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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.

Nitrogen-fixing bacteria (NFB) can reduce nitrogen at ambient pressure and temperature. In this study, we treated effluent from a paper mill in sequencing batch reactors (SBRs) and monitored the abundance and activity of NFB with a view to producing a sludge that could work as a biofertilizer. Four reactors were inoculated with activated sludge enriched with NFB and fed with a high C/N waste (100:0.5) from a paper mill. Though the reactors were able to reduce the organic load of the wastewater by up to 89%, they did not have any nitrogen-fixing activity and showed a decrease in the putative number of NFB (quantified with qPCR). The most abundant species in the reactors treating high C/N paper mill wastewater was identified by Illumina MiSeq 16S rRNA gene amplicon sequencing as Methyloversatilis sp. (relative abundance of 4.4%). Nitrogen fixation was observed when the C/N ratio was increased by adding sucrose. We suspect that real-world biological nitrogen fixation (BNF) will only occur where there is a C/N ratio ≤100:0.07. Consequently, operators should actively avoid adding or allowing nitrogen in the waste streams if they wish to valorize their sludge and reduce running costs. PRACTITIONER POINTS: Efficient biological wastewater treatment of low nitrogen paper mill effluent was achieved without nutrient supplementation. The sludge was still capable of fixing nitrogen although this process was not observed in the wastewater treatment system. This high C/N wastewater treatment technology could be used with effluents from cassava flour, olive oil, wine and dairy industries.

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.

A suitcase laboratory was used for 16S rRNA amplicon sequencing to assess microbial water quality in the holy Bagmati River, Kathmandu, Nepal. SourceTracker analysis and Volcano plots revealed that microbial communities in the downstream part of the river were mainly contributed by untreated sewage. Seasonal variability in the sewage microbiome was reflected in the downstream river water quality. The bacterial genera Acidovorax, Geobacillus and Caulobacter predominated in the upstream sites, while genera containing putative human pathogens and gut bacteria, such as Clostridium, Prevotella, Arcobacter, Lactobacillus, Enterococcus and Streptococcus become prominent in the downstream sites. Marker gene qPCR assays for total bacteria, total coliforms, Human E. coli, Arcobacter butzleri and Vibrio cholerae confirmed the sequencing data trends. Even though basic sanitation provision is nowadays near universal in Nepal, our findings show how inadequate wastewater management may turn an urban river into an open sewer, which poses a public health risk.

Anthropogenic nitrogen fixation is essential to sustain a global population of 7.7 billion. However, there has been a long-standing desire to find cheaper and more environmentally friendly alternatives to the Haber-Bosch process. In this study, we developed a new strategy of nitrogen fixation by enriching free-living N2-fixing bacteria (NFB) in reactors fed with low nitrogen wastewater, analogous to those usually found in certain industrial effluents such as paper mills. Our reactors fixed appreciable quantities of nitrogen with a rate of 11.8 mg N L-1 day-1. This rate is comparable to recent "breakthrough" nitrogen-fixing technologies and far higher than observed in low C/N reactors (fed with organic matter and nitrogen). NFB were quantified using quantitative polymerase chain reaction (qPCR) of the nifH (marker gene used to identify biological nitrogen fixation) and 16S rRNA genes. The nifH gene was enriched by a factor of 10 in the nitrogen-fixing reactors (compared to controls) attaining 13% of the bacterial population (1:4.2 copies of nifH to 16S rRNA). The Illumina MiSeq 16S rRNA gene amplicon sequencing of reactors showed that the microbial community was dominated (19%) by Clostridium pasteurianum. We envisage that nitrogen-enriched biomass could potentially be used as a biofertilizer and that the treated wastewater could be released to the environment with very little post-treatment.

In principle, high carbon to nitrogen wastes can be used to produce nitrogen-fixing bacteria and thus create a potential biofertiliser. In practice biological nitrogen fixation can be inhibited by the presence of ammonia. We wish to know what concentrations of ammonia are inhibitory. We therefore “challenged” sludge from reactors fed with high carbon to nitrogen wastewater with different ammonia concentrations and measured the effect on nitrogen fixation rates. The sludge was resistant to inhibition by ammonia with a half maximal inhibitory concentration (IC50) of 70.2 mg of NH4+ L−1. Moreover, the presence of modest amounts of ammonium in the reactors did not affect the capacity of the biomass to reduce the organic matter contained in the wastewater. We conclude that high carbon to nitrogen wastes can be used to generate a potential fertiliser that could work in the presence of high levels of ammonia contamination.