Influence of seasonality, wastewater treatment plant process, geographical location and environmental parameters on bacterial community selection in activated sludge wastewater treatment plants treating municipal sewage in South Africa.

This is the first comprehensive study that focusses on the correlation between the bacterial community composition and a range of previously identified selective criteria in activated sludge wastewater treatment plants on the African continent. Multivariate statistical analyses were used to determine the relative significance of the geographical location (factor: site), wastewater treatment plant process (factor: configuration), seasonality (factor: season), and environmental parameters on the bacterial communities in nine wastewater treatments plants from two sites in South Africa using terminal restriction fragment length polymorphism as a screening tool to rationalize the number of samples (to 50 samples) for high throughput (Illumina MiSeq) sequencing. Site was the most significant factor (Global ANOSIM R value = 0.91, p = 0.001), and it was established that the inter-site differences were not climatic in origin but related to differences in the composition of the influent and activated sludge. Previous studies that have reported associations between microbial community structure and environmental parameters have measured influent chemistry, and this is the first time, to our knowledge, that the comprehensive chemical character of activated sludge itself has been included in this type of study. It was found using BEST analysis that the activated sludge ammonia, activated sludge total phosphate and influent chemical oxygen demand were the most significant (p < 0.001) drivers for inter-site bacterial community selection (ANOSIM Global R values of 0.862, 0.782 and 0.428, respectively). This link would not have been established with only influent chemical analyses as there was no significant difference (t-test, p > 0.05) in the average influent phosphate concentrations between the 2 sites, but there was a highly significant difference (p < 0.001, t (15.5)>t-crit (2.01)) in the activated sludge total phosphate concentrations (20.8 ± 17.0 and 127.8 ± 40.2 mg/L). This is notable for all future studies on a global level aimed at identifying factors for selection of microbial communities in activated sludge.

Metagenomics analysis of sewage for surveillance of antimicrobial resistance in South Africa.

Our 24-month study used metagenomics to investigate antimicrobial resistance (AMR) abundance in raw sewage from wastewater treatment works (WWTWs) in two municipalities in Gauteng Province, South Africa. At the AMR class level, data showed similar trends at all WWTWs, showing that aminoglycoside, beta-lactam, sulfonamide and tetracycline resistance was most abundant. AMR abundance differences were shown between municipalities, where Tshwane Metropolitan Municipality (TMM) WWTWs showed overall higher abundance of AMR compared to Ekurhuleni Metropolitan Municipality (EMM) WWTWs. Also, within each municipality, there were differing trends in AMR abundance. Notably, within TMM, certain AMR classes (macrolides and macrolides_streptogramin B) were in higher abundance at a WWTW serving an urban high-income area, while other AMR classes (aminoglycosides) were in higher abundance at a WWTW serving a semi-urban low income area. At the AMR gene level, all WWTWs samples showed the most abundance for the sul1 gene (encoding sulfonamide resistance). Following this, the next 14 most abundant genes encoded resistance to sulfonamides, aminoglycosides, macrolides, tetracyclines and beta-lactams. Notably, within TMM, some macrolide-encoding resistance genes (mefC, msrE, mphG and mphE) were in highest abundance at a WWTW serving an urban high-income area; while sul1, sul2 and tetC genes were in highest abundance at a WWTW serving a semi-urban low income area. Differential abundance analysis of AMR genes at WWTWs, following stratification of data by season, showed some notable variance in six AMR genes, of which blaKPC-2 and blaKPC-34 genes showed the highest prevalence of seasonal abundance differences when comparing data within a WWTW. The general trend was to see higher abundances of AMR genes in colder seasons, when comparing seasonal data within a WWTW. Our study investigated wastewater samples in only one province of South Africa, from WWTWs located within close proximity to one another. We would require a more widespread investigation at WWTWs distributed across all regions/provinces of South Africa, in order to describe a more comprehensive profile of AMR abundance across the country.

Investigating (anti)estrogenic activities within South African wastewater and receiving surface waters: Implication for reliable monitoring.

Natural and synthetic steroid hormones and many persistent organic pollutants are of concern for their endocrine-disrupting activities observed in receiving surface waters. Apart from the demonstrated presence of estrogen- and estrogen-mimicking compounds in surface waters, antagonistic (anti-estrogenic) responses originating from wastewater effluent have been reported but are less known. Estrogenicity and anti-estrogenicity were assessed using recombinant yeast estrogen receptor binding assays (YES/YAES) at ten South African wastewater treatment works (WWTWs) and receiving rivers in two separate sampling campaigns during the summer- and winter periods in the area. Four WWTWs were then further investigated to show daily variation in estrogenic endocrine-disrupting activities during the treatment process. Although estrogenicity was notably reduced at most of the WWTWs, some treated effluent and river water samples were shown to be above effect-based trigger values posing an endocrine-disrupting risk for aquatic life and potential health risks for humans. Furthermore, estrogenicity recorded in samples collected upstream from some WWTW discharge points also exceeded some calculated risk trigger values, which highlights the impact of alternative pollution sources contributing towards endocrine disrupting contaminants (EDCs) in the environment. The YAES further showed variable anti-estrogenic activities in treated wastewater. The current study highlights a variety of factors that may affect bioassay outcomes and conclusions drawn from the results for risk decision-making. For example, mismatches were found between estrogenic and anti-estrogenic activity, which suggests a potential masking effect in WWTW effluents and highlights the complexity of environmental samples containing chemical mixtures having variable endocrine-disrupting modes of action. Although the recombinant yeast assay is not without its limitations to show endocrine-disrupting modulation in test water systems, it serves as a cost-effective tier-1 scoping assay for further risk characterisation and intervention.