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Fate of antibiotic resistance genes in biological treatment systems: Moving bed biofilm reactor (MBBR) and anaerobic digestion
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2025-08
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Environmental Engineering
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https://doi.org/10.34944/43x7-ss62
Abstract
The widespread occurrence of antibiotic resistance genes (ARGs) in wastewater treatment systems constitutes an escalating risk to environmental and public health, especially because of the possibility of horizontal gene transfer (HGT) and their propagation into natural water bodies and biological treatment systems including biofilm-based technologies and anaerobic digestion (AD) systems. Moving bed biofilm reactors (MBBRs) as biofilm-based technologies are extensively used for biological wastewater treatment; however, the factors affecting the persistence and proliferation of ARGs in these systems are not well studied. On the other hand, anaerobic co-digestion (AcoD) systems have been used broadly in waste management and pollution control facilities. This study examined the fate of ARGs in MBBR and AcoD systems under diverse operational settings. The study of ARGs’ fate in MBBR systems revolves around the effects of various biocarrier types and carbon-to-nitrogen (C/N) ratios, as well as the effects of emerging pollutants, notably per- and polyfluoroalkyl substances (PFAS) and antibiotics on the fate of ARGs in two phases.
The first phase of this study investigated the performance of MBBR system and ARG dynamics in the MBBR system utilizing two conventional biocarriers (K3 and sponge biocarrier (SB)) and two modified biocarriers (Fe-Ca@SB and Ze-AC@SB) at C/N ratios of 7, 10, and 20. All systems attained > 90% Chemical Oxygen Demand (COD) elimination, regardless of biocarrier type or C/N ratio. At elevated C/N ratios, modified biocarriers, especially Ze-AC@SB, demonstrated enhanced ammonium nitrogen removal (up to 69.9%) and facilitated simultaneous nitrification and denitrification (SND), while concurrently restricting the proliferation of critical ARGs such as tetA, blaTEM, ampR, and the integrase intl1, which serves as an indicator of HGT potential. Metagenomic analysis identified Proteobacteria as the predominant phylum in all systems, with alterations in microbial community diversity and composition associated with both biocarrier type and C/N ratio.
The second phase evaluated the effects of PFAS (PFOA and PFOS) at concentrations of 10, 100, and 1000 µg/L and antibiotics (tetracycline and ampicillin) at 100 µg/L on the fate of antibiotic resistance genes, biofilm properties, and microbial community dynamics in a MBBR system. The performance of MBBR, achieving 76-79% COD removal and 94% ammonium-nitrogen removal, was not significantly affected by the presence of PFAS or antibiotics at environmentally relevant concentrations. Both pollutants significantly elevated the abundance of specific ARGs, notably tetA, ermB, and qnrS, ranging from 1.6 to 175 times relative to the control system. Also, exposure to PFAS increased extracellular polymeric substance (EPS) formation and caused increased reactive oxygen species (ROS) levels (ranging from 14-82%), potentially contributing to increased stress-induced HGT. Analysis of microbial communities revealed that denitrifying species, including Dechloromonas and Dokdonella, emerged as predominant taxa resilient to pollutant stress and probably acted as reservoirs for ARGs following HGT.
Collectively, these results offer essential insights into the impact of operational parameters, biocarrier design, and emerging contaminants on fate, dynamics, and dissemination potential of ARGs in MBBR systems. The study emphasizes the significance of choosing suitable biocarriers, refining operational parameters, and comprehending the inadvertent effects of new contaminants to reduce ARG proliferation. This research enhances understanding of ARG management in biological wastewater treatment and facilitates the formulation of methods to mitigate the danger of antibiotic resistance spread into the environment.
The study on the fate of ARGs in AcoD included the effects of food waste (FW) to sewage sludge (SS) ratios on the abundance of selected ARGs (qnrS, tetA, emrB, blaTEM, ampR) in the AD system. Initially, the effects of various FW:SS ratios (from 5% FW to 50% FW content) on the performance of AD system were studied. Results showed that FW content of 10% and higher showed significant increase in biogas and methane yield reaching a maximum production of 738 and 393 mL.g-1 volatile solids (VS) at 50:50 (FW:SS) ratio, respectively. No significant change in final TS and VS was observed after adding FW. Furthermore, using Response Surface Methodology (RSM) the results were analyzed to find the optimum FW:SS ratios for better biogas and methane production. The RSM results showed that 42.5% FW is the optimal FW content for maximum biogas and minimum H2S production with quadratic model found to be the best fit for AD data.
The distribution of select ARGs (qnrS, tetA, emrB, blaTEM, ampR) was tracked in the liquid and solid fraction of the digestate. In order to track the abundance of the ARGs, DNA was extracted from the liquid and solid fraction of the final AD digestate, and the results were analyzed using qPCR. Results illustrated a decrease (83%-99% reduction) in the overall abundance of the ARGs in the solid fraction after AD. Similar trend was observed for the ARGs in the liquid fractions (65%-99% reduction), except for ermB which became 1.74-10.6-fold higher in the final digestate. Also, at 50% FW the abundance of intl1 increased in liquid and solid fraction of digestate indicating increased potential of ARG dissemination via horizontal gene transfer. Empirical data on ARGs’ abundance and their fate during the AD processes would be useful towards controlling the generation of antibiotic resistance in the environment from AD processed wastes.
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