Long-term effects of copper nanoparticles on granule-based denitrification systems: Performance, microbial communities, functional genes and sludge properties

Highlights

• The presence of 1 mg L⁻¹ Cu NPs was supposed to be safe in granule-based denitrification systems.

• The system lost 51.9% of nitrogen removal capacity under the stress of 5 mg L⁻¹ Cu NPs.

• The presence of 5 mg L⁻¹ Cu NPs reduced the gene abundance of napA, narG and nosZ.

• The introduction of Cu NPs shifted the community composition of denitrifying bacteria.

Abstract

The widespread use of copper nanoparticles (CuNPs) has attracted increasing concern because of their potential effects on biological wastewater treatment. However, their effect on granule-based denitrification systems is unclear. Hence, the effects of CuNPs on denitrifying granules were investigated during long-term operation. The results showed that 51.9% of nitrogen removal capacity was lost after exposure to 5 mg L⁻¹ CuNPs, with the amount of Cu(II) gradually increasing with elevating CuNP levels. Moreover, the relative abundance of denitrifying bacteria (Castellaniella) and denitrifying functional genes (nirK, napA, narG and nosZ) obviously decreased. Meanwhile, the specific denitrification activity, the content of extracellular polymeric substances and dehydrogenase activity decreased by 44.0%, 15.2% and 99.9%, respectively, compared to their values in the initial sludge. Considering the downtrend in the abundance of copper resistance genes, it was deduced that the toxicity of CuNPs was mainly or at least partially due to the release of Cu(II).

Introduction

Rapidly developing nanotechnology is a new scientific field. Currently, engineered nanoparticles (NPs) have wide applications in many fields, such as those involving industrial and consumer products, in which their fate and behavior in the environment, their distribution and their toxicological effects on biological systems could be determined by their unique properties (Ganesh et al., 2010, Mihalache et al., 2017). For example, CuNPs, which possess good thermal and electrical conductivity and bactericidal properties, are widely applied in fuel cells, fungicides, bioactive coatings, cosmetics, skin products, printers and electronics (Chen et al., 2012a, Tang et al., 2018, Wang et al., 2016a). Therefore, CuNPs are inevitably released into the environment, attracting increasing concern related to their potential toxicities in the environment (Chen et al., 2012a, Gonzalez-Estrella et al., 2017). Wastewater treatment plants (WWTPs) have been considered one of the last barriers prior to the environmental release of CuNPs (Musee et al., 2011).

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). The specific anammox activity (SAA) was obviously inhibited after the addition of 5 mg L⁻¹ CuNPs, and CuNPs at 50 mg L⁻¹ significantly damaged cell membranes after exposure for several hours (Zhang et al., 2017a). Furthermore, the performance of an anammox reactor nearly collapsed under the stress from 5.0 mg L⁻¹ CuNPs (Zhang et al., 2017b). Moreover, hydrolysis and acidification were seriously inhibited by the sudden addition of 17.4 mg L⁻¹ CuNPs, thereby decreasing the production of volatile fatty acids (VFAs) (Chen et al., 2014). A previous study reported that CuNPs inhibited denitrification in an anaerobic sludge system (Gonzalez-Estrella et al., 2015). Another study reported that the total nitrogen (TN) removal was obviously enhanced by the addition of CuNPs in an activated sludge process, indicating that CuNPs had different impacts on various bacterial functions (Chen et al., 2012a).

Some studies illuminate that the released toxic metal ions might play an essential role in inducing toxicity in microbial systems (Wang et al., 2016b, Yang et al., 2013, Zhang et al., 2017c). In addition, a previous study reported that NPs could directly disrupt the microbial membrane to stimulate the increase of lactate dehydrogenase (LDH) levels (Zhang et al., 2017c) and produce intracellular reactive oxygen species (ROS) (Das et al., 2012) to cause protein denaturation or DNA damage (Aruoja et al., 2015). The benefits of NP application need to be weighed against their potential adverse effects on the functional bacteria of ecological systems. Some studies have reported that NPs can be removed via settling, aggregation, precipitation, biosorption, and other biomass-mediated processes (Ganesh et al., 2010).

Biological denitrification, one of the biochemical processes to remove nitrogen in WWTPs, could conduct a sequential reduction from NO₃^–-N to N₂ using denitrifying bacteria under anoxic conditions (Isaka et al., 2012, Lu et al., 2014, Nancharaiah and Venugopalan, 2011). Denitrification, which exhibits low cost and high removal efficiency, was regarded as the most promising technology to convert nitrate to nitrogen gas (Isaka et al., 2012). Granular sludge that shows excellent settling and high biomass retention is composed of self-immobilized microbial aggregates (Wang et al., 2016c), which is responsible for resistance under environmental pressure (Nancharaiah and Venugopalan, 2011). In addition, the use of granular sludge is conducive to improving sludge concentration and enhancing organic loadings. Therefore, granule-based denitrification systems should be of great concern.

Over the long term, the potential risk of CuNPs to granule-based denitrification systems is still unclear. Hence, the performance of granule-based denitrification reactors was determined under the stress of various concentrations of CuNPs and during a subsequent recovery phase. Then, qualitative and quantitative analysis was used to evaluate the variety of extracellular polymeric substances (EPSs). Furthermore, the specific denitrification activity (SDA), abundance of functional genes (nirK, napA, narG and nosZ), dehydrogenase activity (DHA) and Cu(II) content were also evaluated. Finally, shifts in the microbial community were revealed in response to elevated CuNP concentrations in a granule-based denitrification system. The outcomes of this study will provide further reference and meaningful insight into the impact of engineered NPs on biological wastewater treatment.