IAMAW Conference:

Eco-innovative Solutions for Mediterranean Wastes and Wastewaters

IAMAW website: www.iamawaste.org

Ecomondo2014, 8th November 2014, Rimini (Italy)

International Associationof Mediterranean Agro-Industrial Wastes

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WELCOME

The International Association of Mediterranean Agro-industrial Wastes (IAMAW) is proud to welcome you to the “IAMAW 4th International Conference 2014” that has been organized in collaboration with CTS Ecomondo in the context of the biggest environmental fair in Italy, “ Ecomondo 2014”. Specific focus of the conference, perfectly in line with the missions of the Association and the Ecomondo fair, is on “Eco-innovative Solutions for Mediterranean Wastes and Wastewaters”. The conference aims to be an opportunity to update, discuss and compare at industrial research level, new management models, experiences and case studies that are in demonstration stage, in scaling-up or already applied at industrial scale. IAMAW President Maurizio PETRUCCIOLI

IAMAW BOARD ADMINISTRATION BOARD: President Maurizio Petruccioli DIBAF – University of

Tuscia, Viterbo, Italy petrucci@unitus.it

Vice-president Rafael Borja Instituto de la Grasa – CSIC, Sevilla, Spain

rborja@cica.es

Vice-president Milva Pepi Stazione Zoologica Anton Dohrn, Naples

milva.pepi@szn.it

Secretary Francesca Santori ISRIM, Terni, Italy secretary@iamawaste.org Treasurer Ermanno Federici Università degli Studi di

Perugia, Italy ermanno.federici@unipg.it

NATIONAL MEMBERS: Montserrat Sarrà Universitat Autònoma de Barcelona,

Spain Montserrat.Sarra@uab.cat

Nicolas Kalogerakis EE-TU-Crete, Chania, Greece nicolas.kalogerakis@enveng.tuc.gr Erdinc Ikizoglu Ege University, Izmir, Turkey erdincikizoglu@gmail.com Maria Pilar Bernal CEBAS-CSIC, Espinardo-Murcia,

Spain pbernal@cebas.csic.es

José Duarte LNEG-Unidade de Bioenergia, Lisbon, Portugal

zcardoso.duarte@gmail.com

Lorenzo Bertin DICAM – University of Bologna, Italy lorenzo.bertin@unibo.it

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PROGRAM

Eco-innovative Solutions for Mediterranean Wastes and Wastewaters

Saturday, November 8, 2014. 9.30-14.00 Sala Diotallevi 2 Hall Sud In the context of Green Economy, all sustainable innovations in monitoring, recycling and treatment of agro-industrial wastes and wastewaters aimed to recover and produce nutrients, biomolecules, energy and every added-value component have a great potential to help businesses survive the recession, representing an opportunity of a competitive agro-industrial development. In this context, specific focus will be held to Mediterranean wastes and wastewaters where the term Mediterranean refers to climatic condition and not necessarily to geographic location. The conference aims to be an opportunity to update, discuss and compare at industrial research level, new management models, experiences and case studies that are in demonstration stage, in scaling-up or already applied at industrial scale. Session Chairs Maurizio Petruccioli, President of IAMAW Josè Cardoso Duarte, Past President of IAMAW Program 9.30 - Introduction by the Presidents and brief report on the poster presentations 9.45 - Why a Mediterranean agro-industrial wastes cluster? Thoughts and Insights Josè Cardoso Duarte (IAMAW, Lisbon, Portugal) 9.55 - Evaluation of Mediterranean wastes: a case study for Aegean region agro-industrial wastes Sayit Sargın, Fazilet Vardar Sukan (Ege University, Bornova-Izmir, Turkey) 10.20 - Renewable energy from thermophilic anaerobic digestion of winery residues: batch and CSTR lab-scale study David Bolzonella, Franco Cecchi (University of Verona; Interuniversity National Consortium "Chemistry for the Environment"), Cinzia Da Ros, Cristina Cavinato, Paolo Pavan P. (University Ca’Foscari, Venice) 10.40 - Experiences of birth business from the recovery of waste in the urban periphery Graziano Bertogli (MAN.SE.F. Onlus, Milan) Selected oral presentations 11.00 - Measure of biochemical methane potential (BMP) of by-products, residues and alternative crops typical of the Mediterranean area. Mariangela Soldano, Lorella Rossi, Nicola Labartino, Claudio Fabbri, Sergio Piccinini (CRPA Lab, Sezione Ambiente ed Energia, Reggio Emilia, Italy) 11.15 - Effect of ultrasound and microwave pretreatments on two-phase olive mill solid waste anaerobic digestibility

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Barbara Rincón, M. González de Canales, L. Bujalance and Rafael Borja (Instituto de la Grasa – CSIC, Sevilla, Spain) 11.30 - The new role of the Sewage Treatment Works: the LIFE WW-SIP project experience Francesca Santori (IAMAW, Terni, Italy) 11.45 - CarbGrowth: maximisation of greenhouse horticulture production with low quality irrigation waters Francisco M. del Amor Saavedra (IMIDA, La Alberca, Murcia, Spain) 12.00 - Fungal laccases production using agro-food wastes: a factorial design approach Federica Spina, Annalisa Nanni, Alice Romagnolo, Giovanna Cristina Varese (University of Turin, Italy), Marcello Fidaleo (University of Tuscia, Viterbo, Italy) 12.15 - An agro-industrial waste valorization: biopolymer production from dephenolized and fermented grape pomace Gonzalo A. Martinez, Joana B. Domingos, Stefano Rebecchi, Lorenzo Bertin, Fabio Fava (DICAM, University of Bologna, Italy) 12.30 - Thermal application for agro industrial wastes utilization Guillermo Rodríguez-Gutiérrez, Juan Fernández-Bolaños, Ana Jimenez-Araujo, Rocio Rodríguez-Arcos, Aranzazu García-Borrego, Fátima Rubio-Senent, Antonio Lama-Muñoz, Abdessalem Mrabet, Rafael Guillén-Bejarano (Instituto de la Grasa – CSIC, Sevilla, Spain) 12.45 - Valorization of corn-silage anaerobic digestate through the cultivation of the white-rot fungus Pleurotus ostreatus. Guglielmo Santi, Simona Proietti, Stefano Moscatello, Alberto Battistelli (IBAF-CNR, Porano, Italy), Valerio G. Muzzini, Emanuela Galli (IBAF-CNR, Monterotondo, Rome, Italy) 13.00 - Effects of dilution ratio, inoculum of yeasts and concentration of ammonium sulphate on the bioremediation of olive mill wastewater Antonio Bevilacqua, Nilde Di Benedetto, Milena Sinigaglia, Maria Rosaria Corbo (University of Foggia, Italy) 13.15 - An approach to split 2-phase olive pomace into a polyphenol liquid and a valuable solid phase Steffen Hruschka (GEA Westfalia Separator Group GmbH, Germany) 13.30 - Need to have a uniform law in the Mediterranean basin for the development of eco-friendly solutions for waste, wastewater and renewable energy Mariagrazia Chianura (Studio Legale Chianura, Grottaglie, TA, Italy) 13.45 - Concluding remarks Poster Session E4-1 Degradation of 2-chlorophenol by laccase-zeolite biocatalyst / Emanuela Galli1, C.M. Polcaro2, P. Ciccioli2, E. Donati2; 1IBAF-CNR and 2IMC-CNR, Monterotondo, Rome, Italy

E4-2 IGAN ECO-POT project / Renzo Spagnesi1, Martina Lotti1, Maurizia Seggiani2, Monica Puccini2, Sandra Vitolo2, Roberto Altieri3, Alessandro Esposito3, Francesco Castellani3,Vitale Stanzione3, Ermanno Federici4, Laura Fidati4, Elena Montalbani4, Roberta Pret4, Masetti Gianna5, Vincenzo Tropiano6, Michele Bellandi6, Mario Romiti7; 1Vivaio Sandro Bruschi, Pistoia, 2University of Pisa, 3ISAFOM-CNR, Perugia, 4University of Perugia, 5Vivai Piante Masetti Sabino s.s.a, Pistoia, 6Impresa Verde Pistoia, Pistoia, 7Romiti F.lli Mario & Marco soc.agr., Pistoia, Italy

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E4-3 The PUMAN Model. Methodology for the quantification of the wastewater volume in dry weather. A concrete solution to elaborate a standardized definition of priorities for actions and investments into the wastewater collection and treatment fields / Mario Chiarugi, Oberdan Cei, Roberto Salvadori, Gianluca Baronti, Simone Lippi; Acque Spa, Pisa, Italy E4-4 Orange peel wastes pretreatment by acid-catalyzed steam explosion for enhancing bioethanol production / Guglielmo Santi, Julia Jasiulewicz, Alessandro D’Annibale, Silvia Crognale, Maurizio Petruccioli, Mauro Moresi; DIBAF, University of Tuscia, Viterbo, Italy E4-5 Bioremediation as alternative solution for wastes management: study on potential phenol-degrading microorganisms / Daniela Campaniello, Antonio Bevilacqua, Milena Sinigaglia, Maria Rosaria Corbo; University of Foggia, Italy E4-6 Fungi isolated from olive mill wastewater: Identification, phylogenetic aspects and preliminary assessment of their effluent-degrading abilities / Vassiliki Fryssouli1, Evangelos Dagres1, Io Kefalogianni1, Milton A. Typas2, Georgios I. Zervakis1, 1Agricultural University of Athens (Lab of General and Agricultural Microbiology) and 2National and Kapodistrian University of Athens, (Dept. of Genetics and Biotechnology), Athens, Greece E4-7 Biohydrogen production from glucose, molasses and cheese whey by suspended and attached cells of hyperthermophilic bacteria belonging to Thermotoga genus / Martina Cappelletti1, Davide Pinelli2, Stefano Fedi1, Davide Zannoni1, Dario Frascari2; 1Dept. of Pharmacy and BioTechnology and 2DICAM, University of Bologna, Italy E4-8 Production of ethanol from xerofile and halo-tolerant plant biomass. The case of Tamarix jordanis grown in the desert and irrigated with wastewater or brackish waters / Guglielmo Santi1, Alessandro D’Annibale1, Silvia Crognale1, Amram Eshel2, Aviah Zilberstein2, Maurizio Ruzzi1, Riccardo Valentini1, Mauro Moresi1, Maurizio Petruccioli1; 1DIBAF, University of Tuscia, Viterbo, Italy and 2Tel Aviv University, Israel E4-9 Next generation sequencing of bacteria involved in the static composting of olive mill waste / Laura Fidati1, Giovanni Cenci1, Alessandro Esposito2, Roberto Altieri2, Ermanno Federici1; 1University of Perugia, and 2ISAFOM-CNR, Perugia, Italy E4-10 Biogas production by mesophilic phase-separated anaerobic digestion of corn-DDGS / Botond Ráduly1, Silvia Crognale2, László Gyenge1, István Máthé1, Beáta Ábrahám1; 1Sapientia Hungarian University of Transylvania, Miercurea Ciuc, Romania and 2DIBAF, University of Tuscia, Italy E4-11 Mediterranean fruits wastes as biorefinery feedstocks / Ana Dionísio, Rute Neves, Pedro Branco, Ivone Torrado, Patricia Moniz, Talita Fernandes, Florbela Carvalheiro, Luís C. Duarte; LNEG-Unidade de Bioenergia, Lisbon, Portugal E4-12 Potential of Opuntia ficus-indica residues for bioethanol production / T. Mesquita1,2, M. S. Romanovich1,2, H. T. Chaves2, F. Carvalho2, M. C.Fernandes1; 1Centro de Biotecnologia Agricola y Agro-Alimentar do Alentejo (CEBAL), Beja, Portugal, 2Departamento de Tecnologias y Ciencias Aplicadas, ESAB-IP, Beja, Portugal E4-13 Eco-innovatve studies in Turkey for olive waste valorisation and OMWW treatment / Erdinc Ikizoglu, Ege University, Bornova-Izmir, Turkey An English-Italian simultaneous translation service is available

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Why a Mediterranean agro-industrial wastes cluster?

Thoughts and Insights

José Cardoso Duarte IAMAW

“Mediterranean” it is a relatively difficult concept to use due to its different implications from the point of view of geography, climate, the territory, sociological or political among others. Besides the geographical concept-the Mediterranean sea-and its evolutionary role, either historical either civilisational we have nowadays quasi-universal concepts using the characteristic adjective “mediterranean” from physical phenomena like “climate” to more prosaic and familiar things such as “kitchen” all important for defining a desired and optimal way of living. So, how this translates into action it is a recurrent problem to avoid misusing and misunderstanding of the Mediterranean adjective. In fact the word “Mediterranean” is used nowadays within many pratical and important initiatives calling for a rigorous definition of its field of application. As an example we notice that for the first time, the word Mediterranean appears on the subtitle of ECOMONDO even if it was more or less explicitly assumed in previous organizations of the Fair. The problem becomes even more complex when we try to apply it to Wastes and in particular to Agro-industrial wastes of Mediterranean origin or character. In particular we try to elaborate in the concept for deducing its importance and for a better definition and arriving to a reasonable consensus on the need of keeping an active cluster within the topic of Agro-industrial wastes for the Mediterranean type agricultural productions. This cluster should work together to promote the science, technology and sustainability behind the exploitation of these productions. We will conclude with recommendations for signalling its importance to the European Commission and other important scientific and development forums.

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EVALUATION OF MEDITERRANEAN WASTES: A CASE STUDY FOR AEGEAN REGION AGROINDUSTRIAL WASTES

Sayit Sargın, Fazilet Vardar SUKAN

Ege University Department of Bioengineering , Bornova-İzmir, TURKEY

The economies of many Mediterranean countries are agricultural and

agro-industry based and because of rapid increase in urban populations as well as industrialization in these countries, vast amounts of residues and wastes are generated leading to a high turnover rate of organic material with an undisputable economic potential which is greatly undervalued. Therefore, it is very important to identify feasible recycling and reutilization strategies, taking into consideration the availability, convertibility, cost, existing and potential usages of these waste materials.

Being a part of the Mediterranean basin the Aegean Region of Turkey contributes immensely to the Turkish economy with its agro-industrial infrastructure and consequently enormous amounts of agricultural wastes and surpluses are produced annually. Presently, these materials are very rarely reutilized. The fact that agro-industrial wastes contain economically valuable or microbially metabolizable components enable them to be converted to high value added products through biotechnological methods. The objective of this study was to develop a data base and to determine the sources, quantities and qualities, the physical, chemical and biological characteristics of the Region’s agro-industrial wastes. The recycling and reutilization potentials were investigated with respect to the information obtained through questionnaires and the comprehensive literature survey conducted. Wastes were grouped on the basis of their main components, industrial sector and possible products to be produced. Existing conventional methods of reutilization were compared with novel methods used in other countries. Waste/Product ratios and estimated amounts of waste produced annually were presented for all the industrial sectors included in the study. The study clearly shows that the Aegean Region agro-industrial wastes and surpluses have a very promising potential for reutilization. However, the success of reutilization depends on the regional distribution, seasonality, quantity, economic value, pretreatment requirements of the wastes as well as the scientific and technological know-how within the country, public awareness, governmental regulations and incentives.

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Renewable energy from thermophilic anaerobic digestion of winery residues: batch and CSTR lab-scale study.

Bolzonella D. 1,2, Da Ros C.3,Cavinato C.3, Cecchi F. 1,2, Pavan P. 3

1 University of Verona, Department of Biotechnology, Strada le Grazie 15-37134, Verona, Italy. 2 Interuniversity National Consortium "Chemistry for the Environment", Via delle Industrie, 21/8-30175 Venice, Italy. 3 University Ca’ Foscari of Venice, Department of Environmental Sciences, Informatics and Statistics, Calle Larga Santa Marta, Dorsoduro 2137-30123, Venice, Italy. (cavinato@unive.it) The production of one hectolitre of wine generates about 18 kg of marcs, 4 kg of stalks, 6 kg of lees, and 1 kg of sewage sludge from winery wastewater treatment (ANPA, 2001). These kind of bio-wastes are characterized by high biodegradability, but at the same time a low nutrient content. Anaerobic digestion (AD) represents the most suitable technology for these substrates in terms of energy saving and CO2 emission. In this study, potential methane production of the different bio-waste was evaluated by standard biological methanisation tests. The study shows that the most interesting substrate is wine lees, with specific a methane production that reached 0.333 m3/kgCOD, while the methane production from grape marc can be increased from 0.150 to 0.301 m3/kgVS after a fermentation step. Considering the amount of grape marc available this substrate seemed to be the most interesting winery by-product. Thus, lab-scale semi-continuous reactor was used to evaluate feasibility of AD of grape marc alone and with sewage sludge. The process fed with grape marc alone showed a not suitable solution: it wasn't stable at 20 days of HRT, mainly due to the presence of slowly biodegradable compounds with low hydrolytic rates and to the low nitrogen content. The hydrolysis can be improved by using a pre-fermentation step, or higher HRT can be applied to stabilise the process. Thus, a second test at HRT=40 days, feeding the reactor with fermented grape marc showed higher biogas production (SGP), 0.3 m3/kgVS. Also in this case, ammonium release was not enough to maintain an adequate buffer condition. Adoption of a co-substrate as sewage sludge seems to be the most promising approach to follow to obtain a stable process thanks to the supply of missing nutrients, even if lower overall SGP can be obtained due to low biogas production of sludge.

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Experiences of birth business from the recovery of waste in the urban periphery

Graziano Bertogli

(MAN.SE.F. Onlus, Milan) The study RECYCLING – FROM E-WASTE TO RESOURCES prepared by UNEP in 2009 underlines that an appropriate handling of e-waste can both prevent serious environmental damage and also recover valuable materials, especially ferrous and non-ferrous metals and precious metals. In fact, S.Chatterjee and Krishna Kumar, estimated in 230 kg the volume of saleable material (steel, glass, plastic, copper, aluminum, etc) recovered from 1,000 kg of Personal Computers (PCs) and Printed Circuit Boards (PCBs) with an estimated value of around 12.000 $ only from PCBs contained in 1,000 kg of PCs. In addition to usual components, in the last 5/6 years another important group of elements became economically suitable to be recovery from e-waste: the Rare Earth (REM) that are used in a wide range of applications, such as automotive catalytic converters, permanent magnets, and rechargeable batteries, petroleum cracking catalysts, flints for lighters, pigments for glass and ceramics etc. The incresing utilization of REM coupled with the increased reduction of export from Cina, the main producer, resulted in a growing price. For intance, the price FOB China of cerium increase from 3.04 $/kg in 2007 till 140,6 $/kg in May 2011, opening an important opportunity for the recycling plants. The recycling chain for e-waste is classified into three main subsequent steps: (i) collection, (ii) sorting/dismantling and pre- processing (including sorting, dismantling and mechanical treatment) and (iii) end- processing. Typically, the end-processing takes place at three main destinations. Ferrous fractions are directed to steel plants for recovery of iron, aluminum fractions are going to aluminum smelters, while copper/lead fractions, circuit boards and other precious metals containing fractions are going to e.g. integrated metal smelters, which recover precious metals, copper and other non-ferrous metals, while isolating the hazardous substances Developing and emerging economies can play an important role in this field so several pilote projects, easlely scalable, can be implemented mainly in urban peripheries taking into account the present level of technology, performance of the recycling chains of both informal and formal recyclers and other economic and social aspects and parameters.

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Misura del potenziale metanigeno (test BMP) di sottoprodotti, scarti e

colture di integrazione tipiche dall’area del Mediterraneo. Measure of Biochemical Methane Potential (BMP) of by-products, residues

and alternative crops typical of the Mediterranean area.

Mariangela Soldano, Lorella Rossi, Nicola Labartino, Claudio Fabbri, Sergio Piccinini

Centro Ricerche Produzioni Animali – CRPA Lab, Sezione Ambiente ed Energia -Reggio Emilia

Il CRPA Lab, sezione Ambiente ed Energia, ha sviluppato un sistema per determinare il potenziale metanigeno, definito con l’acronimo inglese BMP (Biochemical Methane Potential), di diverse matrici utilizzate negli impianti di biogas. Il test misura la produzione massima di metano ottenibile per degradazione anaerobica della sostanza organica contenuta nelle biomasse, secondo la norma UNI EN ISO 11734:2004, ed espressa in Nm3 per kg solidi volatili. Dal 2011 il laboratorio CRPA ha effettuato oltre 1000 analisi BMP di scarti, sottoprodotti dall’agroindustria e colture energetiche. Nei paesi dell’aerea Mediterranea, dove è in crescita il numero di impianti di biogas e di conseguenza l’interesse verso questo settore, l’attività agroindustriale produce quantità significative di sottoprodotti e scarti, molti dei quali inutilizzati e che spesso rappresentano un notevole problema ambientale. Inoltre colture “alternative” alle coltivazioni energetiche, come l’Arundo Donax, Nicotina tabacum, Miscanthus giganteus, Jatropha curcas, prodotte in quest’area, riscuotono sempre più interesse in digestione anaerobica. L’articolo riporta i risultati di diversi test BMP di sottoprodotti e scarti organici provenienti dall’area del Mediterraneo, quali sanse di olive, pastazzo di agrumi, scarti della lavorazione del pomodoro, colture energetiche “alternative” ecc… Dai risultati ottenuti si conferma che l’uso di queste matrici è molto promettente in quanto di buona qualità in termini di sostanza organica e resa in metano e quindi idonei alla digestione anaerobica. La loro trasformazione in biogas permette di incrementare il potenziale metanigeno di piccoli impianti di digestione anaerobica che solitamente utilizzano solo effluenti zootecnici e diminuire l’uso delle colture energetiche dedicate nei grandi impianti.

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EFFECT OF ULTRASOUND AND MICROWAVE PRETREATMENTS ON TWO-PHASE OLIVE MILL SOLID WASTE ANAEROBIC DIGESTIBILITY

B. Rincón*, M. González de Canales, L. Bujalance and R. Borja Instituto de la Grasa (CSIC). Avenida Padre García Tejero, 4, 41012, Sevilla, Spain. This study investigated the effect of ultrasound (US) and microwave (MW) pretreatments on two-phase olive mill solid waste (OMSW) with a view to enhancing its anaerobic digestibility. The effect of US and MW pretreatment on OMSW composition and subsequent anaerobic biodegradation was evaluated by chemical oxygen demand (COD) solubilization and biochemical methane potential (BMP) tests. Two-phase OMSW was ultrasonically pretreated at a power of 200W and a frequency of 24 kHz for different time periods. These time periods correspond to specific energies of 11367 (US1), 21121 (US2), 34072 (US3), 51284 (US4), 68557 (US5) and 106003(US6) kJ/kg TS, respectively. The MW pretreatment was carried out at a power of 800 W and a targeted temperature of 50ºC using different heating rates, ramp and holding times. The following specific energies were applied: 4377 (MW1), 4830 (MW2), 7170 and 7660 (MW3) kJ/kg TS. The US pretreatments that reached the highest COD solubilizations were US1, US4 and US6, with a COD solubilization between 48-57%, these pre-treatments were selected for BMP tests, obtaining methane yields of: 311±15, 393±14 and 370±20 mL CH4/g VSadded respectively, while the untreated OMSW gave a methane yield of 367±4 mL CH4/g VSadded. For the MW pretreatment the COD solubilizations achieved were very similar in all the cases and they were in the range of 41.1%-43.1%. BMP tests revealed that US2 (pretreated at 21121kJ/kg TS) and MW3 (pretreated at 7660kJ/kg TS) gave maximum methane yields very similar (393-395 ± 1 mL CH4/g VSadded), which were around 7% higher than that obtained for untreated OMSW. Methane yield for the US pretreatment increased when the specific energy increased from 11367 to 51284 kJ/kg TS. Nevertheless, for a specific energy of 106003 kJ/kg TS a decrease in the methane yield was observed. For MW the maximum value was obtained for the maximum energy tested (MW3).

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The new role of the Sewage Treatment Works: the LIFE WW-SIP project experience

Francesca Santori

f.santori@lifewwsip.it IAMAW, Perugia - Italy

Rethink the common notion of the sewage treatment plant, from a site devoted to the urban waste water collection, treatment and discharge to an integrated platform for the sustainable and profitable refining of the organic liquid waste, should be considered a priority to face the current emergency in terms of water scarcity, energy recovery, economic and managerial sustainability of the whole water and waste water treatment sector. Notably, the sewage works of the future should integrate different approaches in order to reach environmental and economic sustainability; in actual fact, whilst in the past the works was put in operation merely to treat urban wastewater, losing energy and valuable components in its waste materials, at present, due to the introduction of the anaerobic digestion process inside the works, energy is recovered from the process residues, reducing cost and the carbon footprint of the whole process, going towards energy neutral targets. Based on these premises, a wastewater refinement platform, co-financed by the LIFE+ programme, is going to be built by integrating infrastructures, processes and workforce of the ordinary UWWTP. It will be fed with organic liquid waste available in the surrounding area, carried via truck and pre-treated into the Works by using the electro-coagulation process. The sludge obtained after primary and secondary treatment, will be digested into the already constructed anaerobic reactor for biogas production. The biogas will be conveyed to the CHP station for energy production. The carbon dioxide produced by the CHP station will then be directed to the PBR, fed with N, P and K present in the sewage works effluents. The algal biomass from the PBR will be redirected to the anaerobic reactor or used to generate algal fuel. The final products of the whole process will be reusable water and energy; any process residue and effluent will be re-circulated within the treatment chain.

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CarbGrowth: Maximisation of greenhouse horticulture production with low

quality irrigation waters

Francisco M. del Amor Saavedra - Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA)

Agriculture in the European Union faces some serious challenges in the coming decades: competition for water resources, rising costs, competition for international markets, changes in climate and uncertainties in the effectiveness of current European policies as adaptation strategies. Greenhouse production appears as an alternative to face some of the upcoming challenges. In 2011, the surface dedicated to greenhouse production at worldwide level was up to 3000kha, from which 5% belongs to Europe. This production is characterised by a climate conditions associated to fourth climate regions, which allows for diversity in technologies and practices used for greenhouse vegetable production. On the other hand, The EU is the main export destination with almost one-half of the world’s imports. In the period 2002-2008, fruit and vegetables imports have experimented a constantly growth (up to 39% for this period).Therefore, in order to ensure their competitiveness against producers from other Non-European countries with lower labour costs as Morocco, or Israel, European growers need to adopt new agricultural technologies to improve net production, ensure quality and reduce production costs. On the other hand, European aquaculture represents 20% of the total fish production. Major environmental impacts of aquaculture have been associated mainly with high-output of wastewater of intensive systems. In 2009, the Commission proposed a strategy for the future of the European aquaculture which includes new wastewater management strategies. In order to satisfy the needs of this two different sectors, Carbgrowth aims to: (1)increase net production and tolerance to salinity through CO2 injection (2)recover CO2 for injection by photocatalysis (3)reusing industrial wastewater for irrigation purposes and reducing the cost associated to water supply, (4)reduce charge loses in irrigation systems.(5)Develop process control to optimise irrigation, CO2 injection and greenhouse climate control.

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Fungal laccases production using agro-food wastes: a factorial design approach

Federica Spina1, Marcello Fidaleo2, Annalisa Nanni1, Alice Romagnolo1,

Giovanna Cristina Varese1

1Department of Life Sciences and Systems Biology, University of Torino, Viale Mattioli 25, 10125 Torino, Italy

2Department of Innovation of Biological Systems, Food and Forestry, University of Tuscia, Via San Camillo de Lellis, 01100 Viterbo, Italy

Fungal laccases are excellent green biocatalysts but their actual exploitation at industrial scale is still limited by the commercial availability of large amount of cheap enzymes of interest. Basidiomycetes are considered great enzymes sources and, during this study, the attention was focused on a Trametes pubescens strain: its laccases were stable, versatile and, when a direct comparison was possible, more active than commercial formulates. The objective was then to stimulate laccases production by controlling medium composition and operative conditions. Traditional C/N sources (glucose, peptone) and model inducers resulted functional for this purpose, but they are not cost effective. For industrial applications, the economic balance should be indeed taken into consideration. Wastes of agrochemical and food processes may be used as alternative nourishment sources for fungi; their lignino-cellulosic matrix mimics the natural growth substrate of ligninolytic fungi, being able to sustain and trigger specific metabolisms. Tomato sauce and orange peel waste were here considered because of the large production within Italian and, more in general, Mediterrean boundary. At first, the concentration of both sources (C) and peptone (N) was evaluated in presence of Cu as inorganic inducer. A 22 plus centre point replicated factorial design was used to investigate the role of these factors and their synergic effect on laccase production. Both C and N concentration gave significant effects, even though N was more important for tomato sauce. An interaction between the two factors was always observed. The highest laccase productivity was achieved in presence of orange peel waste (46 U/ml). These data should be verified, and whether possible, acquiring chemical analysis of the wastes acquired. Further experiments are then necessary to define the optimal medium composition for laccase production. Moreover, the effect of other factors (Cu, pH, inoculum, etc.) has to be evaluated.

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An agro-industrial waste valorization: biopolymer production from dephenolized and fermented grape pomace

Gonzalo A. Martinez1*, Joana B. Domingos1, Stefano Rebecchi1, Lorenzo

Bertin1,2, Fabio Fava1 1 DICAM, University of Bologna, via Terracini 28, I-40131, Bologna, Italy

2 IAMAW - International Association of Mediterranean Agro-industrial Wastes c/o ISAFOM-CNR, Via della Madonna Alta, 128, 06128 – Perugia – Italy

The present work was dedicated to verify the possibility of extensively replacing the utilization of simple sugars for the biotechnological production of polyhydroxyalkanoates by pure cultures. To this aim, the utilization of a dephenolized and fermented grape pomace (GP), i.e., a solid organic waste of the winery industry, was proposed. The dephenolization, which represented the first valorization step, consisted on high valuable organic molecules recovery by super-critic CO2 extraction. Thereafter, the pomace was fermented under anaerobically acidogenic conditions for obtaining a volatile fatty acids (VFAs)-rich effluent (GPAcid), mainly containing (g/L): acetic (14.69±0.57), propionic (0.77±0.04), iso-butyric (0.83±0.03), butyric (4.67±0.21) and caproic (0.55±0.02) acids. Finally, a grown culture of Cupriavidus necator was fed with the GPAcid. Experiments were carried out in 500-mL Erlenmeyer flasks (working volume: 150-mL) at 30ºC and 180 rpm. The whole PHA production process was separated into two stages, namely: (a) a balanced cell growth phase, using DSMZ-81 mineral medium amended with 5 g/L of fructose, and (b) an accumulation phase, where harvested cells were suspended in a NH4 free-DSMZ-81 medium containing 20 or 40% of GPAcid (v/v). Higher GPAcid were not tested since VFAs concentrations would inhibit PHA accumulation. A control experiment aimed at indirectly evaluate water matrix inhibition effect was performed by substituting GPAcid with a VFAs water solution, wherein VFAs concentrations were the same of GPAcid. First results showed a higher polymer production when employing 40% of GPAcid, with a final polyhydroxybutyrate content of 60% (cell dry weight bases). Thermo-gravimetric analyses confirmed gas-chromatography ones and identical results were obtained in the control experiment. To the very best of our knowledge, this work represents the first attempt to produce polyhydroxyalkanoates with a pure culture of Cupriavidus necator and a dephenolized and fermented GP as an alternative carbon source.

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Thermal application for agro industrial wastes utilization

Rodríguez-Gutiérrez, Guillermo; Fernández-Bolaños, Juan; Jimenez-Araujo,

Ana; Rodríguez-Arcos, Rocio; García-Borrego, Aranzazu; Rubio-Senent, Fátima, Lama-Muñoz, Antonio; Mrabet, Abdessalem; and Guillén-Bejarano,

Rafael. Instituto de la Grasa: Spanish National Research Council (CSIC). Avda.

Padre García Tejero nº 4, Seville-41012, Spain. The agro industrial wastes generated in the Mediterranean basis countries are based in lignocellulosic matrix that makes difficult their utilization. A novel system based in thermal pretreatment by steam has been successfully tested to get the olive oil waste solid utilization. Nowadays, the technology is being studied for the utilization of other agro industrial wastes like asparagus, grapes, tomato or dates, besides others. Although these studies are still ongoing, some of results are showing the formation of antioxidant extracts from the liquid phase and antioxidant fibres from the final solid. The main advantages found using the thermal technology are based in the solubilization of interesting compounds, like phenols and sugars to the liquid phase, the formation of antioxidant fibres in the solid which is concentrated in other components like proteins and oil, the easier solid-liquid separation and the possibility of further application of bioprocess to both liquid and solid phases. In the case of asparagus and date wastes, the use of the thermal system allows to obtain a liquid extract with antioxidant properties and a final solid rich in fibre, part of them with also antioxidant activities. The olive oil wastes from the two phase extraction system have been widely studied, and the use of the thermal treatment is being transferred to industrial scale for the production of phenols extract and a final solid rich in oil and cellulose. The application of a physical pre-treatment, like the thermal ones, gives us the opportunity to get a real total utilization of agro industrial wastes that are actually causing environmental problems.

19

Valorization of corn-silage anaerobic digestate through the cultivation of

the white-rot fungus Pleurotus ostreatus

Guglielmo Santi1, Valerio G. Muzzini2, Emanuela Galli2, Simona Proietti1, Stefano Moscatello1, Alberto Battistelli1

1Istituto di Biologia Agro-ambientale e Forestale, via Marconi 2, 05010 Porano (TR) Italy

2Istituto di Biologia Agro-ambientale e Forestale, UOS di Montelibretti, Via Salaria Km 29.300; 00015 Monterotondo (Roma) Italy

The white-rot fungus Pleurotus ostreatus SMR 684 was cultivated on the solid digestate obtained from biogas production at commercial scale from corn silage (namely corn silage digestate, CSD), with the aim of i) producing edible mushrooms and ii) degrading lignin. The pure fungal species was grown in sterilized Petri dishes containing 3% malt extract and 1.5% agar. When the mycelium formed an abundant biomass, discs of 9 mm were cut and used for inoculation of the digestate. Each circle was placed in the center of a Petri dish (11 cm diameter) containing 15 g of wheat straw (WS, control) or CSD. Moisture was fixed at 70% in each substrate. The fungus was incubated for 42 days and samples were taken approximately twice per week. The mycelium in CSD grew as fast as in the control. Each sample was extracted with 0.1 M potassium phosphate buffer, filtered and centrifuged. The superrnatant was analyzed for the determination of proteins, ligninolytic activities (laccase, peroxidases) and cellulolytic and hemicellulolytic activities (endoglucanase, cellobiohydrolase, xylanase), while the solid residue was used for the determination of lignin. It is worth noting that the highest activities (total peroxidases: 4.06 IU/mg protein after 24 days; xylanase: 3.03 IU/mg protein after 17 days) were found in the mycelium grown on CSD. Lignin was reduced by 12% in CSD and 11% in WS after 42 days. This study indicated that CSD might represent a suitable feedstock for the production of fungal fruiting bodies. Moreover, the reduction of lignin percentage suggests that spent CSD after mushroom cultivation, enriched with fungal biomass, might be re-introduced into the anaerobic digester with the consequent increase of biogas yield, since fungal biomass is reported to represent a viable feedstock for biogas production.

20

:

Effects of dilution ratio, inoculum of yeasts and concentration of ammonium sulphate on the bioremediation of Olive Mill Wastewater

Antonio Bevilacqua, Nilde Di Benedetto, Milena Sinigaglia, Maria Rosaria Corbo Department of the Science of Agriculture, Food and Environment, University of

Foggia Yeasts can play a significant role in the bioremediation of Olive Mill Wastewater (OMW), as some strains are able to remove phenols, thus they decrease the impact of the wastes on the soil. This research focused on a cocktail of 5 yeast strains (Candida boidinii and Pichia membranifaciens) inoculated into a mixture of water and OMW to reduce the content of phenol. The ratio water/OMW, the amount of the inoculum and the concentration of ammonium sulphate (a coadjuvant previously shown to increase the yield in phenol removal) were modulated and combined by a centroid. Namely, the amount of ammonium sulphate varied from 0 to 18 g/l, the volume of inoculum from 1 to 10% and the ratio water/OMW from 0 (no dilution) to 3 (OMW: water, 1:3). The samples were stored at 25°C and the viable cell count and the concentration of phenols were evaluated periodically; the results were used as input values for multivariate analysis through the DoE approach (Design of Experiments). The statistical analysis highlighted that the three factors of the design affected the yield of phenol removal as single but not as interactive terms. Namely, the effect of dilution was linear and positive, i.e. the yield of phenol removal increased with increasing the dilution of the samples, whilst the effect of inoculum was quadratic and the targets experienced a higher yield when the volume of the inoculum increased up to a threshold level (2.5-4%), after which a further increase of the inoculum caused a reduction of the yield. In conclusion, the results of this research suggest that a suitable combination of the targets, by modulating their level in the system , the addition of ammonium sulphate and the dilution of OMW with water, could increase the yield and the effectiveness of bioremediation.

21

An approach to split 2-phase olive pomace into a polyphenol liquid and a valuable solid phase :

Steffen Hruschka,

Product Technology Manager, Olive Oil Recovery, GEA Westfalia Separator Group GmbH

Nowadays, the olive oil extraction process is becoming increasingly based on centrifugal technology. Today, the majority of the oil is produced in the so called “environmentally friendly system”, the two phase technology. Even if the amount of mills in three phases is higher, more and more tones of olives are being processedby separation into oil and wet pomace instead of oil water and semi dry pomace. It is well known that the oil quality is better due to the higher polyphenol content inside. But it is well known, too, that the wet pomace is difficult to handle besides the industrial processing of cogeneration for thermal and electrical energy production after drying the sludge. But until today after 20 years of the application of this 2-phase-process the wet pomace remains a problem for small and medium sized olive mills. A new patented pending process from GEA Westfalia Separator can be used for the de-pitted pomace in order to split this pomace into dry cake with about 60-65% dry matter (20% of the pomace), into decanter solids with 40-45% dry matter (also 20% of the pomace) and into a liquid without solids, only with 5% soluble materials (60% of the pomace). But the most interesting issue is that: about 80% of the polyphenols are found in the liquid. Because this liquid does not contain any diluting water, the polyphenol concentration in this fruit water is higher than in any previous process; for immediate and easy use.

22

Necessità di una normativa uniforme nel bacino del Mediterraneo per lo sviluppo di soluzioni eco innovative per rifiuti, reflui e energie rinnovabili

Need to have a uniform law in the Mediterranean basin for the development of eco-friendly solutions for waste, wastewater and renewable energy

Avv. Mariagrazia Chianura, Studio Legale Chianura via Giordano 23

Grottaglie (TA) Tel e Fax: 099.561.02.35

Cell: 349.71.60.314; e –mail: avv.chianura@gmail.com Il bacino del Mediterraneo rappresenta una delle zone potenzialmente di maggior sviluppo dal punto di vista della green economy. La vicinanza tra l’europa e la sponda sud del Mediterraneo, unita a condizioni climatiche che ne favoriscono attività antropiche similari, come alcuni tipi di coltivazioni – vite, ulivo, ortaggi – favoriscono la produzione di categorie di rifiuti che nella parte sud non trovano reimpiego, ma che invece possono essere molto appetibili per l’europa. Le condizioni climatiche di tutto il Bacino, favorirebbero poi lo sviluppo del settore delle rinnovabili. Ma spesso ciò che è consentito in un Paese non lo è in un altro, col rischio che lo scambio di rifiuti - ma anche di tecnologie per il recupero o l’utilizzo delle fonti rinnovabili - da potenziale strumento di crescita diventi un problema. Il mercato dei rifiuti da recupero può costituire un settore di crescita sia per l’europa che per i Paesi dell’altra sponda, sia in termini di risparmio di risorse vergini con lo scambio di rifiuto differenziato di buona qualità, sia come allargamento del mercato delle tecnologie per il trattamento dei rifiuti. Stesso discorso per le rinnovabili. Ma l’ostacolo maggiore è costituito dalla normativa frammentaria, complessa, che spesso si modifica nel breve periodo non consentendo di attrarre investimenti stranieri di medio lungo termine. Occorre sviluppare un sistema di norme che abbia un nucleo comune, con lo scopo di tutelare il bacino come ecosistema, ma anche di essere sostegno e occasione di uno sviluppo sostenibile in settori, quali quello dei rifiuti e delle rinnovabili, che presentano ancora molte opportunità di crescita. Una specie di carta delle buone regole ambientali: una serie di norme con principi comuni e condivisi sulle quali sviluppare accordi bi o pluri laterali. L’europa avrebbe il vantaggio di allargare le possibilità di sviluppo per settori che conosciamo già e che abbiamo imparato a sviluppare (recupero rifiuti, tutela degli ecosistemi, produzione di energia da fonti rinnovabili) e i Paesi che si affacciano sul bacino potrebbero trovare in questo un’opportunità di sviluppo economico e democratico, dando impulso a temi molto sentiti come quello della tutela dell’ambiente al fine di creare e mantenere un contatto proficuo con l’Europa.

23

E4-1

DEGRADATION OF 2-CHLOROPHENOL BY LACCASE-ZEOLITE

BIOCATALYST

E. GALLI1, C.M. POLCARO2, P. CICCIOLI2, E. DONATI2

1 Institute of Agro-Environmental and Forest Biology (IBAF), 2Institute of Chemical Metodologies (IMC); National Research Council (CNR), Research

Area of Rome 1, via Salaria Km 29,300; 00015 Monterotondo, Rome, Italy

Laccases (E.C.1.10.3.2.) have great biotechnological potential due to their ability to oxidize a broad range of substrates employed in several industrial sectors. These enzymes show broad specificity, high oxidation capacity, and reduced formation of by-products. Several authors immobilized laccases on different supports, synthetic and natural ones, obtaining improvements for enzyme applications due to the enhancement of storage and operational stabilities. For industrial application, carrier must be chip and highly disposable. With this aim, we have studied the possibility of using natural zeolites as a carrier for laccase immobilization. Zeolites are crystalline porous solids with a tridimensional structure consisting of SiO4 tetraedra, with some tetravalent Si atoms substituted by a trivalent Al, so producing a deficit of positive charge in the reticulum, which is compensated by cations. The hydrophobicity is higher with a high Si/Al ratio and corresponds to an improved proteins binding capacity at or around their isoelectric point (pI). Clinoptilolite is a natural zeolite with a very high Si/Al2 ratio and therefore we used it for laccase immobilization, studying both adsorption and covalent binding. Results obtained showed that covalent binding was the best method for laccase immobilization, showing pH and temperature stability greater than free laccase, an activity of about 50 U laccase g-1 of clinoptilolite (100-200 µm). When stored in water at 4°C, the biocatalyst retained 70% of activity after three months and 30% of activity after six months. For a practical application, the biocatalyst was used for degradation of 2-chlorophenol (0.1 mM) in batch at 25°C with 40 mg biocatalyst. Samples were periodically collected up to 48 h and analyzed by HPLC. After this time, only a 25% residual 2-chlorophenol remained in the solution.

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E4-2

IGAN ECO-POT project

Renzo Spagnesi, Martina Lotti, Vivaio Sandro Bruschi – Pistoia

Maurizia Seggiani, Monica Puccini, Sandra Vitolo, Dipartimento di Ingegneria Civile e Industriale (DICI) - Università di Pisa

Roberto Altieri, Alessandro Esposito, Francesco Castellani,Vitale Stanzione, Istituto per i Sistemi Agricoli e Forestali del Mediterraneo (ISAFOM) Consiglio

Nazionale delle Ricerche (CNR), Perugia Ermanno Federici, Laura Fidati, Elena Montalbani, Roberta Pret, Dipartimento di

Chimica, Biologia e Biotecnologie (DCBB) Università degli Studi di Perugia Masetti Gianna, Vivai Piante Masetti Sabino s.s.a, Pistoia

Vincenzo Tropiano, Michele Bellandi, Impresa Verde Pistoia, Pistoia Mario Romiti, Romiti F.lli Mario & Marco soc.agr., Pistoia

Plant nursery industry makes extensive use of non-biodegradable plastic pots (polypropylene, PP); in Italy, it is estimated an annual consumption of about 440 million PP pots which, at the end of their use, represent an expensive waste to be disposed of; therefore, the replacement of PP pot with similar products based on biodegradable materials, may reduce significantly the environmental impact of this sector. ECO-POT project aims at a cost-effectively production and successive field validation of biodegradable pots realized in different sizes; the mechanical properties of pots, i.e. resistance to lateral deformation and crushing, and rate of biodegradation should match needs of both operators and consumers. In particular, the biodegradation will be tested on plantable pot which are left intact on the root ball and planted into the field. In this case pots are designed to allow root to grow through the container walls and to decompose rapidly after being planted. In addition, the rate of biodegradation will be also tested on pots which are designed to be removed before final planting, thus speculating on the compostability of the used containers (test ISO UNI 14855-1) Different commercial biodegradable products, alone or mixed, will be tested. ECOPOT project involves different plant nursery farms located in Pistoia; up to 10,000 ornamental plants, placed in pots of different sizes, in different environments and belonging to 10 different species / varieties, will be tested. The most important expected results are: reduction in the consumption of non-renewable resources; economic benefits for nurseries gained in the international markets thanks to the adoption of recognizable low environmental impact trademarks (ECOLAB).

25

E4-3 The PUMAN Model. Methodology for the quantification of the wastewater volume in dry weather. A concrete solution to elaborate a standardized definition of priorities

for actions and investments into the wastewater collection and treatment fields.

Mario Chiarugi, Oberdan Cei, Roberto Salvadori, Gianluca Baronti, Simone Lippi. Acque Spa, Pisa, Italy

La Direttiva 91/271/CEE indicava gli obiettivi di qualità degli scarichi da raggiungere sul trattamento delle acque reflue urbane (assimilata dal D.Lgs 152/2006, Parte Terza del T.U.A.). L’Italia, inadempiente, è stata oggetto di più procedure di infrazione (2004/2034, 2009/2034 e 2014/2059) inerenti la depurazione de gli scarichi civili ed industriali in agglomerati con oltre 2000 Abitanti Equivalenti (A.E.) al fine di tutelare la qualità dei corpi idrici ricettori, soprattutto nelle aree c.d. “sensibili”. Una delle problematiche tecniche legate all’adempimento della direttiva è la quantificazione del carico inquinante afferente nei vari punti di scarico che spesso viene stimato con criteri non omogenei. Attraverso il modello matematico proposto, denominato “PUMAN” (Portate Utenze Media Annua Nera) è possibile ottenere una stima affidabile della ripartizione idraulica delle acque reflue tra zone fognate e non fognate ed una quantificazione in Abitanti Equivalenti di ogni punto di scarico del sistema, sia esso un impianto di depurazione o uno scarico non depurato. Per stimare la portata nera in tempo asciutto, ovvero il carico idraulico prodotto nei singoli agglomerati o in ogni unità geografica, vengono considerati i dati relativi al fatturato di ogni singola utenza georeferenziata presente all’interno dei confini dell’unità geografica applicando poi un coefficiente di afflusso in fognatura variabile in base alla tipologia di utenza. Sovrapponendo le varie utenze con il grafo del reticolo fognario è possibile associare ad ogni ramo fognario , con criteri logici di prossimità, il carico ivi afferente e di conseguenza, il carico afferente in ogni punto di scarico del reticolo fognario. Il modello “PUMAN” è flessibile ed esportabile in altri contesti dove non è possibile procedere a una misura diretta delle portate nere. Il sistema può fornire una mappatura puntuale anche su scala nazionale, potrebbe costituire una base-dati comune per definire i contesti critici e quantificare gli impatti in termini sociali, ambientali ed economici. Gli amministratori delle Aziende del Servizio e il Legislatore stesso potrebbero dotarsi di questo strumento per individuare classificare gli interventi in base a una scala di priorità e quantificarne gli aspetti economici per gli investimenti. Il modello, implementato su scala reale di ATO da un gestore del S.I.I., potrebbe essere prossimamente esteso su tutto il territorio della Regione Toscana come metodo per censire gli scarichi diretti; se esteso su scala nazionale potrebbe contribuire ad uniformare il settore del Servizio Idrico e consentire un’oggettiva valutazione delle necessità, delle emergenze e delle relative priorità d’intervento.

26

E4-4 :

Orange peel wastes pretreatment by acid-catalyzed steam explosion for enhancing bioethanol production

Guglielmo Santi, Julia Jasiulewicz, Alessandro D’Annibale, Silvia Crognale,

Maurizio Petruccioli, Mauro Moresi Department for Innovation in Biological, Agro-Food and Forest Systems (DIBAF),

University of Tuscia, Via S. Camillo De Lellis, 01100 Viterbo, Italy Although bioethanol production from cereal grains and molasses is a mature technology, its application raises growing socio-economic concerns due to fuel/food competition. The second generation approach, relying on the use of lignocellulosic wastes, might mitigate the aforementioned competition. In this study, orange peel waste was converted into bioethanol via a process scheme involving acid-catalyzed steam-explosion (ACSE), enzymatic saccharification and fermentation with the industrial strain Saccharomyces cerevisiae F15. The first step was carried in a lab-scale Direct Steam-Injection Apparatus under constant reaction temperature (180 °C), time (150 s) and sulphuric acid content (0.5 % v/v) and using two consistency levels, namely a solid loading of either 160 or 480 g/L. At the latter solid loading, the solubilization of complex sugars and the concentrations of degradation products were lower and higher, respectively, than those found with the former consistency level. The enzymatic hydrolysis (EH) of residual solids from ACSE pretreatment at 480 g/L with a cocktail containing cellulase (12 FPU/ g cellulose) and pectinase (25 U/g DM) enabled the attainment of a hydrolysate with a fermentable sugars concentration of 34.0 g/L. The sugar concentration of these syrups were 3-fold higher than those obtained after enzymatic hydrolysis of samples derived from ACSE at the lower solid loading. Fermentation processes were then conducted on a 1-L Stirred Tank Reactor (STR) operated under repeated batch mode to compare the conversion efficiency of sugar syrups derived from ACSE and subsequent EH at the two consistency levels under study. STR experiments showed that the use of the higher solid loading in the ACSE step enabled to significantly increase the ethanol content in fermentation broths with respect to that obtained from the hydrolyzate from ACSE at the lower consistency (15.38 vs. 7.41 g/L) with a limited loss in the gravimetric ethanol yield (0.48±0.01 vs. 0.49±0.04 g/ g) and productivity (4.07 ± 0.04 vs. 4.23 ± 0.39 g/ L h).

27

E4-5 Bioremediation come soluzione alternativa per la gestione dei rifiuti: studio su

potenziali microrganismi “phenol-degrading” Bioremediation as alternative solution for wastes management: study on

potential phenol-degrading microorganisms

Campaniello Daniela, Bevilacqua Antonio, Sinigaglia Milena, Corbo Maria Rosaria Dipartimento di Scienze Agrarie, degli Alimenti e dell'Ambiente, Università di Foggia

Nel presente lavoro è stato eseguito uno screening di 119 ceppi isolati da suolo (46 identificati più generalmente come mesofili, 44 afferenti al genere Bacillus e 25 al genere Pseudomonas spp.) al fine di individuare batteri “phenol degrading” per la detossificazione delle acque di vegetazione dell'industria olearia. A tale scopo sono state allestite 2 prove sperimentali. Una prima prova avente come obiettivo quello di verificare la capacità dei batteri di degradare i composti fenolici ed una seconda prova avente lo scopo di individuare ceppi i cui enzimi, capaci di rimuovere i fenoli, venissero indotti da un contatto prolungato con il substrato. In entrambi i casi è stato utilizzato un substrato colturale sintetico minimale (Mineral Salt Medium, MSM) addizionato di acido vanillico e acido cinnamico (a 500 e 1000mg/l) come unica fonte di carbonio. In presenza dell’acido cinnamico nessuno dei 119 ceppi testati era capace di svilupparsi. Al contrario, in presenza dell’acido vanillico 13 ceppi rispondevano positivamente. Relativamente alla prova di induzione è stato osservato che in presenza dell’acido vanillico 48 ceppi erano in grado di svilupparsi già alla concentrazione più bassa (500 mg/l); diversamente, in presenza dell’acido cinnamico solo 3 ceppi rispondevano positivamente. Ulteriori indagini verranno effettuate al fine di individuare ceppi batterici da impiegare nel trattamento delle acque di vegetazione a livello industriale.

28

E4-6

Fungi isolated from olive mill wastewater: Identification, phylogenetic aspects and preliminary assessment of their effluent-degrading abilities

Vassiliki Fryssouli1, Evangelos Dagres1, Io Kefalogianni1, Milton A. Typas2 and

Georgios I. Zervakis1 1Agricultural University of Athens, Laboratory of General and Agricultural

Microbiology, Iera Odos 75, 11855 Athens, Greece (e-mail: zervakis@aua.gr) 2National and Kapodistrian University of Athens, Faculty of Biology, Department

of Genetics and Biotechnology, Panepistemiopolis, 15701 Athens, Greece Disposal of olive mill wastewater (OMW) constitutes an important environmental issue in olive-oil producing countries since it could significantly deteriorate quality of water and soil receptors. OMW is generated in huge quantities within a short period of time, and presents high contents in polyphenolics and total organic matter, which complicates treatment by either conventional physicochemical or biological approaches. This study focused at the diversity assessment of OMW’s cultivated mycobiota (adapted to effectively colonize it), and at the establishment of its potential in degrading and detoxifying the effluent. Fungi were isolated by serial dilutions under different temperatures and in various selective media; the morphological characteristics of their colonies were further examined for identifying them (at least) to genus level. Then, representative isolates from each taxon and/or morphotype were studied on the basis of their ITS1-5.8S-ITS2 rRNA sequences. Phylogenetic analysis of the resulting data revealed the existence of four major complexes, including Aspergillus fumigatus, A. niger, A. tubingensis, A. flavus, Geotricum reessii, G. candidum, Penicillium crustosum, P. commune, P. brevicompactum, Mucor racemosus, M. circinelloides and M. fragilis. Selected strains from each species were further evaluated by examining COD, total phenolics reduction, decolorization and phytotoxicity values of the biotreated OMW. Preliminary results indicated that several isolates achieved medium to high COD decrease within a two weeks cultivation period, while for a few strains a satisfactory phenolics reduction was combined with enhancement of plant-seed germinability. Further studies are oriented towards optimization of individual conditions related to the effluent’s treatment process by indigenous fungi. Acknowledgements: This research has been co-financed by European Union (ESF) and Greek national funds (NSRF) through the project titled “Metagenomics of ligninolytic microorganisms – Bioconversion of plant by-products into high-added value products” (THALIS–UOA–MIS377062).

29

E4-7

Biohydrogen production from glucose, molasses and cheese whey by suspended and attached cells of hyperthermophilic bacteria belonging to

Thermotoga genus

Martina Cappelletti1, Davide Pinelli2, Stefano Fedi1, Davide Zannoni1, Dario Frascari2

1 Dept. of Pharmacy and BioTechnology, University of Bologna, Via Irnerio 42,

40126 Bologna, Italy 2 Dept. of Chemical, Mining and Environmental Engineering, University of

Bologna, Via Terracini 28, Bologna, Italy Abstract: Hydrogen (H2) gas is a clean energy source with no CO2 emissions and high energy content. As a viable alternative fuel, H2 is considered the “energy carrier” of the future with a wider range of combustible properties than other fuels. The major problem in the utilization of H2 is its unavailability in nature and the need for non-expensive production methods. Fermentative production of H2 from carbohydrate-rich organic wastes has recently received considerable attention thanks to its possible implementation in cost-effective processes. Hyperthermophilic anaerobic bacteria belonging to Thermotoga genus are considered ideal organisms for H2 production because of their ability to utilize a wide range of carbohydrates, their tolerance to moderate oxygen (O2) amount (up to 1%) and a better resistance to high H2 partial pressure. In this work, the feasibility of a biological H2 production process from food industry wastes was evaluated under thermophilic conditions. The H2-producing performances of 4 Thermotoga strains (T. neapolitana, T. petrophila, T. naphtophila, T. maritima) were compared at 77 ºC using glucose, molasses or cheese whey as carbon source in 120 mL batch bioreactors. Optimization of growth medium composition, biofilm support type and substrate concentration was performed along with the analysis of inhibitory effects exerted by H2 gas accumulation and O2 presence in fermentative cultures. Experiments testing suspended- and attached-cells growth conditions showed a significant advantage of biofilms over planktonic cells with glucose and molasses. Kinetic model of H2 production was developed for the three substrates under analysis and the suspended-cell processes were successfully scaled-up to a 19-L bioreactor. The development of efficient H2 production processes from industrial residues will combine economic and sustainable energy generation with waste management.

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E4-8

Production of ethanol from xerofile and halo-tolerant plant biomass. The case of Tamarix jordanis grown in the desert and irrigated with wastewater

or brackish waters G. Santi1, A. D’Annibale1, A. Eshel2, A. Zilberstein2, S. Crognale1, M. Ruzzi1, R.

Valentini1, M. Moresi1, M. Petruccioli1 1Department for Innovation in Biological, Agro-Food and Forest systems

(DIBAF), University of Tuscia, Via S. Camillo De Lellis, 01100 Viterbo, Italy 2Department of Molecular Biology and Ecology of Plants, Tel Aviv University,

Tel-Aviv 69978, Israel Although the production of bioethanol from cereal kernels and molasses is now a mature technology, the consequent and possible subtraction of resources for human consumption continues to raise large socio-economical concerns. The approach of the second generation, based on the use of lignocellulosic biomasses as sugar source, appears to be the way out to mitigate the problems mentioned above. This is even more interesting if the production of plant biomass takes place on lands that are marginal, due to soil and climate. In the present work, 3 different Tamarix species, namely T. aphylla, T. aphylla “Erect”-type and T. jordanis, were grown in an experimental field under extreme desert conditions and irrigated with either reclaimed sewage or brackish water. Depending on both species and source of irrigation, the above-ground biomass production ranged from 18 to 36 Mg ha-1 in the first year. Among the three chemically characterized Tamarix species, T. jordanis was selected due to its higher cellulose content, and lower hemicellulose and phenol contents so as to outline a preliminary process flow sheet for ethanol production. This included steam-injection heating under acidic conditions (200 °C; 90 s; 0.5% H2SO4; 160 g L-1 solid loading) using a novel lab-scale Direct Steam Injection Apparatus, enzymatic saccharification (50 °C; pH 5.0; 200 g L-1 solid loading; 20 FPU g-1 cellulose) and subsequent ethanolic fermentation (30 °C; Saccharomyces cerevisiae F-15 as the inoculum). Lab-scale fermentation runs were carried out in a 3-L stirred bioreactor in repeated-batch mode and showed an almost quantitative conversion of glucose into ethanol (0.507±0.006 g g-1), thus leading to a satisfactory overall process yield of about 145 L ethanol Mg-1 Tamarix biomass.

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E4-9

NEXT GENERATION SEQUENCING OF BACTERIA INVOLVED IN THE STATIC COMPOSTING OF OLIVE MILL WASTE

Fidati L.1, Cenci G.1, Esposito A.2, Altieri R.2, Federici E.1

1 University Of Perugia - Chimica, Biologia e Biotecnologie - Perugia, Italy

2 Consiglio Nazionale Delle Ricerche - Istituto Per I Sistemi Agricoli e Forestali Del Mediterraneo - Perugia, Italy

Static composting of olive mill waste (OMW) can be regarded as an easy and convenient way for the disposal and valorisation of such agroindustrial by-product. We investigated the bacterial community dynamics during OMW composting by applying a culture-independent molecular approach based on denaturing gradient gel electrophoresis (DGGE) profiling and Illumina next generation sequencing (NGS) of amplified 16S rRNA genes. DGGE analysis indicated the presence of a very high bacterial diversity in both the OMW and the composting mixture and the occurrence of a rapid succession of different bacterial populations throughout the process. Indeed, only few populations were shared among all the samples, while most seemed peculiar of the different phases of biotransformation of OMW into mature compost. NGS analysis allowed the identification of these populations. Actinobacteria were abundant in the OMW, declined at the beginning of the active/thermophilic phase and then represented the predominant populations during the curing/mesophilic phase. Interestingly, while in the OMW most Actinobacteria belonged to the family Microbacteriaceae, the compost in the curing phase featured a high abundance of Pseudonocardiaceae, mostly represented by the moderately halophilic Prauserella genus. As expected, the most abundant populations found in the active phase, when the temperature reached 60°C, were the Bacilli, particularly those belonging to the thermophilic, spore-forming Geobacillus genus. Finally, the compost in the maturing phase was mainly characterized by an even distribution of bacteria belonging to the orders Actinomycetales, Sphingobacteriales, Burkholderiales, Xanthomonadales and Rhizobiales.

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E4-10 Biogas production by mesophilic phase-separated anaerobic digestion of

corn-DDGS

Botond Ráduly1, Silvia Crognale2, László Gyenge1, István Máthé1, Beáta Ábrahám1

(1) Dept. of Bioengineering, Sapientia Hungarian University of Transylvania, Miercurea Ciuc, Romania

(2) DIBAF, Università degli Studi della Tuscia, Viterbo, Italia Anaerobic digestion (AD) is a promising valorization method of bioethanol processing byproducts. By converting these byproducts into biogas, which can be used on-site in the ethanol production line, AD is able to better the net energy balance of the bioethanol fuel. Key parameters to the success of the adoption of AD in the bioethanol industry are the obtainable specific methane yield, and the concentration of methane (CH4) in the produced biogas. On these parameters will depend the quantity of the CH4 that can be produced by the digestion of the byproducts, as well as the required technology level of the biogas upgrading equipment. This study addresses both aspects by proposing phase-separated fermentation of the byproducts of bioethanol processing. By creating optimal conditions for the different phases of the AD process in two distinct reactors, phase separation allows for shorter retention times and better conversion rates of the organic matter, as well as for higher CH4 content of the produced biogas. In our experiments laboratory-scale phase-separated digestion of corn- DDGS (distillers dried grains with solubles) has been accomplished under mesophilic conditions. A specific methane yield of 330 mL CH4/g VS has been obtained, at an organic loading rate of 5.21 g VS/L/d and a total hydraulic retention time of 21 days (5.7 days in the acid, and 15.3 days in the methane reactor). The average CH4 concentration during the experiments was 76.4 %, with a peak value of 81.4 % CH4. Such high methane content may enable the use of the produced biogas in cogeneration units as is, eliminating the expensive biogas upgrading operations. The relatively short retention time also makes the technology appealing for industrial applications.

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E4-11

Mediterranean fruits wastes as biorefinery feedstocks

Ana Dionísio, Rute Neves, Pedro C. Branco, Ivone Torrado, Patrícia Moniz,

Talita Fernandes, Luísa Roseiro, Florbela Carvalheiro, Luís C. Duarte LNEG – Unidade de Bioenergia, Estrada do Paço do Lumiar, 22, Edifício K2,

1649-038 Lisboa, Portugal luis.duarte@lneg.pt

Many Mediterranean fruits are currently used in several agro-food industries, yielding significant amounts of wastes. The majority of these waste materials do not have any significant utilization, being mainly used as low value energy sources, as is the case of e.g. Extracted Olive Pomace (EOP). Due to their putative large amounts and geographical concentration at industrial sites, the use of these residues as feedstock within the biorefinery framework can be a potentially advantageous alternative for their valorisation, which must be explored and validated. As such, the available quantities of fruit wastes in Portugal and other Mediterranean countries were estimated and are discussed. Furthermore, five different typical wastes, namely pine nuts shells (Pinus pinea), EOP (Olea europaea), peach pits (Prunus persica), cherimoya seeds (Annona cherimola) and watermelon rinds (Citrullus lanatus) are used as model materials. The materials were subjected to partition analysis and chemically characterized, taking special interest on their extractives and hemicelluloses content and composition. The comparative advantages of these materials as biorefineries feedstocks are presented and discussed using the Biotechnological Valorization Potential Indicator (1) and guidelines are set for their management. Acknowledgments The financial support of Fundação para a Ciência e a Tecnologia (FCT, Portugal) within research contract PTDC/AGR-ALI/122261/2010 is gratefully acknowledge. References 1. Duarte LC, Esteves MP, Carvalheiro F, Gírio FM. 2007. Biotechnological Valorization Potential Indicator for lignocellulosic materials. Biotechnol. J. 2:1556–1563.

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E4-12

Potential of Opuntia ficus-indica [L.] Miller residues for bioethanol

production

T. Mesquita1,2, M. S. Romanovich1,2, H. T. Chaves2, F. Carvalho2, M. C.Fernandes1 (maria.fernandes@cebal.pt)

1Centro de Biotecnologia Agricola y Agro-Alimentar do Alentejo (CEBAL), Beja, Portugal, 2Departamento de Tecnologias y Ciencias Aplicadas, ESAB-IP, Beja,

Portugal

Opuntia fícus-indica [L.] Miller (OFI) is a culture of domesticated cactus from very ancient times, which is important for the agricultural economies throughout arid and semiarid parts of the world. OFI plantation has been growing in the lasts decade in Europe, especially in the exploitation of marginal areas or lands with very dry climates.

The fruit of this plant, known as Indian-fig or prickly-pear fruit is used in the food industry for the production of jams and juices. From this activity two residues are produced, the peels and the seeds. Seeds constitute about 10–15% of the edible pulp and are usually discarded as waste after extraction of the pulp. From the seed can be extracted oil with high value. In this work the OFI fruit peel and the oil extracted seeds were characterized according to NREL Protocols. Also, we assessed bioethanol production, with 5% solid loading, by direct fermentation and by Sequential Hydrolysis and Fermentation (SHF) of both residues.

Extracted seeds presented higher glucan and Klason lignin content than the peels. Conversely peels presented higher water extractable content that was very rich in sugars. For the extracted seeds, the enzymatic hydrolysis, allowed a yield of 40% of glucan conversion to glucose. On the contrary, for peels, there was no appreciable increase in the production of glucose in the presence of enzymes. The bioethanol production with extracted seeds was lower than with peels. The highest bioethanol concentration achieved was 17.1 ± 0.1 g/L, for peels in SHF mode.

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E4-13

Eco-innovative Studies in Turkey for Olive Waste Valorisation and OMWW

Treatment

Erdinc Ikizoglu Ege University, Bornova-Izmir, Turkey

At the last 20 years, 165 studies at the Universities of Turkey regarding to “Olive Waste” have been published in several International Journals. However, there are only 2 patents belonging to Universities and 3 patents belonging to the companies. Beside the laboratory studies at Universities, 4 pilot and industrial plants were constructed for olive waste valorization and OMWW treatment. All these plant studies will be reviewed at this presentation.

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From the IAMAW Statute

Article I: Name and Concept … IAMAW is an international non-profit scientific association, working in the public interest with only social solidarity objectives in the sector of instruction, training and RTD, to boost issues related to treatment and recycling of Mediterranean waste streams and by-products of agro-industrial activities, in an environmental-sensitive manner. The term Mediterranean refers to climatic/edaphic condition and not to geographic location. The Association is established for an indefinite period of time and it will not promote and perform activities different from those listed in the statute and closely related to it. …… Article II: Objectives The objectives of IAMAW are: to foster environmental solutions for agro-industrial, forestry and municipal organic by-products, residues, waste and effluents through reduction, treatment, reuse and amelioration; to promote scientific research in the above-mentioned fields; to organize working groups that will advance knowledge and expertise through knowledge exchange; to promote knowledge dissemination and transfer of know-how among members, end-users, stakeholders and to the scientific world, mainly through professional conferences and symposia; to improve and demonstrate the feasibility and economic sustainability of the proposed solutions; to create working relations in order to foster international research with institutes/scientists working in the field beyond the Mediterranean region and to access financial and technology resources; … to assist policy-makers in defining more rational and uniform laws and regulations in the field of Mediterranean waste management and in the identification of Best Available Technologies (BAT). Article III – Activities The Association will: 1. Hold International Congresses at regular intervals ….2. Arrange international workshops and symposia as well as other international meetings. 3. Establish Working Groups according to the main scientific disciplines involved in its scope of interests. 4. Form relationships and cooperate with other governmental and nongovernmental organizations in its field of interest. 5. Edit, produce and distribute information, reports and scientific or technical publications, reserving the exclusive rights and copyrights to the Association according to Italian law. 6. Use other legal means to achieve the objectives of the Association. 7. To the aim of collect money through no-profit actions, the association can perform minor economical activities according to the Italian d.m. del 25/5/1995 in the direction of pursue institutional objectives of the association without the use of professional tools developed for competing on the market.

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