Combining Short- and Long-Read Sequencing Technologies to Identify SARS-CoV-2 Variants in Wastewater.

During the COVID-19 pandemic, the monitoring of SARS-CoV-2 RNA in wastewater was used to track the evolution and emergence of variant lineages and gauge infection levels in the community, informing appropriate public health responses without relying solely on clinical testing. As more sublineages were discovered, it increased the difficulty in identifying distinct variants in a mixed population sample, particularly those without a known lineage. Here, we compare the sequencing technology from Illumina and from Oxford Nanopore Technologies, in order to determine their efficacy at detecting variants of differing abundance, using 248 wastewater samples from various Quebec and Ontario cities. Our study used two analytical approaches to identify the main variants in the samples: the presence of signature and marker mutations and the co-occurrence of signature mutations within the same amplicon. We observed that each sequencing method detected certain variants at different frequencies as each method preferentially detects mutations of distinct variants. Illumina sequencing detected more mutations with a predominant lineage that is in low abundance across the population or unknown for that time period, while Nanopore sequencing had a higher detection rate of mutations that are predominantly found in the high abundance B.1.1.7 (Alpha) lineage as well as a higher sequencing rate of co-occurring mutations in the same amplicon. We present a workflow that integrates short-read and long-read sequencing to improve the detection of SARS-CoV-2 variant lineages in mixed population samples, such as wastewater.

The microbiome of two strategies for ammonia removal with the sequencing batch moving bed biofilm reactor treating cheese production wastewater.

IMPORTANCE: Cheese production facilities must abide by sewage discharge bylaws that prevent overloading municipal water resource recovery facilities, eutrophication, and toxicity to aquatic life. Compact treatment systems can permit on-site treatment of cheese production wastewater; however, competition between heterotrophs and nitrifiers impedes the implementation of the sequencing batch moving bed biofilm reactor (SB-MBBR) for nitrification from high-carbon wastewaters. This study demonstrates that a single SB-MBBR is not feasible for nitrification when operated with anerobic and aerobic cycling for carbon and phosphorous removal from cheese production wastewater, as nitrification does not occur in a single reactor. Thus, two reactors in series are recommended to achieve nitrification from cheese production wastewater in SB-MBBRs. These findings can be applied to pilot and full-scale SB-MBBR operations. By demonstrating the potential to implement partial nitrification in the SB-MBBR system, this study presents the possibility of implementing partial nitrification in the SB-MBBR, resulting in the potential for more sustainable treatment of nitrogen from cheese production wastewater.

Kinetic effects of anaerobic staging and aeration rates on sequencing batch moving bed biofilm reactors: Carbon, nitrogen, and phosphorus treatment of cheese production wastewater.

The food and beverage industry produces wastewaters containing high concentrations of organic carbon and nutrients, which when discharged leads to eutrophication and algal blooms. Given recent stringencies in effluent regulations, industries are required to treat their wastewater on-site. There is a critical need for compact, high-rate, cost-effective wastewater technologies to treat industrial wastewaters, such as the sequencing batch moving bed biofilm reactor. The aim of this study is to investigate the potential and evaluate the performance of the sequencing batch moving bed biofilm reactor cycling between anaerobic and aerobic stages to treat high-strength food and beverage wastewaters. Specifically, this study focuses on the effects of anaerobic staging times and enhanced aeration on the removal of carbon, nitrogen, and phosphorous from cheese production wastewaters. Increasing anaerobic staging times was found to improve the removal rates of carbon beyond previously reported moving bed biofilm reactor results. Increasing the anaerobic stage however decreased the total nitrogen removal, with organic nitrogen undergoing ammonification during the anaerobic stage. This study demonstrates an optimum anaerobic staging time of 138 min; with a carbon removal rate of 31.1 g-sCOD·m-2d-1 and a nitrogen removal rate of 1.3 g-N·m-2d-1. Enhanced aeration was found to be detrimental to phosphorous removal, where a moderate aeration rate demonstrated a net total phosphorous removal of approximately 22 mg-P·l-1 with the phosphorous-content of the suspended solids being approximately 4%. Finally, the sequencing batch moving bed biofilm reactor shows potential for on-site treatment of carbon, nitrogen, and phosphorous from cheese production wastewater.

Investigation of copper inhibition of nitrifying moving bed biofilm (MBBR) reactors during long term operations.

Copper, a prevalent heavy metal in industrial mining wastewaters, has been shown to inhibit nitrification in wastewater treatment systems. Biofilm treatment systems have an inherent potential to reduce inhibition. This study investigated the effects of copper concentration on nitrifying biofilms in moving bed biofilm reactor (MBBR) systems across long term operation using influent ammonia concentrations representative of gold mining wastewater. Conventional isotherm models did not adequately model the attachment of copper to the biofilm. Long term nitritation was shown to be uninhibited at influent copper concentrations between 0.13 and 0.61 mg Cu/L. Nitratation was inhibited with influent copper concentrations of 0.28-0.61 mg Cu/L. There was no statistical difference in biofilm characteristics, including biofilm thickness, mass and density, across all copper concentrations tested, however, changes in biofilm morphology were observed. The demonstrated resistance of the nitrifying biofilm to copper inhibition makes the MBBR system a promising technology for treating ammonia in mining wastewaters.