Single and combined effects of divalent copper and hexavalent chromium on the performance, microbial community and enzymatic activity of sequencing batch reactor

Science of the Total Environment 719 (2020) 137289

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Single and combined effects of divalent copper and hexavalent chromium on the performance, microbial community and enzymatic activity of sequencing batch reactor Shanshan Li a,b, Shuyan Wu a, Bingrui Ma a,c, Mengchun Gao a,⁎, Yuanyuan Wu a, Zonglian She a,c, Yangguo Zhao a,c, Liang Guo a, Chunji Jin a, Junyuan Ji a,c,⁎ a b c

Key Lab of Marine Environment and Ecology, Ministry of Education, Ocean University of China, Qingdao 266100, China College of Resource and Environment, Qingdao Agricultural University, Qingdao 266109, China College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Combined Cu2+ and Cr6+ inhibited SBR performance more than Cu2+ or Cr6+. • Combined Cu2+ and Cr6+ showed more inhibition on nitrifying rate than Cu2+ or Cr6+. • Combined Cu2+ and Cr6+ decreased enzymatic activity more than Cu2+ or Cr6 + . • Combined Cu2+ and Cr6+ affected microbial community more than single Cu2+ or Cr6+.

a r t i c l e

i n f o

Article history: Received 19 December 2019 Received in revised form 11 February 2020 Accepted 11 February 2020 Available online xxxx Editor: Huu Hao Ngo Keywords: Divalent copper Hexavalent chromium Nitrogen removal rate Nitrifying enzymatic activity

a b s t r a c t Divalent copper (Cu2+) and hexavalent chromium (Cr6+) are often encountered in industrial wastewater and municipal wastewater, the effect of combined Cu2+ and Cr6+ on biological wastewater treatment systems has cause wide concern. In the present research, the performance, microbial community and enzymatic activity of sequencing batch reactors (SBRs) were compared under the single and combined Cu2+ at 20 mg/L and Cr6+ at 10 mg/L. The chemical oxygen demand (COD) and ammonia nitrogen (NH+ 4 -N) removal efficiencies under the combined Cu2+ and Cr6+ were less than those under the single Cu2+ and Cr6+. The combined Cu2+ and Cr6+ displayed more inhibition effects on the oxygen uptake rate, nitrification rate and denitrification rate of activated sludge than the single Cu2+ and Cr6+. The inhibitory effects of the combined Cu2+ and Cr6+ on the activities of dehydrogenase, ammonia monooxygenase, nitrite oxidoreductase, nitrite reductase and nitrate reductase showed significant increases by comparison with the single Cr6+. However, the combined Cu2+ and Cr6+ had a little more inhibitory effects on the enzymatic activities than the single Cu2+. The microbial richness and

⁎ Corresponding authors at: 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 addresses: [email protected] (M. Gao), [email protected] (J. Ji).

https://doi.org/10.1016/j.scitotenv.2020.137289 0048-9697/© 2020 Elsevier B.V. All rights reserved.

2 Denitrifying enzymatic activity Microbial community

S. Li et al. / Science of the Total Environment 719 (2020) 137289

diversity displayed some obvious changes under the single and combined Cu2+ and Cr6+ by comparison the absence of Cu2+ and Cr6+. The relative abundances of nitrifying genera (e.g. Nitrosomonas and Nitrospira) under the combined Cu2+ and Cr6+ was less than those under the single Cu2+ and Cr6+. These findings will be helpful to better understand the combined effects of multiple heavy metals on biological wastewater treatment systems. © 2020 Elsevier B.V. All rights reserved.

1. Introduction Heavy metals (HMs) in wastewater mainly come from metal finishing, mining, chemical manufacture, electroplating and mineral processing. Due to their potential biotoxicity, the adverse impacts of HMs on different exosystemic environments have attracted widespread concerns in recent years. Divalent copper (Cu2+) and hexavalent chromium (Cr6+) are two kinds of most common HMs. Apart from low Cu2+ concentration, Cu2+ has the potential toxicity to living microorganisms (Trevors and Cotter, 1990; Pamukoglu and Kargi, 2007). Cr6+ is non-essential and a toxic metal ion to microorganisms, which is considered a serious environmental pollutant (Jobby et al., 2018). Some researchers have reported that single Cu2+ and Cr6+ can exert evident toxicity to some microorganisms, such as Pseudomonas fluorescens (Wang et al., 2014), denitrifying bacteria (Mazierski, 1994; Chen et al., 2016), nitrifying bacteria (Kapoor et al., 2016; He et al., 2018) and yeast (Dönmez and Aksu, 1999). Industrial wastewater containing Cu2+ and Cr6+ mainly comes from the processes of electroplating, metal finishing, tanneries, chemical manufacturing, ceramic production and dyeing. Due to the variety of wastewater collection systems and policies in many countries, industrial wastewater might totally (or partially) enters municipal wastewater treatment systems. The combined treatment of industrial water and municipal wastewater inevitably increase the possibility of high Cu2+ or Cr6+ concentration in the influent of biological wastewater treatment systems. Earlier researches demonstrated that Cu2+ or Cr6+ produced the inhibitory effects on the microbial metabolic activity and nitrogen removal rates of activated sludge (Stasinakis et al., 2003; Jiang et al., 2013; Tang et al., 2018; Li et al., 2019). Zhou et al. (2019) demonstrated that the present Cu2+ could affect the nutrient removal from wine wastewater in duckweed systems. Li et al., 2018 reported that Cu2+ could inhibit the nitrogen removal of nitritation-anammox process. Feng et al. (2019) found that the existence of Cr6+ inhibited the biodegradation of tetrabromobisphenol A by pycnoporus sanguineus. The presence of Cu2+ or Cr6+ in the influent could affect the anaerobic ammonia-oxidizing ability of Anammox systems (Yang et al., 2013; Jiang et al., 2018; Ma et al., 2018; Yu et al., 2019). Sun et al. (2016) reported that the microbial richness and diversity of activated sludge displayed obvious changes with the increase of influent Cu2+ concentration from 0 to 40 mg/L. Samaras et al. (2009) demonstrated that the presence of Cr6+ at 1–50 mg/L resulted in the variations in the abundance and diversity of protozoan species in an activated sludge process. As a matter of fact, various HMs usually coexist in industrial wastewater and municipal wastewater due to their different sources. The coexistence of different HMs might produce the synergistic, antagonistic or additive effects on the bioreactor performances for treating wastewater (Gikas, 2008). Aslan and Sozudogru (2017) found that the combined Cu2+ and divalent nickel (Ni2+) inhibited the nitrifying activity of submerged biofilter more than the single Cu2+ or Ni2+. Dilek et al. (1998) demonstrated that the combined Ni2+ and Cr6+ displayed the synergistic impact on the microbial growth rate. Cu2+ and Cr6+ often coexist in industrial wastewater and domestic sewage. However, no systematic report has been found in evaluating the impacts of combined Cu2+ and Cr6+ on the nitrogen removal rate, enzymatic activity and microbial community of sequencing batch reactor (SBR). As Cu2+ and Cr6+ often coexist in industrial wastewater or municipal sewage, it is necessary to investigate whether the combined Cu2+ and Cr6 + produce the synergistic inhibitory effects on biological wastewater treatment systems. The objects of the present research focused on

(a) investigating the effects of single and combined Cu2+ and Cr6+ on the SBR performance, (b) comparing the oxygen uptake rate, nitrifying rate and denitrifying rate under single and combined Cu2+ and Cr6+ stress, (c) evaluating the activities of dehydrogenase, nitrifying enzymes and denitrifying enzymes under single and combined Cu2+ and Cr6+, and (d) analyzing the microbial community under single and combined Cu2+ and Cr6+. 2. Materials and methods 2.1. SBR and synthetic wastewater Four identical SBRs were operated at the same operation mode in the present research, which were marked as SBR0, SBRCu, SBRCr and SBRCu+Cr. The initial sludge for each SBR derived from a secondary sedimentation tank of municipal wastewater treatment plant in Qingdao city, China. The mixed liquor suspended solids (MLSS) of each SBR was about 4.00 × 103 mg/L by regularly discharging excess sludge. The synthetic wastewater was input each SBR through a measuring pump, and the effluent drained through a water outlet in the middle of SBR. Each SBR was sequentially operated in four 6-h cycles every day. Every cycle was successively comprised of 5 min influent, 1 h anoxic period, 3 h oxic period, 1 h anoxic period, 0.5 h settling, 5 min effluent and 20 min idle. An aeration pump was adopted to provide oxygen during the aerobic period, and a magnetic agitator was used to stir the mixed liquids of SBR during the anoxic period. The composition of synthetic wastewater was listed as follows (mg/L): CH3COONa (641), NH4Cl (115), KH2PO4 (8.5), K2HPO4 (9.4), NaHCO3 (113), FeCl3·6H2O (1.5), H3BO3 (0.15), KI (0.03), NiSO4 (0.15), MnCl2·4H2O (0.12), ZnSO4·7H2O (0.12), CoCl2·6H2O (0.15), and Na2MoO4·2H2O (0.06). Prior to the occurrence of Cu2+ or Cr6+, the above-mentioned four SBRs have been operated for 15 days under the same operating conditions. The SBR0 was operated from day 1 to 75 under the absence of Cu2+ or Cr6+. The influent of SBRCu, SBRCr and SBRCu+Cr contained 20 mg/L Cu2+, 10 mg/L Cr6+, and 20 mg/L Cu2+ and 10 mg/L Cr6+ from the 16th day, respectively. The Cu2+ and Cr6+ in the synthetic wastewater were provided from CuSO4·5H2O and K2Cr2O7, respectively. 2.2. Analytical methods The measurements of chemical oxygen demand (COD), ammonia ni− − trogen (NH+ 4 -N), nitrite nitrogen (NO2 -N) and nitrate nitrogen (NO3 -N) were performed by potassium dichromate sulfuric acid-mercuric sulfate method, phenate method, colorimetric method and ultraviolet spectrophotometric screening method, respectively (APHA, 1998). The MLSS were measured by drying the activated sludge at 103–105 °C for 1 h in a drying oven, and the mixed liquor volatile suspended solids (MLVSS) were determined by igniting the activated sludge at 550 °C for 15 min in a muffle furnace (APHA, 1998). According to earlier report (Wang et al., 2016), the measurement methods of specific oxygen uptake rate (SOUR), specific ammonia-oxidizing rate (SAOR), specific nitriteoxidizing rate (SNOR), specific nitrate-reducing rate (SNRR), and specific nitrite-reducing rate (SNIRR) were described in Text S1 of Supporting Information. The determination of dehydrogenase activity (DHA), ammonia monooxygenase (AMO) activity, nitrite oxidoreductase (NOR) activity, nitrite reductase (NIR) activity and nitrate reductase (NR) activity was depicted in Text S2 and S3 of Supporting Information according to earlier report (Li et al., 2018b). The microbial richness and diversity of

S. Li et al. / Science of the Total Environment 719 (2020) 137289

3

higher toxicity to ammonia oxidizing bacteria than the single Cu2+ and Cr2+. Aslan and Sozudogru (2017) reported that the simultaneous presence of Ni2+ and Cu2+ produced more inhibition on the activity of nitrifying bacteria than the single Ni2+ and Cu2+. The effluent NO− 2 -N concentration of SBRCu, SBRCr and SBRCu+Cr was less than 2 mg/L except for SBRCr from day 28 to 40, which demonstrated that the single and combined Cu2+ and Cr6+ might have no obvious inhibitory effect on the nitrite oxidation of nitrifying process or nitrite reduction of denitrifying process. The effluent NO− 3 -N concentration of SBRCu, SBRCr and SBRCu+Cr sharply decreased to nearly zero on day 20, 19 and 20, respectively. Subsequently, the effluent NO− 3 -N of SBRCu and SBRCu+Cr kept nearly zero in the subsequent operation time, whereas that of SBRCr increased from day 21 and then kept at about 5.40 mg/L from day 52 to 75. The present results demonstrated that the presence of single and combined Cu2+ at 20 mg/L and Cr6+ at 10 mg/L affected the COD and nitrogen removal performance of SBR.

activated sludge were analyzed by high throughput sequencing in accordance with Text S4 of Supporting Information. 2.3. Statistical analysis The SOUR, SAOR, SNOR, SNIRR, SNRR, DHA, AMO activity, NOR activity, NIR activity, NR activity and microbial community of activated sludge were measured in triplicate, and the results were expressed as the mean ± SD (standard deviation). The above-mentioned data were subjected to one-way analysis of variance (ANOVA) in SPSS 16.0 for windows. The statistical significance was tested using the least significant difference (LSD) at the p b 0.05 level. The statistical significance of SOUR, nitrogen removal rate and microbial enzymatic activity was obtained through comparing with the absence of Cu2+ and Cr6+ on day 15, and that of Table 1 was acquired by comparison with the seed sludge. 3. Results and discussion 3.1. SBR performance under the single and combined Cu2+ and Cr6+

3.2. COD and nitrogen compound concentrations in SBR during a circle under single and combined Cu2+ and Cr6+

Fig. 1 illustrates the SBR performance under the single and combined Cu2+ and Cr6+. Prior to the occurrence of Cu2+ and Cr6+ in the influent, the COD and nitrogen removals of SBR0, SBRCu, SBRCr and SBRCu+Cr was almost the same from day 1 to 15. From the 16th day, the influent of SBRCu, SBRCr and SBRCu+Cr was added 20 mg/L Cu2+, 10 mg/L Cr6+, and 20 mg/L Cu2+ and 10 mg/L Cr6+, respectively. Due to the absence of Cu2+ and Cr6+, the COD and nitrogen removal efficiencies of SBR0 was almost unchanged from day 1 to 75. Compared with the absence of Cu2+ and Cr6+, the COD removal efficiency of SBRCu, SBRCr and SBRCu+Cr gradually decreased from day 15 to day 26, day 21 and day 26 due to the sudden shock-loading of single and combined Cu2+ and Cr6+, respectively. Subsequently, the COD removal efficiency of SBRCu, SBRCr and SBRCu+Cr gradually increased with the increase of operation time owing to their adaption to the biotoxicity of Cu2+ and Cr6+. The COD removal efficiency of SBRCu, SBRCr and SBRCu+Cr kept relatively stable values from day 35, 26 and 35, respectively. However, the descending order of COD removal efficiency was SBR0, SBRCr, SBRCu and SBRCu+Cr, which suggested that the combined Cu2+ and Cr6+ exerted more inhibitory effects on the COD removal than the single Cu2+ and Cr6+. Jiang et al. (2013) reported that Cu2+ at 20 and 40 mg/L obviously decreased the COD removal efficiency of SBR. Additionally, Stasinakis et al. (2003) demonstrated that the critical Cr6+ concentration of affecting COD removal was in range of 5 and 50 mg/L due to the dependency of operation parameters for different bioreactor. The NH+ 4 -N removal efficiency of SBRCu, SBRCr and SBRCu+Cr sharply decreased to 37.2% on day 24, 9.2% on day 21 and 5.1% on day 21 due to the sudden presence of single and combined Cu2+ and Cr6+, respectively. Subsequently, the NH+ 4 N removal efficiency of SBRCu, SBRCr and SBRCu+Cr gradually increased and then reached relatively stable values. The NH+ 4 -N removal efficiency of SBRCu was lower than that of SBRCr at the same operation time, suggesting that 20 mg/L Cu2+ produced more inhibition on ammonia oxidation than 10 mg/L Cr6+. The combined Cu2+ and Cr6+ had 2+ more inhibitory effect on the NH+ 4 -N oxidation than the single Cu or Cr6+, suggesting that the coexistence of Cu2+ and Cr6+ might cause

The COD and nitrogen compound concentrations in SBR0, SBRCu, SBRCr and SBRCu+Cr during a circle were investigated on day 75 (Fig. 2). At the influent stage (0–5 min), the COD and NH+ 4 -N concentrations were involved with the influent concentration, their residue from previous circle and the dilution effect of 50% volume exchange rate. In the 5th min, the COD and NH+ 4 -N concentrations in SBR0, SBRCu, SBRCr and SBRCu+Cr were 253 and 14.3 mg/L, 286 and 23 mg/L, 280 and 16.8 mg/L, and 288 − and 25.2 mg/L, respectively. The NO− 2 -N and NO3 -N concentrations in SBR at the influent stage were related to their residues from previous circle, the dilution effect of volume exchange rate and denitrification pro− cess. In the 5th min, the NO− 2 -N and NO3 -N concentrations in SBR0, SBRCu, SBRCr and SBRCu+Cr were 1.67 and 1.62 mg/L, 0 and 0.79 mg/L, 0.15 and 1.77 mg/L, and 0.02 and 0.29 mg/L, respectively. The COD concentration in SBR0, SBRCu, SBRCr and SBRCu+Cr gradually decreased from the 5th min to the 335th min (Fig. 2a). However, the ascending order of COD concentration in four SBRs was shown as follows: SBR0, SBRCr, SBRCu and SBRCu+Cr, which suggested that the combined Cu2+ and Cr6+ could produce higher inhibitory effect on the organic matter removal than the single Cu2+ and Cr6+. The NH+ 4 -N concentration in SBR0, SBRCu, SBRCr and SBRCu+Cr was almost unchanged from 6 to 65 min at the first anoxic stage (Fig. 2b), which suggested that the process of ammonia oxidation did not happen owing to the deficiency of dissolved oxygen. At the aerobic stage (66–245 min), the NH+ 4 -N concentration in SBR0, SBRCu, SBRCr and SBRCu+Cr gradually decreased from 14.1, 22.6, 16.2, 24.4 mg/L in the 65th min to 0.17, 16, 4.12 and 17.6 mg/L in the 245th min due to the happen of ammonia oxidation process, respectively. The inhibitory effect of Cu2+ at 20 mg/L on the ammonia oxidation were significantly higher than that of Cr6+ at 10 mg/L. However, the inhibitory effect of the combined Cu2+ and Cr6+ on the ammonia oxidation showed a slight increment by comparison with the single Cu2+. At the second anoxic stage (246–305 min) and setting stage (306–335 min), the NH+ 4 -N concentration in the above-mentioned four SBRs had no obvious change, suggesting that the process had not happen owing to the deficiency of dissolved oxygen. The NO− 2 -N concentration in SBR0, SBRCu, SBRCr and SBRCu

Table 1 Microbial richness and diversity of seed sludge and sludge samples under single and combined Cu2+ and Cr6+ on day 75. Samples

Effective sequences (×104)

OTU number (×103)

Seed sludge S0 SCu SCr SCu+Cr

7.799 8.022 8.502 8.029 8.238

0.983 1.028 0.204 0.745 0.185

± ± ± ± ±

1.128 0.011 0.427 0.009 0.207

± ± ± ± ±

Chao1 index (×103) 0.036 0.034 0.001* 0.010* 0.013*

1.027 1.092 0.276 0.797 0.230

± ± ± ± ±

ACE index (×103) 0.060 0.029 0.021* 0.06* 0.014*

1.040 1.102 0.300 0.821 0.250

± ± ± ± ±

0.060 0.035 0.007* 0.017* 0.011*

Shannon index

Simpson index

7.36 7.67 2.83 6.68 3.08

0.983 0.986 0.752 0.975 0.799

± ± ± ± ±

0.10 0.07 0.42* 0.06* 0.06*

Notes: Asterisks indicate the statistical difference (p b 0.05) from the seed sludge. OTC represents operational taxonomic unit.

± ± ± ± ±

0.002 0.001 0.080* 0.002 0.002*

PD whole tree

Good's coverage

80.0 86.3 24.8 65.7 21.4

0.998 0.997 0.998 0.998 0.999

± ± ± ± ±

4.2 2.4* 3.0* 2.2* 2.1*

± ± ± ± ±

0.001 0.000* 0.000 0.001 0.000*

S. Li et al. / Science of the Total Environment 719 (2020) 137289 SBR0 (Cu 2++Cr6+=0+0 mg/L) SBRCu (Cu 2++Cr6+=20+0 mg/L) SBRCr (Cu 2++Cr6+=0+10 mg/L) SBRCu+Cr (Cu 2++Cr6+=20+10 mg/L)

300

a

500

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400 300

200 150

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100

50

0

0

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Influent NH4+ -N concentration (mg/L)

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SBR0 (Cu 2++Cr6+=0+0 mg/L) SBRCu (Cu 2++Cr6+=20+0 mg/L) SBRCr (Cu 2++Cr6+=0+10 mg/L) SBRCu+Cr (Cu 2++Cr6+=20+10 mg/L)

Cu 2+=0mg/L Cr6+=0mg/L

Effluent COD concentration(mg/L)

Influent CO D concent rat ion (mg/L)

Cu 2+=0mg/L Cr6+=0mg/L

35

35

b

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30 Influent SBR0 SBRCu SBRCr SBRCu+Cr

25

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2

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4

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7

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6 5 4

SBR0 SBRCu SBRCr SBRCu+Cr

3 2 1 0

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− − Fig. 1. Variations of COD and nitrogen compound concentrations in the influent and effluent under the single and combined Cu2+ and Cr6+. (a) COD, (b) NH+ 4 -N, (c) NO2 -N, and (d) NO3 − − N. COD, Cu2+, Cr6+, NH+ -N, NO -N, and NO -N represent chemical oxygen demand, divalent copper, hexavalent chromium, ammonia nitrogen, nitrite nitrogen and nitrate nitrogen, 4 2 3 respectively.

+Cr was zero during most of operational time in a circle except for some minor temporary fluctuations in SBR0 and SBRCr at the aerobic stage (Fig. 2c). Considering the variation of NH+ 4 -N concentration in four SBRs, the variation of NO− 2 -N concentration in a circle might be explained as follows: (1) As most of ammonia in SBR0 and SBRCr was transformed to nitrite or nitrate during the aerobic stage, the nitrite or nitrate might be reduced to nitrogen gas during the anoxic stage, which resulted in low + NO− 2 -N concentration in SBRs. (2) As only a small fraction of NH4 -N was oxidized to NO− 2 -N in SBRCu and SBRCu+Cr, it could conclude that al− most all NO− 2 -N was further oxidized as NO3 -N during the aerobic stage or be reduced as nitrogen gas during the anoxic stage. As shown in Fig. 2d, the NO− 3 -N concentration in SBRCu and SBRCu+Cr was close to zero during a circle. However, the NO− 3 -N concentration in SBR0 and SBRCr decreased with the increase of operation time at the first anoxic stage, and it displayed an increasing trend at the aerobic stage due to the happen of nitrifying process. Subsequently, the NO− 3 -N concentrations of SBR0 and SBRCr decreased with the increase of operation time at the second anoxic stage and setting stage, which was related to the happen of denitrification process due to the deficiency of dissolved oxygen.

3.3. SOUR and specific nitrogen removal rate under single and combined Cu2+ and Cr6+ Fig. 3 illustrates the SOUR and specific nitrogen removal rate of SBR0, SBRCu, SBRCr and SBRCu+Cr. The SOUR, specific nitrification rate and specific denitrification rate of above-mentioned four SBRs had no obvious difference on day 15. The SOUR and nitrogen removal rate of SBR0 were almost unchanged during the whole operation time due to the absence of Cu2+ and Cr6+, whereas those of SBRCu, SBRCr and SBRCu+Cr

reduced with increase of operation from day 16 to 75. Compared with the absence of Cu2+ and Cr6+ on day 15, the SOUR of SBRCu, SBRCr and SBRCu+Cr reduced by 49.7%, 18.4% and 51.4% on day 75, respectively (Fig. 3a). Kapoor et al. (2016) reported that the inhibitory effect of Cr6 + on the SOUR of activated sludge increased with the increase of influent Cr6+ concentration from 0 to 35 mg/L. SOUR is associated with the organic compound biodegradation and nitrifying process. The decrease of SOUR suggested that the single and combined Cu2+ and Cr6+ inhibited the metabolic activities of aerobic heterotrophic bacteria, ammonia-oxidizing bacteria and nitrite-oxidizing bacteria due to their potential biotoxicity. Tang et al. (2018) also demonstrated that Cu2+ at 86.6 mg/L could significantly inhibit the SOUR of nitritation process. The combined Cu2+ and Cr6+ exerted a significant inhibitory effect on the SOUR by comparison with the single Cr6+ on day 35, 55 and 75, whereas the inhibitory effect of combined Cu2+ and Cr6+ on the SOUR was slightly more than that of single Cu2+. Compared with the absence of Cu2+ and Cr6+ on day 15, the SAOR and SNOR of SBRCu, SBRCr and SBRCu+Cr reduced by 47.1% and 48.0%, 16.7% and 21.2%, and 48.6% and 54.1% on day 75, respectively (Fig. 3b and 3c). The variation of SAOR was agreement with the report of Jiang et al. (2013), which demonstrated that the SAOR of SBR at 20 mg/L Cu2+ decreased by 27.33% by comparison with 0 mg/L Cu2+. The single and combined Cu2+ and Cr6 + caused slightly higher inhibitory effects on the SAOR than the SNOR at the same operation time. The decrease of SAOR and SNOR illustrated that the presence of single and combined Cu2+ at 20 mg/L and Cr6+ at 10 mg/L exerted different inhibitory effects on the processes of ammonia oxidation and nitrite oxidation. The inhibitory effect of single Cu2+ at 20 mg/L on the SAOR and SNOR had a significant increase by comparison with that of single Cr6+. Nevertheless, the combined Cu2+ and Cr6+

S. Li et al. / Science of the Total Environment 719 (2020) 137289

influent period anoxic Phase period

a

b

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SBR0 (Cu 2++Cr6+=0+0 mg/L) SBRCu (Cu 2++Cr6+=20+0 mg/L) SBRCr (Cu 2++Cr6+=0+10 mg/L) SBRCu+Cr (Cu 2++Cr6+=20+10 mg/L)

300 250

NH4 +-N concentration (mg/L)

COD co n cen tratio n (mg /L)

350

settling period effluent period influent period idle anoxic anoxic period Phase aerobic period period period

200 150 100 50 0

settling period effluent period idle anoxic period aerobic period period SBR0 (Cu 2++Cr6+=0+0 mg/L) SBRCu (Cu 2++Cr6+=20+0 mg/L) SBRCr (Cu 2++Cr6+=0+10 mg/L) SBRCu+Cr (Cu 2++Cr6+=20+10 mg/L)

30 25 20 15 10 5 0

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influent period anoxic Phase period

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7

settling period

aerobic period

effluent period influent period idle anoxic anoxic period Phase period period

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NO3 --N concentration (mg/L)

SBR0 (Cu 2++Cr6+=0+0 mg/L) SBRCu (Cu 2++Cr6+=20+0 mg/L) SBRCr (Cu 2++Cr6+=0+10 mg/L) SBRCu+Cr (Cu 2++Cr6+=20+10 mg/L)

6

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NO2 --N co n cen tratio n (mg /L)

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5 4 3 2 1

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SBR0 (Cu 2++Cr6+=0+0 mg/L) SBRCu (Cu 2++Cr6+=20+0 mg/L) SBRCr (Cu 2++Cr6+=0+10 mg/L) SBRCu+Cr (Cu 2++Cr6+=20+10 mg/L)

10 8 6

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− Fig. 2. Variation of COD and nitrogen compound concentrations in the SBR under the single and combined Cu2+ and Cr6+ during one cycle on day 75. (a) COD, (b) NH+ 4 -N, (c) NO2 -N, and (d) NO− 3 -N. One circle includes 5 min influent period, 60 min anoxic period, 180 min aerobic period, 60 min anoxic period, 30 min settling period, 5 min effluent period and 20 min idle − − period. COD, Cu2+, Cr6+, NH+ 4 -N, NO2 -N, and NO3 -N represent chemical oxygen demand, divalent copper, hexavalent chromium, ammonia nitrogen, nitrite nitrogen and nitrate nitrogen, respectively.

produced a little more inhibitory effect on the SAOR and SNOR than the single Cu2+. Aslan and Sozudogru (2017) found that the coexistence of Cu2+ and Ni2+ could significantly increase the ammonia oxidizing rate and nitrite oxidizing rate of a submerged biofilter. As shown in Fig. 3d and 3e, the SNIRR and SNRR of SBRCu, SBRCr and SBRCu+Cr reduced by 27.0% and 25.8%, 9.41% and 9.77%, and 29.1% and 29.0% on day 75 by comparing with the absence of Cu2+ and Cr6+ on day 15, respectively. The inhibitory effect of single Cu2+ on the SNIRR and SNRR was significantly higher than that of single Cr6+, whereas the combined Cu2+ and Cr6+ displayed a little more inhibitory effect on the SNIRR and SNRR than the single Cu2+. Chen et al. (2016) reported that the specific denitrifying rate of denitrifying biogranules at 100 mg/L Cu2+ decreased by 57.34% by comparison with 0 mg/L Cu2+. The present results demonstrated that inhibitory effects of combined Cu2+ and Cr6+ on the SOUR, SAOR, SNOR, SNIRR and SNRR were more than those of single Cu2+ or Cr6+, whereas the coexistence of Cu2+ and Cr6+ did not produce a synergistic inhibition effect on the oxygen uptake rate and nitrogen removal rate. 3.4. Microbial enzymatic activities under the single and combined Cu2+ and Cr6+ Fig. 4 shows the variations of DHA, nitrifying enzymatic activity and denitrifying activity under the single and combined Cu2+ and Cr6+. The

enzymatic activities of SBR0, SBRCu, SBRCr and SBRCu+Cr displayed no distinct difference on day 15. The DHA, nitrifying enzymatic activity and denitrifying activity of SBR0 were almost unchanged during the whole operation time due to the absence of Cu2+ and Cr6+, whereas those of SBRCu, SBRCr and SBRCu+Cr illustrated a gradual decreasing trend from day 16 to 75. Compared with the absence of Cu2+ and Cr6+ on day 15, the DHA of SBRCu, SBRCr and SBRCu+Cr reduced by 34.4%, 12.4% and 34.5% on day 75, respectively (Fig. 4a). The inhibitory effect of combined Cu2+ and Cr6+ on the DHA was significantly more than that of single Cu2+. However, the combined Cu2+ and Cr6+ had almost the same inhibitory effect on the DHA as the single Cu2+. Tan et al. (2017) have reported that DHA plays an important role to promote the dehydrogenation from organic compounds. The reduction of DHA under the single and combined Cu2+ and Cr6+ might affect the organic matter removal performance of SBR. As shown in Fig. 4b and 4c, the AMO and NOR activities of SBRCu, SBRCr and SBRCu+Cr reduced by 43.2% and 46.0%, 14.9% and 17.9%, and 44.4% and 48.3% on day 75 by comparison with the absence of Cu2+ and Cr6+ on day 15, respectively. The inhibitory effect of combined Cu2+ and Cr6+ was significantly higher than that of Cr6+. However, the combined Cu2+ and Cd6+ exerted a little more inhibitory effect on the AMO and NOR activities than the single Cu2+. Additionally, the single and combined Cu2+ and Cr6+ had slightly higher inhibitory effect on the NOR activity than the AMO activity. As is known to all, AMO and NOR have the ability of

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S. Li et al. / Science of the Total Environment 719 (2020) 137289

a

SOUR (mg O2 /(g MLVSS·h))

120

SBR0 (Cu 2++Cr6+=0+0 mg/L) SBRCu (Cu 2++Cr6+=20+0 mg/L) SBRCr (Cu 2++Cr6+=0+10 mg/L) SBRCu+Cr (Cu 2++Cr6+=20+10 mg/L)

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SBR0 (Cu 2++Cr6+=0+0 mg/L) SBRCu (Cu 2++Cr6+=20+0 mg/L) SBRCr (Cu 2++Cr6+=0+10 mg/L) SBRCu+Cr (Cu 2++Cr6+=20+10 mg/L)

*

*

*

*

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*

*

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SNOR (mg N/(g MLVSS·h))

SA OR (mg N/(g M LVSS·h ))

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c

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*

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SBR0 (Cu 2++Cr6+=0+0 mg/L) SBRCu (Cu 2++Cr6+=20+0 mg/L) SBRCr (Cu 2++Cr6+=0+10 mg/L) SBRCu+Cr (Cu 2++Cr6+=20+10 mg/L)

20

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* *

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12 8 4 0

SNRR (mg N/(g MLVSS·h))

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SBR0 (Cu 2++Cr6+=0+0 mg/L) SBRCu (Cu 2++Cr6+=20+0 mg/L) SBRCr (Cu 2++Cr6+=0+10 mg/L) SBRCu+Cr (Cu 2++Cr6+=20+10 mg/L)

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Fig. 3. Variations of SOUR and nitrogen removal rate under the single and combined Cu2+ and Cr6+. (a) SOUR, (b) SAOR, (c) SNOR, (d) SNIRR, and (e) SNRR. Asterisks indicate the statistical difference (p b 0.05) from SOUR and nitrogen removal rates at the absence of Cu2+ and Cr6+ on day 15. Error bars represent standard deviations of triplicate measurements. SOUR, Cu2+, Cr6+, SAOR, SNOR, SNIRR and SNRR represent specific oxygen uptake rate, divalent copper, hexavalent chromium, specific ammonia-oxidizing rate, specific nitrite-oxidizing rate, specific nitrate-reducing rate, and specific nitrite-reducing rate, respectively. − − − catalyzing the transformation of NH+ 4 -N to NO2 -N and NO2 -N to NO3 N, respectively. The reduction of AMO and NOR activities under the single and combined Cu2+ and Cr6+ could affect the processes of ammonia oxidization and nitrite oxidization. The AMO and NOR activities had the similar varying tendency to the SAOR and SNOR with the increase of operation time, respectively. It speculated that the reduction of AMO and NOR activities might firstly inhibit the nitrifying rate and further affect 2+ the removal of NH+ and Cr6+ 4 -N. Compared with the absence of Cu on day 15, the NIR and NR activities of SBRCu, SBRCr and SBRCu+Cr decreased by 23.7% and 25.6%, 8.99% and 11.3%, and 26.0% and 30.2% on day 75, respectively (Fig. 4d and 4e). The combined Cu2+ and Cr6+ produced significant inhibitory effects on the NIR and NR activities by comparison with that of single Cr6+. However, the inhibitory effect of combined Cu2+ and Cr6+ was a little more than that of single Cu2+. In addition, the NR was more susceptible to the biotoxicity of single and combined Cu2+ and Cr6+ than the NIR. As everyone knows, NIR and NR can catalyze the reduction processes of nitrite to nitrogen gas and nitrate to nitrite, respectively. The decrease of NIRR and NR activities can affect the denitrifying process under the single and combined Cu2+ and Cr6+. The NIR and NR activities in four SBRs displayed similar changing

trends to the SNIRR and SNRR with the increase of operation time, respectively. It was concluded that the decrease of NIR and NR activities could restrain the denitrifying rate and further impact the nitrite and nitrate reduction of SBR. The present results demonstrated that the inhibitory effects of combined Cu2+ and Cr6+ on the DHA, AMO and NOR activity, and NIR and NR activity were a little more than that of Cu2+, which suggested that the coexistence of Cu2+ and Cr6+ did not result in a synergistic inhibition effect on the microbial enzymatic activity of SBR. 3.5. Microbial community under the single and combined Cu2+ and Cr6+ The sludge samples from SBR0, SBRCu, SBRCr and SBRCu+Cr on day 75 were named as S0, SCu, SCr and SCu+Cr, respectively. As depicted in Table 1, the microbial richness and diversity of S0 displayed had no obvious variation on day 75 by comparison with seed sludge, whereas those of SCr, SCu and SCu+Cr displayed some obvious reduction on day 75, which illustrated that the single and combined Cu2+ and Cr6+ decreased the microbial richness and diversity. The microbial richness and diversity were in a descending order as follows: S0, SCr, SCu and

S. Li et al. / Science of the Total Environment 719 (2020) 137289

DHA (mg TF /(g MLVSS∙h))

SBR0 (Cu 2++Cr6+=0+0 mg/L) SBRCu (Cu 2++Cr6+=20+0 mg/L) SBRCr (Cu 2++Cr6+=0+10 mg/L) SBRCu+Cr (Cu 2++Cr6+=20+10 mg/L)

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Operation time (d) SBR0 (Cu 2++Cr6+=0+0 mg/L) SBRCu (Cu 2++Cr6+=20+0 mg/L) SBRCr (Cu 2++Cr6+=0+10 mg/L) SBRCu+Cr (Cu 2++Cr6+=20+10 mg/L)

b

0.24 0.2

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A M O (μg NO2 --N/(mg p ro tein ·min ))

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SBR0 (Cu 2++Cr6+=0+0 mg/L) SBRCu (Cu 2++Cr6+=20+0 mg/L) SBRCr (Cu 2++Cr6+=0+10 mg/L) SBRCu+Cr (Cu 2++Cr6+=20+10 mg/L)

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Operation time (d) SBR0 (Cu 2++Cr6+=0+0 mg/L) SBRCu (Cu 2++Cr6+=20+0 mg/L) SBRCr (Cu 2++Cr6+=0+10 mg/L) SBRCu+Cr (Cu 2++Cr6+=20+10 mg/L)

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SBR0 (Cu 2++Cr6+=0+0 mg/L) SBRCu (Cu 2++Cr6+=20+0 mg/L) SBRCr (Cu 2++Cr6+=0+10 mg/L) SBRCu+Cr (Cu 2++Cr6+=20+10 mg/L)

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Fig. 4. Variations of microbial enzymatic activity under the single and combined Cu2+ and Cr6+. (a) DHA, (b) AMO activity, (c) NOR activity, (d) NIR activity, and (e) NR activity. Asterisks indicate statistical differences (p b 0.05) from the microbial enzymatic activity at the absence of Cu2+ and Cr6+ on day 15. Error bars represent standard deviations of triplicate measurements. Cu2+, Cr6+, DHA, AMO, NOR, NIR and NR represent divalent copper, hexavalent chromium, dehydrogenase activity, ammonia monooxygenase, nitrite oxidoreductase, nitrite reductase and nitrate reductase, respectively.

Proteobacteria Bacteroidetes Chloroflexi Planctomycetes Actinobacteria Acidobacteria Armatimonadetes Verrucomicrobia Gemmatimonadetes unidentified_Bacteria Others

SCr

S0 Seed sludge SCu+Cr SCu Unweighted unifac distane

0 0

0.1

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0.3

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1

Relative abundance in phylum level

Fig. 5. Unweighted pair-group method with arithmetic (UPGMA) mean analysis of microbial communities under the single and combined Cu2+ and Cr6+ on day 75. Cu2+ and Cr6+ represent divalent copper and hexavalent chromium, respectively.

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S. Li et al. / Science of the Total Environment 719 (2020) 137289

SCu+Cr. In addition, the PD whole tree indexes of seed sludge, S0, SCr, SCu and SCu+Cr were 80, 86.3, 65.7, 24.8 and 21.4, respectively. The variation of PD tree indexes illustrated that the microbial community of SCu and SCu+Cr had better genetic relation. According to unweighted pairgroup method with arithmetic (UPGMA) mean analysis (Fig. 5), the above-mentioned five sludge samples could be divided into three groups as follows: (a) seed sludge and S0, (b) SCr, and (c) SCu and SCu +Cr, which further indicated that the microbial community had some evident difference under the single and combined Cu2+ and Cr6+. Fig. 6 shows the similarity and difference of microbial community under the single and combined Cu2+ and Cr6+ through Venn diagram. The shared operation taxonomic unit (OTU) number of abovementioned five samples was 177, which suggested that some microorganisms always existed in seed sludge, S0, SCu, SCr, and SCu+Cr. At the phylum level, the shared OTUs were categorized as Proteobacteria (46.3%), Bacteroidetes (33.9%), Acidobacteria (7.34%), Chloroflexi (6.21%), Gemmatimonadetes (1.69%), Planctomycetes (1.13%), Nitrospirae (0.562%), Melainabacteria (0.56%), Hydrogenedentes (0.56%), Firmicutes (0.556%), and others (1.69%). The unique OTU number of seed sludge, S0, SCr, SCu and SCu+Cr was 207, 129, 66, 6 and 4, respectively, which demonstrated that very few special microorganisms were found in SCu and SCu+Cr. The microbial community under the single and combined Cu2+ and 6+ Cr was analyzed on day 75 at the genus level (Fig. 7). Compared with seed sludge, the relative abundances of different genera in S0, SCu, SCr and SCu+Cr displayed some evident difference. The relative abundances of Nitrosomonas and Nitrospira were in a descending order as follows: S0, SCr, SCu and SCu+Cr, which suggested that the presence of single and combined Cu2+ and Cr6+ affected the growth, development and reproduction of Nitrosomonas and Nitrospira due to their biotoxicity. The present results demonstrated that Nitrosomonas and Nitrospira were more sensitive to the toxicity of combined Cu2+ and Cr6+ than the single Cu2+ and Cr6+. Nitrosomonas and Nitrospira can transform NH+ 4 -N − − into NO− 2 -N and NO2 -N into NO3 -N by utilizing inorganic matter as carbon source, respectively. The decrease of relative abundance for Nitrosomonas and Nitrospira under the single and combined Cu2+ and Cr6+ could decrease the activities of AMO from Nitrosomonas and NOR from Nitrospira, which could result in the reduction of SAOR and SNOR and further affect the nitrifying process. Compared with seed sludge and S0, the relative abundance of some denitrifying genera (e.g. Thauera, Flavobacterium, Dechloromonas and Defluviimonas) in SCu, SCr and SCu+Cr displayed obvious changes, which had the ability of reducing NO− 2 -N and NO− 3 -N under anoxic or anaerobic conditions (Horn et al., 2005; Foesel et al., 2011; Liu et al., 2014). The relative abundance of Flavobacterium was in a descending order as follows: SCr, SCu+Cr and SCu. Compared with S0, the relative abundance of Defluviimonas was almost unchanged in SCr, whereas it displayed a significant reduction in Seed sludge 207 c

SCu+Cr

S0

2

72 275

The combined Cu2+ at 20 mg/L and Cr6+ at 10 mg/L exerted significant inhibitory effects on the oxygen uptake rate, nitrification rate, denitrification rate, DHA, nitrifying enzymatic activity and denitrifying enzymatic activity of SBR by comparison with the single Cr6+ at 10 mg/L. However, the inhibitory effects of combined Cu2+ and Cr6+ on the oxygen uptake rate, nitrogen removal rate and microbial enzymatic activity was a little more than those of single Cu2+, suggesting that the coexistence of Cu2+ and Cr6+ did not produce a synergistic effect on the SBR performance. The microbial richness and diversity displayed some obvious changes under the single and combined Cu2+ and Cr6+ by comparison with the absence of Cu2+ and Cr6+. The relative abundances of nitrifying genera under the combined Cu2+ and Cr6+ was less than those under the single Cu2+ and Cr6+. The present results will be beneficial to better understand the combined effects of multiple HMs on biological wastewater treatment systems. CRediT authorship contribution statement Shanshan Li: Formal analysis, Data curation, Writing - original draft. Shuyan Wu: Conceptualization, Data curation. Bingrui Ma: Data curation. Mengchun Gao: Funding acquisition, Supervision. Yuanyuan Wu: Data curation. Zonglian She: Formal analysis, Investigation. Yangguo Zhao: Methodology. Liang Guo: Resources. Chunji Jin: Formal analysis, Investigation. Junyuan Ji: Visualization, Project administration.

0.56% 0.56% 1.69% 0.56% 0.56%

Proteobacteria

1.13% 1.69%

Bacteroidetes Actinobacteria

6.21%

Chloroflexi

22

1

8

7.34%

4

Gemmatimonadetes

2 15

Planctomycetes

177

46.33%

1 0

1

18

11

Melainabacteria 22

15

73 6

6

Nitrospirae

5

33.33%

6

SCu

4. Conclusions

2

350

65

129

SCu and SCu+Cr, which demonstrated that Defluviimonas was more susceptible to the biotoxicity of Cu2+ at 20 mg/L than that of Cr6+ at 10 mg/L. The relative abundance of Dechloromonas in SCu+Cr was significantly more than that in S0, SCu and SCr due to its good adaption to the biotoxicity of Cu2+ and Cr6+, which was speculated that Dechloromonas might be a Cr6+ and Cu2+ resistant genera. Some reports also demonstrated that Dechloromonas had higher tolerant ability to other HMs including Cd2+, Pb2+, Zn2+ and Ni2+ (Zou et al., 2015; Kasemodel et al., 2019; Zhang et al., 2019). The relative abundances of many genera in SCu+Cr were obviously lower than those in SCu and SCr, such as Haliscomenobacter, Longilinea, Longilinea, Phaeodactylibacter, Aquimonas, Stenotrophobacter, Candidatus Competibacter and Ohtaekwangia, which indicated that the combined Cu2+ and Cr6+ led to more reduction in relative abundance than single Cu2+ or Cr6+. However, some genera (e.g. Acidovorax, Rhodobacter, Lysobacter, Algoriphagus and Arenimonas) in SCu+Cr were more in relative abundance than those in SCu and SCr. The present results further demonstrated that the presence of single and combined Cu2+ at 20 mg/L and Cr6+ in the influent could affect the microbial richness of SBR.

4

4

68

Hydrogenedentes Firmicutes

66 SCr

Others

Fig. 6. Venn diagram of microbial community under single and combined Cu2+ and Cr6+. The shared OTU number was analyzed at phylum level. The OTU number was the mean value of triplicate samples. Cu2+, Cr6+ and OTU represent divalent copper, hexavalent chromium and operational taxonomic unit, respectively.

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Fig. 7. Microbial community analysis through heat-map of the classified genera of seed sludge and sludge samples under from different SBRs on day 75. The relative abundance of genera was the mean value of triplicate samples. SBR represents sequencing batch reactor.

Acknowledgements The work was supported by the Fundamental Research Funds for the Central Universities (No. 201964003).

Chen, H., Chen, Q.Q., Jiang, X.Y., Hu, H.Y., Shi, M.L., Jin, R.C., 2016. Insight into the shortand long-term effects of Cu(II) on denitrifying biogranules. J. Hazard. Mater. 304, 448–456. Dilek, F.B., Gokcay, C.F., Yetis, U., 1998. Combined effects of Ni(II) and Cr(VI) on activated sludge. Water Res. 32, 303–312.

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