Human activities can have a significant negative impact on the environment, particularly by affecting the global biogeochemical cycles of carbon, nitrogen and phosphorus. Water supplies to urban areas and subsequent sanitation and wastewater systems – even when conventional treatment occurs –produce enormous punctual loadings (point sources pollution) of nitrogen and phosphorus. Such water pollution leads to eutrophication, resulting in a loss of plant and animal species, along with negative impacts on water for human consumption and other purposes. Furthermore, marine ecosystems are similarly affected by excess nutrients and thus the impact of eutrophication extends beyond inland waters to coastal and oceanic regions, too. Advanced wastewater treatment facilities are thus vital to remove these nutrients and meet the main objective of the Urban Waste Water Treatment Directive (91/271/EEC). This directive established minimum requirements for waste water collection and treatment depending on the size of the agglomeration and the characteristics of the discharge area. Water pollution caused by wastewater persists despite three decades of attempts to address the problem and despite the requirements of the abovementioned directive. Current concentrations of orthophosphate and nitrate are still above ‘background’ natural levels.

The LIFE + TL-BIOFER project aims to address the environmental problem of wastewater produced by small- and medium-size urban agglomerations. To meet this aim, the project plans to implement two actions. First, it will develop and demonstrate a wastewater treatment plant using a Twin-Layer (TL) system: an advanced nutrient removal technology based on immobilised cultivation of microalgae. In this technology, microalgae are immobilised by self-adhesion on a wet, microporous ultrathin substrate (the substrate layer). A second layer, which consists of a macroporous fibrous structure (the source layer), will provide and distribute the growth medium. Secondly, the project also plans to address the shortage of P by developing produced and testing biofertilisers derived from the remaining microalgae. The fertiliser will meet high agronomical standards of sustainable farming as well as the requirements of current and future EU regulations. The trials will be conducted in microplots for two different crops in northern Italy and four different crops in Spain.



1.- The main project’s objective is to demonstrate the technical, economical and environmental feasibility of an advanced nutrient removal technology, the TWIN-LAYER system, to address the environmental problem of point source pollution and to foster the compliance with European policies in small and medium size agglomerations, providing new solutions for EU environmental problems and contributing to sustainable development with a remarkable European added value for development EU policy and legislation.


2.- LIFE + TL-BIOFER project aims to close the global biogeochemical cycle of nitrogen and phosphorus, deeply affected by anthropogenic activities, simultaneously addressing water pollution and mineral fertilizers substitution. Key drivers are:

Protection of natural water from eutrophication reducing N and P discharges from small and medium size waste water treatment plants.

To foster the compliance of the Directive 91/271/EEC on urban wastewater treatment for small and medium size waste water treatment plants and for discharges on sensitive areas through advanced microalgae culture technology for tertiary treatment.

To facilitate the reuse of treated waste water and the recovery of nutrients through advanced microalgae based biofertilizers.


3.- The project also aims to serve as a model for an extensive deployment on many other sites shifting the progress in the nutrient removal step on wastewater treatment in medium and small size populations and wastewater treatment plants, which is a big challenge along the next decades in the European Union.




The present proposal is organised in the following actions to reach the objectives:

Preparatory action: a careful planning that will be carried out in a meeting at the beginning of the Project.

Implementation actions:

a) Strain selection by isolation of strains from test site and pre-screening and nutrient uptake capacities tests.

b) Demonstration of the feasibility and potential of the TWIN LAYER prototype for nutrient removal (N and P) through:

i. Design, construction and on-site installation of TWIN LAYER prototype at the WWTP.

ii. Operation, maintenance and optimization of the TWIN LAYER prototype for two years.

iii. Validation of the obtained results and emulation of the TWIN LAYER technology potential for replication and deployment in the EU and other geographical areas.

c) Development of biofertilizers from the microalgal biomass collecteed operating the TL-prototype, by means of:

i. After harvesting, drying and conditioning, full analytical characterization of microalgal biomass to evaluate its potential as biofertilizer.

ii. Evaluation of different microalgal biofertilizers formulations: lab test, pot greenhouse test and agronomical field trials.

iii. Design and production tests of new microalgae based biofertilizers (at least 3 new products) based on the results obtained in the previous characterization and agronomical testing.

Communication and dissemination of the Project by:

a) Dissemination plan, project's logo design, notice boards, specific website, Layman's report, press releases to mass media, participation in conferences, scientific papers, organization of workshops,...

b) Project management and monitoring of the Project progress through:

c) Careful planning, a clear and concise work plan of control and oversight activities and the development of a contingency plan among others means.



a.- Demonstration of the TWIN LAYER (TL) prototype for treatment of 12 m3/day wastewater from the secondary treatment along the two years of planned operation to produce a quality final effluent compliant with stringent requirements for discharge in sensitive zones (estuaries, reservoirs, protected areas...) according to the Directive 91/271/EEC, the general guidelines of the World Health Organization on water reuse and the Spanish Decree R.D. 1620/2007 on water reuse.

Total treated wastewater 7.300 m3 during the project

b.- Uptaking of 90-100% of nutrients (N and P) from wastewater (previous analytical shows Total N 38.33 mg/L, and Total Phosphorus 5.07 mg/L present in wastewater):

248 to 276 Kg of N captured by microalgae during the project

32.9 to 36.5 Kg of P captured by microalgae during the project

c.- Development a concept for transformation of TL microalgae harvested into a biofertilizer marketable product.

d.- Formulation and production of biofertilizers from microalgae in two seasons, compatible with high agronomical standards of sustainable farming and present and future EU regulations (Biowaste, End of Waste, New Harmonized Fertilizers Regulation in preparation and Organic Farming regulations (Regulation 2006/799/CE ecolabel for organic amendments, Regulation 2007/64/CE ecolabel for substrates and growing media, Regulation (EC) 834/2007 of fertilizers and soil conditioners suitable for EU organic farming)

Total amount of biofertilizers produced during the project: 20.000 kg

At least 3 new products will be formulated from 3 biofertilizers lines with at least 1 product at commercial stage for each:

300 L for Suspensions/Foliar product

100 Kg for powdered product

50 Kg for micro-granulated product

e.- Agronomical evaluation of the microalgae derived biofertilizers by the performance of two seasons laboratory and greenhouse tests, and two seasons agronomical microplots field trials (in 2 different crops in Northern Italy and 4 different crops in Spain) by comparison with organic, integrated and conventional (chemicals and pesticides inputs) agricultural systems.


f.- Development of a water reuse trial for the irrigation of the green areas in “El Viso WWTP” with the TWIN-LAYER treated effluent monitoring the water quality according to the Spanish Decree RD 1670/2007 on Water reuse (Human nematodes, Escherichia coli, Total suspended solids, Turbidity, Total N and P)