Effect of anti-inflammatory drug micropollutants in hybrid biofilm nitrification systems on microbial community population

Abstract

Non-steroidal Anti-inflammatory Drugs (NSAIDs) are one of the most frequently used pharmaceuticals, as they are available over the counter worldwide. They are known among top 10 persistent pollutants with a significant environmental concern because of their incomplete degradation in wastewater treatment plants (WWTPs) and long-term toxic effects of NSAIDs even at low concentrations on human health and ecosystem. Hybrid treatment technologies such as moving bed biofilm reactors (MBBRs) can be a promising alternative for conventional activated sludge system. Hybrid MBBR systems which, contain suspended and attached biomass, are capable of tolerating environmental fluctuations and have been reported to sustain effective removal activity in fluctuating organic loading conditions. There is no clear trend in literature about impact of organic loading rate (OLR) on removal efficiency of NSAIDs, and also their effect on microbial profile requires further investigation. The objective of this study is to investigate the removal efficiency of targeted NSAIDs including diclofenac, ibuprofen, ketoprofen, naproxen, indomethacin, and mefenamic acid in hybrid systems, operated under low OLR condition, and to assess their impact on the structure and diversity of microbial communities. For this purpose, laboratory scale sequencing batch reactors (SBRs) were operated as hybrid system by adding biofilm carriers to the reactors to achieve both suspended and attached biomass. The filling ratio for both reactors were 30%. Control and micropollutant (MP) reactors were operated under same operational conditions. The activated sludge was obtained from the aeration tank of an advanced biological WWTP in Istanbul. The chemical oxygen demand (COD) was 150 mg/L COD and the initial ammonium concentration was 60 mg/L. Sludge age was adjusted as 10 days. The micropollutant reactor were fed with six selected NSAIDs including diclofenac, ibuprofen, ketoprofen, naproxen, indomethacin, and mefenamic acid at environmentally relevant concentrations ranging from 1-10 µg/L. In order to track the performance of the reactor, COD, Ammonium Nitrogen (NH4-N), Soluble Chemical Oxygen Demand (sCOD), Nitrite (NO2−), Nitrite (NO3−), Total Suspended Solid (TSS), Volatile Suspended Solid (VSS) analysis were conducted and they quantified. Sample collection was done after reaching steady state condition in the hybrid system to measure targeted NSAIDs concentrations. Microbial analyses were started by DNA extraction from biomass. The V3–V4 region (widely used variable region for species identification) of the 16S ribosomal RNA gene were amplified by PCR. Then Next-generation sequencing (NGS) applied and finally the obtained sequencing went through bioinformatic analysis. The results of effluent concentrations obtained to assess the hybrid reactor performance, showed a carbon removal of approximately 81% in hybrid control and 87% in hybrid MP reactor. The ammonium removal (NH4+) was measured as approximately 77% in hybrid control reactor and approximately 90% in presence of selected NSAIDs. Significant removal efficiencies of ibuprofen (100%), indomethacin (100%), naproxen (99%), mefenamic acid (98%), ketoprofen (96%) and diclofenac (69%) were achieved. The results showed that selected NSAIDs did not inhibit nitrification process and carbon removal, and also low OLR did not adversely affect the removal of selected NSAIDs. The dominant phyla observed in the hybrid MP reactor were Bacteroidota (42.40%), Proteobacteria (35.13%) for suspended biomass and Proteobacteria (29.28%) and Bacteroidota (28.31%) for attached biomass. At the genus level, in the hybrid MP reactor, the genera including uncultured genus belonging to the Chitinophagaceae family (10.08%), Ferruginibacter (7.82%), Comamonas (6.77%) dominated the suspended biomass, while Fimbriimonadaceae (8.73%) and Parafilimonas (6.46%) were seen as dominant genera in attached biomass. Shannon index shows the diversity of microbial community. An increase in shannon index was observed in the attached biomass after addition of selected NSAIDs to the hybrid reactor, while diversity decreased in the suspended biomass of hybrid MP reactor, suggesting higher diversity in attached biomass under NSAIDs stress. Moreover, operational taxonomic units (OTUs) increased in the attached biomass after NSAIDs addition but a decrease was observed in the suspended biomass of hybrid MP reactor, implying that attached biomass had richer microbial profile than suspended biomass after NSAIDs addition. The case for the Pielou's evenness was opposite. This index decreased for both reactors after exposure to the selected NSAIDs. NSAID stress might contribute to the decrease of evenness in microbial community. Higher biodiversity and richness in attached biomass comparing to suspended biomass, might be attributed to an increase in microbial complexity which may enhance the coexistence of various functional bacterial in the biofilm as reported in a comparable study. The difference in biodiversity of hybrid control and hybrid MP reactors of suspended and attached biomass, might be due to the NSAIDs stress as seen in various studies. Uncultured genus belonging to Saprospiraceae family and Saccharimonadale decreased significantly in both suspended and attached biomass of hybrid control reactor comparing to the inoculum sludge. On the other hand, a significant increase was detected for the suspended biomass of hybrid control reactor comparing to inoculum sludge in some genera such as Niabella and Paracoccus. For the attached biomass, significant increase was seen in Paracoccus, Ottowia and Fimbriiglobus. These observed significant changes were probably in response to the lab-scale experimental conditions, as well as selected operational conditions for this study such as low OLR. uncultured genus belonging to Chitinophagaceae family, Ferruginibacter (belongs to Chitinophagaceae family) and Comamonas (belongs to Comamonadacea family) that dominated suspended biomass of hybrid MP reactor, and also genus Fimbri- imonadaceae, Parafilimonas (belongs to Chitinophagaceae family) and Rickettsia which dominated the attached biomass of hybrid MP reactor, might contribute to the nitrification in hybrid system rather than common nitrifiers. Families Chitinophagaceae and Comamonadacea in similar studies, have been identified to be effective in nitrogen removal within MBBR. Moreover, Bacteroidota and Proteobacteria have been reported to play an important role in nitrogen and MP removal. Further research may be conduct by detailed qPCR and NGS with longer target gene region or alternative molecular techniques to more effectively investigate the relationship between microbial diversity, NSAID removal, and also its correlation with nitrification.