Response of wastewater biofilm to CuO nanoparticle exposure in terms of extracellular polymeric substances and microbial community structure
Graphical abstract
Introduction
With the development of the production and application of metallic/metal oxide nanoparticles (NPs) in diverse commercial products (e.g., semiconductors, cosmetics, textiles, and pigments) (Chang et al., 2012, Zhou et al., 2006), increasing amounts of NPs, for example, CuO NPs, are released to sewer systems and reach wastewater treatment plants (WWTPs) (Gottschalk et al., 2009, Gottschalk et al., 2013, Keller et al., 2013, Sun et al., 2014). NPs have already been detected in wastewater streams, for example, TiO2 up to 10 μg/L from WWTPs in Europe (Westerhoff et al., 2011). Recently, many studies have been conducted to investigate the influence of released NPs on the performance of WWTPs due to the potential ecotoxicological impacts of NPs on microorganisms in biological systems (Garner and Keller, 2014). Typically, activated sludge (including aerobic and anaerobic biological treatments) is the main process of sewage treatment in WWTPs. The effects of NPs on activated sludge processes have been extensively studied and mainly focused on the community structure (Yang et al., 2014), wastewater biological nutrient removal (Chen et al., 2012, Garner and Keller, 2014) and waste-activated sludge anaerobic digestion (Brar et al., 2010). The antibacterial mechanisms of metallic/metal oxide NPs include cell membrane permeability disruption, release of metal ions, and accumulation of intracellular radicals (Sharifi et al., 2012).
In addition to activated sludge, biofilms (such as in rotating biological contactors (RBC)), as an alternative biotechnology, have attracted intensive attention due to their excellent physical characteristics (Hassard et al., 2015). In comparison to activated sludge, biofilms have a denser and stronger microbial aggregate structure, a higher biomass concentration, better settling capacity and the ability to withstand shock loads (Hassard et al., 2015). In addition, systems based on biofilms are less sensitive to fluctuation than activated sludge in the presence of inhibitory or toxic compounds because biofilm architecture causes diffusion gradients that protect sensitive bacteria (Flemming and Wingender, 2010). Biofilms are aggregated populations of microorganisms embedded in a matrix of extracellular polymeric substance (EPS) mainly made up of proteins and polysaccharides (Sheng et al., 2010). EPS is usually considered the first barrier for preventing toxic substances from diffusing into a wastewater biofilm, consequently reducing the toxic effects (Flemming and Wingender, 2010, Sheng and Liu, 2011). According to the literature, studies have been conducted to investigate the potential impacts of metallic/metal oxide NPs on biofilm or granular sludge (Gu et al., 2014, Herrling et al., 2016, Walden and Zhang, 2016). The interaction and accumulation of NPs by biofilms involved physical, chemical, and biological processes, such as transport of NPs to biofilms, biosorption of NPs by biofilms and chemical transformations of NPs in biofilms (Ikuma et al., 2015). Based on our previous studies (Hou et al., 2014, Miao et al., 2016), metallic oxide nanoparticles (ZnO NPs and CuO NPs) could cause a decrease in the microbial activity of bacteria in the aerobic biofilm obtained from an RBC system. However, pollutant-induced changes in the content and composition of EPS in biofilms have not been extensively researched. The integrity of biofilms is mediated by species-specific EPS, the major component of biofilms (Sheng et al., 2010). The secretion of EPS is a response to a stress situation and seems to be extremely dependent on the existing environmental conditions (Stewart and Franklin, 2008). Therefore, characterizing different EPS fractions loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) (Hou et al., 2015) in the presence of CuO NPs is crucial to understanding the toxic response mechanism of wastewater biofilms to CuO NPs in a wastewater environment.
In addition, the microbial community structure, diversity and fluctuation will ultimately affect the performance of WWTPs. The potential impacts of NPs on the microbial community in activated sludge have been investigated using clone library analysis, terminal restriction fragment length polymorphism (T-RFLP) (Yuan et al., 2015) and denaturing gradient gel electrophoresis (DGGE) targeting 16S ribosomal RNA (rRNA) genes (Zheng et al., 2011), providing limited information due to the small number of sequences analyzed. Considering that wastewater treatment is a microbial-ecologically driven process, insight into the impacts of NPs on the broader microbial community is needed. Recently, high-throughput sequencing (HTS) techniques have demonstrated considerable advantages for the analysis of microbial communities in association with their unprecedented sequencing depth (Caporaso et al., 2012) and have been used to detect the impacts of NPs on the microbial community structure in activated sludge (Yang et al., 2014) and granular sludge (Pan et al., 2015). Thus, this method should be conducted to characterize the response of the overall microbial communities in wastewater biofilms to CuO NPs under wastewater treatment conditions.
Based on the discussion above, the biochemical effects of NPs on biofilms should be addressed further to better understand the interaction between NPs and biofilms. The objective of this study is to investigate the response of aerobic wastewater biofilm to CuO NP exposure in terms of EPS and microbial community. Intact wastewater biofilms from a lab-scale RBC system were treated with CuO NPs, and the integrity of cells and the oxidation stress induced by CuO NPs were determined by lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) production. In response, the physicochemical characteristics of different EPS fractions (LB-EPS, TB-EPS) including composition, three-dimensional excitation and emission matrix (3D-EEM), and functional groups were determined. The changes of microbes in the biofilms were evaluated by high-throughput microbial community analysis.
Section snippets
Rotating biological contactor system (RBC)
A laboratory scale RBC system (Fig. S1) was housed in a plexiglass semi-cylinder with a liquid volume of 4.8 L. A horizontal steel shaft supported nine disks (each 22 cm in diameter, 2 cm spacing between disks) made of polypropylene, providing a surface area of [11 cm × 11 cm × π × 2] × 9 disks = 6838 cm2. A total of 45% of the disks was submerged in the liquid with disc rotation speed of 3 rpm throughout the experimental period. The RBCs were inoculated with activated sludge obtained from Jiangning WWTP
CuO NP adsorption by biofilms and the toxic effects
As determined by DLS measurements (Fig. 1), obvious aggregations of CuO NPs (10 and 50 mg/L) in the synthetic wastewater were observed within 30 min in a dose-dependent manner, with < Dh > of 486 ± 74 nm and 710 ± 95 nm, respectively. SEM images confirmed the presence of aggregates of CuO NPs in the wastewater. Similar trends of ZnO NPs and TiO2 NPs were also observed in wastewater by Zhou et al. (2015). In this study, the zeta potential of the CuO NPs was determined to be − 22.3 ± 2.1 mV in the synthetic
Conclusions
In response to CuO NP exposure, the EPS content in biofilm was significantly increased, and EPS components were changed. At 50 mg/L, CuO NP treatment significantly altered the composition and structure of wastewater biofilm microbial communities. Comamonas, Zoogloea, and Flavobacterium were significantly shifted, which might be responsible for the change of the content and components of EPS. Further studies are needed to understand the mechanisms by which NPs specifically affect microbial
Acknowledgments
We are grateful for the grants from projects supported by National Science Funds for Creative Research Groups of China (No. 51421006), National Natural Science Foundation of China (No. 51479047, 51109058, 51209069), National Science Funds for Distinguished Young Scholars (No. 51225901), Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13061), the Fundamental Research Funds for the Central Universities (No.2015B22014, No.2015B05714) and PAPD.
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