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Spread of Antibiotic Resistance in Anaerobic Sludge Digestion

Project Number # 2058

Understanding and Reducing the Spread of Antibiotic Resistance in Anaerobic Sludge Digestion

Background:

Wastewater treatment plants have been recognized as the hotspots of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This project aims to understand and reduce the spread of antibiotic resistance in anaerobic sludge digestion. This project will utilize 16S rRNA sequencing, qPCR and metagenomics to uncover the changes in the occurrence, abundance and diversity of ARB and ARGs in anaerobic sludge digestion under various operating conditions. This will understand the fate of ARGs and ARB in anaerobic digestion, and will identify the optimal operating conditions for reducing the spread of antibiotic resistance in anaerobic digestion. In addition, based on the results of this study, the control and management strategy for antibiotic resistance will also be proposed.

Industry Relevance:

The intensive use of antibiotics results in the continuous release of antibiotics into the environment and subsequently the widespread occurrence of antibiotic resistance in both natural and engineered ecosystems. Antibiotic compounds, antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are widely found in soil, surface water, groundwater, and even deep ocean sediments. Many ARGs are also found to be transcribed in natural environments. The widespread occurrence and control of ARB and ARGs is a major public health issue and an emerging challenge to deal with worldwide.

Wastewater treatment plants (WWTPs) have been recognized as the hotspots of ARGs and ARB. In the WWTPs, large amounts of wastewater sludge are produced during biological wastewater treatment. Anaerobic digestion is the commonly used methods to treat wastewater sludge in the WWTPs. Unfortunately, little information is available on the fate of ARGs and ARB in anaerobic digestion in Australian WWTPs. Therefore, it is quite important to understand the fate of ARGs and ARB in anaerobic sludge digestion under various operating conditions to identify the optimal operating conditions for reducing the spread of ARGs and ARB in Australia.

Objectives:

This project aims to understand and reduce the spread of antibiotic resistance in anaerobic sludge digestion. In addition, based on the results of this study, the control and management strategy for antibiotic resistance will also be proposed.

Methodology:

Task 1: Changes in the occurrence, abundance and diversity of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in anaerobic sludge digestion (Months 1-24)

This task will reveal the changes in the occurrence, abundance and diversity of ARGs and ARB in anaerobic sludge digestion.

The sludge samples before and after anaerobic digestion will be collected from the water authority which sponsors this project. Afterwards, the occurrence, abundance and diversity of ARGs and ARB will be analysed using the methods described below.

To identify the optimal operating conditions for reducing the spread of antibiotic resistance in anaerobic digestion, the waste activated sludge and anaerobically digested sludge (i.e. inoculum sludge) will be collected from the water authority which sponsors this project. Four lab-scale semi-continuous aerobic digesters (1 L each) will be set up and inoculated with anaerobically digested sludge. Afterwards, the waste activated sludge will be added to the anaerobic digesters on a daily basis. The effect of sludge retention time and pH on the fate of ARG/ARB will be evaluated. The sludge samples before and after anaerobic digestion will be collected.

DNA from the above sludge samples will be extracted for 16S rRNA sequencing to uncover the profiles of ARB, and high-throughput qPCR will be applied to determine and quantify the occurrence of typical ARGs for these sludge samples. These ARGs will include tetracycline resistance genes (tetA, tetG, tetM, tetX, tetQ and tetW), erythromycin resistance genes (ermB and ermF) and sulfonamide resistance genes (sulI and sulII).  They were selected according to types of antibiotics and main resistance mechanisms. In addition, some selected mobile genetic elements (MGEs) represent the potential of the horizontal gene transfer, including the class 1 integrase gene (intI1), the conjugative transposon Tn916-Tn1545 family (Tn916/1545) and one insertion sequence common region I gene (ISCR1) will also be quantified to (HGT). Furthermore, some sludge samples before and after anaerobic digestion will also be selected for high-throughput metagenomics. This approach will be applied to investigate the fate of the broad-spectrum profiles of ARGs in anaerobic digestion.

In addition, the concentrations of total solids (TS), volatile solids (VS), chemical oxygen demand (COD), and methane production in anaerobic digestion will also be measured.

Task 2: Control and management strategy for antibiotic resistance (Months 25-36)

Based on the results of this study, the control and management strategy for antibiotic resistance will also be proposed.

Latest Project Developments:

Professor Qilin Wang’s (Centre for Technology in Water and Wastewater, School of Civil & Environmental Engineering, University of Technology Sydney) PhD student Huan Liu won 2022 AWA NSW Student Water Prize on 4 March 2022. Her prize-winning project is co-funded by WaterRA and the Australia Research Council. WaterRA is supporting this research work via Project #2058 “Understanding and Reducing the Spread of Antibiotic Resistance in Anaerobic Sludge Digestion” supported (cash and in-kind funding) by SEW (Dr Li Gao). She is developing a technology for improving antibiotic resistance genes and pathogen removal, enhancing anaerobic sludge digestion and boosting dewaterability of digested sludge. Water Research Australia was mentioned on the AWA Linkedln and Facebook official accounts while announcing the Student Water Prize Winner "The project is part of collaborative research projects sponsored by Water Research Australia and the Australia Research Council and will bring economic and environmental benefits to the water industry."