Wastewater-based epidemiology (WBE) has been employed by many countries globally since the beginning of the Covid-19 pandemic in order to assess the benefits of this surveillance tool in the context of informing public health measures. WBE has been successfully employed to detect SARS-CoV-2 at wastewater treatment plants for community-wide surveillance, as well as in smaller catchments and institutions for targeted surveillance of Covid-19. In addition, WBE has been successfully used to detect new variants, identify areas of high infection levels, as well as to detect new infection outbreaks. The usefulness of wastewater samples as a means of accurately quantifying SARS-CoV-2 infection levels among a population is less clear at present, however, due to the large number of inherent uncertainties in the WBE process, including the inherent intricacies of the sewer network, decay of the virus en route to a monitoring point, levels of recovery from sampling and quantification methods, levels of faecal shedding among the infected population, as well as population normalisation methods. The current WBE programmes in place globally will help to identify new areas of research aimed at reducing the levels of uncertainty in the WBE process, thus improving WBE as a public health monitoring tool for future pandemics. In the meantime, such programmes can provide valuable comparisons to clinical testing data and other public health metrics, as well being an effective early warning tool for new variants and new infection outbreaks. This review includes a case study of sampled wastewater from the sewer network in Dublin, Ireland, during a peak infection period of Covid-19 in the city, which evaluates the different uncertainties in the WBE process.

The growth of microbial mats or “biomats” has been identified as an essential component in the attenuation of pollutants within the soil treatment unit (STU) of conventional on-site wastewater treatment systems (OWTSs). This study aimed to characterize the microbial community which colonizes these niches and to determine the influence of the pre-treatment of raw-domestic wastewater on these communities. This was achieved through a detailed sampling campaign of two OWTSs. At each site, the STU areas were split whereby half received effluent directly from septic tanks, and half received more highly treated effluents from packaged aerobic treatment systems [a coconut husk media filter on one site, and a rotating biodisc contactor (RBC) on the other site]. Effluents from the RBC had a higher level of pre-treatment [~90% Total Organic Carbon (TOC) removal], compared to the media filter (~60% TOC removal). A total of 92 samples were obtained from both STU locations and characterized by 16S rRNA gene sequencing analysis. The fully treated effluent from the RBC resulted in greater microbial community richness and diversity within the STUs compared to the STUs receiving partially treated effluents. The microbial community structure found within the STU receiving fully treated effluents was significantly different from its septic tank, primary effluent counterpart. Moreover, the distance along each STU appears to have a greater impact on the community structure than the depth in each STU. Our findings highlight the spatial variability of diversity, Phylum- and Genus-level taxa, and functional groups within the STUs, which supports the assumption that specialized biomes develop around the application of effluents under different degrees of treatment and distance from the source. This research indicates that the application of pre-treated effluents infers significant changes in the microbial community structure, which in turn has important implications for the functionality of the STU, and consequently the potential risks to public health and the environment.

<p>There has been a large output of genomic data in ecological studies of centralised wastewater treatment plants over the past number of years. One significant collaboration of Danish and Swedish research institutions lead to the development of the Microbes of Activated Sludge and Anaerobic Digesters (MiDAS 4) global taxonomic database. The database has been an effective tool in understanding centralised systems, however, there has been no known application of this tool in understanding the ecology of organisms in the on-site wastewater treatment systems. The growth of microbial mats or "biomats" has been identified as an essential component in the attenuation of pollutants within the soil treatment unit (STU) of conventional on-site wastewater treatment systems (OWTSs). Two research sites were employed to determine the influence of the pre-treatment of raw-domestic wastewater on these communities. The STUs at each of the two sites were split, whereby half received effluent directly from septic tanks, and half received more highly treated effluents from packaged aerobic treatment systems [a coconut husk media filter on one site, and a rotating biodisc contactor (RBC) on the other site]. Effluents from the RBC had a higher level of pre-treatment [~90% Total Organic Carbon (TOC) removal], compared to the media filter (~60% TOC removal).  These sites' biomat were sampled two-dimensionally in respect of distance and depth, to configure ecological data with changes in the volumetric water content values which had been used successfully as an indicator of the location of the biomat. A total of 92 samples were obtained from both STU locations and characterized by MiDAS taxonomic database. Our study has shown that the biomats receiving primary or untreated effluent have less pronounced increases in denitrifiers compared to the biomats receiving treated or partially treated effluent. but biomats receiving primary effluents have been found to be capable of removing six times the amount of total nitrogen. This suggests that the increases in functional richness within the STU are secondary to bioclogging, as metabolic rates could be limited by hydraulic conductivity.</p>

<p>Soil Treatment units (STU) receiving domestic wastewater from on-site wastewater treatment systems (ONWTS), such as septic tanks, rely on the development of microbial biomat at the infiltrative surface.  Community ecology analysis was conducted on two separate STUs, each receiving both primary (SE) and secondary effluent (SE) in parallel trenches under identical hydrogeological and environmental conditions.  At Site A SE was produced by a Ecoflo Coco Filter (Premier Tech Aqua Ltd., Ireland) and in Site B SE was produced from a Rotating Biodisc Contactor (Klargester BioDisc,Kingspan Ltd., UK). A total 92 samples were taken from both STU locations, (n= 51) samples were taken at the infiltrative surface of the STUs and (n=24) subsurface samples were taken above the STU system across a range distances and depths for both effluent types. Additional samples were taken of PE and SE effluent (n=5), distribution boxes (n=2), and of adjacent control soils (n=10).</p><p>Samples were characterized by 16S rRNA gene sequencing analysis. Data from water quality (ammonia, chloride, E. coli, nitrate, nitrite, non-purgeable organic carbon, phosphate, sulphate) were also taken on this sampling day using lysimeters installed at both sites. Inter-site phylogenetic analysis showed that there was little to no difference in phylogenetic composition between the control microcosm soil samples at each site. The impact of effluent characteristics on the microbial community’s present within the STU microcosms resulted in the STU receiving SE at Site A being richer in species (ACE) and a greater diversity in species (Shannon) when compared to the SE in Site B.  Further analysis of Site A showed that both species richness and diversity were at their highest in the SE trench at the sampling point closest to the effluent inlet, whereas for PE the opposite was noted as richness and diversity increased downstream of the inlet. This was confirmed with principle component analysis (PCOA) showing a clustering of PE STU samples located at the inlet of the trench. The STU receiving SE at Site B showed a notable lack of species and richness when compared to the PE counterpart across all distances and depth. Again, clear clustering of SE STU samples was present in PCOA results.</p><p>Samples were screened for the abundances of particular sequences corresponding to target organisms (i.e. nitrifiers, denitrifiers, methanotrophs, denitrifying methanotrophs, gut flora, Extracellular Polymeric Substances producing bacteria). STUs in both sites contained a greater abundance of target sequences than the controls. In the case of denitrifiers, EPS producers, methanogens and methanotrophs these sequences were absent from the deep soil control samples taken at both sites. In Site B the number of denitrifying bacteria, EPS bacteria and methanogens sequences counted in the STU receiving SE was on average by an order of magnitude of 2, 3, 2, and 1 greater than its PE STU counterpart respectively, and by an order of magnitude of 2 respectively when compared to SE STU in Site A. It is evident, therefore, that the application of secondary effluent is conferring phylogenetic changes to the composition of the microbiomes within the studied biomats.  </p>