The extensive usage of antibiotics in healthcare and animal husbandry has accelerated the proliferation of antibiotic resistance genes (ARGs) worldwide. ARGs can be classified into the intracellular form existing inside antibiotic resistant bacteria (ARB) and the extracellular form occurring outside bacteria, which are generated from the secretion of living cells or released from dead cells. However, researches about intracellular ARGs have been developed extensively considering ARB, while less attention has been paid to extracellular ARGs (eARGs). Concerning the persistence and unique horizontal gene transfer via transformation, eARGs would seriously increase the environmental risk of antibiotic resistance. Wastewater treatment plants (WWTPs) are regarded as the source and sink of ARGs. Therefore, this PhD study aimed to investigate the occurrence of eARGs in extracellular polymeric substances (EPS-associated ARGs) in WWTPs, to explore the influence of various natural and anthropogenic factors (i.e., EPS, chlorination and temperature) on the transformation of eARGs, and to study the fate of eARGs based on mineral-mediated adsorption and photodegradation in aqueous environment. It provided comprehensive understanding of eARGs, which was valuable for the risk assessment and effective control of eARGs in the environment. Main results of this study are as follows:

1. An efficient extraction method for EPS-associated ARGs was established and the occurrence of EPS-associated ARGs in WWTPs was clarified. EPS exist outside of microbial cells and in the interior of microbial aggregates. The eARGs could be adsorbed by EPS or escape into the supernatant. The former was called EPS-associated ARGs, while the latter was called cell-free ARGs. The occurrence of cell-free ARGs was widely reported, whereas EPS-associated ARGs were ignored previously, leading to the underestimation of eARGs in WWTPs. Therefore, an efficient extraction method for EPS-associated ARGs was developed with higher extraction efficiency without cell lysis (i.e., heat at 60 ℃ for 60 min). Based on the method, seven commonly detected ARGs (i.e., sulI, sulII, tetA, tetO, tetQ, tetW, and bla_TEM-1) in EPS-associated form were extracted and quantified in two commonly used biological treatment processes for WWTPs (SBR and AAO). Results revealed that EPS-associated ARGs were only about one order of magnitude lower than intracellular ARGs, but they were even four orders of magnitude higher than cell-free ARGs in various WWTPs of different treatment processes, indicating the importance of EPS-associated ARGs to eARGs.

2. Transformation of eARGs affected by EPS was explored. The transformation is achieved by direct uptake of eARGs into competent cells. Since microbial cells are surrounded by EPS, passing through EPS is a necessary step for eARGs to enter the intracellular matrix of competent cells and complete the transformation process. The interaction between eARGs and EPS might alter the transformation ability of eARGs. Results showed that transformation efficiency was inhibited by 60.4−92.0% by EPS compared to the control with the absence of EPS. The inhibited transformation was attributed to less adsorption of eARGs onto cells, enhanced permeable barrier of cells, and binding strength between plasmid DNA (Log K_A=9.7 L/mol) and EPS. Compared with oxidized EPS, the reduced EPS caused stronger inhibition, suggesting that EPS in anaerobic environments could cause stronger inhibition of horizontal gene transfer via transformation of eARGs. The enzymatic hydrolysis experiment showed that EPS could protect eARGs from degradation by deoxyribonuclease I (DNase I), leading to the persistence of ARGs in the environment. Therefore, EPS would be a double-edged sword for the distribution and proliferation of eARGs in the environment.

3. Transformation of eARGs influenced by chlorine disinfection was investigated. As a widely used treatment of wastewater system, chlorine disinfection is available for removal of pathogens. Whether ARGs of ARB were available to be transformed after chlorine disinfection was investigated and the inactivation efficiency of ARB was explored simultaneously. Results revealed that chlorine disinfection with the commonly used dose was insufficient for removal of ARGs. However, the transformation of ARGs was greatly inhibited by chlorination, attributing to the reduced abundance and altered structure of eARGs. In addition, a complete inactivation of culturable ARB was observed, and no reactivation was found in wastewater system. These results verified the effectiveness of chlorination in antibiotic resistance control.

4. Transformation of eARGs affected by temperature was studied. Thermal disinfection is also a widely used disinfection method. High temperature can effectively inactivate antibiotic resistant bacteria, but it is not clear whether temperature has an impact on the transformation of eARGs. After treatment with water bath at various temperatures in the 25−100 ℃ range, eARGs were transformed into competent cells. Effects of heating time on transformation were explored at the same time. Results showed that the transformation efficiency decreased as the temperature increased from 25 ℃ to 50 ℃, enhanced later at 50−70 ℃ and then declined with the further increase in temperature at 70−100 ℃. The highest transformation efficiency was achieved at 70 ℃. Increased heating time led to decreased transformation efficiency. The lower transformation efficiency at 50 ℃ was found to be recoverable after eARGs being reheated at 70 ℃, while that of 100 ℃ was unrecoverable. Further experiments by quantitative polymerase chain reaction, gel electrophoresis and atomic force microscope (AFM) revealed that the alteration of supercoiled structure of eARGs resulted in the variation of transformation efficiency. The larger percentage of supercoiled structure caused the higher transformation efficiency.

5. The degradation behavior and environmental fate of eARGs in actual aqueous environment were explored. The eARGs in the effluent of WWTPs are directly discharged into receiving waters, leading to the spread of antibiotic resistance in aquatic environments (e.g., rivers). However, the influence of mineral particles on the environmental fate of eARGs in natural water is unknown. Iron containing particles, such as hematite nanoparticles, are ubiquitous semiconductors in natural water. They show good photodegradation activity under visible light irradiation. Results showed that 40 mg/L hematite nanoparticles could reduce the abundance of eARGs by 4−5 logs in the river water under visible light. The eARGs were adsorbed onto hematite nanoparticles and photodegraded by hydroxyl radical (HO·) generated on the surface of nanoparticles under visible light. Classic facets of hematite include (001) and (100). Hematite plate nanoparticles with a predominant exposure of (001) facets had higher removal rate of eARGs than hematite rod nanoparticles with a predominant exposure of (100) facets. This could be ascribed to the stronger adsorption and higher HO· generation on the (001) facet compared with that on the (100) facet. The gel electrophoresis and AFM imaging further verified that the more serious damage of eARGs was caused by the (001) facet exposed hematite plate nanoparticles compared with the (100) facet exposed hematite rod nanoparticles. This study showed the self-purification ability of the river to eARGs due to the presence of hematite under visible light.