ECOTREAT Project
Novel Ecology-Based Solution to Optimize the Treatment of Urban Sewage Waters
Abstract: Treated urban sewage water is a lost resource, mainly because its quality is too low for reuse. ECOTREAT offers a new low-cost treatment based on a holistic ecological approach to the removal of microbiological and chemical contaminants in the last clarifier. By defining and modifying the microbial community and the water retention time in the final clarifier it will ensure water re-naturalization before its release into the environment or its sustainable reuse, according to current legislation.
Keywords: wastewater reuse - microbial ecology - one-health
State of the art: The treatment of urban sewage waters is currently a problem for Italy (Infraction of the EU Directive 91/271/CEE, with 620 urban areas in 16 Italian Regions not correctly treating wastewater, 1). Technical, bureaucratic, and financial limitations are behind this national failure, resulting in a significant proportion of treated waters too low in quality to allow reuse. This causes the loss of a potential resource of increasing strategic importance (2) and, through the release of chemical and microbiological pollutants into the environment, the reduction of the ecological quality of the receiving water bodies, troubling human health (one-health concept, 3). In the last decades, urban wastewater treatment plants (WWTPs) have applied sophisticated systems to abate nutrients and chemical pollutants through different chemical and biological processes, at extremely high efficiency (4). Yet, the removal of pathogenic organisms is still almost entirely devoted to a final disinfection of the treated effluent (5). The disinfection technologies in use present some drawbacks in terms of sustainability, efficacy and costs (6). Traditional chemical disinfection (e.g. chlorine or peracetic acid) are generally cost-effective and efficient in terms of E. coli removal, but much less so in reducing microbiological risk factors of emerging concern (e.g. antibiotic resistance genes, ARGs) and release into the effluent a number of byproducts with toxic or cancerogenic effects on humans (7). Ultraviolet radiation and ozone disinfection are more efficient in damaging bacterial DNA, but their efficacy towards ARGs is still debated, with running costs that are higher than those of traditional disinfection technologies (8). Novel systems of microbiological control (e.g. activated carbon, nano and micro membranes, advanced oxidation processes) seem to be more efficient but their application is generally experimental and limited to very small WWTPs (9).This scenario calls for a novel approach to remove microbiological contaminants in wastewater balancing costs and efficiency abating the release of harmful byproducts. Alternative disinfection technologies through a classical biotechnological approach are currently under scrutiny, whereas we suggest a novel ecology-based solution to remove human-derived microorganisms from the effluents, concomitantly raising efficiency in chemicals sequestration. The features of the bacterial communities in WWTP effluents are known and understood (10,11), but very little is known on the processes occurring in the communities in the final sedimentation tank (the so called secondary, or final clarifier) of a WWTP (12). The gap is clear in scientific production: only 42 studies on microbiological or ecological parameters in the final clarifiers have been published, in contrast to more than 3200 papers on the same parameters in the activated sludges (data from WoS, Dec 2020). The optimization of secondary clarifiers is a traditional topic in wastewater management, but the considered parameters are related only to hydraulics and flocculation (13). The final clarifier is a small artificial pond, with an aquatic ecosystem maintained by nutrients upload, artificial mixing and oxygenation, and periodical renewal of the sediments. The few studies targeting bacterial communities just out of the secondary clarifiers demonstrated a strong effect of water retention time in the tank and load of nutrients on the microbial composition, with ‘better’ aquatic communities at high retention time and constant inflow (14).
ECOTREAT will analyze, model, and use the aquatic community (bacteria, fungi, phytoplankton, and meiofauna) of a secondary clarifier, applying ecological theory to define the key points where, through direct interventions, a natural microbial community will be stabilized raising its intrinsic resistance and resilience, preventing human pathogens and other human-derived bacteria to survive, without further need for disinfection.
Objectives: The goal of ECOTREAT is to shift paradigm in wastewater treatment towards systems in which the abatement of the harmful microbes is devoted to “nature” once put in the ideal conditions to succeed. Applying Nature-Based-Solutions (15) on this issue guarantees higher efficiency by challenging more directly the harmful microbes, while reducing the treatment costs and the release of chemicals into the environment.
ECOTREAT will set up a model secondary clarifier, identifying the ideal conditions and microbial communities for a complete abatement of human derived bacteria. The lab-based experimental model final output of ECOTREAT will be then directly available for test and application at real scale in small-medium urban wastewater treatment plants (WWTPs) (up to 120K population equivalent, PE) in Northern Italy.
To reach the over-arching objective of ECOTREAT, four consequent research goals are identified as project milestones (Ms), and as specific objectives to be finalized. M1: to assess the microbial communities of the final clarifier of different urban sewage treatment plants; M2: to define the key interactions and the key actors in the microbial communities and to design modified communities in order to optimize the stabilization of typical freshwater organisms; M3: test the removal efficiency of the new communities in lab based experiments in microcosms; and M4: to quantitatively model the system, in order to define general settings for the application in real scale WWTPs.
RESEARCH GOAL 1 (M1). Given the current missing knowledge on the microbial community composition in WWTP final clarifiers we will assess it in 3 small-medium sized urban WWTPs with an overall well-functioning treatment efficiency, sized 20, 60 and 120K PE and with an urban sewage inflow and, if present, a limited industrial inflow (from 0 to 20% of the total inflow). We will sample the final clarifier of each WWTP in winter and in summer, identifying the limits of the water temperature range of the water in the tank. Water, floccules, and sediments will be collected in order to define their associated viral, bacterial and eukaryotic community (i.e. fungi, protozoans, phytoplankton, and meiofauna) using a combination of shotgun metagenomics and gene target metagenomics, epifluorescence, confocal and light microscopy, and flow-cytometry. Fungi will be characterized via isolation and lab cultures. Technical treatment parameters (water retention time, classical WWTP chemical and physical parameters) will be assessed as well. The different microbial communities will be thus defined, identifying the species characterizing natural aquatic communities, and those belonging to the inlet sewage communities, according to existing knowledge in our research group.
RESEARCH GOAL 2 (M2). Identification of the key interactions and key actors in the newly described microbial communities. The whole microbial network and (in absence of larger organisms) the whole tank trophic network will be assessed. The human derived microbiome and other pathogenic microorganisms will be considered as target to be excluded in the ideal community to be considered as objective of the research. By defining the relative importance of the different limiting factors in determining the composition of the different communities, the importance of nutrients availability, and the presence of species with peculiar characteristics (e.g. super-predators, important K and R-strategists, bacteria-fungi positive associations) the most important interactions will be identified. A collection of those species will be then formed by isolating them and keeping them in pure cultures. Microbial communities with enhanced performances in resisting the introduction of allochthonous bacterial strains will be designed, and used in the next research phase.
RESEARCH GOAL 3 (M3). The efficiency of newly designed microbial communities, where the results from M2 have been used to implement changes in order to stabilize the resident communities will be tested in lab-based continuous cultures using microcosms. Here, the microbial communities will be kept in atmospheric conditions mimicking those from the original tank and fed with fresh wastewater from the original inlet of each respective clarifier. These systems can then be considered as specific ecosystems where the natural equilibrium will tend towards an aquatic community, constantly disturbed by the inoculum of allochthonous strains (e.g. human derived microorganisms) with the inlet, and the elimination of the less adapted ones, with the outflow. The results from the microcosms will allow an experimental validation of the theoretical key interactions we identified in M2 and to experimentally identify those ecological parameters are protecting the stability of the resident pathogen-free community. The experiments will also allow us to constantly modify specific interactions, artificially supporting those key-species that are more effective in removing the allochthonous strains, even when the system itself is not able to maintain them in the requested abundances. The most important factors determining composition and abundance of aquatic (bacterial) communities are competition for resources (mainly available forms of carbon, phosphorus and nitrogen), predation (in its different forms, performed by protists, micro- and macro-zooplankton), and viral lysis (16). This means that the aquatic bacteria in the tank and in the mesocosms have, in thousands of centuries of co-evolution, developed specific strategies to optimize the access to resources, to escape predation, and to reduce the impact of viral blooms (17). The same is not true for human derived bacteria, generally well adapted to very different ecosystems (e.g. the animal gut, 18). Artificially modifying the number of predators or of competitors in the community will end up in the composition of an extremely stable (in a dynamic equilibrium) and resistant community, where the permanence of the introduced allochthonous human derived bacteria is a matter of time (19,11). The manipulation will be thus optimized in order to reduce this time, until it will be shorter than the retention time of the tank in the original WWTP. The optimal settings for each consortium will be then reproduced for the different WWTPs and for low and high water temperatures.
RESEARCH GOAL 4 (M4). Ecological modelling will be used to quantify the impact of the different strains introduced on the base of their abundances and will allow defining the parameters to be quantified for the use of these new Nature-Based Tools. We will generate ecological network (19) to reconstruct the architecture of the biotic interactions (predation, competition, facilitation, mutualism) among the community members. Interaction will be defined using key functional traits of each species (20). The centrality of each node (species) in the network will allow us to target the key actors involved in determining the stability of the system, under different set of abiotic conditions (21). Using simulations, we will then explore the effect of removing different nodes in changing the general architecture of the whole network, thereby identifying the most effective and stable community composition in providing effective bioremediation of the urban sewage waters. Then, the cost of the treatment will be quantified and compared to those of the disinfection technologies nowadays in use. The models of utilization will be made available for test in real scale WWTPs, as already planned with the support of Acqua Novara.VCO, the leading water cycle company of Eastern-Piedmont.
If the project ECOTREAT will be successful, the produced WWTP effluent will be at the highest microbiological standard, and this result will be achieved without any harmful addition of chemicals to the water and without applying costly final treatments.
References:
1. EU Commission. 2019. Commission takes Italy to Court over air pollution and failure to properly treat urban waste water. Brussels. 2019. https://ec.europa.eu/commission/presscorner/detail/en/IP_19_1475
2. Sabater S and Barceló D. 2010. Water Scarcity in the Mediterranean, Perspectives Under Global Change. Series: The Handbook of Environmental Chemistry 8. Springer-Verlag Berlin Heidelberg
3. O'Brien E and Xagoraraki I. 2019. A water-focused one-health approach for early detection and prevention of viral outbreaks. One Health (Amsterdam, Netherlands), 7, 100094. https://doi.org/10.1016/j.onehlt.2019.100094
4. Bourgin M, et al. 2018. Evaluation of a full-scale wastewater treatment plant upgraded with ozonation and biological post-treatments: Abatement of micropollutants, formation of transformation products and oxidation by-products. Wat Res 129: 486-498.
5. Al-Gheethi AA, et al. 2018. Removal of pathogenic bacteria from sewage-treated effluent and biosolids for agricultural purposes. Appl Water Sci 8: 74.
6. Marano RBM, et al. 2020. A global multinational survey of cefotaxime-resistant coliforms in urban wastewater treatment plants. Env Int 144: 106035.
7. Di Cesare A, et al. 2016. Fitness and Recovery of Bacterial Communities and Antibiotic Resistance Genes in Urban Wastewaters Exposed to Classical Disinfection Treatments. Environ Sci Tech 50(18): 10153-10161.
8. Alexander J, et al. 2016. Ozone treatment of conditioned wastewater selects antibiotic resistance genes, opportunistic bacteria, and induce strong population shifts. Sci Total Environ 15(559): 103-112.
9. Di Cesare A, et al. 2020. Combination of flow cytometry and molecular analysis to monitor the effect of UVC/H2O2 vs UVC/H2O2/Cu-IDS processes on pathogens and antibiotic resistant genes in secondary wastewater effluents. Wat Res 184: 116194.
10. Wakelin SA, et al. 2008. Effect of wastewater treatment plant effluent on microbial function and community structure in the sediment of a freshwater stream with variable seasonal flow. Appl Environ Microbiol 74: 2659-2668.
11. Corno G, et al. 2019. Effluents of wastewater treatment plants promote the rapid stabilization of the antibiotic resistome in receiving freshwater bodies. Wat Res 158: 72-81.
12. Xu D, et al. 2017. Microbial community compositions in different functional zones of Carrousel oxidation ditch system for domestic wastewater treatment. AMB Express 7(1): 40.
13. Krebs P. 1991. The hydraulics of final settling tanks. Water Science and Technology, 23(4-6): 1037-1046.
14. Gerrity D and Neyestani M. 2019. Impacts of solids retention time and antibiotic loading in activated sludge systems on secondary effluent water quality and microbial community structure. Water Environ Res 91(6): 546-560.
15. Cohen-Shacham E, et al. 2016. Nature-based Solutions to address global societal challenges. Gland, Switzerland: IUCN. ISBN: 978-2-8317-1812-5
16. Thingstad T. 2000. Control of bacterial growth in idealized food webs. in Microbial Ecology of the Oceans, Kirchman DL (ed), Wiley-Liss New York 239–260.
17. Corno G and Jürgens K. 2008. Structural and functional patterns of bacterial communities in response to protist predation along an experimental productivity gradient. Environ Microbiol 10 (10): 2857-2871.
18. Scheuerl T, et al. 2020. Bacterial adaptation is constrained in complex communities. Nat Commun 11: 754.
19. Csardi G and Nepusz T. 2006. The igraph software package for complex network research. Complex Systems 1695.
20. Lindo Z. 2015. Linking functional traits and network structure to concepts of stability. Comm Ecol 16(1): 48-54.
21. Delmas E, et al. 2019. Analysing ecological networks of species interactions. Biol Rev 94(1): 16-36.
Work Plan: ECOTREAT is organized in 5 work packages (WPs), 4 of them designed for the achievement of each specific milestone (see chapter Obiettivi) and a fifth one for the dissemination of the results. The 4 operative WPs articulate along the 2 years duration of the Project with limited research activities overlap and are designed to allow plasticity in the study according to the output of the previous WPs.
WP1: ASSESSMENT OF THE MICROBIAL COMMUNITIES IN THE FINAL CLARIFIERS - 7 months
The final clarifiers of three fully functional wastewater treatment plants (WWTP) have been selected for the assessment of the resident microbial communities. The WWTPs are located in Novara, Verbania, and Cannobio (VB). They have been selected because of an existing detailed knowledge of their microbiological parameters by the PI (e.g. about 20 scientific publications on those systems), and because of diversity in water load and treatment parameters between them: Novara is a large WWTP (160K PE) treating mainly urban wastewater (WW), with important loads of hospital and industrial WW, applying ultraviolet radiation as final disinfection. Verbania is a medium size WWTP (60K PE) treating urban WW only, with a limited inflow from the local hospital, and applies chlorine for the final disinfection. Cannobio is a small plant (15K PE) treating urban and industrial WW, and applying a final disinfection with peracetic acid.
At the beginning of the Project (month 1, cold season) and after 5 months (month 5, warm season) we will collect from each final clarifier samples of water and sludges (the sediments in the final clarifier, composed by floccules from the activated sludge and of other particles) to define, for the two compartments:
Bacteria: total number and aggregational state (number of microcolonies and aggregates) by flow cytometry and epifluorescence microscopy; community composition with specific focus on the pathobiome by 16SrRNA gene amplicon sequencing, resistome (e.g. number and distribution of antibiotic and heavy metal resistance genes, mobile genetic elements) by shotgun metagenomics. All bioinformatics and statistical analyses on the metagenomic and amplicon data from bacteria and other organisms will be performed in house, thanks to the long experience on these analyses by the PI and the research team.
Micro-eukaryotic organisms: number and general classification (flow cytometry and microscopy), community composition by 18SrRNA gene amplicon sequencing.
Fungi: characterization and quantification of the communities by isolation, lab culturing, and by ITS genetic fragment amplicon sequencing.
Viruses: total number by flow cytometry
Water and sludge chemistry (e.g. nutrients, toxic compounds, heavy metals, etc.) will be assessed by fine analyses periodically performed by the WWTPs owner (Acqua Novara.VCO) and made available for this study. The technical design of the WWTPs is available, and includes the description of the different treatment steps and, very important, the water retention times.
The analysis of the microbial communities in the clarifiers will allow to identify: The pathobiome, namely the genera that are (potentially) harmful for humans and their relative abundances; the resistome and other genetic elements of interest in terms of risk for human health; the presence of numerically dominating species, of microbial predators and their importance in terms of prey numbers control, the presence of interspecific interactions (e.g. fungi-bacteria), the viral load.
This represents an extremely detailed knowledge of the clarifier communities, which to date it was never assessed in dedicated studies. It is also the base for the subsequent manipulations together with the analysis of water and sludge chemistry and of physical parameters (turbidity, temperature, water retention time, etc.).
The results of WP1 will allow to define the degree of “naturalization” (e.g. the relative importance of environmental and harmless species already established in the clarifier in relation to those still belonging from the original WW).
M1: Fine assessments of the original microbial communities in the clarifiers.
WP2: IDENTIFICATION OF KEY ORGANISMS AND INTERACTIONS - 5 months
We will collect water and sludge samples from the clarifiers and, in lab controlled environment at the CNR-IRSA we will expose them to specific stresses in batch cultures, to evaluate the response of the communities. The test will be performed reproducing the same conditions of light, temperature, and turbulence as in the original clarifier. 5 replicates for each treatment and for a control treatment where no stressors are applied, will be assessed. The exposure time will be the same than in the clarifiers.
Treatment 1: original microbial community + 10% in abundance of a bacterial supercompetitor under the clarifier settings (a harmless strain, e.g. Pseudomonas putida, others with limited survival rate once released into the environment, inoculated at T0)
Treatment 2: original microbial community + 1% in abundance of a protozoan predator (e.g. Poterioochromonas sp., able to remove up to 80% of non-grazing-resistant aquatic bacteria in a few hours - pathogenic bacteria are generally not able to develop anti-grazing strategies and are thus ideal prey)
Treatment 3: original microbial community + Trichoderma harzianum previously isolated from a river and available from our collection. Members of the genus Trichoderma are present in the natural communities occurring in final clarifiers where they are important producers of antagonistic compounds (proteins, enzymes, and antibiotics) and micronutrients. Trichoderma harzianum has been widely reported as one of the most effective antagonists of pathogens on agar plates. This fast growing species is also approved as a biocontrol agent allowed also in biological agriculture and does not pose risks to human health or to the environment. Secondarily, we will evaluate the efficacy of autochthonous strains isolated directly from the resident community of the final clarifiers. We will exclude slow-growing, mycotoxigenic, harmful, or pathogenic strains during the selection process.
Treatment 4: the original community will be diluted 2:1 with water from the (WWTP effluent) receiving water body collected just before of the WWTP inlet. This can favour the adaptation of natural predators and competitors in the clarifier, enhancing the elimination of WW derived species.
Treatment 5: the retention time will be prolonged by 50% and 100% without additional manipulations, in order to define the natural reduction of potential pathogens by simply leaving them in the clarifier conditions for longer time.
From each treatment replicate we will assess the microbial abundances and the presence of aggregates (flow cytometry and microscopy), the number of faecal indicator bacteria (FIB) by cultivation, quantitative Real Time PCR (qPCR), and (by qPCR) the number of 3-4 specific resistance genes selected on the base of the results of shotgun metagenomic analysis in WP1. The output of these analyses represents the Project’s M2.
The most promising treatments in terms of abatement of human derived potentially threating species will be then tested in microcosms in the following WP.
WP3: TEST OF THE SELECTED TREATMENT(S) AT REDUCED SCALE IN LAB AND IN SITU - 8 months
The most promising treatment will be tested on lab-based microcosms (5 treatment replicates + 5 control) in chemostat vessels at the CNR-IRSA under conditions mimicking those in the original clarifiers. Each vessel will be filled with 750 ml of water from the clarifier’s inlet, and diluted with the same medium (freshly collected every day). A few gr of the original clarifier sludge will be displaced at the bottom of the continuous culture vessels. The microcosms will run for 30 days, to evaluate the long-term adaptation of the communities and the efficiency of the treatment on longer periods.
The water in the treatments will be monitored for the overall bacterial, protozoan and fungal abundance, as well as FIB and other species of interest applying the previously described methodologies.
The community composition, the resistome, and the overall metagenome will be measured (shotgun metagenomics and target gene amplicon) at day 0, 7, 14, 21, and 30. The sludge communities will be tested by shotgun metagenomics and target gene amplicon at day 0 and 30.
In case of need (e.g. some unexpected changes in the microbial community) other stressors tested in WP2 could be applied to the communities as well, to maximize the efficiency of the system.
The experiment in microcosms will allow a detailed understanding of the dynamics of the microbial communities of water and sludges and possible modifications of the treatment protocols will be then implemented if needed.
The same experiment will be repeated in situ (Verbania and Nocera WWTPs), using 50 litres mesocosm vessels (5 treatment replicates + 5 control) constantly refilled with the original inlet of the clarifier ensuring the same conditions of the real one. Also for these experiments the duration and the measured variables will be assessed as described for the microcosms experiment in lab, and detailed chemical analyses of nutrients and pollutant will be measured.
The WWTP of Nocera (Salerno, 150K-300K PE) is available in collaboration with the University of Salerno. It is of particular interest as it presents peculiar changes in water load and quality during the year (30000 m3/d in winter and 60000 m3/d in summer) due to the additional load of agro-food industry in the area. The amount of human derived bacteria in the final effluent (treated by an activated sludge process with a final disinfection by peracetic acid) is 1-2 orders of magnitude higher than those in the other selected WWTPs, and can thus be considered as a test in extreme conditions for our ecology-based-tool.
The analysis of the data on microbial diversity, the efficiency in the abatement of human derived strains and the adjustments to the treatment protocol adopted during the tests will allow to reach the M3 (the definition of the ideal treatment for a fully natural abatement of human derived potentially threatening microbes from a WWTP effluent).
WP4: ECOLOGICAL MODELLING FOR A FULL SCALE IMPLEMENTATION OF THE NEW NATURE-BASED-TOOL - 4 months
Data originating from the previous WPs will be used to fed ecological models aimed at exploring and mechanistically understanding the generalities of the study system—that is, to quantify the impact of the different strains and define the relevant parameters to support the practical implementation of these new Nature-Based Tools. To this end, we will rely on ecological networks, analytical tools for representing and modeling the behavior of complex systems characterized by relational data (interactions) and for exploring the emerging properties deriving from the sum of these interactions, according to a recent publication by Delmas et al. (2019, Biol Rev). All analyses will be conducted in R (libraries network and igraph).
The first step in the analysis will be to build a reference ecological network for a theoretical clarifier community, starting from in-situ amplicon sequencing data (WP1) and information on key species and interactions (WP2). Following the approach by Perreira (2019, Ecol Model), nodes will represent groups of species with similar traits (i.e., species performing analogous metabolic/ecological functions) and connected by fuzzy directed edges (links) representing potential interactions (e.g., predation, competition, cooperation). Grouping species together according to trait similarity is needed both to reduce the dimensionality of the network (and thus computation time) and ease data interpretation.
Different properties of the ecological network will be measured via structural and functional indexes calculated from the number of nodes, links, and the overall architecture. Beside standard statistics, we will describe the network in terms of:
i) Individual nodes centrality. We will use three measure of centrality (Closeness centrality, Degree centrality, Betweenness centrality) to derive the importance of any given node in a network. Closeness centrality is used for finding highly inter-connected nodes, namely those that are likely to hold most information or that can quickly connect with the wider network. Degree centrality tells the nodes that mostly influence the flow around a given network. Betweenness centrality targets nodes that are best placed to influence the entire network most quickly. Therefore, these indexes cut through noisy data, revealing parts of the network that require attention; in our case, they will tell us what are the key actor (i.e., complexes of functionally similar species) supporting the overall network architecture and influencing the most the system behaviour.
ii) Connectance of the food web (Latham, 2006). Is related to the statistical distribution of the interactions per species and it measures the proportion of possible links between species that are noticed, with the formula:
Connectance = L/m2
where L is the number of links in the network and m is the number of compartments.
iii) Global efficiency. This is the ratio between the number of nodes (n) and the product of the number of connection (I) by the network diameter (D):
Global efficiency = n / I*D
This index ranges from 0 to 1 and the higher its value, the more efficient a network is.
Once the reference ecological network representing the community in the clarifier will be constructed and comprehensively explored mathematically, we will use simulations to predict risk and performance scenarios depending on variations in the network structure. Specifically, we will test the effect of removing different nodes in altering the general architecture of the network and its properties. We will simulate a set of different scenarios where, in turn, we will remove different nodes (and consequently their links with other nodes) depending on their values of Closeness centrality, Degree centrality, and Betweenness centrality. Thereby, we will explore the effect of individual(s) removal in terms of Connectance and Global efficiency of the overall network. The significance of these changes will be assessed with null models, whereby we will resample n times the network by shuffling randomly the nodes position and compare the observed values against the null distribution.
Overall, this simulation exercise will allow us to identify the most effective and stable community in providing effective bioremediation of urban sewage waters, thus translating the analysis into real world information for the correct application of this approach (M4).
Finally, the cost of the treatment will be quantified and compared to those of the disinfection technologies nowadays in use. The models of utilization will be made available for test in real scale WWTPs, as already planned with the support of Acqua Novara.VCO, the leading water cycle company of Eastern-Piedmont, managing the WWTPs used in ECOTREAT.
WP5: DISSEMINATION
Towards scientists: At least 4 publications in leading journals in the fields of environmental sciences and microbial ecology are expected. The results of ECOTREAT will be also presented in at least three international conferences in the field.
Towards stakeholders: The main results of ECOTREAT, with a focus on technical implementation of the eco-based clarification of waters, and on legislations, will be presented to WWTP managers, legislators and administrators at regional, national and European level in 2 dedicated workshops hosted at the CNR-IRSA and at the Acqua Novara.VCO headquarter and in specific events organized by the Water JPI and AMR JPI actions of the European Union. This is of particular interest as, due to the presence in the effluents of disinfection by-products, and by the incoming implementation of limitations for contaminants of emerging concerns (including determinants of antibiotic resistance) in national and European directives, the (agricultural/human) reuse of treated WW is in Italy will be almost impossible, given that the final disinfection is nowadays mandatory. The availability of an alternative eco-based technology to abate microbiological and genetic parameters will allow a more efficient reuse of water, and a strong spin for a water-based circular economy net at the national level.
Towards citizens and students: 2 Master thesis will be activated with the University of Girona (Spain) and with one of the Italian Universities (Milan, Salerno, etc.) currently involved in collaborations with our group. A dedicated space for ECOTREAT will be dedicated on the group webpage (www.meg.irsa.cnr.it) and a the group Twitter account (@MEG_Verbania) will be used to update a large crowd on the Projects update. Short videos with interviews of members of the team and on the different experiments will be produced and uploaded on the website, or used during dedicated lectures to high school students at the CNR-IRSA and in a specific ECOTREAT Open Day planned in the second year, where the results of the Project will be presented to the population and to the press, at the historical Conference Room (Aula Tonolli) of the CNR-IRSA in Verbania.
Feasibility and contingency plan: To reach M1 (WP1) no specific risks are identified. For WP2 a potential problem is given by the risk that none of the selected treatments will produce significant abatement of the threatening microbes. In this case different species of competitors, predators, or commensal bacteria fungi or predators (or mixed communities of 2 or more of them) will be tested. The delay expected to solve the issue is of 2-5 weeks. For the WP3 the main identifiable risk is the drop of the microbial communities once exposed to lab conditions in microcosms, or possible disruptive viral blooms once the community is growing in small vessels. To avoid the risks the experiments will be run in fully climate chambers with controlled radiation, temperature, and humidity. Further 5 replicates are designed to reduce the risk of a loss of a replicate (e.g. in case of viral blooms). The limited risk of losing more than one replicate will be thus mitigated by running the experiment a second time. In this case a delay of 5-6 weeks is expected. As long as the data deriving from previous WPs will be successfully acquired/tabulated, there will be few concrete risks that can hamper the progress of WP4. The simulation part of WP4 is likely to be the most complex enterprise, involving a design, implementation, parametrization, testing, and analysis. Within the narrow time frame of the project, setting up a simulation framework is feasible both because of the simplified experimental setting (which is unlikely to pose any particular problem in terms of computation time) and the expertise of the team, having experience e in developing simulation exercise. The risk of getting stuck in one of the tasks of simulation development and testing can be mitigated by focusing on simplified settings to fine tune the parameters, e.g., simplified networks of representative communities with <10–15 nodes.
A general problem that can cause delay in the Project and in the dissemination of the results is the development of the pandemic in the next years. In case of lockdown and limited or no access to the Institute a correspondent delay will be caused. At the same time all the dissemination activities in person will be transformed in virtual ones. Note that, in facing a similar event, we will follow recommendation to maximise lab performance and resilience by Rillig et al. (2020, PLoS Comp Biol).
Expected results: ECOTREAT will produce a straightforward operative output: the methodology and the technology to produce, applying ecology-based-tools, high quality treated WW without the need of additional expensive or polluting chemical and physical disinfection processes. This apparently simple approach was never implemented in the past because of strong technical limitations in the tools (genetics and metagenomics) necessary for its development. In fact, thanks to the implementations of novel sequencing technologies, to the abatement of costs per analysis, and to the development of new and more powerful computational bioinformatic tools, it is now possible to evaluate as a whole and at the same time specific features of a simplified microbial community as those in WWTP final clarifiers. This allows the implementation of ecology-based-tools to modify these communities in the way we prefer, and thus in abating all those microbiological parameters can reduce the overall quality of the treated WW.
The high quality treated WW ECOTREAT is targeting as output of the Project will match the microbiological parameters imposed by the national and international legislations for the agricultural reuse of waters, even considering as limit those imposed by the latest, extremely restrictive, EU Regulation on Wastewater Reuse (2020/741) (e.g. 10 E. coli colony forming units in 100 ml of effluent). Further, it will target and abate microbiological contaminants of emerging concerns (e.g. antibiotic resistant human derived bacteria and resistance genes) that are still not limited in the legislations, but are listed by many international organizations (e.g. the EU, the WHO, the G7 and G20 target groups) as important pollutants with a direct impact on human health, on the base of a plethora of scientific evidences: the EU Regulation on Wastewater Reuse (2020/741) dedicated attention (despite no limit is established) towards determinants of antibiotic resistance and CECs (Annex II), within the framework of a risk management plan (art.5).
By reaching this output we could promote an ecological revolution in the WW treatment, tackling one of the most problematic issue in the currently applied technology: the removal of microbiological pollutants. In fact, the application of ECOTREAT output will strongly reduce the need of disinfection of the effluent, applying a technology that, differently than others nowadays already available (e.g. nanomembranes) will not reduce the productivity of the treatment plant and will be economically sustainable.
The final output is indeed expected at the end of the project, and despite the risk of failure for a never implemented technology is there, we are convinced that the quality of the research team, and the different experiences and qualifications of the researchers involved are the best insurance for its mitigation. A number of intermediate scientific outputs will be produced in order to achieve the final goal:
Research Output 1 (M1): for the first time the overall microbial communities, their interactions and the relations between trophic levels in WW clarifiers will be assessed. This is already a very important result in order to better understand how “nature plays its role” in this artificial systems where microbes of very different origin get in contact in generally non-limiting conditions. Such totally disturbed environments where disturbances can be permanently applied or appear with different frequency and intensity, are not only an ideal theoretical model where to apply system ecology theories, but also to establish deterministic traits that can influence the community composition and services, thus, in general terms, the quality of the effluent. This study will be published in (at least) one scientific article on a leading journal in the field of microbial ecology.
This part of the research is technically established and fully feasible respecting the time devoted to it (7 months). The potential delay in the production of the result caused by an unexpected delay in the sequencing procedures is already considered and mitigated within the 7 months dedicated to the activity.
Research Output 2 (M2): by testing the microbial community of the clarifier in aerated batch cultures in laboratory, where they will be exposed to different stress factors targeting the human derived microbes (thus indirectly favouring the so called “environmental microbes”) we will be able to define the different ecological interactions underlying the modifications imposed by the applied stress. Being the nature of the stress factors applied very different (invasion by supercompetitors, by predators, by an alternative aquatic community, of an eukaryotic commensal, or the time of exposure) the response of the communities will be indeed different and will allow a theoretical observation of the impact caused by the different stressors. The most efficient stress factor (in abating our target groups) will be selected for further studies, and at the same time the whole experiment will ensure the publication of (at least) one scientific article on a leading journal in the field of microbial ecology.
This output is potentially the most problematic to achieve, as we do not only want to select the most efficient stressors, but the selected stressor should also ensure a high performance in the abatement of the target species. Being the tests performed of the same duration of water residence time in a real clarifier (8 hours), we will be allow, without important delay, to test several stressors, and in case far more than the 5 initially selected, in order to find the ideal one(s). For this reason no delay is expected in the output of this part of the research.
Research Output 3 (M3): by assessing microcosms in continuous culture were the microbial community of the clarifier will be then exposed for a longer term (30 days) to the most promising stress factor in conditions mimicking the natural ones, we will be able to define the efficiency of the nature-based-tool. The periodical check of the community conditions will allow to possibly modify the treatments with further disturbances if needed (e.g. other stress factors).
Once optimized the system the further step will be to test it in situ, in mesocosms working in parallel to the real secondary clarifiers. This output, will be ready to be modeled in order to define the final output of the research (see WP4). Also for this prat of the research 1-2 articles on a leading journal in the field of environmental sciences are expected.
The output is pretty much straightforward, and no specific delays are expected, the feasibility of the output is extremely high and safe. Possible postponements can be caused by adverse meteorological conditions, or other technical issues already defined in the previous chapter, still, the time frame devoted to the research ensures a correct respect of the deadlines.
Research Output 4 (M4): Output of WP4 will serve to provide statistical support to previous WPs. Furthermore, the simulation exercise will allow us to rule out the most effective and stable community composition in providing effective bioremediation of urban sewage waters. This is a key result to translate the analysis into real world application of this approach. As previously discussed, there is a risk of getting stuck in one of the tasks of simulation development and testing. This can be mitigated by focusing on simplified settings to fine tune the parameters, e.g., simplified networks of representative communities with <10–15 nodes. Even such simulations would be innovative and publishable, and still provide the basis for more full-fledged models, that could be completed in follow-up projects or by PostDoc students acquired during the project.
All in all, as the simulation exercise based on ecological networks represent an innovative enterprise within the frame of WP4, it could potentially lead to an additional methodological-oriented publication suitable for journals such as Ecological modelling, Methods in Ecology and Evolution, or similar.
Infrastructures: Organisms to develop the ecology based-tools will be selected from the communities of the water bodies receiving the effluents of the selected WWTPs available at the MEG at CNR-IRSA (MEGIC culture collection), but some specific harmless strains of potential interest will be obtained from the pan-European Microbial Resource Research Infrastructure (MIRRI, www.mirri.org) for culture collections. All the sequences (genes, genomes, metagenomes) produced in the project will be deposited and made accessible for other researchers in public databases such as genbank (www.ncbi.nlm.nih.gov/genbank). Bioinformatics and statistics resources for sequence analyses will be either used from the MEG online service (MEGIt, https://services.d4science.org/explore) developed on the d4science platform in collaboration with CNR-ISTI Pisa. For larger metagenomic analyses the research infrastructure ELIXIR (elixir-europe.org) will be used. The MEG is already in collaboration with ELIXIR members (i.e. CINECA).
Future perspectives: The output of ECOTREAT is an ecology-based-tool readily applicable to full scale WWTPs. The application of the tool will allow, without important structural modifications, to eliminate the final disinfection treatment from the wastewater plant and, depending, of the disinfection (alternative removal procedure) in place, will reduce or will not impact the running costs of the treatment.
For the development of the tool, additional experiments and tests, at a full scale in selected WWTPs, is necessary, and can last up to 1-2 years for its optimization. The company Acqua Novara.VCO, and AOP4Water (academic spin-off of the University of Salerno) are interested in a collaboration with the CNR-IRSA for the development and a possible further commercialization of the technology. For example, a larger number of WWTPs applying different biological processes (e.g. biofilm based processes such as MBBR, moving bed biofilm reactor) will be tested as well in order to generalize and optimize the tools.
This technology can have a significant impact in the treatment of wastewaters in Europe, maximizing the possibility for agricultural treated WW reuse, an additional resource of strategic importance due to the scarcity of water for agriculture (especially in warm seasons) caused by climatic changes.
Further, it can become of primary importance in poor and developing countries where the costs of WW treatment are often resulting in poor or missing treatment of the sewages. For this aspect a possible future collaboration with the UN, and with FAO, is already forecasted.
The results of ECOTREAT can help also in the current discussion on the usefulness of a final disinfection treatment of WW. Several legislations (including the Italian law) define as mandatory the disinfection of the WW and, because of the high cost of alternative treatments (UV radiation, ozone, nanomembranes, activated carbons, others) most of the treatment plants add chlorine to their effluents. This can result in the presence of toxic and cancerogenic by-products in the effluent, preventing any reuse, and in fact causing a depletion of the water quality in the receiving river, lake, or coastal water. For this reason, other countries are strongly limiting the final disinfection to the largest WWTPs and to the most problematic ones (e.g. UK, Israel).
ECOTREAT tool can introduce a new variable in this discussion at the national and continental scale, offering an alternative and sustainable tool to achieve the same (and better) results than disinfections in terms of microbiological quality of the WWTP effluent.
ECOTREAT Research team:
Gianluca Corno (MEG Verbania): Principal Investigator.
Luigi Rizzo (University of Salerno): Prof. Rizzo is one of the leading experts in water purification at the European level. His studies on alternative disinfection systems, including those in collaboration with CNR-IRSA, are among the most cited in the last decade. His collaboration with the MEG group of the CNR-IRSA, which began in 2017, is still ongoing, and his experience in WWTP engineering, as well as the deep knowledge of the systems used in this project, make him a crucial professionalism for the success of the research.
Carles Borrego (ICRA Girona): Prof. Borrego is a world recognized expert in the study of microbial communities in rivers, streams and sediments, and his expertise is fundamental for a high quality analysis and a deep understanding of the interactions between species in the final wastewater clarifier. His group, the MMEG at ICRA Girona has already a long-term successful collaboration running with the MEG at the CNR-IRSA. He is Ass.Prof at Uni Girona, and he will involve a dedicated Master student in ECOTREAT.
Jakob Pernthaler (Zurich University): Prof. Pernthaler will be involved in the study of the results from WP1, in the definition and the modelling of the microbial communities in WP2 and 3, and will make available the culture collection of the Limnological Station at UniZH for selecting strains of potential interests. He will participate in the final assessment of the methodology and in the assessment of potential impacts on natural communities.
Andrea Di Cesare (MEG Verbania)
Laura Garzoli (MEG Verbania)
Stefano Mammola (MEG Verbania)
Tomasa Sbaffi (MEG Verbania)
Collaborations: ECOTREAT is a project where microbial ecology, molecular biology and evolution, wastewater treatment and management and treatment plants engineering merge to produce the necessary knowledge to achieve the expected output. In this terms ECOTREAT can be considered an highly interdisciplinary research, where the expertise of the CNR-IRSA needs to be flanked by other specialists, concurring in building the required know-how. For this reason, two external groups at the highest international standards and a leading WWTP management company the academic spin-off AOP4Water are involved/interested in the Project, and their coordinators are part of the Research Team, with direct responsibility on specific aspects of the research.
The University of Salerno, with Prof Luigi Rizzo, will supervise all aspects related to WWTP engineering and on the management and the feasibility at full scale of the proposed modifications. This is of primary importance as the output of ECOTREAT is not only a production of basic scientific knowledge on the ecology of the primary clarifiers and of the WWTP effluents, but it is also, and mainly, an ecology-based-tool to implement in the treatment, and (environmental and economic) sustainability and feasibility are mandatory parameters for a successful application. Uni Salerno will be involved in the definition of the parameters in WP1, in the experimental WP3, in the evaluation of the results in WP4, and indeed in the dissemination (WP5) with a possible shared Master Thesis student in environmental engineering formed on the project.
The ICRA Girona, with Prof Carles Borrego, will participate in the selection of the species to be introduced, in the evaluation of their impacts on the communities, on the environment, and in the evaluation of their efficiency in outcompeting unwanted human derived microorganisms. Their knowledge will perfectly complement the CNR-IRSA knowledge of the microbial community interactions in disturbed environments, and especially in the ecology and evolution of the microbial communities in the sludges.
The Limnological Station at the University of Zurich, leaded by Prof. Jakob Pernthaler, is also from several years actively collaborating with the PI, and it is an historical centre of excellence for the study of aquatic ecology in Europe. They possess the necessary knowledge to define and to correctly evaluate the potential interactions between the different compartments of the microbial communities, and thus to define which modifications can optimize the process. They will be involved WP2, 3 and 4, as well as in the dissemination of the results.
Acqua Novara.VCO is the leading company managing the cycle of water and wastewater in Eastern Piedmont. With more than 180 WWTPs directly managed is the second largest group in the field, in North West Italy. They directly manage the three WWTPs of Verbania, Novara and Cannobio. With the CNR-IRSA they have a long term collaboration for the implementation of new technologies in the wastewater treatment, for the abatement of antibiotic resistances and for the optimization of their systems. Acqua Novara.VCO ensures full support for the Project ECOTREAT and full accessibility of their WWTPs for the purposes of ECOTREAT.
AOP4Water (academic spin-off of the University of Salerno) is involved in the study of alternative technologies for a better disinfection of wastewaters, with a focus on advanced oxidation processes, but not only. The spin-off can support our experimental study at the WWTP of Nocera (WP3) and the dissemination activities towards stakeholders.