The impact of pre-treatment on microbiome within soil treatment units in a temperate region

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).

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.

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.