No abstract

Wastewater treatment plants (WWTPs) are host to diverse microbial communities and receive a constant influx of microbes from influent wastewater, however the impact of immigrants on the structure and activities of the activated sludge (AS) microbial community remains unclear. To gain insight on this phenomenon known as perpetual community coalescence, the current study utilised controlled manipulative experiments that decoupled the influent wastewater composition from the microbial populations to reveal the fundamental mechanisms involved in immigration between sewers and AS-WWTP. The immigration dynamics of heterotrophs were analysed by harvesting wastewater biomass solids from 3 different sewer systems and adding to synthetic wastewater. Immigrating influent populations were observed to contribute up to 25 % of the sequencing reads in the AS. By modelling the net growth rate of taxa, it was revealed that immigrants primarily exhibited low or negative net growth rates. By developing a protocol to reproducibly grow AS-WWTP communities in the lab, we have laid down the foundational principals for the testing of operational factors creating community variations with low noise and appropriate replication. Understanding the processes that drive microbial community diversity and assembly is a key question in microbial ecology. In the future, this knowledge can be used to manipulate the structure of microbial communities and improve system performance in WWTPs.

The United Nations Environment Assembly (UNEA-3) have recognised the importance of the environment in the development, spread and transmission of antimicrobial resistance (AMR) to humans and animals. Such recognition calls for wider surveillance of antimicrobial resistance genes (ARG) in wastewater and other environmental reservoirs. For ARG surveillance to be valuable to regulators, it must enable source tracking and risk assessment. Adequate surveillance also requires the processing of a large number of samples at a relatively low cost, and a low detection limit to allow quantification of the riskiest ARGs. However, current methods for tracking ARGs have various limitations. The current study presents a multiplexed targeted amplicon sequencing approach for the detection of sequence variants of ARGs in environmental samples. To demonstrate the application of this technique, wastewater samples collected from the inlet to 16 treatment plants located along a 440-km transect of the St-Lawrence river in the province of Quebec (Canada) were analysed. Among the ARGs examined, between 3 and 45 nucleic acid sequence variants were detected demonstrating the high sequence diversity that occurs within genes originating from a single sample type and the information that is missed using traditional techniques. Using the PLSDB and Comprehensive Antibiotic Resistance Database (CARD), the risk of ARG sequence variants was inferred based upon their reported mobility and detection in pathogens. Results suggest that sequence variants within a single ARG class present different risks to public health. In the future, targeted amplicon sequencing could be a valuable tool in environmental studies for both risk assessment purposes and in AMR source tracking.

Wastewater influent microorganisms are part of the total chemical oxygen demand (COD) and affect the activated sludge (AS) microbial community. Precise modeling of AS processes requires accurate quantification of influent microorganisms, which is missing in many AS models (ASMs). In this study, influent microorganisms in COD unit were determined using a fast quantification method based on DNA yield and was compared with conventional respirometry method. The actively growing influent microorganisms were identified. A mass-flow immigration model was developed to quantify the influent-to-AS immigration efficiency (m_i) of specific taxon i using mass balance and 16S rRNA gene high-throughput sequencing data. The modelled average m was 0.121-0.257 in site 1 (LaPrairie), and 0.050-0.126 in site 2 (Pincourt), which were corrected to 0.111-0.186 and 0.048-0.109 respectively using a constrain of m_i≤1. The model was further developed to calculate contributions to organic substrate consumption by specific taxa. Those genera with zero or negative net growth rates were not completely immigration dependent (m_i<1) and contributed to 2.4% - 5.4% of the substrate consumption. These results suggest that influent microbiome may be important contributors to AS microbiome assembly and system performance (substrate consumption), which may help to improve future AS process modelling and design.

Microbial community composition has increasingly emerged as a key determinant of antibiotic resistance gene (ARG) content. However, in activated sludge wastewater treatment plants (AS-WWTPs), a comprehensive understanding of the microbial community assembly process and its impact on the persistence of antimicrobial resistance (AMR) remains elusive. An important part of this process is the immigration dynamics (or community coalescence) between the influent and activated sludge. While the influent wastewater contains a plethora of ARGs, the persistence of a given ARG depends initially on the immigration success of the carrying population, and the possible horizontal transfer to indigenously resident populations of the WWTP.The current study utilised controlled manipulative experiments that decoupled the influent wastewater composition from the influent microbial populations to reveal the fundamental mechanisms involved in ARG immigration between sewers and AS-WWTP. A novel multiplexed amplicon sequencing approach was used to track different ARG sequence variants across the immigration interface, and droplet digital PCR was used to quantify the impact of immigration on the abundance of the targeted ARGs. Immigration caused an increase in the abundance of over 70 % of the quantified ARGs. However, monitoring of ARG sequence variants at the immigration interface revealed various immigration patterns such as (i) suppression of the indigenous mixed liquor variant by the immigrant, or conversely (ii) complete immigration failure of the influent variant. These immigration profiles are reported for the first time here and highlight the crucial information that can be gained using our novel multiplex amplicon sequencing techniques. Future studies aiming to reduce AMR in WWTPs should consider the impact of influent immigration in process optimisation and design.