- Email: [email protected]
N ratio influent
Journal of Hazardous Materials 394 (2020) 122583
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Enhancement of PPCPs removal by shaped microbial community of aerobic granular sludge under condition of low C/N ratio influent
T
Zhuodong Yua, Ye Zhanga, Zhiming Zhanga, Jingjing Donga, Jiashen Fua, Xiangyang Xua,b,c, Liang Zhua,b,c,* a
Institute of Environmental Pollution Control and Treatment, Zhejiang University, Hangzhou 310058, China Zhejiang Province Key Laboratory for Water Pollution Control and Environmental Safety, Hangzhou 310058, China c Zhejiang Provincial Engineering Laboratory of Water Pollution Control, 388 Yuhangtang Road, Hangzhou 310058, China b
G R A P H I C A L A B S T R A C T
Mechanism of enhanced denitrification and PPCP removal by aerobic granular sludge.
A R T I C LE I N FO
A B S T R A C T
Editor: Zhu Yu
The frequent occurrence of pharmaceuticals and personal care products (PPCPs) in domestic wastewater has caused great concern. In this study, the removal of two typical pharmaceuticals (Roxithromycin, ROX; Sulfamethoxazole, SMZ) in aerobic granular sludge (AGS) reactors was investigated under condition of different C/N (carbon to nitrogen) ratios. Results showed that higher removal efficiencies of ROX and SMZ (95.2 % and 92.9 %) were achieved in the AGS reactor fed with low C/N influent. Batch experiments further revealed that the removal of ROX was influenced by the adsorption ability of the AGS while SMZ removal was mainly enhanced by biodegradation process. Analysis of extracellular polymeric substances (EPS) showed that the humic acid-like substances were enriched under low C/N condition, which was in accordance with dynamic change of microbial community. The microbes, like Thauera spp. and Xanthomonadaceae, were highly enriched in the reactor with high nitrogen loading rate and functioned as refractory organics degrader. Overall, the AGS process could achieve enhanced pharmaceuticals removal performance by the regulation of microbial community under low C/N influent, which provides insights into a feasible solution for simultaneous removal of nitrogen and trace organic pollutants in AGS reactor.
Keywords: Aerobic granular sludge Pharmaceuticals removal C/N ratio Microbial community
1. Introduction Pharmaceutical and Personal Care Products(PPCPs)are a large
⁎
group of organic compounds, including antibiotics, anti-inflammatories, sanitizer, preservatives, spices, sunscreen and cosmetics (Arp, 2012; Liu and Wong, 2013). The extensive use of PPCPs has
Corresponding author at: Department of Environmental Engineering, Zhejiang University, No. 866 Yuhangtang Road, Hangzhou 310058, PR China. E-mail address: [email protected] (L. Zhu).
https://doi.org/10.1016/j.jhazmat.2020.122583 Received 20 November 2019; Received in revised form 10 March 2020; Accepted 23 March 2020 Available online 27 March 2020 0304-3894/ © 2020 Elsevier B.V. All rights reserved.
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
2. Materials and methods
caused great concern in recent years, especially in developing countries (Bu et al., 2013; Rehman et al., 2015). Asian pharmaceutical market is growing at a rate of 10–15 % annually compared to 5–7 % growth in G7 countries (Rehman et al., 2015). As the world's largest producer and consumer of PPCPs, China exports more than 60 % of the total active pharmaceutical ingredients to the global pharmaceutical industry, and consumes more than 25,000 tons of antibiotics annually (Ma et al., 2017; Liu et al., 2020). These trace pollutants have been continually released and frequently detected in the aquatic environment, which are threatening the aquatic environment and drinking water security (Boxall Alistair et al., 2012; Zhang et al., 2015; Ebele et al., 2017; Qiao et al., 2018). Thus, increasing attention from China has been paid to prevention and control of PPCPs in environment. Wastewater treatment plants (WWTPs) are important facilities to remove the pollutants in wastewater and count for much to water security. However, current WWTPs are mainly designed for biological removal of the nutrients and unsatisfying in removal of the trace pollutants like pharmaceuticals (Sun et al., 2014; Thomaidi et al., 2015; Yang et al., 2017). Compared to the complementary physic-chemical process in WWTPs, bio-treatments are most convenient and low-cost methods to prevent those emerging trace pollutants from the natural environment (Luo et al., 2014b; Ahmed et al., 2017). Thus, advanced bio-treatment technology coupled with emerging trace pollutants and nutrient removal is desired in WWTPs. The aerobic granular sludge (AGS) technology is a novel biological treatment process with several advantages such as high biomass retention, excellent settling properties, and high removal efficiency of total nitrogen (de Kreuk et al., 2005d; Pronk et al., 2015; Corsino et al., 2016). Meanwhile, previous studies have showed that the aerobic granular sludge reactor is a promising biotechnology to withstand shock loading and remove trace pollutants (Moreira et al., 2015; Gómez-Acata et al., 2018). Although the AGS process has obtained fast progress in last decade, the industrial AGS application was very limited compared to traditional sewage treatment process (Pronk et al., 2015). There still is a lack of knowledge for AGS systems in practical influent condition, which becomes the bottleneck and impedes its further development (Derlon et al., 2016). In recent years, the influent of most municipal WWTPs in China and other countries shows the characteristics of low ratio of carbon to nitrogen (Sun et al., 2010; Derlon et al., 2016; Zhang et al., 2018). The relationship between the nitrogen loading rate and microbial community becomes an important factor to optimize the performance of WWTPs (Ye et al., 2011; Luo et al., 2014a; Wang et al., 2018; Zhang et al., 2018). Previous works reported that the microbes could enhance the removal of trace pollutants via co-metabolic degradation, which provided an insight to the enhancement of PPCPs removal in aerobic bio-treatment process (Tran et al., 2009; Suarez et al., 2010; FernandezFontaina et al., 2012). Thus, the aims of this study are to evaluate the pollutants (COD, TN, pharmaceuticals) removal performance under different conditions of C/N ratio influent in AGS reactors. Two typical antibiotics (Roxithromycin, ROX; Sulfamethoxazole, SMZ) with different toxicity were selected in this study. ROX is targeted to grampositive bacteria, which is low toxic to the microbes (dominated by gram-negative bacteria) in WWTPs. The SMZ is a broad-spectrum antibiotic and is a hardly degradable pollutant in WWTPs (FernandezFontaina et al., 2012; Kassotaki et al., 2016; Pamphile et al., 2019). Sludge characteristics like the extracellular polymeric substances (EPS) and sludge loading rate are monitored, and their influences on microbial community and pollutants removal were analyzed at the same time. Finally, the redundancy analysis (RDA) was used to illustrate the effect of the pharmaceuticals and C/N ratio on the dynamic change of functional microbes. This study provides insights into a feasible solution for the simultaneous removal of nitrogen and antibiotics in AGS reactors.
2.1. Operation of AGS reactors Three identical sequencing batch reactors (SBRs) (R1, R2, R3) were seeded with activated sludge from A2O in Qige WWTP (Hangzhou, China). The WWTP is designed for municipal wastewater and the SRT is about 10∼20 days. The COD fluctuates at 350∼550 mg/L and the COD to TN ratio is about 10. Each reactor had the volume of 4 L with the diameter to height ratio of 5. The aeration rate was controlled to maintain the superficial upflow air velocity of the reactors at 1.5 cms−1 and the dissolved oxygen (DO) was 7.5–8.5 mg/L in reactors. The SBR operation mode comprised 5 min feeding, 225 min aeration, 5 min settling and 5 min discharge from the middle of the reactor. The sludge retention time (SRT) of reactors was determined by the sludge discharged with the effluent. The C/N ratio was set at 30 (R1),15 (R2), and 7.5 (R3) with initial total nitrogen (TN) concentration of 26, 52, and 106 mg/L in the influent (Table S1.a). Synthetic wastewater was used as feeding water, comprising the following: Sodium acetate, 1008.3 mg/L; NH4Cl, variable; KH2PO4, 27.17 mg/L; K2HPO4·5H2O, 34.72 mg/L; yeast, 16.15 mg/L; peptone, 24.2 mg/L; CaCl2, 80 mg/L; MgSO4, 30 mg/L. The ambient temperature in the laboratory was 25 ± 2 °C. The sampling of water quality and was conducted two or three times a week. The influent pH was 7.1 ± 0.1 during the whole operation, as the pH was regulated by the addition of KH2PO4 and K2HPO4·5H2O. There were three stages during the reactor operation, including the stage I (the formation of AGS), stage II (the addition of ROX) and stage III (the addition of SMZ). The ROX (lower toxicity) was firstly added into the AGS and the concentration of antibiotics (influent, 0.01−0.1 mg/L) was gradually increased to prevent the shock loading from the reactors. The time and concentration of the antibiotic addition are shown in Table S1.b and Fig. 4. 2.2. Batch test for antibiotics removal The batch tests of antibiotics removal were conducted in four batch reactors with 800 mL working volume. The air upflow rate was controlled at 0.16 m3 h−1. The control group and experiment group were seed with the AGS (4000 mg/L) taken from R1 (C/N = 30) and R3 (C/ N = 7.5) reactor at day 133. In every group, the AGS in inactive batch was further treated with 0.1 % (w/v) sodium azide solution to eliminate biological activity of microbes according to Jadhav et al. (2008). The ROX and SMZ were added in the start of the experiment and the removal process was maintained for 2 days. The concentrations of the antibiotic addition in four batches are shown in Table S1.c. 3 × 5 ml mixture was sampled at 0, 0.33, 0.5, 2, 8, 16, 22, 30, 42, and 48 h, and centrifuged at 1000010,000g for 5 min. The supernatant was filtered by 0.22um filter membrane (Millipore, USA) and stored at 4 °C for detection. The data was fitted by the pseudo-second-order rate equation (Huang et al., 2014) to calculate the proportion of biodegradation and bio-adsorption between the AGS cultivated in different C/N ratio. The PPCP removal was fitted by the equation as follows:
qt =
Ktqe2 1 + Ktqe
Where qt is equilibrium removal capacity and qe represents maximum removal ability of the aerobic granular sludge. The equation can also be transformed to the following formula for linear fitting:
1 1 t = + ∙t qt Kqe 2 qe The absorption of the AGS was represented by the qe in inactive group (the sludge was treated by sodium azide), and the proportion of biodegradation was represented by the variation of the maximum removal ability between the active group and inactive group. 2
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
Fig. 1. Variation of sludge characteristics during reactor operation: biomass concentration (mixed liquor suspended solids, MLSS; mixed liquor volatile suspended solid, MLVSS) and Sludge Volume Index(SVI); in R1 (a), R2 (b), and R3 (c); sludge COD loading (d); sludge NH4+-N loading (e).
performance liquid chromatography-mass spectrometry (LC–MS/MS, Agilent 6460, USA). The Column ZorHax SB C18 (150 × 2.1 mm ID, 3.5 microns) was used and Column temperature maintained 40 °C. The mobile phase A is 2 mM ammonium acetate (0.1 % formic acid). The mobile phase B was acetonitrile with a flow rate of 0.4 mL min−1. During the operation, mobile phase B was gradually increased to 95 % in 10 min (table S3.a), and then phase B was then decreased to 2% from 10 to 15 min and held at 2% until the separation process stopped at 15 min. The MS/MS analysis was operated in positive electrospray ionization with multiple reactions monitoring (MRM) mode (table S3.b). All tests were conducted in triplicate and the quality assurance of the extraction protocol was evaluated by spiking 20 μg of antibiotic standards of interest into effluent with an overnight equilibration. The
2.3. Analytic methods 2.3.1. Water quality The effluent of three reactors was centrifuged at 4000 rpm for 10 min to test total nitrogen (TN) and chemical oxygen demand (COD) according to Zhang at al. (2018). The effluent for detection of NH4+-N, NO2–-N and NO3–-N was filtered by 0.45 μm membrane (Millipore, USA) and tested by standard methods (APHA, 2005). 2.3.2. Analysis of antibiotics The effluent water was filtered through a 0.45-μm membrane (Millipore, USA). The antibiotics in filtered water were enriched and purified by Solid-Phase extraction (SPE) and measured by high3
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
sludge loading rates of R3 reactor maintained above 0.35 mg COD mg−1 VSS and 0.03 mg N mg−1 VSS, which were about 2-fold and 8fold higher than the sludge loading rates in R1. Previous studies suggested that the sludge loading rate could be an important factor to the stability and performance of the AGS via influencing the microbial community (Hamza et al., 2018; Wu et al., 2018). In this study, the microbes in R3 were affected by higher ammonia loading rate and F/M rate during the domestication, which may contribute to the enrichment of ammonium oxidizing and denitrifying bacteria. The antibiotics were added to the reactors when the granulation degree of the AGS in reactors was more than 90 % (day 59) in stage II (the ROX addition) and stage III (the SMZ and ROX addition). The MLSS concentrations in three reactors were decreased to 68.1 %, 85.7 % and 81.25 % with the fluctuation of SVI30 in R1 and R3 from day 59 to day 80. The shock of antibiotics lead to sludge loss (day 80) but gradually recovered within two weeks. The MLSS concentration in high C/N groups (R1, R2) keep growing until the end of the experiment. The result suggested that the MLSS concentrations were affected by the C/N conditions and the occurrence of antibiotics in AGS reactors. The C/N conditions could be a main cause for the biomass concentration and sludge settleability (Luo et al., 2014a,b). The SVI30 of R3 fluctuated obviously after addition of antibiotics, indicating there might be significant alteration of microbes in R3. The variation of settleability and the different level of the biomass under this situation could be serious problem to the stability of SBR reactors (Dong et al., 2017; Zhang et al., 2018). As the sludge loss is mainly resulted by variation of settleability, the control of MLSS became more complicated during the occurrence of antibiotics with different condition in three reactors. Flexible settling time could be considered to avoid the excessive biomass loss or accumulation.
spiking recovery rates ranged from 85 to 105 %. 2.3.3. Analysis of EPS components The EPS was extracted from the sludge sample by a heating method (Li and Yang, 2007). The polysaccharide (PS) content in the EPS was quantified by the phenol–sulfuric acid method (Dubois et al., 1956), and the protein (PN) content in sludge EPS was determined by a modified Lowry colorimetric method with bovine albumin serum as the standard(Lowry et al., 1951). The EPS components were identified by the fluorescence excitation-emission matrix (EEM) using a fluorospectrophotometer (Shimadzu F-4500) according to Luo et al. (2014): Scanning emission spectra was obtained from 250 to 550 nm, and the excitation wavelength varied from 200 to 400 nm. Excitation and emission slits were both kept at 5 nm, and the scanning speed was set at 1200 nm min−1 and the data was displayed as contour maps of intensity. 2.4. Molecular characterization Sludge samples were taken from three AGS reactors at day 25, 59, 93, and 120. The DNA of sludge samples was extracted with the Power Soil DNA extraction kit (MO BIO Laboratories Inc.). Concentrations and quality of the extracted DNA was checked by spectrophotometric analysis on a Nano Drop ND-1000 (Thermo Fisher Scientific, USA). Then the DNA extracted from sludge samples was stored at −80 °C until analysis. The total DNA from sludge samples were used to amplify the V3/V4 region of the 16S rRNA (Primers:338 F; 806R). The PCR reactions were carried out in mixtures (25 μL) containing primer (2.5 μL), genomic DNA (10∼25 ng), PCR Premix (12.5 μL), and PCR-grade water to adjust the volume. All PCR reactions were performed in a Master Cycler Gradient thermocycler (Eppendorf, Hamburg, Germany). The conditions were: initial denaturation (98 °C,30 s); 35 cycles of denaturation (98 °C, 10 s), annealing (53 °C, 30 s), and extension (72 °C, 45 s); and final extension (72 °C, 10 min). Then the PCR products were sequenced by the Illumina MiSeq platform (PE300, CA, USA). The 16S rRNA amplicon sequences were aligned with the SILVA database, and then a distance matrix was generated between the aligned sequences. All sequences were clustered to operational taxonomic units (OTUs) at 97 % sequence similarity.
3.1.2. Removal of COD and TN under different C/N ratio The removal of COD in the three reactors is shown in Fig. 2. The COD in effluent was mostly less than the limit of detection (100 % removal efficiency) in R1 and R2 reactor after 20 days’ operation. Meanwhile, the fluctuation of COD was more obvious in R3. But removal efficiency of COD was stable over the 96 %. All reactors also showed high removal capacity to nitrogen. The ammonia nitrogen was also almost removed completely in three reactors (Fig. S2). However, there were significant differences in TN removal among the reactors. Higher removal efficiencies in R1 and R2 were achieved with high C/N ratio in influent during the domestication period. The average removal efficiencies of TN in first week were 87.8 ± 2.4 %, 76.0 ± 0.7 % and 65.6 ± 0.5 %, respectively. The TN removal efficiencies of R1 and R2 were stable at around 85 % and 74 % during the experiment while the TN removal efficiency of R3 showed an obvious growth tendency and reached 82.4 ± 2.9 % at the end of stage III. The TN removal efficiencies in AGS reactor were obvious effected by the C/N ratio. The concentrations of NH4+ and NO2− were mostly close to the detection limit in effluent in R3, which indicated that the NH4+ in influent was most transformed to NO3− (Fig. S2). Thus, the growth tendency of TN removal efficiency indicated that the advantageous condition was achieved for denitrifiers via higher nitrogen loading rate in R3 reactor.
2.5. Statistical analysis The RDA analysis was performed to assess the relationship between the abiotic/biotic conditions and bacterial communities using R version 3.4.0, (R Core Team 2017) with the CRAN package 'vegan' for the analysis of ecological communities (http://vegan.r-forge.r-project.org/ ). To better identify dynamic change of the dominant microorganism under different condition, the relative abundance of OTUs were represented in figures by the radius of the circle. 3. Results and discussion 3.1. Performance of AGS reactors
3.2. Removal of pharmaceuticals under different C/N ratio 3.1.1. Effects of the C/N ratio on sludge characteristics The experiment was divided into three stages: Stage I (formation of AGS), Stage II and Stage III (the SMZ and ROX removal). As is shown in Fig. 1, the concentration of inoculation sludge was 4000 ± 205 mg L−1. The mixed liquor suspended solids (MLSS) concentration increased rapidly in three reactors, and reached 21,240 mg L-1 (R1), 13,630 mg L-1 (R2) and 8250 mg L−1 (R3) at the end of stage I. It showed that the C/N ratio had significant effects on biomass concentration. The biomass accumulated fast in R1 (C/N = 30) and R2 (C/N = 15), which lead to low sludge loading rate (or Food/Microorganism ratio, F/M) in AGS reactors fed with high C/N influent. As is shown in Fig. 1d and e, the
The profile of pharmaceuticals removal efficiency is shown in Fig. 3. When the SBRs were started to feed with 10 μg L−1 of ROX at the beginning of stage II, the removal efficiencies of ROX in reactors were 91.2 ± 3.3 %、87.3 ± 4.8 % and 88.1 ± 4.1 %, respectively. But as the inflow ROX concentration was further increased to 100 μg L−1, the removal efficiencies of the reactors were 73.0 ± 0.1 % (R1), 85.0 ± 0.6 %(R2) and 80 ± 0.5 % (R3), respectively. The removal efficiencies of R1 and R2 were decreased to 68 % and 58 % while the R3 remained a high removal efficiency up to 95.2 ± 0.5 % on day 110. The SMX, a typical broad-spectrum antibiotic was started to feed in 4
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
Fig. 2. COD and TN removal efficiencies during reactor operation: COD in R1 (a), R2 (c) and R3 (e); TN in R1 (b), R2 (d) and R3 (f).
reactors on day 90. When the SMZ was added with 10 μg L-1, there was no obvious difference in SMZ removal efficiency (93.5 ± 0.6 %, R1; 95.1 ± 1.1 %, R2; 97.0 ± 2.0 %, R3). But when the SMZ increased, the removal efficiency of R1 and R2 were decreased to 78.2 ± 1.8 %, 84.5 ± 1.0 %. The R3 reactors maintained the highest removal efficiency (91.0 ± 0.8 %) throughout the experiment. Thus, there was a notable difference in the removal efficiency under different C/N conditions. The result showed the ROX and SMZ were effectively removed in AGS reactors and the AGS cultivated in low C/N group maintained highest removal efficiency. It should be noted that the R3 reactor remained the lowest MLSS throughout the experiment, which suggested that high biomass might not contribute to the removal of refractory pollutants like pharmaceuticals (Bu et al., 2013; Evgenidou et al.,
2015). Several researches have indicated that the removal of pharmaceuticals could be enhanced by the low-growth microbes like ammonia oxidizing bacteria (AOB) (Suarez et al., 2010; Fernandez-Fontaina et al., 2012; Yang et al., 2016). Suarez et al. (2010) reported that the SMZ showed higher resistance to the biological transformation (21 %) than ROX (91 %) in activated sludge due to the high hydrophilic property of SMZ. The enhanced SMZ removal also was achieved in the complementary physic-chemical process like membrane bioreactor via high sorption of the SMZ by the activated sludge. Similarly, Kang et al. (2018) reported that the granules sludge obtained 84 % removal efficiency of the SMZ than 73 % removal efficiency in suspended sludge. The adsorption capacity of the biomass could be another factor to influence the pharmaceutical removal in WWTPs. 5
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
Fig. 3. Removal efficiencies (R.E.)of ROX and SMZ in aerobic granular sludge reactor: removal efficiency of ROX in R1 with C/N = of 30 (a), R2 with C/N = of 15 (c), and R3 with C/N = of 7.5 (e); removal efficiency of SMZ in R1 (b), R2 (d) and R3 (f).
resulted in high PN/PS ratio in R3 during the time. The EPS components in day 65 were further analyzed by three-dimensional excitation-emission fluorescence spectra matrix (3D-EMM). The results showed that there were mainly three fluorescence peak area in excitation wavelengths (Ex) and emission wavelengths map of the EPS: Peak A (230–235/305−330 nm), Aromatic Protein; Peak B (290/ 345 nm), soluble microbial by-product-like substances; Peak C (345–350/430−440 nm), humic acid-like substances (Chen et al., 2003; Zhang et al., 2018). The fluorescent intensities of 3D-EEM suggested that the tryptophan and protein-like substances (peak B) were the main EPS component in reactors. The fluorescent intensities of humic acid-like substances (Peak C) in R3 was higher than the those in R1 and R2, indicating the humic acid-like substances were enriched in R3 reactors under the condition of low C/N influent. Tryptophan, protein-like substances and humic acid-like acids were reported to be abundant in the EPS of mature granules, which may contribute to resist the drug toxicity and maintain the structure stability (Wei et al., 2016; Dong et al., 2017; Zhang et al., 2018).
Thus, the adsorption and degradation of antibiotics by aerobic granular sludge were further analyzed to explore the removal mechanism in AGS reactors. According to the experimental and fitting results (Fig S3.a; b), the removal rate of the AGS after inactivation declined slightly, indicating the proportion of biodegradation were low during the process of ROX removal. In contrast, the fitting results (Fig S3.c;.d) showed that there was a difference of SMZ removal capacity between AGS from R1 and R3. The highest removal efficiencies were 41.0 % in R1 and 72.6 % in R3. Meanwhile, the biodegradation proportion of AGS from R3 accounted for 42.2 % while the biodegradation proportion of AGS from R1 was negligible. The results suggested the enhanced removal of SMZ in R3 should be credited to biodegradation process. And the removal of ROX in R3 was influenced by the adsorption ability of the AGS. 3.3. Dynamic component variation of EPS The EPS contents were monitored to better understand the characteristics of AGS under different C/N ratios. As is shown in Fig. 4, the PN content was 180 mg g−1 VSS at the beginning of Stage I and the PN/ PS ratio was about 2.75−3.15. The PN content in three reactors declined to 120 ± 11 mg g−1 VSS in the first three weeks. During Stage I and Stage II, The PN content further decreased to 59.2 and 65.2 mg g−1 VSS in R1 and R2 while R3 remain the high PN content. Compared to reactors of high C/N ratio (R1, R2), the R3 showed a higher concentration of PN content during stage II and stage III, which also
3.4. The shift of functional microbes in AGS The sludge samples from three reactors during different stages were sequenced on the Illumina Miseq platform. A total of 427,237 effective sequence tags were yielded from 10 sludge samples and clustered to 3492 Operational Taxonomic Units (OTUs) numbers (Table S2). The identical units accounted for only 32.3 %, 48.2 % and 44.7 % of total 6
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
Fig. 4. Variation of sludge characteristics during reactor operation: variation of EPS in R1 (a), R2 (c) and R3 (e); excitation wavelengths (Ex) and emission wavelengths (Em) of EPS in R1 (b), R2 (d) and R3 (f) on day 59.
iron cast pipe (Ren et al., 2015; Ni et al., 2017; Perrin et al., 2019). In R3 reactor with a lower C/N ratio influent, the relative abundances of Thauera spp., Gammaproteobacteria (unclassified), Proteobacteria (unclassified), Defluviicoccus Xanthomonadaceae and Sphingomonadaceae increased from 0.7 %, 1.4 %, 1.0 %, 0.3 %, 0.6 % and 0.4 % at inoculation to 14.0 %, 8.9 %, 7.0 %, 5.9 %, 4.7 % and 3.2 % on day 93. After the addition of SMZ at stage III, Gammaproteobacteria (unclassified), Proteobacteria (unclassified) and Defluviicoccus decreased to 1.6 %, 1.8 % and 1.3 %, while Thauera spp., Xanthomonadaceae and Nitrospira further increased to 31.3 %, 4.3 % and 5.8 %, respectively. Previous studies indicated that Thauera spp. was the representative bacteria, which has the capability to degrade complex aromatic compounds and the function of denitrification and EPS-production, and plays an important role in the formation and stability in aerobic granular sludge (Mechichi et al., 2005; Holmes et al.,
OTUs of R1, R2 and R3 reactors, indicating the changes of microbial community were significant in reactors (Fig. S4). The succession of microbial community at genus level was described in Fig. 5. Defluviicoccus, Chryseolinea, Hyphomicrobium were the dominant microbes in R1 during the whole operation. Thereinto, Defluviicoccus was the most abundant OTU in R1 (C/N = 30), which increased from 0.3 % at inoculation to 29.0 % on day 93, but decreased to 23.0 % after SMZ addition on day 120. The relative abundances of Hyphomicrobium and Chryseolinea were negligible (0.7 % and 0.1 %) on day 25, but increased to 8.0 % and 6.3 % on day 120, respectively. Defluviicoccus was reported to have a high growth rate with the advantages of taking the intermittent available carbon in SBRs (Pronk et al., 2017; Onetto et al., 2019). Previous study found Hyphomicrobium and Chryseolinea could take the labile organic substances as the main carbon source, and contributed much to biofilm formation in manual environment such as 7
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
Fig. 5. Succession of microbial community during reactor operation (Genus level).
2011; Yang et al., 2016). In this study, Thauera spp. was the main functional microbe in R3 reactor and may contribute much to the good biodegradability of SMZ with the aromatic ring (Fig. S1). Nitrospira and Xanthomonadaceae were reported to degrade complex organic compounds like xenobiotizs in nitrate-reducing conditions (Adav et al., 2010; Fitzgerald et al., 2015; Han et al., 2019; Kowalska et al., 2019).
Of all, the study showed that the C/N ratio played a key role in dynamic change of microbial community in AGS reactors via influencing the sludge property and nutritional condition. Meanwhile, the removal performance of pharmaceuticals and nitrogen removals were highly connected to the mixed microbes with function of denitrification and refractory organics oxidation in AGS system.
3.5. The role of influent C/N ratio in the AGS system
4. Conclusion
The RDA analysis was conducted to describe the interaction between microbial community and operation parameters in Fig. 6. The relative abundance of microbes was represented by the diameter of OTUs to visualize response of microbial community to C/N ratio. The biomass concentration (MLVSS), the nitrogen loading rate (NLR), PN and PS contents in sludge EPS, ROX and SMZ concentrations were selected as functional indicators in RDA plots. The date showed the ROX and SMZ addition were independent with the MLVSS and NLR, but significantly related to the dynamic change of PS content (P < 0.05). Meanwhile, the NLR and MLVSS were highly connected, confirming that the C/N ratio was the main factor influencing the sludge concentration. The MLVSS and NLR showed their influence on microbial community succession. The microbes which associated with PS content (Fig. 6a, c; orange zone) decreased with the PPCPs addition. The results suggested that the microbes capable of secreting polysaccharides were lost their advantages during the pharmaceutical addition. The result was in accordance with other studies, which reported that the removal of the SMZ would be influenced greatly by the adsorption of aromatic substances (Xu et al., 2013; Zhao et al., 2015; Hu et al., 2018). The niches were occupied by the microbes positive connected with the MLVSS (R3, blue zone), and the microbes negative connected with the NLR (R1, grey zone), respectively. It suggested that the MLVSS was an influential parameter for the microbes such as Defluviicoccus, Hyphomicrobium and Chryseolinea in R1 while the microbes capable of nitrogen removal were enriched in reactor with higher nitrogen loading in R3. Thus, results suggested that C/N ratio was the determining factor which influencing both the sludge character and nutritional condition for functional microbes in AGS.
Aerobic granular sludge cultivated in low C/N ratio influent would suffer higher ammonia loading and F/M ratio during the domestication, which created the environment in favor of the ammonium oxidizing and denitrifying microbes. Several fast-growing microbes lost their niches. Some functional microbes such as Thauera spp. were enriched obviously in AGS and promoted the degradation of refractory organics and secretion of EPS. The enhanced removal of ROX and SMZ were finally achieved under condition of low C/N influent. CRediT authorship contribution statement Zhuodong Yu: Writing - original draft. Ye Zhang: Visualization. Zhiming Zhang: Data curation. Jingjing Dong: Investigation. Jiashen Fu: Methodology. Xiangyang Xu: Supervision. Liang Zhu: Project administration. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was financially supported by the Major Science and Technology Program for Water Pollution Control and Treatment (2017ZX07201003), the National Natural Science Foundation of China (No. 51961125101), and Science and Technology Project of Zhejiang 8
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
Fig. 6. RDA analysis of microbial community shift, pollutants loading, and sludge characters.
Province (2018C03003).
granules for the treatment of real and low-strength municipal wastewater using a sequencing batch reactor operated at constant volume. Water Res. 105, 341–350. Dong, J., Zhang, Z., Yu, Z., Dai, X., Xu, X., Alvarez, P.J.J., et al., 2017. Evolution and functional analysis of extracellular polymeric substances during the granulation of aerobic sludge used to treat p-chloroaniline wastewater. Chem. Eng. J. 330, 596–604. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Ebele, A.J., Abou-Elwafa Abdallah, M., Harrad, S., 2017. Pharmaceuticals and personal care products (PPCPs) in the freshwater aquatic environment. Emerg. Contam. 3, 1–16. Evgenidou, E.N., Konstantinou, I.K., Lambropoulou, D.A., 2015. Occurrence and removal of transformation products of PPCPs and illicit drugs in wastewaters: a review. Sci. Total Environ. 505, 905–926. Fernandez-Fontaina, E., Omil, F., Lema, J.M., Carballa, M., 2012. Influence of nitrifying conditions on the biodegradation and sorption of emerging micropollutants. Water Res. 46, 5434–5444. Fitzgerald, C.M., Camejo, P., Oshlag, J.Z., Noguera, D.R., 2015. Ammonia-oxidizing microbial communities in reactors with efficient nitrification at low-dissolved oxygen. Water Res. 70, 38–51. Gómez-Acata, S., Vital-Jácome, M., Pérez-Sandoval, M.V., Navarro-Noya, Y.E., Thalasso, F., Luna-Guido, M., et al., 2018. Microbial community structure in aerobic and fluffy granules formed in a sequencing batch reactor supplied with 4-chlorophenol at different settling times. J. Hazard. Mater. 342, 606–616. Hamza, R.A., Sheng, Z., Iorhemen, O.T., Zaghloul, M.S., Tay, J.H., 2018. Impact of foodto-microorganisms ratio on the stability of aerobic granular sludge treating highstrength organic wastewater. Water Res. 147, 287–298. Han, P., Yu, Y., Zhou, L., Tian, Z., Li, Z., Hou, L., et al., 2019. Specific micropollutant biotransformation pattern by the comammox bacterium Nitrospira inopinata. Environ. Sci. Technol. 53, 8695–8705. Holmes, D.E., Risso, C., Smith, J.A., Lovley, D.R., 2011. Genome-scale analysis of anaerobic benzoate and phenol metabolism in the hyperthermophilic archaeon Ferroglobus placidus. ISME J. 6, 146.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jhazmat.2020.122583. References Adav, S.S., Lee, D.J., Lai, J.Y., 2010. Microbial community of acetate utilizing denitrifiers in aerobic granules. Appl. Microbiol. Biotechnol. 85, 753–762. Ahmed, M.B., Zhou, J.L., Ngo, H.H., Guo, W., Thomaidis, N.S., Xu, J., 2017. Progress in the biological and chemical treatment technologies for emerging contaminant removal from wastewater: a critical review. J. Hazard. Mater. 323, 274–298. Arp, H.P.H., 2012. Emerging decontaminants. Environ. Sci. Technol. 46, 4259–4260. Boxall Alistair, B.A., Rudd Murray, A., Brooks Bryan, W., 2012. Pharmaceuticals and personal care products in the environment: what are the big questions? Environ. Health Perspect. 120, 1221–1229. Bu, Q., Wang, B., Huang, J., Deng, S., Yu, G., 2013. Pharmaceuticals and personal care products in the aquatic environment in China: a review. J. Hazard. Mater. 262, 189–211. Chen, W., Westerhoff, P., Leenheer, J.A., Booksh, K., 2003. Fluorescence excitation emission matrix regional integration to quantify spectra for dissolved organic matter. Environ. Sci. Technol. 37, 5701–5710. Corsino, S.F., Capodici, M., Morici, C., Torregrossa, M., Viviani, G., 2016. Simultaneous nitritation-denitritation for the treatment of high-strength nitrogen in hypersaline wastewater by aerobic granular sludge. Water Res. 88, 329–336. de Kreuk, M., Heijnen, J.J., van Loosdrecht, M.C.M., 2005d. Simultaneous COD, nitrogen, and phosphate removal by aerobic granular sludge. Biotechnol. Bioeng. 90, 761–769. Derlon, N., Wagner, J., da Costa, R.H.R., Morgenroth, E., 2016. Formation of aerobic
9
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
extracellular polymeric substance of aerobic granular sludge dominated by Defluviicoccus sp. Water Res. 122, 148–158. Qiao, M., Ying, G.-G., Singer, A.C., Zhu, Y.-G., 2018. Review of antibiotic resistance in China and its environment. Environ. Int. 110, 160–172. Rehman, M.S.U., Rashid, N., Ashfaq, M., Saif, A., Ahmad, N., Han, J.-I., 2015. Global risk of pharmaceutical contamination from highly populated developing countries. Chemosphere 138, 1045–1055. Ren, H., Wang, W., Liu, Y., Liu, S., Lou, L., Cheng, D., et al., 2015. Pyrosequencing analysis of bacterial communities in biofilms from different pipe materials in a city drinking water distribution system of East China. Appl. Microbiol. Biotechnol. 99, 10713–10724. Suarez, S., Lema, J.M., Omil, F., 2010. Removal of Pharmaceutical and Personal Care Products (PPCPs) under nitrifying and denitrifying conditions. Water Res. 44, 3214–3224. Sun, S.-P., Nàcher, C.Pi., Merkey, B., Zhou, Q., Xia, S.-Q., Yang, D.-H., et al., 2010. Effective biological nitrogen removal treatment processes for domestic wastewaters with low C/N ratios: a review. Environ. Eng. Sci. 27, 111–126. Sun, Q., Lv, M., Hu, A., Yang, X., Yu, C.-P., 2014. Seasonal variation in the occurrence and removal of pharmaceuticals and personal care products in a wastewater treatment plant in Xiamen, China. J. Hazard. Mater. 277, 69–75. Thomaidi, V.S., Stasinakis, A.S., Borova, V.L., Thomaidis, N.S., 2015. Is there a risk for the aquatic environment due to the existence of emerging organic contaminants in treated domestic wastewater? Greece as a case-study. J. Hazard. Mater. 283, 740–747. Tran, N.H., Urase, T., Kusakabe, O., 2009. The characteristics of enriched nitrifier culture in the degradation of selected pharmaceutically active compounds. J. Hazard. Mater. 171, 1051–1057. Wang, B., Zhao, M., Guo, Y., Peng, Y., Yuan, Y., 2018. Long-term partial nitritation and microbial characteristics in treating low C/N ratio domestic wastewater. Environ. Sci. Water Res. Technol. 4, 820–827. Wei, D., Li, M., Wang, X., Han, F., Li, L., Guo, J., et al., 2016. Extracellular polymeric substances for Zn (II) binding during its sorption process onto aerobic granular sludge. J. Hazard. Mater. 301, 407–415. Wu, D., Zhang, Z., Yu, Z., Zhu, L., 2018. Optimization of F/M ratio for stability of aerobic granular process via quantitative sludge discharge. Bioresour. Technol. 252, 150–156. Xu, J., Sheng, G.-P., Ma, Y., Wang, L.-F., Yu, H.-Q., 2013. Roles of extracellular polymeric substances (EPS) in the migration and removal of sulfamethazine in activated sludge system. Water Res. 47, 5298–5306. Yang, F.C., Chen, Y.L., Tang, S.L., Yu, C.P., Wang, P.H., Ismail, W., et al., 2016. Integrated multi-omics analyses reveal the biochemical mechanisms and phylogenetic relevance of anaerobic androgen biodegradation in the environment. ISME J. 10, 1967–1983. Yang, Y., Ok, Y.S., Kim, K.-H., Kwon, E.E., Tsang, Y.F., 2017. Occurrences and removal of pharmaceuticals and personal care products (PPCPs) in drinking water and water/ sewage treatment plants: a review. Sci. Total Environ. 596-597, 303–320. Ye, F., Ye, Y., Li, Y., 2011. Effect of C/N ratio on extracellular polymeric substances (EPS) and physicochemical properties of activated sludge flocs. J. Hazard. Mater. 188, 37–43. Zhang, Q.-Q., Ying, G.-G., Pan, C.-G., Liu, Y.-S., Zhao, J.-L., 2015. Comprehensive evaluation of antibiotics emission and fate in the River Basins of China: source analysis, multimedia modeling, and linkage to bacterial resistance. Environ. Sci. Technol. 49, 6772–6782. Zhang, Z., Yu, Z., Dong, J., Wang, Z., Ma, K., Xu, X., et al., 2018. Stability of aerobic granular sludge under condition of low influent C/N ratio: correlation of sludge property and functional microorganism. Bioresour. Technol. 270, 391–399. Zhao, X., Chen, Z., Wang, X., Li, J., Shen, J., Xu, H., 2015. Remediation of pharmaceuticals and personal care products using an aerobic granular sludge sequencing bioreactor and microbial community profiling using Solexa sequencing technology analysis. Bioresour. Technol. 179, 104–112.
Hu, J., Xu, Q., Li, X., Wang, D., Zhong, Y., Zhao, J., et al., 2018. Sulfamethazine (SMZ) affects fermentative short-chain fatty acids production from waste activated sludge. Sci. Total Environ. 639, 1471–1479. Huang, B., Li, Z., Huang, J., Guo, L., Nie, X., Wang, Y., et al., 2014. Adsorption characteristics of Cu and Zn onto various size fractions of aggregates from red paddy soil. J. Hazard. Mater. 264, 176–183. Jadhav, U.U., Dawkar, V.V., Ghodake, G.S., Govindwar, S.P., 2008. Biodegradation of direct Red 5B, a textile dye by newly isolated Comamonas sp. UVS. J. Hazard. Mater. 158, 507–516. Kang, A.J., Brown, A.K., Wong, C.S., Yuan, Q., 2018. Removal of antibiotic sulfamethoxazole by anoxic/anaerobic/oxic granular and suspended activated sludge processes. Bioresour. Technol. 251, 151–157. Kassotaki, E., Buttiglieri, G., Ferrando-Climent, L., Rodriguez-Roda, I., Pijuan, M., 2016. Enhanced sulfamethoxazole degradation through ammonia oxidizing bacteria cometabolism and fate of transformation products. Water Res. 94, 111–119. Kowalska, K., Felis, E., Sochacki, A., Bajkacz, S., 2019. Removal and transformation pathways of benzothiazole and benzotriazole in membrane bioreactors treating synthetic municipal wastewater. Chemosphere 227, 162–171. Li, X.Y., Yang, S.F., 2007. Influence of loosely bound extracellular polymeric substances (EPS) on the flocculation, sedimentation and dewaterability of activated sludge. Water Res. 41, 1022–1030. Liu, J., Wong, M., 2013. Pharmaceuticals and personal care products (PPCPs): a review on environmental contamination in China. Environ. Int. 59, 208–224. Liu, N., Jin, X., Feng, C., Wang, Z., Wu, F., Johnson, A.C., et al., 2020. Ecological risk assessment of fifty pharmaceuticals and personal care products (PPCPs) in Chinese surface waters: a proposed multiple-level system. Environ. Int. 136, 105454. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Luo, J., Hao, T., Wei, L., Mackey, H.R., Lin, Z., Chen, G.-H., 2014a. Impact of influent COD/N ratio on disintegration of aerobic granular sludge. Water Res. 62, 127–135. Luo, Y., Guo, W., Ngo, H.H., Nghiem, L.D., Hai, F.I., Zhang, J., et al., 2014b. A review on the occurrence of micropollutants in the aquatic environment and their fate and removal during wastewater treatment. Sci. Total Environ. 473-474, 619–641. Ma, R., Wang, B., Yin, L., Zhang, Y., Deng, S., Huang, J., et al., 2017. Characterization of pharmaceutically active compounds in Beijing, China: occurrence pattern, spatiotemporal distribution and its environmental implication. J. Hazard. Mater. 323, 147–155. Mechichi, T., Patel, B.K.C., Sayadi, S., 2005. Anaerobic degradation of methoxylated aromatic compounds by Clostridium methoxybenzovorans and a nitrate-reducing bacterium Thauera sp. strain Cin3,4. Int. Biodeterior. Biodegradation 56, 224–230. Moreira, I.S., Amorim, C.L., Ribeiro, A.R., Mesquita, R.B.R., Rangel, A.O.S.S., van Loosdrecht, M.C.M., et al., 2015. Removal of fluoxetine and its effects in the performance of an aerobic granular sludge sequential batch reactor. J. Hazard. Mater. 287, 93–101. Ni, N., Song, Y., Shi, R., Liu, Z., Bian, Y., Wang, F., et al., 2017. Biochar reduces the bioaccumulation of PAHs from soil to carrot (Daucus carota L.) in the rhizosphere: a mechanism study. Sci. Total Environ. 601-602, 1015–1023. Onetto, C.A., Grbin, P.R., McIlroy, S.J., Eales, K.L., 2019. Genomic insights into the metabolism of "Candidatus Defluviicoccus seviourii’, a member of Defluviicoccus cluster III abundant in industrial activated sludge. FEMS Microbiol. Ecol. 95, 12. Pamphile, N., Xuejiao, L., Guangwei, Y., Yin, W., 2019. Synthesis of a novel core-shellstructure activated carbon material and its application in sulfamethoxazole adsorption. J. Hazard. Mater. 368, 602–612. Perrin, Y., Bouchon, D., Delafont, V., Moulin, L., Héchard, Y., 2019. Microbiome of drinking water: a full-scale spatio-temporal study to monitor water quality in the Paris distribution system. Water Res. 149, 375–385. Pronk, M., de Kreuk, M.K., de Bruin, B., Kamminga, P., Kleerebezem, R., van Loosdrecht, M.C.M., 2015. Full scale performance of the aerobic granular sludge process for sewage treatment. Water Res. 84, 207–217. Pronk, M., Neu, T.R., van Loosdrecht, M.C.M., Lin, Y.M., 2017. The acid soluble
10
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Enhancement of PPCPs removal by shaped microbial community of aerobic granular sludge under condition of low C/N ratio influent
T
Zhuodong Yua, Ye Zhanga, Zhiming Zhanga, Jingjing Donga, Jiashen Fua, Xiangyang Xua,b,c, Liang Zhua,b,c,* a
Institute of Environmental Pollution Control and Treatment, Zhejiang University, Hangzhou 310058, China Zhejiang Province Key Laboratory for Water Pollution Control and Environmental Safety, Hangzhou 310058, China c Zhejiang Provincial Engineering Laboratory of Water Pollution Control, 388 Yuhangtang Road, Hangzhou 310058, China b
G R A P H I C A L A B S T R A C T
Mechanism of enhanced denitrification and PPCP removal by aerobic granular sludge.
A R T I C LE I N FO
A B S T R A C T
Editor: Zhu Yu
The frequent occurrence of pharmaceuticals and personal care products (PPCPs) in domestic wastewater has caused great concern. In this study, the removal of two typical pharmaceuticals (Roxithromycin, ROX; Sulfamethoxazole, SMZ) in aerobic granular sludge (AGS) reactors was investigated under condition of different C/N (carbon to nitrogen) ratios. Results showed that higher removal efficiencies of ROX and SMZ (95.2 % and 92.9 %) were achieved in the AGS reactor fed with low C/N influent. Batch experiments further revealed that the removal of ROX was influenced by the adsorption ability of the AGS while SMZ removal was mainly enhanced by biodegradation process. Analysis of extracellular polymeric substances (EPS) showed that the humic acid-like substances were enriched under low C/N condition, which was in accordance with dynamic change of microbial community. The microbes, like Thauera spp. and Xanthomonadaceae, were highly enriched in the reactor with high nitrogen loading rate and functioned as refractory organics degrader. Overall, the AGS process could achieve enhanced pharmaceuticals removal performance by the regulation of microbial community under low C/N influent, which provides insights into a feasible solution for simultaneous removal of nitrogen and trace organic pollutants in AGS reactor.
Keywords: Aerobic granular sludge Pharmaceuticals removal C/N ratio Microbial community
1. Introduction Pharmaceutical and Personal Care Products(PPCPs)are a large
⁎
group of organic compounds, including antibiotics, anti-inflammatories, sanitizer, preservatives, spices, sunscreen and cosmetics (Arp, 2012; Liu and Wong, 2013). The extensive use of PPCPs has
Corresponding author at: Department of Environmental Engineering, Zhejiang University, No. 866 Yuhangtang Road, Hangzhou 310058, PR China. E-mail address: [email protected] (L. Zhu).
https://doi.org/10.1016/j.jhazmat.2020.122583 Received 20 November 2019; Received in revised form 10 March 2020; Accepted 23 March 2020 Available online 27 March 2020 0304-3894/ © 2020 Elsevier B.V. All rights reserved.
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
2. Materials and methods
caused great concern in recent years, especially in developing countries (Bu et al., 2013; Rehman et al., 2015). Asian pharmaceutical market is growing at a rate of 10–15 % annually compared to 5–7 % growth in G7 countries (Rehman et al., 2015). As the world's largest producer and consumer of PPCPs, China exports more than 60 % of the total active pharmaceutical ingredients to the global pharmaceutical industry, and consumes more than 25,000 tons of antibiotics annually (Ma et al., 2017; Liu et al., 2020). These trace pollutants have been continually released and frequently detected in the aquatic environment, which are threatening the aquatic environment and drinking water security (Boxall Alistair et al., 2012; Zhang et al., 2015; Ebele et al., 2017; Qiao et al., 2018). Thus, increasing attention from China has been paid to prevention and control of PPCPs in environment. Wastewater treatment plants (WWTPs) are important facilities to remove the pollutants in wastewater and count for much to water security. However, current WWTPs are mainly designed for biological removal of the nutrients and unsatisfying in removal of the trace pollutants like pharmaceuticals (Sun et al., 2014; Thomaidi et al., 2015; Yang et al., 2017). Compared to the complementary physic-chemical process in WWTPs, bio-treatments are most convenient and low-cost methods to prevent those emerging trace pollutants from the natural environment (Luo et al., 2014b; Ahmed et al., 2017). Thus, advanced bio-treatment technology coupled with emerging trace pollutants and nutrient removal is desired in WWTPs. The aerobic granular sludge (AGS) technology is a novel biological treatment process with several advantages such as high biomass retention, excellent settling properties, and high removal efficiency of total nitrogen (de Kreuk et al., 2005d; Pronk et al., 2015; Corsino et al., 2016). Meanwhile, previous studies have showed that the aerobic granular sludge reactor is a promising biotechnology to withstand shock loading and remove trace pollutants (Moreira et al., 2015; Gómez-Acata et al., 2018). Although the AGS process has obtained fast progress in last decade, the industrial AGS application was very limited compared to traditional sewage treatment process (Pronk et al., 2015). There still is a lack of knowledge for AGS systems in practical influent condition, which becomes the bottleneck and impedes its further development (Derlon et al., 2016). In recent years, the influent of most municipal WWTPs in China and other countries shows the characteristics of low ratio of carbon to nitrogen (Sun et al., 2010; Derlon et al., 2016; Zhang et al., 2018). The relationship between the nitrogen loading rate and microbial community becomes an important factor to optimize the performance of WWTPs (Ye et al., 2011; Luo et al., 2014a; Wang et al., 2018; Zhang et al., 2018). Previous works reported that the microbes could enhance the removal of trace pollutants via co-metabolic degradation, which provided an insight to the enhancement of PPCPs removal in aerobic bio-treatment process (Tran et al., 2009; Suarez et al., 2010; FernandezFontaina et al., 2012). Thus, the aims of this study are to evaluate the pollutants (COD, TN, pharmaceuticals) removal performance under different conditions of C/N ratio influent in AGS reactors. Two typical antibiotics (Roxithromycin, ROX; Sulfamethoxazole, SMZ) with different toxicity were selected in this study. ROX is targeted to grampositive bacteria, which is low toxic to the microbes (dominated by gram-negative bacteria) in WWTPs. The SMZ is a broad-spectrum antibiotic and is a hardly degradable pollutant in WWTPs (FernandezFontaina et al., 2012; Kassotaki et al., 2016; Pamphile et al., 2019). Sludge characteristics like the extracellular polymeric substances (EPS) and sludge loading rate are monitored, and their influences on microbial community and pollutants removal were analyzed at the same time. Finally, the redundancy analysis (RDA) was used to illustrate the effect of the pharmaceuticals and C/N ratio on the dynamic change of functional microbes. This study provides insights into a feasible solution for the simultaneous removal of nitrogen and antibiotics in AGS reactors.
2.1. Operation of AGS reactors Three identical sequencing batch reactors (SBRs) (R1, R2, R3) were seeded with activated sludge from A2O in Qige WWTP (Hangzhou, China). The WWTP is designed for municipal wastewater and the SRT is about 10∼20 days. The COD fluctuates at 350∼550 mg/L and the COD to TN ratio is about 10. Each reactor had the volume of 4 L with the diameter to height ratio of 5. The aeration rate was controlled to maintain the superficial upflow air velocity of the reactors at 1.5 cms−1 and the dissolved oxygen (DO) was 7.5–8.5 mg/L in reactors. The SBR operation mode comprised 5 min feeding, 225 min aeration, 5 min settling and 5 min discharge from the middle of the reactor. The sludge retention time (SRT) of reactors was determined by the sludge discharged with the effluent. The C/N ratio was set at 30 (R1),15 (R2), and 7.5 (R3) with initial total nitrogen (TN) concentration of 26, 52, and 106 mg/L in the influent (Table S1.a). Synthetic wastewater was used as feeding water, comprising the following: Sodium acetate, 1008.3 mg/L; NH4Cl, variable; KH2PO4, 27.17 mg/L; K2HPO4·5H2O, 34.72 mg/L; yeast, 16.15 mg/L; peptone, 24.2 mg/L; CaCl2, 80 mg/L; MgSO4, 30 mg/L. The ambient temperature in the laboratory was 25 ± 2 °C. The sampling of water quality and was conducted two or three times a week. The influent pH was 7.1 ± 0.1 during the whole operation, as the pH was regulated by the addition of KH2PO4 and K2HPO4·5H2O. There were three stages during the reactor operation, including the stage I (the formation of AGS), stage II (the addition of ROX) and stage III (the addition of SMZ). The ROX (lower toxicity) was firstly added into the AGS and the concentration of antibiotics (influent, 0.01−0.1 mg/L) was gradually increased to prevent the shock loading from the reactors. The time and concentration of the antibiotic addition are shown in Table S1.b and Fig. 4. 2.2. Batch test for antibiotics removal The batch tests of antibiotics removal were conducted in four batch reactors with 800 mL working volume. The air upflow rate was controlled at 0.16 m3 h−1. The control group and experiment group were seed with the AGS (4000 mg/L) taken from R1 (C/N = 30) and R3 (C/ N = 7.5) reactor at day 133. In every group, the AGS in inactive batch was further treated with 0.1 % (w/v) sodium azide solution to eliminate biological activity of microbes according to Jadhav et al. (2008). The ROX and SMZ were added in the start of the experiment and the removal process was maintained for 2 days. The concentrations of the antibiotic addition in four batches are shown in Table S1.c. 3 × 5 ml mixture was sampled at 0, 0.33, 0.5, 2, 8, 16, 22, 30, 42, and 48 h, and centrifuged at 1000010,000g for 5 min. The supernatant was filtered by 0.22um filter membrane (Millipore, USA) and stored at 4 °C for detection. The data was fitted by the pseudo-second-order rate equation (Huang et al., 2014) to calculate the proportion of biodegradation and bio-adsorption between the AGS cultivated in different C/N ratio. The PPCP removal was fitted by the equation as follows:
qt =
Ktqe2 1 + Ktqe
Where qt is equilibrium removal capacity and qe represents maximum removal ability of the aerobic granular sludge. The equation can also be transformed to the following formula for linear fitting:
1 1 t = + ∙t qt Kqe 2 qe The absorption of the AGS was represented by the qe in inactive group (the sludge was treated by sodium azide), and the proportion of biodegradation was represented by the variation of the maximum removal ability between the active group and inactive group. 2
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
Fig. 1. Variation of sludge characteristics during reactor operation: biomass concentration (mixed liquor suspended solids, MLSS; mixed liquor volatile suspended solid, MLVSS) and Sludge Volume Index(SVI); in R1 (a), R2 (b), and R3 (c); sludge COD loading (d); sludge NH4+-N loading (e).
performance liquid chromatography-mass spectrometry (LC–MS/MS, Agilent 6460, USA). The Column ZorHax SB C18 (150 × 2.1 mm ID, 3.5 microns) was used and Column temperature maintained 40 °C. The mobile phase A is 2 mM ammonium acetate (0.1 % formic acid). The mobile phase B was acetonitrile with a flow rate of 0.4 mL min−1. During the operation, mobile phase B was gradually increased to 95 % in 10 min (table S3.a), and then phase B was then decreased to 2% from 10 to 15 min and held at 2% until the separation process stopped at 15 min. The MS/MS analysis was operated in positive electrospray ionization with multiple reactions monitoring (MRM) mode (table S3.b). All tests were conducted in triplicate and the quality assurance of the extraction protocol was evaluated by spiking 20 μg of antibiotic standards of interest into effluent with an overnight equilibration. The
2.3. Analytic methods 2.3.1. Water quality The effluent of three reactors was centrifuged at 4000 rpm for 10 min to test total nitrogen (TN) and chemical oxygen demand (COD) according to Zhang at al. (2018). The effluent for detection of NH4+-N, NO2–-N and NO3–-N was filtered by 0.45 μm membrane (Millipore, USA) and tested by standard methods (APHA, 2005). 2.3.2. Analysis of antibiotics The effluent water was filtered through a 0.45-μm membrane (Millipore, USA). The antibiotics in filtered water were enriched and purified by Solid-Phase extraction (SPE) and measured by high3
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
sludge loading rates of R3 reactor maintained above 0.35 mg COD mg−1 VSS and 0.03 mg N mg−1 VSS, which were about 2-fold and 8fold higher than the sludge loading rates in R1. Previous studies suggested that the sludge loading rate could be an important factor to the stability and performance of the AGS via influencing the microbial community (Hamza et al., 2018; Wu et al., 2018). In this study, the microbes in R3 were affected by higher ammonia loading rate and F/M rate during the domestication, which may contribute to the enrichment of ammonium oxidizing and denitrifying bacteria. The antibiotics were added to the reactors when the granulation degree of the AGS in reactors was more than 90 % (day 59) in stage II (the ROX addition) and stage III (the SMZ and ROX addition). The MLSS concentrations in three reactors were decreased to 68.1 %, 85.7 % and 81.25 % with the fluctuation of SVI30 in R1 and R3 from day 59 to day 80. The shock of antibiotics lead to sludge loss (day 80) but gradually recovered within two weeks. The MLSS concentration in high C/N groups (R1, R2) keep growing until the end of the experiment. The result suggested that the MLSS concentrations were affected by the C/N conditions and the occurrence of antibiotics in AGS reactors. The C/N conditions could be a main cause for the biomass concentration and sludge settleability (Luo et al., 2014a,b). The SVI30 of R3 fluctuated obviously after addition of antibiotics, indicating there might be significant alteration of microbes in R3. The variation of settleability and the different level of the biomass under this situation could be serious problem to the stability of SBR reactors (Dong et al., 2017; Zhang et al., 2018). As the sludge loss is mainly resulted by variation of settleability, the control of MLSS became more complicated during the occurrence of antibiotics with different condition in three reactors. Flexible settling time could be considered to avoid the excessive biomass loss or accumulation.
spiking recovery rates ranged from 85 to 105 %. 2.3.3. Analysis of EPS components The EPS was extracted from the sludge sample by a heating method (Li and Yang, 2007). The polysaccharide (PS) content in the EPS was quantified by the phenol–sulfuric acid method (Dubois et al., 1956), and the protein (PN) content in sludge EPS was determined by a modified Lowry colorimetric method with bovine albumin serum as the standard(Lowry et al., 1951). The EPS components were identified by the fluorescence excitation-emission matrix (EEM) using a fluorospectrophotometer (Shimadzu F-4500) according to Luo et al. (2014): Scanning emission spectra was obtained from 250 to 550 nm, and the excitation wavelength varied from 200 to 400 nm. Excitation and emission slits were both kept at 5 nm, and the scanning speed was set at 1200 nm min−1 and the data was displayed as contour maps of intensity. 2.4. Molecular characterization Sludge samples were taken from three AGS reactors at day 25, 59, 93, and 120. The DNA of sludge samples was extracted with the Power Soil DNA extraction kit (MO BIO Laboratories Inc.). Concentrations and quality of the extracted DNA was checked by spectrophotometric analysis on a Nano Drop ND-1000 (Thermo Fisher Scientific, USA). Then the DNA extracted from sludge samples was stored at −80 °C until analysis. The total DNA from sludge samples were used to amplify the V3/V4 region of the 16S rRNA (Primers:338 F; 806R). The PCR reactions were carried out in mixtures (25 μL) containing primer (2.5 μL), genomic DNA (10∼25 ng), PCR Premix (12.5 μL), and PCR-grade water to adjust the volume. All PCR reactions were performed in a Master Cycler Gradient thermocycler (Eppendorf, Hamburg, Germany). The conditions were: initial denaturation (98 °C,30 s); 35 cycles of denaturation (98 °C, 10 s), annealing (53 °C, 30 s), and extension (72 °C, 45 s); and final extension (72 °C, 10 min). Then the PCR products were sequenced by the Illumina MiSeq platform (PE300, CA, USA). The 16S rRNA amplicon sequences were aligned with the SILVA database, and then a distance matrix was generated between the aligned sequences. All sequences were clustered to operational taxonomic units (OTUs) at 97 % sequence similarity.
3.1.2. Removal of COD and TN under different C/N ratio The removal of COD in the three reactors is shown in Fig. 2. The COD in effluent was mostly less than the limit of detection (100 % removal efficiency) in R1 and R2 reactor after 20 days’ operation. Meanwhile, the fluctuation of COD was more obvious in R3. But removal efficiency of COD was stable over the 96 %. All reactors also showed high removal capacity to nitrogen. The ammonia nitrogen was also almost removed completely in three reactors (Fig. S2). However, there were significant differences in TN removal among the reactors. Higher removal efficiencies in R1 and R2 were achieved with high C/N ratio in influent during the domestication period. The average removal efficiencies of TN in first week were 87.8 ± 2.4 %, 76.0 ± 0.7 % and 65.6 ± 0.5 %, respectively. The TN removal efficiencies of R1 and R2 were stable at around 85 % and 74 % during the experiment while the TN removal efficiency of R3 showed an obvious growth tendency and reached 82.4 ± 2.9 % at the end of stage III. The TN removal efficiencies in AGS reactor were obvious effected by the C/N ratio. The concentrations of NH4+ and NO2− were mostly close to the detection limit in effluent in R3, which indicated that the NH4+ in influent was most transformed to NO3− (Fig. S2). Thus, the growth tendency of TN removal efficiency indicated that the advantageous condition was achieved for denitrifiers via higher nitrogen loading rate in R3 reactor.
2.5. Statistical analysis The RDA analysis was performed to assess the relationship between the abiotic/biotic conditions and bacterial communities using R version 3.4.0, (R Core Team 2017) with the CRAN package 'vegan' for the analysis of ecological communities (http://vegan.r-forge.r-project.org/ ). To better identify dynamic change of the dominant microorganism under different condition, the relative abundance of OTUs were represented in figures by the radius of the circle. 3. Results and discussion 3.1. Performance of AGS reactors
3.2. Removal of pharmaceuticals under different C/N ratio 3.1.1. Effects of the C/N ratio on sludge characteristics The experiment was divided into three stages: Stage I (formation of AGS), Stage II and Stage III (the SMZ and ROX removal). As is shown in Fig. 1, the concentration of inoculation sludge was 4000 ± 205 mg L−1. The mixed liquor suspended solids (MLSS) concentration increased rapidly in three reactors, and reached 21,240 mg L-1 (R1), 13,630 mg L-1 (R2) and 8250 mg L−1 (R3) at the end of stage I. It showed that the C/N ratio had significant effects on biomass concentration. The biomass accumulated fast in R1 (C/N = 30) and R2 (C/N = 15), which lead to low sludge loading rate (or Food/Microorganism ratio, F/M) in AGS reactors fed with high C/N influent. As is shown in Fig. 1d and e, the
The profile of pharmaceuticals removal efficiency is shown in Fig. 3. When the SBRs were started to feed with 10 μg L−1 of ROX at the beginning of stage II, the removal efficiencies of ROX in reactors were 91.2 ± 3.3 %、87.3 ± 4.8 % and 88.1 ± 4.1 %, respectively. But as the inflow ROX concentration was further increased to 100 μg L−1, the removal efficiencies of the reactors were 73.0 ± 0.1 % (R1), 85.0 ± 0.6 %(R2) and 80 ± 0.5 % (R3), respectively. The removal efficiencies of R1 and R2 were decreased to 68 % and 58 % while the R3 remained a high removal efficiency up to 95.2 ± 0.5 % on day 110. The SMX, a typical broad-spectrum antibiotic was started to feed in 4
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
Fig. 2. COD and TN removal efficiencies during reactor operation: COD in R1 (a), R2 (c) and R3 (e); TN in R1 (b), R2 (d) and R3 (f).
reactors on day 90. When the SMZ was added with 10 μg L-1, there was no obvious difference in SMZ removal efficiency (93.5 ± 0.6 %, R1; 95.1 ± 1.1 %, R2; 97.0 ± 2.0 %, R3). But when the SMZ increased, the removal efficiency of R1 and R2 were decreased to 78.2 ± 1.8 %, 84.5 ± 1.0 %. The R3 reactors maintained the highest removal efficiency (91.0 ± 0.8 %) throughout the experiment. Thus, there was a notable difference in the removal efficiency under different C/N conditions. The result showed the ROX and SMZ were effectively removed in AGS reactors and the AGS cultivated in low C/N group maintained highest removal efficiency. It should be noted that the R3 reactor remained the lowest MLSS throughout the experiment, which suggested that high biomass might not contribute to the removal of refractory pollutants like pharmaceuticals (Bu et al., 2013; Evgenidou et al.,
2015). Several researches have indicated that the removal of pharmaceuticals could be enhanced by the low-growth microbes like ammonia oxidizing bacteria (AOB) (Suarez et al., 2010; Fernandez-Fontaina et al., 2012; Yang et al., 2016). Suarez et al. (2010) reported that the SMZ showed higher resistance to the biological transformation (21 %) than ROX (91 %) in activated sludge due to the high hydrophilic property of SMZ. The enhanced SMZ removal also was achieved in the complementary physic-chemical process like membrane bioreactor via high sorption of the SMZ by the activated sludge. Similarly, Kang et al. (2018) reported that the granules sludge obtained 84 % removal efficiency of the SMZ than 73 % removal efficiency in suspended sludge. The adsorption capacity of the biomass could be another factor to influence the pharmaceutical removal in WWTPs. 5
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
Fig. 3. Removal efficiencies (R.E.)of ROX and SMZ in aerobic granular sludge reactor: removal efficiency of ROX in R1 with C/N = of 30 (a), R2 with C/N = of 15 (c), and R3 with C/N = of 7.5 (e); removal efficiency of SMZ in R1 (b), R2 (d) and R3 (f).
resulted in high PN/PS ratio in R3 during the time. The EPS components in day 65 were further analyzed by three-dimensional excitation-emission fluorescence spectra matrix (3D-EMM). The results showed that there were mainly three fluorescence peak area in excitation wavelengths (Ex) and emission wavelengths map of the EPS: Peak A (230–235/305−330 nm), Aromatic Protein; Peak B (290/ 345 nm), soluble microbial by-product-like substances; Peak C (345–350/430−440 nm), humic acid-like substances (Chen et al., 2003; Zhang et al., 2018). The fluorescent intensities of 3D-EEM suggested that the tryptophan and protein-like substances (peak B) were the main EPS component in reactors. The fluorescent intensities of humic acid-like substances (Peak C) in R3 was higher than the those in R1 and R2, indicating the humic acid-like substances were enriched in R3 reactors under the condition of low C/N influent. Tryptophan, protein-like substances and humic acid-like acids were reported to be abundant in the EPS of mature granules, which may contribute to resist the drug toxicity and maintain the structure stability (Wei et al., 2016; Dong et al., 2017; Zhang et al., 2018).
Thus, the adsorption and degradation of antibiotics by aerobic granular sludge were further analyzed to explore the removal mechanism in AGS reactors. According to the experimental and fitting results (Fig S3.a; b), the removal rate of the AGS after inactivation declined slightly, indicating the proportion of biodegradation were low during the process of ROX removal. In contrast, the fitting results (Fig S3.c;.d) showed that there was a difference of SMZ removal capacity between AGS from R1 and R3. The highest removal efficiencies were 41.0 % in R1 and 72.6 % in R3. Meanwhile, the biodegradation proportion of AGS from R3 accounted for 42.2 % while the biodegradation proportion of AGS from R1 was negligible. The results suggested the enhanced removal of SMZ in R3 should be credited to biodegradation process. And the removal of ROX in R3 was influenced by the adsorption ability of the AGS. 3.3. Dynamic component variation of EPS The EPS contents were monitored to better understand the characteristics of AGS under different C/N ratios. As is shown in Fig. 4, the PN content was 180 mg g−1 VSS at the beginning of Stage I and the PN/ PS ratio was about 2.75−3.15. The PN content in three reactors declined to 120 ± 11 mg g−1 VSS in the first three weeks. During Stage I and Stage II, The PN content further decreased to 59.2 and 65.2 mg g−1 VSS in R1 and R2 while R3 remain the high PN content. Compared to reactors of high C/N ratio (R1, R2), the R3 showed a higher concentration of PN content during stage II and stage III, which also
3.4. The shift of functional microbes in AGS The sludge samples from three reactors during different stages were sequenced on the Illumina Miseq platform. A total of 427,237 effective sequence tags were yielded from 10 sludge samples and clustered to 3492 Operational Taxonomic Units (OTUs) numbers (Table S2). The identical units accounted for only 32.3 %, 48.2 % and 44.7 % of total 6
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
Fig. 4. Variation of sludge characteristics during reactor operation: variation of EPS in R1 (a), R2 (c) and R3 (e); excitation wavelengths (Ex) and emission wavelengths (Em) of EPS in R1 (b), R2 (d) and R3 (f) on day 59.
iron cast pipe (Ren et al., 2015; Ni et al., 2017; Perrin et al., 2019). In R3 reactor with a lower C/N ratio influent, the relative abundances of Thauera spp., Gammaproteobacteria (unclassified), Proteobacteria (unclassified), Defluviicoccus Xanthomonadaceae and Sphingomonadaceae increased from 0.7 %, 1.4 %, 1.0 %, 0.3 %, 0.6 % and 0.4 % at inoculation to 14.0 %, 8.9 %, 7.0 %, 5.9 %, 4.7 % and 3.2 % on day 93. After the addition of SMZ at stage III, Gammaproteobacteria (unclassified), Proteobacteria (unclassified) and Defluviicoccus decreased to 1.6 %, 1.8 % and 1.3 %, while Thauera spp., Xanthomonadaceae and Nitrospira further increased to 31.3 %, 4.3 % and 5.8 %, respectively. Previous studies indicated that Thauera spp. was the representative bacteria, which has the capability to degrade complex aromatic compounds and the function of denitrification and EPS-production, and plays an important role in the formation and stability in aerobic granular sludge (Mechichi et al., 2005; Holmes et al.,
OTUs of R1, R2 and R3 reactors, indicating the changes of microbial community were significant in reactors (Fig. S4). The succession of microbial community at genus level was described in Fig. 5. Defluviicoccus, Chryseolinea, Hyphomicrobium were the dominant microbes in R1 during the whole operation. Thereinto, Defluviicoccus was the most abundant OTU in R1 (C/N = 30), which increased from 0.3 % at inoculation to 29.0 % on day 93, but decreased to 23.0 % after SMZ addition on day 120. The relative abundances of Hyphomicrobium and Chryseolinea were negligible (0.7 % and 0.1 %) on day 25, but increased to 8.0 % and 6.3 % on day 120, respectively. Defluviicoccus was reported to have a high growth rate with the advantages of taking the intermittent available carbon in SBRs (Pronk et al., 2017; Onetto et al., 2019). Previous study found Hyphomicrobium and Chryseolinea could take the labile organic substances as the main carbon source, and contributed much to biofilm formation in manual environment such as 7
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
Fig. 5. Succession of microbial community during reactor operation (Genus level).
2011; Yang et al., 2016). In this study, Thauera spp. was the main functional microbe in R3 reactor and may contribute much to the good biodegradability of SMZ with the aromatic ring (Fig. S1). Nitrospira and Xanthomonadaceae were reported to degrade complex organic compounds like xenobiotizs in nitrate-reducing conditions (Adav et al., 2010; Fitzgerald et al., 2015; Han et al., 2019; Kowalska et al., 2019).
Of all, the study showed that the C/N ratio played a key role in dynamic change of microbial community in AGS reactors via influencing the sludge property and nutritional condition. Meanwhile, the removal performance of pharmaceuticals and nitrogen removals were highly connected to the mixed microbes with function of denitrification and refractory organics oxidation in AGS system.
3.5. The role of influent C/N ratio in the AGS system
4. Conclusion
The RDA analysis was conducted to describe the interaction between microbial community and operation parameters in Fig. 6. The relative abundance of microbes was represented by the diameter of OTUs to visualize response of microbial community to C/N ratio. The biomass concentration (MLVSS), the nitrogen loading rate (NLR), PN and PS contents in sludge EPS, ROX and SMZ concentrations were selected as functional indicators in RDA plots. The date showed the ROX and SMZ addition were independent with the MLVSS and NLR, but significantly related to the dynamic change of PS content (P < 0.05). Meanwhile, the NLR and MLVSS were highly connected, confirming that the C/N ratio was the main factor influencing the sludge concentration. The MLVSS and NLR showed their influence on microbial community succession. The microbes which associated with PS content (Fig. 6a, c; orange zone) decreased with the PPCPs addition. The results suggested that the microbes capable of secreting polysaccharides were lost their advantages during the pharmaceutical addition. The result was in accordance with other studies, which reported that the removal of the SMZ would be influenced greatly by the adsorption of aromatic substances (Xu et al., 2013; Zhao et al., 2015; Hu et al., 2018). The niches were occupied by the microbes positive connected with the MLVSS (R3, blue zone), and the microbes negative connected with the NLR (R1, grey zone), respectively. It suggested that the MLVSS was an influential parameter for the microbes such as Defluviicoccus, Hyphomicrobium and Chryseolinea in R1 while the microbes capable of nitrogen removal were enriched in reactor with higher nitrogen loading in R3. Thus, results suggested that C/N ratio was the determining factor which influencing both the sludge character and nutritional condition for functional microbes in AGS.
Aerobic granular sludge cultivated in low C/N ratio influent would suffer higher ammonia loading and F/M ratio during the domestication, which created the environment in favor of the ammonium oxidizing and denitrifying microbes. Several fast-growing microbes lost their niches. Some functional microbes such as Thauera spp. were enriched obviously in AGS and promoted the degradation of refractory organics and secretion of EPS. The enhanced removal of ROX and SMZ were finally achieved under condition of low C/N influent. CRediT authorship contribution statement Zhuodong Yu: Writing - original draft. Ye Zhang: Visualization. Zhiming Zhang: Data curation. Jingjing Dong: Investigation. Jiashen Fu: Methodology. Xiangyang Xu: Supervision. Liang Zhu: Project administration. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was financially supported by the Major Science and Technology Program for Water Pollution Control and Treatment (2017ZX07201003), the National Natural Science Foundation of China (No. 51961125101), and Science and Technology Project of Zhejiang 8
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
Fig. 6. RDA analysis of microbial community shift, pollutants loading, and sludge characters.
Province (2018C03003).
granules for the treatment of real and low-strength municipal wastewater using a sequencing batch reactor operated at constant volume. Water Res. 105, 341–350. Dong, J., Zhang, Z., Yu, Z., Dai, X., Xu, X., Alvarez, P.J.J., et al., 2017. Evolution and functional analysis of extracellular polymeric substances during the granulation of aerobic sludge used to treat p-chloroaniline wastewater. Chem. Eng. J. 330, 596–604. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Ebele, A.J., Abou-Elwafa Abdallah, M., Harrad, S., 2017. Pharmaceuticals and personal care products (PPCPs) in the freshwater aquatic environment. Emerg. Contam. 3, 1–16. Evgenidou, E.N., Konstantinou, I.K., Lambropoulou, D.A., 2015. Occurrence and removal of transformation products of PPCPs and illicit drugs in wastewaters: a review. Sci. Total Environ. 505, 905–926. Fernandez-Fontaina, E., Omil, F., Lema, J.M., Carballa, M., 2012. Influence of nitrifying conditions on the biodegradation and sorption of emerging micropollutants. Water Res. 46, 5434–5444. Fitzgerald, C.M., Camejo, P., Oshlag, J.Z., Noguera, D.R., 2015. Ammonia-oxidizing microbial communities in reactors with efficient nitrification at low-dissolved oxygen. Water Res. 70, 38–51. Gómez-Acata, S., Vital-Jácome, M., Pérez-Sandoval, M.V., Navarro-Noya, Y.E., Thalasso, F., Luna-Guido, M., et al., 2018. Microbial community structure in aerobic and fluffy granules formed in a sequencing batch reactor supplied with 4-chlorophenol at different settling times. J. Hazard. Mater. 342, 606–616. Hamza, R.A., Sheng, Z., Iorhemen, O.T., Zaghloul, M.S., Tay, J.H., 2018. Impact of foodto-microorganisms ratio on the stability of aerobic granular sludge treating highstrength organic wastewater. Water Res. 147, 287–298. Han, P., Yu, Y., Zhou, L., Tian, Z., Li, Z., Hou, L., et al., 2019. Specific micropollutant biotransformation pattern by the comammox bacterium Nitrospira inopinata. Environ. Sci. Technol. 53, 8695–8705. Holmes, D.E., Risso, C., Smith, J.A., Lovley, D.R., 2011. Genome-scale analysis of anaerobic benzoate and phenol metabolism in the hyperthermophilic archaeon Ferroglobus placidus. ISME J. 6, 146.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jhazmat.2020.122583. References Adav, S.S., Lee, D.J., Lai, J.Y., 2010. Microbial community of acetate utilizing denitrifiers in aerobic granules. Appl. Microbiol. Biotechnol. 85, 753–762. Ahmed, M.B., Zhou, J.L., Ngo, H.H., Guo, W., Thomaidis, N.S., Xu, J., 2017. Progress in the biological and chemical treatment technologies for emerging contaminant removal from wastewater: a critical review. J. Hazard. Mater. 323, 274–298. Arp, H.P.H., 2012. Emerging decontaminants. Environ. Sci. Technol. 46, 4259–4260. Boxall Alistair, B.A., Rudd Murray, A., Brooks Bryan, W., 2012. Pharmaceuticals and personal care products in the environment: what are the big questions? Environ. Health Perspect. 120, 1221–1229. Bu, Q., Wang, B., Huang, J., Deng, S., Yu, G., 2013. Pharmaceuticals and personal care products in the aquatic environment in China: a review. J. Hazard. Mater. 262, 189–211. Chen, W., Westerhoff, P., Leenheer, J.A., Booksh, K., 2003. Fluorescence excitation emission matrix regional integration to quantify spectra for dissolved organic matter. Environ. Sci. Technol. 37, 5701–5710. Corsino, S.F., Capodici, M., Morici, C., Torregrossa, M., Viviani, G., 2016. Simultaneous nitritation-denitritation for the treatment of high-strength nitrogen in hypersaline wastewater by aerobic granular sludge. Water Res. 88, 329–336. de Kreuk, M., Heijnen, J.J., van Loosdrecht, M.C.M., 2005d. Simultaneous COD, nitrogen, and phosphate removal by aerobic granular sludge. Biotechnol. Bioeng. 90, 761–769. Derlon, N., Wagner, J., da Costa, R.H.R., Morgenroth, E., 2016. Formation of aerobic
9
Journal of Hazardous Materials 394 (2020) 122583
Z. Yu, et al.
extracellular polymeric substance of aerobic granular sludge dominated by Defluviicoccus sp. Water Res. 122, 148–158. Qiao, M., Ying, G.-G., Singer, A.C., Zhu, Y.-G., 2018. Review of antibiotic resistance in China and its environment. Environ. Int. 110, 160–172. Rehman, M.S.U., Rashid, N., Ashfaq, M., Saif, A., Ahmad, N., Han, J.-I., 2015. Global risk of pharmaceutical contamination from highly populated developing countries. Chemosphere 138, 1045–1055. Ren, H., Wang, W., Liu, Y., Liu, S., Lou, L., Cheng, D., et al., 2015. Pyrosequencing analysis of bacterial communities in biofilms from different pipe materials in a city drinking water distribution system of East China. Appl. Microbiol. Biotechnol. 99, 10713–10724. Suarez, S., Lema, J.M., Omil, F., 2010. Removal of Pharmaceutical and Personal Care Products (PPCPs) under nitrifying and denitrifying conditions. Water Res. 44, 3214–3224. Sun, S.-P., Nàcher, C.Pi., Merkey, B., Zhou, Q., Xia, S.-Q., Yang, D.-H., et al., 2010. Effective biological nitrogen removal treatment processes for domestic wastewaters with low C/N ratios: a review. Environ. Eng. Sci. 27, 111–126. Sun, Q., Lv, M., Hu, A., Yang, X., Yu, C.-P., 2014. Seasonal variation in the occurrence and removal of pharmaceuticals and personal care products in a wastewater treatment plant in Xiamen, China. J. Hazard. Mater. 277, 69–75. Thomaidi, V.S., Stasinakis, A.S., Borova, V.L., Thomaidis, N.S., 2015. Is there a risk for the aquatic environment due to the existence of emerging organic contaminants in treated domestic wastewater? Greece as a case-study. J. Hazard. Mater. 283, 740–747. Tran, N.H., Urase, T., Kusakabe, O., 2009. The characteristics of enriched nitrifier culture in the degradation of selected pharmaceutically active compounds. J. Hazard. Mater. 171, 1051–1057. Wang, B., Zhao, M., Guo, Y., Peng, Y., Yuan, Y., 2018. Long-term partial nitritation and microbial characteristics in treating low C/N ratio domestic wastewater. Environ. Sci. Water Res. Technol. 4, 820–827. Wei, D., Li, M., Wang, X., Han, F., Li, L., Guo, J., et al., 2016. Extracellular polymeric substances for Zn (II) binding during its sorption process onto aerobic granular sludge. J. Hazard. Mater. 301, 407–415. Wu, D., Zhang, Z., Yu, Z., Zhu, L., 2018. Optimization of F/M ratio for stability of aerobic granular process via quantitative sludge discharge. Bioresour. Technol. 252, 150–156. Xu, J., Sheng, G.-P., Ma, Y., Wang, L.-F., Yu, H.-Q., 2013. Roles of extracellular polymeric substances (EPS) in the migration and removal of sulfamethazine in activated sludge system. Water Res. 47, 5298–5306. Yang, F.C., Chen, Y.L., Tang, S.L., Yu, C.P., Wang, P.H., Ismail, W., et al., 2016. Integrated multi-omics analyses reveal the biochemical mechanisms and phylogenetic relevance of anaerobic androgen biodegradation in the environment. ISME J. 10, 1967–1983. Yang, Y., Ok, Y.S., Kim, K.-H., Kwon, E.E., Tsang, Y.F., 2017. Occurrences and removal of pharmaceuticals and personal care products (PPCPs) in drinking water and water/ sewage treatment plants: a review. Sci. Total Environ. 596-597, 303–320. Ye, F., Ye, Y., Li, Y., 2011. Effect of C/N ratio on extracellular polymeric substances (EPS) and physicochemical properties of activated sludge flocs. J. Hazard. Mater. 188, 37–43. Zhang, Q.-Q., Ying, G.-G., Pan, C.-G., Liu, Y.-S., Zhao, J.-L., 2015. Comprehensive evaluation of antibiotics emission and fate in the River Basins of China: source analysis, multimedia modeling, and linkage to bacterial resistance. Environ. Sci. Technol. 49, 6772–6782. Zhang, Z., Yu, Z., Dong, J., Wang, Z., Ma, K., Xu, X., et al., 2018. Stability of aerobic granular sludge under condition of low influent C/N ratio: correlation of sludge property and functional microorganism. Bioresour. Technol. 270, 391–399. Zhao, X., Chen, Z., Wang, X., Li, J., Shen, J., Xu, H., 2015. Remediation of pharmaceuticals and personal care products using an aerobic granular sludge sequencing bioreactor and microbial community profiling using Solexa sequencing technology analysis. Bioresour. Technol. 179, 104–112.
Hu, J., Xu, Q., Li, X., Wang, D., Zhong, Y., Zhao, J., et al., 2018. Sulfamethazine (SMZ) affects fermentative short-chain fatty acids production from waste activated sludge. Sci. Total Environ. 639, 1471–1479. Huang, B., Li, Z., Huang, J., Guo, L., Nie, X., Wang, Y., et al., 2014. Adsorption characteristics of Cu and Zn onto various size fractions of aggregates from red paddy soil. J. Hazard. Mater. 264, 176–183. Jadhav, U.U., Dawkar, V.V., Ghodake, G.S., Govindwar, S.P., 2008. Biodegradation of direct Red 5B, a textile dye by newly isolated Comamonas sp. UVS. J. Hazard. Mater. 158, 507–516. Kang, A.J., Brown, A.K., Wong, C.S., Yuan, Q., 2018. Removal of antibiotic sulfamethoxazole by anoxic/anaerobic/oxic granular and suspended activated sludge processes. Bioresour. Technol. 251, 151–157. Kassotaki, E., Buttiglieri, G., Ferrando-Climent, L., Rodriguez-Roda, I., Pijuan, M., 2016. Enhanced sulfamethoxazole degradation through ammonia oxidizing bacteria cometabolism and fate of transformation products. Water Res. 94, 111–119. Kowalska, K., Felis, E., Sochacki, A., Bajkacz, S., 2019. Removal and transformation pathways of benzothiazole and benzotriazole in membrane bioreactors treating synthetic municipal wastewater. Chemosphere 227, 162–171. Li, X.Y., Yang, S.F., 2007. Influence of loosely bound extracellular polymeric substances (EPS) on the flocculation, sedimentation and dewaterability of activated sludge. Water Res. 41, 1022–1030. Liu, J., Wong, M., 2013. Pharmaceuticals and personal care products (PPCPs): a review on environmental contamination in China. Environ. Int. 59, 208–224. Liu, N., Jin, X., Feng, C., Wang, Z., Wu, F., Johnson, A.C., et al., 2020. Ecological risk assessment of fifty pharmaceuticals and personal care products (PPCPs) in Chinese surface waters: a proposed multiple-level system. Environ. Int. 136, 105454. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Luo, J., Hao, T., Wei, L., Mackey, H.R., Lin, Z., Chen, G.-H., 2014a. Impact of influent COD/N ratio on disintegration of aerobic granular sludge. Water Res. 62, 127–135. Luo, Y., Guo, W., Ngo, H.H., Nghiem, L.D., Hai, F.I., Zhang, J., et al., 2014b. A review on the occurrence of micropollutants in the aquatic environment and their fate and removal during wastewater treatment. Sci. Total Environ. 473-474, 619–641. Ma, R., Wang, B., Yin, L., Zhang, Y., Deng, S., Huang, J., et al., 2017. Characterization of pharmaceutically active compounds in Beijing, China: occurrence pattern, spatiotemporal distribution and its environmental implication. J. Hazard. Mater. 323, 147–155. Mechichi, T., Patel, B.K.C., Sayadi, S., 2005. Anaerobic degradation of methoxylated aromatic compounds by Clostridium methoxybenzovorans and a nitrate-reducing bacterium Thauera sp. strain Cin3,4. Int. Biodeterior. Biodegradation 56, 224–230. Moreira, I.S., Amorim, C.L., Ribeiro, A.R., Mesquita, R.B.R., Rangel, A.O.S.S., van Loosdrecht, M.C.M., et al., 2015. Removal of fluoxetine and its effects in the performance of an aerobic granular sludge sequential batch reactor. J. Hazard. Mater. 287, 93–101. Ni, N., Song, Y., Shi, R., Liu, Z., Bian, Y., Wang, F., et al., 2017. Biochar reduces the bioaccumulation of PAHs from soil to carrot (Daucus carota L.) in the rhizosphere: a mechanism study. Sci. Total Environ. 601-602, 1015–1023. Onetto, C.A., Grbin, P.R., McIlroy, S.J., Eales, K.L., 2019. Genomic insights into the metabolism of "Candidatus Defluviicoccus seviourii’, a member of Defluviicoccus cluster III abundant in industrial activated sludge. FEMS Microbiol. Ecol. 95, 12. Pamphile, N., Xuejiao, L., Guangwei, Y., Yin, W., 2019. Synthesis of a novel core-shellstructure activated carbon material and its application in sulfamethoxazole adsorption. J. Hazard. Mater. 368, 602–612. Perrin, Y., Bouchon, D., Delafont, V., Moulin, L., Héchard, Y., 2019. Microbiome of drinking water: a full-scale spatio-temporal study to monitor water quality in the Paris distribution system. Water Res. 149, 375–385. Pronk, M., de Kreuk, M.K., de Bruin, B., Kamminga, P., Kleerebezem, R., van Loosdrecht, M.C.M., 2015. Full scale performance of the aerobic granular sludge process for sewage treatment. Water Res. 84, 207–217. Pronk, M., Neu, T.R., van Loosdrecht, M.C.M., Lin, Y.M., 2017. The acid soluble
10