Current status of microbial source tracking applications in constructed wetlands serving as nature-based solutions for water management and wastewater treatment.

Microbial source tracking (MST) has been recognised as an effective tool for determining the origins and sources of faecal contamination in various terrestrial and aquatic ecosystems. Thus, it has been widely applied in environmental DNA (eDNA) surveys to define specific animal- and human-associated faecal eDNA. In this context, identification of and differentiation between anthropogenic and zoogenic faecal pollution origins and sources are pivotal for the evaluation of waterborne microbial contamination transport and the associated human, animal, and environmental health risks. These concerns are particularly pertinent to diverse nature-based solutions (NBS) that are being applied specifically to secure water safety and human and ecosystem well-being, for example, constructed wetlands (CWs) for water and wastewater treatment. The research in this area has undergone a constant evolution, and there is a solid foundation of publications available across the world. Hence, there is an early opportunity to synthesise valuable information and relevant knowledge on this specific topic, which will greatly benefit future work by improving NBS design and performance. By selecting 15 representative research reports published over 20 years, we review the current state of MST technology applied for faecal-associated contamination measures in NBS/CWs throughout the world.

Introducing Semi-Interpenetrating Networks of Chitosan and Ammonium-Quaternary Polymers for the Effective Removal of Waterborne Pathogens from Wastewaters.

The present work aims to study the influence of ammonium-quaternary monomers and chitosan, obtained from different sources, upon the effect of semi-interpenetrating polymer network (semi-IPN) hydrogels upon the removal of waterborne pathogens and bacteria from wastewater. To this end, the study was focused on using vinyl benzyl trimethylammonium chloride (VBTAC), a water-soluble monomer with known antibacterial properties, and mineral-enriched chitosan extracted from shrimp shells, to prepare the semi-IPNs. By using chitosan, which still contains the native minerals (mainly calcium carbonate), the study intends to justify that the stability and efficiency of the semi-IPN bactericidal devices can be modified and better improved. The new semi-IPNs were characterized for composition, thermal stability and morphology using well-known methods. Swelling degree (SD%) and the bactericidal effect assessed using molecular methods revealed that hydrogels made of chitosan derived from shrimp shell demonstrated the most competitive and promising potential for wastewater (WW) treatment.

Molecular Identification of Infectious Enteropathogens in Faeces of Healthy Horses.

Zoogenic faecal contamination of the environment is one of the indices included in the evaluation of ecological threats, health hazards and adverse impacts on various ecosystems. The risks and environmental concerns are associated with the fact that faeces of wild and domesticated animals constitute the largest source of environmental loading of enteropathogens associated with transmission of zoonotic diseases (enteric zoonoses). Although sick animals are more likely to transmit pathogens, healthy ones can also be the carriers and defecate them into the environment. This is of particular importance given the close human-animal interactions and health effects resulting from human and ecological exposures to faecal hazards from companion and farm animals. We have therefore set out to investigate whether healthy equines can carry and defecate human infectious pathogens. For this purpose, we set up a pilot study to examine the faecal DNA of horses using culture-independent molecular diagnostics - fluorescent probe-based quantitative real-time PCR. Our results revealed that among a total of 23 horses, 6 were found to carry Campylobacter jejuni (C. jejuni), and 5 had Salmonella enterica serovar Typhimurium (S. Typhimurium). Moreover, Enterococcus faecalis (E. faecalis) was found in 14 horses, while 19 were positive for Clostridium perfringens (C. perfringens). Furthermore, the frequently reported protozoan parasites in livestock, Cryptosporidium parvum (C. parvum) and Giardia lamblia (G. lamblia), were discovered in 8 and 7 samples, respectively. This pilot study shed new light on the phenomenon of healthy horses carrying C. jejuni and other human-health-related enteropathogens.

Mitigating Antibiotic Resistance Genes in Wastewater by Sequential Treatment with Novel Nanomaterials.

Wastewater (WW) has been widely recognized as the major sink of a variety of emerging pathogens (EPs), antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which may disseminate and impact wider environments. Improving and maximizing WW treatment efficiency to remove these microbial hazards is fundamentally imperative. Despite a variety of physical, biological and chemical treatment technologies, the efficiency of ARG removal is still far from satisfactory. Within our recently accomplished M-ERA.NET project, novel functionalized nanomaterials, i.e., molecularly imprinted polymer (MIP) films and quaternary ammonium salt (QAS) modified kaolin microparticles, were developed and demonstrated to have significant EP removal effectiveness on both Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB) from WW. As a continuation of this project, we took the further step of exploring their ARG mitigation potential. Strikingly, by applying MIP and QAS functionalized kaolin microparticles in tandem, the ARGs prevalent in wastewater treatment plants (WWTPs), e.g., blaCTXM, ermB and qnrS, can be drastically reduced by 2.7, 3.9 and 4.9 log (copies/100 mL), respectively, whereas sul1, tetO and mecA can be eliminated below their detection limits. In terms of class I integron-integrase I (intI1), a mobile genetic element (MGE) for horizontal gene transfer (HGT), 4.3 log (copies/100 mL) reduction was achieved. Overall, the novel nanomaterials exhibit outstanding performance on attenuating ARGs in WW, being superior to their control references. This finding provides additional merit to the application of developed nanomaterials for WW purification towards ARG elimination, in addition to the proven bactericidal effect.

Cross-tracking of faecal pollution origins, macronutrients, pharmaceuticals and personal care products in rural and urban watercourses.

This study describes microbial and chemical source tracking approaches for water pollution in rural and urban catchments. Culturable faecal indicator bacteria, represented by Escherichia coli, were quantified. Microbial source tracking (MST) using host-specific DNA markers was applied to identify the origins of faecal contamination. Chemical source tracking (CST) was conducted to determine contaminants of emerging concern (CEC) of human/anthropogenic origin, including pharmaceuticals and personal care products (PPCPs) and endocrine-disrupting chemicals (EDCs). In addition, the eutrophication-causing macronutrients nitrogen and phosphorus were studied. MST tests revealed both anthropogenic and zoogenic faecal origins, with a dominance of human sources in the urban stream; non-human/environmental sources were prevalent in the rural creek. CST analyses revealed a higher number of CECs in the urban stream than in the rural watercourse. Positive correlations between PPCPs and both E. coli and the human DNA marker were uncovered in the urban stream, while in the rural creek, PPCPs were only highly correlated with the anthropogenic marker. Interestingly, macronutrients were strongly associated with primary faecal pollution origins in both watercourses. This correlation pattern determines the main pollutant contributors (anthropogenic or zoogenic) to eutrophication.

Seasonal dynamics of lotic bacterial communities assessed by 16S rRNA gene amplicon deep sequencing.

Aquatic microbial diversity, composition, and dynamics play vital roles in sustaining water ecosystem functionality. Yet, there is still limited knowledge on bacterial seasonal dynamics in lotic environments. This study explores a temporal pattern of bacterial community structures in lotic freshwater over a 2-year period. The aquatic bacterial communities were assessed using Illumina MiSeq sequencing of 16S rRNA genes. Overall, the communities were dominated by α-, ß-, and γ-Proteobacteria, Bacteroidetes, Flavobacteriia, and Sphingobacteriia. The bacterial compositions varied substantially in response to seasonal changes (cold vs. warm), but they were rather stable within the same season. Furthermore, higher diversity was observed in cold seasons compared to warm periods. The combined seasonal-environmental impact of different physico-chemical parameters was assessed statistically, and temperature, suspended solids, and nitrogen were determined to be the primary abiotic factors shaping the temporal bacterial assemblages. This study enriches particular knowledge on the seasonal succession of the lotic freshwater bacteria.

DNA-based faecal source tracking of contaminated drinking water causing a large Campylobacter outbreak in Norway 2019.

During June 2019, an outbreak of campylobacteriosis occurred in Askøy, an island northwest of Bergen, Norway. According to the publicly available records, over 2000 residents fell ill and 76 were hospitalised, and two deaths were suspected to be associated with Campylobacter infection. By investigating the epidemic pattern and scope, an old caved drinking water holding pool was identified that had been faecally contaminated as indicated by the presence of Escherichia coli (E. coli). Furthermore, Campylobacter bacteria were found at several points in the water distribution system. In the escalated water health crisis, tracking down the infectious source became pivotal for the local municipality in order to take prompt and appropriate action to control the epidemic. A major task was to identify the primary faecal pollution source, which could further assist in tracking down the epidemic origin. Water from the affected pool was analysed using quantitative microbial source tracking (QMST) applying host-specific Bacteroidales 16S rRNA genetic markers. In addition, Campylobacter jejuni, Enterococcus faecalis, Clostridium perfringens and Shiga toxin-producing E. coli were detected. The QMST outcomes revealed that non-human (zoogenic) sources accounted predominantly for faecal pollution. More precisely, 69% of the faecal water contamination originated from horses.

Faecal pollution affects abundance and diversity of aquatic microbial community in anthropo-zoogenically influenced lotic ecosystems.

The aquatic microbiota is known to be an important factor in the sustainability of the natural water ecosystems. However, the microbial community also might include pathogens, which result in very serious waterborne diseases in humans and animals. Faecal pollution is the major cause of these diseases. Therefore, it is of immense importance to assess the potential impact of faecal pollution, originating from both anthropogenic and zoogenic sources, on the profile of microbial communities in natural water environments. To this end, the microbial taxonomic diversity of lotic ecosystems in different regions of Norway, representing urban and rural areas, exposed to various levels of faecal pollution, was investigated over the course of a 1-year period. The highest microbial diversity was found in rural water that was the least faecally polluted, while the lowest was found in urban water with the highest faecal contamination. The overall diversity of the aquatic microbial community was significantly reduced in severely polluted water. In addition, the community compositions diverged between waters where the dominant pollution sources were of anthropogenic or zoogenic origin. The results provide new insight into the understanding of how faecal water contamination, specifically that of different origins, influences the microbial diversity of natural waters.

Aquatic microbial diversity associated with faecal pollution of Norwegian waterbodies characterized by 16S rRNA gene amplicon deep sequencing.

Faecal contamination is one of the major factors affecting biological water quality. In this study, we investigated microbial taxonomic diversity of faecally polluted lotic ecosystems in Norway. These ecosystems comprise tributaries of drinking water reservoirs with moderate and high faecal contamination levels, an urban creek exposed to extremely high faecal pollution and a rural creek that was the least faecally polluted. The faecal water contamination had both anthropogenic and zoogenic origins identified through quantitative microbial source tracking applying host-specific Bacteroidales 16S rRNA genetic markers. The microbial community composition revealed that Proteobacteria and Bacteroidetes (70-90% relative abundance) were the most dominant bacterial phyla, followed by Firmicutes, especially in waters exposed to anthropogenic faecal contamination. The core archaeal community consisted of Parvarchaeota (mainly in the tributaries of drinking water reservoirs) and Crenarchaeota (in the rural creek). The aquatic microbial diversity was substantially reduced in water with severe faecal contamination. In addition, the community compositions diverge between waters with dominant anthropogenic or zoogenic pollution origins. These findings present novel interpretations of the effect of anthropo-zoogenic faecal water contamination on microbial diversity in lotic ecosystems.

Norwegian study on microbial source tracking for water quality control and pollution removal in constructed wetland treating catchment run-off.

This study describes the first Norwegian microbial source tracking (MST) approach for water quality control and pollution removal from catchment run-off in a nature-based treatment system (NBTS) with a constructed wetland. The applied MST tools combined microbial analyses and molecular tests to detect and define the source(s) and dominant origin(s) of faecal water contamination. Faecal indicator bacteria Escherichia coli and host-specific Bacteroidales 16 s rRNA gene markers have been employed. The study revealed that the newly developed contribution profiling of faecal origin derived from the Bacteroidales DNA could quantitatively distinguish between human and non-human pollution origins. Further, the outcomes of the MST test have been compared with the results of both physicochemical analyses and tests of pharmaceutical and personal care products (PPCPs). A strong positive correlation was discovered between the human marker and PPCPs. Gabapentin was the most frequently detected compound and it showed the uppermost positive correlation with the human marker. The study demonstrated that the NBTS performs satisfactorily with the removal of E. coli but not PPCPs. Interestingly, the presence of PPCPs in the water samples was not correlated with high concentrations of E. coli. Neither has the latter an apparent correlation with the human marker.

Preservation of nutrients during long-term storage of source-separated yellowwater.

Source separation of human urine (yellowwater) enhances the sustainability of wastewater management and efficiency of nutrient recovery and recycling. Storage of source-separated yellowwater is recommended prior to agronomic reuse. At this point, it is of immense interest to determine the effect of storage time on quality of yellowwater. Therefore, this study focused on examining changes in some chemical properties of raw, undiluted, freshly collected, source-separated yellowwater stored for a period of 1 year under different temperature regimes: cold (4 °C), mild (10 °C) and warm (22 °C). Chemical parameters (biochemical oxygen demand (BOD(5)), N-tot, N-NO(2), N-NO(3), N-NH(4), P-tot, K, S, and pH), with the main focus on fertiliser nutrient compounds intended for agricultural utilisation, were tested. The outcomes revealed that both nitrification and denitrification processes took place in the stored yellowwater, and an increase in the pH level of up to pH greater than 9 was observed. The study found that the main macronutrients can be well preserved in yellowwater, as there were no substantial changes in the contents of these elements over a 1 year storage period at the three temperatures tested.

Long-term hygienic barrier efficiency of a compact on-site wastewater treatment system.

The long-term use of a filter-based, on-site wastewater treatment system increases nutrient discharge to receiving waters and may reduce its hygienic barrier efficiency. The main purpose of this research was to assess the hygienic barrier efficiency and the associated health risks of an on-site system that had exceeded its 5-yr design capacity with respect to phosphorus (P) removal. The system was investigated for bacteria and virus removal and assessed with respect to potential health risks in relation to reuse of effluent for irrigation. The system consists of a septic tank, a pressure-dosed vertical flow biofilter, and an up-flow filter unit with lightweight clay aggregates. The total P concentration in the effluent had increased gradually from initially <0.1 mg P L(-1) during the first 2 yr of operation to 1.8 mg P L(-1) after 5.3 yr. Escherichia coli was used as an indicator organism for fecal bacteria removal, whereas bacteriophages phiX174 and Salmonella typhimurium phage 28B (S.t. 28B) were used to model enteric virus removal. An overall decrease in E. coli removal occurred from a complete (approximately 5.6 log10) reduction during the first 3 yr of operation to 2.6 log10 reduction. The removal amounts of the bacteriophages phiX174 and S.t. 28B were 3.9 and 3.7 log10, respectively. Based on removal of S.t. 28B, the risks of rotavirus infection and disease for the investigated scenarios were above the acceptable level of 10(-4) and 10(-3), respectively, as defined by the World Health Organization.

Treatment of tunnel wash waters--experiments with organic sorbent materials. Part I: Removal of polycyclic aromatic hydrocarbons and nonpolar oil.

Tunnel wash waters characterize all waters that run off after washing procedures of tunnels are performed. These waters represent a wide spectrum of organic and inorganic pollutants, such as polycyclic aromatic hydrocarbons (PAHs) and toxic metals. Removal of such contaminants from water runoff was investigated using laboratory tests after washing procedure was performed on two road tunnels in eastern Norway (Hanekleiv and Bragernes). Due to diverse character of both, treatment media and treated wash waters, the whole investigation was divided into two separate laboratory experiments. The treatment efficiencies were established based on the levels of concentrations and reductions of the measured contaminants in the effluents released from the tested media. In the first part of the article, the contents of nonpolar oil (NPO), 16 individual PAHs, and total PAHs are described. This part revealed that the combination of two organic sorbent materials provided the highest treatment efficiency for wash waters released from the road tunnel and from electrostatic filters. The greatest reduction levels reached 97.6% for NPO, 97.2% for benzo[a]pyrene, and 96.5% for the total PAHs. In the second part of the article, the concentrations and the removal rates of toxic metals are reported.

Treatment of tunnel wash waters--experiments with organic sorbent materials. Part II: Removal of toxic metals.

In the first part of the article, the column and the bag experiments concerning removal of polycyclic aromatic hydrocarbons (PAHs) and nonpolar oil (NPO) from tunnel wash waters using organic sorbent materials have been described. This part presents the results of removal of toxic metals. The metals of concern (Al, As, Cd, Cr, Cu, Fe, Pb, Mo, Ni, and Zn) were selected based on the priority toxicant pollutants defined in surface water quality criteria. Concentrations of these metals in the collected effluents varied more than the concentrations of PAHs and NPO, and thus only metal contents were considered for statistical analyses. These analyses determined significant differences (P < 0.05, P < 0.01, and P < 0.001) between the mean metal concentrations in the column effluents and those in applied wash water of road tunnel. The results obtained during both experiments revealed that the organic sorbents, and in particular their combination, removed toxic metals more effectively from wash water of road tunnel than from wash water of tunnel electrostatic filters. Among the investigated toxicants, Al and Fe showed the highest levels of reduction in the column experiment, 99.7% and 99.6%, respectively. The lowest reduction levels of 66.0% and 76.2% were found for Pb and Mo, respectively. The results of the bag experiment showed that even one day treatment of wash waters from tunnel electrostatic filters could reduce concentration of some toxicants by more than 70% (Al and Fe) and 80% (Cu).