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Fate of Antibiotic Resistance Genes in Activated Sludge Processes and Anaerobic Digestion at Six Wastewater Treatment Plants in California


Antibiotic resistance has been a topic of increasing concern for several decades. Wastewater treatment plants are one of the potential sources of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria. Activated sludge processes (ASP) are the most widely used biological secondary treatment processes for wastewater while anaerobic digestion (AD) processes are used for treating biosolids, both of which play an import role in degrading and removing most of the organic wastes. In our study, we investigated six wastewater treatment plants located near Los Angeles operated at long and short solids retention times (SRTs) for the activated sludge processes and under thermophilic and mesophilic conditions for the anaerobic digestion processes. The prevalence and dynamics of five selected ARGs (sul1, sul2, tetA, tetW, and ermB), class 1 integron (intI1), and the total 16S rRNA were selected and evaluated by means of quantitative polymerase chain reaction (qPCR) in influent and effluent samples in six activated sludge processes and in digester feed and digested sludge in four anaerobic digestion processes.

Among the six full-scale activated sludge processes, the average removal efficiency of all target ARGs was 99% (1.98 log removals) at the three long SRT ASPs, which was higher than the 95% (1.29 log removals) observed at three short SRT ASPs. The relative gene abundance per gram volatile suspended solids (VSS) was introduced to study the relationship between organic materials and ARG levels and reinforced the hypothesis that both long and short SRT ASPs can remove ARGs and long SRT ASP also showed higher removal efficiencies. In addition, significant (p < 0.05), strong, and positive (r > 0.7) correlations were found between VSS concentrations and the total 16S rRNA. Furthermore, potential horizontal gene transfer (HGT) was indicated at all the six plants’ ASPs in terms of the correlations between intI1 and target ARGs. These correlations were found more frequently at short SRT plants than at long SRT plants. In general, our results concluded that ASPs could remove the absolute gene abundance of selected ARGs but might in some cases increase the relative gene abundance. Also, ASP operating at long SRT achieved higher and less variable removal efficiencies than at short SRT.

Among the four full-scale anaerobic digestion processes, most of the ARGs’ absolute abundance were significantly (p < 0.05) removed up to 1.58 logs. The lowest absolute gene abundance of all target ARGs and 16S rRNA were found at AD3, the only thermophilic anaerobic digestion process. And the relative gene abundance per VSS for wastewater samples were similar to the abundance gene abundance per g ds for sludge samples. There was evidence for HGT at all the four AD processes, especially sul1, sul2, and ermB, which were all found to be significantly and positively correlated with intI1. Therefore, anaerobic digestion processes can remove the target ARGs but potential HGT may have occurred.

Ten heavy metal contents (barium, copper, iron, manganese, lead, rubidium, strontium, titanium, zinc, and zirconium) during three anaerobic digestion processes were also studied and the relationships between these heavy metal contents as well as phosphorus and five ARGs as well as intI1 were evaluated. In the results: Fe was observed with the highest heavy metal concentration while Rb was the lowest heavy metal concentration at all the three AD processes; 14 out of 30 observations were decrease observations with the highest removal efficiency of 61% in AD4 for Mn but 16 out of 30 observations were increase observations with the highest increase percentage of 124% in AD6 for Sr; redundancy analysis showed that Zr and Ba significantly (p < 0.05) explained the changes in ARGs, which accounted for 54.9% of the total variation in the environmental factors.

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