Relating microbiome profiles to removal of non-steroidal anti-inflammatory drugs in sequencing batch reactors along a sludge retention time

Abstract

Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) are medications commonly used for pain and inflammation treatment. The residues generated from their use can contaminate water sources. The primary concern regarding these pollutants is their insufficient removal in wastewater treatment plants, leading to their release into the environment. In recent years, studies investigating the presence of NSAIDs in aquatic environments at critical levels and their long term effects have increased. The environmental impacts of these pollutants are not yet fully understood, raising concerns. In March 2015, the Water Framework Directive initiated by the European Commission listed some NSAIDs on the Watch List under Decision 2015/495/EU. The concept of harnessing the activities of adaptable microorganisms in low-cost and conventional systems, instead of expensive advanced wastewater treatment plants, is important for the treatment of micropollutants. Activated sludge based treatment processes have the potential to partially remove micropollutants; however, there is insufficient knowledge regarding which microorganisms play an active role. Furthermore, there have been limited studies on determining the appropriate sludge retention time for the biological treatment of micropollutants. The microbial communities in activated sludge can vary depending on the different chemicals present in the wastewater, which can adversely affect the system's performance. In this study, the impact of a mixture of six NSAIDs (diclofenac, ibuprofen, ketoprofen, naproxen, indomethacin, and mefenamic acid) on microbial population and composition in activated sludge systems was investigated using advanced molecular biotechnology and bioinformatics tools. This study mainly focused on (i) the impact of sludge age (SRT) on the treatability of the selected mixture of NSAIDs in activated sludge systems, (ii) a comparison of the microbiome analysis of samples collected from an advanced wastewater treatment plants in Istanbul and three lab scale sequential batch reactors operated at sludge retention times (SRTs) of 5, 10, and 20 days, (iii) the impact of reference database selection on the analysis of the activated sludge microbiome, (iv) the analysis of the impact of the selected NSAIDs on the microbial population and composition using bioinformatics tools and software, and (v) the correlation of the changing microbial population and composition with the treatment efficiency of the wastewater and selected NSAIDs. In the laboratory scale study, a total of six parallel sequential batch activated sludge reactors were operated with three different SRT: 5, 10, and 20 days. Among these reactors, three served as control reactors, while the remaining three were designated as micropollutant added reactors. Each SRT had a corresponding pair of control and micropollutant added reactors. The control reactors were operated under normal conditions without the addition of micropollutants, while the micropollutant added reactors were operated with the addition of a specific mixture of selected NSAIDs in predetermined proportions. The operating performance of the reactors was monitored regularly by measuring various parameters including suspended solids (SS), volatile suspended solids (VSS), pH in the reactor and chemical oxygen demans (COD), ammonia (NH4+), nitrite (NO2-) and nitrate (NO3-) in the effluent. The result of these analysis showed that these chemicals did not have a long term inhibitory effect on biological degradation in all six reactors. There was no significant change in COD removal efficiency in reactors where NSAIDs were added. The nitrification efficiencies were found to be better in the micropollutant added reactors with sludge retention times of 5 and 20 days compared to the control reactors. The removal efficiencies of the six NSAIDs were individually determined in all sludge retention time reactors. Ibuprofen exhibited removal efficiencies exceeding 99% in all micropollutant added reactors operated for 5, 10, and 20 days. Ketoprofen showed average removal efficiencies above 75% in reactors operated for 5 and 20 days. Naproxen had removal efficiency above 90%, with the highest removal efficiency observed in the reactor operated for 20 days. Indomethacin exhibited removal efficiencies above 90% in all micropollutant added reactors. Mefenamic acid had the highest removal efficiency observed in the reactor operated for 5 days, but its removal efficiency decreased in reactors with longer sludge retention times. Diclofenac removal efficiency was adversely affected in reactors with longer sludge retention times. This study aimed to understand the impact of micropollutants on the diversity and activity of microbial communities within activated sludge, which play a crucial role in wastewater treatment. Using next generation sequencing technology, the microbial communities involved in the degradation of six NSAIDs were investigated in laboratory scale activated sludge reactors, along with the potential adverse effects of these drugs on the communities. The sequenced data obtained from the Illumina MiSeq platform were analyzed using the QIIME2 software package to assess microbial diversity. A comparison between the NCBI and SILVA databases revealed that the NCBI database provided more taxonomic information at the species level. The addition of NSAIDs was found to affect microbial diversity in the 5 and 10 day sludge retention time systems, reducing species richness at the taxonomic level. However, in the micropollutant added 20 day sludge retention time system, an increase in diversity was observed. The observed operational taxonomic unit (OTU) numbers in the clone libraries for the sludge samples collected from the control and micropollutant added reactors were 203 and 145 for 5 day sludge retention time, 171 and 106 for 10 days sludge retention time, and 100 and 182 for 20 days sludge retention time, respectively. In this study, it was determined that Proteobacteria, Actinobacteria, and Bacteroidetes phyla were dominant in raw sludge, control, and micropollutant added reactors. The presence of NSAIDs at a sludge retention time of 5 days increased the abundance of the Verrucomicrobia phylum, while it decreased at a sludge retention time of 10 days. In the control reactor, Verrucomicrobia was not detected at a sludge retention time of 20 days, but its abundance increased with the addition of NSAIDs. In the micropollutant added reactors, different species became dominant at different sludge retention times. Prosthecobacter and Paracoccus species were dominant at a sludge retention time of 5 days, Chryseobacterium and Niabella species at a sludge retention time of 10 days, and Niabella and Parafilimonas species at a sludge retention time of 20 days. The presence of these species can be associated with their capacity to degrade NSAIDs in the micropollutant added reactors. In contrast, the presence of NSAIDs led to the disappearance of more than 80% of species such as Frigidibacter albus and Prosthecobacter vanneervenii in the reactor with a sludge retention time of 5 days, Thauera terpenica in the reactor with a sludge retention time of 10 days, and Intrasporangium oryzae in the reactor with a sludge retention time of 20 days. These species were unable to adapt to the new conditions created by the addition of NSAIDs. Changes in bacterial abundance in functional groups were also observed in the presence of NSAIDs. Specifically, an increase in the abundance of species involved in the nitrification process was observed in the reactor with a sludge retention time of 20 days with NSAID addition.