Introduction

Due to high toxicity, accumulation, and stability, heavy metals (Cu, Cd, Cr, Hg, Pb, As, etc.) not only harm the growth, metabolism, and reproduction of animals, plants, and microorganisms (Sall et al. 2020; Shuaib et al. 2021), but also can pollute water and land resources and further cause human’s health and ecological security (Briffa et al. 2020; Gupta et al. 2012). The discharge of heavy metal-containing wastewater in developing countries was still very large and has induced many serious environmental problems (Li et al. 2022a, b). According to a global survey, concentration and type of heavy metals have increased significantly in water bodies, and the situation in developing country was more serious than developed country (Gupta et al. 2012). In China, a lot of water bodies, such as Taihu Lake and Baiyangdian Lake, have been polluted by heavy metals (Ding et al. 2019; Rajeshkumar et al. 2018; Rao et al. 2018; Rodríguez-Lado et al. 2013; Yi et al. 2011). Therefore, heavy metal pollution has been widely concerned by environment researchers.

Heavy metal-containing industrial wastewater was considered to be the main source of heavy metal pollution. Studies have found that heavy metal-containing wastewater was mainly discharged from mining, metal processing, and electroplating industries (Yi et al. 2011; Zhou et al. 2020). In China, heavy metal-containing wastewater accounts for about 20% of the total wastewater (Li et al. 2022a, b), and therefore it is significant for the water environment protection and human health to treat them effectively.

Conventional wastewater treatment methods for heavy metal removal include physicochemical and biological methods (Fu and Wang 2011; Jacob et al. 2018). Compared with physicochemical methods, biological methods have many advantages such as low cost, easy operation, and can remove COD, nitrogen, and phosphorus synchronously and efficiently (Jacob et al. 2018). Biological methods mainly rely on the adsorption of activated sludge. Heavy metals can be adsorbed on the surface of activated sludge or enriched in microbial cells and removed from wastewater through discharging of excess sludge. The biological adsorption mechanism includes electrostatic adsorption, ligand exchange, surface complexation, and precipitation. Microorganisms such as bacteria, fungi, and algae in activated sludge have a large number of functional groups (e.g., phenols, alcohol hydroxides, and carboxylic acids), which can provide biosorption sites to adsorb heavy metals in wastewater (Barros et al. 2006; Eccles 1995; Fomina and Gadd 2014). In technical specification for electroplating industry wastewater treatment (HJ 2002–2010) in China, biological treatment method was recommended for the treatment of electroplating wastewater (Li et al. 2022a, b). Furthermore, the treatment of heavy metals in wastewater using activated sludge does not involve extra energy or chemical consumption, showing great potential for the application in treating heavy metal-containing wastewater (Matyja et al. 2021).

As common domestic sewage treatment technology, the activated sludge process can adsorb heavy metals by continuously producing activated sludge. However, previous studies paid more attention to heavy metal toxicity to activated sludge rather than removal of heavy metals. Liu et al. (2022) found that the number of Gram-negative bacteria in activated sludge increased obviously under the stress condition of heavy metals Pb, As, and Hg. Bhat et al. (2020) found that activated sludge had tolerance to heavy metals, but treatment efficiency was significantly reduced when the concentration of heavy metals was high. The report from You et al. (2009) indicated that heavy metal removal could be inhibited in the complex heavy metal-containing wastewater, and the inhibited degree was Ni > Cd > Pb. Some studies have confirmed the great potential of activated sludge process to remove heavy metals from wastewater. Laurent et al. (2009) found that activated sludge can remove Cu and Cd through proton exchange, ligand exchange, and precipitation. Pagnanelli et al. (2009) found that activated sludge systems had many carboxylic and amino groups which could adsorb Cd and Pb. Activated sludge pretreated with alkali was used to adsorb heavy metals, and the maximum adsorption capacity of Cu and Cd achieved 131.6 and 93.4 mg·g−1, respectively, while the adsorption capacity of Ni was low. However, these studies did not investigate the removal efficiency by activated sludge during long-term operation, and the researches on treatment efficiency and adsorption mechanism of composite heavy metal-containing wastewater were neglected. In the long-term operation process of activated sludge, heavy metals will change the microbial community structure of activated sludge and then have an impact on the removal effect of heavy metals (Zhang et al. 2022). Therefore, the exploration of these two aspects shows great value to the increasingly serious situation of compound heavy metal wastewater pollution.

In this study, the SBR was continuously operated for 123 days to investigate the long-term treatment effects of activated sludge process in removing three kinds of heavy metals Pb2+, Cu2+, and Ni2+ from wastewater. And the adsorption mechanism was revealed based on static adsorption experiment and microbial community analysis.

Materials and methods

SBR equipment

During the dynamic removal experiment, sequencing batch reactor (SBR) was selected as the form of activated sludge system, because it can well adapt to the characteristics of large fluctuation of the hydraulic load of industrial wastewater (Uygur and Kargı 2004). The reactor consisted of a cylindrical plexiglass tank with an effective volume of 6 L (Fig. 1). Five circular holes were set on the tank wall for aeration, water entry and exit, and sample collection. The peristaltic pump was used to regulate the water in and out. The activated sludge used in SBR and static adsorption experiments was taken from a pilot-scale domestic sewage treatment system in the Renmin University of China. The sludge concentration was around 3000 mg·L−1, and the sludge sedimentation ratio was 20 ~ 30%.

Fig. 1
figure 1

Diagram of SBR equipment

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Long-term operation of SBR

The simulated wastewater was designed based on the quality of the actual heavy metal-containing industrial wastewater. Table 1 showed the operation conditions of the SBR activated sludge system. The operation phase was set as follows: filling (6 min)—reaction (9.9 h)—settling (1 h)—discharging (6 min)—idling (54 min). Aeration took place only in the reaction stage.

Table 1 SBR operation parameter settings
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The composition of the simulated wastewater containing heavy metals is shown in Table 2. The SBR dynamic removal experiment was divided into four stages: culture (0 mg·L−1), 3 mg·L−1, 5 mg·L−1, and 10 mg·L−1. During the culture stage, the influent had no heavy metals so that the activated sludge can adapt and reproduce. In latter three stages, the concentration of various heavy metals was 3 mg·L−1, 5 mg·L−1, and 10 mg·L−1, respectively.

Table 2 Composition of heavy metal wastewater
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Static adsorption experiment

The pH of Pb2+, Cu2+, and Ni2+ solution (20 mg·L−1) was adjusted to 6.0 using KOH and HCl. Each 25 mL of heavy metal-containing solution and 0.4 mL of dewatered activated sludge (by centrifuge) were added into 50-mL conical flasks. The activated sludge concentration was 1.0 ± 0.1 g·L−1. The mixture was then put in a shaking table to shake for 2 h at 200 r/min at 25 ℃. The sampling interval in the first 30 min was 3 min, and the sampling interval between 30 and 60 min was 10 min, and the sampling interval in the last 60 min was 15 min. Then 10 mL mixture was quickly collected, and activated sludge was separated by high-speed refrigerated centrifugation (4℃, 8000 rpm, 5 min), and the concentrations of Pb2+, Cu2+, and Ni2+ in the supernatant were measured using flame atomic absorption spectrophotometer (Z-2000). The pH, sludge concentration, adsorption time, heavy metal solution concentration, temperature, and coexisting heavy metal were adjusted to investigate the influence of different factors on the adsorption effect.

Analytical determination

The concentration of heavy metals was measured by atomic absorption spectroscopy (AAS) with flame atomic absorption spectrophotometer (Z-2000); chemical oxygen demand (COD) was measured by rapid digestion spectrophotometry with Lianhua digestion instrument and multi-parameter water quality tester. Specific measurement methods of mixed liquid suspended solids (MLSS) and sludge volume index (SVI) were provided in Text S1.

To explore the adsorption mechanisms of activated sludge, the analyses including kinetic model fitting, isothermal adsorption model fitting, Fourier transform infrared spectroscopy (FT-IR), energy-dispersive x-ray spectroscopy (EDS), zeta potential analysis, scanning electron microscope (SEM), and 16S rDNA amplicon sequencing were carried out.

Results

Long-term treatment effect of heavy metal in the SBR system

Long-term treatment of heavy metals in the SBR system was divided into four stages including culture stage (0–4d), domestication stage I (3 mg·L−1, 5–11 d), domestication stage II (5 mg·L−1, 12–72 d), and domestication stage III (10 mg·L−1, 73–123 d). The operation results are shown in Fig. 2. It was obvious that the removal rate for three kinds of heavy metals was the highest at low heavy metal concentration and gradually decreased with the increase of concentration. In the stable period of three operation stages (3 mg·L−1, 5 mg·L−1, and 10 mg·L−1), the removal rate of Pb2+ achieved 100%, 91.4 ~ 96.2%, and 83.1 ~ 90.0%, respectively, the removal rate of Cu2+ was 87.7 ~ 90%, 81.8 ~ 85.6%, and 74.3 ~ 80.6%, respectively, and the removal rate of Ni2+ was 13.3 ~ 33.6%, 0.8 ~ 22.7%, and 0 ~ 6.2%, respectively. The removal efficiency of Pb2+ was the highest, followed by Cu2+ and Ni2+. This result was similar to the results reported by Pagnanelli et al. (2009). This may be related to the high toxicity of Ni (You et al. 2009). Although COD removal rate, MLSS, and SVI decreased under the influence of heavy metals, they tended to be stable during the long-term operation process, indicating that the activated sludge system had adaptability and recovery capacity.

Fig. 2
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Operation effect of the SBR activated sludge system

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Static adsorption experiment

Adsorption effect of activated sludge on single heavy metals

As shown in Fig. 3, the removal efficiency of Pb2+ was the highest, followed by Cu2+ and Ni2+. The adsorption process of Pb2+, Cu2+, and Ni2+ by activated sludge could be divided into rapid adsorption stage (0 ~ 4 min) and slow adsorption stage (4 ~ 120 min). The actual equilibrium adsorption capacities of activated sludge for Pb2+, Cu2+, and Ni2+ at initial concentration of 20 mg·L−1 were 18.55 mg·g−1, 17.27 mg·g−1, and 8.37 mg·g−1, respectively. As the initial heavy metal concentration increased from 5 to 200 mg·L−1, the adsorption capacity of Pb2+, Cu2+, and Ni2+ increased from 3.71 mg·g−1, 4.62 mg·g−1, and 4.65 mg·g−1 to 119.1 mg·g−1, 42.5 mg·g−1, and 44.5 mg·g−1, respectively. The increase gradually slowed down, and the removal rate of heavy metals decreased (Fig. S1). It was possible that the adsorption sites were saturated or the active adsorption of microorganisms was inhibited by the toxicity of heavy metals. With the increase of sludge concentration, the adsorption capacity decreased gradually, but the removal rate of three kinds of heavy metals showed an upward trend (Fig. S2). Increased adsorption dose led to increased adsorption sites and further led to increased removal rate of heavy metals. However, this also causes decreased adsorption capacity of unit adsorbent, suggesting that the trends in the adsorption capacities of activated sludge and removal rates of heavy metals were opposite. The activated sludge had the best removal effect on Pb2+, followed by Cu2+, while the removal effect on Ni2+ was poor. This result was consistent with the operation results in the SBR system.

Fig. 3
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Effect of adsorption time on heavy metal removal by activated sludge

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Adsorption effect of activated sludge on composite heavy metals

As shown in Fig. 4, the Pb2+ removal capacity of activated sludge was almost unaffected by the existence of Cu2+ and Ni2+ (Fig. 4a). The adsorbed Cu2+ capacity by activated sludge was mainly affected by Pb2+, showing a decrease of 16.6% (Fig. 4b). However, the Ni2+ removal capacity of activated sludge was greatly inhibited by composite Pb2+ and Cu2+, which caused a sharp decline of Ni2+ removal rate by 80.3% (Fig. 4c). In addition, it can be found from the adsorption effect experiments that Cu2+ was a main inhibition factor that caused the decrease in Ni2+ adsorption capacity by activated sludge. In the composite wastewater, there was competitive adsorption between different heavy metals and activated sludge, which might be closely related to the charge, atomic weight, atomic size of heavy metals, and their affinity to functional groups on the surface of activated sludge (Zhang et al. 2022).

Fig. 4
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Adsorption effect of heavy metals by activated sludge in different systems

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Effects of initial pH on heavy metal adsorption

With increasing pH value, the adsorption capacity of heavy metals generally increased (Fig. 5). The pH range with the best adsorption effect was from 4.0 to7.0, and the maximum adsorption capacities of Pb, Cu, and Ni were 19.37 mg·g−1 (at pH 4.0), 18.27 mg·g−1 (at pH 6.0), and 8.76 mg·g−1 (at pH 6.0), respectively. The increased pH reduced the zeta potential of the activated sludge (Fig. S3). Because activated sludge surface was negatively charged, under the existence of electrostatic attraction, activated sludge showed high efficiency to adsorbing positively charged heavy metal (Morfesis et al. 2009). In acidic environment, excessive hydrogen ions will compete with heavy metals to adsorb on the surface of activated sludge, and acidic environment can also lead to the desorption of heavy metals adsorbed through ligand exchange, which can inhibit the adsorption of heavy metals (Wei et al. 2021).

Fig. 5
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Effect of pH on adsorption of heavy metal ions by activated sludge

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Adsorption kinetic of heavy metals by activated sludge

The pseudo-first-order and pseudo-second-order models were used to fit the kinetic of the adsorption process of Pb2+, Cu2+, and Ni2+ using activated sludge (Wei et al. 2021; Xu et al. 2022). The fitting results are shown in Table 3.

Table 3 Fitting results of kinetic models
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The R2 of each type of heavy metal fitted by the pseudo-second-order kinetic equation was higher than that fitted by the pseudo-first-order equation. In addition, the theoretical equilibrium adsorption capacity (Qe) fitted by the pseudo-second-order kinetic equation was closer to the actual equilibrium adsorption capacity (Mentioned in 3.1) than that obtained by the pseudo-first-order equation. Therefore, it was considered that the pseudo-second-order kinetic equation was more suitable to describe the adsorption process of three kinds of heavy metals. It was concluded that the adsorption was mainly controlled by chemical adsorption (Aman et al. 2008), and the theoretical equilibrium adsorption capacities of Pb2+, Cu2+, and Ni2+ at the initial concentration of 20 mg·L−1 were 18.35 mg·g−1, 17.06 mg·g−1, and 8.37 mg·g−1, respectively.

Adsorption isotherm of heavy metals by activated sludge

Langmuir and Freundlich models were used to fit the adsorption data, and various isotherm parameters were obtained through calculation (Pagliaccia et al. 2022; Wei et al. 2021). The fitting results are shown in Table 4. Although the isothermal adsorption of Pb2+ was not well fitted by the two models, the adsorption process of Cu2+ could be fitted by Langmuir equation, and the Ni2+ was fitted by the Freundlich equation. With the increase of temperature, the R2 value decreased, which was likely due to the unstable adsorption caused by the increase of temperature. The theoretical saturated adsorption capacity of three kinds of heavy metals at different temperatures could be obtained from the Langmuir model parameters. The saturated adsorption capacity of Pb2+ was the largest. The saturated adsorption capacities of Pb2+, Cu2+, and Ni2+ at 25 ℃ were the highest and achieved 136.21 mg·g−1, 41.13 mg·g−1, and 65.37 mg·g−1, respectively.

Table 4 Fitting results of adsorption isothermal models
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Role of sludge characteristics in adsorb heavy metals

There are sufficient polysaccharides, proteins, lipids, and other substances on the surface of microbial cells and extracellular polymeric substances (EPS) in the outer layer of activated sludge, which was beneficial to heavy metal adsorption. The previous studies showed that the functional groups in activated sludge such as carboxyl, hydroxyl, amino, amide, carbonyl, and sulfhydryl had complexation reactions with Pb2+, Cu2+, and Ni2+ (Aslan et al. 2018; Wang et al. 2006), which was also a major contribution to heavy metal removal from wastewater. The activated sludge surface before and after the adsorption was analyzed by FT-IR. Results are shown in Table 5 and Fig. S4. There were a large number of amido, hydroxyl, methylene, amide, and carboxyl groups on the surface of activated sludge, which played an essential role in the complex adsorption of Pb2+, Cu2+, and Ni2+ (Wang et al. 2006). The peak intensity of -NH2 and -NH decreased obviously on activated sludge surface after the adsorption of Pb2+ and Cu2+, indicating the action of ligand exchange. Due to the organic complexation and electrostatic adsorption of saccharides, the peak intensity of C-O and O–H on the activated sludge surface decreased after the adsorption of Pb2+, Cu2+, and Ni2+. The results of FT-IR analysis showed that Pb2+, Cu2+, and Ni2+ could be removed by organic complexation and electrostatic adsorption. On the surface of activated sludge, Pb2+ and Cu2+ could also exchange coordination with amino groups, but Ni2+ was not found the process. At the same time, the saccharide component of activated sludge played an important role in the removal of the three kinds of heavy metals.

Table 5 The FT-IR changes of activated sludge before and after adsorption of heavy metals
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When activated sludge adsorbed heavy metal, other metal cations such as K+, Na+, Ca2+, and Mg2+ may exchange with heavy metals, which caused that the adsorbed heavy metal released into the solution (Yuncu et al. 2006; Zhou et al. 2016). Activated sludge was analyzed by EDS before and after the adsorption, and the results are shown in Fig. 6. The activated sludge contained elements such as K, Na, Ca, and Mg before the adsorption. After the adsorption of heavy metals, the peaks corresponding to Pb2+, Cu2+, and Ni2+ appeared, which proved that heavy metals have been adsorbed on activated sludge. The peaks corresponding to K, Na, Ca, and Mg decreased significantly or even disappeared, and atomic percentages also decreased significantly, which showed that K, Na, Ca, and Mg cations exchanged with heavy metals during the adsorption process.

Fig. 6
figure 6

EDS results of activated sludge before and after the adsorption of heavy metals. (a): Before adsorption; (b): After Pb2+adsorption; (c) After Cu2+ adsorption; (d): After Ni2+ adsorption

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Changes of SBR system after heavy metal adsorption

The activated sludge images at the end of the culture stage and 10 mg·L−1 domestication stage was analyzed using SEM, and the sludge morphology is shown in Fig. 7. The microbial cells in activated sludge in the culture stage were loose and porous, and many microbial cells were distributed on the surface of the activated sludge with and obvious outlines. After the absorption of heavy metals, the microbial cells became dense, and micropores decreased greatly. The distribution of micelles decreased, and the contour was not obvious. Bacteria in the activated sludge had serious agglomeration after the absorption of heavy metals.

Fig. 7
figure 7

SEM results of activated sludge in the SBR system. (a): Culture stage, (b):10 mg·L−1 domestication stage

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The 16S rDNA amplicon sequencing was performed for microbes in the activated sludge at the end of the culture stage and 10 mg·L−1 domestication stage using the NovaSeq sequencing platform (Fig. 8). At culture stage, although genus Dokdonella, Dechloromonas, and Zoogloea were dominant at the genus level, the relative abundance was only 22%, indicating a good biodiversity in the activated sludge. After the long-term adsorption of heavy metals, the relative abundance of Rhodobacter reached 66%, while that of other types decreased to 14%, indicating biological diversity of activated sludge decreased significantly. The same conclusion could also be illustrated by the comparison of rarefaction curves (Fig. S5).

Fig. 8
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Relative abundance of microorganisms in activated sludge at the genus level

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Discussion

Activated sludge process has been proven to be effective in removing heavy metals from the wastewater in the present study. The removal of Pb2+ and Cu2+ was more effective than that of Ni2+ in the SBR activated sludge system. Firstly, the highly effective removal of Pb2+ was attributed to its small hydration ion radius. The hydration ion radius formed by heavy metal dissolved in aqueous solutions can reflect the degree of difficulty of adsorption by activated sludge. The smaller the hydration ion radius was, the easier it can be adsorbed by activated sludge (Maslova et al. 2020). The radius relationship between Pb2+, Cu2+, and Ni2+ hydration ions was rH(Pb2+) < rH(Cu2+) ≈ rH(Ni2+) (Volkov et al. 1997). Therefore, Pb2+ can be adsorbed easier than Cu2+ and Ni2+. Secondly, it was related to the differences in adsorption mechanism. Pb2+ and Cu2+ could perform ligand exchange with amino groups on the surface of activated sludge, which was also the main reason that the removal efficiency of Pb2+ and Cu2+ was much higher than Ni2+. Although Ni2+ was found to have ligand exchange process with the other metal ions such as Mg2+, Na+, and K+ on the surface of activated sludge in this study, the amount of these metal ions was much lower than that of amino groups. The adsorption process of Cu2+ was more consistent with the Langmuir model, indicating a monolayer adsorption, which can prove that electrostatic adsorption and ligand exchange were the main mechanisms of Cu2+ adsorption by activated sludge. The adsorption process of Ni2+ was more consistent with the Freundlich model, indicating that a single ion can occupy more than one active site (Wei et al. 2021). Therefore, with the same number of active sites, the amount of Ni2+ adsorbed was less than that of Cu2+ when the adsorption was nearly saturation. On the other hand, the biotoxicity of Cu2+ was lower than Ni2+, which made activated sludge easier to retain Cu2+ than Ni2+. Therefore, the adsorption of Ni2+ by activated sludge was more difficult than Cu2+. In addition, due to the high concentration of phosphate in the wastewater, part of Pb2+ or Ni2+ may be removed through the inorganic micro-precipitation mechanism, which can be proved by the higher P and O peaks (Fig. 6) after Pb2+ adsorption by activated sludge (Daskalakis et al. 2013).

Activated sludge mainly removes Pb2+ and Cu2+ from wastewater through ligand exchange, organic complexation, and electrostatic adsorption in the SBR system, and the removal of Ni2+ mainly depended on the organic complexation and electrostatic adsorption. In the treatment process of Pb2+-, Cu2+-, and Ni2+-containing wastewater by activated sludge in the SBR activated sludge system, Pb2+ could quickly combine with the adsorption site on activated sludge and complete the process of adsorption, and thus the reaction of Pb2+ was faster than Cu2+ and Ni2+. This was mainly because the hydration radius of Pb2+ was smaller than Cu2+ and Ni2+. Ni2+ was hardly exchange coordination with -NH2, -NH, or -OH on the surface of activated sludge, and thus Ni2+ has disadvantages in competitive adsorption with Pb2+ and Cu2+ during the organic complexation and electrostatic adsorption processes. Therefore, the adsorption capacity of activated sludge to Ni2+ was the weakest.

However, the SBR activated sludge system was significantly affected by heavy metals, showing that heavy metal showed biological toxicities to activated sludge. Heavy metals produced toxicity to activated sludge by affecting intracellular enzymes, inducing generation of reactive oxygen species (ROS), affecting cell membrane structure, and so on, which could further affect activated sludge shape and microbial community structure (Kelly et al. 2004; Li et al. 2022a, b). Generally, activated sludge enabled to be protected by secreting more EPS to adapt to heavy metal stress (Pagliaccia et al. 2022). This led to better agglomeration and settleability of the activated sludge in the SBR system. In addition, microbial community in activated sludge changed during the long-term impact of heavy metals. It was found that microbial diversity decreased significantly under the condition of the influence of heavy metals, and genus Rhodobacter became the dominant in activated sludge, which was a heterotrophic microorganism that can perform photosynthesis. Genus Rhodobacter can degrade organic matter in wastewater under the conditions of anaerobic light or aerobic dark, and rich pigment in their cells makes them have strong antioxidant properties to cope with heavy metal stress (Lu et al. 2022; Talaiekhozani and Rezania 2017).

Conclusions

The long-term treatment of Pb2+-, Cu2+-, and Ni2+-containing wastewater by activated sludge a SBR activated sludge system was continuously carried out for 123 days, and the mechanism of heavy metal removal by activated sludge was discussed through static adsorption experiments and sludge characteristics. Activated sludge process was effective in removing heavy metals (Pb2+ and Cu2+) from the wastewater in the present study, activated sludge and the removal rate of Pb2+ and Cu2+ in SBR activated sludge system were 83.1 ~ 90.0% and 74.3 ~ 80.6%, respectively, at the 10 mg/L of initial concentration, while the removal rate of Ni2+ was less than 6.2%. The equilibrium adsorption capacities of Pb2+, Cu2+, and Ni2+ at the initial concentration of 20 mg·L−1 were 18.35 mg·g−1, 17.06 mg·g−1, and 8.37 mg·g−1, respectively. Activated sludge removed Pb2+ and Cu2+ by the ligand exchange, electrostatic adsorption, and surface organic complexation processes, while Ni2+ was removed by the electrostatic adsorption and surface organic processes. The Ni2+ removal was inhibited by Pb2+ and Cu2+ in composite heavy metal-containing activated sludge system. In the long-term treatment process of heavy metal-containing wastewater, microbial diversity of activated sludge decreased, and genus Rhodobacter became the dominant community with a relative abundance of 66%.