Evaluating the effect of antibiotics on aerobic granular sludge treatment of pharmaceutical wastewater

Antibiotics play a crucial role in clinical treatment and poultry farming, contributing significantly to modern human health and economic development (Pazda et al. 2019). However, the excessive use of antibiotics imposes substantial pressure on wastewater treatment. Improper treatment of wastewater from antibiotic manufacturing can lead to secondary environmental pollution, and there are potential risks such as the spread of antibiotic resistance genes (Watkinson et al. 2007). Effectively addressing the treatment of wastewater from antibiotic manufacturing has become a critical research imperative.

Compared to traditional activated sludge processes, aerobic granular sludge (AGS) exhibits advantages such as high biomass retention, excellent settling performance, and a diverse microbial population (Purba et al. 2020). The unique structure of AGS provides it with a stable internal environment and external resistance, making it a potential treatment technology for specialized industrial wastewater (Wan et al. 2022; Zhao et al. 2023). In recent years, AGS has been applied in the treatment of pharmaceutical wastewater containing antibiotics (Jiang et al. 2021). Wan et al. (2018) investigated the impact of different concentrations of sulfamethoxazole on the granular sludge system, finding that sulfamethoxazole had minimal effects on granular sludge, with removal efficiencies exceeding 90%. Tetracycline (TE), as an antibiotic often detected in wastewater, has a certain ecological toxicity, which can affect the biological treatment process of wastewater (Scaria et al. 2021). As the target species of antibiotics, coupled with the characteristics of prokaryotes, bacteria are more sensitive to TE. TE can change the structure, composition, and functional properties of bacteria (Muñoz-Palazon et al. 2022). Previous studies on TE on AGS are mostly focused on the treatment of urban low C/N wastewater, while AGS treatment of pharmaceutical wastewater containing TE antibiotics is rare (Kim et al. 2007; Zhang et al. 2016; Wang et al. 2018a, 2018b). However, the influence of TE on AGS in treating pharmaceutical wastewater remains largely unexplored, and the effects of TE on the characteristics of AGS sludge and the variations in intracellular/extracellular polymers are not well understood. Examining the impact of TE on AGS in the treatment of pharmaceutical wastewater will contribute to the efficient operation of AGS systems.

Therefore, this work investigated the impact of TE on the behavior of AGS in the treatment of pharmaceutical wastewater and revealed the relevant mechanisms. Firstly, the long-term exposure of AGS to different concentrations of TE was analyzed to assess its operational efficiency. Subsequently, the influence of TE on the characteristics of AGS sludge was investigated. Finally, the effect of TE on the activities of key enzymes during the operation of AGS was analyzed to reveal the relevant mechanism. The research findings provide data support and a theoretical basis for the application of AGS in treating pharmaceutical wastewater containing TE.

The inoculum sludge was sourced from an AGS system in a laboratory, which demonstrated efficient performance in treating urban wastewater with low C/N ratios, achieving removal efficiencies of over 92% for chemical oxygen demand (COD) and 70% for total nitrogen (TN). The main characteristics of the inoculum were as follows: total suspended solids (TSS) 6.5 ± 0.2 g/L, volatile suspended solids (VSS) 4.8 ± 0.3 g/L, and sludge volume index (SVI) 64.2 ± 0.3 mL/g.

The sequencing batch reactor (SBR) used in the experiments was a cylindrical reactor with a resin structure, having an effective volume of 10.0 L, and a bottom radius and height of 8.0 and 50 cm, respectively. The SBR was equipped with a mechanical stirrer, with a stirring speed of 200–300 rpm during the aeration phase to ensure thorough mixing of the wastewater.

The experimental water was obtained from the pharmaceutical wastewater generated by a TE production plant, and its water quality characteristics are listed in Table 1. The pharmaceutical wastewater used in the experiment was treated with adsorption before the experiment, so the inherent concentration of TE in the wastewater can be ignored. Additionally, to enhance microbial metabolic activity, a trace element stock solution (2.0 mL/L) was added to the pharmaceutical wastewater. The main composition of the trace element stock solution can be found in the documentation (Zhao et al. 2021; Yuan et al. 2023; Zhang et al. 2023).

Table 1

Main characteristics of experimental water

Index .	Unit .	Value .
pH	/	6.7–7.1
COD	mg/L	342–482
	mg/L	54–62
TN	mg/L	67–82
SOP	mg/L	8.2–13.5

Index .	Unit .	Value .
pH	/	6.7–7.1
COD	mg/L	342–482
	mg/L	54–62
TN	mg/L	67–82
SOP	mg/L	8.2–13.5

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The experiment was conducted in five sets of identical reactors, each comprising three similar reactors. Initially, 6.0 L of pharmaceutical wastewater and 4.0 L of inoculum sludge were pumped into each reactor of the five sets. Subsequently, different concentrations of TE were added to the wastewater to achieve the controlled concentrations of 0, 0.1, 2.0, 4.0, and 6.0 mg/L, with the selected concentrations slightly modified based on previous literature (Hu et al. 2022). Finally, these reactors were transferred to an air-conditioned room with a temperature maintained between 25 and 30 °C for the reaction. Throughout the reaction period, water quality characteristics of the effluent were regularly measured to assess the impact of TE on the operational efficiency of AGS.

COD, TSS, and VSS were determined according to national standard methods (APHA 1998). was measured using the Nessler's reagent method. TN was analyzed using the ultraviolet spectrophotometric method. Soluble phosphate (SOP) was determined using the molybdenum blue spectrophotometric method. The extraction of extracellular polymeric substances (EPS) from granules was performed using cation exchange resin technology. The protein (PN) and polysaccharide (PS) within EPS were quantified using the Folin phenol method and the anthrone colorimetric method, respectively, with bovine serum protein and glucose as standard substrates. Polyhydroxyalkanoates (PHA) were analyzed using gas chromatography, and the specific measurement methods can be found in the literature (Tran et al. 2023). Glycogen was determined using the anthrone colorimetric method, with glucose as the standard substrate. The determination of key enzyme activity related to nutrient removal during AGS operation can be found in the literature (Zheng et al. 2023).

In contrast to the impact on -N, low-dose TE significantly reduces SOP removal. In the 0.1 mg/L group, SOP removal efficiency has already decreased to 84.5–89.6%, much lower than the blank group. Further elevating TE exposure concentration to 6.0 mg/L results in a further decline in SOP removal efficiency to 71.6–75.8%. The experimental results confirm that even at low concentrations, such as 0.1 mg/L, TE significantly reduces SOP removal. The notable inhibition of AGS biological phosphorus removal by TE is attributed to the absence of an anaerobic zone specifically designated for phosphorus-accumulating microorganisms, which results in the suppression of polyphosphate microbial metabolism (Dall'Agnol et al. 2020). Additionally, the removal efficiency of SOP in the process of treating pharmaceutical wastewater in this study is lower than that in traditional urban wastewater. This difference is related to the presence of other pollutants in pharmaceutical wastewater. Table 2 further compares the effects of TE on the operational efficiency of different biological wastewater treatment processes. The presence of TE has been identified to impart a notable negative influence on the biological processes within wastewater treatment systems. Consequently, the management of wastewater streams containing TC warrants careful consideration and strategic optimization in subsequent treatment.

The impact of TE on the settling performance of AGS is depicted in Figure 4. It is observed that the SVI in both the blank group and low-dose TE group initially stabilizes and then decreases. During the initial phase, microorganisms within the AGS gradually adapt to the influent conditions, leading to an accelerated particle formation process in the later stages, resulting in improved sludge settling (Zhao et al. 2019; Nancharaiah & Sarvajith 2023; Qin et al. 2023). However, in groups with high concentrations of TE, the change in SVI is closely associated with TE concentration. In the 2.0 and 4.0 mg/L TE groups, the final SVI remains in the range of 73.6–82.3 and 79.2–85.6 mL/g, slightly lower than the initial values. However, in the 6.0 mg/L TE group, the final SVI ranges from 88.5 to 94.8 mL/g, significantly higher than the blank group and low-concentration TE groups, indicating a deterioration in sludge settling in the high-concentration TE group. High concentrations of TE exert toxic effects on microorganisms and disrupt sludge structure, leading to the disintegration of granular sludge (Yang et al. 2021). However, in this study, no sludge bulking phenomenon was found in the high-concentration TE group.

Glycogen is also an important intracellular polymer in AGS, and its metabolism is closely related to glycogen-accumulating organisms (GAOs). GAOs are recognized as a group of microorganisms with metabolic characteristics similar to polyphosphate-accumulating organisms (PAOs) but with no contribution to phosphate removal (Zhou et al. 2008; Fu et al. 2024). Similarly, when TE exposure concentration is low, glycogen content remains unaffected. However, when TE concentration increases to 2.0 mg/L, glycogen content significantly increases, and the higher the TE concentration, the higher the glycogen content. Particularly at 6.0 mg/L, glycogen content increases to 6.51–6.62 mmol/g, approximately 0.21–0.38 mmol/g, higher than the blank group. The vigorous metabolism of glycogen implies a high abundance of GAOs. Although GAOs do not contribute to nutrient removal, they can compete with denitrifying bacteria and polyphosphate-accumulating bacteria for limited carbon sources, thereby reducing phosphate removal. The promotion of GAO metabolism within the high-concentration TE group consumes a large amount of carbon source, resulting in unsatisfactory nutrient removal.

Key enzymes play an important role in nutrient removal, and this study also investigated the activity of key enzymes under TE exposure. The activity of exopolyphosphatase (PPX) and polyphosphate kinase (PPK) is related to the removal of phosphate, while the activity of nitric oxide reductase (NOR) and nitrate reductase (NIR) is related to biological denitrification, and the activity of dehydrogenase is related to microbial metabolism. As shown in Figure 7, the effect of TE on the key enzymes mentioned above is closely related to its exposure dose. When the concentration of TE is below 0.1 mg/L, the activity of the key enzymes mentioned above is not significantly different from the blank group. However, when the TE concentration exceeds 2.0 mg/L, the activity of the key enzymes mentioned above is inhibited, and there is a change pattern where the higher the TE concentration, the more significant the inhibition of the key enzyme activity. For example, in the 4.0 and 6.0 mg/L groups, the relative activities of PPX were 90.8 and 82.6%, and the relative activities of NOR were 91.5 and 85.6%, respectively. Similar experimental results were also found in the detection of other key enzymes. The inhibition of key enzyme activities related to nutrient removal by high-concentration TE is also one of the reasons for the inhibition of nutrient removal.

The effect of TE on the treatment of pharmaceutical wastewater by AGS was systematically studied. The results showed that the effect of TE on the operational efficiency of AGS was dose-dependent, and less than 0.1 mg/L TE had almost no effect on the operation of AGS, while more than 2.0 mg/L TE reduced the removal of pollutants and nutrients in the process of AGS treatment of pharmaceutical wastewater, and reduced the concentration of sludge and the proportion of organic matter in AGS. A high concentration of TE increased the content of EPS but decreased the content of PHA, which limited denitrification and biological phosphorus removal. Enzyme analysis revealed that a high concentration of TE decreased the activities of key enzymes related to nutrient removal. Furthermore, it is essential to delve into the specific degradation mechanisms of TE within the AGS system. Concurrently, the influence exerted by the degradation products of TE on the performance of AGS across different temperature conditions should be closely monitored.

This research was funded by the Major Project of Science and Technology Research Program of Henan Education Commission of China. (KJZD-K201958506).

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.