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Final Report Summary - WATER4CROPS (Integrating bio-treated wastewater with enhanced water use efficiency to support the Green Economy in EU and India)

Executive Summary:
Through its 7th Framework Program the European Commission on 2012 co-funded with about six millions of euros the project “Integrating biotreated wastewater reuse with enhanced water use efficiency to support the Green Economy in EU and India” whose acronym is “Water4Crops”.
According to the call, the similar but not identical twin project “Integrating bio-treated wastewater reuse with enhanced water use efficiency to support the Green Economy in EU and India” was contemporarily funded with about three millions of euros by the Indian Government through its Department of Biotechnology. The Water4Crops-EU project started on August 2012 and regularly ended, as scheduled, after four years. The twin projects had similar objectives, i.e. The European consortium included 21 partners form eight Countries: 5 Universities, 8 Research Institutes, 4 agro-industrial companies, 2 spin-off companies and 2 consultant companies. The main objectives of W4Cs can be synthetically summarized as in the following:
• Valorizing agro-food-industry wastewater by recovering and/or producing valuable substances;
• Increasing water availability by treating and reusing treated wastewater in agriculture;
• Saving water in agriculture by improving water use efficiency (WUE) at field level through: improved agronomics, plant breeding and locally adapted innovative irrigation strategies and techniques;
• Enhancing stakeholders participation by their real involvement in co-creation processes with the help of two innovative tools developed just in W4Cs, i.e. Mirror Cases and INNOVA platforms.

To achieve its objectives W4Cs was structured in seven work packages, namely:
• WP1-Valorization, treatment and reuse of agrofood industry wastewaters
• WP2-Innovative municipal wastewater bio-treatment for agricultural reuse
• WP3-Efficient water use in Irrigated Agriculture
• WP4-Improving WUE and drought tolerance of maize, sorghum, millet and tomato via genomics approaches and modelling
• WP5-Identifying business opportunities and integration of solutions
• WP6-Dissemination and technology transfer
• WP7-Management and Coordination

The W4Cs objectives were all essentially aimed at enhancing and supporting the Green Economy in Europe and India. At the end of the project, its outcomes actually contributed to achieve such a goal. Within Water4Crops, in fact, it was developed and implemented an original and new modular biotechnological process aimed at fully exploiting the use of wastewater water and its content of the organic carbon and nutrients. This lead to an innovative cascade approach with creation of added value compounds (e.g., organic acids, alcohols, PHA) from agrofood industry wastewater and additional water resources from reusing treated wastewater for irrigation purposes. Then, nutrients and wastewaters go back to the land and create opportunities to increase crop yield and to allow new crops to grow (spreading harvest periods and processing times). Finally the new crops and higher yields will allow more activities such as food processing and biorefinery. The co-creation of these new product combinations will lead to enhanced business opportunities. Water4Crops provided for the first time an innovative combination of several technical and procedural improvements to bridge bio-treatment of wastewater and increase water productivity with a transdisciplinary identification of agri-business opportunities and the related requirements for tailoring technological innovations.

Project Context and Objectives:
The world’s population is expected to increase dramatically from 7 billion at present to 9 billion by the year 2050. This growth is expected to be matched with an increased water demand and subsequently, increased wastewater volume. Many regions of the world are approaching, or have already reached, the limits of their available water supplies. Efforts to offset the declining surface water availability due to droughts and reduced precipitation and worsened by the reduced groundwater recharge in water stressed regions are hampered by the rapid increase in population and water demand (Kundzewicz et al. 2007; UNDP 2006; CIHEAM-IAMB 2007).
India has a strong emerging bio-based economy (highlighted by the fact that India already completed a detailed country report in preparation for the Rio+20). However, there are significant challenges in linking Bio-technology to the rural development and provision of crops and fibres production from the agricultural sector.

Since irrigated agriculture in is the main consumer of fresh water there is a need to acquire further knowledge on how to mitigate and adapt to this serious situation. Currently, around 205 million hectares of agricultural land in developing countries is irrigated and it provides about 40% of crop production in these countries. Developing countries are expected to expand their irrigated area by 40 million hectares by 2030. There are several projections of world irrigation-water demand and supply by 2025. The most accredited and reliable studies suggest that the area equipped for irrigation expand by a rate of 0.6% per annum. In parallel, the global potential irrigation-water demand will rise by 9.5% in 2021-25 (Rosegrant et al. 2002). The challenge of irrigated agriculture is tightly related to water scarcity and droughts, hence it needs to be addressed both as an essential environmental issue and also as a precondition for sustainable economic growth [EU- COM (2007) 414].
Climate changes will exacerbate an already critical situation. Not only Mediterranean and arid and semi-arid region will suffer higher temperature and reduced precipitation, with more severe drought periods, but also temperate and humid areas in northern Europe are expected to cope with possible increasing water scarcity (Bates, et al, 2008). Crop yields could drop sharply as temperatures rise and water becomes scarcer. Yield losses could range from 10 to 30% in many large areas of the South (EU-SEC 2007).

The huge demand for irrigation (70% of global water consumption) combined with water scarcity encourage the reuse of wastewater as a water resource. However, the use of such alternative resource is often linked to the problem of gastro-intestinal diseases caused by using the untreated wastewater for irrigating food crops. In 2003, more than 30 % of the stomach infections in the US were caused by this problem that needs to be matched.

In order to cope with the imbalance between water supply and demand, we need to adopt new approaches for sustainable and efficient use of water resources, using nonconventional water resources, adopting correct demand and integrated management of water resources, minimizing input to agriculture through precise agriculture practices, minimizing wastes, recycling and reuse of natural resources in sustainable manner. However, traditional concepts to increase wastewater reuse in irrigated agriculture are not expected to bring a breakthrough in economic developments at rural areas. According to the World Bank, the greatest challenge in the water and sanitation sector over the next two decades will be the implementation of low cost sewage treatments that will at the same time permit selective reuse of treated effluents for agricultural (or industrial purposes). The comparable high costs for treated wastewater, its spatially restricted availability and the limited return on investment in irrigating field crops call for new approaches and combinations of products. Time has come to explore new ways for development both in Europe as in India.
The Europe 2020 strategy, promoted under “Horizon 2020”, intended to build a smart, sustainable and inclusive economy. In order to reach this, concrete targets are set within the next decade in areas such as innovation, energy use, employment and education to overcome the impact of the economic crisis and put Europe back on track for economic growth. The EU CAP 2020 strategy demands to foster green growth through innovation that requires adopting new technologies, developing new products, changing production processes, and supporting new patterns of demand, notably in the context of the emerging bioeconomy. The Strategic Forum for International Science and Technology Cooperation (SFIC) identified India as the first partner country with which to initiate a pilot initiative on collaboration in S&T on water, biomass, energy and health.
New approaches are required to exploit additional product market combinations, which address the economic challenge, the optimal use of resources and to cover the demand for water and food in an integrated way.
Treated wastewater reuse should be not only considered as an instrument for producing alternative water resources, but also as a central source for recycling high value elements and input for integrated bio-refinery processes. Already accepted and endorsed by the public in many urban and agricultural areas, properly implemented non-potable reuse projects can help communities to meet water demand and supply challenges without significant health risks. Under the broad definition of water reclamation and reuse, the sources of reclaimed water may range from industrial process waters to the tail waters of agricultural irrigation systems. The use of reclaimed water for non-potable purposes offers the potential for exploiting a “new” resource that can replace existing use of potable water sources in some sectors such as agriculture and forestry.
Climate change and increasing resources scarcity (e.g. fossil fuels) are forcing the world population to search for alternatives resources. Nowadays of the best renewable resources leading to a lower CO2 footprint are bio-fuel, energy crops biomass, biowastes and bioresidues. As a result, agriculture worldwide is becoming more intensive not only to provide the necessary food for the growing population, but also to provide biomass as resources for food, chemicals/materials and energy. Intensive agriculture with high crop yields is only possible under perfect nutrient and water management. Water4Crops addresses the above mentioned challenges and provides sustainable solutions through: valorizing agrofood and domestic wastewater by recovering valuable substances and nutrients; increasing water resources supplies by developing new technologies to treat and reuse wastewater in agriculture; using water more efficiently in irrigation through modern techniques and systems as well as better management of water, land and crops; improving water use efficiency by genomics and breeding; involving potential stakeholders in the processes leading towards a viable and stronger green economy.

Technologies developed in India and Europe, both in the field of bio-treatment and increased water use efficiency are basically comparable but their applications are context specific and would require new adaptations and integration. In order to boost the bio-based economy both in Europe and India Water4Crops is based on providing a comprehensive set of individual key technologies (reflecting the highest state of the art in Europe and India), to understand the differences (at processing and application levels) and finally to identify best possible modifications which would allow a higher and combined use of technological advances from both at both regions. Water4Crops aims not at the simple further development of an individual technology, but also at understanding their added value in up-to-now less exploited fields of application (both in India and Europe).
The concept of the project Water4Crops follows similar principles as the InfoDev (World Bank-hosted programme focused on building local capacity in developing countries to create and accelerate innovative technology SMEs) launch of “Climate Innovation Centers” (CIC). In a dedicated process views and experiences of around 100 climate technology stakeholders in India had been used to prioritize amongst others Water, Agriculture, and Bio-based technologies. By addressing the variety of experiences of key technologies from Europe and India in total, Water4Crops provides the required critical mass, both for true in-depth know how and to think completely out of the box for new product market combinations.

Water4Crops is based on an innovative modular biotechnological process dedicated to fully exploit the use of water and its content of the organic carbon and nutrients. This leads to an innovation triangle with creation of extra added value compounds (e.g., organic acids, alcohols, PHA) besides nutrients, water (both necessary to increase crop yield) and energy as last recovery in a cascade approach. Nutrients and water will go back to the land and create opportunities to increase crop yield and to allow new crops to grow (spreading harvest periods and processing times). Finally the new crops and higher yields will allow more activities such as food processing and biorefinery. The co-creation of these new product combinations will lead to enhanced business opportunities.

Water4Crops provides for the first time an innovative combination of several technical improvements to bridge bio treatment of waste water and increase water productivity with a transdisciplinary identification of agri-business opportunities and the related requirements for tailoring technological innovations.

This takes into account that there is still a transition period and a co-learning process required before understanding the true potential of transdisciplinary approaches both in the EU as well as in India. There are still open technical questions in the field of improving the applicability of biotreatment in wastewater re-use for agricultural production, which need to be elaborated at laboratory scale. At the same time the support of green economy and the actual implementation of technological innovations will benefit to a large extend from a transdisciplinary identification of new agri-business opportunities.

Water4Crops recognizes the economic challenges of rural areas in Europe as well as in India. To strengthen the identification of Agribusiness opportunities, Water4Crops will focus on wastewater treatment and reuse for agro industries at community level. By this it will follow the processing chain from primary production to first processing levels (diary, olive manufactures, sugar factories, distilleries etc.). Moreover this focus will enable the consideration of a sufficient collection level of waste water (both from households as well as from processing factories/manufactures).

Combining an identification of possible solid and aquatic wastes from agricultural products processing with the innovative extraction of high valuable chemicals (e.g. phenols) and other materials for biorefinery, these products are expected to provide new market opportunities, and hence economic opportunities.

In the following the main Water4Cops objectives are listed:

• Production of water apt for irrigation from wastewater (food-processing, domestic or biorefineries) and return the nutrients as fertilizer to the land.
✓ Recovery of specific high added value products from agrofood and domestic wastewater (e.g. polyphenols), anaerobic conversion of waste water components into organic acids, alcohols coupled with in situ product recovery, production of bioplastics (PolyHydroxyAlcanoates) from high carbon wastewater, and energy recovery from the final treatment.
✓ Development of simple and cheap microbial monitoring methods and/or protocols to control the irrigation water quality in terms of pathogens coontent.
✓ Optimized domestic wastewaters treatment for enhancing their safe agricultural reuse paying particular attention to minimize sludge production, and to the management of sustainable plant-based systems such as constructed wetlands in terms of improved purification capacity and suitable plants selection.
✓ Development of improved irrigation products, systems and strategies, coupling of irrigation systems with soil moisture control and modeling in saline conditions, and provide an accurate estimation of crop water requirements using new technologies for area based actual evaporation and soil moisture measurements.
✓ Modeling the impact of using poor quality water on crop and soil quality.
✓ Improved water use efficiency at field level through genomics and breeding;
✓ Development of a green economy by transdisciplinary co-creation of agri-business opportunities and water biotreatment and evaluation and optimization of the proposed combinations of water processing from a perspective of supporting the green economy;
✓ Stimulate cross-fertilization and knowledge transfer between the individual work packages and activities in Europe and India;
✓ Disseminate the newly developed technologies, the new economical concepts and local businesses demands and exchange the experience between India and Europe on advancing the Green Economy.

• References

- CIHEAM-IAMB, 2007, Vision document, Priorities, goals and targets for future research on water management in Mediterranean agriculture, International Conference on Water Saving in Mediterranean Agriculture and future Research needs. WASAMED (Water Saving in MEDiterranean Agriculture) EC- RTD Framework Programme 5th.
- EU COM (2007) 414 final. COMMUNICATION FROM THE COMMISSION TO THE EUROPEAN PARLIAMENT AND THE COUNCIL. Addressing the challenge of water scarcity and droughts in the European Union. {SEC(2007) 993}{SEC(2007) 996}
- Kundzewicz, Z.W., L.J. Mata, N.W. Arnell, P. Döll, P. Kabat, B. Jiménez, K.A. Miller, T. Oki, Z. Sen and I.A. Shiklomanov, 2007. Freshwater resources and their management. Climate Change 2007: Impacts, Adaptation and Vulnerability. Contribution of Working Group II to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change, M.L. Parry, O.F. Canziani, J.P. Palutikof, P.J. van der Linden and C.E. Hanson, Eds., Cambridge University Press, Cambridge, UK, 173-210.
- Rosegrant, M., X. Cai, S. Cline. 2002. World Water and Food to 2025. Dealing with Scarcity. -Washington D.C. International Food Policy Institute. International Water Management Institute (IWMI). 2000. World water and climate atlas. http://www.atlas.htm
- UNDP, 2006: Beyond Scarcity: Power, Poverty, and the Global Water Crisis. Human Development Report 2006. United Nations Development Program, New York.

Project Results:

NOTE. All the results obtained within each work package of the project concerning specific issue investigated have been reported in detail in the associated deliverables. Below, they have been just summarized.

WP1 - Valorization, treatment and reuse of agrofood industry wastewaters

Main objectives of WP1 were: Development of integrated processes for the treatment and valorization of wastewaters heavily loaded in BOD/COD and nutrients, resulting from biomass treatment processes (e.g., food processing, biorefineries), leading to the obtainment of (a) effluents suitable for irrigation purposes, (b) nutrients originally occurring in the treated wastewaters (to be employed in land fertilization) and (c) valuable chemicals (e.g., organic acids, bioplastics (PHA) and/or alcohols),originally occurring in the wastewater or obtained by dedicated biotransformation processes of the wastewater organic matter.

To achieve its objectives WP1 was structured in four Tasks:

Task 1.1 Evaluation on the exploitation of the target wastewaters
Task 1.2 Combinatorial polymers for the specific recovery of valuable phenols from water
Task 1.3 Assessment of reductive treatment train for valorization of biorefinery wastewater
Task 1.4 Development of a multipurpose and modular biorefinery for the obtainment of suited water for irrigation and valuable products for agriculture from olive mill wastewaters


Two types of wastewater were selected for further study: olive mill wastewater (OMW) and biorefinery wastewater (BRW). Their exploitation potential was evaluated through the collection and assessment of physical, chemical, biochemical and microbiological features, data related to disposal costs and the possibility of employing the wastewater as the feedstock for full-scale applications. The information was compiled in two literature reviews.
Usually, anaerobic digestion is applied as a first treatment step for these highly loaded waters yielding biogas. While accumulation of the intermediate volatile fatty acids (VFA) or carboxylates is not desirable in regular anaerobic digestion, it can be exploited in the carboxylate platform for the bio-based production of fuels and biochemicals. This represents a more advanced valorization strategy from practical, economic and sustainability viewpoints as it can generate multiple valuable products from a single feedstock. This cascaded approach was investigated for both selected wastewaters.

Biorefinery wastewater (BRW)
Experimental work proved that the following approach was well suited for BRW valorization:

• production of VFA and their further conversion to medium chain fatty acids (MCFA)
• recovery of target products through integrated membrane-based reactive extraction.

Production of short and medium chain carboxylates through acidogenic fermentation
In view of efficient recovery of VFA from a complex wastewater stream, the product concentration should be as high as possible. Therefore, undiluted thin stillage was selected as feedstock for the continuous fermentation tests, which were executed under varying operational and environmental conditions. Maximum VFA levels of 20 g/L were reached at a Hydraulic Retention Time (HRT) of 15 d and a pH of 4.9. Taking into account that no protein hydrolysis seemed to take place and that sugar conversion stabilized at about 80%, it was assumed that no higher VFA levels can be reached on this substrate.
In addition to total VFA levels, their composition is important as well in view of recovery and purification. A higher organic loading rate typically led to a more equal distribution of products, which may be undesirable from a product recovery and purification point of view. Two periods were identified with 1 major product: production of 120 mM propionate (10 g/L) at HRT 15d and pH 4.9, and production of 80 mM of acetate (5 g/L) at HRT 15d and pH 5.4. From a separation perspective, the first case would be the optimal one.
Interestingly, chain elongation towards more valuable MCFA, e.g. valerate and caproate was temporarily obtained under certain conditions. Because some inhibition effects can be expected at elevated VFA levels (particularly for the MCFA and depending on the degree of dissociation), integration of in-situ product recovery (ISPR) techniques was attempted to alleviate inhibition effects and simultaneously achieve maximal product recovery.

Recovery of carboxylates through extraction
Taking into account the characteristics of the product, the desired selectivity and the operational environment, extraction approaches are well suited for organic acid recovery. Reactive extractants based on extractant-diluent combinations demonstrated higher extractive yields and extraction efficiency increased with increasing chain length. Aliquat 336 resulted in the highest extraction efficiency among the tested extractants. In addition, this quaternary ammonium was able to extract both the undissociated and dissociated forms of the acids. Differences among the tested diluents were not significant. The optimal VFA:extractant molar ratio was defined as 1:2. Among the tested regeneration solutions, VFA recovery was most successful using 1 M NaOH. At pH 4.5, 82% of VFA was extracted using 0.5 M Aliquat 336 in methyl octanoate of which 77% could be regenerated from the organic loaded phase. Because of potential toxic effect of the reactive extractants on the fermentation, membrane-based reactive extraction (MBRE) was proposed as ISPR concept. The coupling of MBRE with the fermentation was initially not successful and only little extraction of VFAs was observed. The solution to the problem consisted of strongly increasing the membrane surface area. The total VFAs level in the fermentor then gradually decreased after multiple integrated tests, with a spectrum enriched in 63% acetic acid and 37% propionic acid. The MCFA were enriched in the organic phase. This approach thus allows to introduce selectivity in acid removal.
Further experimental work showed that adiabatic extraction could be an alternative for VFA recovery, though only at acidic pH levels.

Olive Mill wastewater (OMW)
A modular integrated concept for decontamination and valorisation of OMWs was investigated aimed at obtaining irrigation waters together with bio-energy and chemicals, i.e. phenolic compounds (PCs), polyhydroxyalkanoates (PHAs), and fertilizing materials. A simplified flow sheet of the integrated process has been developed and designed. The flow sheet has some degree of flexibility that allows adapting to different conditions and economical constraints.

Recovery of polyphenol mixtures through adsorption
Based on its high selectivity and a high adsorption capacity for polyphenols, the commercial resin AMBERLITE XAD16 was selected for the development of an optimized pilot scale process. The adsorption process was characterized through the determination of isotherms and the study of the fluid dynamic behavior. For the desorption step, the use of acidified ethanol as the solvent led to high polyphenol recovery from the sorbent. A continuous plant was then designed and assembled. Smooth operation of the packed bed column required an effective OMW pre-treatment with high removal of suspended solids and the implementation of a newly developed column packing method. Optimization of the adsorption/desorption process led to: i) a 4-fold increase in adsorption column length and ii) the choice of the appropriate superficial velocity to increase the efficiency of the process. Mass balances over a sorption/desorption cycle at a chosen breakpoint of 0.2 showed 92% and 74% absorption and desorption yields respectively with an adsorption bed utilization efficiency of 42%. Separation performances were stable during 4 sorption/desorption cycles. A 1-D axial dispersion model with mass transfer described the processes well. Process simulation is therefore expected to allow a further optimisation of the system and a model-based identification of the optimal operational conditions for the industrial process.
The mix of PCs obtained after the desorption step showed a very high antioxidant capacity, which increases the value of the obtained product. However, despite the good selectivity of the chosen sorbent, the purity of the final product is expected to be <30%, possibly requiring a further purification step depending on the specifications requested.

Specific recovery of valuable individual phenols through combinatorial polymers
Since the extraction process with commercial resins does not allow differentiation between the different phenolic compounds, there is potential to further refine it by employing molecular imprinting polymers (MIPs) to extract specific polyphenols from raw OMW or from PCs mixtures, obtained after desorption from XAD16. Several nanostructured cyclodextrin-based polyurethanes (CDPs) were tested as recognition materials for specific recovery of phenolic compounds. The production protocol was successfully optimized at lab-scale and several adjustments were carried out to allow the sustainable production of the polymer including: reduction of toxic catalyst, change of toxic solvent, change of isocyanate monomer, reduction of energy consumption. These modifications allow the production at pilot scale (Kg) of the desired CDP at low cost with a simple synthetic procedure.
The process of adsorption and desorption of phenolic compounds with CDPs was effective at pilot scale. Raw OMW was used after a centrifugation and filtration step and no problem of CDP saturation was observed over several adsorption/desorption cycles. The CDP showed a high selectivity towards tyrosol, so it can be used to separate this valuable compound from complex organic mixtures. The use of a solvent gradient for desorption was a good strategy to increase tyrosol and hydroxytyrosol purity to >60%. The COD level in dephenolized wastewaters was reduced 5 times, making them relevant for irrigation purposes.
The fractional recovery of phenolic compounds allowed not only to select the samples with higher purity but also to discard samples with very low concentration of phenolic compounds of interest. The latter can then be used for irrigation purpose or for production of e.g. PHA.

Production of PHA through an integrated anaerobic-aerobic process from dephenolized OMW using a single bacterial strain or a mixed microbial culture
The first step of the process consists of bioconverting the organic load of (dephenolized) OMW into VFA, which represent a suitable substrate for PHA accumulation in bacteria. To this aim, wastewaters were anaerobically digested under acidogenic conditions. Both immobilized and freely suspended cell systems resulted in significant OMW acidification, but the continuously operated biofilm system could achieve this at lower residence times. Each acidogenic process led to a comparable VFA product spectrum.
Conversion of acidified OMW into PHA was investigated at bench-top scale by employing either a pure Cupriavidus necator strain or mixed microbial cultures (MMC), enriched in PHA accumulating organisms due to a feast-famine regime selection pressure. While pure cultures typically achieve higher PHA content and productivities, MMC do not require sterile conditions and are better adapted to changes in operational (e.g. seasonal) conditions.
The feasibility of both approaches was demonstrated. The pure culture could accumulate PHA in a two-stage batch process, in which pregrown cells of C. necator were fed with an acidified dephenolized OMW. The maximal PHA content of 47% (w/w) of cell dry weight, was obtained when the pretreated wastewater was 4-fold diluted in the accumulation phase.
PHA production from OMWs using MMC yielded very high PHA concentrations, corresponding to 50% (w/w) polymer content in the biomass. Interestingly, PHA storage also occurred in the presence of phenols, provided that both the step of MMC selection and of PHA accumulation were fed with non-dephenolized OMW. This offers the possibility to consider this strategy as an alternative process for PHA production from OMW, because the phenolics removal step leads to a loss of part of the COD contained in OMWs which could be potentially diverted towards further PHA production.
Finally, an alternative solvent-free method was developed to extract and purify PHAs. The application of acidic pre-treatment enhanced the purification degree.

Biomethanization of residues
Liquid and solid side-streams from the different process steps could be exploited for biomethane production. Dilution of the solids using a low concentrated liquid effluent, e.g. dephenolized water, allowed to limit phenol inhibition phenomena. The anaerobic digestion step then produces methane, a dephenolized water with low COD content that is suitable for irrigation and a solid that can be used as soil amendment and fertilizer.
WP2 - Innovative municipal wastewater bio-treatment for agricultural reuse

WP2 was aimed at developing innovative biotechnologies for the treatment of municipal wastewater to produce water suitable for reuse in agriculture.
Objectives of WP2 were to develop new and sustainable solutions based on technological and natural plant-based systems to obtain effluents with adequate quality for irrigation. Solutions particularly suited for decentralized applications at small/rural communities or villages were specifically addressed. Pathogens monitoring, minimization of sludge production and field scale testing were also targeted.

WP2 was structured in two technical Tasks: Task 2.1 (“Technology based treatments”) and Task 2.2 (“Plant based systems”). In turn, each task was organized in four Sub-tasks.


Sub-task 2.1.1 (Pilot scale investigation on biotreatment coupled to surface filtration for agricultural effluent reuse) tested pilot scale biotreatment coupled to surface filtration for agricultural effluent reuse. Activities were aimed at testing non-conventional technologies for the treatment of municipal sewage to produce effluents suitable for reuse in irrigation. Tests were performed at the pilot scale, and two experimental plants were operated in order to evaluate the suitability of the adopted technologies and the quality of produced effluents with respect to current national water quality standards for reuse in agriculture. Both the tested technologies were based on coupling biological processes and surface filtration, and two large experimental installations located at the municipal wastewater treatment plant of Castellana Grotte (Apulia Region, Southern Italy) were operated. Each of the two pilots was made of two parts: 1) a process based on surface filtration which treated a continuous flow; 2) an UV disinfection system treating only the effluent fraction used for irrigation and operated on demand.
The first plant was based on the new technology IFAS-MBR (Integrated Fixed-film Activated Sludge – Membrane BioReactor), and treated raw sewage after preliminary screening. The second pilot plant was based on the FDG (Gravity Disk Filter) technology, and treated a fraction of the effluent taken downstream the secondary settling tank of the main wastewater treatment plant. The effluents of both plants were accumulated into storage tanks and then submitted to UV disinfection, operated on demand (when the irrigation line was switched on). Considering the importance of the faecal indicator Escherichia coli for the reuse of treated wastewater in agriculture, some experiments were also performed at the lab scale to evaluate the fate of this parameter in soil. A set of soil columns was operated and monitored for E. coli concentration over time and along depth.
The adopted technologies were selected based on two prevalent criteria: The possibility of nutrient conservation, and the sustainability in terms of cost of operation and management. In particular, the IFAS-MBR technology was considered appropriate for the complete treatment of municipal sewage and the production of effluents suitable for irrigation. Indeed this technology limits the sludge production by favouring the growth of biomass onto the carriers as biofilm, thus reducing the operational costs related to sludge handling and disposal. At the same time, appropriate management of the biological processes may ensure nitrogen conservation in the form of nitrate, and provide high removal of suspended solids, COD, and microbial contamination. IFAS-MBR is a highly technological wastewater treatment option, considering the need of accurate monitoring and automation for the operation of the biological and membrane filtration processes. On the other hand, the FDG technology was selected for the tertiary treatment of secondary effluents due to its conceptual simplicity and operational cost-effectiveness. As a matter of facts this “passive” filtration system mostly operates under gravity, thus it has extremely limited electric power requirements. It can be considered a low-tech treatment technology, nevertheless providing good performance with respect to the main parameters that are relevant for effluent reuse. Both these filtration-based technologies were coupled with a disinfection system. The selection of the most suitable disinfection process was made on the basis of two main requirements: Operational flexibility and no disinfection by-products generation. As a matter of facts, when nitrogen and organic concentrations are considered acceptable (and beneficial) in water for irrigation, the possible formation of toxic chlorinated by-products (chloro-amines, trihalomethanes, other chlorinated organics) needs to be avoided. Therefore chlorine-based disinfection systems should not be adopted in these cases. Furthermore, considerations related to cost and dosage (or over-dosage) drove the choice towards UV based disinfection. The well-known limited persistence of the UV disinfection effects over time suggested to apply this tool under a non-conventional operational scheme. Therefore, the UV systems were connected to the irrigation pumps, allowing the lamps to be switched on only when the treated water was sent to the field (“on-demand” disinfection). This operational strategy was very effective both for contrasting the bacterial regrowth possibly occurring within the effluent storage tanks placed downstream the IFAS MBR, and ensuring disinfection to the water stream produced by the FDG, provided sufficient quality of the latter in terms of clearness and transparency.
It is relevant to stress that the whole approach in Sub-task 2.1.1 was oriented towards the production of water, rather than the disposal of effluents. The adoption of technologies allowing for an effective removal of the main chemical and microbiological contaminants possibly providing nutrient conservation was critical with specific reference to reuse in irrigation.
Long term testing at the pilot scale showed the effectiveness of both tested technologies in producing effluents suitable for reuse in agriculture and complying with the local standards. Soil column tests showed the decay rate of the faecal indicator as the dominant mechanism with respect to accumulation and possible leaching through the deeper soil layers.
The experimental activities carried out within the project provided very relevant insights on the effectiveness of these technologies, their suitability to the objectives, and operational sustainability. The IFAS-MBR confirmed high effectiveness in producing effluents of excellent quality and suitable for irrigation in terms of chemical and microbiological characteristics. Nevertheless, the overall complexity of the combined treatment and the difficulties encountered in maintaining steady operation under variable wastewater characteristics suggested that this technology could be appropriate for very high added value applications, where higher water production costs can be sustained. The FDG technology confirmed its robustness and showed operational reliability even when the upstream processes (full scale wastewater treatment plant) did not perform perfectly. However, the performance of this technology is strongly related to the quality of filter materials and their assemblage. Cloths should be preliminary tested for their filtering characteristics and their fitting to the system’s mechanical parts requires particular care. The “on demand” UV disinfection revealed very effective in removing the faecal contamination indicators included in the current regulations for treated wastewater reuse in irrigation. As a matter of fact, parallel agronomic investigations revealed no microbial contamination of soil and crops in parcels irrigated with the reclaimed effluents. This strategy ensuring lower operation cost and higher effectiveness of the disinfection process can be considered optimal for all situations of discontinuous delivery of treated wastewater. The investigation on the fate of the faecal indicator Escherichia coli also provided interesting results and indicated limited persistence of this indicator in the environment. These results should drive the attention towards the suitability of current quality standards in the field of treated wastewater reuse, and suggest that possible revisions of current regulations should carefully consider aspects including sustainability of strategies and technologies, resource conservation, environmental protection, and public health.

Sub-task 2.1.2 (Wastewater reuse by an innovative reactor with low environmental impact) has evaluated a recently developed innovative reactor whose acronym is SBBGR (Sequencing Batch Biofilter Granular Reactor). Compared with conventional wastewater treatment systems, SBBGR is more compact and flexible and reduces sludge production and treatment costs up to 80 and 40%, respectively. The overall objective of this sub-task was the evaluation of SBBGR effectiveness for treating and reusing in agriculture wastewater produced by small and/or rural communities. The effectiveness of the SBBGR system and its integration with different disinfection strategies (UV irradiation, PAA addition) was evaluated at the pilot scale. The main results obtained are below summarized:

✓ Biological treatment by SBBGR was able to produce an effluent with a physical and chemical quality that always conforms to the stringent standards required in Italy for agricultural reuse.
✓ Despite the compactness and simple scheme, the SBBGR effluent showed a microbiological quality higher or at least comparable with that of conventional municipal wastewater treatment plants.
✓ The average E. coli content in the effluent of SBBGR was lower than 103 MPN/100mL. Thus, the SBBGR effluent was in line with the quality criteria indicated by the WHO guidelines for agricultural reuse (lower than 103 CFU/100mL; WHO 2006) and with the Italian requirements for discharge in water bodies (5 103 CFU/100mL).
✓ The integration of SBBGR with SSF allowed a further increase of microbiological quality of the water to be achieved for all the monitored parameters. This treatment was able to produce an effluent quality compatible with its safety reuse in agriculture and also complying with several present regulations about wastewater reuse without the addition of further disinfection treatments. Nevertheless, it still did not meet the very strict limit established by Italian regulation for E. coli (10 CFU/100mL).
✓ Both investigated disinfection processes (i.e., UV and PAA) were very effective and showed similar results for total coliforms, E. coli and somatic coliphages. However, they had no effect on Clostridium perfringens spores.
✓ UV radiation and PAA doses as low as 40 mJ/cm2 and 1 mg/L respectively were able to reduce E. coli content in the final effluent below the limit for agricultural reuse in Italy (i.e., 10 CFU/100 mL).

Municipal wastewaters represent an important source of micropollutants, such as pharmaceuticals, personal care products or pesticides. In order to minimize the introduction of these chemicals into the environment, technologies for their elimination beyond the capabilities of the conventional biological treatments are being developed in recent years.

Within Sub-task 2.1.3 (Immobilized enzymes as biocatalysts in tertiary treatments), the removal of bisphenol-A (BPA) from the effluent of a municipal wastewater treatment plant (WWTP) by immobilized laccase enzyme was tested in a continuous pilot unit. The immobilization of the cheap laccase enzymes onto mesoporous nanomaterials was investigated in order to produce nanobiocatalysts (benchmark 100-150 CHF/kg) suited for the treatment of “cocktails” of micropollutants in biologically treated wastewater. The nanobiocatalysts were applied in a pilot scale post-treatment membrane bioreactor system at the wastewater treatment plant Birsfelden (Switzerland) to remove micropollutants from biologically treated municipal wastewater and upgrade the effluent quality to a level suitable for reuse.
The membrane allowed efficient and reliable retention of the nano-material carrying the enzyme into the reactor. It was possible to operate the MBR at high concentration of suspended solids (up to 12 g L-1 total solids) with low membrane fouling rate. Correspondingly, the bioreactor could be operated at high enzymatic activity. However, certain loss of activity could not be avoided and addition of new immobilized enzyme was necessary. The concentration of BPA in the bioreactors was always low but the removal rates could not be directly and precisely evaluated, most probably due to the transformation of the compound to another form during the storage of the samples and/or to possible contamination due to tubing system.

The definition and evaluation of bio-molecular methods for the quantification of biological contaminations in treatment systems for wastewater reuse was performed within Sub-task 2.1.4 (Microbiological quality assessment of treated wastewater). In particular, the main results achieved are related to the definition and evaluation of complete protocols for the quantitative estimation by Q-PCR of ARGs and pathogens in water samples with different contamination levels, including the following steps:
✓ Evaluation of sample concentration and DNA recovery protocols for different water contamination levels;
✓ Evaluation and monitoring of Q-PCR assays performances (amplification efficiencies sensitivity and precision);
✓ Analysis of the factors affecting Q-PCR sensitivity raw and treated wastewaters (assays sensitivity, endogenous DNA, QPCR inhibitory compounds);
✓ Definition of Salmonella and ARGs lower quantifiable level by QPCR in water samples with different contamination levels;
✓ Assessment of ARGs and Salmonella presence and diffusion in raw and treated wastewater from some of the treatment systems tested for water reuse within the project;
✓ The defined and optimized methods contributed to assess the performance of some of the wastewater treatment solutions tested within the project in terms of required hygienic aspects.
✓ In conclusion, Q-PCR efficiently quantified ARGs over a wide range of concentrations (102-109 GC/ml) in water with different levels of contamination. Q-PCR was instead not sensitive enough for monitoring less concentrated target genes. The lower quantifiable Salmonella level in raw and treated wastewater ranged between 102-103 and 10-102 GC/ml respectively. Sample characteristics influenced the QPCR quantification limit that could not be reduced any further unless inhibiting compounds are removed or target DNA is specifically enriched (i.e. immunomagnetic separation).
✓ The following advantages and limits of QPCR analysis for the quantitative monitoring of biological contamination in wastewater treatment systems were shown:
✓ Good performance of the optimized concentration extraction protocols were obtained (high DNA extraction and sufficient volume could be concentrated on 45mm filters for a large number of Q-PCR reaction).
✓ The volume of untreated or treated wastewater samples that can be analysed by QPCR is limited by the sample DNA content; therefore, concentration of larger sample volumes will not improve the sensitivity of the method.
✓ Inhibitory compounds and endogenous DNA can largely affect Q-PCR quantitative detection of target gene in the analysed samples. High variation of inhibition was observed indicating that inhibition should be checked for each the analysed samples
✓ Q-PCR inhibition could be efficiently removed by sample dilutions but with a consequent reduction of method sensibility (10-100 times) and increased cost of the analysis.
✓ Minimal requirements for an efficient and reliable QPCR quantitative detection of biological contaminants (filterable volumes, QPCR inhibition monitoring, maximum non inhibitory template DNA, lower detectable level) were defined for the analysed raw and treated wastewater.

Sub-task 2.2.1 (Improved hygienization of Constructed Wetlands) assessed the effectiveness of constructed wetlands (CW) as hygienisation system for pre-treating municipal wastewater to be upgraded for reuse purposes. Moreover, technological variations within CW systems (e.g. different wastewater flow regimes and aeration) and treatment enhancements via downstream additions such as lagooning, slow sand filtration, black peat filtration, UV illumination were addressed. Further aspects of hygienization such as water loss via evapotranspiration as well as the fate of antibiotic resistant bacteria were also investigated. The performances of various types of CW and systems integrated with other advanced technologies were evaluated also on the basis of the removal of faecal indicators rather than of specific etiological agents.
Partner UNICT assessed the hygienization efficiency of a horizontal sub-surface flow constructed wetland (HSSF-CW) unit in Sicilia. Its effluent was treated by either UV disinfection or lagooning. The system was monitored during 12-month sampling campaigns; four types of samples were collected: (1) raw wastewater; (2) CW effluent; (3) tertiary effluent after lagooning and (4) tertiary effluent after UV treatment. The UV treatment achieved substantial removal of faecal indicators (around 6 log units) while some improvement in microbiological water quality was achieved by combining CW with the lagooning system (about 3 log units).
Partner TUC investigated the same system used by Partner TMS (ex Envinhealth) also concerning Enterococci removal. Furthermore, partner TUC developed a fast and sensitive methodology for the detection of enteroviruses and adenoviruses in raw sewage and treated effluents. The procedure is being further tested on samples from the Heraklion CW system, and the removal efficiency of the system for these viruses is still being investigated. This system was also examined for the presence of bacteriophages (MS2 Coliphages) and their correlation with viruses.
Partner TMS also examined the removal of faecal indicators (Total coliforms and E. coli) in novel system (HSSF-slow sand filter-inactivate unit) as well as in the new established constructed wetlands [free-water surface (FWS) and vertical flow wetland (VFW)]. In the novel system, results showed an overall reduction of about 2 log units for both examined microbiological parameters. Total coliforms concentration in the inlet of the system was 6.1 log units (MPN/100 ml) while in the outlet it was 4 log units (MPN/100 ml). Similar the concentration of E. coli reduced from 5.4 in the inlet to 3.3 log units (MPN/100 ml) in the outlet of the system. Results from free water surface wetland planted with Juncus acutus showed limited removal efficiency for both pathogens. On the other hand VFW planted with Atriplex halimus reduced total coliforms and E. coli by about 2 log units.
Partners Vita34 and UFZ worked on improving wastewater treatment by CW via integrating slow sand filtration. Vita34 observed a germ reduction below detection limit within the pre-treated wastewater after passing the SSF. Partner UFZ completed the evaluation of the 4 various pilot-scale slow sand filter (SSF) units that were set-up during the first reporting year. Results showed that only the static cascade and the rotating cascade systems complied with European standards for E. coli and Enterococci water concentrations, achieving mean log removal of 2.7-4.7 and 2.1-2.4, respectively. The important contribution of the “Schmutzdecke” to the removal of faecal indicators was highlighted in a second study on standard SSF columns of various grain sizes. Partner UFZ completed the characterization of predatory microbes that contribute to pathogen removal in such treatment systems. Furthermore, in-depth molecular microbiological evaluation of wastewater treatment in standard horizontal sub-surface flow (HSSF) constructed wetlands and intensified HSSF constructed wetlands continued. In conclusion, adequate removal of faecal indicators may be achieved by (i) combining domestic wastewater treatment in traditional CW types (i.e. HSSF, VFW and FWS) with additional treatment such as UV irradiation or slow sand/black-peat filtration; (ii) in newly developed CW systems with different configurations and operational strategies, like aerated and tidal operated CW. The results obtained show remarkable bacteria removal efficiencies. Further assessments and mechanistic interpretations of indicator bacteria removal will guide the way to significant pathogens removal in these newly developed units. Of course, now detailed cost analyses need to be carried out.

Sub-task 2.2.2 (Investigation of CW hydraulics and hydrology) was aimed at investigating hydraulic and hydrological aspects of constructed wetlands and, in particular, at quantifying the evapotranspiration dynamics (with regards to weather conditions, seasons, vegetation types and growth) and at determining the mechanisms and consequences of clogging in CW (with regards to substrates and operation conditions). Specific objectives of were: -) evaluation of the dynamics and the effect of the evapotranspiration in CWs; -) characterization of clog material; -) evaluation of clogging’s effects on the wetland hydraulic properties by the analysis of residence time distribution functions (RTDs) and measure of hydraulic conductivity of gravel substrates. The following experimental activities were performed:

✓ UNICT carried out research activities both at full-scale and pilot-scale horizontal sub surface flow (H-SSF), located in Eastern Sicily, used for the tertiary treatment of secondary effluent from a conventional wastewater treatment plant.
✓ TUC – ENVINHEALTH carried out the research activity in HSSF CW, followed by a long slow sand filter, located in the City of Heraklion (Greece).
✓ UFZ carried out the research activity in H-SSF CW (aerated and non) located at the UFZ Ecotechnology Research Facility in Langenreichenbach.
✓ PHYTOREM carried out the research activity in Bamboo Vegetation Filter (BVF), located in the city of Saint-Philippe (Reunion Island).

The methods applied for the evaluation of clogging phenomena and for the evaluation of water losses in CW by each partner have been the following:

✓ UNICT estimated and compared the saturated hydraulic conductivity by the Falling Head method in the three H-SSF CWs at full-scale and in five H-SSF CWs at pilot-scale. Solid samples were taken and were analysed once in 12 sampling points in each of the three H-SSF CWs at full-scale. Three tracer tests were performed in the three H-SSF CWs at full scale (one for each bed) by using NaCl as tracer. From the experimental breakthrough curves of each tracer test, actual residence times (τ), variance (σ2) and dispersion number (D) were calculated. Finally, two methods (simplified water balance and energy balance) were used to estimate the evapotranspiration both at full-scale and pilot-scale horizontal sub surface flow (H-SSF).
✓ TUC and ENVINHEALTH analysed the influence of clogging phenomena by means of one tracer test by using NaCl as tracer.
✓ UFZ estimated the evapotranspiration by simplified water balance.

The main outcomes are synthetically reported below:
Hydraulic aspects
Hydraulic conductivity measurements, clog matter characterization and flow paths visualization provided useful information on the extent of clogging and on hydraulic aspects of constructed wetland systems. In particular, even if the older CW (H-SSF2) seems to be rather clogged, since:
- its mean hydraulic conductivity was lower than both the younger CWs (H-SSF3 and H-SSF4),
- the Total Solids (TS), Volatile solids (VS) and Belowground plant biomass (BGB) concentrations are higher than those of younger CWs,
- several stagnant zones and preferential flow paths were detected by means of tracer tests,
- its treatment capacity remained largely unchanged after eight years of operation confirming the high reliability of CWs for wastewater treatment.
Hydrological aspects
In Langenreichenbach, during summer time (June/July), ET of Phragmites australis amounted to 21% (28.7 L m-2 d-1) of the total water for the aerated CW and to 45% for the non-aerated CW.
In Sicily, during summer time, the water loss through ET process was 4% (13 L m-2 d-1) at M. giganteus bed, 5% (16 L m-2 d-1) at V. zizanoides and at Arundo donax bed and 9% (29 L m-2 d-1) at P. australis beds, regard to a mean influent flow rate. The simplified water balance method and the energy balance method to estimate ET showed very similar results.

As for the Sub-task 2.2.3 (Optimizing heavy metals and N removal in CW), it was aimed at assessing the performances of the rhizofiltration technology for the removal of heavy metals from polluted waters through CW pilots using two selected halophytic wetland plants. The results from the constructed wetlands pilots revealed that both halophytes-CW are able to reduce hexavalent chromium, cadmium, nickel and zinc concentrations from waters to levels well beyond the limits for wastewater reuse in EU.
Referring to Cr(VI) removal, both systems were able to reduce the hexavalent chromium concentration of the medium below the detection limit of the analytical method with the highest and fastest efficiency performed by Juncus acutus-CW, followed by Halimione portulacoides-CW. Cr content analysis in plant tissues revealed that J. acutus accumulated around 43% of the metal added and retained by the system while H. portulacoides accumulated 14.7% of the total Cr(VI) added and retained by the system and the main accumulation site of both plant were their roots. Visual observation of both plants species health status during the experiments supported that they both are Cr(VI)-tolerant plants.
These results lead to the investigation of J. acutus endophytic bacteria role on plant’s tolerance to Cr(VI) toxicity, and it was found that the plant’s endophytic community consists of high-Cr(VI) tolerant strains which also have the remarkable ability to reduce Cr(VI) to Cr(III) within the plant tissues, and hence, contribute to plant detoxification from Cr(VI) toxicity stress. For typical non-redox sensitive heavy metals (Cd, Ni, Zn) removal, again both CWs were able to treat heavy metal polluted waters and they both achieved similar efficiencies. J. acutus-CW was found able to achieve 84% average reduction of the influent Ni and 92% average reduction of the influent Zn while for Cd it achieved total reduction. H. portulacoides-CW achieved 79% average reduction of the influent Ni, 92% average reduction of the influent Zn and again total reduction of the influent Cd. Moreover, visual observation for toxicity symptoms of both plants species during the experiments proves that they both are heavy metal tolerant plants. Moreover, further investigation of the J. acutus capacity to receive and accumulate typical non redox sensitive metals through the performed J. acutus wetland soil experiments revealed that the plant is not a heavy metal hyper-accumulator, but it is a highly heavy metal-tolerant plant that can accumulate the metals to some extent and could be used in phytoremediation strategies of mixed heavy metal pollution.
As for the work done regarding N removal in CW, the following aspects were investigated: i) the role of vegetation (unplanted, Phragmites australis, Typha latifolia) in treatment efficiency, ii) the variations in the abundance of the main functional groups involved in N cycling (ammonia oxidizing archaea (AOA) and bacteria (AOB), denitrifiers, anammox) to understand and evaluate treatment processes and iii) the variation of total microbial community structure in order to identify possible treatment and seasonal effects.
The findings indicated nitrification-denitrification as the principal route of N removal in CWs, while anammox did not have a strong contribution. Evidence was also arisen that ammonia oxidizing archaea (AOA) contributed on NH3 oxidation. Overall, plant species had a weak effect on the abundance of N functional genes (amoA of AOA), but it strongly affected the performance of CWs in terms of N removal in the following order: unplanted < Phragmites communis < Typha latifolia. These findings suggest that plant species stimulate N removal by upregulating the rates that the responsible biochemical pathways operate, probably by increasing O2 supply. In addition, our study revealed differences in indicators linked to N2O emissions. The abundance of clade II nosZ genes remained low across the season scaling down a strong contribution in the reduction of the emitted N2O. The increasing ratios of nosZ/nirS and nirS/nirK with the progress of season indicate a shift in the composition of denitrifiers towards strains with a lower genetic potential for N2O release. Similar trends were observed among the treatments but the mechanisms differed. The planted treatments stimulated an increase in the nosZ/nirS ratio, while the unplanted an increase in the nirS/nirK ratio. With regard to total microbial community structure and composition, the application of the influent disturb the composition of the community which in turn started to re-stabilized, at the new nutrient conditions, at least after 3 months (between 3rd and 4th sampling). As a consequence, any plantation influence in the community composition compressed under that nutrient "shock". After the influent application γ-proteobacteria dominated in the community regardless treatment, while after three months, unplanted tanks and those planted with P.australis had a rapid increase for β-proteobacteria. T.latifolia planted tanks reached this levels of β-proteobacteria after five months. Studies have suggested that members of both bacterial phyla are key players in denitrification in municipal and industrial activated sludge processes. With regard to β-diversity, sampling time found to explain up to 50% of the community variably, while COD and NO3--N were the main variables that influence the variation of the microbial community composition.

Within Subtask 2.2.4 (Innovation in CW design) partners TMS and Vita34 investigated on two novel CW systems the performances of: 1.Halophytes plants (TMS) and 2.Floating mats (Vita 34).

1. Halophytes (TMS). Activities included testing of novel systems with halophytes for their potential to remove organic matter, nitrogen, phosphorus and pathogens (Total coliforms and E.coli as indicators) from domestic wastewater. In particular the tests included:

✓ three different types of CW: Free water surface (FWS), horizontal subsurface (HSF) and a vertical (VF) CW,
✓ five different halophytes: Tamarix Parviflora, Limoniastrum Monopetalum, Junkus Acutus, Sarcocornia Perennis and Atriplex Halimus,
✓ post-treatment with a slow sand filter and a novel inactivation Unit with TiO2-coated materials (pumice and expanded clay aggregate).

All experiments took place at an experimental pilot plant located in Heraklion, Crete, South Greece.
At the beginning, a literature search was performed to select appropriate halophytes for horizontal subsurface CW. In addition, previous results obtained by Technical University of Crete (TUC) about the potential of several halophytes on heavy metal phytoremediation were took under consideration during selection phase.
In order to polish the effluent from HSSF CW, a slow sand filter (SSF) and an inactivation unit (IU) was designed and operated. SSF was constructed according to suggestions by UFZ. The IU was designed and constructed based on the technology of solar photocatalytic disinfection. It contained a series of low-depth plastic canals containing TiO2-coated low cost material (pumice and expanded clay aggregate). At the end of 2014, a free water surface and a vertical flow CW were planted with halophytes. These systems operated receiving primarily treated wastewater from wastewater treatment plant of the city of Heraklion, Greece. Monitoring started in January 2015 and ended in May 2016. The main results obtained during this study were:

✓ CW planted with halophytes efficiently remove organic matter and suspended solids from domestic wastewater.
✓ Vertical flow CW shown better performance in comparison with Horizontal flow CW.
✓ The combination of CW planted with halophytes with slow sand filter and inactivation unit with TiO2-coated expanded clay aggregate achieved international threshold of WHO for waste water reuse for agricultural irrigation (1 ,000 CFU/100 ml for E. coli).
✓ Investigated technologies are more cost efficient in terms of operation and investment costs in comparison to conventional wastewater treatment process (activated sludge and chlorination).

2. Floating Mats (Vita 34). The activities focused on novel systems with floating plant mats for their potential for removal of pathogens (esp. E.coli as indicator) from municipal wastewater and for subsequent agricultural reuse. This contribution should lead to innovative design of Constructed Wetlands. A set of tests (pre-test in lab, secondary test in greenhouse and pilot test in field) were performed according to the three phases described below.
After an initial literature research on pathogen reduction using plants, suitable plant species were selected for pretesting in lab – phase 1. In preliminary tests in greenhouse different plant species were investigated for their potential to reduce coliform bacteria from pre-treated municipal wastewater with respect to international water quality standards for agricultural reuse. Therefore hydroponic systems in technical scale (in greenhouse) were used for monitoring effects on coliform removal according to chosen plant species – phase 2. Additionally dependence of efficiency on contact time of plant roots and associated microbes (rhizosphere) with pre-treated municipal wastewater was investigated.
Beside these pre-tests suitable floating matrices were evaluated for establishing pilot test in field, started in May 2015 and ended up in December 2015 – phase 3. For construction of plant mats a frame made from bamboo and carrier matrix of tissue made from coconut fibres were chosen by their advantages of cost-efficiency, availability, eco friendliness and slowly rotting. Outcomes of testing suitable plant species for removal of E. coli from municipal waste water showed best results for Carex acuta (acute sedge) and Scirpus lacustris (true bulrush) as they were chosen for pilot-scaled application. Both selected species indicated high potential for germ reduction. Although maximum effective log reduction for E. coli was reached after longer time compared to other tested species, both species showed highest cleaning efficiency. For selection suitable material for construction of floating plant mats several materials were evaluated regarding their stability, environmental acceptability, disposability and costs efficiency. Following for the frame bamboo sticks and for carrier material for plants coconut tissue was chosen.
The following are results refer to pilot testing of floating plant mats as well as to their efficiency for removal of pathogens from pre-treated municipal wastewater.
Pilot experiment was located at main sewage treatment plant of city Leipzig, Germany, where Vita 34 installed three parallel test systems. Each system consisted of three containers connected in series. Every system was equipped with floating plant mats and charged continuously with pre-treated wastewater from secondary clarifier. First system composed of floating plant mats planted with Carex acuta (acute sedge) and second system planted with Scirpus lacustris (true bulrush) since they showed best results in preliminary tests for reduction of E. coli. Third system was installed with floating plant mats without plants (control).
The pilot experiment provided reliable results for suitability of chosen materials for floating plant mats and on the removal of coliform bacteria. For the investigation on efficiency retention time of pre-treated municipal wastewater within the systems was decreased from 4 to 2 days. Obtained results show 100% efficiency for removal of E. coli. Irrespective an inoculum was developed for artificial increase of bacterial load within the influent. Treatment effect amounted up to 2 log-steps (CFU/100 mL) for system with Carex acuta and 2.5 log-steps (CFU/100 mL) for system with Scirpus lacustris. However, also the control system without plant mats showed hygienisation effect as inter alia potential removal of pathogens by UV-disinfection was assumed.
There was no significant trend within course of COD. Moreover, concentration of COD within inflow was very low. Concentration of total nitrogen was reduced significantly in both systems.

WP3 - Efficient water use in Irrigated Agriculture

WP2 focused on the improvement of water use efficiency in irrigated agriculture.

The main WP3 objectives were:

✓ to adapt advanced water saving irrigation technologies and strategies to water use and reuse at field scale,
✓ to provide technology tailored for field scale agriculture,
✓ to further develop, test and adapt actual evapotranspiration and soil water sensor technologies relevant for treated waste water reuse problems,
✓ to increase water use efficiency and productivity as a step forward towards a better local green economy, to model the impact of the proposed irrigation technologies and strategies on crop, soil at field scale
✓ to assess the possibilities of water saving at field and basin scale.

To achieve its objectives WP3 have been structured in six technological Tasks:

Task 3.1: Proper selection of the irrigation systems
Task 3.2: Proper selection of the irrigation strategies
Task 3.3: Improving irrigation performance to increase water use efficiency, prevent adverse impacts and assure durability
Task 3.4: New Technologies for actual evapotranspiration (Eta) and soil moisture measurements
Task 3.5: Impact modelling at field scale
Task 3.6: Improvement of water use efficiency at basin scale

This package identified the drip irrigation system and the deficit irrigation, PRD strategy, as a good management practice for water saving and improving water use efficiency and productivity. The new technologies such as Eddy Covariance and Scintillometers for actual evaporation measurement proved to be suitable for crop water irrigation estimation and could lead to significant improvement in water use efficiency and water saving of almost 50 % when compared with the globally used crop water requirement that is based on Penman Monteith equation. The new soil moisture probe of Cosmos rays proved to be able to sense soil moisture up to 60 cm and an external extent beyond 100 meters, thus could be used to accurately determine the soil moisture deficit of the root zone and subsequently could lead to water saving and increase in water use efficiency. The work package has also produced a new drip emitter that is suitable for poor quality water as well as quantified the drift in water application when using sprinkler irrigation. Moreover, the modelling work using the SALTMED model, supported by the field measurements at field scale, showed that the deficit irrigation scheme used (PRD) was suitable for treated waste water and quantified the water saving and the increase in water use efficiency and productivity when using the PRD as strategy with drip irrigation system. The results of upscaling from field to basin scale using the APEX model suggested the amount of potential water saving for maize to be 9.7% and 7.8% for potato.


WP3 activities were carried out at CER Experimental farm in Italy (Po valley) as well as in Irstea laboratory and experimental fields in Montpellier, France. Cash-crops with a high water demand were selected (maize, potato and tomato). As for the water quality, treated wastewater (TWW) spiked with salt was used and compared with fresh. Water saving and efficient water use have been achieved through proper selection and performance optimization of irrigation systems and strategies, accurate estimation of crop water requirements using new technologies, and model simulations, as described below.

Proper selection of the irrigation systems study was carried out at the field experiments site of CER, Bologna, Italy. The proper selection of irrigation system dealt with the issue of matching the irrigation system with the reclaimed wastewater quality. Therefore, the selection process has been implemented into a user friendly Decision Support System (SelSys DSS). SelSys DSS has been made operational (Deliverable D3.1). The DSS has been built in order to work as a web service, thus increasing its potential dissemination to the end users.

Proper selection of the irrigation strategies was carried out on the CER site with the aim to maximize water use efficiency and water productivity using two water qualities and two deficit irrigation strategies. The water sources utilized in the field experiments were: surface water from irrigation/drainage canal; secondary treated waste water, spiked with salt to increase the electric conductivity, ECw up to 4.0 dS m-1. Over the three experimental years (2013-2015), TWW and surface water, SW (irrigation canal) were utilized jointly with deficit irrigation strategies, regulated deficit irrigation, RDI and partial root drying method, PRD. Fertirrigere DSS was utilized to manage Irrigation and Fertigation scheduling. The data obtained over the 3 years rotation were used as input to the SALTMED model.

Detailed test and analysis on drip emitters clogging due to particle deposits and biofilm development using laboratory experiments were performed to identify the impact of hydrodynamic conditions (velocity, shear stress) on biofilm growth and particles deposits in both pipes and emitters. It was found that, dissolved limestone precipitates in pipes leading to an increase of biofilm dry matter content when pumps were turned off. When using a combination of synthetic effluent and mineral particle, pressure-compensated (PC) emitters tend to clog faster than Non-PC. Simulations were conducted using Computer Fluid Dynamics software and validated against Particle Image Velocimetry (PIV) measurement in transparent wavy milli-channels to understand the impact of hydrodynamic conditions on clogging. Velocity profiles were plotted along the ten labyrinth channel. Turbulence was close to zero in the vortex zones which facilitate particle deposit and emitter clogging.

To extend the wastewater reuse possibilities to very roughly treated wastewaters (e.g. for bioenergy trees production), a pressure compensating emitter has been developed. This emitter proved to be tolerant to high contaminant content effluent after a simple filtration. Mechanical tests and Computer Fluid Dynamic (CFD) modelling were also performed to study membrane compartment and improve emitter geometry. The new generation of emitters were tested during two months in the field. The plastic material used for emitters 3D printing too fragile causing leakages under the influence of successive operating phases.

To simulate the influence of operating parameters on wastewater dispersion when using sprinklers irrigation, field tests under different climate conditions were conducted to investigate this dispersion. Water was collected at different distances from the sprinkler. The volume collected decreased with the distance. An empirical model was established to predict drift volume as a function of different parameters (distance from the sprinkler, average wind speed, evaporation, to name just a few). Numerical simulations were performed to model this dispersion.

New Technologies for actual evapotranspiration (Eta) and soil moisture measurements were implemented at CER experimental farm, Bologna, Italy. The aim of this task was to test the robustness, reliability and suitability of new technologies to determine the crop water requirements based on measurements of actual evaporation and soil moisture deficit, SMD using Cosmos rays technology. Large Aperture Scintillometer (LAS) and Eddy covariance instruments were installed to measure the actual evaporation. A novel method to measure soil moisture and SMD, at area based scale using Cosmic rays was carried out. In addition, transect base scale to study the soil moisture distribution and irrigation application efficiency under different irrigation water qualities and strategies was conducted using the Electric Resistivity Tomography, ERT.

The results showed significant differences between actual evaporation values measured by the new technologies of Eddy Covariance and Scintillometer when compared with the worldwide used potential reference evaporation, ETo calculated from meteorological data using Penman-Monteith equation, and the crop potential evaporation, ETc (based on the ETo and the crop coefficient, Kc). The ETc and ETo showed higher values than those of Eta Eddy and Eta Scintillometer. On average, the actual evaporation of Eddy Covariance and Scintillometers for the cropping seasons 2014 and 2015 represented 45% and 35% of the ETo, respectively. These are quite significant differences.

These results indicate that there is a potential for water saving in irrigation, should the crop water requirement be based on actual measured evapotranspiration rather than the calculation based on the widely used Penman –Monteith equation and possibly other methods of calculating potential evaporation not the actual evaporation. Calculating the reference evapotranspiration, Eto, or the crop evapotranspiration, Etc, from meteorological data produces potential evaporation that would represent the atmospheric demand for water rather than the crop demand for water. Accurate crop water requirement should be based on crop and soil demand but not on atmospheric demand for water.

The exact percentage of water saving by using these new technologies, the Eddy Covariance and Scintillometer will differ between seasons and crops but will always be actual irrigation water requirement. However, at present, with only two cropping seasons’ results, the indication is that the actual crop water requirement based on the new technology could save at least 50% of irrigation water estimated by the commonly used methods for potential evaporation such as Penman – Monteith equation.

The COSMOS technology is one step in the right direction as it provides continuous, integrated area based values and solves the problem of spatial variability often found in point measurements in relation to the soil spatial heterogeneity. This method could also be used to determine the soil moisture deficit, SMD, hence determine when and how much to irrigate. The Root Mean Square Error, RMSE obtained by sensors, soil cores, and by profile probes, when compared with Cosmos, was reasonable with an overall average of 0.0394. The results showed that the COSMOS soil moisture falls within the band of the top 50-60 cm soil layer soil moisture measured by sensors, soil cores and profile probes supported by the model. This indicates that there is a possibility that COSMOS probe’s effective depth could be within the top 50-60 cm of the irrigated lands, particularly during the summer crop seasons. In such case, knowing that almost 80% of the crop root system is accommodated within the top 50-60 cm, the COSMOS measurement could be useful for monitoring the soil water status and subsequently soil moisture deficit in the root zone. The Cosmos results could be made operational for irrigation managers to determine when and how much to irrigate to avoid harmful water stress. In summary, these results support the use of Cosmos as an integrated area based, non-destructive and hazard free method of measuring soil moisture.

The results of the ERT indicated different behaviour between soil irrigated with fresh channel water and brackish treated water. When fresh irrigation water is used, significant changes in soil water content have been observed, instead, when brackish-treated water is used for irrigation, changes in soil water content were less evident. The different irrigation techniques (PRD and RDI) didn't show any significant changes in soil electrical resistivity, probably due to the high clay content, hence high surface conductivity of the soil.

Both, the modelling and field results showed that when considering only the irrigation amount for potato in 2013 (excluding the rainfall), the PRD-SW received 15% less irrigation water than RDI-SW and produced a yield only 6% less yield than RDI-SW while RDI-TWW received a similar amount of irrigation and produced equal yield to RDI-SW. Moreover, PRD-TWW used 12% less irrigation water amount and produced a yield only 6% less than RDI-SW. This indicates that the treated wastewater with salinity of 4 dSm-1 is as good as the fresh water for potato production and could be a good alternative to the river water for that site. In addition, PRD seems to be a good water saving strategy in this case.

For maize 2014, RDI-SW and RDI-TWW received the same amount of irrigation water (but different qualities) and produced similar yields. Moreover, PRD-SW received 17% less irrigation water but produced similar yield to RDI-SW. In addition, PRD-TWW received 15% less irrigation water but produced similar yield to RDI-SW. Once again this confirms the results of potato that the treated wastewater with salinity of 4 dSm-1 is as good as the fresh water for maize production and could be a good alternative to the river water for that site. In addition, PRD seems to be a good water saving strategy also in this second case.

For tomato 2015, RDI-SW and RDI-TWW received a similar amount of irrigation water (but different qualities) and produced similar yields. Meanwhile, PRD-SW received 28% less irrigation water and produced a yield only 9% less than the RDI-SW. Another confirmation that the treated wastewater with salinity of 4 dSm-1 is as good as the fresh water for tomato production and could be a good alternative to the river water for that site. In addition, PRD seems to be a good water saving strategy also in this third case.

One should note here that the irrigation water amount used for TWW was slightly higher in both RDI and PRD when using treated wastewater as this treatment requires application of extra water for leaching of salts from the root zone to prevent excessive accumulation.

The SALTMED model showed a good fit for the soil moisture, soil salinity, dry matter, yield, and water productivity over three years period for the potato, maize and tomato crops.
The water productivity was calculated as the yield produced, in kg, per cubic meter of water (including rainfall and irrigation) used. The field and modelling results show that the water productivity was generally slightly higher in PRD for potato, maize and tomato crops during the years 2013, 2014 and 2015, respectively. Overall high water productivity of 2.27 kg m-3, 3.56 kg m-3, 2.29 kg m-3 was observed for the PRD with surface water for potato, maize and tomato, respectively. Similar results were also observed for the total dry matter for the three crops. In most cases, the lowest productivity was observed for the RDI with treated wastewater.

Although, the two irrigation strategies, RDI and PRD, are based on the concept of deficit irrigation, one can see from the results that the PRD with 12 to 28% less water than the RDI, produced similar or only slightly lower yield. In addition, using different water qualities did not seem to have a large impact on crop yield and the biomass. The model not only showed a good correlation with the observed soil moisture, soil salinity and the crop yield but also the intermediate values of biomass during the growth stages. The field experiment and the modelling study suggest that the partial root drying (PRD) and regular deficit irrigation (RDI) irrigation strategies have a huge potential to save irrigation water in comparison to the full irrigation. The finding of this study suggests that the treated wastewater produced a similar amount of crop yield as was produced by the freshwater. The outcome of this study also suggests that crop water productivity could be increased by implementing more innovative irrigation systems and proper irrigation strategies, like partial root drying.

Overall the modelling results are good and the model has shown a strong relationship between the observed the simulated soil moisture and salinity profiles, total dry matter and final crop yield. This illustrates the model’s ability to simulate the biomass and the crop yield of C3 and C4 crops, as well as to simulate different water qualities and different irrigation strategies. Considering this, the model can run now with “what if” scenarios depicting several water qualities, crops and irrigation system strategies without the need to try them all in the field. It has the potential to reduce the cost and the labour.

Improvement of water use efficiency at basin scale modelling study was carried out on the Idice River basin, located in the Emilia-Romagna region, Bologna, Italy. The results of APEX model simulation suggest that, maintaining a fixed irrigation volume, the use of drip instead of sprinkler system ensures a water saving effect and a low crop yield decrease both for maize and potato. The more encouraging result for maize showed a decrease of irrigation water of 9.6 % was accompanied by a crop yield decrease of 4.2 %, while a water decrease of 7.8 % for potato was accompanied by 4.7 % yield decrease. Unexpectedly the different scenarios gave, under the simulated conditions and assumptions, similar results with the exception of drip irrigation at mild water stress of -65 kPa that showed a lower performance in potential water saving (and for potato also in yield loss) compared to more severe scenarios (drip/-90 kPa and drip/-185 kPa).

In scaling up the model output to the catchment scale, it was possible to calculate the total highest water saving that could be obtained in the whole Idice basin, if the selected best performing irrigation practices were applied to all the maize and potato fields in the area. The results indicate that a large amount of potential water saving is associated with the maize fields, 9.7% while for potato, it is 7.8%.

WP4 - Improving WUE and drought tolerance of maize, sorghum, millet and tomato via genomics approaches and modelling

Given the tight connection of crop production with temperature, water availability and other environmental factors, agriculture is one of the human activities mostly affected by climate change and by the overexploitation of soil and environmental resources. Of course, good agronomic practices contribute to crop resilience in presence of negative growing conditions. However, equally or even more powerful means are those provided by genetic improvement of cultivated varieties in modern plant breeding. A comprehensive and causal knowledge of the genetic control of the traits related to water acquisition and movement in the plants is fundamental to any plant breeding program.
Because of this, within Water4Crops, a specific WP (i.e. WP4) was aimed at identifying the genomic regions involved in the control of plant traits involved in drought tolerance and developing model systems able to simulate the effects of the genetic variability on traits linked to water use efficiency and leaf growth on plant productions in multiple climatic scenarios and irrigation strategies.

Specific WP4 objectives were:

✓ identify and evaluate major QTLs (Quantitative Trait Locus) influencing WUE (Water Use Efficiency) and drought tolerance for biomass production in cereals and for root architecture in tomato
✓ produce genetic materials and knowledge useful for the improvement of WUE and drought tolerance at field level through genomics-assisted approaches
✓ provide elements for modeling growth and biomass production as well as for optimizing the choice of the species and genotypes to be grown in typical European and Indian cropping systems under rainfed and irrigated conditions.

To achieve its objectives WP4 was structures in six Tasks:

Task 4.1. Molecular characterization of maize isogenic materials
Task 4.2. Mapping and characterization of QTLs for roots and WUE in maize and tomato
Task 4.3. Testing the effect of a major QTL for seminal roots on WUE and agronomic traits in maize
Task 4.4. Fine mapping of a major QTL for seminal roots in maize
Task 4.5. Comparative analysis of physiology and agronomic performance of maize, sorghum and millet under different water regimes
Task 4.6. Modelling the interaction between irrigation techniques and genotypes of maize, sorghum and millet in typical climates of Europe and India


The focus species of WP4 were maize and tomato for the genetic part and maize and sorghum for the modeling part. In maize and tomato the main object of the study were roots. Despite of the agronomic importance of roots, little is known about root system genetics and root analysis has only recently became a phenotyping target of top interest. Roots are an extremely complex system and strong influenced by the environment. In some cases, we studied the root system in controlled environment (paper roll, rhizotron) but we also carried out a full set of experiments in the open field, in order to better understand the relation between physiological traits, yield and drought. For this, during this project we set up a protocol for high-throughput phenotyping of maize roots at adult stage in the field, which led us screening more than 5,000 plants in the project. An important part of the project was to model and testing by simulation the effect of different plant architecture (e.g. root) and/or feature (e.g. flowering time), agronomic practice and environmental conditions. This is a compulsory step in the knowledge acquisition process given the great complexity of the high number of interacting factors.

For the study of the genetic control of root development and architecture in maize and their role in affecting production under water stress we utilized a type of plant experimental population known as introgression library (IL). The IL is composed by 73 inbred lines each one differing for one or few chromosome substitutions from a common background line (inbred B73, which is the reference line in the maize genetic community). The limited and known differences in chromosome structure between the IL lines (and between each line with the reference B73) enable (at least ideally) to easily map the genetic causal loci (QTLs = quantitative trait loci, or ultimately, genes) responsible for the different in phenotypic expression between the lines. The 73 IL lines were deep characterized for traits related to phenology, yield and root system architecture (RSA) in relation to drought in close collaboration between UNIBO and HORTA. We identified significant variability for the tested traits and high plasticity in drought response that let us to identify several QTLs involved in their control. In many cases, overlaps of root and yield QTLs were identified. The identification of QTL overlaps is a first indication that the specific QTL/gene not only would affect the morphological trait but also crop productivity (in this case, maize grain yield). One of the most promising QTL refers to the architecture of brace and crown roots (nodal roots above and below the soil level, respectively), including differences in angle of root insertion on the main stem. This QTL was located on chromosome 2, bin 2.04.

The entire IL population was also screened in a lysimeter platform in collaboration with ICRISAT (India) for traits related to water use efficiency at different Vapour Pressure Deficit (VPD: an index of the strength to which plants would tend to lose water from leaf) and thus it was possible to identify overlaps between QTLs for roots, yield related traits and QTLs for water uptake and transpiration efficiency.
The effect of the most interesting QTLs was verified in additional two years of field experiments in collaboration between UNIBO and HORTA by testing lines per se and F1 hybrids. One of the main results of this experiment was the confirmation of chrom. 2 QTL role on affecting crown and brace root angle even in hybrid background, with a correlated response on grain yield. Moreover, in order to better characterize the role of the tested QTLs on drought tolerance, the F1 hybrids were tested at UNIBO in an artificially sloped field with a homogeneous gradient of soil moisture as a function of the water table depth (WTD). The different drought scenarios provided by the slope-field (only the longer/deeper rooted lines would reach the water table at the extreme of the field) were an ideal setting to test the effect of root architecture QTLs on drought tolerance. Very interestingly, lines carrying the “flat angle root” allele at the chrom 2 QTL suffered a drastic grain yield reduction when tested at deeper water table depth.

Another objective of our work within W4Cs was to characterize and clone a gene affecting the number of seminal roots in maize. Whether variation in number and architecture of seminal roots does impact crop performance in terms of final yield is still unclear. In W4Cs we carried on the phenotypic characterization and the fine mapping of qSRN-1.02, a locus previously identified on chromosome 1 affecting the number of seminal root. The QTL was precisely mapped and shown to correspond with the gene rtcs, a transcription factor known to affect seminal and brace roots development in maize. Intriguingly, the root phenotype shown by qSRN-1.02 was quite different from the one caused by the mutation rtcs. This very likely suggests the presence of a new milder rtcs allele in the IL line. From a deep characterization of qSRN-1.02 at adult stage it was possible to confirmed the role in the significant reduction in roots, mainly in WS condition, associated to an effect on stomatal conductance. The effect on root and stomatal conductance was confirmed also in other genetic backgrounds by testing F1 hybrids. A strong correlation (r = 0.9) was observed between root mass and yield in WW condition.

One of the mechanisms utilized by plants to enhance the tolerance to water stress is osmotic adjustment (OA). In W4Cs we aimed to explore the genetic variability of soluble sugars (SS) concentration in the leaf and its relationships with OA and other drought-related traits, in the maize introgression line (IL) population. Our results confirmed the presence of (genetically controlled) difference in the capacity of changing leaf osmotic potential (OP) when maize plants growing in well-watered conditions undergo drought stress. Interesting statistically significant correlations were identified between SS and OP or OA, and with other traits, suggesting that physiological mechanisms are in place in maize, linking sugar content in the leaves, osmotic potential and tolerance to drought. However, the complexity of the SS accumulation prevented us to pick up a precise genetic signal in terms of lines and markers associated with the trait. The results also highlighted one IL line (121-6-6-6) deserving further analysis because of its performance in terms of SS, OA and drought tolerance.

A collection of 119 accessions of cultivated tomato (Solanum lycopersicum L.) and wild-related tomato species was screened for root system architecture (RSA) traits at 21 days after sowing in controlled environment using a plant root observation chamber (rhizotron box). The collection consisted of 67 Italian landraces (mainly from Sardinia), 40 landraces from other countries, 10 vintage cultivars and two wild species and was provided by “Centro Interdipartimentale per la Conservazione e Valorizzazione della Biodiversità Vegetale” (CBV), University of Sassari, Italy. In Sardinia, tomato landraces were widely cultivated until the introduction of modern cultivars with adaptation to very different soil and climate scenario. Even if Sardinia is a quite small region, it is characterized by a wide range of environments ranging from Mediterranean climate with high soil salinity along the coasts to continental conditions in the inner mountain area.

The phenotypic data underwent a statistical genetic analysis called Genome-wide association (GWA), which also exploited previously available molecular genotypic data (by University of Sassari), in order to identify the main loci responsible for variation of roots and shoots in this tomato collection. Based on GWA, sixteen regions located on ten chromosomes were identified as involved in the control of root traits. For seven of the identified regions overlaps were observed between root and shoot related traits, suggesting a common role of ‘vigor’ loci on both root and shoot dimension. Nine chromosome regions specifically affected root architectural traits only. Overall, our study, showed the presence of interesting genetic variation of root architecture traits in tomato, and open the way to the localization of genes/QTLs to be potentially exploited in marker-assisted selection of new tomato cultivars with enhanced water use efficiency. To the best of our knowledge, this was one of the first investigations of RSA variation in tomato seedling grown in soil-like substrate.

A new multi-species, multi-genotypes model based on APSIM (Agricultural Production Systems sIMulator,, able to simulate the effects of the genetic variability on traits linked to WUE and leaf growth on plant productions in multiple climatic scenarios and irrigation strategies was developed.
At first, three micro panels (20-30 genotypes / species) of maize (tropical and temperate accessions), sorghum and pearl millet were selected in collaboration with ICRISAT (India) taking into account their origins, groups, and responses to evaporative demand and/or soil water deficit. Subsequently a framework was elaborate for plant and leaf development, transpiration and leaf expansion analysis. Different controlled environmental conditions enabled to test the sensitivity of these traits to soil water deficit and evaporative demand in the three species. This was based on four experiments carried out by INRA in both the PhenoDyn and PhenoArch phenotyping platforms of Montpellier. Based on the phenotyping of three micropanels in three experiments in the PhenoDyn platform a new model of leaf development and sensitivities to soil water potential and evaporative demand valid in the three species for genotypes with different numbers of leaves was developed. This model was tested over Europe with maize temperate accessions in a network of field trials of a companion project (EU FP7 DROPS) and with data of a field trial performed by Horta in Italy with genotypes of both sorghum and maize in well watered and water deficit conditions.
Finally, using this new model, the effect on yield was simulated for nine genotypes under typical climates of Europe and considering various irrigation technique. Simulations were run for 59 Europeans sites during 36 years in two soil depths (60 cm and 150 cm). For the 59 European sites variation in soil depths was entered in the simulations.
In addition to the intrinsic value of such data, these results could have a large impact for breeding by indicating the best combination of trait values in each combination of site by irrigation strategy.

Main WP4 outcomes:

✓ A protocol for high-throughput phenotyping of maize adult root in the field was implemented.
✓ QTLs for root traits in adult plants of maize were identified. Overlaps between QTLs for root architectural traits and QTLs for grain yield in relation to drought, were identified for five genomic regions.
✓ The most stable QTL was detected on chromosome 2 (root angle, yield).
✓ A QTL for seminal root number on chromosome 1 was fine mapped and shown to correspond to the gene rtcs. The effect of this QTL on seminal roots and other agronomically important traits was studied both in controlled environment and in the field.
✓ An investigation on osmotic adjustment and soluble sugar concentration in maize leaves was carried out showing the presence of statistically significant, albeit low, genetic variation for osmotic adjustment in this maize population.
✓ A protocol for tomato root phenotyping in soil-filled rhizotrons was optimized. QTLs for root architecture variability were mapped in tomato.
✓ A new multi-species, multi-genotypes model based on APSIM, able to simulate the effects of the genetic variability on traits linked to WUE and leaf growth on plant productions in multiple climatic scenarios and irrigation strategies was developed.

WP5 - Identifying business opportunities and integration of solutions

Integrating biotechnology with innovations in water use efficiency requires the close collaboration of different communities of practice and different scientific communities. Bridging these communities in order to identify business opportunities and integration of solutions has been the main aim of this work package.

Main WP5 objectives were:
✓ To identify boundary conditions and perspectives for enabling green economy
✓ To identify the existing knowledge level relating to the project objectives and knowledge gaps of the key stakeholders that should be bridged.
✓ To facilitate a transdisciplinary co-creation process and identification of business opportunities to increase the use of (bio)treatment technologies and water saving technology in irrigated agriculture
✓ To stimulate the cross-fertilization and knowledge transfer between the individual work packages and activities in Europa and India
✓ To evaluate and optimize the proposed combinations of biotreatment and waste water reuse from a perspective of supporting green growth

To achieve its objectives WP4 was structured in six Tasks:

Task 5.1 Establishment of INNOVA co-creation platforms for the Mirror case
Task 5.2 Future trends and boundary conditions
Task 5.3 Co-creation and innovation at INNOVA platforms: defining input to technology development
Task 5.4 Evaluation of shortlisted business opportunities
Task 5.5 Synthesis of results from technology research to INNOVA at mirror case: defining input to business development
Task 5.6 Screening of the identified stakeholders

Where traditional research focuses on generating new knowledge, innovations are concerned with the practical use of new knowledge. To support innovation in W4Cs, WP5 developed, designed and organized a co-creation process for identifying and developing business opportunities for W4Cs (bio)technologies.

The co-creation process was called the INNOVA process involving both the developers of W4Cs technologies as well as potential users, clients, input suppliers and investors. By bringing these actors together in innovation platform meetings, WP5 facilitated the development of W4Cs technologies into marketable innovations.
To achieve its objectives, three interrelated types of activities were carried out:

1. A literature and modelling study to identify boundary conditions (technical, health, policy) to wastewater treatment and reuse in Europe and India;
2. Conducting two online surveys, one at the start of the INNOVA process and one at the end, to identify: Technology Readiness Levels (TLRs), involvement of actors and to assess perceived barriers and opportunities for bringing W4Cs technologies to the market;
3. Organization of three INNOVA platform meetings both in Europe and in India, focussing on:

- (First INNOVA meeting): assessing the business potentials of the technologies using criteria including: maturity (technical feasibility), attractiveness, policy feasibility and financial viability.

- (Second INNOVA meeting): shortlisting and focus on the ‘societal and financial costs and benefits’ of W4Cs technologies;

- (Third INNOVA meeting): exploring the concept of a cascaded bio-refinery, which emerged from the INNOVA process and involved a combination of multiple W4C technologies to valorise olive mill wastewater.

Boundary conditions
Potential of waste water reuse. Although the relevance of wastewater reuse (WRR) in water scarce peri-urban areas is beyond doubt, the expected overall contribution of WWR to irrigation water supply strongly depends on catchment characteristics i.e. proximity of irrigation areas to urban centers, seasonal distribution of rainfall and the type of additional fresh water resources.
W4Cs found stark regional differences. In Italy, domestic and industrial wastewater could contribute significantly to fulfill agricultural water demand. In several places it is still an untapped potential. In India, reuse of treated waste water will still only contribute a fraction of overall agricultural water use. That said, with growing urbanization and an increasing population competition will increase and it is an amount of water that India’s agriculture and ecosystems can ill afford to lose. With wastewater already being reused, but untreated in many regions, the value of waste water treatment and reuse is not so much an issue of quantities (in liters, m3 or hectares), but especially one of quality, by reducing negative health impacts on farmers and consumers, by increasing the value of products and by reducing pollution and degradation of soils and water bodies and the environment at large.

The survey and INNOVA meetings revealed in addition to ‘financial resources mobilization’, i.e. lack of investments or lack of pricing mechanisms that hamper the uptake of the technologies, several other barriers [“Costs & investments” (29%), “Constraining legislations and regulations” (25%), “Piloting possibilities” (17%), “Acceptance” (12%), “Other” (17%)]. In addition, the developers acknowledged the poor relationship with potential clients as important barrier for the marketing of W4Cs technologies.

In response to these identified barriers, the INNOVA process paid specific attention to assessing legislation and, issues around health and perceptions in the mirror cases in Italy and India, which resulted in two factsheets (and one deliverable report). Regarding regulations and legislation, the main conclusion was that conditions in Europe and India are rather opposite, but with the same negative effect: in Europe regulations for reuse of wastewater are often so strict that they limit technology uptake, while in India, lack of regulation or enforcement of existing regulation means the incentive to invest in treatment is less. The challenge for any government is to strike a balance between being strict and lenient in setting water quality standards. Too strict will not encourage stakeholders to improve or to invest in developing innovative solutions, as the norms will never be met anyhow. Too lenient will not assist in developing wastewater treatment technologies either. Improving legislation and regulations is identified as a necessary but slow and lengthy process, with several W4C researchers being involved in workgroups and advisory boards.
To address financial barriers costs and benefits of technologies were explicitly addressed in the INNOVA meetings in order to be able to better identify how potential financial resources could be mobilized in a later stage.

Developing bio-technologies into marketable innovations
The main aim of an innovation-focused research oriented project like W4Cs was to bring ideas, concepts and new technologies to the market, to a level that they become available to users. W4Cs combined a total set of 50 technologies with potential for commercialization of product/market combinations.
During INNOVA process this long list was reduced to a short list of 8 technologies, all of which were in a further state of maturity and/or of high attractiveness to potential investors.
Overall, over the course of the project the Technological Readiness Level has improved greatly. Where the majority of technologies had a TLR around 2 at the start of the project – so in the concept and experiment phase - by the end of the project this has shifted, with TLR 3 – experimental proof of concept – being the minimum and six technologies being available for users.

Furthermore, two integrative ideas, the CASCADEBR and LOTUSHR, emerged from WP5 and were further explored. CASCADEBR was based on the idea that costs can be reduced and benefits might be increased if several technologies around value extraction from olive oil waste water could be combined. When integrated, ISPR – In situ recovery of Volatile fatty acids through electro-dialysis (i), Olive Mill Wastewater polyphenols through an SPE procedure (ii), and Anaerobic Digestion (iii) become a cascaded bio-refinery. LOTUSHR is a pilot concept and proposal, based on a set of technologies - several of them derived from W4Cs - to treat and reuse drain water in a drain in New Delhi. It has been approved just before the end of W4Cs and is starting up.

Lessons learned
Innovation is anything but business as usual. It is about expecting the unexpectedness which requires opportunities to bring in new actors, to adjust technologies or to take new paths. The way an FP7 project is structured meant, however, that the process of innovation still remained largely supply driven: technologies were pre-selected, linked to project partners with fixed budgets, and with their expected output fixed in milestones and deliverables. The INNOVA process tried to explore a mixed approach, supporting both innovation around existing individual technologies as well as integration of various W4Cs technologies, like in the CASCADEBR.
Bringing together universities, research institutes and SMEs did diversify the scope of W4C and brought in useful experience in marketing technologies, but cooperation on innovation still remained largely confined to those partners that had cooperated before. A flexibly allocable budget to allow for new partners to come allow for even more demand driven innovation.
A large number of skills are required for innovation, ranging from technical skills to “soft” skills and the ability to learn. Researchers tend to be highly qualified in academic and technical matters. The INNOVA process revealed that researchers tend to lack the entrepreneurial type of skills. The INNOVA process addressed the development of these competences via training and support. However, these efforts could be complemented by for example innovation hubs within the participating research organisations or training on the job.
The INNOVA platform approach was conducted in both the EU and India and appeared useful in both contexts. EU PIs participated in the Indian INNOVA meeting and vice versa. One of the outcomes was the joint proposal on establishing a pilot around drain water treatment in Delhi, which has very recently been accepted for funding; LOTUSHR
While the INNOVA platform meetings and WP5 in general helped to give some structure to the co-creation process, facilitated researchers to open-up to the ‘outside world’, and helped to record some of that progress on innovation, many individual partners were of course conducting various functions of innovation already by themselves. The surveys tried to capture some of that autonomous co-creation and indicated both overall increased Technological Readiness Levels as well as a better understanding of the demands of innovation, e.g. marketing skills.

Main WP5 outcomes:

✓ A tested and proven INNOVA process around waste water treatment, valorisation and reuse and increased water use efficiency /strengthened relationships between developers of W4Cs technologies and (potential) clients;
✓ Strengthened entrepreneurial skills
✓ 4 deliverables being converted into 2 papers: one on potential and boundary conditions, one on the INNOVA process.
✓ 2 integrative WWR solutions of which one is close to funding
✓ Several patenting explorations, i.e. on maize traits and drip irrigation emitters.

WP6 - Dissemination and technology/knowledge transfer

The dissemination of project results is the main task of this WP.
The principle methods for the dissemination are the active involvement of relevant stakeholders and local businesses in targeted workshops and open day visits to the pilot sites. Targeted groups were identified and relevant dissemination and training programs established. The members of the W4Cs consortium shared their results with wider audiences via regional, national and international workshops and conferences.

Main WP6 objectives were:

✓ To identify and convey the local businesses demands to the project’s consortium
✓ To convey the technology developed within the project to the local businesses
✓ To disseminate and exchange the experience between India and Europe on: advancing Green Economy in cooperation with EBCT and the use of new technologies and models for efficient water use in agriculture.
✓ To disseminate project results to EBCT, the scientific and wider public community, ensuring maximum use of the project results by a broad audience (scientists, policymakers, planners)
✓ To provide tailor made capacity building program to train users on the new technologies and approaches for efficient water use in agriculture.
✓ To disseminate the project results to wider communities through workshops, conferences and open day visits to pilot sites.

To achieve its objectives WP6 was structured in six Tasks:

Task 6.1 Exchange of experiences and results within the INNOVA innovation platforms
Task 6.2 Organization of special local businesses and SME knowledge brokerage events
Task 6.3 Providing Mass Media dissemination
Task6.4 Dialogue with EU delegations on Green Economy
Task 6.5 Capacity building
Task 6.6 Disseminate the project results to wider communities
Task 6.7 IPR safeguarding


First of all, a dissemination and exploitation plan has been worked out and adapted in cooperation with India and EU-Partners. Along of this, the dissemination of project results was organized in close coordination with the related complementary activities in India, aiming at providing a sound synthesis of the overall project achievements.

Business demands

Resulting from researching the wastewater treatment situation in Europe it may be concluded for the demand of wastewater treatment (WWT) technologies following facts:

- Most of the European countries have implemented technologies for the collection of wastewater, complying with the Urban Waste Water Treatment Directive (UWWTD) by 92%.
- The technologies for secondary treatment, mostly based on biological treatment for removal of BOD by either activated sludge or biological filter bed processes, have reached the total implementation value of 82%. However, there is still remarkable demand on the secondary treatment technologies in future.
- The highest demand on WWT process technologies is accounting at tertiary treatment technologies, such as filtration, activated carbon, ozonation, chlorination, UV, membrane technologies incl. MBRs (Membrane Bio Reactors). These technologies are widely discussed as one of the most promising options for the mitigation of micro-pollutants (emerging contaminants, including pharmaceuticals and personal-care products, other industrial chemicals) entering the aquatic environment. The remaining 23% of the compliance gap will force the tertiary wastewater treatment technologies for “more stringent treatment” in Europe and Italy.
- In two mirror cases, Emilia Romagna, Italy and Hyderabad, India, wastewater is discharged in rivers and canals and is indirectly reused during the irrigation season. But there are significant differences. While most water in Italy is treated at least to secondary levels, regulations prevent direct reuse of wastewater in agriculture. In Hyderabad, wastewater, both treated and untreated is discharged into a dried up river were already 90% is used in agriculture closely downstream, making it almost a form of direct use.
- Despite these differences the potential for waste water treatment and improved reuse is large in both mirror cases. In Italy, increased usage of wastewater, an important European objective and a step towards a greener economy, will require cost effective technologies to bring wastewater to such a level that it complies with existing strict laws. In India large investments are needed to increase even still primary treatment. To improve the health situation of producers and consumers and the ecosystem, existing plans seem insufficient. Rapid urbanization and growing middle class with rising living standards might put additional pressure on quality standards and regulation with regard to waste water reuse in agriculture. To supply the increasing urban demand for fresh produce, more treatment will be needed.

Conveying and disseminating of developed technology and the project

In total 22 technologies have been part of the W4C technology offer. Within them, new research has been done in many different areas. In total this lead to some 51 subcomponents of technologies being developed or improved. For example, within the technology ‘Constructed Wetlands’, some 9 sub-components were further developed within the consortium, from Bamboo systems to a new substrate design to reduce clogging.
The focus of the different W4Cs technology-development was on wastewater reuse in agriculture (18), wastewater treatment (21), water reuse (11).

Overall W4Cs offered a balanced approach integrating development on treatment technologies with new water reuse systems and water use efficiency improvements. Within a survey, most researchers identified opportunities for implementation of their technology in both the EU and India, while 14% of the technology developers stated that they do not know yet. What became clear from a questionnaire distributed is that most technology developers are still in an early or middle stage of developments. This phase consists of the initial concept development, the lap phase and the prototype development. Only 23% of technology developers indicated that they were involved in the later stage of testing, up-scaling and market expansion.

The common webpage for both projects (W4Cs-India & W4Cs-EU) conveyed to outsiders that W4Cs is a joint project between EU & India and both sides were working together. For stakeholders the website offered information on conducted technologies and activities. Moreover, it enabled better connections among the project partners and was used as an internal platform. Every partner got the same information, so that all were informed about the progresses going on in India and Europe. In addition, the communication between Indian and European partners was supported.

Various ways in order to disseminate new developments of technologies or activities within the W4Cs project were approached. Flyers, posters, presentations of various kinds, events, videos, and the participation of partners to international conferences were ways to distribute outcomes of the project’s work. Annual newsletters with the aim to open the project to public and private stakeholders diffusing W4Cs’s scientific research results with a problem-solving approach were published as well.
Among the dissemination activities, exhibition and fairs appeared as the best and most efficient platforms. Thus, in the future, more attention should be paid on dissemination events using the platforms of such related fairs and exhibitions where most of the stakeholders come together.
A successful dissemination activity always requires the establishment of a good basis for tuned communication skills between the partners, which was successfully achieved in the case of W4Cs.

An event to be highlighted was the IFAT India 2014, framing two sessions on W4Cs, which included a brokerage event, where both representatives of the public and private sector participated. It provided an opportunity and a platform to learn and discuss about the promising technologies within the W4Cs project. Innovative technologies for treatment of wastewater, irrigation technologies, technologies for valorization of wastewater and suitability of treated wastewater for agricultural use were discussed during the event. Besides, the event offered an opportunity to interact and network with Indian and International experts representing Premier Indian and European research organizations. Within the frame of the event a questionnaire was distributed to all present participants, whereby the general interest of participants could be identified for further planning of the dissemination and exploitation activities.

Capacity building

As a main objective of Work Package (WP) 6 was to provide tailor made training with a special focus to train the specific needs emerging from the water scarcity issue, substantial resources to the training and capacity building were devoted to this manner.
Field tours, “Open Day’s” of fields, organized by various partners, made it possible to demonstrate new developments of various technologies to international and local audience. Thus, also the exchange between the Indian and European partners could be covered.

Within INNOVA-meetings the following steps were subsequently implemented:

- Learning and explaining local bottlenecks in economic development
- Identification of locally existing frameworks to advance the waste water use in irrigated agriculture
- Presenting technological potentials and experience from the “technology development spots”
- Facilitating the cross fertilization of ideas to promote technology development
- Innovation brainstorming
- Meeting of Indian and European delegations to exchange ideas
- Give local impulses for technology use and business development opportunities
- Therefore, the participating partners could exchange their experiences and results of recent work. Also a cost benefit analysis of W4C technologies could be conducted.
- Furthermore, two workshops in order to teach participants how to use the SALTMED model were carried out in Bengaluru, India and Montpellier, France. The SALTMED model, which was further developed within W4C can be used as irrigation management tool to predict the impact of different management of crops, soils and water qualities.

In order to distribute the knowledge of the use of the SALTMED model to even wider communities and make it accessible to amateurs, a video tutorial as distant learning tool was created. Altogether 17 Tutorials were made with the help of the professional film-makers Cross Culture Film ( All tutorials are easily accessible on YouTube. Through the given link,,
the playlist of 9 Tutorials may be accessed, in which the functionality and operation of the SALTMED model is being explained step-by-step. Within 8 further videos, frequently asked questions (FAQs) are covered and answered as well.

Main WP6 outcomes:

- Establishment of dissemination and exploitation plan
- IPR safeguarding
- Data collection and evaluation of knowledge level and knowledge gaps
- Identification of stake holders
- Screening of the identified stakeholders
- Exchange of experiences and results in collaboration with ALTERRA (INNOVA Platform Meetings)
- Co-Organization of W4Cs Session and brokerage event at IFAT India 2014 in Mumbai
- Organization of special local businesses and SME knowledge brokerage events
- Providing Mass Media dissemination in collaboration with EIRC and other WP6-Partners (Newsletters, Flyers, Posters, Proceedings, Technical Scientific Papers, contributions to the W4Cs web page)
- Dialogue with EU delegations on Green Economy in collaboration with ALTERRA (Water Group Meeting at Brussels, Meeting with EU water experts at WssTP events in Brussels)
- Capacity Building in collaboration with other W4Cs Partners (SALTMED Workshops in Bengaluru & Montpellier, Open Field Day, Exchange with India)
- Development and realization of the distant learning tools such as SALTMED.
- Highlighting relevant niches for new technology transfer (with regards to biotreatment and water use efficiency) between India and Europe.


The Exploitable Foreground is specified in detail in TABLE-B2 (OVERVIEW TABLE WITH EXPLOITABLE FOREGROUND) reported in section B of the following paragraph 4.2-Use and dissemination of foreground.

Potential Impact:
Within Water4Crops innovative technologies and best practices have been developed and assessed for wastewater reuse and water use efficiency in irrigated agriculture both for Europe and India.
Innovation has been considered as turning inventions into practice. The participation of industries has been essential to provide and apply technologies in new application areas and to identify relevant new business opportunities as a central tool to bring a market oriented innovation and to stimulate jobs creation in rural areas of Europe and India. This helped to achieve not only strong integration between research and practice, but also an increased utilization of results. In this way Water4Crops contributes to environmental and economic benefits optimizing the use of water in agriculture including water saving by domestic wastewater reuse. Wastewater reuse, in fact, is an important European objective and a key step towards a greener economy. However, while most wastewater in Europe is treated at least to secondary level, in force very strict local regulations frequently hinder direct reuse of wastewater in agriculture. In any case, cost effective technologies are required to bring wastewater to a quality level acceptable for irrigation. As for India, large investments are needed to increase even still primary treatment. To improve producers‘ and consumers‘ health and the ecosystem, existing plans seem insufficient. Rapid urbanization and the emergence of a growing middle class with rising living standards put additional pressure on quality standards and regulations with regard to agricultural wastewater reuse. To supply the increasing urban demand for fresh produces, more treatment would be necessary.
W4Cs addresses the above challenges in Europe and in India. It’s impact aims to go beyond purely developing and disseminating new technologies. W4CSs aims to help to innovate the way to treat and reuse waste water – i.e. „the use of new ideas, new technologies or new ways of doings things in a place or by people where they have not been used before“.
Thus, W4Cs idea is that innovative technologies as well as new best practices should not only work; they have to be safe, legal and cost-effecive and applicable within diverse and demanding socio-economic contexts.

Major impacts of Water4Crops are and/or could be:

• Clear environmental and economic benefits by optimising water saving and use of water in agriculture.

• W4CSs research and development activities provides solutions for sustainable processes and products as well as for preventing and cleaning pollution.

• Making use of the fact that the link between bio-treatment and water use efficiency is not sufficiently exploited, involving businesses players since the beginning of the project into W4Cs business development platforms, helped to define and identify potential new application areas as well as to ensure technologies commercialization and job creation.

• Participation of industry, including SMEs, has contributed to bringing market oriented innovations in the reference field, addressing the social dimension of the project.

• The diversity of participants in the W4Cs project brought together researchers and industry partners that might otherwise not have interacted, creating diversity in knowledge and opinions. Inclusion of SMEs in the project from the start brought in useful experience in marketing technologies. W4Cs also explored the concept of new kind of ‘agent’ or ‘connector’ in the innovation process, i.e. a person or an organisation working on the interface between science and technology. This connector should be able to push, pull and bring forward integrative innovative designs, a complex step due to the variety of investigated technologies, multiple suppliers of raw products and different clients and stakeholders. Being able to deal with the complexity of such a process is a challenge and requires specific transdisciplinary, and cross-cultural skills and expertise.

• Greater integration of research actors and activities across Europe and India. A wide co-ordination of research activities in the investigated areas between EU and India, which are both major players in the reference fields, has contributed to step up the EU-India collaboration in scope and scale. During the W4Cs duration the project’s consortium has evolved from a group of individuals to a network of researchers and entrepeneurs spread over more than 40 resaearch organizations and SMEs. More than half of W4Cs researchers identified opportunities for implementation of their technology in both the EU and India (while an additional 14% of the technology developers stated that they are still exploring).

• Using the “on-the-ground” experience from the INNOVA platforms at MIRROR cases level has helped to reform national (and regional) R&D and innovation systems to foster excellence and smart specialisation, revitalize cooperation between universities, research and business, implement joint programming and enhance cross-border co-operation in areas with EU value added and adjust national funding procedures accordingly, to ensure the diffusion of technology across the EU territory. Experiences of the Water4Crops project have been exchanged within the 'European Innovation Partnerships' (EIP) between the EU and national levels, in particular to 'building the bio-economy by 2020'.

• Two integrative ideas, the CASCADEBR and LOTUSHR, emerged from W4Cs and were further explored. CASCADEBR is based on the idea that costs can be reduced and benefits might be increased if several technologies, e.g. for extracting valuable products from olive oil wastewater, could be combined. When integrated, “in situ” recovery of Volatile fatty acids through electro-dialysis (i), Olive Mill Wastewater polyphenols through an SPE procedure (ii), and Anaerobic Digestion (iii), becomes a cascaded bio-refinery. LOTUSHR is a pilot concept and proposal, based on a set of technologies - several of them derived from W4Cs - to treat and reuse drain water in a drain in New Delhi. It has been selected for funding and is currently in the start-up phase.

• For sure W4Cs outputs will have a relevant impact on food-processing and biorefinery industrial development. Food processing is strongly established in Europe and biorefineries are under strong development and expansion. However, food-processing wastes is in Europe not that efficiently developed. It has certainly not yet reached a high sustainability level. In India food-processing is expected to strongly increase in the future as urban consumption needs will increase leading to the need for better conservation and processing of food in the rural areas also to make agriculture more efficient and sustainable (nowadays more that 40% of the agricultural crops are lost in India).

• Technologies aimed at valorizing wastewater from food processing and biorefinery industry, as those investigated by the partners of the Work Package1 (WP1) of W4Cs, allow to integrate these industries in agricultural areas and to create benefits to agriculture (recovering valuable products, nutrients and water) and to industry (providing chemicals of added value). The return of water and nutrients will keep in first instance the yields at the existing levels (at the moment sugar cane yields are declining) and it will be the start of a strong process helping to improve the yields. This cyclic strategy completely changes the value chain of agriculture to products. Presently, biomass and wastewater that are discharged unprocessed cause, in addition to environmental pollution, large emissions of the greenhouse gas methane. The W4C proposed technologies will lead immediately to greenhouse gas reductions.

• Other relevant impacts of W4Cs on industrial activities are those on agricultural water use efficiency and wastewater treatments (WWT).

• As for WWT, this sector has an increasing worldwide market (6% annual growth). However, nowadays, the sector, particularly in developed countries, mostly relies on activated sludge systems that are featured by high costs, elevated energy demand, huge production of bio-sludge to be disposed of, and need of large footprint. In emerging and/or undeveloped countries the costs of such systems are prohibitive and, over there, most domestic wastewater are discharged untreated causing frequent and serious health problems to local populations.

• In such a context, the impact of W4Cs is evident looking at the outputs from WP1 involved in the development and/or optimization of: i) reliable technologies aimed at treating domestic wastewater up to a quality level appropriate for a healthy-safe agricultural reuse; ii) a very innovative system (i.e. SBBGR) that produces up to 90% less of bio-sludge and needs very small footprint; iii) economically and environmentally sustainable natural plants based systems (CW).

• Referring to water use efficiency, it is known that greater crop yields will be worldwide necessary to support the progressive and continuous increasing food demand. It is also clear that solving and/or mitigating water stress is only way to increase the yields. Just for India, according to several calculations, the yields could be increased by a factor of 3 if appropriate irrigation and nutrient management could be implemented.

• Within W4Cs, WP3 and WP4 outputs provided rather impacting results in the area of water stress mitigation in agriculture by: -) proper selection of irrigation systems and strategies, -) optimized management of use of water and nutrients, -) best agricultural practices for increasing water use efficiency, -) genetic approach to enhance water use efficiency and drought tolerance of high water demanding crops.

• By exploiting such results farmers will not work only just for their self-subsistence but could become commercial actors in a green economy.

• Due to higher crop yields also new crops could be planted, e.g. tropical sugar beet can be added to sugar cane plantations leading to better results and to extension of the sugar milling season (two harvests a year). In Europe new plants will be used that are multipurpose (leading to food but also to chemicals or materials). Furthermore, India is sitting on a future market of medicinal plants with a potential 15 to 20 000 plant species with a worldwide market of 4 M €. Even in the US medicinal plant products count for 25 % of the market whereas in emerging countries like India this is 80%.

• W4CSs:
- supports the ambition of Europe and India to get a world-leading role in Knowledge Based Bio-Economy (KBBE).
- addresses the principles of European Knowledge Based Bio-Economy and Europe 2020: A strategy for smart, sustainable and inclusive growth.
- is central to the KBBE program and potentially even for the entire Europe 2020 strategy, the flagship initiative "Innovation Union”.
- addresses biotechnology in the full scope of the targeted development of Key Enabling Technologies. Especially the application of new bioproducts to bring cleaner and sustainable process alternatives for industrial and agro-food have been in the scope of Water4Crops.

• The outputs of W4Cs will certainly impact relevant Indian water policies, under definition or future, such as the Indian National Water Policy and the National Urban Sanitation Policy with important related Sate Sanitation Policy as well as the EU Water for Life Initiative and the achievement of the Millennium Development Goals, by providing new opportunities for extending the treatment of municipal and industrial wastewater in India.

• Referring to wider societal impact and in particular to rural development in India and Europe, Both Europe and India face the challenge of severe societal and economic transformation at rural areas. Water4Crops specifies new opportunities by considering the potential of rural and/or urban interfaces. The co-creation of new opportunities provided by the application of the new W4Cs technologies and practices as well as new food processing bio-products, helps to create job opportunities at rural areas. Information sessions with stakeholders have been organized in India, particularly with organizations representative of local farmers and small farmers communities., to stimulate the up-scaling W4Cs approach and to intensify the economic development further.

• As for gender issue, according to recent worldwide estimations, women constitute 40 per cent of the agricultural workforce and this percentage is comtinuously rising. The Millennium Development Goals (MDGs) recognize the need to promote gender equality and empowerment of women, the need to alleviate poverty and ensure sustainable environmental management.

• Water4Crops project addressed the gender aspects by encouraging women to involve in undertaking small-scale agricultural activities. In particular in India, the process of wastewater treatment and reuse in rural areas helps women members of small and marginal farming community to undertake vegetable cultivation, developing kitchen gardens, etc. Large available quantity of domestic wastewater treated by simple and sustainable systems such as constructed wetlands, could support women diversify the cropping systems from low-value to high-value crops which provide ample opportunities to achieve food, employment and income security. This should reduce the drudgery of women, improving the quality of life, and increasing nutrition status of the family members. Wastewater treatment and reuse should also encourage women members to start micro-enterprises activities for enabling water led development through inclusive market oriented development.

Main dissemination activities

The specific objectives of Dissemination and Technology Transfer in Water4Crops have been to disseminate the newly developed technologies, the new economical concepts and local businesses demands and exchange the experience between India and Europe on advancing the Green Economy. These aims have been addressed by the targeted dissemination activities at:

• Identifying and conveying the local businesses demands to the project’s consortium;
• Conveying the technology developed within the project to the local businesses;
• Disseminating and exchanging the experience between India and Europe on advancing Green Economy and the use of new technologies and models for efficient water use in agriculture;
•Disseminating project results to the scientific and wider public community, ensuring maximum use of the project results by a broad audience (scientists, policymakers, planners);
• Providing tailor made capacity building programs to train users on the new technologies and approaches for efficient water use in agriculture;
• Disseminating project results to wider communities through workshops, conferences and open day visits to pilot sites.

The dissemination of project results has been organized in close coordination with the related complementary activities in India, aiming at providing a sound synthesis of the overall project achievements. In addition, Water4Crops highlighted relevant niches for new technology transfer (with regards to bio-treatment and water use efficiency) between India and Europe.

-Technical/Business events
Within several dissemination activities, carried out both in Europe and in India, the technology producers in India and Europe have been basically informed about the general potential and also particular possibilities and as well as upcoming process applications. Those are important to boost the efforts of Europe and India for reaching a world position in high innovative biotechnology. A relevant role has been played by the W4Cs participating SMEs, which supported the dissemination via fairs and professional associations/dissemination platforms (EBCT) and chambers of commerce. Parallel to this, Technology users have been pro-actively sought in the field of bio-refinery and bio production, making in particular the upmost used of valorized products (carbons, nutrients, energy) and apply technologies to improve the water use efficiency in agriculture and producing of bio-products. Wate4Crops dissemination activities have addressed also enterprises in trading innovative bio-products and stakeholder associations beyond the production process to promote innovative bio-products. In order to boost the technology transfer and to disseminate the project results within the relevant business partners effectively, the bilateral technology platforms and dedicated networks of EBTS, EIRC, and GIZ in India have been used. On the basis of the ccooperation with EBTC the project dissemination plan was discussed and established. EBTC agreed to support the W4Cs activities. These align well with EBTC activities (like EBTCs Water Partnership, EWP) and goals. EBTC has supported by updating the W4Cs consortium on upcoming relevant events and initiatives, supporting for the INNOVA platform meetings in India and invitation and dissemination of the project results in relevant events under IWDP (Indian Water Development Program) and India Water Innovation Cluster - two initiatives and platforms that EBTC has partnered with.
Although ETBC was not officially involved in W4Cs, the EU project’s partner GIZ-India, with a technology-transfer role, took care of having regular contacts with EBTC that was always informally updated about W4Cs progresses.
An event to be mentioned was the IFAT India 2014, framing two sessions on W4Cs, which included a brokerage event. Dr.-Ing. Süleyman Yüce (STEP) moderated the event, where both representatives of the public and private sector participated. It provided a grat opportunity and a platform to learn and discuss about the promising technologies within the W4Cs project. Innovative technologies for treatment of wastewater, irrigation technologies, technologies for valorization of wastewater and suitability of treated wastewater for agricultural use were discussed during the event. Besides, the event offered an opportunity to interact and network with Indian and International experts representing Premier Indian and European research organizations. Within the frame of the event a questionnaire was distributed to all present participants, whereby STEP was enabled to identify and evaluate the general level of interest of participants.

-Capacity building
As one objective of Work Package (WP) 6 was to provide tailor made training with a special focus to train the specific needs emerging from the water scarcity issue, substantial resources to the training and capacity building were devoted to this..
Field tours, “Open Day’s” of fields, organized by various partners, made it possible to demonstrate new developments of various technologies to international and local audience. Thus, also the exchange between the Indian and European partners could be covered.

Within INNOVA meetings, the following steps were subsequently implemented:

- Learning and explaining local bottlenecks in economic development
- Identification of locally existing frameworks to advance the waste water use in irrigated agriculture
- Presenting technological potentials and experience from the “technology development spots”
- Facilitating the cross fertilization of ideas to promote technology development
- Innovation brainstorming
- Meeting of Indian and European delegations to exchange ideas
- Giving local impulses for technology use and business development opportunities
- Therefore, the participating partners could exchange their experiences and results of recent work. Also a cost benefit analysis of W4CS technologies could be conducted.

Moreover, two workshops in order to teach participants how to use the SALTMED model were carried out in Bengaluru, India and Montpellier, France. The SALTMED model, which was developed by Dr. Ragab (CEH Wellington) can be used as irrigation management tool to predict the impact of different management of crops, soils and water qualities.
In order to distribute the knowledge of the use of the SALTMED model to even wider communities and make it accessible to amateurs, a video tutorial as distant learning tool was created. Altogether 17 Tutorials were made with the help of the professional film-makers Cross Culture Film ( All tutorials are easily accessible on YouTube. Through the given link,,
the playlist of 9 Tutorials may be accessed, in which Dr. Ragab step-by-step explains the functionality and operation of the SALTMED model. Within 8 further videos, frequently asked questions (FAQs) are covered and answered by Dr. Ragab. Through discussing the best ways the software could be presented, great videos could be produced, while Dr. Ragab visited STEP partner in Aachen.

-Scientific events
Special attention has been given to address the scientific community in a way that might stimulate in particular the cooperation between Europe and India. Relevant dissemination channels of the project results to the scientific community in the field of biotechnology and green economy have been the numerous project’s results presentations at scientific conferences and papers published in relevant scientific journals. Special attention had been paid to organisation of project meeting or joint project meeting parallel to international conference on W4Cs close topics with a high potential for the scientific exchange among the participating researchers and also potential users of the technological results of the project.
Particularly worthy to be highlighted is the participation of the W4Cs-EU project coordinator (Dr A. Lopez) in the International Conference ISWATS (“Innovations in Sustainable Water and Wastewater Treatment Systems”) held in Pune-India on 21-23 April, 2016. This conference was jointly supported and organized by the European DG-Environment and the Indian governmental Department of Science & Technology (DST). The specific aim of ISWATS was to evaluate and compare the results of four EU-India projects, all focused on water and wastewater related issues and jointly funded during the EC 7th Framework Program by the Indian DST and the European DG-Environment, namely: “NaWaTech: Natural water systems and treatment technologies to cope with water shortages in urbanized areas in India” (, “Saraswati: Supporting consolidation, replication and up-scaling of sustainable wastewater treatment and reuse technologies for India” (, “SWINGS: Safeguarding Water Resources in India with Green and Sustainable Technologies” (, “ECO-India: Energy-efficient, Community-based Water & Wastewater-treatment systems for India” (
Although financed by different European DG and governmental Indian Department (Department of BioTechnologies), the Water4Crops project was inserted within the ISWATS conference for being evaluated and compared as several of its investigated topics were similar to those of the above four projects. At the end of the evaluation-comparison step carried out by a panel of experts, both from EC and DST, Dr. Vivek Dham, member of the Delegation of the European Union to India, sent a mail to Dr Lopez, saying: “....I must mention that results/output of Water4Crops were much higher standards and innovative compared to the other 4 projects”.

-Political events/activities
Water4Crops has elaborated relevant policy questions at EU / India level related to the follow up of Rio+20 and Europe 2020. These questions were addressed in individual policy briefs, promoting the potential solutions within the Innovation initiative. During participation in several meetings organised by the Water Group of the European Parliaments in Brussels, the essential political measures in the context of Water4Crops have been discussed with the high level politicians and other decision makers.
Finally, it should be observed that not everything is in the hands of researchers, just as an example, In Europe legislations on agricultural wastewater reuse are often so strict that they limit technology uptake, while in India, lack of reliable regulations or enforcements means the incentive to invest in treatment is less, The challenge for any government or regulator is to strike a balance between being strict and lenient in setting water quality standards, and the W4Cs project has sought to aids policy makers in making these calls.

-Dissemination material
Various ways in order to disseminate new developments of technologies or activities within the W4Cs project were approached. Flyers, posters, presentations of various kinds, events, videos, and the participation of partners to international conferences were ways to distribute outcomes of the project’s work. Annual newsletters with the aim to open the project to public and private stakeholders diffusing W4Cs’s scientific research results with a problem-solving approach were published as well.
Furthermore, together with updated news, Water4Crops yearly Newsletters and the final factsheet-booklet have been uploaded on the project web site as well as on the IGEP website:
The Water4Crops project updates have also been provided as input to the existing information hubs of the Indo German Environment Partnership (IGEP) Programme
In order to distribute the knowledge of the use of the SALTMED model to wider communities and make it accessible to professionals, a video tutorial as distant learning tool was created. Within a workshop organised on "Managing irrigation with fresh and saline water using the SALTMED model as a management tool" several useful dissemination tools have been developed. These tools are enabling the professionals of the SALTMED to use the model worldwide and provides them with distant learning materials, useful tools for being able to work with this software, further developed within the Water4Crops project. All tutorials are easily accessible on YouTube through the given link,
An intensive use of such model will increase the water use efficiency, avoid the excess fertilization enabling more affordable food in water stressed and high population regions of the world.

Exploitation of results
It must me preliminarily pointed out that several activities carried out within Water4Crops were “fundamental” researches, e.g. the investigations on plant breeding in WP4. Accordingly, their practical implementation at large scale in real fields need some time to occur.
Nevertheless, as reported below, some developed technologies have good chances to be promptly implemented.
As for results implementation, an event to be highlighted is the IFAT India 2014 in Mumbai, framing two sessions on W4CS, which included a brokerage event, where both representatives of the public and private sector participated. This event was particularly for the dissemination of the project outputs high efficient and very useful. Such platforms are providing ideal conditions attracting most of the targeted groups such as technology developers, owners one side and end-users, customers, traders on the other side. It provided an opportunity and a platform to learn and discuss about the promising technologies within the W4CS project. Innovative technologies for treatment of wastewater, irrigation technologies, technologies for valorisation of wastewater and suitability of treated wastewater for agricultural use were discussed during the event. Besides, the event offered an opportunity to interact and network with Indian and International experts representing Premier Indian and European research organizations.
In total 22 technologies are being part of the W4Cs technology offer, equally balanced between treatment technologies and water use efficiency improvements. On these technologies new research was done in many different areas. In total this has lead to some 51 subcomponents of technologies being developed or improved. For example, within the technology ‘Constructed Wetlands’, 9 sub-components are further developed within the consortium, from Bamboo systems to a new substrate design to reduce clogging.
Overall, over the course of the project the Technological Readiness Level has improved greatly. Where the majority of W4Cs technologies had a TLR around 2 at the start of the project – so in the concept and experiment phase - by the end of the project this has shifted, with TLR 3 – experimental proof of concept – being the minimum and six technologies in the highest category, being available for users.
Over the course of the project, then, the technical readiness of several technologies improved significantly. What is now necessary are larger pilot projects specifically devoted to validate these technology clusters. I would be great to see these pilots build upon technologies for which proof of concept has already been established. Such technologies could be then applied, improved and developed for actual use. In the long term, W4Cs developed solutions will lead both Europe and India towards realizing a better green economy.

List of Websites:

Relevant contact details

Role / Name / Email Address / Institution / web site

Coordinator / Antonio Lopez / / IRSA-CNR /

WP1 Leader / Ludo Diels / / VITO /

WP2 Leader / Pollice Alfieri / / IRSA-CNR /

WP3 Leader / Ragab Ragab / / NERC /

WP4 Leader / Silvio Salvi / / UNIBO /

WP5 Leader / Christian Siderius / / ALTERRA /

WP6 Leader / Suleyman Yuce / / STEP /

WP7 Leader / Antonio Lopez / / IRSA-CNR

Additional contact details are reported in the attached file

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