cycle, the
average N2O emission factors in control group and “GO 10 mg/L00 group were 0.73% and 0.68%, while it was merely 0.13% in “GO 100 mg/L00 group (Fig. 2). The unstable microorganism activity in a new growing environment might be the reason for N2O generation in the first beginning (Massara et al., 2017). In the following cycles, N2O emission factors gradually decreased in control group and “GO 10 mg/L00 group but it kept rising in the “GO 100 mg/L00 group and gradually climbed up to 0.46% in the 30 th cycle. Two approaches
Fig. 2. N2O emission factors in 1st, 8th, 15th, 22nd and 30 th operation cycle under different GO concentrations.
Fig. 1. The variations of NHþ 4-N, NO- 2-N and NO- 3-N concentrations under different GO concentrations at the 1st (a, c, e) and 30th operation cycle (b, d, f).
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were responsible for N2O emission during nitrification process: (I) nitrite was taken as the alternative electron acceptor by AOB instead of O2 (Ni et al., 2013); and (II) the inordinate oxidation of hydroxylamine by nitrite (Wunderlin et al., 2012). Therefore, N2O generation always showed positive correlation with nitrite accumulation (Campos et al., 2009), considering which, it was tempting to speculate that the increasing nitrite concentration in the high GO concentration was the key factor in stimulating N2O accumulation. 3.2. Effect of GO on EPS contents As the essential composition of activated sludge, EPS was an essential indicator of microorganism activities as well as sludge dewaterability (Liu, 2019). Therefore, polysaccharides (PS) and protein (PN) as the two main compositions of EPS were quantified under different GO concentrations in the 1 st and 30 th cycle. In the 1 st cycle, the concentration of PN in “GO 100 mg/L00 group was 106.77 ± 7.90 mg/g VSS, slightly higher than “GO 10 mg/L00 group (90.00 ± 1.66 mg/g VSS) and control group (77.647 ± 1.66 mg/g VSS). EPS contents showed significant increase under all the GO concentrations in the 30 th cycle. However, the PN concentration in “GO 100 mg/L00 group was 253.53 ± 0.83 mg/g VSS, which turned to be remarkably lower than control group at 312.94 ± 3.33 mg/g VSS (Fig. 3 a). This result was consistent with the previous study that GO could reduce the generation of EPS by directly affecting the expression of protein (Blazer-Yost et al., 2010). PS concentrations were found slightly lower in GO-contained groups when compared with the control group, but not in significant difference at either the 1st or the 30th cycle (Fig. 3 b). EPS is the microbial secretions adhered to sludge flocs, playing the role as the storage and passageway of NHþ 4-N before reaching the surface of the bacterial cells. Besides, EPS can also serve as the adsorbent by itself, contributing to the improvement of the nitrogen removal efficiency by adsorbing NHþ 4-N, NO- 2-N and NO- 3N with a large amount of neutral, polyanionic and polycationic macromolecules in it (Yan et al., 2016). An adsorption amount of about 1.7 mg NHþ 4-N/g VSS was achieved by EPS of aerobic granular sludge through ion exchange with ammonium (Bassin et al., 2011). In the present study, the significant increase of EPS level was probably contributed to the acceleration of NHþ 4-N removal as less time was taken for completing the nitrogen conversion in the 30 th cycle than the initial cycle (Fig. 1). Moreover, much lowered EPS contents in “100 mg/L GO” group might be responsible for the decline of NHþ 4-N removal efficiency. Therefore, the suppression on EPS content by GO was inferred to be the probable cause for the declined nitrogen removal efficiency.
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3.3. Effect of GO on oxidative stress and antioxidant activities The reactive oxygen species (ROS) levels generated by the nitrifying activated sludge at the two exposure time were determined. In the 1 st cycle, no significant difference was found in generated ROS level among three GO concentrations, indicating that the toxicity of 100 mg/L GO cause no serious oxidative damage to bacteria in short terms. Generated ROS level increased in all the experimental groups when the operation reached the 30th cycle and significantly higher ROS level in the “GO 100 mg/L” group occurred than the control group, demonstrating that the bacteria exposed to 100 mg/L of GO had to face more serious oxidation stress risk (Fig. 4). ROS was produced by the respiration and metabolic reactions of bacteria and the moderate ROS level was essential for maintaining intracellular homeostasis and serving as the transmitter of cellular signals (Winterbourn, 2018). However, excessive superoxide and hydrogen peroxide tended to damage iron-sulphur clusters in intracellular proteins and hydroxyl radicals could directly damage DNA or indirectly by oxidizing the deoxynucleotide pool (Van Acker and Coenye, 2017). Besides, overproduction of ROS could also lead to the change of cell motility, unregulated cell signaling and protein denaturation, eventually resulted in apoptosis (Fu et al., 2014).
Fig. 4. Effects of GO concentrations and exposure time on ROS level. Error bars represent standard deviations of triplicate batch tests, and asterisks indicate statistical differences (p < 0.05) between two groups.
Fig. 3. Effects of GO concentrations and exposure time on PN (a) and PS (b) concentrations in EPS. Error bars represent standard deviations of triplicate batch tests, and asterisks indicate statistical differences (p < 0.05) between two groups.
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Fig. 5. Effects of GO concentrations and exposure time on SOD (a) and CAT (b) level. Error bars represent standard deviations of triplicate batch tests, and asterisks indicate statistical differences (p < 0.05) between two groups.
Bacteria cells can produce endogenous antioxidant enzymes like superoxide dismutase (SOD) and catalase (CAT) to construct the enzymatic antioxidant defense system to eliminate the excess ROS when facing oxidative stress (Ullah et al., 2018). SOD was produced to convert superoxide radical anion into H2O2 and an oxygen molecule, and CAT was mainly responsible for H2O2 elimination, of which the enzymatic activities could also be taken as the significant index of cell viability (Truong et al., 2018). In the initial cycle, enzymatic activities of SOD and CAT were maintained at a relatively higher level (Fig. 5 a b), contributing to the reduction of ROS level in the beginning. The remarkable discrepancy of SOD level between the control group (14.94 ± 0.01 U/mgprot) and “GO 100 mg/L00 group (4.83 ± 0.26 U/mgprot) in the 30 th cycle was inferred to be the reason for the diffference of the ROS level. Thus, it could be
concluded that long-term exposure to GO could impair the SOD and CAT activities, which thereby led to the ascent of ROS level and caused damage to the nitrifying bacteria. Except for the antioxidant enzymes, the oxidation stress caused by GO could also weaken the activities of nitrogen conversion enzymes, which was bound to influence the normal function of the activated sludge like nitrogen removal and N2O generation (Wang et al., 2018b). 3.4. Effect of GO on the abundance of the total and nitrifying bacteria The abundances of 16S rRNA, CAOB, AOB and NOB in the activated sludge samples in the two exposure times were quantified with qPCR. The total bacterial amount showed remarkable growth
Fig. 6. Effect of GO on abundance of 16S rRNA (a), CAOB amoA (b), AOB amoA (C) and NOB 16S rRNA (d) gene. Error bars represent standard deviations of triplicate batch tests. ‘*’denotes p-value < 0.05 and ‘**’denotes p-value < 0.01 between two groups.
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during the 10 days’ incubation in the control and “GO 10 mg/L00 group (Fig. 6 a). However, in the “GO 100 mg/L00 group, the total biomass significantly decreased in the 30th cycle, which would potentially weaken the nitrification capability of activated sludge. Moreover, the decreased EPS content ascribed from the biomass reduction was another essential factor for the decreased NHþ 4-N and NO- 2-N removal performance. Considering the exposure of activated sludge to GO could also affect microbial communities and bacterial metabolic activity in wastewater treatment system (Ahmed and Rodrigues, 2013), the abundance of the key nitrifying bacteria including CAOB, AOB and NOB in 1 st cycle and 30 th cycle was investigated. The results indicated that the existence of GO exert no obvious effect on AOB amoA gene during experimental period in this research (Fig. 6 c), but 100 mg/L of GO significantly inhibited the growth of CAOB amoA and NOB 16S rRNA, from 1.99 108 to 1.17 108 and 1.25 109 to 4.60 108 copies g1 SS, respectively (Fig. 6 b, d). Traditional nitrification process was long-held perceived to be catalyzed separately by AOB and NOB, i.e., NHþ 4-N was firstly oxidized to NO- 2-N by AOM, and then NOB turned NO- 2-N to NO3-N (Zheng et al., 2015). However, the discovery of CAOB belonging to genus Nitrospira confirmed that nitrification can be solely carried out by one single bacterium (Daims et al., 2015). As shown in the results, CAOB and NOB were more sensitive to GO toxicity than AOB, and the inhibitions of 100 mg/L GO to CAOB would inevitably influence the ammonia-oxidizing process, leading to the reduction of NHþ 4-N removal and increase of NO- 2-N accumulation under long-term exposure time. Likewise, NO- 2-N removal capability would also be impaired since the decrease of NOB abundance (Fig. 1). Therefore, it could be inferred that the decline of CAOB and NOB abundance caused by GO toxicity was the underlying cause for the significant promotion of N2O generation in the high GO concentration. Considering the high water solubility of GO and the capability to tightly bond with nitrifying bacteria in activated sludge, it is of great significance to avoid the leakage of GO to wastewater treatment plants. 4. Conclusions GO showed no obviously negative effect on nitrifying capability of the activated sludge in 4 h but caused toxicity effects in 10 days’ exposure time. The lower EPS contents and bacterial abundance caused by 100 mg/L GO was mainly responsible for the reduction of NHþ 4-N and NO- 2-N removal efficiency. High concentration of GO also inhibited the activities of endogenous antioxidant enzymes including SOD and CAT and result in the promotion of ROS level generated by the activated sludge, leading to higher oxidative stress to microorganisms. The significant decrease of CAOB and NOB abundance owing to GO exposure resulted in the NO- 2-N accumulation and further stimulated N2O generation. This study highlighted the significance of avoiding the release of GO to wastewater treatment plants. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 41701278) and the Fundamental Research Funds for the Central Universities (2019MS041). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.06.009.
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The effects of graphene oxide on nitrification and N2O emission: Dose and exposure time dependent Nan Zhou a, Zhirong Zhao a, Huihui Wang a, Xiangyu Chen a, Mingyuan Wang a, Shishi He a, Wen Liu b, Maosheng Zheng a, * a
College of Environmental Science and Engineering, North China Electric Power University, The Key Laboratory of Resources and Environmental Systems Optimization, Ministry of Education, Beijing, 102206, China College of Environmental Sciences and Engineering, Peking University, The Key Laboratory of Water and Sediment Sciences, Ministry of Education, Beijing, 100871, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 February 2019 Received in revised form 9 May 2019 Accepted 3 June 2019 Available online 10 June 2019
With the extensive application of graphene oxide (GO), its leakage and release into wastewater treatment plants become inevitable. However, the toxicity of graphene oxide (GO) on nitrification process and the underlying mechanisms still remain unclear. In this study, the toxic effects of GO at concentration of 10 and 100 mg/L in 4 h and 10 days were evaluated with sealed reactors operated in sequencing batch mode. In the initial 4 h, both GO concentrations showed no negative effect on nitrogen conversion. However, the exposure to 100 mg/L GO significantly weakened the NHþ 4-N and NO- 2-N conversion capabilities and intensified the nitrous oxide (N2O) generation after 10 days. Extracellular polymeric substance (EPS) analysis suggested that 100 mg/L GO decreased the protein content of the nitrifying activated sludge. Moreover, reactive oxygen species (ROS) level was promoted by 100 mg/L GO owing to the impaired endogenous antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT), which caused oxidative stress to bacteria. Finally, quantitative PCR results confirmed that nitriteoxidizing bacteria (NOB) and complete ammonia oxidizing bacteria (CAOB) were more sensitive to GO, which was the primary cause for the significant promotion of N2O generation in the high GO concentration. This study offered new insights in the toxicity of GO on nitrification and N2O generation in the terms of dose and exposure time. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Graphene oxide Nitrification Nitrous oxide Comammox
1. Introduction Graphene oxide (GO) has received wide attention owing to its distinctive physiochemical properties including outstanding compressibility (Chen and Chen, 2018), ideal compatibility in biological behaviors (Chung et al., 2013), superior adsorption capacity (Alqadami et al., 2017) and high thermal conductivities (Chen et al., 2018). Graphene productions market is projected to be worth approximately $1.3 billion by 2023 (Mcwilliams, 2011). Ubiquitous commercial or industrial applications would inevitably result in the exposure and release of these nanomaterials, and eventually accumulate in wastewater treatment plant (Kuhlbusch et al., 2011). Thus, stringent exposure assessments of wastewater disposal
* Corresponding author. Main Building G619, North China Electric Power University, Beijing, 102206, China. E-mail address: [email protected] (M. Zheng). https://doi.org/10.1016/j.envpol.2019.06.009 0269-7491/© 2019 Elsevier Ltd. All rights reserved.
process to GO must be taken into account. Nitrification is the central and rate-limited process in biological nitrogen removal (BNR) in wastewater treatment plant (WWTP), which is mainly contributed by diverse groups of microorganisms including ammonia-oxidizing microbes (AOM), nitrite-oxidizing bacteria (NOB) and complete ammonia oxidizing bacteria (CAOB) (Daims et al., 2015; Zheng et al., 2017). Nitrous oxide (N2O) is one of the most significant greenhouse gases with 298 times of global warming potential than CO2 (Mannina et al., 2018) and BNR is the primary source of N2O generation in WWTP (Mampaey et al., 2016). Toxic substances in receiving water can weaken the activity of these microorganisms and reduce the nitrogen removal efficiency (Zheng et al., 2019; Liu et al., 2019) and even accelerate the generation of nitrous oxide (N2O) which can result in the aggravation of greenhouse effect and depletion of ozone layer (Ahn et al., 2010). The antibacterial activity of GO could raise the oxidative stress and membrane stress by direct contact with bacterial cultures (Liu et al., 2011). Apart from this, low concentration of GO exposure
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could also lead to the differential expression of functionalized proteins and eventually result in cell death (Guo and Mei, 2014; Lv et al., 2018). Acute exposure to GO concentration at 50 ~ 300 mg/L could be accumulated inside the activated sludge and deteriorate the effluent quality through impacting the water turbidity and sludge dewaterability (Ahmed and Rodrigues, 2013). However, existing reports also argued that nano-GOs functionalized with polyethylene glycol was a positive regulator to stimulate the growth of E. coli by enhancing the production of recombinant proteins (Luo et al., 2016). Moreover, 0.06 g/L of GO could instantly improve the bioactivity of both ammonia-oxidizing bacteria (AOB) and NOB in 4 h, posing negligible negative effect on microbial functions of activated sludge (Guo et al., 2018). According to all, the discrepancy of GO toxicity mainly depends on exposure time and GO dose. However, the potential toxicity of GO to nitrifying bacteria under long-term exposure time still remains unclear. Considering the easy combination and coexistence of GO and activated sludge, the risk of long exposure of activated sludge to GO is essential to be evaluated. In this study, the effect of 10 mg/L and 100 mg/L GO on nitrification process and N2O generation was successively evaluated. Moreover, the underlying mechanisms of GO toxicity were explained by analyzing the reactive oxygen species (ROS), extracellular polymeric substance (EPS) and metabolic activity of the nitrifying activated sludge. Finally, the total and the nitrifying bacteria (AOB, NOB and CAOB) was quantified to reveal the variation of the bacterial abundance under the exposure of GO. 2. Materials and methods 2.1. Preparation and characterization of GO GO was made from the flake graphite based on modified Hummers' method (Chen et al., 2015). The concentration of dispersed GO solution was measured at 3.75 g/L. The typical morphology of GO was observed by Scanning Electron Microscopy (Hitachi, Japan) and Transmission Electron Microscope (JEOL-2100, Japan). Moreover, the molecular structure and functional groups were also identified by X-ray Diffraction (Rikagu-XDS-2000, Japan) and Fourier Transform Infrared (Nicolet 8700, USA). The characterization results can be found in Supplementary Material. 2.2. Batch experiments setting Nitrifying activated sludge was taken from a membrane bioreactor (MBR) which has been stably operated in laboratory for more than one year and equally divided into nine sealed shaking bottles with working volume of 300 ml. 10 mL nutrient medium was supplemented into each bottle to start the reaction containing 1.00 g/L K2HPO4, 0.10 g/L MgCl2$6H2O, 0.02 g/L CaCl2$H2O, 0.005 g/ L FeSO4$7H2O, 0.50 g/L KCl, 0.80 g/L NaHCO3 and 1.21 g/L NH4Cl. The mixed liquid suspended solids (MLSS) was about 1700 mg/L and the initial NHþ 4-N concentration was approximately 20 mg/L. Three GO concentrations (0 mg/L, 10 mg/L and 100 mg/L) were triply set in a total of nine bottles. NHþ 4-N, NO- 2-N and NO- 3-N concentrations were measured each hour in the first cycle until NHþ 4-N and NO- 2-N were completely oxidized. Following cycles were manually operated in sequencing batch reactor (SBR) mode with each cycle of 8 h including 6 h of shaking and 2 h of idleness. 10 mL of liquid supernatant was replaced by new nutrient medium before the new cycle to keep the total volume at 160 mL in the shaking bottles. The experiment was operated for ten days and the gas samples were retained in the end of each cycle every two days. N2O concentration was detected using a gas chromatography equipped with an electron capture detector (GC-6890N, Agilent, USA). The
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N2O emission factors were calculated by the percentage of N2O emission accounting for the removed NHþ 4-N amount in one cycle (Zheng et al., 2019). Sludge from the 1 st cycle in the 1 st day and the 30 th cycle in the 10 th day was sampled and stored for further analysis. 2.3. EPS extraction and analysis EPS was extracted by the modified heating method (Adav and Lee, 2008; C.F.Forster, 1971). 5 mL of activated sludge in 1 st cycle and 30 th cycle were taken from the shaking bottles. Sludge samples were centrifuged at 10000g for 20 min, the supernatant was filtered through 0.22 mm to achieve soluble EPS (SEPS). The precipitate was re-suspended with 5 mL of 0.85% NaCl solution and heated in water bath at 80 C for 30 min, then repeating the above procedure to gain bound EPS (BEPS). Total EPS was the sum of SEPS and BEPS (Nielsen et al., 1997). As the two main compositions of EPS, polysaccharides (PS) content in both SEPS and BEPS was measured by the Anthrone method and the protein (PN) content was detected by TGem Spectrophotometer (Tiangen OSE-260, China) using glucose and bovine serum albumin (BSA) as the respective standards. 2.4. Measurement of ROS production and antioxidant activities Overproduction of ROS was regarded as the main cause of oxidative stress, leading to the disruption of the homeostasis inside the cells (Fu et al., 2014). In this study, intracellular ROS measurement was carried out by chemical fluorescence method with 2, 7dichlorofuorescin diacetate (DCFH-DA) as the fluorescence probe (Nanjing Jiancheng Bioengineering Institute, China). DCFH-DA can be hydrolyzed by intracellular esterase to dichlorofluorescin (DCFH), and then be oxidized to dichlorofluorescein (DCF) inside the cells, producing strong green fluorescence. 10 mm of DCFH-DA was added to 2 mL of activated sludge samples, incubating in water bath at 37 C for 30 min. The mixture was washed three times by phosphate buffer saline (PBS) before detection. Fluorescence intensity was measured using a fluorescence spectrometer (PerkinElmer, LS 55, USA) at emission wavelength of 527 nm and excitation wavelength of 488 nm. The enzymatic activities of superoxide dismutase (SOD) and catalase (CAT) from activated sludge under different GO exposure time were determined by assay kit purchased from Nanjing Jiancheng Bioengineering Institute. The enzymatic activities were normalized against the protein content according to a previous study (Wu et al., 2018). 2.5. DNA extraction and real-time quantitative PCR (qPCR) Activated sludge samples were extracted from the bottles in 4 h and 10 d and stored at 20 C before DNA extraction. A total of 2 mL samples were used for DNA extraction using FastDNA™ Spin Kit for Soil (MPbio, USA) according to the instruction manual. The abundances of AOB, CAOB, NOB and 16S rRNA were quantified with StepOne™ Real-time PCR System (Applied Biosystems, USA). Primer pairs and amplifying conditions for qPCR referred to previous studies (Pester et al., 2012; Wang et al., 2018a). 2.6. Statistical analysis All the results were expressed as mean ± SD values. The statistical analyses were conducted by Statistical Product and Service Solution (SPSS) software (IBM Inc. USA). Values of P < 0.05 were considered to be statistically significant.
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3. Results and discussion 3.1. Effect of GO on nitrogen variations and N2O generation The variations of NHþ 4-N, NO- 2-N and NO- 3-N concentrations in the 1 st and 30 th operation cycle were detected every 1 h until NHþ 4-N and NO- 2-N were completely consumed. Compared with the control group in the 1st cycle, 10 mg/L GO showed no obvious impacts in terms of NHþ 4-N and NO- 2-N conversion rates. Intriguingly, 100 mg/L GO significantly promoted NHþ 4-N removal rate and NO- 2-N degradation efficiency. The acceleration occurred in the short-term interaction was probably attributed to the closer interaction between the nitrifying bacteria and the positive ammonia ion through surface chemistry and nano-bio interfaces (Luo et al., 2016). Similar results were also found in previous study that nitrifying bacteria could be positively stimulated by 60 mg/L of GO and thereby facilitated the conversion of NHþ 4-N and NO- 2-N (Guo et al., 2018). Moreover, within the short exposure time the maximal GO concentration used in this study (100 mg/L) could rise trivial ROS generation and cause little oxidative stress to bacteria (discussed in Section 3.3). Nevertheless, when the operation reached the 30 th cycle, the facilitation of nitrification by 100 mg/L GO turned to be inhibited with decreased NHþ 4-N and NO- 2-N conversion rates compared with the control group (Fig. 1 b, d), suggesting that the chronic exposure to GO would probably weaken the ammonia and nitrite oxidizing capabilities of nitrifying bacteria in the activated sludge. N2O was an unwanted by-product generated in biological nitrogen removal (BNR) process which could not be ignored due to its significant greenhouse effect (Xie et al., 2018). In the 1 st cycle, the
average N2O emission factors in control group and “GO 10 mg/L00 group were 0.73% and 0.68%, while it was merely 0.13% in “GO 100 mg/L00 group (Fig. 2). The unstable microorganism activity in a new growing environment might be the reason for N2O generation in the first beginning (Massara et al., 2017). In the following cycles, N2O emission factors gradually decreased in control group and “GO 10 mg/L00 group but it kept rising in the “GO 100 mg/L00 group and gradually climbed up to 0.46% in the 30 th cycle. Two approaches
Fig. 2. N2O emission factors in 1st, 8th, 15th, 22nd and 30 th operation cycle under different GO concentrations.
Fig. 1. The variations of NHþ 4-N, NO- 2-N and NO- 3-N concentrations under different GO concentrations at the 1st (a, c, e) and 30th operation cycle (b, d, f).
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were responsible for N2O emission during nitrification process: (I) nitrite was taken as the alternative electron acceptor by AOB instead of O2 (Ni et al., 2013); and (II) the inordinate oxidation of hydroxylamine by nitrite (Wunderlin et al., 2012). Therefore, N2O generation always showed positive correlation with nitrite accumulation (Campos et al., 2009), considering which, it was tempting to speculate that the increasing nitrite concentration in the high GO concentration was the key factor in stimulating N2O accumulation. 3.2. Effect of GO on EPS contents As the essential composition of activated sludge, EPS was an essential indicator of microorganism activities as well as sludge dewaterability (Liu, 2019). Therefore, polysaccharides (PS) and protein (PN) as the two main compositions of EPS were quantified under different GO concentrations in the 1 st and 30 th cycle. In the 1 st cycle, the concentration of PN in “GO 100 mg/L00 group was 106.77 ± 7.90 mg/g VSS, slightly higher than “GO 10 mg/L00 group (90.00 ± 1.66 mg/g VSS) and control group (77.647 ± 1.66 mg/g VSS). EPS contents showed significant increase under all the GO concentrations in the 30 th cycle. However, the PN concentration in “GO 100 mg/L00 group was 253.53 ± 0.83 mg/g VSS, which turned to be remarkably lower than control group at 312.94 ± 3.33 mg/g VSS (Fig. 3 a). This result was consistent with the previous study that GO could reduce the generation of EPS by directly affecting the expression of protein (Blazer-Yost et al., 2010). PS concentrations were found slightly lower in GO-contained groups when compared with the control group, but not in significant difference at either the 1st or the 30th cycle (Fig. 3 b). EPS is the microbial secretions adhered to sludge flocs, playing the role as the storage and passageway of NHþ 4-N before reaching the surface of the bacterial cells. Besides, EPS can also serve as the adsorbent by itself, contributing to the improvement of the nitrogen removal efficiency by adsorbing NHþ 4-N, NO- 2-N and NO- 3N with a large amount of neutral, polyanionic and polycationic macromolecules in it (Yan et al., 2016). An adsorption amount of about 1.7 mg NHþ 4-N/g VSS was achieved by EPS of aerobic granular sludge through ion exchange with ammonium (Bassin et al., 2011). In the present study, the significant increase of EPS level was probably contributed to the acceleration of NHþ 4-N removal as less time was taken for completing the nitrogen conversion in the 30 th cycle than the initial cycle (Fig. 1). Moreover, much lowered EPS contents in “100 mg/L GO” group might be responsible for the decline of NHþ 4-N removal efficiency. Therefore, the suppression on EPS content by GO was inferred to be the probable cause for the declined nitrogen removal efficiency.
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3.3. Effect of GO on oxidative stress and antioxidant activities The reactive oxygen species (ROS) levels generated by the nitrifying activated sludge at the two exposure time were determined. In the 1 st cycle, no significant difference was found in generated ROS level among three GO concentrations, indicating that the toxicity of 100 mg/L GO cause no serious oxidative damage to bacteria in short terms. Generated ROS level increased in all the experimental groups when the operation reached the 30th cycle and significantly higher ROS level in the “GO 100 mg/L” group occurred than the control group, demonstrating that the bacteria exposed to 100 mg/L of GO had to face more serious oxidation stress risk (Fig. 4). ROS was produced by the respiration and metabolic reactions of bacteria and the moderate ROS level was essential for maintaining intracellular homeostasis and serving as the transmitter of cellular signals (Winterbourn, 2018). However, excessive superoxide and hydrogen peroxide tended to damage iron-sulphur clusters in intracellular proteins and hydroxyl radicals could directly damage DNA or indirectly by oxidizing the deoxynucleotide pool (Van Acker and Coenye, 2017). Besides, overproduction of ROS could also lead to the change of cell motility, unregulated cell signaling and protein denaturation, eventually resulted in apoptosis (Fu et al., 2014).
Fig. 4. Effects of GO concentrations and exposure time on ROS level. Error bars represent standard deviations of triplicate batch tests, and asterisks indicate statistical differences (p < 0.05) between two groups.
Fig. 3. Effects of GO concentrations and exposure time on PN (a) and PS (b) concentrations in EPS. Error bars represent standard deviations of triplicate batch tests, and asterisks indicate statistical differences (p < 0.05) between two groups.
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Fig. 5. Effects of GO concentrations and exposure time on SOD (a) and CAT (b) level. Error bars represent standard deviations of triplicate batch tests, and asterisks indicate statistical differences (p < 0.05) between two groups.
Bacteria cells can produce endogenous antioxidant enzymes like superoxide dismutase (SOD) and catalase (CAT) to construct the enzymatic antioxidant defense system to eliminate the excess ROS when facing oxidative stress (Ullah et al., 2018). SOD was produced to convert superoxide radical anion into H2O2 and an oxygen molecule, and CAT was mainly responsible for H2O2 elimination, of which the enzymatic activities could also be taken as the significant index of cell viability (Truong et al., 2018). In the initial cycle, enzymatic activities of SOD and CAT were maintained at a relatively higher level (Fig. 5 a b), contributing to the reduction of ROS level in the beginning. The remarkable discrepancy of SOD level between the control group (14.94 ± 0.01 U/mgprot) and “GO 100 mg/L00 group (4.83 ± 0.26 U/mgprot) in the 30 th cycle was inferred to be the reason for the diffference of the ROS level. Thus, it could be
concluded that long-term exposure to GO could impair the SOD and CAT activities, which thereby led to the ascent of ROS level and caused damage to the nitrifying bacteria. Except for the antioxidant enzymes, the oxidation stress caused by GO could also weaken the activities of nitrogen conversion enzymes, which was bound to influence the normal function of the activated sludge like nitrogen removal and N2O generation (Wang et al., 2018b). 3.4. Effect of GO on the abundance of the total and nitrifying bacteria The abundances of 16S rRNA, CAOB, AOB and NOB in the activated sludge samples in the two exposure times were quantified with qPCR. The total bacterial amount showed remarkable growth
Fig. 6. Effect of GO on abundance of 16S rRNA (a), CAOB amoA (b), AOB amoA (C) and NOB 16S rRNA (d) gene. Error bars represent standard deviations of triplicate batch tests. ‘*’denotes p-value < 0.05 and ‘**’denotes p-value < 0.01 between two groups.
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during the 10 days’ incubation in the control and “GO 10 mg/L00 group (Fig. 6 a). However, in the “GO 100 mg/L00 group, the total biomass significantly decreased in the 30th cycle, which would potentially weaken the nitrification capability of activated sludge. Moreover, the decreased EPS content ascribed from the biomass reduction was another essential factor for the decreased NHþ 4-N and NO- 2-N removal performance. Considering the exposure of activated sludge to GO could also affect microbial communities and bacterial metabolic activity in wastewater treatment system (Ahmed and Rodrigues, 2013), the abundance of the key nitrifying bacteria including CAOB, AOB and NOB in 1 st cycle and 30 th cycle was investigated. The results indicated that the existence of GO exert no obvious effect on AOB amoA gene during experimental period in this research (Fig. 6 c), but 100 mg/L of GO significantly inhibited the growth of CAOB amoA and NOB 16S rRNA, from 1.99 108 to 1.17 108 and 1.25 109 to 4.60 108 copies g1 SS, respectively (Fig. 6 b, d). Traditional nitrification process was long-held perceived to be catalyzed separately by AOB and NOB, i.e., NHþ 4-N was firstly oxidized to NO- 2-N by AOM, and then NOB turned NO- 2-N to NO3-N (Zheng et al., 2015). However, the discovery of CAOB belonging to genus Nitrospira confirmed that nitrification can be solely carried out by one single bacterium (Daims et al., 2015). As shown in the results, CAOB and NOB were more sensitive to GO toxicity than AOB, and the inhibitions of 100 mg/L GO to CAOB would inevitably influence the ammonia-oxidizing process, leading to the reduction of NHþ 4-N removal and increase of NO- 2-N accumulation under long-term exposure time. Likewise, NO- 2-N removal capability would also be impaired since the decrease of NOB abundance (Fig. 1). Therefore, it could be inferred that the decline of CAOB and NOB abundance caused by GO toxicity was the underlying cause for the significant promotion of N2O generation in the high GO concentration. Considering the high water solubility of GO and the capability to tightly bond with nitrifying bacteria in activated sludge, it is of great significance to avoid the leakage of GO to wastewater treatment plants. 4. Conclusions GO showed no obviously negative effect on nitrifying capability of the activated sludge in 4 h but caused toxicity effects in 10 days’ exposure time. The lower EPS contents and bacterial abundance caused by 100 mg/L GO was mainly responsible for the reduction of NHþ 4-N and NO- 2-N removal efficiency. High concentration of GO also inhibited the activities of endogenous antioxidant enzymes including SOD and CAT and result in the promotion of ROS level generated by the activated sludge, leading to higher oxidative stress to microorganisms. The significant decrease of CAOB and NOB abundance owing to GO exposure resulted in the NO- 2-N accumulation and further stimulated N2O generation. This study highlighted the significance of avoiding the release of GO to wastewater treatment plants. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 41701278) and the Fundamental Research Funds for the Central Universities (2019MS041). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.06.009.
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