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Impacts of silver nanoparticles on performance and microbial community and enzymatic activity of a sequencing batch reactor
Journal of Environmental Management 204 (2017) 667e673
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Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Research article
Impacts of silver nanoparticles on performance and microbial community and enzymatic activity of a sequencing batch reactor Qiaoyan Xu a, c, Shanshan Li a, Yiping Wan a, Sen Wang b, Bingrui Ma a, Zonglian She a, Liang Guo a, Mengchun Gao a, *, Yangguo Zhao a, Chunji Jin a, b, Junwei Dong a, Zhiwei Li a a b c
Key Lab of Marine Environment and Ecology, Ministry of Education, Ocean University of China, Qingdao, 266100, China School of Environmental Science and Engineering, Qingdao University, Qingdao, 266071, China Shandong Provincial Key Laboratory of Marine Environment and Geological Engineering, Ocean University of China, Qingdao, 266100, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 August 2017 Received in revised form 14 September 2017 Accepted 18 September 2017
The performance, microbial community and enzymatic activity of a sequencing batch reactor (SBR) were evaluated under silver nanoparticles (Ag NPs) stress. Over 5 mg/L Ag NPs inhibited the COD and phosphorus removals, whereas the NHþ 4 removal kept stable during the whole operational period. The organic matter, nitrogen and phosphorus removal rates were obviously inhibited under Ag NPs stress, which showed similar varying trends with the corresponding microbial enzymatic activities. The change of Ag content in the activated sludge indicated that some Ag NPs were absorbed by the sludge. The presence of Ag NPs promoted the increase of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) of microorganism due to the microbial response to the Ag NPs toxicity, which could impact on the microbial morphology and physiological functions. The presence of Ag NPs could produce some evident changes in the microbial community. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Ag NPs Biotoxicity Microbial community Sequencing batch reactor Reactive oxygen species
1. Introduction Silver nanoparticles (Ag NPs) have been applied in room fresheners, food production, shampoos, bioengineering, biomedical products, laundry products and textiles (Lombi et al., 2013; Cushen et al., 2014). Ag NPs unavoidably enter the terrestrial and aquatic environment due to their increasing production and extensive applications. The presence of Ag NPs in the environment has recently attracted public concerns regarding their potential effects on the biota and human health. Some researches reported that Ag NPs had obvious ecotoxicity to microorganisms and aquatic organisms, such as bacteria (Sheng and Liu, 2017), algae (Oukarroum et al., 2012) and fish (Chen et al., 2017). Ag NPs are also reported to have adverse effects on human health (Sthijns et al., 2017; Marambio-Jones and Hoek, 2010). The Ag NPs release during the production, transport and utilization will end up in biological wastewater treatment systems. Ag NPs content in municipal sewage is the microgram level owing to
* Corresponding author. Key Lab of Marine Environment and Ecology, Ministry of Education, Ocean University of China, No. 238 Songling Road, Qingdao, Shandong Province, 266100, China. E-mail address: [email protected] (M. Gao). https://doi.org/10.1016/j.jenvman.2017.09.050 0301-4797/© 2017 Elsevier Ltd. All rights reserved.
the mixture of domestic sewage and industrial wastewater (Gottschalk et al., 2009). Nevertheless, high Ag NPs-containing industrial wastewater will result in Ag NPs at the milligram grade (Zhang et al., 2014). Considering the biotoxicity of Ag NPs, it is essential to evaluate the Ag NPs effect on bioreactors treating wastewater. Ag NPs could obviously inhibit the activity of Nitrosomonas europaea (Yuan et al., 2013) and denitrifying bacteria (Chen et al., 2014), indicating that Ag NPs could impact on the processes of nitrification and denitrification. Qiu et al. (2016) reported that less than 5 mg/L Ag NPs did not affect the COD removal, nitrification and denitrification of a sequencing batch reactor (SBR), whereas the presence of Ag NPs caused significant shifts in microbial community of SBR. Gu et al. (2014) demonstrated that Ag NPs at over 50 mg/L could significantly inhibit the biological nitrogen removal. Ag NPs concentration at less than 5 mg/L did not adversely affect biological phosphorus removal (Chen et al., 2012). Giao et al. (2017) evaluated the inhibition impacts of Ag NPs on ammonia oxidation kinetics and mechanism of nitrifying sludge. As is known to all, the bioreactor performances treating wastewater are closely associated with the microbial community and enzymatic activity. Therefore, it is interesting to investigate the microbial community and enzymatic activity of SBR under Ag NPs stress. In this study, the SBR performance and its pollutant removal rates were evaluated under Ag NPs stress. The key microbial
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enzymatic activity relating to the organic matter, nitrogen and phosphorous removals were compared at 0e30 mg/L Ag NPs. The Ag NPs toxicity to activated sludge was analyzed under Ag NPs stress. The variation of microbial community under Ag NPs stress was explored at the phylum and genus levels. 2. Materials and methods 2.1. Apparatus setup and influent composition A lab-scale SBR was applied in the present study, which had 7.7 L effective volume. The influent and air were introduced into the SBR by an influent pump and aeration pump, respectively. The solid retention time was approximately 20 d during the whole experimental period. Each cycle consisted of 6 min influent, 144 min anoxic stage, 240 min aerobic stage, 78 min settling and 12 min discharge, which was controlled by a time controller. The dissolved oxygen concentration was maintained above 2 mg/L and less than 0.5 mg/L during the aerobic stage and anoxic stage, respectively. Prior to the addition of Ag NPs, the SBR had operated for 117 d without Ag NPs addition and the SBR performance had reached a steady state. Ag NPs in the present study was approximately 30 nm particle diameter and 99.9% purity. 500 mg/L Ag NPs stock suspension was prepared in the light of Keller et al. (2010). The influent composition was similar to our previous research (Ma et al., 2017). The influent COD, soluble orthophosphate (SOP) and NHþ 4 -N were 414.3 ± 8.3, 22.4 ± 0.7 and 9.9 ± 0.3 mg/L during the operational period, respectively. 2.2. Analytical methods COD, NHþ 4 , NO3 , NO2 , SOP and mixed liquor volatile suspended
solids (MLVSS) were measured in light of standard methods (APHA, 1998). The specific ammonia oxidation rate (SAOR), specific nitrite oxidation rate (SNOR), specific oxygen utilization rate (SOUR), specific nitrate reduction rate (SNRR), specific nitrite reduction rate (SNIRR), specific phosphorus release rate (SPRR), and specific phosphorus uptake rate (SPUR) were analyzed as described in previous research (Wang et al., 2016). Reactive oxygen species (ROS) and lactate dehydrogenase (LDH) were measured according to Wang et al. (2017). Nitrite oxidoreductase (NOR), dehydrogenase (DHA), nitrite reductase (NIR), nitrate reductase (NR), polyphosphate kinase (PPK), ammonia monooxygenase (AMO), and exopolyphosphatase (PPX) activities were determined in the light of Wang et al. (2016). High throughput sequencing was applied to analyze the microbial community (Gao et al., 2014). 3. Results and discussion 3.1. SBR performance evaluation under Ag NPs stress Fig. 1 shows the COD, nitrogen and phosphorus removals under Ag NPs stress. The COD removal retained steady under less than 2 mg/L Ag NPs (Fig. 1a), suggesting that low Ag NPs concentration displayed a negligible inhibition influence on the activities of heterotrophic microorganisms owing to their tolerance ability to Ag NPs toxicity (Liang et al., 2010). Previous researches also illustrated that low Ag NPs concentration had no adverse effect on the COD removal in some bioreactors (Hou et al., 2012; Qiu et al., 2016). However, the COD removal efficiency in the present study reduced from 91.70 ± 0.84% to 87.76 ± 0.66% with the increase of Ag NPs concentration from 2 to 30 mg/L, illustrating that high Ag NPs concentration exerted a slight inhibition impact on COD removal. The NHþ 4 removal efficiencies were 98.28 ± 1.19% during the
Fig. 1. Impact of Ag NPs on the SBR performance. (a) COD, (b) NHþ 4 -N, (c) NO2 -N and NO3 -N, and (d) SOP.
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whole operational period (Fig. 1b), indicating that less than 30 mg/L Ag NPs had no obvious influence on the ammonia oxidization. Qiu et al. (2016) reported that less than 5 mg/L Ag NP had no distinct influence on the nitrification process. Contrarily, some researches demonstrated that Ag NPs inhibited the NHþ 4 oxidization in different biological wastewater treatment processes (Alito and Gunsch, 2014; Liang et al., 2010; Hou et al., 2012). The discrepancy between the present results and previous researches could be associated with exposure time, wastewater quality, Ag NPs addition dose, bioreactor type, MLVSS, hydraulic retention time and sludge retention time. The effluent NO 2 -N concentration varied between 0 and 0.83 mg/L during the whole operational period (Fig. 1c). Nevertheless, the effluent NO 3 -N gradually reduced with the increase of Ag NPs concentration. The present results might be explained that the adsorption of Ag NPs on the sludge surface decreased the mass transfer efficiency of oxygen into the interior of sludge flocs and led to more favorable anaerobic environment for the denitrification process of activated sludge. The SOP removal efficiency slightly decreased at 0e5 mg/L Ag NPs (Fig. 1d), which had similar results to previous report (Chen et al., 2012). Nevertheless, the SOP removal efficiency reduced from 75.99 ± 1.59% to 58.08 ± 4.89% with the increase of Ag NPs concentration from 5 to 30 mg/L, illustrating the biological phosphorous removal was obviously inhibited by Ag NPs.
3.2. Nitrogen and phosphorus removal rates under Ag NPs stress Fig. 2 shows the nitrogen and phosphorus removal rates and SOUR under Ag NPs stress. The SOUR decreased from 48.00 ± 1.79 to 31.88 ± 1.33 mg O2/(g MLVSS$h) with the increase of Ag NPs concentration from 0 to 30 mg/L (Fig. 2a). Compared with the
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absence of Ag NPs, the SOUR reduced by 33.58% at 30 mg/L Ag NPs, demonstrating that Ag NPs inhibited the metabolic activities of heterotrophic bacteria and then affect the organic matter removal rate. García et al. (2012) also found that Ag NPs had evident inhibitory effect on the SOUR of ordinary heterotrophic organisms by respiratory experiments. Both the SAOR and SNOR reduced with the increase of Ag NPs concentration from 0 to 30 mg/L (Fig. 2b). Compared with the absence of Ag NPs, the SAOR and SNOR reduced by 26.50% and 23.53% at 30 mg/L Ag NPs, respectively. The changes of SAOR and SNOR demonstrated that Ag NPs could exert more inhibition effect on the SAOR than the SNOR. Michels et al. (2017) illustrated that the specific nitrite production rate of ammonia oxidizing bacteria had an evident reduction under Ag NPs stress. As shown in Fig. 2c, the SNIRR and SNRR decreased from 10.49 ± 0.39 and 11.14 ± 0.42 mg N/(g MLVSS$h) at 0 mg/L Ag NPs to 8.30 ± 0.25 and 9.28 ± 0.47 mg N/(g MLVSS$h) at 30 mg/L Ag NPs, respectively. The SNIRR and SNRR decreased by 20.88% and 16.70% at 30 mg/L Ag NPs by comparison with 0 mg/L Ag NPs, respectively, indicating that Ag NPs could inhibit the SNIRR more than the SNRR. The SPUR and SPRR of activated sludge reduced from 14.39 ± 0.54 and 13.66 ± 0.51 mg P/(g MLVSS$h) at 0 mg/L Ag NPs to 9.57 ± 0.68 and 8.50 ± 0.35 mg P/(g MLVSS$h) at 30 mg/L Ag NPs with the increase of Ag NPs concentration from 0 to 30 mg/L (Fig. 2d), respectively. Compared with the absence of Ag NPs, the SPUR and SPRR decreased by 33.50% and 37.78% at 30 mg/L Ag NPs, respectively, illustrating that Ag NPs inhibited the biological phosphorus release and uptake.
3.3. Variation of microbial enzymatic activity under Ag NPs stress The organic matter, nitrogen and phosphorus removals of
Fig. 2. Impacts of Ag NPs on the microbial activities of activated sludge. (a) SOUR, (b) SAOR and SNOR, (c) SNIRR and SNRR, and (d) SPUR and SPRR. Asterisks indicate the statistical difference (p < 0.05) from the microbial activities at 0 mg/L Ag NPs. Error bars represent standard deviations of triplicate measurements.
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wastewater biological treatment systems are closely associated with microbial enzymatic activity. The DHA decreased from 15.138 ± 0.565 to 9.799 ± 0.770 mg TF/(mg MLVSS$h) with the increase of Ag NPs concentration from 0 to 30 mg/L (Fig. 3a). The DHA had a similar varying tendency to the SOUR, suggesting that the decrease of SOUR was related to the variation of DHA under Ag NPs stress. Both the AMO and NOR activities decreased with the increase of Ag NPs concentration from 0 to 30 mg/L (Fig. 3b), respectively. The varying trends of the AMO and NOR activities were similar to those of the SAOR and SNOR, respectively, demonstrating that the reduction of SAOR and SNOR were related to the inhibition impact of Ag NPs on the AMO and NOR. Compared with the absence of Ag NPs, the AMO and NOR activities decreased by 28.29% and 28.90% at 30 mg/L Ag NPs, respectively, indicating that Ag NPs had similar inhibition rates on the enzymatic activities of NHþ 4 -oxidization microorganism and NO 2 -oxidization microorganism. The NIR and NR activities decreased from 5.423 ± 0.154 and 0.372 ± 0.011 mg NO 2 -N/(min$mg protein) at 0 mg/L Ag NPs to 3.895 ± 0.162 and 0.274 ± 0.011 mg NO 2 -N/(min$mg protein) at 30 mg/L Ag NPs (Fig. 3c), respectively. Compared with the absence of Ag NPs, the NIR and NR activities reduced by 28.18% and 26.34% at 30 mg/L Ag NPs, respectively, demonstrating that Ag NPs had higher inhibition rate on the enzymatic activities of NO 2 -reduction microorganism than NO 3 -reduction microorganism. Additionally, the NIR and NR activities showed similar variation trends to the SNIRR and SNRR, respectively. As shown in Fig. 3d, the PPX activity decreased from 0.0587 ± 0.0017 to 0.0407 ± 0.0017 mmol pnitrophenol/(min$mg protein) with the increase of Ag NPs concentration from 0 to 30 mg/ L, and the PPK activity reduced from 0.0892 ± 0.0025 mmol NADPH/ (min$mg protein) at 0 mg/L Ag NPs to 0.0681 ± 0.0028 mmol NADPH/(min$mg protein) at 30 mg/L Ag NPs. The PPX and PPK activities had similar varying tendencies with the SPUR and SPRR,
respectively. Compared with the absence of Ag NPs, the PPX and PPK activities decreased by 30.66% and 23.65% at 30 mg/L Ag NPs, respectively, demonstrating that Ag NPs could exert more inhibition impact on the microbial enzymatic activities related to phosphorus release than those related to phosphorus uptake. 3.4. Biotoxicity evaluation of Ag NPs Ag NPs biotoxicity is associated with intact nanoparticles and Ag ion released from Ag NPs (Gliga et al., 2014; Liu et al., 2012). Before investigating the biotoxicity of Ag NPs, the Ag contents of the effluent and activated sludge were measured at each Ag NPs concentration (Supplementary Material, Fig. S1). The Ag content of sludge showed a remarkable increasing tendency with the increase of Ag NPs concentration, suggesting that some Ag NPs was absorbed by the sludge. Additionally, the effluent Ag content also slightly increased with an increase in Ag NPs concentration. Feng et al. (2000) have reported that Ag ion can also damage the cell membrane, DNA, protein and respiratory chain. ROS generation is acted as a significant indicator for evaluating NPs biotoxicity in the environment, whereas LDH release is regarded as a marker to evaluate the integrity of cytomembrane. As illustrated in Fig. 4, the ROS production was 8.58%, 18.89%, 28.30% and 39.78% at 2, 5, 10 and 30 mg/L Ag NPs by comparing to the absence of Ag NPs, respectively. The change of ROS indicated that Ag NPs promoted the ROS production of the microorganisms and then exerted the biotoxicity of Ag NPs. Hao and Chen (2012) reported that the increase of ROS production might be related to the imbalance of oxidation process and anti-oxidation process, which could overwhelm the defense capacity of the organisms and lead to the enzymatic inactivation, protein oxidization and DNA destruction. Compared to the absence of Ag NPs, the LDH release increased with the increase of Ag NPs concentration, indicating that Ag NPs resulted in destroying the microbial cytomembrane integrity. The destruction to the
Fig. 3. Impacts of Ag NPs on the microbial enzymatic activities of activated sludge. (a) DHA, (b) AMO and NOR, (c) NIR and NR, (d) PPX and PPK. Asterisks indicate statistical differences (p < 0.05) from the key enzymatic activity at 0 mg/L Ag NPs. Error bars represent standard deviations of triplicate measurements. The enzymatic activity at 0, 2, 5, 10 and 30 mg/L was determined on day 117, 179, 215, 247 and 290, respectively.
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some evident variations during the operational period, suggesting that Ag NPs distinctly impacted on the microbial richness and diversity of SBR. The rarefaction curve under each Ag NPs concentration trended to level off along the X-axis direction and further explained that the obtained OTU number was reasonable in the present study (Supplementary Material, Fig. S2). The distinct separation of microbial community was observed among the abovementioned four samples through the principal coordinate's analysis (Supplementary Material, Fig. S3). The principal component 1 (PC1) and PC2 of microbial communities were 42.86% and 31.91% of the variability in total microbial community, respectively. Venn diagram was applied to investigate the difference and similarity of microbial community (Fig. 5). The common OTU number (819) among the above-mentioned four samples constituted 47.12% of the total OUT number, suggesting that some microorganisms existed at all times during the whole operational period. The dominant phyla in the shared OTUs included 42.2% Proteobacteria, 19.4% Bacteroidetes, 8.3% Chloroflexi, 8.3% Planctomycetes, 4.1% Actinobacteria, 3.9% Acidobacteria, 3.0% Verrucomicrobia, 2.4% Firmicutes and 2.1% Chlorobi. The sum of special OTU among the above-mentioned four samples was 207, which only constituted 11.91% of total OTUs. The microbial community under Ag NPs stress was further explored at the phylum and genus levels. At the phylum level (Fig. 6a), the relative abundances of dominant phyla at 0e30 mg/L Ag NPs were 37.85e54.72% Proteobacteria, 27.06e36.42% Bacteroidetes, 2.94e6.63% Chloroflexi, 1.44e2.77% Planctomycetes, 1.12e2.46% Chlorobi, 0.90e2.37% Verrucomicrobia, 1.19e2.19% Acidobacteria, 0.59e1.43% Armatimonadetes, 0.38e1.39% Elusimicrobia and 0.79e1.12% Nitrospirae, suggesting that Ag NPs obviously impacted on the microbial richness. At the genus level (Fig. 6b), 32 dominant genera were chosen to analyze the changes of microbial richness under Ag NPs stress. Compared with the absence of Ag NPs, the relative abundance of Nitrosospira slightly increased at 5 mg/L Ag NPs, whereas decreased at 10e30 mg/L Ag NPs. However,
Fig. 4. Relative ROS production and LDH release of activated sludge at different concentrations of Ag NPs. Asterisks indicate the statistical difference (p < 0.05) from 0 mg/ L Ag NPs. Error bars represent standard deviations of triplicate measurements.
cytomembrane resulted in the leakage of intracellular substances, which could impact on the microbial morphology and physiological functions. 3.5. Impact of Ag NPs on microbial community High throughput sequencing was adopted to investigate the microbial community. As illustrated in Table 1, the effective sequence at 0, 5, 10 and 30 mg/L Ag NPs was classified as 1,432, 1,375, 1389 and 1495 operational taxonomic units (OTU), respectively. All the Good's coverage was greater than or equal to 0.995, suggesting that the sequence library covered the microbial diversity. The Chao 1, Shannon, Simpson and Ace indices displayed
Table 1 Microbial richness and diversity of activated sludge at different Ag NPs concentrations. Ag NPs concentration (mg/L)
Raw reads
Raw sequences
Effective sequences
OTUs
Chao1 index
Shannon index
Simpson index
ACE index
Good's coverage
0 5 10 30
45,547 49,904 47,887 56,756
44,562 48,859 46,958 55,654
43,058 47,144 45,644 51,692
1432 1375 1389 1495
1363.9 1370.9 1366.6 1466.3
7.995 7.237 7.371 7.446
0.986 0.966 0.974 0.976
1394.0 1374.6 1381.0 1484.6
0.997 0.995 0.995 0.995
Fig. 5. Venn diagram based on high-throughput sequencing of the microbial community at different Ag NPs concentrations (OTUs at 3% distance). The shared OTUs were analyzed at phylum level.
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Fig. 6. Taxonomic classification of 16S rDNA paired-end sequencing from activated sludge exposed to different Ag NPs concentrations at the phylum (a) and genus (b) levels.
the relative abundance of Nitrosomonas reduced with the increase of Ag NPs concentration from 0 to 30 mg/L. Nitrosomonas and Nitrosospira have the ability to oxidize ammonia to nitrite under aerobic condition. The relative abundance of Nitrococcus reduced from 10.30% at 0 mg/L Ag NPs to 2.55% at 30 mg/L Ag NPs. Nitrococcus has the capacity to oxidize nitrite to nitrate under aerobic condition. The relative abundances of Paracoccus, Devosia, Hyphomicrobium and Azoarcus maintained the decreasing trends with the increase of Ag NPs concentration, and these genera were reported to have the denitrifying ability (Baumann et al., 1996; Rivas et al., 2003; Green et al., 2010; Lee and Wong, 2014). Compared to the absence of Ag NPs, the relative abundances of Dechloromonas decreased at 10 mg/L Ag NPs and subsequently increased at 30 mg/ L Ag NPs, whereas Azospirillum had no obvious variation in the relative abundance during the operational period. Some researches have reported that Dechloromonas and Azospirillum have the ability to reduce nitrite or nitrate (Horn et al., 2005; Qiu and Ting, 2013). The present results demonstrated that Ag NPs affected the growth, development and reproduction of denitrifying bacteria. The relative abundance of Candidatus accumulibacter decreased from 2.49% to 1.3% with the increase of Ag NPs concentration from 0 to 10 mg/L and rose to 18.28% at 30 mg/L Ag NPs. He et al. (2007) have reported that Candidatus accumulibacter is related to the biological phosphorus removal. Additionally, the relative abundances of some
genera increased or reduced at each Ag NPs concentration by comparing with the absence of Ag NPs, such as Haliscomenobacter, Fimbriimonas, Sediminibacterium, Chitinophaga, Arcobacter, Ignavibacterium and Phaselicystis. 4. Conclusions Ag NPs concentration at over 5 mg/L inhibited the COD and phosphorus removal, whereas the NHþ 4 removal efficiency kept stable during the whole operational period. The SOUR and nitrogen and phosphorus removal rates could be inhibited by Ag NPs, which showed similar variation tendencies to their corresponding microbial enzymatic activities. The presence of Ag NPs promoted the ROS production and LDH release due to the microbial response to the Ag NPs toxicity, which could impact on the microbial morphology and physiological function. The presence of Ag NPs exerted evident changes in the microbial richness and diversity. Acknowledgements The work was funded by the National Natural Science Foundation of China (No. 51178437) and Province Key Technologies R & D Program of Shandong (Grants 2007GG10006002).
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Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jenvman.2017.09.050. References Alito, C., Gunsch, C.K., 2014. Assessing the effects of sliver nanoparticles on biological nutrient removal in bench-scale activated sludge sequencing batch reactors. Environ. Sci. Technol. 49, 970e976. APHA, 1998. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, DC. Baumann, B., Snozzi, M., Zehnder, A.J.B., Meer, J.R.V.D., 1996. Dynamics of denitrification activity of Paracoccus denitrificans in continuous culture during aerobicanaerobic changes. J. Bacteriol. 178, 4367e4374. Chen, J., Tang, Y.Q., Li, Y., Nie, Y., Hou, L., Li, X.Q., Wu, X.L., 2014. Impacts of different nanoparticles on functional bacterial community in activated sludge. Chemosphere 104, 141e148. Chen, S.X., Yang, X.Z., Deng, Y., Huang, J., Li, Y., Sun, Q., Yu, C.P., Zhu, Y., Hong, W.S., 2017. Silver nanoparticles induce oocyte maturation in zebrafish (Danio rerio). Chemosphere 170, 51e60. Chen, Y., Chen, H., Zheng, X., Mu, H., 2012. The impacts of silver nanoparticles and silver ions on wastewater biological phosphorous removal and the mechanisms. J. Hazard Mater. 239e240, 88e94. Cushen, M., Kerry, J., Morris, M., Cruz-Romero, M., Cummins, E., 2014. Evaluation and simulation of silver and copper nanoparticle migration from polyethylene nanocomposites to food and an associated exposure assessment. J. Agric. Food Chem. 62 (6), 1403e1411. Feng, Q.L., Wu, J., Chen, G.Q., Cui, F.Z., Kim, T.N., Kim, J.O., 2000. A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J. Biomed. Mater. Res. 52 (4), 662e668. Gao, C., Wang, A., Wu, W.M., Yin, Y., Zhao, Y.G., 2014. Enrichment of anodic biofilm inoculated with anaerobic or aerobic sludge in single chambered air-cathode microbial fuel cells. Bioresour. Technol. 167, 124e132. , J.A., Casals, E., Gonz García, A., Delgado, L., Tora alez, E., Puntes, V., Font, X., Carrera, J., S anchez, A., 2012. Effect of cerium dioxide, titanium dioxide, silver, and gold nanoparticles on the activity of microbial communities intended in wastewater treatment. J. Hazard Mater. 199e200, 64e72. Gliga, A.R., Skoglund, S., Wallinder, I.O., Fadeel, B., Karlsson, H., 2014. Size-dependent cytotoxicity of silver nanoparticles in human lung cells: the role of cellular uptake, agglomeration and Ag release. Part. Fibre Toxicol. 11 (1), 1. Giao, N.T., Limpiyakorn, T., Kunapongkiti, P., Thuptimdang, P., SiripattanakulRatpukdi, S., 2017. Influence of silver nanoparticles and liberated silver ions on nitrifying sludge: ammonia oxidation inhibitory kinetics and mechanism. Environ. Sci. Pollut. Res. 24 (10), 9229e9240. Gottschalk, F., Sonderer, T., Scholz, R.W., Nowack, B., 2009. Modeled environmental concentrations of engineered nanomaterials (TiO2, ZnO, Ag, CNT, fullerenes) for different regions. Environ. Sci. Technol. 43, 9216e9222. Green, S.J., Prakash, O., Gihring, T.M., Akob, D.M., Jasrotia, P., Jardine, P.M., Watson, D.B., Brown, S.D., Palumbo, A.V., Kostka, J.E., 2010. Denitrifying bacteria isolated from terrestrial subsurface sediments exposed to mixed-waste contamination. Appl. Environ. Microbiol. 76 (10), 3244e3254. Gu, L., Li, Q., Quan, X., Cen, Y., Jiang, X., 2014. Comparison of nanosilver removal by flocculent and granular sludge and short- and long-term inhibition impacts. Water Res. 58, 62e70. Hao, L., Chen, L., 2012. Oxidative stress responses in different organs of carp (Cyprinus carpio) with exposure to ZnO nanoparticles. Ecotoxicol. Environ. Saf. 80, 103e110. He, S.M., Gall, D.L., McMahon, K.D., 2007. “Candidatus Accumulibacter” population structure in enhanced biological phosphorus removal sludges as revealed by polyphosphate kinase genes. Appl. Environ. Microbiol. 73, 5865e5874. Horn, M.A., Ihssen, J.L., Matthies, C., Schramm, A., Acker, G., Drake, H.L., 2005.
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Dechloromonas denitrificans sp. nov., Flavobacterium denitrificans sp. nov., Paenibacillus anaericanus sp. nov. and Paenibacillus terrae strain MH72, N2O-producing bacteria isolated from the gut of the earthworm Aporrectodea caliginosa, 2005. Int. J. Syst. Evol. Microbiol. 55, 1255e1265. Hou, L.L., Li, K.Y., Ding, Y.Z., Li, Y., Chen, J., Wu, X.L., Li, X.Q., 2012. Removal of silver nanoparticles in simulated wastewater treatment processes and its impact on COD and NH4 reduction. Chemosphere 87, 248e252. Keller, A.A., Wang, H., Zhou, D., Lenihan, H.S., Cherr, G., Cardinale, B.J., Miller, R., Ji, Z., 2010. Stability and aggregation of metal oxide nanoparticles in natural aqueous matrices. Environ. Sci. Technol. 44 (6), 1962e1967. Lee, D.J., Wong, B.T., 2014. Denitrifying sulfide removal and nitrososulfide complex: Azoarcus sp. NSC3 and Pseudomonas sp. CRS1 mix. Bioresour. Technol. 166, 616e619. Liang, Z., Das, A., Hu, Z., 2010. Bacterial response to a shock load of nanosilver in an activated sludge treatment system. Water Res. 44 (18), 5432e5438. Liu, J., Wang, Z., Liu, F.D., Kane, A.B., Hurt, R.H., 2012. Chemical transformations of nanosilver in biological environments. ACS Nano 6 (11), 9887e9899. Lombi, E., Donner, E., Taheri, S., Tavakkoli, E., J€ amting, Å.K., McClure, S., Naidu, R., Miller, B.W., Scheckel, K.G., Vasilev, K., 2013. Transformation of four silver/silver chloride nanoparticles during anaerobic treatment of wastewater and postprocessing of sewage sludge. Environ. Pollut. 176, 193e197. Ma, B.R., Wang, S., Li, Z.W., Gao, M.C., Li, S.S., Guo, L., She, Z.L., Zhao, Y.G., Zheng, D., Jin, C.J., Wang, X.J., Gao, F., 2017. Magnetic Fe3O4 nanoparticles induced effects on performance and microbial community of activated sludge from a sequencing batch reactor under long-term exposure. Bioresour. Technol. 225, 377e385. Marambio-Jones, C., Hoek, E.M.V., 2010. A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment. J. Nanopart. Res. 12, 1531e1551. Michels, C., Perazzoli, S., Soares, H.M., 2017. Inhibition of an enriched culture of ammonia oxidizing bacteria by two different nanoparticles: silver and magnetite. Sci. Total Environ. 586, 995e1002. Oukarroum, A., Bras, S., Perreault, F., Popovic, R., 2012. Inhibitory effects of silver nanoparticles in two green algae, Chlorella vulgaris and Dunaliella tertiolecta. Ecotoxicol. Environ. Saf. 78, 80e85. Qiu, G.L., Ting, Y.P., 2013. Osmotic membrane bioreactor for wastewater treatment and the effect of salt accumulation on system performance and microbial community dynamics. Bioresour. Technol. 150, 287e297. Qiu, G.L., Wirianto, K., Sun, Y.L., Ting, Y.P., 2016. Effect of silver nanoparticles on system performance and microbial community dynamics in a sequencing batch reactor. J. Clean. Prod. 130, 137e142. Rivas, R., Willems, A., Subba-Rao, N.S., Mateos, P.F., Dazzo, F.B., Kroppenstedt, R.M., Martínez-Molina, E., Gillis, M., Vel azquez, E., 2003. Description of Devosia neptuniae sp. nov. that nodulates and fixed nitrogen in sysbiosis with neptunia natans, an aquatic legume from India, 2003. System. Appl. Microbiol. 26, 47e53. Sheng, Z., Liu, Y., 2017. Potential impacts of silver nanoparticles on bacteria in the aquatic environment. J. Environ. Manag. 191, 290e296. Sthijns, M.M.J.P.E., Thongkam, W., Albrecht, C., Hellack, B., Bast, A., Haenen, G.R.M.M., Schins, R.P.F., 2017. Silver nanoparticles induce hormesis in A549 human epithelial cells. Toxicol. Vitro 40, 223e233. Wang, S., Gao, M.C., She, Z.L., Zheng, D., Jin, C.L., Guo, L., Zhao, Y.G., Li, Z.W., Wang, X.J., 2016. Long-term effects of ZnO nanoparticles on nitrogen and phosphorus removal, microbial activity and microbial community of a sequencing batch reactor. Bioresour. Technol. 216, 428e436. Wang, S., Li, Z.W., Gao, M.C., She, Z.L., Ma, B.R., Guo, L., Zheng, D., Zhao, Y.G., Jin, C.J., Wang, X.J., Gao, F., 2017. Long-term effects of cupric oxide nanoparticles (CuO NPs) on the performance, microbial community and enzymatic activity of activated sludge in a sequencing batch reactor. J. Environ. Manag. 187, 330e339. Yuan, Z., Li, J., Cui, L., Xu, B., Zhang, H., Yu, C.P., 2013. Interaction of silver nanoparticles with pure nitrifying bacteria. Chemosphere 90 (4), 1404e1411. Zhang, C.Q., Liang, Z.H., Hu, Z.Q., 2014. Bacterial response to a continuous long-term exposure of silver nanoparticles at sub-ppm silver concentrations in a membrane bioreactor activated sludge system. Water Res. 50, 350e358.
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Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Research article
Impacts of silver nanoparticles on performance and microbial community and enzymatic activity of a sequencing batch reactor Qiaoyan Xu a, c, Shanshan Li a, Yiping Wan a, Sen Wang b, Bingrui Ma a, Zonglian She a, Liang Guo a, Mengchun Gao a, *, Yangguo Zhao a, Chunji Jin a, b, Junwei Dong a, Zhiwei Li a a b c
Key Lab of Marine Environment and Ecology, Ministry of Education, Ocean University of China, Qingdao, 266100, China School of Environmental Science and Engineering, Qingdao University, Qingdao, 266071, China Shandong Provincial Key Laboratory of Marine Environment and Geological Engineering, Ocean University of China, Qingdao, 266100, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 August 2017 Received in revised form 14 September 2017 Accepted 18 September 2017
The performance, microbial community and enzymatic activity of a sequencing batch reactor (SBR) were evaluated under silver nanoparticles (Ag NPs) stress. Over 5 mg/L Ag NPs inhibited the COD and phosphorus removals, whereas the NHþ 4 removal kept stable during the whole operational period. The organic matter, nitrogen and phosphorus removal rates were obviously inhibited under Ag NPs stress, which showed similar varying trends with the corresponding microbial enzymatic activities. The change of Ag content in the activated sludge indicated that some Ag NPs were absorbed by the sludge. The presence of Ag NPs promoted the increase of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) of microorganism due to the microbial response to the Ag NPs toxicity, which could impact on the microbial morphology and physiological functions. The presence of Ag NPs could produce some evident changes in the microbial community. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Ag NPs Biotoxicity Microbial community Sequencing batch reactor Reactive oxygen species
1. Introduction Silver nanoparticles (Ag NPs) have been applied in room fresheners, food production, shampoos, bioengineering, biomedical products, laundry products and textiles (Lombi et al., 2013; Cushen et al., 2014). Ag NPs unavoidably enter the terrestrial and aquatic environment due to their increasing production and extensive applications. The presence of Ag NPs in the environment has recently attracted public concerns regarding their potential effects on the biota and human health. Some researches reported that Ag NPs had obvious ecotoxicity to microorganisms and aquatic organisms, such as bacteria (Sheng and Liu, 2017), algae (Oukarroum et al., 2012) and fish (Chen et al., 2017). Ag NPs are also reported to have adverse effects on human health (Sthijns et al., 2017; Marambio-Jones and Hoek, 2010). The Ag NPs release during the production, transport and utilization will end up in biological wastewater treatment systems. Ag NPs content in municipal sewage is the microgram level owing to
* Corresponding author. Key Lab of Marine Environment and Ecology, Ministry of Education, Ocean University of China, No. 238 Songling Road, Qingdao, Shandong Province, 266100, China. E-mail address: [email protected] (M. Gao). https://doi.org/10.1016/j.jenvman.2017.09.050 0301-4797/© 2017 Elsevier Ltd. All rights reserved.
the mixture of domestic sewage and industrial wastewater (Gottschalk et al., 2009). Nevertheless, high Ag NPs-containing industrial wastewater will result in Ag NPs at the milligram grade (Zhang et al., 2014). Considering the biotoxicity of Ag NPs, it is essential to evaluate the Ag NPs effect on bioreactors treating wastewater. Ag NPs could obviously inhibit the activity of Nitrosomonas europaea (Yuan et al., 2013) and denitrifying bacteria (Chen et al., 2014), indicating that Ag NPs could impact on the processes of nitrification and denitrification. Qiu et al. (2016) reported that less than 5 mg/L Ag NPs did not affect the COD removal, nitrification and denitrification of a sequencing batch reactor (SBR), whereas the presence of Ag NPs caused significant shifts in microbial community of SBR. Gu et al. (2014) demonstrated that Ag NPs at over 50 mg/L could significantly inhibit the biological nitrogen removal. Ag NPs concentration at less than 5 mg/L did not adversely affect biological phosphorus removal (Chen et al., 2012). Giao et al. (2017) evaluated the inhibition impacts of Ag NPs on ammonia oxidation kinetics and mechanism of nitrifying sludge. As is known to all, the bioreactor performances treating wastewater are closely associated with the microbial community and enzymatic activity. Therefore, it is interesting to investigate the microbial community and enzymatic activity of SBR under Ag NPs stress. In this study, the SBR performance and its pollutant removal rates were evaluated under Ag NPs stress. The key microbial
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enzymatic activity relating to the organic matter, nitrogen and phosphorous removals were compared at 0e30 mg/L Ag NPs. The Ag NPs toxicity to activated sludge was analyzed under Ag NPs stress. The variation of microbial community under Ag NPs stress was explored at the phylum and genus levels. 2. Materials and methods 2.1. Apparatus setup and influent composition A lab-scale SBR was applied in the present study, which had 7.7 L effective volume. The influent and air were introduced into the SBR by an influent pump and aeration pump, respectively. The solid retention time was approximately 20 d during the whole experimental period. Each cycle consisted of 6 min influent, 144 min anoxic stage, 240 min aerobic stage, 78 min settling and 12 min discharge, which was controlled by a time controller. The dissolved oxygen concentration was maintained above 2 mg/L and less than 0.5 mg/L during the aerobic stage and anoxic stage, respectively. Prior to the addition of Ag NPs, the SBR had operated for 117 d without Ag NPs addition and the SBR performance had reached a steady state. Ag NPs in the present study was approximately 30 nm particle diameter and 99.9% purity. 500 mg/L Ag NPs stock suspension was prepared in the light of Keller et al. (2010). The influent composition was similar to our previous research (Ma et al., 2017). The influent COD, soluble orthophosphate (SOP) and NHþ 4 -N were 414.3 ± 8.3, 22.4 ± 0.7 and 9.9 ± 0.3 mg/L during the operational period, respectively. 2.2. Analytical methods COD, NHþ 4 , NO3 , NO2 , SOP and mixed liquor volatile suspended
solids (MLVSS) were measured in light of standard methods (APHA, 1998). The specific ammonia oxidation rate (SAOR), specific nitrite oxidation rate (SNOR), specific oxygen utilization rate (SOUR), specific nitrate reduction rate (SNRR), specific nitrite reduction rate (SNIRR), specific phosphorus release rate (SPRR), and specific phosphorus uptake rate (SPUR) were analyzed as described in previous research (Wang et al., 2016). Reactive oxygen species (ROS) and lactate dehydrogenase (LDH) were measured according to Wang et al. (2017). Nitrite oxidoreductase (NOR), dehydrogenase (DHA), nitrite reductase (NIR), nitrate reductase (NR), polyphosphate kinase (PPK), ammonia monooxygenase (AMO), and exopolyphosphatase (PPX) activities were determined in the light of Wang et al. (2016). High throughput sequencing was applied to analyze the microbial community (Gao et al., 2014). 3. Results and discussion 3.1. SBR performance evaluation under Ag NPs stress Fig. 1 shows the COD, nitrogen and phosphorus removals under Ag NPs stress. The COD removal retained steady under less than 2 mg/L Ag NPs (Fig. 1a), suggesting that low Ag NPs concentration displayed a negligible inhibition influence on the activities of heterotrophic microorganisms owing to their tolerance ability to Ag NPs toxicity (Liang et al., 2010). Previous researches also illustrated that low Ag NPs concentration had no adverse effect on the COD removal in some bioreactors (Hou et al., 2012; Qiu et al., 2016). However, the COD removal efficiency in the present study reduced from 91.70 ± 0.84% to 87.76 ± 0.66% with the increase of Ag NPs concentration from 2 to 30 mg/L, illustrating that high Ag NPs concentration exerted a slight inhibition impact on COD removal. The NHþ 4 removal efficiencies were 98.28 ± 1.19% during the
Fig. 1. Impact of Ag NPs on the SBR performance. (a) COD, (b) NHþ 4 -N, (c) NO2 -N and NO3 -N, and (d) SOP.
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whole operational period (Fig. 1b), indicating that less than 30 mg/L Ag NPs had no obvious influence on the ammonia oxidization. Qiu et al. (2016) reported that less than 5 mg/L Ag NP had no distinct influence on the nitrification process. Contrarily, some researches demonstrated that Ag NPs inhibited the NHþ 4 oxidization in different biological wastewater treatment processes (Alito and Gunsch, 2014; Liang et al., 2010; Hou et al., 2012). The discrepancy between the present results and previous researches could be associated with exposure time, wastewater quality, Ag NPs addition dose, bioreactor type, MLVSS, hydraulic retention time and sludge retention time. The effluent NO 2 -N concentration varied between 0 and 0.83 mg/L during the whole operational period (Fig. 1c). Nevertheless, the effluent NO 3 -N gradually reduced with the increase of Ag NPs concentration. The present results might be explained that the adsorption of Ag NPs on the sludge surface decreased the mass transfer efficiency of oxygen into the interior of sludge flocs and led to more favorable anaerobic environment for the denitrification process of activated sludge. The SOP removal efficiency slightly decreased at 0e5 mg/L Ag NPs (Fig. 1d), which had similar results to previous report (Chen et al., 2012). Nevertheless, the SOP removal efficiency reduced from 75.99 ± 1.59% to 58.08 ± 4.89% with the increase of Ag NPs concentration from 5 to 30 mg/L, illustrating the biological phosphorous removal was obviously inhibited by Ag NPs.
3.2. Nitrogen and phosphorus removal rates under Ag NPs stress Fig. 2 shows the nitrogen and phosphorus removal rates and SOUR under Ag NPs stress. The SOUR decreased from 48.00 ± 1.79 to 31.88 ± 1.33 mg O2/(g MLVSS$h) with the increase of Ag NPs concentration from 0 to 30 mg/L (Fig. 2a). Compared with the
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absence of Ag NPs, the SOUR reduced by 33.58% at 30 mg/L Ag NPs, demonstrating that Ag NPs inhibited the metabolic activities of heterotrophic bacteria and then affect the organic matter removal rate. García et al. (2012) also found that Ag NPs had evident inhibitory effect on the SOUR of ordinary heterotrophic organisms by respiratory experiments. Both the SAOR and SNOR reduced with the increase of Ag NPs concentration from 0 to 30 mg/L (Fig. 2b). Compared with the absence of Ag NPs, the SAOR and SNOR reduced by 26.50% and 23.53% at 30 mg/L Ag NPs, respectively. The changes of SAOR and SNOR demonstrated that Ag NPs could exert more inhibition effect on the SAOR than the SNOR. Michels et al. (2017) illustrated that the specific nitrite production rate of ammonia oxidizing bacteria had an evident reduction under Ag NPs stress. As shown in Fig. 2c, the SNIRR and SNRR decreased from 10.49 ± 0.39 and 11.14 ± 0.42 mg N/(g MLVSS$h) at 0 mg/L Ag NPs to 8.30 ± 0.25 and 9.28 ± 0.47 mg N/(g MLVSS$h) at 30 mg/L Ag NPs, respectively. The SNIRR and SNRR decreased by 20.88% and 16.70% at 30 mg/L Ag NPs by comparison with 0 mg/L Ag NPs, respectively, indicating that Ag NPs could inhibit the SNIRR more than the SNRR. The SPUR and SPRR of activated sludge reduced from 14.39 ± 0.54 and 13.66 ± 0.51 mg P/(g MLVSS$h) at 0 mg/L Ag NPs to 9.57 ± 0.68 and 8.50 ± 0.35 mg P/(g MLVSS$h) at 30 mg/L Ag NPs with the increase of Ag NPs concentration from 0 to 30 mg/L (Fig. 2d), respectively. Compared with the absence of Ag NPs, the SPUR and SPRR decreased by 33.50% and 37.78% at 30 mg/L Ag NPs, respectively, illustrating that Ag NPs inhibited the biological phosphorus release and uptake.
3.3. Variation of microbial enzymatic activity under Ag NPs stress The organic matter, nitrogen and phosphorus removals of
Fig. 2. Impacts of Ag NPs on the microbial activities of activated sludge. (a) SOUR, (b) SAOR and SNOR, (c) SNIRR and SNRR, and (d) SPUR and SPRR. Asterisks indicate the statistical difference (p < 0.05) from the microbial activities at 0 mg/L Ag NPs. Error bars represent standard deviations of triplicate measurements.
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wastewater biological treatment systems are closely associated with microbial enzymatic activity. The DHA decreased from 15.138 ± 0.565 to 9.799 ± 0.770 mg TF/(mg MLVSS$h) with the increase of Ag NPs concentration from 0 to 30 mg/L (Fig. 3a). The DHA had a similar varying tendency to the SOUR, suggesting that the decrease of SOUR was related to the variation of DHA under Ag NPs stress. Both the AMO and NOR activities decreased with the increase of Ag NPs concentration from 0 to 30 mg/L (Fig. 3b), respectively. The varying trends of the AMO and NOR activities were similar to those of the SAOR and SNOR, respectively, demonstrating that the reduction of SAOR and SNOR were related to the inhibition impact of Ag NPs on the AMO and NOR. Compared with the absence of Ag NPs, the AMO and NOR activities decreased by 28.29% and 28.90% at 30 mg/L Ag NPs, respectively, indicating that Ag NPs had similar inhibition rates on the enzymatic activities of NHþ 4 -oxidization microorganism and NO 2 -oxidization microorganism. The NIR and NR activities decreased from 5.423 ± 0.154 and 0.372 ± 0.011 mg NO 2 -N/(min$mg protein) at 0 mg/L Ag NPs to 3.895 ± 0.162 and 0.274 ± 0.011 mg NO 2 -N/(min$mg protein) at 30 mg/L Ag NPs (Fig. 3c), respectively. Compared with the absence of Ag NPs, the NIR and NR activities reduced by 28.18% and 26.34% at 30 mg/L Ag NPs, respectively, demonstrating that Ag NPs had higher inhibition rate on the enzymatic activities of NO 2 -reduction microorganism than NO 3 -reduction microorganism. Additionally, the NIR and NR activities showed similar variation trends to the SNIRR and SNRR, respectively. As shown in Fig. 3d, the PPX activity decreased from 0.0587 ± 0.0017 to 0.0407 ± 0.0017 mmol pnitrophenol/(min$mg protein) with the increase of Ag NPs concentration from 0 to 30 mg/ L, and the PPK activity reduced from 0.0892 ± 0.0025 mmol NADPH/ (min$mg protein) at 0 mg/L Ag NPs to 0.0681 ± 0.0028 mmol NADPH/(min$mg protein) at 30 mg/L Ag NPs. The PPX and PPK activities had similar varying tendencies with the SPUR and SPRR,
respectively. Compared with the absence of Ag NPs, the PPX and PPK activities decreased by 30.66% and 23.65% at 30 mg/L Ag NPs, respectively, demonstrating that Ag NPs could exert more inhibition impact on the microbial enzymatic activities related to phosphorus release than those related to phosphorus uptake. 3.4. Biotoxicity evaluation of Ag NPs Ag NPs biotoxicity is associated with intact nanoparticles and Ag ion released from Ag NPs (Gliga et al., 2014; Liu et al., 2012). Before investigating the biotoxicity of Ag NPs, the Ag contents of the effluent and activated sludge were measured at each Ag NPs concentration (Supplementary Material, Fig. S1). The Ag content of sludge showed a remarkable increasing tendency with the increase of Ag NPs concentration, suggesting that some Ag NPs was absorbed by the sludge. Additionally, the effluent Ag content also slightly increased with an increase in Ag NPs concentration. Feng et al. (2000) have reported that Ag ion can also damage the cell membrane, DNA, protein and respiratory chain. ROS generation is acted as a significant indicator for evaluating NPs biotoxicity in the environment, whereas LDH release is regarded as a marker to evaluate the integrity of cytomembrane. As illustrated in Fig. 4, the ROS production was 8.58%, 18.89%, 28.30% and 39.78% at 2, 5, 10 and 30 mg/L Ag NPs by comparing to the absence of Ag NPs, respectively. The change of ROS indicated that Ag NPs promoted the ROS production of the microorganisms and then exerted the biotoxicity of Ag NPs. Hao and Chen (2012) reported that the increase of ROS production might be related to the imbalance of oxidation process and anti-oxidation process, which could overwhelm the defense capacity of the organisms and lead to the enzymatic inactivation, protein oxidization and DNA destruction. Compared to the absence of Ag NPs, the LDH release increased with the increase of Ag NPs concentration, indicating that Ag NPs resulted in destroying the microbial cytomembrane integrity. The destruction to the
Fig. 3. Impacts of Ag NPs on the microbial enzymatic activities of activated sludge. (a) DHA, (b) AMO and NOR, (c) NIR and NR, (d) PPX and PPK. Asterisks indicate statistical differences (p < 0.05) from the key enzymatic activity at 0 mg/L Ag NPs. Error bars represent standard deviations of triplicate measurements. The enzymatic activity at 0, 2, 5, 10 and 30 mg/L was determined on day 117, 179, 215, 247 and 290, respectively.
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some evident variations during the operational period, suggesting that Ag NPs distinctly impacted on the microbial richness and diversity of SBR. The rarefaction curve under each Ag NPs concentration trended to level off along the X-axis direction and further explained that the obtained OTU number was reasonable in the present study (Supplementary Material, Fig. S2). The distinct separation of microbial community was observed among the abovementioned four samples through the principal coordinate's analysis (Supplementary Material, Fig. S3). The principal component 1 (PC1) and PC2 of microbial communities were 42.86% and 31.91% of the variability in total microbial community, respectively. Venn diagram was applied to investigate the difference and similarity of microbial community (Fig. 5). The common OTU number (819) among the above-mentioned four samples constituted 47.12% of the total OUT number, suggesting that some microorganisms existed at all times during the whole operational period. The dominant phyla in the shared OTUs included 42.2% Proteobacteria, 19.4% Bacteroidetes, 8.3% Chloroflexi, 8.3% Planctomycetes, 4.1% Actinobacteria, 3.9% Acidobacteria, 3.0% Verrucomicrobia, 2.4% Firmicutes and 2.1% Chlorobi. The sum of special OTU among the above-mentioned four samples was 207, which only constituted 11.91% of total OTUs. The microbial community under Ag NPs stress was further explored at the phylum and genus levels. At the phylum level (Fig. 6a), the relative abundances of dominant phyla at 0e30 mg/L Ag NPs were 37.85e54.72% Proteobacteria, 27.06e36.42% Bacteroidetes, 2.94e6.63% Chloroflexi, 1.44e2.77% Planctomycetes, 1.12e2.46% Chlorobi, 0.90e2.37% Verrucomicrobia, 1.19e2.19% Acidobacteria, 0.59e1.43% Armatimonadetes, 0.38e1.39% Elusimicrobia and 0.79e1.12% Nitrospirae, suggesting that Ag NPs obviously impacted on the microbial richness. At the genus level (Fig. 6b), 32 dominant genera were chosen to analyze the changes of microbial richness under Ag NPs stress. Compared with the absence of Ag NPs, the relative abundance of Nitrosospira slightly increased at 5 mg/L Ag NPs, whereas decreased at 10e30 mg/L Ag NPs. However,
Fig. 4. Relative ROS production and LDH release of activated sludge at different concentrations of Ag NPs. Asterisks indicate the statistical difference (p < 0.05) from 0 mg/ L Ag NPs. Error bars represent standard deviations of triplicate measurements.
cytomembrane resulted in the leakage of intracellular substances, which could impact on the microbial morphology and physiological functions. 3.5. Impact of Ag NPs on microbial community High throughput sequencing was adopted to investigate the microbial community. As illustrated in Table 1, the effective sequence at 0, 5, 10 and 30 mg/L Ag NPs was classified as 1,432, 1,375, 1389 and 1495 operational taxonomic units (OTU), respectively. All the Good's coverage was greater than or equal to 0.995, suggesting that the sequence library covered the microbial diversity. The Chao 1, Shannon, Simpson and Ace indices displayed
Table 1 Microbial richness and diversity of activated sludge at different Ag NPs concentrations. Ag NPs concentration (mg/L)
Raw reads
Raw sequences
Effective sequences
OTUs
Chao1 index
Shannon index
Simpson index
ACE index
Good's coverage
0 5 10 30
45,547 49,904 47,887 56,756
44,562 48,859 46,958 55,654
43,058 47,144 45,644 51,692
1432 1375 1389 1495
1363.9 1370.9 1366.6 1466.3
7.995 7.237 7.371 7.446
0.986 0.966 0.974 0.976
1394.0 1374.6 1381.0 1484.6
0.997 0.995 0.995 0.995
Fig. 5. Venn diagram based on high-throughput sequencing of the microbial community at different Ag NPs concentrations (OTUs at 3% distance). The shared OTUs were analyzed at phylum level.
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Fig. 6. Taxonomic classification of 16S rDNA paired-end sequencing from activated sludge exposed to different Ag NPs concentrations at the phylum (a) and genus (b) levels.
the relative abundance of Nitrosomonas reduced with the increase of Ag NPs concentration from 0 to 30 mg/L. Nitrosomonas and Nitrosospira have the ability to oxidize ammonia to nitrite under aerobic condition. The relative abundance of Nitrococcus reduced from 10.30% at 0 mg/L Ag NPs to 2.55% at 30 mg/L Ag NPs. Nitrococcus has the capacity to oxidize nitrite to nitrate under aerobic condition. The relative abundances of Paracoccus, Devosia, Hyphomicrobium and Azoarcus maintained the decreasing trends with the increase of Ag NPs concentration, and these genera were reported to have the denitrifying ability (Baumann et al., 1996; Rivas et al., 2003; Green et al., 2010; Lee and Wong, 2014). Compared to the absence of Ag NPs, the relative abundances of Dechloromonas decreased at 10 mg/L Ag NPs and subsequently increased at 30 mg/ L Ag NPs, whereas Azospirillum had no obvious variation in the relative abundance during the operational period. Some researches have reported that Dechloromonas and Azospirillum have the ability to reduce nitrite or nitrate (Horn et al., 2005; Qiu and Ting, 2013). The present results demonstrated that Ag NPs affected the growth, development and reproduction of denitrifying bacteria. The relative abundance of Candidatus accumulibacter decreased from 2.49% to 1.3% with the increase of Ag NPs concentration from 0 to 10 mg/L and rose to 18.28% at 30 mg/L Ag NPs. He et al. (2007) have reported that Candidatus accumulibacter is related to the biological phosphorus removal. Additionally, the relative abundances of some
genera increased or reduced at each Ag NPs concentration by comparing with the absence of Ag NPs, such as Haliscomenobacter, Fimbriimonas, Sediminibacterium, Chitinophaga, Arcobacter, Ignavibacterium and Phaselicystis. 4. Conclusions Ag NPs concentration at over 5 mg/L inhibited the COD and phosphorus removal, whereas the NHþ 4 removal efficiency kept stable during the whole operational period. The SOUR and nitrogen and phosphorus removal rates could be inhibited by Ag NPs, which showed similar variation tendencies to their corresponding microbial enzymatic activities. The presence of Ag NPs promoted the ROS production and LDH release due to the microbial response to the Ag NPs toxicity, which could impact on the microbial morphology and physiological function. The presence of Ag NPs exerted evident changes in the microbial richness and diversity. Acknowledgements The work was funded by the National Natural Science Foundation of China (No. 51178437) and Province Key Technologies R & D Program of Shandong (Grants 2007GG10006002).
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Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jenvman.2017.09.050. References Alito, C., Gunsch, C.K., 2014. Assessing the effects of sliver nanoparticles on biological nutrient removal in bench-scale activated sludge sequencing batch reactors. Environ. Sci. Technol. 49, 970e976. APHA, 1998. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, DC. Baumann, B., Snozzi, M., Zehnder, A.J.B., Meer, J.R.V.D., 1996. Dynamics of denitrification activity of Paracoccus denitrificans in continuous culture during aerobicanaerobic changes. J. Bacteriol. 178, 4367e4374. Chen, J., Tang, Y.Q., Li, Y., Nie, Y., Hou, L., Li, X.Q., Wu, X.L., 2014. Impacts of different nanoparticles on functional bacterial community in activated sludge. Chemosphere 104, 141e148. Chen, S.X., Yang, X.Z., Deng, Y., Huang, J., Li, Y., Sun, Q., Yu, C.P., Zhu, Y., Hong, W.S., 2017. Silver nanoparticles induce oocyte maturation in zebrafish (Danio rerio). Chemosphere 170, 51e60. Chen, Y., Chen, H., Zheng, X., Mu, H., 2012. The impacts of silver nanoparticles and silver ions on wastewater biological phosphorous removal and the mechanisms. J. Hazard Mater. 239e240, 88e94. Cushen, M., Kerry, J., Morris, M., Cruz-Romero, M., Cummins, E., 2014. Evaluation and simulation of silver and copper nanoparticle migration from polyethylene nanocomposites to food and an associated exposure assessment. J. Agric. Food Chem. 62 (6), 1403e1411. Feng, Q.L., Wu, J., Chen, G.Q., Cui, F.Z., Kim, T.N., Kim, J.O., 2000. A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J. Biomed. Mater. Res. 52 (4), 662e668. Gao, C., Wang, A., Wu, W.M., Yin, Y., Zhao, Y.G., 2014. Enrichment of anodic biofilm inoculated with anaerobic or aerobic sludge in single chambered air-cathode microbial fuel cells. Bioresour. Technol. 167, 124e132. , J.A., Casals, E., Gonz García, A., Delgado, L., Tora alez, E., Puntes, V., Font, X., Carrera, J., S anchez, A., 2012. Effect of cerium dioxide, titanium dioxide, silver, and gold nanoparticles on the activity of microbial communities intended in wastewater treatment. J. Hazard Mater. 199e200, 64e72. Gliga, A.R., Skoglund, S., Wallinder, I.O., Fadeel, B., Karlsson, H., 2014. Size-dependent cytotoxicity of silver nanoparticles in human lung cells: the role of cellular uptake, agglomeration and Ag release. Part. Fibre Toxicol. 11 (1), 1. Giao, N.T., Limpiyakorn, T., Kunapongkiti, P., Thuptimdang, P., SiripattanakulRatpukdi, S., 2017. Influence of silver nanoparticles and liberated silver ions on nitrifying sludge: ammonia oxidation inhibitory kinetics and mechanism. Environ. Sci. Pollut. Res. 24 (10), 9229e9240. Gottschalk, F., Sonderer, T., Scholz, R.W., Nowack, B., 2009. Modeled environmental concentrations of engineered nanomaterials (TiO2, ZnO, Ag, CNT, fullerenes) for different regions. Environ. Sci. Technol. 43, 9216e9222. Green, S.J., Prakash, O., Gihring, T.M., Akob, D.M., Jasrotia, P., Jardine, P.M., Watson, D.B., Brown, S.D., Palumbo, A.V., Kostka, J.E., 2010. Denitrifying bacteria isolated from terrestrial subsurface sediments exposed to mixed-waste contamination. Appl. Environ. Microbiol. 76 (10), 3244e3254. Gu, L., Li, Q., Quan, X., Cen, Y., Jiang, X., 2014. Comparison of nanosilver removal by flocculent and granular sludge and short- and long-term inhibition impacts. Water Res. 58, 62e70. Hao, L., Chen, L., 2012. Oxidative stress responses in different organs of carp (Cyprinus carpio) with exposure to ZnO nanoparticles. Ecotoxicol. Environ. Saf. 80, 103e110. He, S.M., Gall, D.L., McMahon, K.D., 2007. “Candidatus Accumulibacter” population structure in enhanced biological phosphorus removal sludges as revealed by polyphosphate kinase genes. Appl. Environ. Microbiol. 73, 5865e5874. Horn, M.A., Ihssen, J.L., Matthies, C., Schramm, A., Acker, G., Drake, H.L., 2005.
673
Dechloromonas denitrificans sp. nov., Flavobacterium denitrificans sp. nov., Paenibacillus anaericanus sp. nov. and Paenibacillus terrae strain MH72, N2O-producing bacteria isolated from the gut of the earthworm Aporrectodea caliginosa, 2005. Int. J. Syst. Evol. Microbiol. 55, 1255e1265. Hou, L.L., Li, K.Y., Ding, Y.Z., Li, Y., Chen, J., Wu, X.L., Li, X.Q., 2012. Removal of silver nanoparticles in simulated wastewater treatment processes and its impact on COD and NH4 reduction. Chemosphere 87, 248e252. Keller, A.A., Wang, H., Zhou, D., Lenihan, H.S., Cherr, G., Cardinale, B.J., Miller, R., Ji, Z., 2010. Stability and aggregation of metal oxide nanoparticles in natural aqueous matrices. Environ. Sci. Technol. 44 (6), 1962e1967. Lee, D.J., Wong, B.T., 2014. Denitrifying sulfide removal and nitrososulfide complex: Azoarcus sp. NSC3 and Pseudomonas sp. CRS1 mix. Bioresour. Technol. 166, 616e619. Liang, Z., Das, A., Hu, Z., 2010. Bacterial response to a shock load of nanosilver in an activated sludge treatment system. Water Res. 44 (18), 5432e5438. Liu, J., Wang, Z., Liu, F.D., Kane, A.B., Hurt, R.H., 2012. Chemical transformations of nanosilver in biological environments. ACS Nano 6 (11), 9887e9899. Lombi, E., Donner, E., Taheri, S., Tavakkoli, E., J€ amting, Å.K., McClure, S., Naidu, R., Miller, B.W., Scheckel, K.G., Vasilev, K., 2013. Transformation of four silver/silver chloride nanoparticles during anaerobic treatment of wastewater and postprocessing of sewage sludge. Environ. Pollut. 176, 193e197. Ma, B.R., Wang, S., Li, Z.W., Gao, M.C., Li, S.S., Guo, L., She, Z.L., Zhao, Y.G., Zheng, D., Jin, C.J., Wang, X.J., Gao, F., 2017. Magnetic Fe3O4 nanoparticles induced effects on performance and microbial community of activated sludge from a sequencing batch reactor under long-term exposure. Bioresour. Technol. 225, 377e385. Marambio-Jones, C., Hoek, E.M.V., 2010. A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment. J. Nanopart. Res. 12, 1531e1551. Michels, C., Perazzoli, S., Soares, H.M., 2017. Inhibition of an enriched culture of ammonia oxidizing bacteria by two different nanoparticles: silver and magnetite. Sci. Total Environ. 586, 995e1002. Oukarroum, A., Bras, S., Perreault, F., Popovic, R., 2012. Inhibitory effects of silver nanoparticles in two green algae, Chlorella vulgaris and Dunaliella tertiolecta. Ecotoxicol. Environ. Saf. 78, 80e85. Qiu, G.L., Ting, Y.P., 2013. Osmotic membrane bioreactor for wastewater treatment and the effect of salt accumulation on system performance and microbial community dynamics. Bioresour. Technol. 150, 287e297. Qiu, G.L., Wirianto, K., Sun, Y.L., Ting, Y.P., 2016. Effect of silver nanoparticles on system performance and microbial community dynamics in a sequencing batch reactor. J. Clean. Prod. 130, 137e142. Rivas, R., Willems, A., Subba-Rao, N.S., Mateos, P.F., Dazzo, F.B., Kroppenstedt, R.M., Martínez-Molina, E., Gillis, M., Vel azquez, E., 2003. Description of Devosia neptuniae sp. nov. that nodulates and fixed nitrogen in sysbiosis with neptunia natans, an aquatic legume from India, 2003. System. Appl. Microbiol. 26, 47e53. Sheng, Z., Liu, Y., 2017. Potential impacts of silver nanoparticles on bacteria in the aquatic environment. J. Environ. Manag. 191, 290e296. Sthijns, M.M.J.P.E., Thongkam, W., Albrecht, C., Hellack, B., Bast, A., Haenen, G.R.M.M., Schins, R.P.F., 2017. Silver nanoparticles induce hormesis in A549 human epithelial cells. Toxicol. Vitro 40, 223e233. Wang, S., Gao, M.C., She, Z.L., Zheng, D., Jin, C.L., Guo, L., Zhao, Y.G., Li, Z.W., Wang, X.J., 2016. Long-term effects of ZnO nanoparticles on nitrogen and phosphorus removal, microbial activity and microbial community of a sequencing batch reactor. Bioresour. Technol. 216, 428e436. Wang, S., Li, Z.W., Gao, M.C., She, Z.L., Ma, B.R., Guo, L., Zheng, D., Zhao, Y.G., Jin, C.J., Wang, X.J., Gao, F., 2017. Long-term effects of cupric oxide nanoparticles (CuO NPs) on the performance, microbial community and enzymatic activity of activated sludge in a sequencing batch reactor. J. Environ. Manag. 187, 330e339. Yuan, Z., Li, J., Cui, L., Xu, B., Zhang, H., Yu, C.P., 2013. Interaction of silver nanoparticles with pure nitrifying bacteria. Chemosphere 90 (4), 1404e1411. Zhang, C.Q., Liang, Z.H., Hu, Z.Q., 2014. Bacterial response to a continuous long-term exposure of silver nanoparticles at sub-ppm silver concentrations in a membrane bioreactor activated sludge system. Water Res. 50, 350e358.