Resistance of anammox granular sludge to copper nanoparticles and oxytetracycline and restoration of performance

Highlights

• Better resistance and resilience were observed in unacclimated anammox granular sludge.

• Anammox system was significantly suppressed by the addition of high level Cu NPs and OTC.

• Dominant bacteria and functional genes coincide with each other.

• The presence of ARGs improved the resistance of anammox sludge to Cu NPs and OTC.

Abstract

Nanoparticles and antibiotics, the two most frequently detected emerging pollutants from different wastewater sources, are eventually discharged into wastewater treatment plants. In this study, the widely used materials CuNPs and oxytetracycline (OTC) were selected as target pollutants to investigate their joint effects on anaerobic ammonium oxidation (anammox). The results indicated that the environmental concentration slightly inhibited the performance of the reactors, while the performance rapidly deteriorated within a week under high-level combined shocks (5.0 mg L⁻¹ CuNPs and 2.0 mg L⁻¹ OTC). After the second shock (2.5 mg L⁻¹ CuNPs and 2.0 mg L⁻¹ OTC), the resistance of anammox bacteria was enhanced, with an elevated relative abundance of Candidatus Kuenenia and absolute abundance of hzsA, nirS, and hdh. Moreover, the extracellular polymeric substance (EPS) content and specific anammox activity (SAA) showed corresponding changes. Improved sludge resistance was observed with increasing CuNP and OTC doses, which accelerated the recovery of performance.

Introduction

Nitrogen, one of the most essential nutrients, promote the growth of organisms, while nitrogen pollution has become a worldwide topic in wastewater treatment (Liu et al., 2019). In nitrogen-rich wastewater treatment, it is extremely important to eliminate nitrogen pollution with high removal efficiency (Zhang et al., 2018a). The anaerobic ammonium oxidation (anammox) is regarded as the preferred nitrogen removal process in the absence of oxygen and organic carbon and has a high nitrogen removal rate (Gao et al., 2014, Kartal et al., 2010); directly ammonium and nitrite can be directly transformed to nitrogen gas (N₂) in an anoxic environment (Kang et al., 2019, Zhang et al., 2019a). However, anammox bacteria are very sensitive and vulnerable to various inhibitors in wastewater, such as heavy metals, antibiotics and nanoparticles (NPs) (Zhang et al., 2015a, Zhang et al., 2018c, Zhang et al., 2018b).

Recently, antibiotics have been widely used as health safeguards in both humans and animals, while they have also been extensively detected in wastewater (Meng et al., 2019, Shi et al., 2017, Sun et al., 2019, Yin et al., 2019). Oxytetracycline (OTC), belonging to the broad-spectrum antibiotic tetracycline compounds, was chosen to investigate the antibiotic selection pressure because it is the most commonly used compound in humans and animals (Liu et al., 2016, Yan et al., 2018). The principal sources of antibiotics in the environment include (i) sewage (treated/untreated), (ii) hospitals, (iii) livestock farms/operations (cattle, swine, and poultry), (iv) aquaculture farms, and (v) pharmaceutical industries. Various groups of antibiotics have been frequently detected in the effluents of municipal wastewater treatment plants, surface water, groundwater, and drinking water (Boy-Roura et al., 2018, Cheng et al., 2015, Giebułtowicz et al., 2018, Hu et al., 2018a, Mirzaei et al., 2018). Extremely high concentrations of 50 mg L⁻¹ OTC was observed in the effluent from an OTC manufacturer (Li et al., 2008), while the OTC concentration was estimated at 2.1 mg L⁻¹ in livestock (swine) farms. Therefore, the anammox process as well as functional bacteria would be largely impacted by varying concentrations of antibiotics in wastewater treatment plants (WWTPs). When the OTC concentration reached 2 mg L⁻¹, a decreased anammox bacterial activity of 80% was observed due to the reduction of heme c content (Shi et al., 2017). Another study found that 5 ± 3.5 mg L⁻¹ OTC could completely suppress anammox activity (Noophan et al., 2012). The key functional genes in the anammox system, such as nirS, hzsA, and hdh, also showed conspicuous variations in response to the addition of OTC (Zhang et al., 2019b). In addition, OTC addition retarded the growth rates of anammox bacteria (Zhang et al., 2016). On the other hand, metal nanoparticles (MNPs) have been widely applied in many fields due to their unique properties (Wang and Chen, 2016). Copper nanoparticles (CuNPs) are widely utilized in smelting, rubbers, fuel cells and power stations (Wang et al., 2015, Zhang et al., 2018b), which inevitably leads to their release and causes a potential risk to the environment. Recent studies found that engineered nanomaterials that result in estimated CuNP levels in wastewater from μg L⁻¹ to mg L⁻¹ might cause negative effects on the microorganisms used in wastewater treatment systems (Ganesh et al., 2010, Musee et al., 2011). Wastewater treatment systems, such as denitrification systems (Cheng et al., 2019) and anammox systems (Li et al., 2019, Zhang et al., 2017a), have been significantly impacted due to the introduction of CuNPs. In addition, the released copper ions from CuNPs have been proven to be a vital reason for the toxicity of NPs (Chen et al., 2012, Zhang et al., 2017a). Both antibiotics and NPs ultimately find their way to wastewater; two or more of these substances have been observed in nitrogen-rich wastewater (Michael et al., 2013). To the best of our knowledge, there are no available data evaluating the combined effects of CuNPs and OTC on the anammox process. The coexistence of CuNPs and OTC might lead to accumulation and result in compound pollution in water because of the extensive application of these materials. It is necessary to adequately understand the joint effects of CuNPs and OTC on anammox systems.

Therefore, this study aims to adequately understand the combined effects of CuNPs and OTC on the anammox process. First, the anammox reactor performance under the stress of CuNPs and OTC was investigated. Second, the dynamic characteristics of extracellular microbial products from three reactors in response to variations in CuNPs and OTC were investigated. Finally, the responses of the anammox microbial communities, functional genes and antibiotic resistance genes (tetC, tetG, tetM, and tetX) were analyzed. The outcomes of this study will provide further reference to and meaningful insight into the combined impact of engineered NPs and antibiotics on biological wastewater treatment.