Final Report Summary - BIOAOPBDIES (Intimate coupling of biological advanced oxidation processes for environmental de-pollution and biodiesel production) The EU funded research project BioAOPBDies focused on the development of novel and effective systems for the elimination of highly stable pollutants that are poorly biodegradable for environmental cleaning and human health protection. In this aspect, the application of biological and “advanced biooxidation” processes were tested together with the potential of using the biomass and sludges developed as an inexpensive and renewable resource for biodiesel production. Both main research objectives constitute hot topics of the present and future science in the fields of environmental protection, sustainable development and sustainable energy production. Based on the above, the importance of the project was based on four principal directions: a) Evaluation of a variety of biological systems on the removal of exogenous chemicals, with emphasis on poorly biodegradable chemicals present in wastewater treatment plants (WWTPs), such as pharmaceutical substances and personal care products (PPCPs), b) Application of advanced biooxidation processes in order to increase the degradation rate of PPCPs, c) Utilization of the biomass generated during the treatment towards biodiesel production and d) Application of the above processes by using real conditions for more practical and realistic applications. Through the successful accomplishment of the research activities of this multidisciplinary research project, the beneficiary obtained substantial expertise and interdisciplinary research profile, integrating her scientific knowledge and competitiveness. Poorly biodegradable pollutants (PPCPs) present in WWTPs: The pharmaceutical compounds that were used for the elimination studies belonged to different therapeutical classes and were selected according to their widespread occurrence in WWTPs. Application of biological systems for PPCPs removal from wastewater: The project considered the use of two different biological systems for PPCPs biodegradation: a) mixed microbial populations enriched from the pilot-scale WWTP located in the host institute and b) commercially available white-rot fungi (Trametes versicolor and Ganoderma lucidum). The research aimed to study the time evolution of PPCPs elimination (degradation kinetics) as well as the biomass mechanisms (sorption, desorption, biodegradation) involved in the removal process in both systems (mixed and fungal). The enzymatic system of fungi, which is known to drive the degradation of PPCPs, was analyzed. The study also focused on the optimization of operating factors in order to maximize the elimination performance. Different types of biological reactors were studied, such as suspended and attached growth reactors, in order to decide on the more effective system. The reduction of the toxicity of the treated wastewater compared to the untreated was determined. The efficiency of the combination of different fungi populations in the absence and presence of microbial culture on the PPCPs removal was examined. Finally, both artificial and real wastewaters contaminated with PPCPs were applied to better determine the effect of the real wastewater matrix on the systems’ performance. Mathematical models were developed in order to simulate the whole biological elimination process in both mixed and fungi cultures either in suspended or in attached growth reactors.Application of advanced biooxidation processes (ABOP) for PPCPs removal from wastewater: The project considered the conjugation of biological with advanced oxidation process (called advanced biooxidation processes - ABOP), in order to enhance the catalytic decomposition of poorly biodegradable PPCPs. The research aimed to study the time evolution of the elimination of PPCPs (degradation kinetics) as well as the biomass mechanisms involved in the removal process. In addition, the aim was to study the mechanistic aspects of this process, such as the quinones-mediators, the reduction of Fe3+ to Fe2+ and the evolution of enzymatic mechanism. Thus, four different quinone molecules were used as mediators and their efficiency on the Fe2+ production was tested. The methodology for the estimation of OH˙ formation was developed. The reduction of the toxicity of the treated wastewater compared to the untreated was determined. The efficiency of the combination of different fungal populations in the absence and presence of microbial culture on the PPCPs removal was examined. Finally, both artificial and real wastewaters contaminated with PPCPs were applied to better determine the effect of the real wastewater matrix on the systems’ performance. Mathematical models were developed in order to simulate the whole elimination process in suspended growth reactors.Biodiesel production from biomass used during the Biological and Advanced Biooxidation Processes: The project considered the potential of using biomass produced during the Biological and Advanced Biooxidation Processes as a lipid feedstock for biodiesel production. The aim was to transform the lipid fraction of the sludge/biomass discharged from reactors (suspended and attached growth) to Fatty acid methyl ester (FAME) through transesterification for biodiesel production. Different types of biomass were used for this purpose: a) mixed microbial culture discharged from reactors performing biological PPCPs removal, b) biomass from fungi produced from reactors during the biological and ABOP PPCPs removal and c) secondary and primary sludge produced and discharged from the pilot-scale WWTP located in Rey Juan Carlos University. Three different solvents were tested and the conditions of extraction were optimized to achieve lipids’ separation from biomass. The characteristics of each lipid source, such as acid content and saponifiable matter were determined. Two different methods for FAME production were studied by using home-made heterogeneous acid-catalyzed transesterification (heterogeneous Zr-SBA-15 acid catalyst): a) the conventional method where the initial separation of lipids was necessary and b) the in situ transesterification process which eliminated the need of lipids extraction. The factors affecting the FAME production were also optimized (time, quantity of solvent). Many different methodologies were used in order to evaluate the quality of the produced material (acid content, saponifiable matter, FAME yield, FAME profile, metal content).Training activities: Within the framework of the scientific objectives of this project, the beneficiary became an expert on biological wastewater treatment, while at the same time she was introduced to the fields of decomposition of pollutants using ABOP and biodiesel production. She gained substantial experience by enhancing her scientific knowledge in many and new for her techniques and methodologies used for both biological and ABO processes as well as for biodiesel production. Moreover, apart from working on the aspects of this project, experience was gained through the participation in other research lines of the host group and collaborations with research groups from the host institute as well as from abroad. The beneficiary increased her leadership and training skills through the supervision of undergraduate and graduate students on their diploma thesis. Finally, the beneficiary improved her communication skills through presentations of her work at group meetings, seminars, workshops and conferences. In conclusion, in the host institute she integrated her professional maturity through the accomplishment of the specific objectives given under the framework of the project.Part of the outcome of the above research activities has been already published, while the rest is under preparation and will be published in the next few months. In most of the scientific articles the beneficiary is the main and corresponding author: 1. I.A. Vasiliadou, R. Molina, F. Martínez, J.A. Melero (2013) Biological removal of pharmaceutical and personal care products by a mixed microbial culture: Sorption, desorption and biodegradation, Biochemical Engineering Journal, 81, 108-119, http://dx.doi.org/10.1016/j.bej.2013.10.010.2. I.A. Vasiliadou, R. Molina, F. Martínez, J.A. Melero (2014) Experimental and modeling study of pharmaceutically active compounds removal in rotating biological contactors, Journal of Hazardous Materials, revised.3. I.A. Vasiliadou, R. Molina, F. Martínez, J.A. Melero, G. Tsiamis, K. Bourtzis (2014) The effect of toxicity of pharmaceutical compounds on microbial community structure, under preparation.4. I.A. Vasiliadou, R. Molina, F. Martínez, J.A. Melero, (2014) Ability of white-rot fungi to biologically remove selected pharmaceuticals under sterilized and non-sterilized conditions, under preparation.5. I.A. Vasiliadou, R. Molina, F. Martínez, J.A. Melero, (2014) Oxidation of pharmaceutical compounds by a biological Fenton-like system mediated by the white-rot fungi Trametes versicolor and Ganoderma lucidum under sterilized and non-sterilized conditions, under preparation.6. R. Sánchez-Vázquez, I.A. Vasiliadou, J. Iglesias, G. Morales, F. Martínez, J.A. Melero (2014) Biodiesel production from municipal sewage sludges, under preparation (Part A).7. I.A. Vasiliadou, R. Sánchez-Vázquez, J. Iglesias, G. Morales, F. Martínez, J.A. Melero (2014) Biodiesel production from mixed microbial and fungal biomass, under preparation (Part B).According to the results obtained, the operation scheme that could be proposed for WWTPs is the following: the mixed microbial culture could be successfully used for PPCPs biological removal (>80%) in WWTPs using attached growth reactors (rotating biological contactors). In order to achieve further PPCPs elimination, the effluent of this system could be treated in a suspended growth reactor (tank) with fungal biomass. The method of the treatment (biological or ABOP) could be selected according to the characteristics of the primary effluent and the operating cost. The ABOP found to enhance PPCPs elimination. At the same time the biomass produce from the WWTP could be used for biodiesel production following the in-situ transesterification, since it was observed that primary sludge and fungal biomass were promising lipid feedstock (16% and 30% FAME/dried biomass, respectively). Note that the mathematical models that have been developed can be used as a guide in designing and assessing the conditions under which PPCPs degradation may be expected in a full scale process.The research achieved within the present project can find easily application in the development and operation of WWTPs, targeting on the degradation of stable pollutants and the simultaneous production of biodiesel. From a fundamental point of view, it will help on the better understanding of the combined biological and chemical process for enhanced pollutants degradation and the utilization of the biomass developed for the concomitant biodiesel production. The research objectives of the project have been planned in an effort to combine both environmental de-pollution and sustainable energy production, exploiting in a more efficient way the concept of sustainable development compared to other alternatives. The results of the present project are expected to have a broad audience with direct social impact through the expected impact on future technological advances in the field of: 1) environmental and human health protection and 2) sustainable energy production. This can be considered grounded since both main concepts of the project are directly related with the needs of the current and future society as well as with the direction of the industry sector towards alternative energy production.