Spatial Variation of the Microbial Community Structure of On-Site Soil Treatment Units in a Temperate Climate, and the Role of Pre-treatment of Domestic Effluent in the Development of the Biomat Community

Abstract

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.

Keywords: bioclogging; microbial community composition; microbial community structure; microbial diversity; on-site wastewater treatment; soil treatment unit.

Graphical Abstract

Influence of pre-treatment on the microbial community structure and biomat development.

Figure 1

(A) Horizontal and (B) vertical profile of the STU (Knappe et al., 2020).

Figure 2

(A) Mean ± SD of relative read abundance of phylum level analysis for Sites A and B for both the Primary (PE), secondary (SE), and control samples for each “system”: control; base (CB), top (CT), STU topsoil (TT), and STU subsoil “trench base” (TB). The STU base is further divided into proximal (P) at 1 m, midpoint (m) at 5 m, and distal (d) at 12 m. (B) The changes of key phylum relative abundance relative to control subsoil communities, and increases are highlighted in blue and reductions in red.

Figure 3

(A) Mean ± SD of relative read abundance of genus-level analysis for Sites A and B for both the Primary (PE), secondary (SE), and control samples for each “system”: control; base (CB), top (CT), STU topsoil (TT), and STU subsoil “trench base” (TB). The STU base is further divided into proximal (P) at 1 m, midpoint (m) at 5 m, and distal (d) at 12 m. (B) 2D spatial profile of changes of key genus relative abundance with respect to the control subsoil communities; increases are highlighted in blue and reductions in red.

Figure 4

Boxplots displaying rarefied data for the observed OTUs, species richness calculated using an abundance-based coverage estimates (Chao1) and alpha diversity (Shannon). Samples were aggregated on the basis of systems' primary, secondary effluent (PE, SE), distribution box biofilms (DB), STU top (TT), STU base (TB), control top (CT), and base (CB).

Figure 5

The Shannon diversity (A) and Chao1 richness (B) across the distance and depth of the sites' primary (PE) and secondary effluent (SE) STUs.

Figure 6

Principal coordinates of beta diversity based on weighted Unifrac distances within STUs at both sites. Each data point represents a sample taken from either PE effluent stream, SE effluent stream, or control soils. Samples are further subdivided based on the position within the system, i.e., at the STU; base, top, and control; base, top, and pure effluent samples.

Figure 7

(A) Mean ± SD of relative read abundance of Anaerobic Methane Oxidizers (AMO), Denitrifying bacteria, polyphosphate-accumulating organisms (PAO), Nitrite Oxidizing Bacteria (NOB), Methanogens, Glycogen Accumulating Organisms (GAO), Ammonia Oxidizing Bacteria (AOB), AMO, and Acetogen functional groups for Site A for both the Primary (PE), secondary (SE), and control samples for each “system”: control; base (CB), topsoil (CT), STU topsoil (TT), and STU subsoil “trench base” (TB). The STU base is further divided into proximal (P) at 1 m, midpoint (m) at 5 m, and distal (d) at 12 m. (B) 2D spatial profile of changes of relative abundances of Anaerobic Methane Oxidizers (AMO), Denitrifying bacteria, Polyphosphate-accumulating organisms (PAO), Nitrite Oxidizing Bacteria (NOB), Methanogens, Glycogen Accumulating Organisms (GAO), Ammonia Oxidizing Bacteria (AOB), AMO, and Acetogen functional groups sequences with respect to the control subsoil communities; increases are highlighted in green and reductions in blue.