II SIMPÓSIO FibEnTech Materiais Fibrosos e Tecnologias Ambientais LIVRO DE ATAS

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2 II SIMPÓSIO FibEnTech 2017 Materiais Fibrosos e Tecnologias Ambientais LIVRO DE ATAS COVILHÃ, UNIVERSIDADE DA BEIRA INTERIOR DE SETEMBRO DE 2017

3 Reservados todos os direitos Título: Atas do II Simpósio Materiais Fibrosos e Tecnologias Ambientais FibEnTech - Universidade da Beira Interior de setembro de 2017, Covilhã Portugal Coordenador da Edição: Manuel José dos Santos Silva Comissão Organizadora: Manuel José dos Santos Silva Ana Maria Matos Ramos Rui Alberto Lopes Miguel Arlindo Caniço Gomes Annabel Dias Barrocas Fernandes Kátia Nuelma Gil Monteiro Comissão Científica: Manuel José dos Santos Silva Ana Maria Carreira Lopes José Mendes Lucas Rogério Manuel Simões Apoio Técnico: Annabel Fernandes Kátia Monteiro ISBN: Apoios:

4 ÍNDICE Electrochemical treatment of cheese whey wastewater: Influence of anode material, chloride concentration and current density M.M. Batista, A. Fernandes, L. Ciríaco, M.J. Pacheco, A. Lopes...1 Recycling of Silicone Coated Paper J.M.R. Curto, A.P. Costa, P.E.M. Videira, M.E. Amaral, R.M.S. Simões, M.J.S. Silva...5 Companies' adaptation through innovation J.A.B. Barata, R.A.L. Miguel, S.G. Azevedo...8 The identity of the Portuguese Fashion through the materials, techniques and technologies C. Rito, M. Pereira...12 Design of nanomaterials for Biomedical applications using 3D Computational Simulation J.M.R. Curto, F.P. Morais, R.M.S. Simões, M.J. Santos Silva...14 Fashion Marginalization and the Feminine F.T.S. Gonçalves, S.R. Fernandes, J.M. Lucas...17 Porosity optimization of nano cellulose drug delivery systems to control the release kinetics F.P. Morais, J.M.R. Curto...21 Women wears men s clothing: a study case for a niche market B. Reis, M. Pereira, S. Azevedo, N. Jerónimo, R. Miguel...26 Entrepreneurship: new models of business in fashion design C. Fernandes, M. Pereira, J. Lucas, R. Miguel, J. Santos Silva...30 PLA/cellulose composite spheres: preparation and characterization S. Sousa, A.P. Costa, R. Simões...33 Influence of Bi 4 Ti 3 O 12 as a secondary phase in the photocatalytic activity of perovskite materials M.J. Nunes, A. Lopes, M.J. Pacheco, P.T. Fiadeiro, L. Ciríaco...37 U.MAKE.ID A Digital Sourcing Platform Project: A Theoretical Approach B. Reis, L. Pina, P. Rafael, M. Pereira, R. Miguel...40 Optimization of EDOT polymerization by using Design Expert P. Baptista, G. Soares, R. Simões, R.Miguel...43 Industrial solid waste landfill leachate treatment by electrochemical processes R. Silva, A. Fernandes, L. Ciríaco, M.J. Pacheco, A. Lopes...48 Theoretical Essay on the Networked Collaborative Consumption S.R. Fernandes, J.M. Lucas, M.J. Madeira, A.I.C. Barreiros, F.T.S. Gonçalves...52

5 Creative textiles design: traditional crafts as source of inspiration L.S. Ribeiro, R.A.L. Miguel...55 Flame Atomic Absorption Spectrometry: parameters optimization L. Matias, A. Marques...58 Quality control techniques for routine analysis used in accredited laboratories: physicalchemical analysis L. Borges, R. Palmeira-de-Oliveira, A. Marques...61 Use of the phytoremediation for tuning cheese wastewater pretreated by chemical precipitation A. Almeida, A. Prazeres, C. Ribeiro, H. Chaves, F. Carvalho...65 Synthesis of carbohydrate derivates with potential activity for the treatment of bipolar disorder M. Gomes, M. Lucas, J.A. Figueiredo, S. Silvestre, M.I. Ismael...69 Phytochemical characterization and determination of the biological activity of bark's extracts of Salix salviifolia M. Lucas, M. Gomes, J.A. Figueiredo, A.P. Duarte, M.I. Ismael...72 Studies on the Solubility of Methyl Orange and Acid Orange 8 in Water C.M.G. Pinto, M.I.A. Ferra, I.M.S.C. Gonçalves, M.J.A. Pacheco, A. Marques...76 Phytochemical composition and assessment of rheological and antioxidant properties of juices of berries Â. Luís, S. Sousa, A.P. Duarte, L. Pereira, F. Domingues...78 Assessment of Cork Boiling Wastewater Treatment by Constructed Wetland at Horizontal Subsurface Flow Configuration A.C. Gomes, M.L. Silva, A. Stefanakis, R. Simões, A. Albuquerque...82 Development of sustainable antimalarial clothing C.I.S. Pinheiro, N.J.R. Belino, P. Roshan...86 A preliminar study on the bioactive compounds present in leaves and stems of Urtica dioica L. M.F.C. Tenreiro, M.E. Amaral...88 Anaerobic mesophilic biodegradation of raw olive pomace V. Guilherme, I.C. Gonçalves, A. Marques, A. Lopes, I. Ferra...92 Essential oils from fennel seeds collected in different regions from Portugal L. Silva, V. Lopes, E. Bettencourt, S. Pombal, J. Rodilla, A. Gomes...96 Production of high performance filaments from nano/microfibrillated vegetal cellulose V.L.D. Costa, J.W. Gomulka, P.D.L. Gaspar, D.J.M. Santos, A.P. Costa, M.E. Amaral, R.M.S. Simões...98

6 FibEnTech - Fiber Materials and Environmental Technologies 2nd Symposium of the FibEnTech Research Unit: Fiber Materials and Environmental Technologies Faculty of Engineering, September 2017 Conference Programme Thursday, 21 September 9:00-9:30 Registration, Anfiteatro 8.1, Faculty of Engineering 9:30-10:00 WELCOME SESSION AND PRESENTATION OF THE RESEARCH UNIT OF FIBER MATERIALS AND ENVIRONMENTAL TECHNOLOGIES Paulo Moniz, Vice Rector - Universidade da Beira Interior Mário Freire, President of the Faculty of Engineering - Universidade da Beira Interior 10:00-11:00 SESSION I Manuel José dos Santos Silva, Coordinator of the FibEnTech research unit - Universidade da Beira Interior Chairpersons: Ana Lopes, José Lucas and Rogério Simões Chairpersons: Arlindo Gomes, Paulo Fiadeiro and Isabel Ismael 10:00-10:20 Electrochemical treatment of cheese whey wastewater: Influence of anode material, chloride concentration and current density M.M. Batista, A. Fernandes, L. Ciríaco, M.J. Pacheco, A. Lopes FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 10:20-10:40 Recycling of Silicone Coated Paper J.M.R. Curto 1,2,3, A.P. Costa 1,2, P.E.M. Videira 1, M.E. Amaral 1,2, R.M.S. Simões 1,2, M.J.S. Silva 1 1 FibEnTech-UBI, Universidade da Beira Interior, Covilhã, Portugal 2 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 3 CIEPQPF, Chemical Process Engineering and Forest Products Research Centre, Faculdade de Ciências e Tecnologia da Universidade de Coimbra, Coimbra, Portugal 10:40-11:00 Companies' adaptation through innovation J.A.B. Barata 1, R.A.L. Miguel 1, S.G. Azevedo 2 1 FibEnTech-UBI, Textile Science and Technology Department, Universidade da Beira Interior, Covilhã, Portugal 2 CEFAGE, Business and Economics Department, Universidade da Beira Interior, Covilhã, Portugal 11:00-11:30 Coffee Break and Poster Session 11:30-12:30 SESSION II Chairpersons: Ana Paula Costa and Rui Miguel

7 11:30-11:50 The identity of the Portuguese Fashion through the materials, techniques and technologies C. Rito 1, M. Pereira 1,2 1 FibEnTech-UBI, Textile Science and Technology Department, Universidade da Beira Interior, Covilhã, Portugal 2 UNIDCOM, IADE, Universidade Europeia, Lisboa, Portugal 11:50-12:10 Design of nanomaterials for Biomedical applications using 3D Computational Simulation J.M.R. Curto 1,2, F.P. Morais 1, R.M.S. Simões 1, M.J. Santos Silva 3 1 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 2 CIEPQPF - Chemical Process Engineering and Forest Products Research Centre, Department of Chemical Engineering, University of Coimbra, Coimbra, Portugal 3 FibEnTech-UBI, Department of Textile Science and Technology, Universidade da Beira Interior, Covilhã, Portugal 12:10-12:30 Fashion Marginalization and the Feminine F.T.S. Gonçalves, S.R. Fernandes, J.M. Lucas FibEnTech-UBI, Textile Science and Technology Department, Universidade da Beira interior, Covilhã, Portugal 12:30-14:00 Lunch and Poster Session 14:00-15:40 SESSION III Chairpersons: Ana Ramos, Maria Emília Amaral and Ana Gomes 14:00-14:20 Porosity optimization of nano cellulose drug delivery systems to control the release kinetics F.P. Morais 1, J.M.R. Curto 1,2 1 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 2 CIEPQPF - Chemical Process Engineering and Forest Products Research Centre, Department of Chemical Engineering, University of Coimbra, Coimbra, Portugal 14:20-14:40 Women wears men s clothing: a study case for a niche market B. Reis 1, M. Pereira 2, S. Azevedo 3, N. Jerónimo 4, R. Miguel 1 1 FibEnTech-UBI, Department of Textile Science and Technology, Universidade da Beira Interior, Covilhã, Portugal 2 FibEnTech-UBI and UNIDCOM, Faculty of Engineering, Universidade da Beira Interior, Covilhã, Portugal 3 Faculty of Social and Human Sciences, CEFAGE-UBI, Universidade da Beira Interior, Covilhã, Portugal 4 Faculty of Social and Human Sciences, LabCom.IFP, Universidade da Beira Interior, Covilhã, Portugal 14:40-15:00 Entrepreneurship: new models of business in fashion design C. Fernandes 1, M. Pereira 1,2, J. Lucas 1, R. Miguel 1, J. Santos Silva 1 1 FibEnTech-UBI, Department of Textile Science and Technology, Universidade da Beira Interior, Covilhã, Portugal 2 UNIDCOM, IADE, Universidade Europeia, Lisboa, Portugal

8 15:00-15:20 PLA/cellulose composite spheres: preparation and characterization S. Sousa, A.P. Costa, R. Simões FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 15:20-15:40 Influence of Bi 4 Ti 3 O 12 as a secondary phase in the photocatalytic activity of perovskite materials M.J. Nunes 1, A. Lopes 1, M.J. Pacheco 1, P.T. Fiadeiro 2, L. Ciríaco 1 1 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 2 FibEnTech-UBI, Department of Physics, Universidade da Beira Interior, Covilhã, Portugal 15:40-16:10 Coffee Break 16:10-17:30 SESSION IV Chairpersons: Joana Curto, Álvaro Vaz, and Jesus Rodilla 16:10-16:30 U.MAKE.ID A Digital Sourcing Platform Project: A Theoretical Approach B. Reis 1, L. Pina 2, P. Rafael 3, M. Pereira 4, R. Miguel 1 1 FibEnTech-UBI, Department of Textile Science and Technology, Universidade da Beira Interior, Covilhã, Portugal 2 Department Textiles, U.MAKE.ID Researcher, Universidade da Beira Interior, Covilhã, Portugal 3 Consultant in the project U.MAKE.ID, Lisbon, Portugal 4 Department Textiles, UNIDCOM, FibEnTech, Universidade da Beira Interior, Covilhã, Portugal 16:30-16:50 Optimization of EDOT polymerization by using Design Expert P. Baptista 1, G. Soares 2, R. Simões 3, R. Miguel 1 1 FibEnTech-UBI, Department of Textile Science and Technology, Universidade da Beira Interior, Covilhã, Portugal 2 Textile Engineering Department, Universidade do Minho, Guimarães, Portugal 3 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 16:50-17:10 Industrial solid waste landfill leachate treatment by electrochemical processes R. Silva, A. Fernandes, L. Ciríaco, M.J. Pacheco, A. Lopes FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 17:10-17:30 Theoretical Essay on the Networked Collaborative Consumption S.R. Fernandes 1, J.M. Lucas 1, M.J. Madeira 2, A.I.C. Barreiros 3, F.T.S. Gonçalves 1 1 FibEnTech-UBI, Textile Science and Technology Department, Universidade da Beira interior, Covilhã, Portugal 2 CIEO, Department of Management and Economics, Universidade da Beira Interior, Covilhã, Portugal 3 Polytechnic Institute of Castelo Branco, Castelo Branco, Portugal 17:30-18:30 Scientific Council of the FibEnTech Research Unit

9 Friday, 22 September 9:30-10:50 SESSION V Chairpersons: Isabel Ferra, Luísa Salvado and Lurdes Ciríaco 9:30-9:50 Creative textiles design: traditional crafts as source of inspiration L.S. Ribeiro, R.A.L. Miguel FibEnTech-UBI, Textile Science and Technology Department, Universidade da Beira interior, Covilhã, Portugal 9:50-10:10 Flame Atomic Absorption Spectrometry: parameters optimization L. Matias 1, A. Marques 2 1 Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 2 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 10:10-10:30 Quality control techniques for routine analysis used in accredited laboratories: physical-chemical analysis L.Borges 1, R. Palmeira-de-Oliveira 1, A. Marques 2 1 Labfit-HPRD: Health Products Research and Development, Ubimedical, Covilhã, Portugal 2 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 10:30-10:50 Use of the phytoremediation for tuning cheese wastewater pretreated by chemical precipitation A. Almeida 1, A. Prazeres 2,3, C. Ribeiro 1, H. Chaves 1, F. Carvalho 1 1 Departamento de Ciências e Tecnologias Aplicadas, Escola Superior Agrária, Instituto Politécnico de Beja, Beja, Portugal 2 Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo (CEBAL) /Instituto Politécnico de Beja, Beja, Portugal 3 Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM), Universidade de Évora, Évora, Portugal 10:50-11:20 Coffee Break and Poster Session 11:20-12:00 SESSION VI Chairpersons: Maria de Fátima Carvalho, Albertina Marques and Maria José Pacheco 11:20-11:40 Synthesis of carbohydrate derivates with potential activity for the treatment of bipolar disorder M. Gomes 1, M. Lucas 1, J.A. Figueiredo 1, S. Silvestre 2, M.I. Ismael 1 1 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 2 CICS-UBI, Health Sciences Research Centre, Universidade da Beira Interior, Covilhã, Portugal 11:40-12:00 Phytochemical characterization and determination of the biological activity of bark's extracts of Salix salviifolia M. Lucas 1, M. Gomes 1, J.A. Figueiredo 1, A.P. Duarte 2, M.I. Ismael 1 1 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 2 CICS-UBI, Health Sciences Research Centre, Universidade da Beira Interior, Covilhã, Portugal

10 12:00-12:30 DISCUSSION and FINAL SESSION Chairpersons: Manuel Santos Silva, Ana Lopes, José Lucas and Rogério Simões POSTER SESSION Studies on the Solubility of Methyl Orange and Acid Orange 8 in Water C.M.G. Pinto, M.I.A. Ferra, I.M.S.C. Gonçalves, M.J.A. Pacheco, A. Marques FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal Phytochemical composition and assessment of rheological and antioxidant properties of juices of berries Â. Luís 1, S. Sousa 2, A.P. Duarte 1, L. Pereira 3, F. Domingues 1 1 CICS-UBI, Universidade da Beira Interior, Covilhã, Portugal 2 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 3 CMA-UBI, Universidade da Beira Interior, Covilhã, Portugal Assessment of Cork Boiling Wastewater Treatment by Constructed Wetland at Horizontal Subsurface Flow Configuration A.C.Gomes 1, M.L. Silva 1, A. Stefanakis 2, R. Simões 1, A. Albuquerque 3 1 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 2 Bauer Resources GmbH 3 FibEnTech-UBI, Department of Civil Engineering and Architecture, Universidade da Beira Interior, Covilhã, Portugal Development of sustainable antimalarial clothing C.I.S. Pinheiro 1, N.J.R. Belino 1, P. Roshan 2 1 Department of Textile Science and Technology, Universidade da Beira Interior, Covilhã, Portugal 2 Indo-German Science and Technology Centre, New Delhi, India A preliminar study on the bioactive compounds present in the leaves and stems of Urtica dioica, L. M.F.C. Tenreiro 1, M.E. Amaral 1,2 1 Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 2 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal Anaerobic mesophilic biodegradation of raw olive pomace V. Guilherme, I.C. Gonçalves, A. Marques, A. Lopes, I. Ferra FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal Essential oils from fennel seeds collected in different regions from Portugal L. Silva 1, V. Lopes 2, E. Bettencourt 3, S. Pombal 1, J. Rodilla 1, A. Gomes 1 1 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 2 Banco Português de Germoplasma Vegetal lniav, I.P., Braga, Portugal 3 Unidade Estratégica de Investigação e Serviços de Biotecnologia e Recursos Genéticos, INIAV, I.P., Portugal Production of high performance filaments from nano/microfibrillated vegetal cellulose V.L.D. Costa, J.W. Gomulka, P.D.L. Gaspar, D.J.M. Santos, A.P. Costa, M.E. Amaral, R.M.S. Simões FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal

11 Electrochemical treatment of cheese whey wastewater: Influence of anode material, chloride concentration and current density M.M. Batista, A. Fernandes, L. Ciríaco, M.J. Pacheco, A. Lopes FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal Abstract The application of anodic oxidation with boron doped diamond (BDD) and lead oxide (PbO 2) anodes was carried out to treat a cheese whey wastewater (CWW). The influence of the applied current density and chloride concentration was also assessed. Results showed that BDD anode is more prone to eliminate the organic load than PbO 2. However, PbO 2 anode showed to be more efficient in the removal of phenolic compounds. For both anode materials used, it was observed that chemical oxygen demand (COD) removal rate increased with applied current density and decreased when chloride concentration was augmented. For the best experimental conditions tested, COD and phenol removals of 96% were achieved, showing that anodic oxidation is a feasible method to treat CWW. Introduction Due to its composition, wastewater coming from the cheese industry represents a serious contamination source. Direct release of these effluents into natural receiving waters can generate an unlimited number of environmental and public health impacts [1]. Based on the potential nutritional value of CWW, which are rich in biodegradable organic matter and nutrients, it has been considered its direct application on land for irrigation. However, a negative impact on soil structure can be caused by the accumulation on soil surface of suspended solids and fats [2]. Conventional treatments, such as anaerobic or aerobic systems, as a single process or integrated with physicochemical technologies, have been widely studied [3-5]. However, some drawbacks have been mentioned in the literature and, in most cases, the treated effluent is not suitable to be discharged into the environment. Thus, the efficient treatment of CWW is one of the greatest challenges in cheese industry. Electrochemical technologies have shown high efficiency in the elimination of persistent pollutants, and several studies have reported their application in wastewater treatment [6,7]. In particular, anodic oxidation (AO) has demonstrated its high potential for the degradation of persistent organic pollutants, especially when BDD anodes are used. This anode material presents exceptional properties and, when applied in electrochemical reactors, generates hydroxyl radicals that have a high oxidation potential and are non-selective oxidants, leading to the complete mineralization of the organic compounds present in wastewaters [8]. However, BDD electrodes high cost and energy required to the complete mineralization of organics hinder the implementation of this technology at full scale [9]. Different low-cost electrode materials have been studied for the AO process, and, among them, metal oxides have shown promising results, even when used to treat complex effluents [10]. Besides the anode material, applied current density and chloride ion concentration also have shown to play an important role in the AO performance. Several authors reported an increase in COD removal by increasing the applied current density or the chloride ion concentration [11]. The aim of this work was to study the electrodegradation of CWW by anodic oxidation. The influence of applied current density and of chloride ion concentration during the degradation process was assessed, and the use of two different anode materials was compared: BDD, which yields the highest organic oxidation rates and the greatest current efficiencies [11], and Ti/Pt/PbO 2, which is a less costly material and presents similar performance to BDD [10]. Experimental The CWW used in this study was collected from a cheese industry located in the Leiria district (Portugal) and was kept refrigerated until use. The results for the CWW characterization are presented in Table 1. Table 1. Physicochemical characteristics of the CWW. Property Mean value ± SD a COD / mg L ± 41 TOC / mg L ± 2 Total nitrogen / mg L ± 0.1 Total phosphorus / mg L ± 0.6 Total phenol / mg L ± 0.04 Chloride ion / mg L ± 4 ph 4.93 ± 0.05 Conductivity / ms cm ± a SD Standard deviation AO experiments were conducted in batch mode, with stirring, using 200 ml of solution. Ti/Pt/PbO 2 (hereinafter designated by PbO 2) and BDD were used as anodes, and stainless steel was used as cathode. PbO 2 electrode was prepared as described in the literature [12], and the BDD electrode was purchased from Adamant Technologies. Both the anode and cathode were used with an immersed area of 10 cm 2, with a 0.5-cm gap between them. Because the CWW samples presented low conductivity values, for most of the AO experiments, sodium chloride (Pronalab, PA) was added as a supporting electrolyte to allow higher current intensities than those obtained when no electrolyte was added. To study the effect of the 1

12 chloride addition, experiments were conducted in the presence of different sodium chloride concentrations. The effect of the current was also studied by varying the applied current intensity. Assays were also performed without adding support electrolyte. The experimental conditions tested are presented in Table 2. All the electrochemical assays were performed at least in duplicate and the values presented for the parameters used to follow the assays are the mean values. Table 2. Experimental conditions for the AO assays. [NaCl]added / g L -1 Applied current intensity / A concentration did not significantly increased the indirect oxidation through chlorine/hypochlorite. Moreover, the addition of NaCl reduced the rate of TOC removal. This behavior must be related with an optimum COD/[Cl ] ratio, and an extra addition of chloride does not result in an increase in COD removal rate because part of the applied current is spent in the chloride oxidation and intermediate compounds more resistant to oxidation may also be formed, in particular at low applied current densities [15]. Degradation tests were followed by COD, total organic carbon (TOC), total phosphorus and total phenol determinations. All the analytical determinations were conducted according to standard procedures [13], except for the total phenol content expressed in terms of tannic acid concentration, which was quantified by methodologies adapted by Makkar et al. [14]. Results and discussion Fig. 1 presents the COD and TOC decays during electrolysis, at an applied current density of 15 ma cm -2, with and without the addition of 5 g L -1 NaCl, for both anode materials studied. As can be seen, for both COD and TOC, BDD anode presented higher removal rates than PbO 2. This BDD better performance in the organic load removal has been reported in the literature [10,15] and is probably due to the stronger adsorption of the hydroxyl radicals, which are responsible for the oxidation, at the metal oxide surface. For both anode materials, TOC removal was lower than COD removal. This behavior has been reported previously in the literature and can be explained by the formation of low molecular mass organic compounds with high degrees of oxidation that resist to complete mineralization [16]. For the experiments performed without NaCl addition, the difference between the COD and TOC removal is less pronounced when BDD anode is used than with PbO 2, suggesting that BDD anode promotes more easily the complete combustion of the organic matter. In fact, due to the inert surface of BDD anodes, hydroxyl radicals, the main species that mediates the oxidation of the organic compounds, are very weakly adsorbed; consequently, they are very reactive toward the complete oxidation of organics. Alternatively, for PbO 2, hydroxyl radicals are expected to be more strongly adsorbed on the surface; consequently, they promote the conversion of the organic matter into more oxidized by-products rather than resulting in mineralization. At the applied current density of 15 ma cm -2, COD removal rate didn t change significantly with the addition of NaCl, meaning that the increment on the chloride Fig. 1. (a) COD and (b) TOC decays during electrolysis, at an applied current density of 15 ma cm -2, with and without the addition of 5 g L -1 NaCl, using BDD and PbO2 as anode material. In a second set of assays, the applied current density was increased to 30 ma cm -2, and two different NaCl additions were tested, 0.5 and 5 g L -1. Fig. 2 shows the COD and TOC decays obtained at these experimental conditions, for both anode materials utilized. Results show that, at higher applied current density, COD removal rate was still not influenced by the increase in chloride concentration when BDD anode was used. Also, a decrease in TOC removal was observed with the increase in chloride concentration, due to the reasons already presented. 2

13 For the experiments performed with PbO 2, an increase in COD removal rate was attained with the increase in chloride concentration, indicating augmented indirect oxidation of the organic matter through chlorine/ hypochlorite species. However, no significant change was observed in TOC removal rate, which means that the organic matter that was indirectly oxidized through chlorine/hypochlorite was converted into more oxidized by-products instead of resulting in mineralization. ma cm -2 a 100% removal was achieved. This finding can be explained considering that hydroxyl radicals are weakly adsorbed on BDD and, consequently, they are more proficient in the oxidation of larger molecules with lower diffusion coefficients than metal oxides, which are more effective in the oxidation of smaller molecules with higher diffusion coefficients, because their oxidation occurs at the electrode surface where the hydroxyl radicals are strongly adsorbed. Table 3. Removals obtained for the different parameters determined after 8-h AO assays, at different experimental conditions. Current density / ma cm [NaCl]added / g L COD / % BDD PbO TOC / % BDD PbO Total phosphorus / % BDD PbO Total phenol / % BDD PbO Conclusions The depuration of CWW by anodic oxidation was successful and, for the best experimental conditions tested, achieved COD values below the discharge limit of 150 mg L -1. When using BDD anode, and especially at the highest applied current density, the increase in chloride concentration showed to be detrimental for COD and TOC removals. On the contrary, with PbO 2 anode, the increase in chloride concentration led to an increase in organic load removal. For both anode materials, the COD and TOC removals increased with applied current density, being the best removal results attained by BDD. Regarding total phenol, the highest removals were achieved when PbO 2 anode was used. However, BDD anode showed to be more prone to phosphorus removal, especially at higher applied current densities. Fig. 2. (a) COD and (b) TOC decays during electrolysis, at an applied current density of 30 ma cm -2, with NaCl additions of 0.5 and 5 g L -1, using BDD and PbO2 as anode material. Table 3 presents the percentage removal obtained for the different parameters determined for the AO assays, after 8 h of electrolysis. It can be seen that COD and TOC removal rates increased with applied current density, for both anode materials. This behavior is explained by the increased production of oxidizing species with the operating current density. Total phosphorus and total phenol removals were also assessed. Regarding total phosphorus, the highest removal, 65%, was achieved with BDD anode, at an applied current density of 30 ma cm -2. On the contrary, higher total phenol removals were accomplished by PbO 2, for all the experimental conditions tested. In fact, at 30 Acknowledgements The authors gratefully acknowledge the financial support received from Fundação para a Ciência e a Tecnologia, FCT, for the funding of the FibEnTech-UBI Research Unit, project UID/Multi/00195/2013, and for the grant awarded to A. Fernandes, SFRH/BPD/103615/2014. References [1] A.R. Prazeres, J. Rivas, U. Paulo, F. Ruas, F. Carvalho, Sustainable treatment of different high-strength cheese whey wastewaters: an innovative approach for atmospheric CO 2 mitigation and fertilizer production, Environ. Sci. Pollut. Res. 23 (2016) [2] A.R. Prazeres, F. Carvalho, J. Rivas, Cheese whey management: a review, J. Environ. Manage. 110 (2012) [3] H.H.P. Fang, Treatment of wastewater from a whey processing plant using activated sludge and anaerobic processes, J. Dairy Sci. 74 (1991)

14 [4] S.V. Kalyuzhnyi, E.P. Martinez, J.R. Martinez, Anaerobic treatment of high-strength cheese-whey wastewater in laboratory and pilot UASB-reactors, Bioresour. Technol. 60 (1997) [5] J. Rivas, A.R. Prazeres, F. Carvalho, F. Beltrán, Treatment of cheese whey wastewater: combined coagulation-flocculation and aerobic biodegradation, J. Agr. Food Chem. 58 (2010) [6] G. Chen, Electrochemical technologies in wastewater treatment, Sep. Purif. Technol. 38 (2004) [7] A. Anglada, A. Urtiaga, I. Ortiz, Contributions of electrochemical oxidation to wastewater treatment: fundamentals and review of applications, J. Chem. Technol. Biotechnol. 84 (2009) [8] E. Brillas, C.A. Martínez-Huitle, Decontamination of wastewaters containing synthetic organic dyes by electrochemical methods. An updated review, Appl. Catal. B Environ. 166 (2015) [9] P. Canizares, P. Rubén, C. Sáez, M.A. Rodrigo, Costs of the electrochemical oxidation of wastewaters: a comparison with ozonation and Fenton oxidation processes, J. Environ. Manage. 90 (2009) [10] A. Fernandes, D. Santos, M.J. Pacheco, L. Ciríaco, A. Lopes, Nitrogen and organic load removal from sanitary landfill leachates by anodic oxidation at Ti/Pt/PbO 2, Ti/Pt/SnO 2-Sb 2O 4 and Si/BDD, Appl. Catal. B Environ. 148 (2014) [11] A. Fernandes, M.J. Pacheco, L. Ciríaco, A. Lopes, Review on the electrochemical processes for the treatment of sanitary landfill leachates: Present and future, Appl. Catal. B Environ. 176 (2015) [12] L. Ciríaco, C. Anjo, J. Correia, M.J. Pacheco, A. Lopes, Electrochemical degradation of Ibuprofen on Ti/Pt/PbO 2 and Si/BDD electrodes, Electrochim. Acta 54 (2009) [13] A. Eaton, L. Clesceri, E. Rice, A. Greenberg, M.A. Franson, Standard methods for examination of water and wastewater, 21 ed., Washington, DC, American Public Health Association, [14] H. Makkar, M. Blummel, M. Brorowy, N. Becker, Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods, J. Sci. Food Agric. 61 (1993) [15] A. Fernandes, D. Santos, M.J. Pacheco, L. Ciríaco, A. Lopes, Electrochemical oxidation of humic acid and sanitary landfill leachate: Influence of anode material, chloride concentration and current density, Sci. Total Environ. 541 (2016) [16] A. Fernandes, M.J. Pacheco, L. Ciríaco, A. Lopes, Anodic oxidation of a biologically treated leachate on a boron-doped diamond anode, J. Hazard. Mater (2012)

15 Recycling of Silicone Coated Paper Curto, J.M.R.¹, ², Costa, A.P.¹, Videira, P.E.M.¹, Amaral, M.E.¹, Simões, R.M.S.¹, Silva, M.J.S.¹ ¹FibEnTech-UBI, Fiber Materials and Environmental Technologies, Departamento de Química, Faculdade de Ciências, Universidade da Beira Interior, Covilhã, Portugal 2 CIEPQPF, Chemical Process Engineering and Forest Products Research Centre, Faculdade Ciências e Tecnologia da Universidade de Coimbra, Coimbra, Portugal Abstract An innovative approach to recycle silicone coated paper is investigated to obtain individualized fibres for posterior integration in the paper recycling industry. The coating formed by silicone polymers is very stable and difficult to extract or disperse. The goal is to develop a green method to recycle this paper. Several analysis techniques such as scanning electron microscopy, optical microscopy, x-ray dispersive spectroscopy, Fourier transformed infrared attenuated total reflectance spectroscopy and contact angle were used to characterize the silicone coating and the cellulose fibres from the silicone paper. After mechanical and chemical operations, recycled paper structures were produced and characterized using mechanical strength tests, like Elmendorf and tensile, and optical properties such as ISO whiteness, ISO opacity and yellowness. The recycled pulps were compared with reference short fibre Eucalyptus globulus and reference long fibre Picea abies. Using an optimized combination of mechanical and chemical process operations the recycled cellulose fibres obtained presented good mechanical strength with tensile index values for recycled sheets of 39,6 Nm/g in the same range of eucalyptus with 42,2 Nm/g and Picea abies with 50,3 Nm/g. Tearing strength Elmendorf test for recycled papers presents values of 8,8 mnm²/g similar to Eucalyptus globulus values. The drainability was assessed by the Schopper-Riegler method and the values obtained for the laboratory scale are in agreement with values of Eucalyptus globulus and Picea abies. A successful Pilot pulper test of 15l was conducted after achieving an effective reaction separately for the green additive and solving mass transfer issues. The structures obtained at pilot scale presented good mechanical properties in the same range of the ones obtained at the laboratorial scale. The ISO standard sheets obtained from the Pilot pulper presented tensile index values of 11,8 mnm²/g and lower yellowness values than the laboratorial essay, however the drainability, for the Pilot pulper decreased when compared with the laboratory scale tests. Introduction The intention to be greener, and to recycle cellulosic based materials currently going to the landfill, is the drive for this project, proposed to Universidade da Beira Interior FibEnTech Research Unit by a traditional printing company and a recycling start-up company. In 2015 the worldwide production of paper and cardboard was of 410 million tons and Europe was responsible for almost a fourth, producing 90,9 million tons [1]. Portugal is the 3 rd largest European producer making 2,2 million tons of paper and cardboard [2]. In Europe, were recycling habits are very enrooted the recycling rates goes around 72%. In Portugal that values are lower around 60% for paper and 66% for cardboard and packaging [2,3]. The worldwide recycling rate is in 58% [3]. The silicone coated paper is difficult to disintegrate due to the silicone layer bonding that aggregates the fibres. In Portugal, the use and production represents 4 tones and goes entirely to landfill [4,5]. One of the first tasks of this project was to chemically characterize the paper using different techniques and understand the chemistry affinity to extract it [6]. To recycle the fibres a combination of renewable sources chemicals and mechanical operations to extract/disperse the silicone coating from the paper were tested and optimized. Materials and Methods The chemical characterization of the silicone coated paper was made using scanning electron microscopy (SEM), Energy dispersive x-ray spectroscopy (EDX), Fourier transform infrared spectroscopy attenuated total reflectance (FTIR-ATR) and contact angle according to T558. The production of the paper sheets was done according the ISO 5264 for refining, ISO 5263 for disintegration, ISO 5269 for formation and pressing. Two types of essays were made, laboratorial essay where the recyclability was accessed using several chemicals and the integration of the recycled pulp in pulps of reference short fibres and reference long fibres. The second type of essays made where in a pulper pilot 15 l to optimize the conditions that are going to be used in the recycling plant. In all the pulps produced in the two types of essays, the physical properties of the papers were accessed using mechanical resistance tests like Elmendorf and tensile strength test, and optical properties such as whiteness, opacity and yellowness. Several steps were done to prepare the paper for recycling and the ones made for the laboratorial essay are specified below. Pre-chemical Treatment Refining Disintegration Extraction/ Coating dispersion Drainage Filtration 5

16 Wash After the silicone removal/dispersion the fibres were characterized and paper sheets were produced according ISO regulations. The fibres obtained were long fibres (length>1cm) (Fig. 3-A) and the FTIR-ATR (Fig. 3-B) results of the recycled paper indicate the removal or dispersion of the silicon coated layer. Mechanical and optical essays Papermaking After the optimization of several steps in the laboratorial essay the upgrade to the pilot pulper 15 l was made and the steps to achieving of the paper sheets is specified below. Pre-chemical Treatment Disintegration Pulper Pulper Extraction A B Mechanical and optical essays Papermaking Results The characterization and quantification of the coated paper silicone layer was done using SEM image analysis (Fig. 2A) A Figure 3. A) Optical microscopy image of the fibres after the treatment (100x), B) FTIR-ATR results of the siliconized paper (red) and after treatment (blue), B Paper Properties Figure 1. A) SEM image of the silicone coated paper; B) FTIRATR results of the coated side from siliconized paper The layer was characterized using scanning electron microscopy, SEM, (Fig. 1-A). The percentage of the silicone coated layer in proportion to the amount of fibres is small, approximately 6% and the fibres present length dimensions in the range of reinforcement fibres. The chemical characterization includes the determination of the functional groups using Fourier transformed infrared attenuated total reflectance spectroscopy, FTIR-ATR, (Fig. 1-B) and contact angle. The main compound of the layer is the siloxane (Fig. 2). The hydrophobicity was measured using contact angle with a value of 96, which corresponds to hydrophobic surface. Pilot Pulper 15l Laboratorial essay Eucalyptus globulus Reference long fiber Picea abies Figure 4. Comparison of different paper properties in different experimental and reference papers. Several paper properties where analysed in the produced paper sheets (Fig. 4). The mechanical resistance tensile index values for recycled sheets 39,6 Nm/g value compared with Eucalyptus globulus 42,2 Nm/g an Picea abies 50,3 Nm/g for the Elmendorf test 8,8 mnm²/g and for Eucalyptus globulus and Picea abies 8,8 mnm²/g and 16,1 mnm²/g respectively. Optical properties such ISO Whiteness for the recycled paper sheets are 68,6, for Picea abies 83,7 and Eucalyptus globulus 85,4. ISO Opacity for Eucalyptus globulus is 81,3 Picea abies 82,0 and for the recycled sheets 91,3. After the results obtained in the laboratorial scale and posterior optimization of operation times and concentrations, the upgrade to pilot scale was done using a pilot pulper with 15l. The values obtained for optical essays agree with those obtained for the laboratorial essays. The tensile index obtained in the pilot pulper was the lowest with value of 29,9 Nm/g but the tearing index was higher than the laboratorial essay and the reference short fibre (Fig. 4). The measure of the yellowness between the pilot Figure 2. Linear silicone polymer structure. 6

17 pulper essay and the laboratorial essay was also assessed and the values obtained for the pilot pulper essay were lower than the laboratorial essay. The drainability was accessed using Schopper-Riegler method and values of 23 ºSR were obtained for the fibres treated at laboratory scale, improving from the 58 ºSR of the original silicon coated paper, however with pilot pulper essay the value obtained were around 44 ºSR. Conclusions The recycling process uses a combination of chemical and mechanical operations to obtain the removal and dispersion of the silicone coating. Pulps drainability accessed by SR, mechanical strength, paper sheet formation and optical properties indicate that the silicon coated paper can be recycled. Acknowledgements This project was financed by Compete Centro an Horizonte 2020 Research and Technological Development Project I&DT RPS-Revalorização do Papel Siliconado References [1] CELPI Key Statistics [2] Boletim Estatístico da CELPA [3] Recycling Paper Council- Monitoring Report [4] FINAT-European association for the self-label industry accessed May [5] Venditti, Richard A. and Chang, Hou-min, Evaluation of Various Adhesive Contaminant Analysis Methods for the Use in Old Corrugated Container Recycling Plants, North Carolina State University, Dept. of Wood and Paper Science Raleigh NC [6] Hentzschel et al., Process for recycling silicone-coated paper United States Patent, Patent Number: 5,567,272; Oct. 22,

18 Companies' adaptation through innovation J.A.B. Barata 1, R.A.L. Miguel 1, S.G. Azevedo 2 1 FibEnTech-UBI, Textile Science and Technology Department, Universidade da Beira interior, Covilhã, Portugal 2 CEFAGE, Business and Economics Department, Universidade da Beira interior, Covilhã, Portugal Abstract This article aims to study organizational innovation. Here innovation is perceived as an opportunity to increase the companies competitiveness and ensure its survival. Organizational adaptation is understood as the pursuit to be apt to face future demands, anticipating future problems. The capacities required for a company to anticipate the market s fluctuations can be called adaptability. In theory creativity is related with the survival instinct, in a way that the human being has constructed/create new solutions to his problems, seeking the common wellbeing. Creativity is perceived as fuel to the innovation pipeline. The interest in design has been augmenting in the last decades, it is associated with the add value for the outcomes, increasing with it the organizations competitiveness. In this document is presented a literature review on what are creativity, innovation, design as innovation, organizational culture and organizational climate. Here are present some studies in innovation regarding the Portuguese industry. Introduction This is a literature review on several topics related to organizational innovation. Starting by presenting a strong pillar of Darwinism that concerns the survival of the fittest individuals within the species. This concept is to be perceived as an analogy to what was found as a common force among the companies desire to be prepared to face the economic and market impositions unpredictability. Follows a presentation of how creativity is analysed nowadays and why it drives innovation. The processes of innovation are also complex and here are presented several visions and models for it. Design is another term to be included in this review, as it is a bridge that conducts creativity until the implementation (innovation) process. Regarding the Portuguese scenario, were reviewed two analyses at a national level (INDINOVA and SOTIP) and two at a European level ([1] correlating creativity, design and innovation comparing to 2008 Summary Innovation Index and 2015 Summary Innovation Index). Some citations in this document were freely translated (f. t.). Darwinism adaptation and the survival of the apt ones Lets suppose a wolf that feeds from different animals, seizing ones by its cunning, others by force and others for its agility. ( ) In these circumstances, the more agile and faster wolves are more likely to survive than others; ( ) (f. t.) [2, p. 84]. This pillar of Darwinism is related to the natural selection and the survival of the fittest within the species. This reference relates, in some well-known examples with the alteration of the surrounding environment. Let s present the case of long-necked giraffes that will survive instead of the other ones in the event of the decrease of low vegetation. Long-necked giraffes will be considered fitted (apt) and so, will resist to the alteration of its environment; they will survive in the natural selection process. These principles from biology are a good starting point to explain some concepts in what respects the environment changing to witch the apt characteristic is considered an attribute that might ensure survival [2]. What are creativity, innovation, design as innovation, organizational culture and organizational climate? Here are presented some definition of the terms above mentioned. Creativity: Regarding creativity, it was not possible to find a consensual definition among the scalars and researchers in the reviewed literature. In fact, it was found this particular association, in the theoretical background, with the survival instinct, in a way that the human being has constructed/create new solutions for his problems, seeking the common well being [3]. The investigations on this subject were reviewed from a series of perspectives, concerning what is creativity, its process and the characteristics of the creative person. The first investigation on the cognitive perspective is associated with the Attitudes Research Project conducted by Guilford (1949 to 1969) [4, 5]. The most recent approach on creativity indicates that it drives from a series of contexts, both physical and nonphysical [6, 13]. Innovation: Creativity and innovation are often mentioned together, the main difference is that innovation is the successful process of implementation of the creative output [13, 15]. If the emergence of new and creative ideas is related to the individual performance, innovation has much to do with implementation, ergo commercial value [14, 16, 18]. In this sense, the innovation activities are scientific, 8

19 technologic, organizational financial and commercial steps guiding the implementation of the innovations (f. t.) [18, p. 56]. According to Oslo Manual [17, 18], the companies may innovate in four elements, product, process, organizational and marketing innovations (please check [18, p. 23] for detailed information). One can innovate with two types of new outcome, radical or incremental innovation. Radical relates to the introduction of a new product or service, never known in the universe of the final client and incremental innovation is presenting improved products or services [13, 14]. Understanding innovation in its dynamic quality, there were detached three models of innovation from the literature, (1) Linear Model, (2) Interactive Model and (3) Systemic Model. (1) Linear Model: The first model is related to classic and neo-classic growing and development theories. It is a combination of two approaches, science push and demand pull. Science push indicates that e social and economics growth come from technologic and scientific innovation while demand pull considers the markets needs as a primary stimulus for the innovation growth [19]. (2) Interactive Model: This model fills the breach proposed by the evolutionary current regarding the interaction principle. The present model refers the constant loops and feedbacks among its participants regarding the individual necessities and contributions to innovation [19, 20]. Organizational Culture and Organizational Climate: Culture is defined, in theory, as the meaning and beliefs of a determined population; these conceptions are to be observed in the production and the artefacts from that same group. To design is the way by witch humans reproduces their cultural reality. Climate relates to behavioural manifestations and the intrinsic values to the population or group in study [12, 23]. This clarification is rather important in this study once from this point forward will be presented models from the literature review that aimed to study organizational climate. Major contributions on Organizational Climate research: Here are presented two models to study organizational climate, [10] (1996) and [24] (1997); these models have been wieldy tested and they have been approved in may empirical researches studying the influence of several factors on creative outcomes in companies. [10] has identified ten factors such as idea time, risktaking, challenge, freedom, idea support, conflicts, debates, playfulness/ humour, trust/ openness and dynamism/liveliness (figure 1). In this study, every factor with exception of conflicts load positively on the creative outcome of teams [10, 25]. (3) Systemic Model: The great contribution of this model is the notion that companies cannot innovate singly rather than a direct or indirect relationship with other entities; it introduces innovation as a result of a networking collaboration. The process of globalization is including faster and growing needs for specialization and differentiation in both national and international innovation systems [14, 19]. Design as innovation: Design is a difficult word to define; even de dictionaries vary in its definition. These conceptions are arbitrary and they change as much as the opinions on what design refers to [13, 14, 21]. In this article, design is used in its important interpretation towards innovation practice; it stands for the process of conscious decision making as a vehicle to a tangible (product) or intangible (service) output. (...) design is defined as the shaping (or transformation) of ideas into new products and processes; and innovation is defined as the exploitation of ideas (...) [1, p. 5]. The interest in design has been augmenting in the last decades; it is associated with the add value for the outcomes, increasing with it the organizations competitiveness [1, 14, 22]. Design s contribution is considered relevant in this differentiation; it is associated with creativity and directs to innovation. Design (...) contributes to the expansion of available ideas (...) [1, p. 6]. Figure 1. Ekval model of creative climate Source: [25, p. 300] [24] developed a componential theory of organizational creativity, with keys to study six scales that will differentiate high-creativity climates and low-creativity climates. These scales are organizational encouragement, supervisory encouragement, work group supports, freedom, sufficient resources and challenge. In her work, Amabile (1996) points out three fundamental components to the creative activity, domain relevant skills, task motivation and relevant processes (figure 2) [6, 11, 12, 24, 25]. 9

20 [1, p. 7] (2009) presented a study correlating creativity, design and innovation for the countries on the EU. It involved three dimensions, the creative climate, creativity and design and innovation (please see [1, p. 7] for further detail). Figure 2. Amabile s componential model Source: [24, p. 53] Innovation studies in Portugal For the creative climate, it is possible to find Portugal in the 23th place. The creative education and tolerance levels have average scores compared to other countries. Personal expression is small in relation to countries with high rates of innovation. When correlating creativity and design in the companies, comparing to the 2008 Summary Innovation Index (SII) results, it is notable that Portugal is in the ring of countries with low innovation, creativity and business innovation index quotation (figure 4). In the 2015 SII, Portugal already has demonstrated improvement indicators, occupying the in the 30th position worldwide when in 2008 was on 40 th [26, 27]. From the literature review was found that the empirical studies in Portuguese industries are recent and scarce. It seems that only recently Portuguese scholars started to pay attention on recognizing innovation as an important force to development [13]. At a national level were found these longitudinal company analyses. The first was INDINOVA project ( ) with the purpose to describe the state of innovation in the Portuguese manufacturing industry; the main conclusions were that there were more process innovations than product innovations and also that little importance was given to R&D activities and to scientific collaborations. The second study was SOTIP Project ( ) and here was founded that companies already detach research centres and universities as an important pillar to innovation as well as the existence of more innovation processes on incremental innovations. Another point of interest in this second study is the proof that innovation works in a network environment as related above in the Systemic Model of innovation. The figure 3 presents the comparison of relevance given to four factors in both studies. Figure 3. Factors of innovation INDINOVA and SOTIP Source: [13, p. 308] Figure 4. Correlation of creativity and design in the companies and the 2008 Summary Innovation Index Source: [1, p. 23] The organizational adaptation [15] indicates that often the companies do not have a structured way to channel the employees creativity and much so are the barriers the workers perceive between their ideas and the management staff. In this scenario, which usually is vertical in terms of organization, employees feel powerless to express creatively. The capacities required for a company to anticipate the market s fluctuations can be called adaptability. This characteristic shows efficiency in a way it can anticipate problems and continue to improve in their results. At the same time, it is important to follow the routines efficiently and this also requires that each one try to adapt as much as possible to the changes imposed from the outside (flexibility), while at the same time pursuing a constant improvement. (f. t.) [13, p. 19] These concerns are also presented in the project for the Portuguese Textile and Apparel Industry; this project concerns the changes in the industry and seeks adaptation 10

21 to unstable macroeconomic frameworks. One of the structuring axes for this goal is innovation and creativity [28, 29]. Conclusions: It is visible that nowadays companies see in innovation an efficient way to face economics uncertainty and impermissibility. Organizational adaptation is understood as the pursuit to be apt to face future demands, anticipating future problems. [6, 10, 15, 17, 25, 28, 31]. A great part of the investigation on organisational innovation started by studying organizational creativity whit a major focus on its creative climate, understanding how this factors enable the workers creative output [12, 25, 31]. The main reason for this fact is that its creativity that fuels the innovation pipeline. [12, p. 240]. Design, R&D and a network ground are very important assets to the process of innovation, and some of these indicators can be perceived when detaching the relevant innovation factors in INDINOVA and SOTIP studies [13]. There was not found any study that focused the Portuguese Textile and Apparel industries in terms of creativity nor innovation. References: [1] H. Hollanders and A. Van Cruysen, Design, Creativity and Innovation : A Scoreboard Approach, [2] C. Darwin, Origem das Espécies, 1 a edição. Porto: Lello & Irmão - Editores, [3] S. Bahia, Promoção de ethos Criativos, in Criatividade e educac a o: conceitos, necessidades e intervenc a o, M. de F. Morais and S. Bahia, Eds. Braga: Equilibrios, 2008, pp [4] R. S. R. de Oliveira, Um Programa de Treino da Criatividade Estudo exploratório com alunos do 1 o Ciclo, Universidade da Madeira, [5] R. J. Sternberg and E. L. Grigorenko, Guilford s Structure of Intellect Model and Model of Creativity : Contributions and Limitations, vol. 13, pp , [6] T. Amabile, Creativity in Context. Oxford: Westview Press, [7] B. A. Hennessey and T. M. Amabile, Creativity, Annu. Rev. Psychol., vol. 61, no. 1, pp , [8] T. Amabile, The Social Psychology of Creativity, 1st ed., vol. 1. Springer Series in Social Psychology, [9] T. M. Amabile, R. Conti, H. Coon, J. Lazenby, T. M. Amabile, H. Coon, and M. Herron, Assessing the Work Environment for Creativity ASSESSING THE WORK ENVIRONMENT FOR CREATIVITY University of Michigan University of Southern California, vol. 39, no. 5, pp , [10] G. Ekvall, Organizational Climate for Creativity and Innovation, Eur. J. Work Organ. Psychol., vol. 5, no. 1985, pp , [11] M. G. Jiménez, Employee Creativity and Culture: Evidence from an examination of culture s influence on perceived employees creativity in Spanish organizations, Universitat Ramon Llull, [12] L. D. Mclean, Organizational Culture s Influence on Creativity and Innovation : A Review of the Literature and Implications for Human Resource Development, vol. 7, no. 2, pp , [13] J. Monteiro-Barata, Innovation in the Portuguese Manufacturing Industry : Analysis of a Longitudinal Company Panel, Int. Adv. Econ. Res., no. 11, pp , [14] B. Von Stamm, Managing innovation, design and creativity, 2 o. Chichester: John Wiley & Sons Ltd, [15] F. Cardoso and I. P. Monteiro, Liderança de Equipas na resolução de problemas complexos, 2 a edição. Lisboa: Edições Sílabo, [16] C. Europeia, Livro Verde sobre a Inovação, [17] OECD, Oslo Manual - GUIDELINES FOR COLLECTING AND INTERPRETING INNOVATION DATA, [18] OECD, Manual de Oslo: Diretrizes para a Coleta e Interpretação de dados sobre Inovação Tecnológica, [19] A. Lobosco, M. Barbosa, D. Moraes, and E. Antonio, Inovação: uma análise do papel da agência USP de inovação na geração de propriedade intelectual e nos depósitos de patentes da Universidade de São Paulo, Rev. Adm. UFSM, vol. 4, no. 3, pp , [20] M. F. Gurgel, CRIATIVIDADE & INOVAÇÃO: Uma Proposta de Gestão da Criatividade para o Desenvolvimento da Inovação, [21] P. Swann and D. Birke, How Do Creativity and Design Enhance Business Performance?: A Framework for Interpreting the Evidence., [22] M. C. 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22 The identity of the Portuguese Fashion through the materials, techniques and technologies Rito, Catarina 1, Pereira, Madalena 2 1,2 Departamento de Ciência e Tecnologia Têxteis, FibEnTech-UBI, Universidade da Beira Interior, Covilhã, Portugal 2 UNIDCOM, IADE, Universidade Europeia, , Lisboa, Portugal Abstract The objective of this work was to assess the potential of Portuguese culture from the point of view of materials, techniques and technologies to create an identity for Fashion Design. The aim of this work is to present and analyse the Portuguese culture associated with materials, techniques and technologies used in fashion design or in other areas but with potential for application in Fashion Design. Introduction One of the main problems of the Portuguese fashion pointed by international experts of the area is the lack of identity of the Portuguese fashion and its scale of business. The effects of globalisation are more and more notorious in the creativity of designers, when developing their products and collections. According to the well known designer Louboutin (2011), the Portuguese fashion has not been yet pressed by the International Fashion Industry, which allows a certain creative freedom, well presented in the works of some designers. As far as I am concerned, I think that the Portuguese fashion, somehow lacks identity. One must not ignore the fact that consumers look for products reflecting the spirit and soul of the country, including the cultural tourism, which has significantly risen specially in Portugal. The designers and the fashion (at both national and international level) aim to develop their own identity, as a way to differ from the market where their products are traded; through creativity and innovation at different levels. Fashion needs a method of analysis that takes into account multiple meanings and interpretations. These methods have been developed by historians, but in a limited way related to the culture of a people. Christopher Breward notes that the model by Melling and Barry (1992) addresses the differences between the new cultural approaches, suggesting a better use of different lines of thought. Fashion and art have convergent elements of visual image communication relative to shapes, silhouette, lines, volume, colors, textures. Both are aimed at "a reflection of their time and society", which makes them open and subject to recreation and re-reading the interpretation (Moura, 1994). Still in the line of thought of this author (Moura 2005), the design has relations of proximity and dialogues with art. In this context "the process of artistic creation is the search for the new, and not for the novelty, for new connections, for new ways of instituting questions". It is the need for constant renewal, and it is also a process of knowledge, which comprises a series of actions interconnected with the creative subject, "which reflects, understands, relates, elaborates, orders, classifies, transforms and creates" (Castilho e Preciosa, 2005). In the era of globalization, the country continues in a recurrent creative and productive backwardness, short of its effective capabilities. Proof of this, is in the way that Portugal still continues to have a purely productive image, where in time comes recognition in other creative areas directly and indirectly associated with fashion design. The recognition of Portugal in the fashion area is still strongly associated with the productive performance, but there is a recent evolution in design, which in a small way begins to create value in the textile, clothing and footwear industry. Associated with these areas, the ATP and APIC- CAPS have developed efforts to promote and develop the sectors, creating strategic plans that outline the future lines for Portugal, such as ATP with the Strategic Textile Plan 2020 (ATP, 2014) and Of APICCAPS with the Strategic Footwear Cluster Plan - Footure 2020 (APICCAPS, 2013). Understand if fashion is a reflection of a local identity, regardless of its origin, or if fashion is a global product, that exists derived from a popular culture that is reflected through a stratified product. In this context, some creators of the national fashion resort to this popular culture in a systematic or punctual way in their seasonal collections, especially Luís Buchinho, Nuno Gama, Alexandra Moura, Nuno Baltazar and Pelcor, Grigi, Vida Portuguesa or Fabrica do Burel. Materials and Methods The search for methods, materials and techniques typically Portuguese, until very recently, relativized by poles of interest, as is the case of fashion designers, in an almost inexpressive way. Many began a process of approaching artisans, learning the rules of construction of each technique, in order to integrate in their creative processes, extolling and promoting a symbiotic relationship between t r a d i t i o n a n d m o d e r n f a s h i o n d e s i g n. The use of techniques and materials that can express the uniqueness of a country ends up being evident in some concepts or specific works of national designers, some already mentioned. Throughout the almost 40 years of Portuguese democracy and the expansion of the profession of stylist and designer, the search for the valorization of typically Portuguese techniques was reneged on other ways of producing and personalizing the creative concept of each project. The truth is that in the last 20 years, gradually, that perception has changed. Although in a punctual way, some names of the national fashion began a path of 12

23 collaboration and knowledge about techniques and fabrics that express national typicity. Variety shows the local wealth, read national, that when applied to collections that follow international trends, can add a stamp. From the burel to the frill, from the potato blanket to the embroidery of Castelo Branco, from the embroidery of the lovers to the cork, Very particular, differentiating from the existing global.the encounter between creativity and technique can enhance, in some way, the work done by each author. In the last years, already in the XXI century, the integration of techniques and typical Portuguese fabrics in seasonal collections is observed, conciliating the contemporary look with the look of the tradition. The combination of both is one of the ways to help enhance an aesthetic of fashion design in Portugal. Part of the interest in these techniques and materials comes from the growing interest of academic institutions, promoting the study and knowledge of local traditions, encouraging the promotion, together with future disseminators and promoters of these same techniques and materials, typical of Portuguese p o p u l a r c u l t u r e. This may be the possible way to expand both the interest in the Portuguese craft tradition and the internal and external dissemination of an enriching mirror product of a unique culture, which is Portuguese culture that is evident in different techniques and materials. Conclusions The discussion about the identity of the Portuguese fashion; If it is important these days to have an identity in national fashion design; If the identity of Portuguese fashion can be acquired through the use of local traditional techniques and materials, it is still a barely consensual but necessary discussion. The path is made through the potentialization of synergies that have as final objective, or not, the valorization of both the product and the brand. It is assumed that this way can start a process of interest of the internal and external market of the creativity and production of the fashion made in Portuguese. References Cabral, M.V. (2003). A Identidade Nacional Portuguesa: Conteúdo e Relevância. Revista de Ciências Sociais, Rio de Janeiro, Brasil. Castilho, K., Preciosa, R.. (2005). A criação e o Design de Moda: apontamentos. Design, Arte e Tecnologia; espaço de trocas. Universidade Anhembi Morumbi, PUC- Rio&Rosario. Colombi, Chiara. (2011). Design Research in Fashion: From Trends to Design Directions. Redige, v.2, n.1. Itália. Melling, J. e Barry, J. (1992). Culture in History. Exeter: Exeter University Press. Gaddi, Rossana (2014). Design for Territorial Enhancement: The cultural value of Fashion Design, Proceddings of 2º CIMODE, Politécnico de Milão, Milão. Lipovetsky, Gilles. (2010). O império do Efémero - A moda e o seu destino nas sociedades modernas. D. Quixote. Portugal. Moura, M. (1994). Arte e Moda. Universidade Aberta. Fascículo No 4, p.4/5. Fundação Demócrito Rocha; Fort - aleza, Brasil. Moura, M. (2005). Design, Arte e Tecnologia. Edições Rosari. Brasil Moreira da Silva. Fernando. (2010). Investigar em design versus investigar pela prática do design - um novo desafio científico. Revista INGEPRO - Inovação, Gestão e Produção, Abril. Brasil. Murani, B. (1979). Artista e Designer. Editorial Presença, Lisboa. 13

24 Design of nanomaterials for Biomedical applications using 3D Computational Simulation J.M.R. Curto 1,2, F.P. Morais 1, R.M.S. Simões 1, M.J. Santos Silva1 3 1 FibEnTech-UBI, Fiber Materials and Environmental Technologies, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 2 CIEPQPF - Chemical Process Engineering and Forest Products Research Centre, Department of Chemical Engineering, University of Coimbra, Coimbra, Portugal 3 FibEnTech_UBI, Fiber Materials and Environmental Technologies, Department of Textile Science and Technology, Universidade da Beira Interior, Covilhã, Portugal Abstract The development of polymeric porous materials in which it is possible to optimize porosity and pore distribution is very important for the development of materials for biomedical applications such as drug delivery systems and scaffolds. An innovative approach is used to develop nano polymeric fibrous materials with optimized porosity and thickness by using a materials computational model simulator to design a new material with optimized properties for biomedical applications. The material structure optimization is fundamental for obtaining functionalities like the transport of molecules or support for cell growing. The validated computational simulator is used to obtain the 3D structure and perform simulation studies to obtain the desired pore dimension and pore distribution. In this study structures made from nanofibers obtained from cellulose and from polyamide-6 and polyvinyl alcohol were produced. The polymeric structures were obtained by electrospinning, which is a technique that allows the creation of fibers with diameters down to nanoscale. Scanning electron microscopy was used to measure fiber dimensions, porosity and thickness of the structure (z dimension). A computational simulator was used to create 3D structures and build several scenarios were the fiber dimension and fiber flexibility were changed and learn about the structures. The use of this computational simulator was used to have information about a large number of structures. From the one thousand computational experiments that were done it was possible to obtain structures with different porosities and pore distributions and select the ones to be made in the laboratory. The computational simulator has proven to be an efficient tool to help design porous structures, allowing the creation of optimized systems, capable to transport therapeutic molecules, or be used in regenerative tissue biomedicine. Introduction The design of polymeric porous materials for biomedical applications, such as drug delivery systems and scaffolds, presents the challenge to obtain the porous materials with control pore dimensions and variability [2-6]. Electrospinning is a technique that allows us to create fibers whit diameters downs to nanoscale. A basic electrospinning system consist in three major components a high voltage power supply, a spinneret and a grounded collecting plate [7]. When a thin string of charged polymer exits from the spinneret, it will be deposit in a metal plate according to an electric field that is generated. A scaffold based on electrospun nanofibers presents a large surface area that allows retaining great volumes of cells or therapeutic molecules [8]. The use of porous structures with the right characteristic such as pore dimensions and pore width variability, as well as the homophilic/heterophilic characteristics of the polymer have been studied [9]. Bones are a 3D material composed by three distinct kinds of cells osteoclasts, osteoblasts and osteocytes. When a bone fractures the construction of the bone is done by the osteoclasts and like every cell it takes time to multiply. The use of 3D structures that can mimic the properties of the bone and improve regeneration can be a turn event in the regeneration medicine. An innovative approach for pore analysis has been used. This approach uses the software Espirit 1.9 to analyze the images obtained by SEM and then analyze them using different image analysis techniques and filters. Using the right threshold for binarization and the filters to obtain images with pores well defined it was possible to measure several properties like the pore diameter, pore area, por dimension distribution. A computational simulator was developed in Matlab to predict and help create 3D structures [10]. The use of this computational tool can be very helpful when doing many experiments. In this study nanofibers of polyamide-6 and polyvinyl alcohol were prepared by electrospinning and analyzed by SEM to obtain measurements of the fiber with, used as in input to the simulator. The porosity of the structures was studied using the computational simulator [10]. 14

25 Materials and Methods 2.1 Materials: Polyamida-6, NH(CH2)5CO)n, was supplied by Acros Organics (Belgium), and the solvent Formic acid CH2O2) at 90% from Fisher Scientific (United States). Polyvinyl Alcohol (C2H4O)n was bought from Sigma-Aldrich (United States). 2.2 Preparation of polyamida-6 and PVA nanofibers The preparation of nanofibers was done using the electrospinning solvent method. For the polyamida-6 polymer solution a 25% w/w solution was prepared. The prepared solution was then placed into needle syringe (0,7mm x 40mm). The flowrate was 0,212 ml/h and a voltage of 30 kv was applied. The collection plate was aluminum foil placed 20 cm from the collector. 2.3 Scanning electron microscopy analysis To obtain pore and fiber measures scanning electron microscopy (SEM) images were obtained in a Hitachi S3400N-II (Tokyo, Japan). The fibers were coated with gold on a Sputter Quorum Q1ORES (Quorum Technologies, United Kingdom). Sixty random images were analyzed and pore and fiber measures were obtained for polyamida-6 and PVA nanofibers. 2.4 Software image analysis To measure the pores dimensions the software Espirit 1.9 (Bruker, Germany) was used. For this method SEM images from the nanofibers were obtained and the pores were measured Figure 1. Scanning electron microscopy images of polyvinyl alcohol nanofibers (left column) and polyamide- 6 nanofibers (right column). To measure the pores dimensions an image software analysis was performed using SEM images (Fig. 2). 2.5 Computational Simulation. The computational simulator validated and implemented in MATLAB [1] was used for the formation of the 3D matrix nanocellulose computational simulation study. Results and Discussion SEM images were used to characterize the 3D structure of the porous materials (Fig. 1) in the xy plane and thickness direction (Z). The polymer matrix presented random pores and fibres segments with dimensions in the nanoscale. This type of structure can be used to transport molecules or as a scaffolds to cell growth, because of its high porosity and mechanical stability. There is an advantage in the design of this materials because it possible to control the pore dimensions and pore variability. SEM images from Polyamida-6 and Polyvinyl alcohol 3D structures, where the first column is Polyamide-6 and the second column is for polyvinyl structures. Images A, B, C and D are xy planes obtained by SEM with the same amplification, 1000x and 4000x, respectively (fig.1). F and E images are thickness cuts of the structure in the z dimension. Figure 2. SEM images analysis of polyamide-6 nanofibers Conclusion The computational simulator has proven to be an efficient tool to help design porous structures, allowing the creation of optimized systems, capable to transport therapeutic molecules, or be used in regenerative tissue biomedicine. 15

26 [1] Curto, J. M. R., Conceição, E. L. T., Portugal, a. T. G., & Simões, R. M. S. (2011). Three dimensional modelling of fibrous materials and experimental validation. Materialwissenschaft Und Werkstofftechnik, 42(5), [2] Liu, J., Huang, Y., Kumar, A., Tan, A., Jin, S., Mozhi, A., & Liang, X. J. (2014). PH-Sensitive nano-systems for drug delivery in cancer therapy. Biotechnology Advances, 32(4), [3] Kreuter, J. (2014). Drug delivery to the central nervous system by polymeric nanoparticles: What do we know? Advanced Drug Delivery Reviews, 71, [4] Han, N., Johnson, J., Lannutti, J. J., and Winter, J. O. (2012). Hydrogel-electrospun fiber composite materials for hydrophilic protein release. Journal of Controlled Release, 158(1), [5] Dvir, T., Timko, B. P., Kohane, D. S., and Langer, R. (2011). Nanotechnological strategies for engineering complex tissues. Nature Nanotechnology, 6(1), [6] Hollister, S. J. (2005). Porous scaffold design for tissue engineering. Nature Materials, 4(7), [7] Liang, D., Hsiao, B. S., & Chu, B. (2007). Functional electrospun nanofibrous scaffolds for biomedical applications. Advanced Drug Delivery Reviews, 59, [8] Liu, W., Thomopoulos, S., & Xia, Y. (2012). Electrospun nanofibers for regenerative medicine. Advanced Healthcare Materials, 1(1), [9] Yu, R., Chen, H., Chen, T., & Zhou, X. (2008). Modeling and simulation of drug release from multi-layer biodegradable polymer microstructure in three dimensions. Simulation Modelling Practice and Theory, 16, [10] Curto, J.M.R., Hekmaty, A.H., Drean, J.Y., Conceição, E.L.T Portugal A.T.G., Simões, R.M.S., Santos Silva, M.J. In Proc. of the 11th World Textile Conference, Autex 2011, Mulhouse, France, 2 (2011)

27 Fashion Marginalization and the Feminine F. T. S. Gonçalves 1, S. R. Fernandes 1, J. M. Lucas 1 1 FibEnTech-UBI, Textile Science and Technology Department, Universidade da Beira interior, Covilhã, Portugal Abstract Fashion was marginalized within the social studies for its connection with the feminine, which is part of the process of indiscernibility of the feminine in the Western tradition. Heteronormativity is a symbolic set of obligations that follows individuals from birth to death, and that society expects and hopes that are respected by all. In a more modern vision, the woman would no longer be considered a man less perfect. In the new model, would then be considered in its specificities (anatomical and biological) that, in turn, would work in a private basis, as opposed to man. In contemporary society and post-modernity, which will be even more unstable, fluid, liquid, fast, where nothing else will be fixed and immutable, there will be no unquestionable truths and fashion, as well as everything that surrounds the human cultural meanings, should also be in change, be fluid, unstable, disorderly and rebellious. Fashion can and should be understood as a legitimate and autonomous field of knowledge. Research supports this understanding in order to define it as field of knowledge. Keywords Marginalized Fashion, Western Standards, Free Fashion Creation, Heteronormativity Introduction Fashion was marginalized within the social studies for its connection with the feminine, which is part of the process of indiscernibility of the feminine in the Western tradition. There are conceptual conflicts about the practice of fashion and what apparel production is. And, on the role of clothing, the setting and differentiation of identities of the genera within the logic of Western standards, is different from the role of free fashion creation. Heteronormativity is a symbolic set of obligations that follows individuals from birth to death, and that society expects and hopes to be respected by all. Thus, heteronormativity is an assembly of "prescriptions" that underlies social processes of regulation and control, that dictates how people should be, must think, should behave, always linked with a match between sex-gender-sexuality. It requires discursively that this link is maintained in the heterosexual logic. Through the heteronormative speeches, fashion was listed as the symbolism of the feminine universe and that, consequently, ended up having "minor" value and being "deprecated" in relation to other fields of knowledge. Fashion, in the sense of cultural practice of free creation of body ornaments, comes as social phenomenon in the West concurrently with the period of the Renaissance, middle of the XIV century, and this is directly linked to the growth of cities, with trade routes and with the new possibilities of social rise of "new money" that came from noble lineages. After the consolidation of industrial capitalism as a form of production in the West, consequently various forms of dissemination and selling these products have been developed, as a kind of "education" to uncontrolled consumption. And, with this, the mass fashion or "ready to wear" was regarded as the democratization of fashion, allowing to public in lower layers to consume and use these products. The marginalization of female Since the Old Testament, the woman was related to the temptations of the flesh and decorations. Within Christianity, these decorations were considered a vice, mingling with the very essence of woman. At the bottom of these attitudes about the woman, there was fear about the body that, in Christian teachings, is the address of desire and the temptations of "evil", which should be regenerated by the spirit. The woman, as a descendant of Eve, was seen as more susceptible to the temptations of the flesh and could also use their bodies to pay attention to the man who deviate from the path of God [1, 170]. This citation of Entwistle, exemplifies how the subjugation of women and the female is a historical and intricate process in the own constitution of Western society. The woman, whether as body, or as a soul and later as female gender, has always been marginalized, punished, discredited, including their cultural productions, who never had a place of prominence and appreciation. In a more modern vision, the woman would no longer be considered a man less perfect. In this new model, would then be considered in its specificities (anatomical and biological) that, in turn, would work in a private basis, as opposed to man. Rousseau's ideas had great impact and spread, in short, according to Nunes, "the woman should "reign" in the home, give up any pretence and personal desire of another order that was not referenced to her performance in the domestic sphere, as opposed to the man who, due to his qualities, supposed to "reign" in the public scene" [2, 37]. Thus, it was formulated that men and women would have specific vocations that would indicate different places. The vocation of female nature was directed to maternal function and to the domestic (private), depending on the target that the "nature" had determined to woman, otherwise would be "unnatural". "That way, the modern view puts the woman as mother, turning positive the so-called female attributes such as fragility, dependence and virtue, suited to the maternal function" [2, 21-22]. 17

28 This interpretation of the female body, which indicated a new "modern" way of perpetuating the subordination and marginalization was evidenced by Foucault, which in addition to inferior, incapable, relegated to the home and the family, she now has also a deviant sexuality and therefore having a pathology concern [3]. This model of woman and of femininity, according to Nunes [2], was taken to the last consequences, which involves the idea that a woman should be able to endure the sufferings, injustice and subjugation. This women's capacity to "endure hardships and injustices", present in the speeches, especially from social speech of doctors, was very useful to justify the confinement of women of the lower classes within the smothering textile factories. Entwistle elucidates that "Breward noted that the manufacture of clothes since the preparation of the raw materials up to their modifications were primarily in the hands of women by the end of the Middle Age" [1, 100]. The marginalization of Fashion Fashion has played a role of challenging the old hierarchies and power structures, that the new capitalist class were planned. Sumptuary laws and discourses on luxury and good taste illustrate the alleged threat fashion was to the old aristocratic order, which saw the decrease of its own power and influence with the emergence of the new-rich [1, 93-94]. One can consider the existence of a dialogue and an interconnection between the issues that involve the emergence of fashion as a disruptive and cultural practice integrated in social life with the processes of "disciplinary measures" (and its standards and normalizing devices and those of sexuality), processes arising from the speeches of modernity that generated the concept of "anomalies" that led to the marginalization of female (in a broadly way) and has resulted in the invention of the homosexual and the marginalization of all types of cultural production linked to the symbolism of the feminine. Therefore, marginalization occurred as a consequence of a historical process that led to consider people, places, and practices as inferior, pathological, or simply unethical, to modern bourgeois logic. The above quote from Entwistle presents two issues of extreme importance. The first, in the sense that fashion was a power of "rebellion" on the part of the new bourgeoisie, as she said: "in particular, it was found that (fashion) played a role of challenging the old hierarchies and power structures" and, secondly, it's about how fashion, after the rebellion of the "nouveau riche" turned out to be marginalized by the theoreticians of various areas and also by media discourses, as well as all other cultural practices linked to the "stigma of female thing". Being the "marginalization" a process that places outside the hegemonic system all those things that don't matter to the dominant power, as the marginal, the offender, the outlaw, the secondary, the misuse, those which does not participate in the political and economic order, hold no power, no duties, no voice. In this sense, Bancroft argues about the marginalization of fashion: Fashion matters because it s the only creative form whose automatic default is feminine. Entwistle says, I have a book, The 100 Greatest Women Artists of the Twentieth Century. There is no companion volume, The 100 Greatest Men Artists of the Twentieth Century, [1, 93-94] because art is assumed to be practiced by men that women artists are a subsection within it. It s the same with literature. Wander around Waterstones or any other large bookshops and you ll see a dedicated section Women s Literature because literature written by women is niche. Female film directors can be counted on your fingers while for male film directors you need to open a spreadsheet, and theatre fares no better [4]. In modernity, with the "containment" of the feminine, there was a cultural and spatial constraint of the types of activities that women could exercise that linked automatically the elements: female and private (home). The inverse restriction appeared, because if men practiced women's and home activities, these were judged, and marginalized, for being "unnatural". Thus, several activities were considered female, and therefore were judged as a "minor" cultural practice, as being less important to the economic and cultural growth of society. Under this perspective, it is possible to notice that textile arts, mostly, embroidery, tapestry, knitting and fashion, all were marginalized and not considered "free arts", therefore not entered as artistic practices in the academies of Fine Arts. In this same sense, Entwistle argues that, for centuries, has been associated with the woman, fickle fashion with the banal exhibitionism and indulgent narcissism. In fact, this association may explain partially the marginalization of fashion within social theory, the reason why being considered frivolous in the sense of ephemeral, so it would be unworthy of scholarly attention [1, 167]. According to Jones' arguments, cited by Entwistle, "in the 19th century with the entrance of women in commercial culture fostered new ideologies on the meaning of woman who ended up trapping the female merchants to places allowed to sew and cook, but never command and control [1, 168]. Entwistle states that "even the aristocratic men to be nearly as decorated as women, for most of the modern period, this simple fact not dissolved the fashion association with femininity". In fact, when it criticized the ornamenting of men, at the principle was being alike to feminine" for showing much interest in fashion. Sometimes these criticisms were based on the idea that the man's interest in fashion infringed the fair division of the genera. On other occasions, this was seen as an image problem for the nation" [1,173]. Clearly, these arguments of Entwistle will meet the Foucault analytics, whereas modern society, more advanced in the industrial capitalism, as said by him, needed disciplined bodies with greater power, greater control of emotions, of motivations and mainly being trained for the production system. Therefore, decorations, embellishments and taste for beauty, depends on having free time, taking energy and this "lost" energy with the 18

29 auto embellishment is not a practice that meets the desires that modernity required for a "respectable" man. What industrial modernity wished for its members is that they were docile, cleaned, organized, trained, and well suited to the industrial production system. As Foucault explains, modernity is not exactly a historical period marked by closed events, but rather a way of thinking, behaving and to explain the world, that from the means of sexuality, has completely separated universes both male and female in wearing clothes, and in the activities and value that each has, "while fashion was historically crystallized as a performance and repetitive space of female roles and men who have these identifications would be considered as deviated from the indicative standards of masculinity" [5, 144]. The modern thought was so rooted and characterizer of the contemporary subject, for example, one can mention the Bauhaus school, founded by Walter Gropius in 1919 in Germany. This school is a great example of how modern discourses marginalized the female (and fashion) of all public spheres and how the female was allowed only in the private domain. Women were regarded as not having enough strength and "not having value, courage, nor the energy needed to create the new world. The major speeches of the modern movement left out women and established men as the protagonists of social life" [5, 60]. In the 20th century, the modernist movement established for men the functions in the areas of productivity-related industrial design. However, women were named to do the practices associated with domesticity, such as decorative arts, jewellery, illustration, textiles and ceramics, among others. As noted by Valdivieso, the great influx of women who experienced Bauhaus was disturbing to the Director, fearing that they could harm the lofty goals of the school" [6, 56]. Only from the 90 s, names like Judy Attfield (1989), Cheryl Buckley (1989) and Pat Kirkham (2000) were the first to denounce the historic invisibility of women in the Design field. Also questioned the legacy of the modern movement to the discipline of Design, especially around the rhetoric of neutrality [7]. Therefore, "from feminist criticism, it became apparent that the canon is designed as a universal and neutral value, denouncing, at the same time, that is a social construction that excludes other interventions, practices and female subjects" [5, 57]. On the fashion and feminine marginalization, Bancroft argues that there s nothing unusual in this. In the real world, women are under-represented in just about every sphere, from business, politics, sport, to medicine, for example [4]. Thus, Bancroft says that, under the normative society point of view, that considers Fashion as a vanity fair. Thus, Fashion is something trivial that is only of interest to women and gay men and thus pointless by virtue of those who are not interested on it. If it were serious, significant, relevant in any way, shape or form, then straight men would take an interest in it. The fact that, on the whole, they don t take an interest on it, and the people that do, are on the whole, minority groups, is enough to move fashion to the back of the queue of cultural and political importance [4]. In contemporary society and post-modernity, which will be even more unstable, fluid, liquid, fast, where nothing else will be fixed and immutable, there will be no unquestionable truths and fashion, as well as everything that surrounds the human cultural meanings, should also be in change, be fluid, unstable, disorderly and rebellious. Thus, what is advocated here must be understood and studied as a cultural practice and a legitimate field of knowledge and with disruptive characteristics. So, when defending that fashion can be understood as a cultural practice, the research refers to the concept of culture developed by the vast field of Cultural Studies to Temer and Tondato the notion of culture (Cultural Studies) goes beyond ideology, encompassing also subjective and everyday practice actions" [8, 154]. Also, when defending that fashion can and should be understood as a legitimate and autonomous field of knowledge, research supports this understanding based on studies of José D'Assunção Barros who argues that to define a "discipline" or "field of knowledge" of any area, first, one must "outline the essential aspects that contribute to define a disciplinary field, whatever it is [9, 206]. Conclusions Fashion must be understood as a disruptive tool. It must internalize, both in practice and in academic studies, values such as representativeness, activism, environmental and human sustainability, aesthetics, and ethics, generate profit without belittling lives and keep its essence of subversion and contestation of social norms of the bodies and of the individual expressions. Fashion can also be a powerful force for promoting new trends, influence and inspire new consumer behaviours and should be adopted as a tool for Western society to move against the dominant system towards an unrestricted, fair and more open-minded future, always focusing on environmental, social and symbolic sustainability. To common sense, fashion is just an industry based on large-scale production, periodic changes in styles, constant innovation and imitation of styles, passed by the celebrities, television channels, magazines and currently the "blogosphere". In contemporary times, fashion is also judged to be a billion-dollar, polluting, slave industry, objectifying and owner of women. Conceptual problems aside, there is another problem with clothing (or with Fashion) in the contemporary scenario. This is the perception, in the 21st century, of the frequent gender separation of the garment, such as skirts and ties, or colours such as pink and blue. In the collective imagination, these items are in a binary and unique form associated with the feminine and the masculine. However, little is discussed about the individual and social consequences that this garment division produces, because it strengthens the impact of prejudiced, stereotyped and separatists based in binaries speeches, that places subjects as being marginal and of lower standing. 19

30 Acknowledgements The authors of this study acknowledge to FCT - Fundação para a Ciência e Tecnologia and to FibEnTech-UBI (Fiber Materials and Environmental Technologies) Research and Development Unit for their technical and financial supports. References [1] Entwistle, J. (2002). El cuerpo y la moda. Una visión sociológica. Barcelona, Espanha: Paidós Contextos. [2] Nunes, A. (2000). O corpo do diabo entre a cruz e a caldeirinha: um estudo sobre a mulher, o masoquismo e a feminilidade. (1ª ed.) Rio de Janeiro, Brasil: Civilização Brasileira. [3] Foucault, M. (1988). A história da sexualidade 1: a vontade de saber. (13ª ed.). Rio de Janeiro, Brasil: Graal. [4] Bancroft, A. (2014). Why Fashion Matters. [post in blog]. Available in /why-fashion-matters/. [Accessed on: 08/01/2017]. [5] Zambrini, L. (2010). Modos de vestir e identidades de género: reflexiones sobre las marcas culturales sobre el cuerpo. In Revista de Estudios de Género Nomadías, (11), pp DOI: / [6] Zambrini, L. (2016). Olhares sobre moda e design a partir de uma perspectiva de gênero. In: Dobras, Associação Brasileira de Estudo e Pesquisas em Moda, 9 (19), pp Available in [Accessed on 19/01/2017]. [7] Butler, J. (2000). Corpos que pesam: sobre os limites discursivos do sexo. In: Louro, G. L. (ed.), O corpo educado: pedagogias da sexualidade (2ª edição, pp ). Belo Horizonte, Brasil: Autêntica. [8] Temer, A. C., Tondato, M. P., (2004). A Tradição dos Estudos Culturais na Perspectiva das Contribuições Latinoamericanas. In Revista Novos Olhares, 3 (2), pp Available in /article/viewfile/90211/ [Accessed on 24/05/2017]. [9] Barros, J. (2010). Contribuição para o estudo dos campos disciplinares. In Revista ALPHA. (11), pp Available in /18125/23730 contribuicao_para_o_estudo_dos_campos. pdf. [Accessed on 24/05/2017]. 20

31 Porosity optimization of nano cellulose drug delivery systems to control the release kinetics F.P. Morais 1, J.M.R. Curto 1,2 1 FibEnTech-UBI, Fiber Materials and Environmental Technologies, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 2 CIEPQPF - Chemical Process Engineering and Forest Products Research Centre, Department of Chemical Engineering, University of Coimbra, Coimbra, Portugal Abstract Highly porous polymeric materials, which are produced from cellulosic sources, represent an important delivery system for various medicinal applications. This study focuses on developing highly porous promising drug delivery systems containing the drug Diclofenac in order to avoid the gastric side effects induced by this active substance. In this study three porous cellulosic sources were used, namely, nanofibrillated cellulose, bacterial cellulose and carboxymethylcellulose. Therapeutic molecule release kinetics was evaluated through an in vitro gastrointestinal model to understand the release mechanism from each of the delivery systems developed. A DDS structural characterization was also performed using the SEM technique and image analysis. Through these studies it was possible to conclude that DDS presented fibers width dimensions in the order of nanoscale In addition, a porosity and pore dimensions structural consolidation of 3D porous structures was performed using a validated computational simulation model implemented using MATLAB. Through these computational studies it was possible to conclude that it was possible to obtain DDS obtained from NFC with different pore dimension and pore distribution. The structural consolidation of the 3D NFC matrix was obtained using filtration process variables modification. The SEM measurements of the pore distribution indicates that the 3D NFC matrix presented a more uniform pore distribution. The DDS release kinetics also was more uniform. The delivery systems produced also prevented the diclofenac drug from being released at the stomach acidic ph, avoiding the stomach mucous attack side effects. Finally, the porous structure that took the most benefit from 3D optimization through computational simulation was nanofibrillated cellulose, obtaining a 3D network with more uniform pore size and consequently more controlled release kinetics. Introduction Porous polymeric materials because of the possibility to optimize porosity and pore dimensions to transport drugs can be used as drug delivery systems (DDS) in various therapeutic applications [1]. DDS are developed for delivering drugs at predetermined rates and predefined time periods or to be targeting drugs to a desirable cells group [2,3]. The goal of this targeted delivery is to transport appropriate drugs amounts to desirable targets while minimizing unwanted drugs side effects on other tissues [2]. An example of a drug advantageous to be incorporated into a DDS is Diclofenac as it is associated with side effects mainly on the gastrointestinal tract, including irritation, bleeding, ulceration and possibly perforation in the gastric wall because. Like other non-steroidal anti-inflammatory drugs, it is not stable in the acidic environment of the stomach [4]. Natural polymers, such as cellulose, are used in the medicinal chemistry field due to the low incidence of side effects, therapeutic efficacy [5] and biocompatibility [6]. Cellulose is a linear, resistant and fibrous polymer composed of glucose units, interconnected by β (1 4) glycosidic bonds and presenting reactive functional hydroxyl groups [7,8]. With the possibility of using cellulose at the nanoscale, as a building block, the development of nanocellulose based materials is being taken into consideration to obtain advanced materials. Cellulosic nanofibrils in the form of nano cellulose can be extracted from the lignocellulosic bioresources by using appropriate chemical, mechanical, enzymatic or the combination of more than one method [9]. Nano cellulose exhibits several excellent properties such as its high aspect ratio, low density of 1.6 g/cm3, very low thermal expansion coefficient, and high tensile strength about 500MPa [10], which has resulted in numerous applications including drug delivery. Within the nano cellulose family are included nanofibrillated cellulose (NFC) and bacterial cellulose (BC). NFC is first obtained through a chemical treatment, TEMPO mediated oxidation, with subsequent mechanical treatment, high pressure homogenization [11]. On the other hand, BC is obtained in culture with abundant glucose medium, aerobic and temperature controlled conditions, through gram-negative bacteria (Gluconacetobacter sacchari) [12]. Another attractive cellulose material in drug delivery field is carboxymethylcellulose (CMC) produced by Williamson reaction, which have good solubility, high chemical stability, are safe, non-toxic, hydrophilic, biocompatible, and biodegradable [13-15]. 3D molecular modeling and computational simulations are important tools in DDS development. The 3D structure and porosity of porous nano cellulosic systems are determinant to optimize the transport and release function of these systems [16]. This optimization is performed using a computer simulator validated and implemented in MATLAB [17]. The use of experimentally validated computer simulations allows optimization of the structural properties that are determinant to the transport of 21

32 therapeutic molecules. Hence, porous materials can be produced using nano cellulose building blocks and functionalized cellulose, all derived from cellulose. The ability to control pore dimensions is a major step in the DDS development process. The use of cellulose in the nano-scaleas to form a 3D matrix, and cellulose derivates to modify water interaction, is innovative in order to obtain the desired drug release kinetics. The main goal of this study was to develop and characterize structurally nano cellulosic DDS, using a porous NFC, BC and CMC 3D matrix. The computational study was also carried out to optimize the porosity and pore dimensions of the DDS produced and to obtain a more uniform Diclofenac release kinetics. Materials and Methods Materials: CMC sodium salt (NaCMC), high viscosity ( )cP in 1% H 2O (25ºC) and Diclofenac sodium salt (NaDCF) (>99%) were purchased from Sigma Aldrich (Germany). Alginate sodium salt (NaAlg), high viscosity acquired by BDH Chemicals Ltd (England). All other chemicals and reagents used in the study were analytical grade. NFC gel was obtained in the FibEnTech Research Unit of Chemistry Department of University of Beira Interior and BC membrane was obtained in the CICECO - Aveiro Institute of Materials of the Chemistry Department, University of Aveiro. Polymeric DDS production: Spherical polymeric DDS are produced by mixing NaAlg solution, as natural crosslinking polymer, NFC gel, BC gel and NaCMC solution, separately, as an internal 3D network, and NaDCF solution, as therapeutic molecule, in a 2:1:2 ratio. This mixture was homogenized for about 30 minutes. After this process, mixture was added dropwise to a 0.2M CaCl2 solution. The resulting beads were left in this solution for 24 h for their hardening [18]. Finally, the beads were filtered, rinsed and washed with distilled water, with subsequent drying. Image analysis: With SEM images, fibers and pores dimensions were obtained using ImageJ and Diameter, with defined proceedings and criteria. Drug release in vitro studies: Kinetic studies were performed in triplicate for 6 hours using an in vitro gastrointestinal model with controlled temperature ((37±0.5)ºC) and homogenization (100 rpm). In the first 2h, a buffer solution HCl ph 2 was used. Within the following 2 hours, a phosphate buffer solution ph 6.6 was used. In last 2h, a phosphate buffer solution ph 7.4 was also used [20]. Throughout the assay several samples were withdrawn over time (volume restored with the same amount of liquid as was removed) and analyzed by UV-Vis spectroscopy method (276 nm) using a Helios Omega UV- Vis spectrophotometer and a quartz cell with an optical path of 1 cm. Computational Simulation: The computational simulator validated and implemented in MATLAB [17] was used for the formation of the 3D matrix nanocellulose computational simulation study. Results and Discussion SEM technique was used to structurally characterize the porous cellulosic materials used in DDS (Fig. 1) in the xy plane. NFC forms a 3D polymer matrix with random pores and fibres in the nanoscale. BC is presented as a layered structure with some separation degree, as well as horizontal fibrils between the layers. The 3D network of this nanocellulose is very open, allowing the active substance to be released more easily, in a prolonged and controlled way, so achieving the desired therapeutic effects quickly and safely. Finally, CMC forms an interlaced, uniform and open 3D network, i.e. there are several layers in 3D structure. This type of structure has the ability to trap large amounts of water as well as therapeutic molecules because of its high porosity. This is an advantage because it makes it possible to releases them in a controlled way. SEM Analysis: To maintain the porous structure for SEM analysis samples were immersed in a solution of glutaraldehyde 2.5% (w/w) overnight. Later, these were treated with ethanol solutions of graded concentrations, during 10 minutes, in order to replace water with ethanol. Subsequently, the samples are dried by CO 2 Critical Point Drying method, using EMS K850 Critical Point Drier equipped with thermo-electronic heating, adiabatic cooling and temperature control of +5ºC in cooling and +35ºC during heating [19]. After this treatment, the samples were placed on an aluminum support using a double-sided adhesive tape. Then the samples were coated with gold using a Sputter Quorum Q 15 OR ES (Quorum Technologies, United Kingdom) in order to become better conductors. Finally, after this coating, samples were analyzed by SEM S-2700 Hitachi (Tokyo, Japan), operating at 20 kv and at different magnifications. Fig. 1. SEM images of NFC, BC and CMC 3D polymer network. 22

33 In addition to structural differences in 3D network, a difference in fibers and pores dimensions is also visible (Table 1 and 2). NFC had a nanofibers average width and length of (68.5 ± ) nm and (174 ± 0.140) nm, respectively. However, BC and CMC presented higher values. From the results obtained, NFC presents values more similar to the BC values because it has a width of (77.2 ± ) nm and a length of (177 ± 0.146) nm, while CMC has a width of (99.6 ± ) nm and a length of (243 ± 0.179). The average NFC diameters are distributed between 10.9 nm and nm. Comparatively, BC presented a similar diameters distribution (between 9.8 nm and nm) while CMC presented a larger diameter distribution (between 16.3 nm and nm). Table 1. Nanocellulosic fibers characterization. Fibers Characterization NFC BC CMC Count Average width (nm) 68.5± ± ± Average length (nm) 174± ± ±0.179 Diameter min. (nm) Diameter max. (nm) The pores found in 3D NFC network had a average diameter of (132±15.6) nm, with a porosity of 35%. Comparatively, the BC pores had an average diameter of (212±23.9) nm, with a porosity of 48%. Finally, CMC pores presented (273±27.0) nm of average diameter, with a porosity of 51%. Table 2. Nanocellulosic fibers characterization. Pores Characterization NFC BC CMC Count Average Diameter min. (nm) 86.5± ± ±20.6 Average Diameter max. (nm) 176± ± ±33.4 Average Diameter (nm) 132± ± ±27.0 Porosity is a property of great importance in DDS because of its influence on therapeutic agent kinetics release, such as Diclofenac. Diclofenac in vitro release studies from the different polymeric DDS (Fig. 2) showed that the release kinetics did not show a continuous and regular release profile over the 6h assay. At ph 2 it is observed that release profile is similar between in three systems, with almost zero release concentrations, although the CMC DDS reaches DCF concentration released higher values. At ph 6.6 a completely abrupt release profile is found for NFC DDS, while in other systems are more regular. However, at ph 7.4 a greater release is noticeable and also more controlled by the three systems. BC DDS is the one that presents a greater and more controlled released (80%), while NFC presented 64% release and CMC presented 53%. Hence, nanocellulosic DDS produced are innovative because they prevent the Diclofenac release into the stomach (ph 2) and release it from the gastrointestinal tract (ph 6.6) preventing the stomach mucous attack side effects. Amount released (%) ph 2 ph 6.6 ph NFC DDS 20 BC DDS 10 CMC DDS Time (min) Fig. 2. Diclofenac release in vitro studies from polymeric DDS at different ph medium. Since a DDS is intended to release controlled drugs, an NFC 3D network properties optimization, through computational simulations, is necessary to improve its release kinetics. The average values of the NFC 3D network fiber properties are used in the computational simulator [17] to subsequently create the porous structures virtually. This analysis allows develop a 3D polymer matrix structure with porosities similar to real ones. Subsequently this porosity and pore dimensions can be optimized in a desired way to obtain a more regular and uniform 3D network to produce a more efficient delivery system. The use of 3D models has proven to be important in the delivery systems development. Initially a computational network of NFC 3D network was obtained (Fig. 3a) and through its analysis it is possible to validate this study, whose pore properties results are described in Table 3. NFC computational 3D network (n=156) has a porosity of 42%. As experimentally we obtained values of 35%, the results obtained for the computational images can be validated experimentally. In addition to this parameter, all other results of this computational porous structure have similarities with the results obtained for the structures developed in the laboratory. With all the results obtained previously, it is possible to experimentally validate this computational method. In this way, this innovative methodology can be used to optimize porous nanomaterials structural properties, in order to extrapolate these computational results to the laboratory, saving time and resources. Previously a computational image was obtained with an NFC 3D network more open and with irregular pore sizes. Through a computational optimization of this structure (Fig. 3b) it is possible to obtain a more closed and regular 3D NFC network with uniformized pore dimensions (Table 3). Through NFC 3D network computational optimization (n=192), an identical porosity was obtained (40%), however, with smaller pore dimensions results. With this 3D information it is possible, through structural consolidation, to obtain an NFC structure with similar porosity and pore distribution experimentally, obtaining 23

34 more controlled drug release kinetics since this therapeutic molecule has to go a path larger to be released, unlike what happened in a more open structure since the 3D network dimensions are larger. Fig. 3. NFC 3D network computational images before (a) and after (b) a porosity and pore dimensions optimization. Table 3. Computational NFC pores properties. 3D Pores Optimized NFC Characterization NFC Count Average Diameter min. (nm) 63.6± ±18.4 Average Diameter max. (nm) 150± ±9.63 Average Diameter (nm) 104± ±14.1 Through structural consolidation it was possible to produce new and improved systems obtaining more controlled Diclofenac release kinetics for DDS with NFC (Fig. 4), in order to achieve the desired therapeutic effect. Amount released (%) ph 2 ph 6.6 ph Optimized NFC DDS 10 Optimized BC DDS Optimized CMC DDS Time (min) Fig. 4. Diclofenac release in vitro studies from optimized polymeric DDS at different ph medium. By comparing the studies before and after the computational simulations, it can be seen that DDS containing NFC presented great benefits of structural consolidation at kinetic level. The release profile was no longer abrupt at ph 6.6 and began to be more controlled. And at ph 7.4 the release profile is more regular for the optimized system. DDS with BC and CMC release kinetics presented a release profile similar to the first in vitro study, mainly at ph 7.4. For three polymeric systems, the final Diclofenac release amount is higher than the first in vitro studies, with no significant drug amount retained in the system remaining. There was an Diclofenac amount released 70%, 88% and 58% in the DDS with NFC, BC and CMC, respectively. In this way, a DDS optimization allows better results, with computational simulations aid. Conclusions 3D porous materials modelling and simulation has been used as a tool to obtain 3D DDS structures. Pore size distribution has been modified and has an impact on the therapeutic molecule release kinetics. The incorporation of nanocellulose and functionalized cellulose, like CMC, have been used to control OH bonding and water affinity. Optimization of 3D porosity, pore dimension and distribution in DDS is determinant to drug transport and release. Biocompatible DDS have been developed to avoid Diclofenac release in the stomach (ph 2) and prevent the stomach mucous attack side effects. And, finally, nano cellulosic DDS applications, using a porous biocompatible materials combination constitute a promising medicinal chemistry research field to improve drug delivery systems. Acknowledgements The authors would like to thank to FCT, Fundação para a Ciência e Tecnologia, for financial support for the Research Unit Fibre Materials and Environmental Technologies FibEnTec (Refª UID/Multi/00195/2013). References [1] S.E. Moulton, G.G. Wallace, 3-dimensional (3D) fabricated polymer based drug delivery systems., J. Control. Release 193 (2014) [2] J. Safari and Z. Zarnegar, Advanced drug delivery systems: Nanotechnology of health design: A review, J. Saudi Chem. Soc. 18 (2014) [3] A. N. Zelikin, C. Ehrhardt, A. M. Healy, Materials and methods for delivery of biological drugs, Nat. Chem. 8 (2016) [4] A. Philip, B. Philip, Colon Targeted Drug Delivery Systems: A Review on Primary and Novel Approaches, Oman Medical Journal 25 (2010) [5] M. F. Oliveira, D. Suarez, J. C. B. Rocha, A. V. N. C. Teixeira, M. E. Cortés. F. B. de Sousa, R. D. Sinisterra, Electrospun nanofibers of polycd/pmaa polymers and their potential application as drug delivery system, Mat. Sci. Eng. C-Mater., 54 (2015) [6] A. Kierys, M. Grochowicz, P. Kosik, The release of ibuprofen sodium salt from permanently porous poly(hydroxyethyl methacrylate-co-trimethylolpropane trimethacrylate) resins, Micropor. Mesopor. Mat., 217 (2015) [7] M. H. Mousa, Y. Dong, I. J. Davies, Recent advances in bionanocomposites: Preparation, properties, and applications, Int. J. Polym. Mater. Po., 65 (2016) [8] M. Jorfi, E. J. Foster, Recent advances in nanocellulose for biomedical applications, J. Appl. Polym. Sci., 132 (2015) [9] S. Mondal, Preparation, properties and applications of nanocellulosic materials, Carbohydr. Polym. 163 (2017) [10] M. H. Mousa, Y. Dong, I. J. Davies, Recent advances in bionanocomposites: Preparation, properties, and applications, Int. J. Polym. Mater. Polym. Biomater. 65 (2016)

35 [11] J. H. Kim, B. S. Shim, H. S. Kim, Y. J. Lee, S. K. Min, D. Jang, Z. Abas, J. Kim, Review of nanocellulose for sustainable future materials, Int. J. Precis. Eng. Manuf. - Green Technol. 2 (2015) [12] I. Sulaeva, U. Henniges, T. Rosenau, A. Potthast, Bacterial cellulose as a material for wound treatment: Properties and modifications: A review, Biotechnol. Adv. 33 (2015) [13] S. Butun, F. G. Ince, H. Erdugan, N. Sahiner, One-step fabrication of biocompatible carboxymethyl cellulose polymeric particles for drug delivery systems, Carbohydr. Polym., 86 (2011) [14] A. Dafe, H. Etemadi, H. Zarredar, G. R. Mahdavinia, Development of novel carboxymethyl cellulose/kcarrageenan blends as an enteric delivery vehicle for probiotic bacteria, Int. J. Biol. Macromol. 97 (2017) [15] J. S. Ferreira, J. M. R. Curto, R. M. S. Simões, M. J. S. Silva, 3D Computational simulation of Drug Delivery Systems (DDS) made from carboxymethyl cellulose (CMC), 1st Symp. FibEnTech Res. Unit (2016) [16] J.M.R. Curto, A. O. Mendes, E. L. T. Conceição, A. T. G. Portugal, P. T. Fiadeiro, A. M. M. Ramos, R. M. S. Simões, M. J. S. Silva, Development of an Innovative 3D Simulator for Structured Polymeric Fibrous Materials and Liquid Droplets, in: A. Öchsner, H. Altenbach, (Eds.), Mechanical and Materials Engineering of Modern Structure and Component Design Advanced Structured Materials, Springer International Publishing, 2015, pp [17] J. M. R. Curto, E. L. T. Conceição, A. T. G. Portugal, R. M. S. Simões, Three dimensional modelling of fibrous materials and experimental validation, Materialwiss. Werkst., 42 (2011) [18] K. Vijayalakshmi, T. Gomathi, P. N. Sudha, Preparation and characterization of nanochitosan/sodium alginate/microcrystalline cellulose beads, Der Pharm. Lett. 6 (2014) [19] M.H.G. Junior, E.V. Sterzo, M. Macari, I.C. Boleli, Use of Scanning Electron Microscopy for the Evaluation of Intestinal Epithelium Integrity, R. Bras. Zootec. 33 (2004) [20] M. L. González-Rodríguez, M. A. Holgado, C. Sánchez-Lafuente, A. M. Rabasco, A. Fini, Alginate/chitosan particulate systems for sodium diclofenac release, Int. J. Pharm. 232 (2002)

36 Women wears men s clothing: A study case for a niche market Reis, Benilde 1 ; Pereira, Madalena 2 ; Azevedo, Susana 3 ; Jerónimo, Nuno A. 4 ; Miguel, Rui 5 1 U.MAKE.ID Researcher, FibEnTech-UBI, Textile Science and Technology Department, Universidade da Beira interior, Covilhã, Portugal 2 FibEnTech-UBI, Textile Science and Technology Department and UNIDCOM, Universidade da Beira interior, Covilhã, Portugal 3 CEFAGE-UBI, Faculty of Social and Human Sciences, Universidade da Beira Interior, Covilhã, Portugal 4 LabCom.IFP, Faculty of Social and Human Sciences, Universidade da Beira Interior, Covilhã, Portugal 5 FibEnTech-UBI, Textile Science and Technology Department, Universidade da Beira interior, Covilhã, Portugal Abstract This research aims to study and clarify ideas of genderless clothing, to contribute later in depth and contemporary knowledge about this subject. Study and analyses the existing impact, at the sociological level, and in what way the genderless clothing is inserted in the contemporary market. Also, understand the relation between the garments and the possible consumers of genderless clothing. It was developed a case study with three persons (women) who was made to an exploratory interview to possible consumers of genderless clothing. To explore gender issues in clothing, it is necessary to cover issues of sociology and fashion. Subsequently verifying if this has a future in the market it is necessary to evaluate the part of management and marketing. Sociologists draw a distinction between sex and gender. Sex refers to biological differences between the male and female bodies, while gender concerns psychological, social and cultural differences between men and women. In fashion, to produce genderless clothing, it is necessary to verify the shapes of the men and women bodies and their measurements. For the modelling of clothing practiced for current stereotypes doesn t apply to many people and is only produced for a niche market. Furthermore, it was possible to establish an association in physical differences between men and women. Knowing that, there is a niche market for genderless clothing, thus enabling the creation of a business model for this target. Keywords: Niche Market; Fashion; Clothing; Gender; Genderless; Sociology; Marketing. Introduction The implications of this investigation can be useful because it will help to a better understanding of genderless clothing and what remains to be done in relation to this type of clothing. The problem is the same garment fitting in man and woman. Placing, so the hypothesis of a garment adapts to the body of a man and a woman, regardless of gender. the present study involves three women from unknow gender. In this sense, this research aims to fill that gap, not only in fashion design and patterns, but also on literature. The results can be useful for the market knows that exits this target in market. The object of study of this investigation is to analyse genderless clothing in all its complexity, which includes an analysis of the phenomena in various areas of study such as sociology, fashion design and marketing. Attributing thus, an interdisciplinary character in this investigation. This research focuses on understanding and analysing current issues and aspects of society regarding gender issues in fashion collections. By making the case study through a qualitative methodology, in which the main objective is to interpret the phenomenon that is observed. Is a freer method of scientific investigation, the purpose is not to count quantities but to understand the behaviours, particularities and individual experiences. Literature Review Sociology Like Simmel (2008), the first theme is that of society as a field of tensions and interactions, that fashion itself, its expression, its variations, its rhythms, its mechanisms, its ambiguity, its meaning, in its specific place within social reality, is a privileged manifestation, because it is always present as factor of socialization and individualization. [1] For Schouten (2011), studies on gender, in particular, the sociology of gender, are not residual scientific categories, but rather a specific mode of observation and analysis of social reality. It attends to the ways of involving people in social relations and their ideas, depending on whether they are male or female. Gender must be studied because of the great importance of this principle in the organization of social life, but also because it is at the root of many situations of disparity and injustice. [2] According to Giddens (2010), at the sociological level, a distinction is made between sex and gender. Sex refers to biological differences between the male and female bodies, while gender concerns psychological, social and cultural differences between men and women. The socialization of gender consists of learning the gender roles with the help of agents such as the family and the media. It is believed that gender socialization begins as soon as a child is born. [3] Children and internalize the norms and expectations that are supposed to correspond to their biological sex. In this sense, they adopt "sexual roles" and the masculine and feminine identities (masculinity and femininity) that accompany them. Usually, we do not even notice them - precisely because they are too diffuse. They have been 26

37 infused into us from birth. Our conceptions of gender identity, as well as the sexual attitudes and inclinations associated with them, are formed so early in life that we consider them, most of the time, as certain in adulthood. But gender does not exist simply; All of us, as some sociologists claim, "doing gender" in the social interactions we establish daily with others. Like Anthony Giddens (2010) before we review these contradictory approaches, it is necessary to make an important distinction between sex and gender. In general, sociologists use the term sex to refer to the anatomical and physiological differences that define the male body and the female body. By contrast, gender is understood as the psychological, social and cultural differences between males and females. Gender is associated with socially constructed notions of masculinity and femininity; It is not necessarily a direct product of the biological sex of an individual. The distinction between sex and gender is fundamental, since many differences between men and women are not of biological origin. Sociological interpretations of gender differences and inequalities have assumed contrasting positions in this issue of gender and gender. [4] However, some authors argue that the aspects of human biology - from hormones to chromosomes, from brain size to genetics - are responsible for the congenital differences in behavior between men and women. These differences, they say, are visible in all cultures, implying that natural factors are responsible for the gender inequalities that characterize most societies. Fashion According to Wilson (1985), in all societies the body is dressed and everywhere clothes and adornments have a symbolic role of communication and an aesthetic role. In general, clothing and adornments seem to fill a several social, aesthetic and psychological function, joining and expressing them all simultaneously. Fashionable dressing implies a person stand out and simultaneously, merge into the crowd, claim the exclusive and follow the herd. [5] Dorfles (1984) argues that the phenomenon of clothing and the fact that it is linked to the very deep dimensions of our character, our mood, our way of being in the world, always seemed a given not to eliminate and considerable Importance, not only for himself but also for others. This same author also mentions that fashion is not only an insignificant and superficial phenomenon, but it is the mirror of habits, psychological behaviour of the individual, profession, political orientation and taste. Thus, giving fashionable fashion, considering its social, aesthetic and cultural weight. [6] In 1905 Simmel (2008), fashion expresses, in the most visible and concrete way, the essentially dialectical and dynamic reality of society, made of inevitable conflicts between individuals, between the multiple and different social formations, between individuals and Groups or classes. [7] Monneyron (2006), also addresses the question of whether, or not, a unisex fashion exists, also referring to the historical facts of Poiret, Chanel and Yves Saint Laurent, mentioning later the dysfunction of sexual dimorphism between woman and man. With this brief approach, one can refer to a contemporary representation of androgyny in clothing, in the present investigation, its popularity and what impact it has on society today. For Wilson (1985), part of the strangeness of clothing is that they link the biological body to the social being, and the public to the private. This makes clothing difficult, because it obliges us to recognize that the human body is more than a biological entity. It is a cultural organism, even a cultural artefact, and its own borders are not well defined. [8] Clothing consumption genderless and marketing Following a sociological and fashion design approach, it is essential to mention how genderless clothing will avenge the market. For this, it is necessary to see that brands and designers produce clothing genderless and what strategy used by each one of them. In 1994, Bohdanowicz and Clamp affirms that it is important to acknowledge the impact of the social and economic environment on the fashion marketing process, since few other industries are so powerfully and directly affected by social and economic trends. The concept of a satisfying exchange relationship is also of major importance. Marketing involves the exchange usually of money, but also of time and effort, in return for goods. Sometimes people may be willing to invest more time or money in acquiring something that is highly desirable because they feel it has a greater value derived from its status as a fashion item. A fashion item is worthless when it is no longer in fashion, as value is implicit in the item being fashionable. At the heart of the marketing concept is the appreciation of the need to satisfy customer s needs, wants and demands. [9] Besides, the factors that differentiate fashion marketing from the marketing of other types of goods services can be broadly classified into three areas: strong influence of environmental pressures; time constraints and role of buyers. To Pinto (2015), the development of genderless clothing is a fashion business attempt, a way to reach different audiences, responding to the needs of an increasingly fluid market niche. [10] According to Monneyron (2006), it raises an issue, which is also pertinent to answer in the present investigation, if and in what way the clothes, or the dress determines our behaviors. Consumer behaviors will be something to consider in this investigation. [11] English (2007), in his conclusions of one of his chapters of "Cultural history fashion, 20th century", leaves several questions in which one adapts in this context: "As with the evolution of modern marketing strategies, in a product that has more commercial value than the product itself? " this is one of the questions that ends up fitting into the present investigation. [12] Other s studies In a study by David Pinto in 2015, which studies the theme of unisex clothing as a response to male and female gender stereotypes, it is a clarification about the creation of unisex clothing in response to male and female gender stereotypes. 27

38 According to the author, androgyny and the unisex are present in the main current trends of fashion, confusing and tearing the tenuous barrier between the masculine and the feminine. A historical analysis is made, a brief synthesis of the body related to Fashion and asks the question: "Unisex, a paradigm or trend?", However after the analysis of his research, ends his investigation without giving a concrete answer to the question Given the complexity of the topic and the need for a greater depth of research carried out indicated in the suggestions for future research. [13] In another study done by Pereira et. Al (2009), about the relationship between apparel attributes and advertising on consumer buying behavior, that refers the consumer behavior approaches and the tendencies in contemporary advertising display the reflux of its former pedagogical and constructive dimension. Advertising s classical model the famous copy strategy - insisted on a message that praised the functional or phycological benefits of a product. Also, the product attributes play an important role in marketing communication for both the consumer and the marketer alike. The study concludes that fashion magazines advertising influence the following attributes: used materials, style, durability, cut, treatment, quality, satisfaction, necessity and function. [14] Study Case For Júnior (2012), the case study method is an empirical investigation that investigates a contemporary phenomenon in its real-life context, even though the boundaries between the phenomenon studied and the context are not clearly defined. Júnior still mentions Yin (2005), the case study lends itself to investigations of contemporary social phenomena in which the researcher cannot manipulate relevant behaviors that influence and / or alter its object of study. The method enables the researcher to deal with a wide variety of evidence, from documentary analysis, field visits, interviews and participating observation. [15] In this sense and according to Coutinho (2011), a case study belongs to a type of qualitative plan, which in addition to understanding and describing in depth the phenomenon investigated, is an intense study of a single subject or group about an emerging problem. The expected results are to understand explanatory factors of behaviours; identify personalities / socialization traits; test and discover concepts; coding cases and discovering patterns. [16] Initially, for the present case study, the participants were asked to share their experience and to answer the questions, to be sincere in expressing their opinion of the experience they were experiencing. The pieces used for this study correspond to the collections produced (2012 and 2013) in the scope of development of men's collections, these were designed and produced by Benilde Reis at the University of Beira Interior. In each piece dressed, the respective photographic record was made. Each participant is physically woman (sex). Each person was asked the following questions in each dressed piece: 1. The appearance of the piece: is it for a man or a woman? 2. How does it feel when wearing this piece? 3. Used as a daily use piece? If not, why? 4. Aesthetically (applications, colors...), what do you think of the piece you have dressed? 5. Forgetting the problems of wearable, did you buy this piece? If not, why? 6. Confusion when tightening? (If justified in the respective piece of clothing). Results of the case study Case I Woman characteristics of CASE I - The woman has 31 years old, the sizes of clothes that she usually wears are a size 36 and S, sometimes M for long sweaters and measures in height 1,59m. In this case, the garments 1, 2, 4 the answer to question 1. was unisex and the garments 3, 5, 6 the answer was man. In question 2. the fifth garment was probably the size of the user, the rest of the garments were comfortable but too large or long. In question 3. the user respond that probably used majority of garments. The answers to question 4. the user just doesn t like the third garment. In question 5. the user only bought the first and second garment. The answers for the question 6. the user just felt confusion wearing the second garment. Observations of CASE I - The woman has short stature, Besides the clothes being bigger in width, is also in length. Case II Woman characteristics of CASE II - The woman has 28 years old, the sizes of clothes that she usually wears are a size 38/40 and M (size L when she like the large fit) and measures in height 1,67 m. In this case, the garments 1 and 6 the answer to question 1. was unisex, to the garments 3 and 5 the answer was woman and for the garments 2 and 4 the user was confusing but maybe more for man. In question 2. the fifth and sixth garment it was fit on the body user, the rest of the garments were comfortable but too large or long. In question 3. the user respond that probably used majority of garments, less the fourth and fifth garment. The answers to question 4. the user like all the garments, but probably changed some colours. In question 5. the user doesn t buy the fourth and fifth garment. The answers for the question 6. the user doesn t felt confusion at all when she wearing the garments. Observations of CASE II - The woman has medium stature, the piece 6 it fitted perfectly. Case III Woman characteristics of CASE III - The woman has 29 years old, the sizes of clothes that she usually wears are a size 34/36 and S/M, and measures in height 1,68 m. In this case, the garments 1, 2 and 3 the answer to question 1. was unisex, to the garments 4, 5 and 6 the answer was man. In question 2. all the garments were comfortable 28

39 but too large or long, only the sixth garment was uncomfortable. In question 3. the user respond that probably used majority of garments, less the fifth garment. The answers to question 4. the user like all the garments. In question 5. the user doesn t buy the fifth garment. The answers for the question 6. the user just felt confusion in the second garment. Observations of CASE III - The woman has medium stature but very thin, besides the clothes being bigger in width, is also in length. Conclusions of the study case In the positive aspects, the results and answers can take this investigation to points that had not previously been thought, since it is an ongoing investigation. It is remarkable in this study case that is possible to exist a niche market, since the clothes that the participants (women) used are of man target. Some negative aspects, the background of the people who wore the clothes in question was not considered (what they do, what they studied and their level of education); only women (referring to sex as a human being) were aged between 28 and 31 years old; they are people close to the author. In this study case, there are some considerations to consider. The pieces 1, 2, 3 and 4 from the "Second Life Replay" project collection in 2013, was purposefully made and designed to fit widest in men s models. For this study were not considered the sizes of the pieces of clothing in relation to the people who wore the pieces of clothing in this study case. Observations Initially in number one question "Is the appearance of the piece for a man or a woman?", however after this case study, it was felt the need to put the term unisex in this investigation. The term unisex have as definition in the dictionary, as an adjective of designed or suitable for both sexes; not distinguishing between male and female; undifferentiated as to sex. Final Reflections and Forthcoming Studies After this study case, it s possible to conclude that for better and other results of this ongoing research, that it s necessary to make an anthropometric survey (at least at European level), it s necessary to make a survey of size tables (man and woman) analyse them and see differences of measures between tables, studying and verifying the relationship between sizes, heights and stature and make a study / survey of attributes of the consumers for genderless clothing. After this study case, came out on newsstands the National Geographic in January 2017, the special issue about gender revolution. In this gender issue was prepared a glossary from the book The Teaching Transgender Toolkit is possible to verify that there are not only the masculine and feminine gender, but it s clear that there are at least 13 types of gender (officially) they are: agender, androgynous, cisgender, gender binary, gender conforming, gender dysphoria, gender expression, genderfluid, gender identity, gender marker, gender nonconforming, genderqueer and nonbinary. In this issue of N.G. the types of gender are defined and what differentiates them. Beyond these 13 types of gender we cannot forgot the genderless and the unisex term. It is necessary to consider these terms to subsequently obtain valid data in this ongoing investigation. [17] References [1, 7] G. Simmel, Filosofia da Moda e Outros Escritos, Edições Textos & Grafia, Lisboa. (2008) [2] M. J. Schouten, UMA SOCIOLOGIA DO GÉNERO, Edições Húmus, Lda. (2011) [3, 4] A. Giddens, SOCIOLOGIA, 8ª edição, Fundação Calouste Gulbenkian. (2010) [5] E. Wilson, ENFEITADA DE SONHOS. MODA E MODERNIDADE, Edições 70, Lisboa. (1985) [6] G. Dorfles, A MODA DA MODA, Edições 70, Lisboa. (1984) [8, 11] F. Monneyron, 50 respuestas sobre la moda, GG moda, Barcelona. (2006) [9] J. Bohdanowicz, L. Clamp, FASHION MARKETING, Routledge, New York. (1994) [10, 13] D. Pinto, Vestuário Unissexo como Resposta aos Estereótipos de Género, University of Beira Interior. (2015) [12], B. English, A cultural history of fashion in the 20th century: from the catwalk to the sidewalk, Berg Publishers. (2007) [14] M. Pereira, S. G. Azevedo, J. Ferreira, R. A. L. Miguel, V. Pedroso, The Relationship between apparel attributes and advertising on consumer buying behaviour, University of Beira Interior, Available in: blication/ _the_relationship_betwee N_APPAREL_ATTRIBUTES_AND_ADVERTISING_O N_CONSUMER_BUYING_BEHAVIOUR/links/56b26f1 a08aed7ba3feddbe7.pdf. (2009) [15] S. S. C. Júnior, Estudo de Caso x Casos para Estudo: Esclarecimentos a cerca de suas características e utilização, Paper presented Anais do VII Seminário de Pesquisa em Turismo do Mercosul, 16 and 17 November of 2012, Universidade de Caxias do Sul, avaible at: rios_semintur/semin_tur_7/gt01/arquivos/01/04_clement e_jr, (2012) [16] C. P. Coutinho, Metodologia de Investigação em Ciências Sociais e Humanas: Teoria e Prática, Edições Almedina, Coimbra. (2011) [17] N. Geographic, Gender Revolution: Special Issue the Shifting Landscape of Gender, January 2017, vol. 231, no.1, Official Journal of the National Geographic Society. (2017) 29

40 Entrepreneurship: new models of business in fashion design Fernandes, Clara 1, Pereira, Madalena 1,2, Lucas; José 1, Miguel, Rui 1, Santos Silva, José 1 1 Departamento de Ciência e Tecnologia Têxteis, FibEnTech-UBI, Universidade da Beira Interior, Covilhã, Portugal 2 UNIDCOM, IADE, Universidade Europeia, , Lisboa, Portugal Abstract The present study inserts itself in a Ph.D research that focuses on Fashion design entrepreneurs in Portugal and abroad. As this article deals with the primary conclusions of the study, final conclusions are not yet available. As unemployment is still an issue identified at this economic stage, many entrepreneurial venture have seen the light of the day, in an attempt to give a new breath to an industry contrasting between tradition and innovation. As new era related to the creation of new businesses in the fashion design industry, this study seeks to focus on people who want to offer other opportunities to fashion designers right after graduation. How do these companies can be helpful to fashion designers and which tools can they implement to make them reach their business goals? These are some of the aspects that will be observed. This study is based on interviews made to new companies that were made to help young designers to launch their first brand. Key words Fashion Design, Entrepreneurship, fashion business. Introduction Each year, many fashion schools around the globe observe their students graduate, and begin a new chapter of their lives. These newly graduates have many options to start this new part and can choose many paths for their future. Most of them will attempt to get a position as intern in a fashion-related company, some will start to work in the fashion industry and others will even decide to proceed their academic formation and gain more knowledge as researchers in the scientific area of fashion design. The current economy, still recovering from one of the roughest crisis has compelled many to take a risk as entrepreneurs. Lately, a new paradigm can be observed in the global apparel industry, as the diversity of professional outcomes in the business of fashion have evolved, as well as the society we live in. Speaking of the fashion industry and starting from students in Fashion Design, it can easily be observed that there is an increase of young people, wanting to increase their chances of a successful future, taking the decision to get a college degree. However, such numbers have decreased throughout the years, since the economic crisis and the need for young people to become financially independent right after their majority [1]. Narrowing this study on Fashion Design courses and also witnessing data released by the Portuguese General Direction of Superior Education [2] in the last academic year of 2014/2015, 131 openings were made for new students in fashion design Courses throughout the country, however, these numbers only consider degrees delivered by public universities and entitled Fashion Design. Facing a still stagnating economy, many complications can come in the way of recently graduated individuals (Kozar & Connell, 2014, p.1). Even if for some students it is not a choice to risk it all to enter the job market, the ones who end up taking a chance can mostly count on their former teachers, who can help them in the job seeking process and even influence it (Kozar & Connell, 2014, p.10). In the UE and more particularly in Portugal, the application of Bolonha in 2006 has been responsible for many more students entering college courses with an augmentation of more students in 2007, comparing to 2006 (Vieira & Marques, 2014, p.90), but also implicating changes in the academic formation of the students. Masters degrees are strongly recommended by superior institutions, as Bolonha was implemented in a continuity logic (Sousa, 2015). After a license degree, students face the difficult situation to choose between entering the job market right away, mostly through an internship, or to improve their knowledge in the field they studied, or furthermore in another complementary field. Unfortunately, if Portugal has presented good results in terms of entrepreneurial and innovative-driven education at schools (GEM, 2015), the contrary is happening in terms of college education, as very few programs prepare future fashion professionals for entrepreneurship, even if it can be agreed that an innovation-driven curriculum is essential for such studies, as referred by Black, Freeman & Stumpo: Most educators would agree that the development of creativity is a crucial part of an apparel design curriculum and an essential component in the success of students. In the face of ever-changing global apparel industry there is a demand for creative graduates (2015, p.131). In Portugal, the Fashion Industry has encountered a series of particular challenges, in a way that its importance for the country is strategic, more specifically because of the development of clusters since the 1990 s (Porter, 2002). These clusters were related to wool manufacturing, textile development, knitting and the footwear industry (Amaral, 2015). However, with the economic crisis that stroke globally in 2008, the situation analyzed and planned by Porter in the 90 s was completely altered, as it was considered that 20 years after the Porter Project, the country is rather worse in terms of international competitively (Bom, 2012). Nevertheless, it is central to refer that the fashion industry is somehow always in crisis, since it is a very unstable and unpredictable sector, responding to many exterior factors related to politics, economy, society and culture, the global market for 30

41 fabrics and raw materials, as well as the constant mutation of fashion, consumerism and lifestyles [3]. For the Portuguese association of Textiles and Clothing (ATP) it is essential to see entrepreneurship as a new way to improve the Portuguese Fashion dynamics but also those who take initiative in daring to take risks and living a project like an adventure [3]. As these people should be encouraged those who have the entrepreneurial experience necessary for them to become successful. Following this quote by the Portuguese Textile and Clothing association and also the idea that there is essential to encourage fashion related ventures, it is important to observe the reality of entrepreneurship and entrepreneurial prospects after a degree in the fashion design field. For that matter and after considering the multiplicity of potential ways to enter the market as a young fashion designer, it seemed important to observe who are these people who want to change the fashion industry and how they use entrepreneurship to do so. Methods The following study proposes to explore the people behind this new reality using a mixed approach, in order to comprehend the risks taken by these designers, as well as the allurement of entrepreneurship in such a tight competition. The use of such methodology is justified by the need to combine both qualitative and quantitative data in order to obtain results from different samples [4;5]. By interviewing these recently graduated fashion designers, this article seeks to understand the point of view and reasons behind the choices they made as young entrepreneurs. Are these young designers, freshly graduated prepared for an entrepreneurial future? Is their entrepreneurial motivation self-taught or is it coming from their scholar background? In a second part of the methodology, this study will use questionnaires directed to fashion design students in their last year of study, considering both Bachelor of Arts (BA) and Masters of Arts (MA). The use of surveys is related with the need to approach a broad sample of fashion design courses seniors and get a wider view of their [6]. By inquiring these future professionals, as they are almost ending their course, the following article seeks to understand the importance of entrepreneurship for young fashion designers, as well as their view on an entrepreneurial career in the industry. By mixing quantitative and qualitative data, the goal is to obtain both personal opinions and numerical data [7]. Findings The finality of this article resides in the efficiency of fashion design courses and their curricula, as they are still very few courses related to the field of fashion that consider entrepreneurship as a possible future for their students. This paradigm is related to the fact that institutions still consider that fashion design students will directly enter in the industry following graduation, as interns or through apprenticeships in medium and large apparel companies, not even considering the fact that many can see entrepreneurship as way in the job market. Moreover, this study finds that many freshly graduated fashion designers began their entrepreneurial career as a side job, as they pursued their academic formation. The research also shows the conception and views of entrepreneurship among students and their doubts and hopes as future professionals. The data obtained by the questionnaires directed to seniors in BA and MA fashion courses also reveal the lack of entrepreneurial teaching in curricula [8], as well as the efforts that are still to be made to improve such courses [9]. Finally, the interviews clearly hint for a need to create more entrepreneurial ventures in creative industries, moreover in fashion, as the industry is growing as fast as technology-driven innovation. However, results also show the need to rejuvenate the traditional textile and clothing industry, contrasted with convention and need for innovation, creating projects and structures to undertake them, such as hubs and other platforms. Value and Originality The following work researches an educational field that is still trying to figure how to implement entrepreneurship in its curricula, following the paradigm of young fashion designers trying to enter the apparel industry on their own. Such paradigm is very contrasting with the tradition of beginning in the industry as an apprentice or through several internships, as a way to promote knowledge from the inside of the industry. However, such industry is very little alluring for millennials, as they often tend to reject the industry and want to be a part of the change [10], by making conscious decisions and turning sustainability as their top-priority [11]. Results and discussion As this article deals with the primary conclusions of the study, final conclusions are not yet available. However, the study can primarily conclude the need for new ventures and innovation in fashion design, as well as one can observe the hunger for entrepreneurial ventures related with creative industries. Such investment and evolution can be observed with the recent creation of creative hubs, among other online platforms created to encourage innovation, creation and new projects involving design. Many solutions are observed in the final study, as well as interviews to specialists of the industry, who have seen this evolution and observed it. As the final conclusions of the study cannot be disclosed at this stage, the goal of this Ph.D research hints for the creation of a solution, based on the results and data obtained through literature review, interviews and surveys. Acknowledgements We would like to thank the University of Beira Interior, and more specifically, the Department of Sciences and Textile Technologies (DCTT) the Informatics Department that both contributed to this venture as well as I&D FibEnTech. Special thanks to all involved with this study. 31

42 References [1] DIREC A O-GERAL DO ENSINO SUPERIOR. (2014). Concurso Nacional de Acesso: 2014 em Nu meros. [PDF]. DGSE Available at: [2] DIREC A O-GERAL DO ENSINO SUPERIOR. (2015). Concurso Nacional de Acesso: 2015 em Nu meros. [PDF]. DGSE Available at: [3] ASSOCIAC A O TE XTIL E VESTUA RIO DE PORTUGAL. (2014). [E-book] Plano Estrate gico Te xtil 2020: Projetar o Desenvolvimento da Fileira Te xtil e Vestua rio ate Available at: ebook.pdf [4] Stokes, D., & Bergin, R. (2006). Methodology or methodolatry? An evaluation of focus groups and depth interviews. Qualitative Market Researh: An International Journal, 9(1), [5] Bernard, H. R. (2000). Social Research Methods: Qualitative and Quantitative. Boston: Pearson Education. [6] Groves, R. M., Fowler Jr., F. J., Couper, M. P., Lepkowski, J. M., Singer, E., & Tourangeau, R. (2004). Survey Methodology. (J. W. & Sons, Ed.). Hoboken, New Jersey: Wiley-Interscience. [7] Wheeldon, J. (2010). Mapping Mixed Methods Research: Methods, Measures, and Meaning. Journal of Mixed Methods Research, 4(2) [8] Breder, M. (2010). Empreendedorismo aplicado ao Design. Ensaio metodológico em prol de um design(er) Proativo., [9] Sousa, G., & Simoes, I. (2015). Fashion Design and Entrepreneurship : a Strategic Model for Higher Education in. Summer Cumulus Conference. [10] Ünay, F. G., & Zehir, C. (2012). Innovation intelligence and entrepreneurship in the fashion industry. Procedia - Social and Behavioral Sciences, 41, [11] Hill, J., & Lee, H. (2012). Young Generation Y consumers perceptions of sustainability in the apparel industry. Journal of Fashion Marketing and Management: An International Journal, 16(4),

43 PLA/cellulose composite spheres: preparation and characterization S. Sousa 1, A.P. Costa 1, R. Simões 1 1 FibEnTech-UBI, Chemistry Department, Universidade da Beira interior, Covilhã, Portugal Abstract For the first time, the solvent evaporation technique was used to obtain poly(lactic acid) (PLA)/cellulose pulp fibers composite spheres. In order to evaluate the effect of cellulose particle size and surface chemistry on its incorporation into PLA spheres, the following tasks were performed: i) unbleached pulp fibers were modified by grinding or surface modification by lignin deposition and grafting of linoleic acid and, ii) the different types of cellulose were incorporated into PLA spheres. The PLA/cellulose composite spheres were characterized regarding its chemical and thermal properties. The highest percentage of cellulose and lignin incorporation (79% and 71%, respectively) was obtained with cellulose pulp fibers with the highest lignin content achieved by lignin precipitation. Microcrystalline cellulose, the most hydrophilic cellulose sample, was poorly incorporated into PLA spheres. Contrarily to surface chemistry, the fiber size is not a key parameter to increase the percentage of cellulose incorporation. Thermal properties assessed by DSC show an increase of glass transition temperature and a slight shift of melting peaks for the PLA/cellulose composite spheres, compared to PLA. Introduction In the packaging industry, plastic materials are customizable, easy to process and cheap. The great majority of plastics materials are made up from nonrenewable petroleum-based fossil fuels. Due to limited nature of fossil fuels and the growing environmental concern on their use, there is a need for finding more environmentally friendly materials. Currently, the development of biodegradable polymer composites combining biodegradable matrices and natural fibers as reinforcement is of great importance [1]. Among the polymer matrices, PLA is one of the most used and at present PLA-based materials find applications in the biomedical, textile and packaging areas [2]. PLA is a commercially available polyester polymerized from lactic acid. On the other hand, the use of natural fibers as reinforcement materials presents some advantages as their low density, high specific strength and modulus, nonabrasive character and low cost [3]. Due to its hydrophilic nature, cellulose is not easily uniformly dispersed into hydrophobic PLA matrix which leads to poor interfacial adhesion, thus reducing the mechanical properties of the composites [4]. To overcome this limitation, many physical and chemical treatments have been applied for cellulose modification. These include plasma treatment [5], static soaking and dynamic ultrasonication with tap water [6], treatment with alkali or enzymes [7], acetylation, silylation or grafting with other hydrophobic monomers [8]. Besides cellulose chemical properties and its dispersion in the PLA matrix, the biocomposite properties are also affected by the processing conditions. Melt-compounding is traditionally used for many types of biocomposites as it is fast and easier to scale-up the process to industrial scale [9]. Solvent casting is a simple and widely used technique to prepare cellulose nanocomposites using different matrices, but it is mostly applied at laboratory scale [9]. The use of PLA microparticles is an alternative to PLA fibers resulting in nanocomposites with good cellulose nano fiber dispersions [10]. The production of PLA microparticles was carried out by solvent evaporation technique which is widely used in pharmaceutical industry for drug microencapsulation [10,11]. In a typical procedure, a volatile organic phase containing dissolved polymers is emulsified in an aqueous phase at continuous constant stirring rate. Then, the volatile solvent is gradually evaporated to form the spherical polymer particles. This technique was also used by Lee and Ji (2014) [12] to encapsulate hydrophilic entities such as cellulose nanowhiskers and microfibrils within polymeric nano/microspheres. However, until now, the solvent evaporation technique was never used to produce PLA spheres incorporating pulp fibers, which have a much higher size. The aim of this work is to produce composite spheres of PLA and different types of cellulose, namely pulp fibers, using emulsion-solvent evaporation. The effectiveness of cellulose incorporation into PLA spheres was analysed taking into account the particle size and chemical composition of cellulose. Spheres of the PLA/cellulose composites were characterized concerning to their chemical and thermal properties. Material and Methods Materials PLA used in this study was Ingeo 2500HP from NatureWorks. Microcrystalline cellulose was Avicell purchased from Merck. Cellulosic fibers from Eucalyptus globulus unbleached kraft pulp and kraft lignin were supplied by Celtejo Empresa de Celulose do Tejo, S.A (Portugal). All other reagents were analytical grade. Cellulose modification Cellulose modification was carried out by grinding pulp fibers, lignin deposition by precipitation and grafting of linoleic acid under cold plasma treatment, according to procedures described by Sousa et al. [13]. Seven cellulose samples were used, referred as: UP unbleached pulp fibers; GUP grinded unbleached pulp fibers; GUP-L25 33

44 and GUP-L61 grinded unbleached pulp fibers with 25% and 61% lignin achieved by lignin precipitation; GUP-LA grinded unbleached pulp fibers grafted with linoleic acid; MCC microcrystalline cellulose; MCC-L61 microcrystalline cellulose with 61% lignin achieved by lignin precipitation. Preparation of PLA/cellulose composite spheres For the preparation of PLA/cellulose composite spheres, a typical laboratorial procedure was: a suitable amount of PVA was dissolved in 110 ml deionized water containing 0.1 g CTAB and stirred. Separately, 0.5 g cellulose fibers was suspended in 50 ml of deionized water, and added to the PVA solution. After homogenization of PVA and cellulose fibers, the solution of 2 g PLA in 40 ml dichloromethane (previously prepared) was poured into the aqueous suspension producing an emulsion. The system was mechanical stirred at constant rate (300 rpm) for 24 h at room temperature. The spheres were recovered from the liquid phase by sedimentation, thoroughly washed with deionized water and dried at 40 ºC for 2 days. Cellulose and spheres characterization The amount of cellulose and lignin incorporated in the composite spheres was estimated by mass balance, after measuring their content in the liquid phase after separation of the spheres. For cellulose determination, the liquid phase was dried at 40 ºC and the solid residue was subjected to acid hydrolysis with 72% H 2SO 4 for polysaccharides depolymerisation and neutral sugar determination according to NREL procedure NREL/TP The percentage of incorporated cellulose into the PLA spheres was calculated by the difference between the initial amount of cellulose charged and the remained amount in the liquid phase. A similar procedure was used to quantify the amount of incorporated lignin into PLA spheres; the lignin in the liquid phase were determined according to TAPPI T236 cm-85. Cellulose samples and PLA/cellulose composite spheres were characterized by ATR/FTIR, water contact angle measurements and DSC. Results and discussion Cellulose and lignin incorporation PLA/cellulose composite spheres were successfully obtained by solvent evaporation technique as shown in Fig. 1. The percentage of cellulose and lignin incorporation into PLA spheres is depicted in Fig. 2. The cellulose incorporation was 31% when unbleached pulp fibers were use. The grinding of the pulp fibers only increased the incorporation to 36%, which allows to conclude that the particle dimension is not the main parameter determining the cellulose incorporation. As seen in Fig. 2, the use of MCC, i.e. cellulose particles with much lower size than GUP, results in lower cellulose incorporation. This result might be attributed to the different chemical composition of the samples, since the GUP has some lignin content (4%) whereas MCC is only constituted by cellulose. For the GUP fibers it was observed an increase in cellulose incorporation into the PLA spheres from 36% to 79% with the increase of lignin content from 4% to 61%. Moreover, the lignin retention exbibits a similar trend. For the MCC, the behaviour of cellulose incorporation is the opposite; there is a decrease when the cellulose is modified with lignin (Fig. 2). This result may be due to lignin desorption from MCC particles, conjugated with its preferential incorporation into PLA. In fact, the amount of lignin incorporated is high (63%). Concerning to GUP fibers grafted with linoleic acid, the cellulose incorporation was lower than with the ungrafted pulp. Although the linoleic acid has a long aliphatic chain (hydrophobic), the grafted chains may be uneven distributed over the fibers surface. Zanini et al. [14] found that the plasma-induced graft polymerization of polyethylene glycol acrylate on polypropylene substrates presents macro- and micro-scale heterogeneity. The uneven distribution of linoleic acid contrary to uniform coverage of the fibers achieved by lignin precipitation may explain the different percentage of cellulose incorporation for these modified pulp fibers. Fig. 1. Optical microscopy images of some PLA/composite spheres. Fig. 2. Cellulose and lignin incorporation into PLA/cellulose spheres. Chemical and thermal characterization of cellulose and PLA/cellulose spheres Surface chemistry induced by the chemical modifications of the cellulose samples as well as of the PLA/cellulose composite spheres were investigated by ATR/FTIR and the recorded spectra are displayed in Fig. 3. When compared with MCC, ATR/FTIR spectrum of unbleached 34

45 pulp fibers showed an additional band at 1590 cm -1 characteristic of lignin which is associated to aromatic ring vibration [15,16]. Both spectra exhibit the typical bands of cellulose between 896 cm -1 and 2899 cm - 1 [14,16]. The spectra of the cellulose samples modified with lignin (GUP-L61 and MCC-L61) shows new peaks at 1605 cm -1, 1515 cm -1, 1455 cm -1 and 1212 cm -1 assigned to lignin components [16,17]. The spectra of the grinded pulp fibers before and after plasma-treatment with linoleic acid were very similar (data not shown) due to the fact that the depth of analysis in ATR/FTIR is too big to account to the small percentage of linoleic acid at surface. PLA spectrum showed distinctive absorption bands assigned to CH 3 stretching at 2997 and 2946 cm -1, C=O stretching at 1748 cm -1, CH 3 bending at 1455 and 1359 cm -1, C-O-C asymmetric stretching at 1180 cm -1 and C- CH 3 stretching at 1083 cm -1 [18]. Concerning to composite spheres spectra, it is possible to observe the presence of bands associated to lignin, cellulose and PLA, except in the case of PLA/MCC whose spectrum is very similar to that of PLA spectrum. This last result is due to the very low incorporation of MCC into the spheres, mainly composed of PLA. Fig. 3. ATR/FTIR spectra of cellulose samples, PLA and composite spheres. The chemical differences of the cellulose samples are reflected in their hydrophilicity, as shown in Fig. 4 for the water contact angle. Fig. 4. Water contact angle of cellulose samples, PLA and composite spheres. MCC presents the lower water contact angle (41º), i.e. it is the more hydrophilic sample because it is essentiality cellulose. In turn, unbleached pulp presents a higher contact angle (48º) due to the presence of residual lignin. The lignin precipitation on the cellulose fibers and MCC increases very significantly the water contact angle. The grafting of linoleic acid over the hydroxyl groups of cellulose fibers under plasma treatment decreases the hydrophilicity very significantly; water contact angle increases from 48º to 79º, indicating the presence of the aliphatic chain of the linoleic acid on fiber surface. Although none chemically modified sample has reached hydrophobic character, they have a contact angle value closer to that of PLA which is 83º. Thus, it would be expected that chemically modified cellulose samples were more compatible with PLA than those unmodified, resulting in higher percentage of cellulose incorporation. This behaviour was in fact confirmed for GUP-L25 and GUP-L61, but not for the MCC-L61 and the GUP-LA. Regarding the contact angles for the PLA/cellulose spheres, the values are always inferior to that of PLA, ranging from 59º to 75º for PLA/MCC and PLA/MCC- L61 samples, respectively. Overall, the cellulose samples with higher contact angle leads to the corresponding PLA/cellulose spheres with slightly higher contact angle. Thermal properties of cellulose samples, PLA and PLA/cellulose spheres were investigated using DSC. Table 1 presents the results for glass transition temperature (T g), melting temperature (T m), endothermic enthalpy of melting ( H m) and degradation temperature (T peak) of the different samples. Table 1. DSC parameters for cellulose samples, PLA and composite spheres. Amostra Tg (ºC) Tm (ºC) Hm (J/g) Tpeak (ºC) UP GUP GUP-L GUP-L ; GUP-LA MCC MCC-L ; PLA PLA/UP PLA/GUP PLA/GUP-L PLA/GUP-L PLA/GUP-LA PLA/MCC PLA/MCC-L The cellulose samples showed thermal degradation at T peak between and ºC. Cellulose samples with higher lignin content (GUP-L61 and MCC-L61) presented a second stage of degradation, at higher temperatures, assigned to lignin [19]. The PLA exhibited a T g at 53.7 ºC and a melting temperature around 180 ºC. Compared to PLA, the increase of T g for composite spheres, excepting for PLA/GUP-LA sample, was observed (Table 1). The decrease in T g for the PLA/GUP-LA sample is probably due to the plasticizing effect of linoleic acid, reported in literature [20]. The slight shift of melting peaks was observed in all the composites spheres and this suggests a little effect of cellulose in decreasing the melting 35

46 temperature of the composites. Melting enthalpy was also slightly reduced for all the composites spheres, excepting for PLA/GUP-LA, being the samples with 61% of lignin (PLA/GUP-L61 and PLA/MCC-L61) those that present the lower enthalpy values. Conclusions For the first time the solvent evaporation technique was used to produce PLA/cellulose fibers composite spheres, which can be easily processed by compression, injection or extrusion. The highest incorporation of cellulose and lignin (79% and 71%) was obtained for the cellulose pulp fibers with 61% lignin achieved by lignin precipitation (PLA/GUP- L61 sample). The opposite was observed for the PLA/MCC sample (20% of cellulose incorporated). The results showed the importance of cellulose surface chemistry on the percentage of cellulose incorporated, as the water contact angles measurements showed that MCC is the most hydrophilic sample, while the precipitation of lignin over cellulose decreases their hydrophilicity. In fact, the effectiveness of cellulose incorporation was improved more than 50% compared to GUP when grinded pulp fibers were chemically modified with 61% lignin (GUP-L61). Although the pulp fibers esterified with linoleic acid exhibit a water contact angle similar to that PLA (79º and 83º, respectively), their incorporation into PLA spheres was poor (29%), probably due to the uneven distribution of grafted chains of linoleic acid over the fibers. It was shown that the reduction of the fiber size by grinding only increased cellulose incorporation from 31% to 36% confirming that the particle dimension is not the main parameter affecting cellulose incorporation. The slight shift of glass transition temperature and melting temperature observed for the composites show a little effect of the cellulose incorporation on thermal properties. Acknowledgements This work was carried out within the scope of the project Thermocel Desenvolvimentos de compósitos de base celulose POCI FEDER co-financed by FEDER funds through COMPETE References [1] B.C. Mitra, Environment friendly composite materials: Biocomposites and green composites, Def. Sci. J. 64 (2014) [2] M., Murariu, P. Dubois, PLA composites: From production to properties, Adv. Drug. Deliv. Rev. 107 (2016) [3] N.C. Loureiro, J.L. Esteves, J.C. Viana, S. Ghosh, Development of polyhydroxyalkanoates/poly(lactic acid) composites reinforced with cellulosic fibers, Compos. Part B Eng. 60 (2014) [4] T. Wang, L.T. Drzal, Cellulose-Nano fiber-reinforced Poly(lactic acid) Composites Prepared by a Water-Based Approach, ACS Appl. Mater. Interfaces 4 (2012) [5] N. Graupner, K. Albrecht, D. Hegemann, J. Müssig, Plasma modification of man-made cellulose fibers (Lyocell) for improved fiber/matrix adhesion in poly(lactic acid) composites, J. Appl. Polym. Sci. 128 (2013) [6] D. Cho, J.M. Seo, H.S. Lee, C.W. Cho, S.O. Han, W.H. Park, Property improvement of natural fiberreinforced green composites by water treatment, Adv. Compos. Mater. 16 (2007) [7] G. Raj, E. Balnois, C. Baley, Y. Grohens, Role of polysaccharides on mechanical and adhesion properties of flax fibres in flax/pla biocomposite, Int. J. Polym. Sci (2011) 11 pages. [8] H.A. Khalil, A. Bath, A. Yusra, Green composites from sustainable cellulose nanofibrils: A review, Carbohydr. Polym. 87 (2012) [9] K. Oksman, Y. Aitomäki, A.P. Mathew, G. Siqueira, Q. Zhou, S. Butylina, et al., Review of the recent developments in cellulose nanocomposite processing, Compos. Part A Appl. Sci. Manuf. 83 (2016) [10] T. Wang, L.T.Drzal, Cellulose-Nano fiber- Reinforced Poly(lactic acid) Composites Prepared by a Water-Based Approach, ACS Appl. Mater. Interfaces 4 (2012) [11] S. Freitas, H.P. Merkle, B. Gander, Microencapsulation by solvent extraction/evaporation: Reviewing the state of the art of microsphere preparation process technology, J. Control Release 102 (2005) [12] I. Lee, S. Ji, Device and method for encapsulation of hydrophilic materials, US patent 2014/ A1 (2014). [13] S. Sousa, A.P. Costa, A.P. Silva, R. Simões, Preparation of PLA/cellulose pulp fibers composite spheres by emulsion-solvent evaporation method, submitted to Compos. Part A Appl. Sci. Manuf. [14] S. Zanini, M. Orlandi, C. Colombo, E. Grimoldi, C. Riccardi, Plasma-induced graft-polymerization of polyethylene glycol acrylate on polypropylene substrates, Eur. Phys. J. D 54 (2009) [15] A.N. Frone, S. Berlioz, J.-F. Chailan, D.M. Panaitescu, D. Donescu, Cellulose fiber-reinforced polylactic acid, Polym. Compos. (2011) [16] L.N. Ludueña, A. Vecchio, P.M. Stefani, V.A. Alvarez, Extraction of cellulose nanowhiskers from natural fibers and agricultural byproducts, Fibers Polym. 14(2013) [17] K.K. Pandey, A study of chemical structure of soft and harwood and wood polymers by FTIR spectrscopy, J. Appl. Polym. Sci. 71 (1999) [18] G. Kister, G. Cassanas, M. Vert, Effects of morphology, conformation and configuration on the IR and Raman spectra of various poly(lactic acid)s, Polymer 39 (1998) [19] P. Ghetti, L. Ricca, L.Angelini, Thermal analysis of biomass and corresponding pyrolysis products, Fuel 75 (1996) [20] M.G.A.Vieira, M.A. da Silva, L.O. dos Santos, M.M. Beppu, Natural-based plasticizers and biopolymer films: A review, Eur. Polym. J. 47 (3) (2011)

47 Influence of Bi4Ti3O12 as a secondary phase in the photocatalytic activity of perovskite materials M.J. Nunes 1, A. Lopes 1, M.J. Pacheco 1, P.T. Fiadeiro 2, L. Ciríaco 1 1 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 2 FibEnTech-UBI, Department of Physics, Universidade da Beira Interior, Covilhã, Portugal Abstract The aim of this work was to determine the influence of a secondary phase (Bi 4Ti 3O 12) in the photocatalytic activity of perovskite materials (SrTiO 3 and Sr 0.95Bi 0.05TiO 3) in the degradation of Acid Orange 7, under visible light. Oxides were synthesized by solid-state reaction method and characterized structurally by X-ray diffraction. The single oxide and phase mixtures films were prepared by slurry-paint method over Ni-foam. The films were characterized morphologically by scanning electron microscopy and applied as photocatalysts in the degradation of Acid Orange 7, under visible light. The results showed significant improvement for the Sr 0.95Bi 0.05TiO 3+Bi 4Ti 3O 12 mixture when compared with both oxides separately. This indicates that the presence of Bi 4Ti 3O 12, as a secondary phase, can improve the photocatalytic activity of some predominant phases, even when in small quantities. Introduction Perovskite are ceramic compounds with general formula ABX 3, where A and B are cations and X is an anion. The A site is commonly occupied by alkaline-earth metal elements or rare earth elements and the B site by transitionmetal elements. Perovskites can occur in many structures, from cubic to triclinic, but the cubic (Fig. 1), space group Pm3m, is the ideal structure for these compounds. Fig. 1. Ideal cubic perovskite structure, ABX3(adapted from [1]) This structure is known for its flexibility, as it can accommodate many cationic combinations without collapsing. This characteristic grants this compound family a large variety of properties and applications, namely as photocatalysts in water decontamination [2]. A photocatalytic reaction begins by the irradiation of a catalyst surface with radiation that possesses energy equal or superior to the distance between the material s valence band (VB) and conduction band (CB), the band gap energy (BG). If this requirement is met the electrons (e - ) in the VB will be excited to the CB, leaving positive vacancies (h + ) in the VB and forming electron-vacancy pairs with powerful reducing and oxidizing properties, respectively [2]. In the photocatalytic degradation process, Fig. 2, the electronvacancy pairs generate highly reactive species, O 2 - and OH, that will react with the organic compounds, forming intermediate degradation metabolites and, ideally, leading to a full mineralization. The BG width is directly associated with the reaction efficiency. Fig. 2. Representation of the photocatalytic degradation of organic compounds Recently, studies on the formation of heterostructures, between two different oxides, in an attempt to enhance the photocatalytic activity, have been reported [3,4]. In a previous work [5], when testing the photocatalytic activity of Sr 1-xBi xtio 3 oxides, on the Acid Orange 7 (AO7) degradation, under UV radiation, it was found that the compounds that exhibited the best results were those where a secondary phase belonging to the tetragonal Bi 4Ti 3O 12 was identified. The occurrence of this phase was not intentional, as it was a result of solubility limitation, but the results implied the possibility that the secondary phase could be partially responsible for the observed catalytic improvement. Considering these results, this work aimed to assess the influence of Bi 4Ti 3O 12 as a secondary phase in the photocatalytic activity of perovskite materials (SrTiO 3 and Sr 0.95Bi 0.05TiO 3) in the degradation of AO7, under visible light. 37

48 Materials and Methods SrTiO 3 (STO), Sr 0.95Bi 0.05TiO 3 (Bi 0.05) and Bi 4Ti 3O 12 (BTO) samples were synthesized via solid-state reaction method. Stoichiometric amounts of SrCO 3 (Merck, 99.9%), TiO 2 (Aldrich, 99.9%) and Bi 2O 3 (Aldrich, 99.9%) were mechanically grinded and heated at 750 ºC, for 24 h. The resulting mixture was regrinded and reheated for 24 h. The powders were structurally characterized by X-ray diffraction (XRD), on a Rigaku, model DMAX III/C with automatic data acquisition (APD Philips v3.5b), equipped with a monochromatized Cu k α radiation (λ= nm), operating at 40 ma and 30 kv. The recording conditions were 2θ between 10 and 90º at a scanning rate of 1.2º/min. The photocatalytic films were prepared via slurry-paint method, by coating a nickel foam support (Goodfellow, 95% porosity, 1.6 mm thickness and 20 pores cm -1 ) with a dispersion of the oxide powders or mixtures in a surfactant, Triton X-100 (Aldrich), followed by its degradation at 400 ºC. The films were morphologically characterized by scanning electron microscopy (SEM), using a Hitachi model S-3400N, operating at 20 kev. All heating procedures were performed in a tubular furnace (Carbolite, model STF, with a Eurotherm 808 temperature controller). The films photocatalytic activity was assessed by the photocatalytic degradation of AO7 (Sigma, C 0=25 mg L -1 ) under visible light radiation, applied by a mercury lamp (250 W, Osram HOL-R). The dye degradation was monitored every hour, for a total assay time of 7 h, by UV- Visible absorption spectrophotometry (Shimadzu UV- 1800). Results and Discussion The XRD patterns for all synthesized powders and oxide mixtures are presented in Fig. 3. The diffractograms for STO and Bi 0.05 show that both oxides exhibit a wellcrystallized phase belonging to the perovskite cubic symmetry, Pm3m space group, (ICDD file PDF# ). For BTO a predominant well-crystallized phase was identified as belonging to the tetragonal symmetry (ICDD file PDF# ). In the case of STO+BTO and Bi 0.05+BTO mixtures the BTO phase was identified by the presence of its most intense peak at 30.0 º. Fig. 4 shows the SEM micrographies of the Ni foam support before and after the immobilization of the oxide powders in. It is possible to observe the pore structure of the foam, on the left, with an average pore size of 200 μm, and the effective result of the applied technique, on the right. The prepared films were tested on the photocatalytic degradation of AO7, under visible light. In the AO7 UV- Vis absorption spectrum is possible to identify two typical bands, at 310 nm and 484 nm, resulting from the conjugated double bonds system of the aromatic rings and the azo bond, respectively. Both bands were monitored and the results obtained after the 7-h assay are presented in Fig. 5 For the STO film a <5% dye degradation was achieved; these results are corroborated with the material broad band gap and known low photocatalytic activity under visible light radiation [2]. When comparing these results with the ones obtained by Bi 0.05 film, an improvement in the degradation is observed for the latter. This shows that doping the STO structure with Bi enhances the material s photocatalytic activity under visible light. Regarding the phase mixture study, no improvement to the STO oxide was observed with the addition of BTO. However, for the Bi 0.05+BTO film, and increase in 10% was observed for the degradation of AO7, after the 7-h assay. These results demonstrate that a two-phase mixture can improve the photocatalytic activity of both materials under specific conditions. This can be associated with the formation of an heterostructure, known to improve the photocatalytic activity by displaying the VB and CB of both phases, in a way that the photo-induced e - and h + pairs are kept separated for a longer period, improving the photocatalytic process [4]. Fig. 3. XRD patterns for the following oxides: STO, Bi0.05 and BTO; and mixtures: STO+BTO and Bi0.05+BTO. Fig. 4. SEM micrographies of the Ni foam (left) and after oxide immobilization (right) 38

49 Acknowledgements The authors acknowledge the financial support from Fundação para a Ciência e a Tecnologia, FCT, for the project Pest-OE/CTM/UI0195/2004 of the FibEnTech- UBI RU and Santander Totta/ICIUBI, for the PhD grant awarded to M. J. Nunes, BID/ICI-FC-BST-UBI2015- Química. Fig. 5. Degradation % of the AO7 solutions submitted to the photocatalytic films under visible light, after 7 h. Conclusions STO, Bi0.05 and BTO oxide powders were successfully synthesized, displaying predominant well-crystallized phases belonging to the cubic symmetry, for the first two, and to the tetragonal symmetry for the latter. The phase mixtures were confirmed by XRD and the powders immobilization in the Ni-foam by SEM. Bi-doped STO exhibited improved photocatalytic activity in the AO7 degradation when compared with the undoped oxide and when mixed with BTO, as a secondary phase, showed a 10% increase in the degradation of the dye. This can be associated with a heterostructure formation between both phases in a way that the photo-induced species are separated for longer times, improving the photocatalytic degradation of AO7 under visible light. References [1] J. B. Goodenough, Electronic and ionic transport properties and other physical aspects of perovskites, Rep. Prog. Phys. 67 (2004) [2] W. Wang, M. O. Tadé, Z. Shao, Research progress of perovskite materials in photocatalysis- and photovoltaicsrelated energy conversion and environmental treatment, Chem. Soc. Rev. 44, (2015) [3] H. Che, J. Chen, K. Huang, W. Hu, X. Liu, G. Che, C. Liu, W. Shi, Construction of SrTiO 3/Bi 2O 3 heterojunction towards to improved separation efficiency of charge carriers and photocatalytic activity under visible light, J. Alloy. Compd. 688 (2016) [4] J. Zhang, L. Huang, P. Liu, Y. Wang, X. Jiang, E. Zhang, H. Wang, Z. Kong, J. Xi, Z. Ji, Heterostructure of epitaxial (001) Bi 4Ti 3O 12 growth on (001) TiO 2 for enhancing photocatalytic activity, J. Alloy. Compd. 654 (2016) [5] M. J. Nunes, A. Lopes, M. J. Pacheco, L. Ciríaco, Preparation, characterization and environmental applications of Sr 1-x(La,Bi) xtio 3 perovskites immobilized on Ni-foam: photodegradation of the Acid Orange 7, Environ. Sci. Pollut. Res. 24 (2017)

50 U.MAKE.ID A digital sourcing platform project: A theoretical approach Reis, Benilde 1 ; Pina, Lilina 2 ; Rafael, Paulo 3 ; Pereira, Madalena 4 ; Miguel, Rui 5 1 Textiles Dept., U.MAKE.ID Researcher, FibEnTech-UBI, Universidade da Beira Interior, Covilhã, Portugal 2 Textiles Dept., U.MAKE.ID Researcher, Universidade da Beira Interior, Covilhã, Portugal 3 Consultant in the project U.MAKE.ID, Lisbon, Portugal 4 Textiles Dept., FibEnTech-UBI; UNIDCOM, Universidade da Beira Interior, Covilhã, Portugal 5 Textiles Dept., Unit Research. FibEnTech-UBI; Universidade da Beira Interior, Covilhã, Portugal Abstract The purpose of this paper is make an approach to the business-to-business in fashion industries, for later introducing the U.MAKE.ID project, which is a platform created for the players in the textile, clothing, jewellery and footwear industries. Is important to understand the digital technologies and agility processes in fashion business, for that was made a literature review. Considering the problems of the fashion industries, is proposed a digital platform that the players in fashion industries can use for find each other, considering the characteristics and specifications of each product, supporting the communication between players and facilitate the sourcing in the fashion field. According to Pina et al. (2017), a B2B strategy allied a professional e-commerce platform, can bring to this business many positive results. Being aware of all the implications and benefits with the purpose to find the best solution, companies have for the first time the opportunity to reach 100% of its potential market, showing and valuing the creative and production process. [1] The limitations of this paper are based in the fact that there is no data from this research, however the U.MAKE.ID is already tested and on-line for the brands, factories and suppliers of fashion industries can start communicating and finding with each other. Key words: fashion industries, easy sourcing, sourcing platform, U.MAKE.ID. Introduction Discovery the precise partners to produce products of fashion industries, that can satisfy the requirements in terms of technical specifications of the products and have appropriate timings for the development and production, within a reasonable price, considering the expected quality, it s a multifaceted and difficult process. Is typically constructed upon knowledges, experiences and relationships assimilated through years of work. As well as from middlemen that not always add value to the entire process. This stands out more in the most fashion brands and designers in the textile, jewellery and footwear industries. For this purpose, it s important and justifiable discovery answers that can overcome the requests inside the manufacturing. Likewise, conjugating in an effective and simple way for offer considering the demand, concentrating on valuable resources while cutting out barriers and structuring processes without harming the ability of negotiation of every intervenient in the process, so that an increase of the acknowledgment while improving the internationalization of the offer of products and services can be conceivable. The U.MAKE.ID project, is an easy sourcing platform and the main goal of this project is get together, in a network platform, the fashion industries: brands, factories and suppliers of textile, clothing, footwear and jewellery. Literature Review Digital technology in fashion industries In 2007, Azevedo et al. refers that with the rapid growth of technologies, our economic society and life are changing significantly in the 21th century. The way to capture their competitive advantage has become the most important issue for enterprises in the rapidly changing and uncertain business environments. [2] According to Hollensen (2004), the internationalization is for a lot of companies a process of development and growing. A several of small and medium size companies try to expand its business to foreign markets so that they can access more profitable markets and so increase its competitiveness. [3] When a firm competes in any industry, it performs a several discrete but interconnected value-creating activities, such as operating a sales force, producing a component, or delivering products, and these activities have points of connection with the activities of suppliers, channels, and customers (Azevedo et. al. 2007). [4] The task of sourcing for fashion brands is something that implies a strong sense of organization and flexibility, in which sometimes the proximity and the communication with the suppliers is left aside. An agile supply chain management approach is designed to create the ability to respond quickly, costing effectively to changeable changes in markets and increasing levels of environmental turbulence, both in terms of volume and variety. (Carvalho H., Susana et Cruz Machado., 2012; Agarwal A., et al. 2007) [5, 6] Saviolo (2002), presents a model with three main focus, for the administration of an effective fashion brand. The first focus concerns that brands should be able to access the best suppliers regarding technology, services and quality. In the second place is essential for a brand to have 40

51 a creative team according to the design, advertisement and promotion. To finalise, the location is always very important in a global perspective. [7] However, the main goal of U.MAKE.ID project platform, is to generate a network of services that can reduction the barriers to access the industries, that can condense the distance between the conception and the production of the products, through the simplification of a series of processes that goes from the sourcing to the production, simplifying the communication and the organization between the different companies. The several of possibilities for a B2B platform can bring benefits to all the stakeholders involved (managers, clients and teams of representatives) is quite vast, it s enough for this that the policy is known by all, as the identification of each request can be made aware of a predetermined universe of the adherent and thus maintain the relationship between companies and representatives. Agility in fashion business According to Christopher et al. (2004), the fashion supply systems are considered by three critical lead-times: timeto-market, time-to serve and time-to-react. These three factors are important for the agility in fashion supply networks. However, agility does require essential changes in organisational structures, strategies and a move away from forecast-driven supply. Market sensitivity, virtual integration, networked logistical systems and process alignment all become important basics to accomplishing the final agility, a quick response capability. The agile supply chains are more likely to be information-based, by their nature, fashion markets are instable and hard to forecast, hereafter the need for agility. [8] For Bharadwaj et. al. (2013), the construction of digital business strategy includes the design of products, services and their communication skills with other complementary platforms, and their deployment as products and services by taking advantage of digital resources. [9] Brands, Factories and supplier s problems In the industries of the textile, clothing, footwear and jewellery (the brands, factories and suppliers) from time to time have difficulties in their sourcing for other companies complement their work, always considering the factor of neediness for agility in the supply chain. According to Christopher (2000), in recent years, there has been a growing interest in the design and implementation of agile supply chain strategies. [10] Several companies are still convinced that the complexity of their business can t be supported convincingly in a medium like the internet. In fact, a B2B e-commerce tend to simplify processes and expand the relationship between suppliers and customers. This is because e-commerce provides a layer of order picking and business rules can be incorporated into the B2B platform when it is a specific platform for B2B e-commerce. In this new era of digital business-to-business it s necessary to develop and optimize the fashion industries players communication. U.MAKE.ID platform is permitting the establishment of new partnerships with factories, brands and suppliers in European and global markets. For most fashion brands and designers, the price should agree with the quality standards of the brand. The problem of brands or designers is finding the precise partner (factory) to produce the clothes, satisfying the necessities in technical specifications of the products, regarding consequently the timings of the development and production of the garments. In the case of most the European factories and suppliers, of fashion products, have a micro or small dimension, usually family owned companies. They have some scarce economical resources to invest in communication and marketing strategies, so that they can reach highly possible customers. Therefore, they have a great quantity of difficulties in forming a varied group of clients that could minimize the risks, instead of being dependent on only a few clients. In the fashion clusters, considering that the players are micro and small companies, their resources to invest in creation, development and production software are very limited. Consequently, most information of the entire cycle of creation, development and production isn't centralized, updated and accessible to the different players which only increases the lack of productivity and mistakes in the production chain and in the end, there aren t agility in the process. The use of a B2B platform is an essential and decisive means to face the challenges and achieve the desired goal, specifically by promoting the image and entrepreneurship in the sector so that it can be positioned superiorly in the value chain and reinforce the row. U.MAKE.ID Platform All the above-mentioned reasons justify the implementation of a B2B solution that integrates, given the existing synergies throughout the fashion cluster. U.MAKE.ID platform is fundamentally an instrument for the improvement of processes presenting itself as a tool that can accelerate the production process, as well as for the internationalization of products and services. The main goal of this project is the easy sourcing that the fashion industries: brands, factories and suppliers of textile, clothing, footwear and jewellery, can use to communicate between them, but not only. U.MAKE.ID intends be a helper of access to the industry, making it simple and accessible, both industrial sourcing and the production process, permitting a brand to set up a customized production line according to its particularly requirements. One key word of this platform is: convenient. Meaning that brands or designers that search for a factory, as well as the manufacturer/factory or a service search for suppliers that are available for both production process. The possibility of the creation of projects is shared confidentially through the platform in the cloud, as well as the visibility of the profiles for suppliers, manufactures and brands can have as appealing tool of marketing, 41

52 promoting communication between the users of the platform. Other of the primary goals of any B2B e- Commerce project is also to sell more. But each company has its objectives that must be well designed and prioritized for the B2B strategy to work. For some customers, the initial focus of B2B is to win over new customers while for others it is to improve the relationship and sell more to the existing customer base. For each strategy, a set of action plans, metrics, and goals must be established. For this, it s considered that the B2B e- commerce platform, U.MAKE.ID, can help a company in its sales goals, namely as: purchase new clients - this is probably the main goal among companies that decide to adopt a B2B strategy, particularly in times of crisis taking new clients directly means opportunities for new incomes; reactivate old clients - most businesses that are already established have an inactive customers, who have already made acquisitions or who were regular customers, but who do not place orders or have a business relationship for a certain period, however reactivating contacts, setting up promotional actions focused exclusively on these clients can bring good and fast results; increasing the relationships with current clients - the greater the level of customer relationships, and the more the company brand is exposed to these customers, the greater the probability of a business relationship with this customer increasing and being constant, because the companies that can establish a constant and efficient communication, sell more; increase the recurrence of sale - as important as getting new customers, is to make customers have a shopping recurrence, sales done by base customers have a much greater return, since the cost of marketing and customer capture involved is considerable lesser. The platform will allow to clients, geographically distant, work in the same way for a client, without this barrier, the corporations can now look for costumers that previously weren t economically viable selling to regions that could never reach before. Other reason for companies use the U.MAKE.ID platform, is that small costumers can require small orders; also the all the products are available, meaning not only the products that sell the most are easier to sell, but the products that don t sell so much are also available, promoting and communicating them. Final Considerations Nowadays, new models of business-to-business platforms in fashion business will be necessary to develop and optimize the communications between players, considering also the internationalization, not only of the brands, but also the factories and suppliers, becoming important because the globalization progressions. With this approach is possible to verify U.MAKE.ID can improve resources for brands, factories and suppliers problems. From these pre-selected needs, they will be able to find the relevant suppliers for their specific needs, the platform will allow brands and designers to understand in a simple and spontaneous way, all the steps of the productive process, since conception to the final product, allowing them to shape, planning and developing their project in their own terms, selecting what they are looking for. With the on-line platform, the process of sourcing and communication is much faster and cheaper for the players in fashion business; the virtual experiences, digital descriptions and the sourcing, doing the work through the internet instead of doing everything in person. During this study databases, have been developed, for in a near future, a deeper research in the field of the fashion industries players, that are brands, manufactures and suppliers of each field in fashion (textile, clothing, footwear and jewelry). The U.MAKE.ID platform, version Beta, is already online and accessible to users, that in this case are the players in fashion industries, being the orientation to find the Best European Factory available in: Acknowledgements This investigation is supported by the project leaded by PICTÓNIO company and sponsored by Project U.MAKE.ID Financed by the Centre s Regional Operational Program, within the scope of Portugal 2020 I&DT PROJECTS IN COPROMOTION ENTERPRISES and also co-financed for European Union. References [1] L. Pina, B. Reis, P. Rafael, M. Pereira, R. Miguel, U.MAKE.ID A DIGITAL SOURCING PLATFORM PROJECT FOR THE FASHION BUSINESS: A THEORETICAL STUDY, (2017). [2, 4] S. G. Azevedo, J. Ferreira, J. Leitão, The role of logistics information and communication technologies in promoting competitive advantages of the firm, Munich Personal RePEc Archive. (2007) [3] S. Hollensen, Global Marketing, Harlow, Prentice Hall. (2004) [5] H. Carvalho, S. G. Azevedo, V. Cruz-Machado, Agile and resilient approaches to supply chain management: influence on performance and competitiveness, Logistics research, Vol.4 (1-2), (2012) [6] A. Agarwal, R. Shankar, M. Tiwari, Modeling agility of supply chain. Ind Mark Manage, Vol. 36 No. 4 pp (2007) [7] S. Saviolo, S. Testa, Strategic Management in the Fashion Companies, 1st ed., Etas, Milano. (2002) [8] M. Christopher, R. Lowson, H. Peck, Creating agile supply chains in the fashion industry, International Journal of Retail & Distribution Management, Vol. 32 Issue: 8, pp (2004) [9] A. Bharadwaj, O. A. E. Sawy, P. A. Pavlou, N. Venkatraman, Digital Business Strategy Includes Digitization of Products and Services and the Information Around Them - MIS Quarterly, Vol. 37 No. 2, pp (2013) [10] M. Christopher, The agile supply chain competing in volatile markets, Industrial Marketing Management, Vol.29, (2000) 42

53 Optimization of EDOT polymerization by using Design Expert P. Baptista 1, G. Soares 2, R. Simões 3 R. Miguel 1 1 FibEnTech-UBI, Textile Science and Technology Department, Universidade da Beira interior, Covilhã, Portugal 2 Textile Engineering Department, Universidade do Minho, Guimarães, Portugal 3 FibEnTech_UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal Abstract The objective of this work was to study the effect of the drying time at 60ºC, the polymerization temperature and the polymerization time on the electrical characteristics of the textile substrates coated with poly (3, 4 ethylenedioxythiophene) (PEDOT). Plain weave fabrics were coated by the vapor phase polymerization method, to provide textile fabrics of polyester with high electrical conductivity for smart textile applications. The method used oxidant solutions of Iron (III) p-toluenesulfonate hexahydrate (FTS) and was optimized by using Design Expert to maximize electrical conductivity. The samples exhibit a lower sheet resistivity with a minimum value of 25 Ohm/sq obtained for a drying time at 60ºC of 16 minutes, a polymerization temperature of 90ºC, and a polymerization time of minutes. Introduction Since the first discovery of polymeric conductors in 1977 by Shikarawa, Heeger and MacDiarmid [1, 2], the research area of "organic electronics" has grown dramatically. Electrically conductive or electro-active fibers are the key components of smart and interactive textiles, which could be used in medical, sports, energy, and military applications. The functionalization of high-performance textile with conjugated polymers can produce conductive fibers with better electro-mechanical properties, which is difficult with commonly used spinning techniques [3]. Conjugated polymers consist of a backbone structure along which delocalized electrons, resulting from p-orbitals overlap, provide charge mobility along the polymer backbone yielding electrical conductance. Metallic conductivity can be achieved through charge injection onto the conjugated macromolecular chains, or doping, which is achieved with salts and redox chemistry [1]. In the 1980 s, Bayer introduced the poly(3.4-ethylenedioxythiophene) (PEDOT), a conjugated polymer that exhibits high chemical and physical stability and high electrical conductivity [4]. The synthesis of PEDOT can be performed by various techniques, but concerning textile substrates, the main relevant ones are the wet polymerization, also referred to as liquid phase polymerization (LPP), and the vapor phase polymerization (VPP) [5]. In the VPP method, the fabrics are firstly immersed in an oxidant solution and secondly are exposed to the monomer in the vapor phase. The exposure of the substrates to an atmosphere of monomer is typically performed in a reactor with controlled vacuum and temperature [5, 6, 7]. Research works conducted with PEDOT coatings on fabrics [5] and yarns [7, 8], showed that the electrical and mechanical characteristics of the samples largely depend on the concentration of the oxidant solution, the type of fibers and the VPP parameters, such as immersion time, drying time and polymerization temperature. In this investigation, the VPP method was used to synthetize coatings of conductive PEDOT on substrates consisting of plain weave fabrics of polyester, using water as solvents for oxidant impregnation. Water solvent is considerably better choice over organic solvents, because is eco-friendly and considerably less expensive. After the previous results obtained by the research group [9, 10], using as oxidant Iron(III) p-toluenesulfate hexahydrate (FTS), the effect of the oxidant concentration, drying time, and reaction time and reaction temperature on the electrical characteristics of the samples was studied, by using a Response surface methods (RSM) with a Design-Expert software for design of experiments. Response surface methodology is a group of statistical and mathematical techniques useful for developing, improving, and optimizing processes [11]. Materials and Methods The textile substrates consisted of plain weave fabrics of polyester with a thickness of 0.22 mm and a specific weight of 1.2 g/cm 2. A large piece of fabric was machine washed at 100 ºC with a mild soap and dried. Small size substrates were then cut off from the large piece to rectangular geometrical shapes, having lateral dimensions of 80 mm 5 mm accurately obtained at a more or less one thread. The substrates were washed in methanol for 20 minutes, rinsed three times in deionized water and left to dry at 23 ºC in a room with conditioned ambient (50% of relative humidity and at 23ºC) for at least 24 hours. For the polymerization of EDOT, EDOT monomer (97%), Iron (III) p-toluenesulfonate hexahydrate, and methanol (99.5%) were purchased from Sigma-Aldrich. Schematics of the VPP method and polymerization reactor unit are shown in Fig. 1. The VPP method uses three steps; firstly, a step of immersion of the substrates in an oxidant solution of specific concentration; secondly, a drying step with conditions depending upon the solvent used; thirdly, the polymerization step, in which the samples are placed into a reactor and exposed to an atmosphere of the monomer, at certain vacuum pressure and temperature. 43

54 Fig. 1. Description of VPP method and schematic of reactor unit: OX SOL=oxidant solution, S=substrate, MVP¼membrane vacuum pump, VV=vacuum valve, SH=sample holder, WCB=water circulating bath, CW=circulating water, M=monomer The oxidant solutions were prepared with Iron (III) p- toluenesulfonate hexahydrate (FTS) dissolved in bidistilled water, with concentration of 0.4M. Samples soaked in oxidant solution were dried in a convection oven at 60 C for different drying times. The polymerization process used a droplet of 15 microliter of monomer, allowing two cycles of polymerization, We used various polymerization times and several polymerization temperatures. The samples were placed on a sample holder and moved into the reactor, which was then sealed and pumped down to a vacuum pressure of 160 mtorr. Next, a vacuum valve was closed and the polymerization cycle initiated. Once the time of the polymerization cycle was completed, the reactor was vented with air, and the samples removed and immediately washed in methanol and rinsed in water. The samples were next put to dry in an ambient conditioned room for at least 24 hours. The drying time and polymerization reaction conditions were carried out under different operating conditions, which were obtained through Design-Expert software. The operating conditions were drying time (10-22 min), reaction temperature ( ºC) and reaction time (20-60 min). A total of 20 runs with different combinations of the variables were used (Table 1) to optimize the polymerization of EDOT. Three independent variables were studied and the set points were selected according to the results obtained with preliminary tests. The results were analyzed using Design Expert software. Both linear and quadratic effects of the three variables under study were calculated, as well as their possible interactions, on the electrical conductivity. Their significance was evaluated by variance analysis (ANOVA). Three-dimensional surface plots were drawn to illustrate the effects of the independent variables on the dependent variable, been described by a quadratic polynomial equation and fitted to the experimental data. The fit of the models was evaluated by the determination of R 2 coefficient. For the best reactions conditions obtained with the Design Expert, the effect of oxidant concentrations was also studied, by testing 0.2 M and 0.8 M of FTS. Table 1. Operating conditions obtained through using the Design- Expert software. Run A:Drying B:Reaction Sheet C:Reaction time Temperature Resistivity time [min] [min] [ºC] [Ohm/sq] , , , , , , , , , , , , , , , , , , , ,85 The electrical characterization of the samples was performed with the two-probe method by measurements of sheet resistivity, R sq, given by the ratio of the electrical resistance by the geometric factor, GF=L/W, in Ohm/sq, (1) where V, is the voltage across the probes, I is the electrical current that flows in the sample, L is the distance between the probes, and W is the width of the sample. From the Ohm s law, the sheet resistivity is related to the ratio of the bulk resistivity, ρ, to the sample thickness, t, and has units of Ohm, The method is suitable for continuous film samples that are homogeneous and have relatively high electrical conductance, but could be applied to the textile samples coated with PEDOT as good degree of measurement repeatability was obtained with relative errors of less than 10%. To address the error involved, samples with a length of 120mm were prepared and the sheet resistivity of the samples measured with the four-probe method for variable inner probes distances in the range of mm. The mean value of sheet resistivity obtained matched the twoprobe typical mean value within an error of less than 10%. The electrical measurements were performed with a Hameg power supply, a Hameg 8012 programmable ampermeter and the data acquisition performed via a RS232 interface and a Labview program that displayed and recorded the sample s I V curves, from mean values of electrical current, I, for each applied voltage, V. For each VPP condition, 6 12 samples were measured. In order to graphically compare different (2) 44

55 samples, I* V curves taking into account the geometric factor of the samples, GF, were used, with I* given by, (3) Results and Discussion In Table 2 we can analyse the information corresponding to variance analysis (ANOVA), for the optimization of polymerization of EDOT Table 2. Analysis of variance. Source Sum of Squares Mean Square F Value p-value Prob>F Model 2,325E+07 2,583E+06 4,75 0,0115 A-Drying time B-Reaction Temperature C-Reaction time 62638, ,59 0,12 0,7412 1,054E+07 1,054E+07 19,39 0,0013 2,486E+05 2,486E+05 0,46 0,5140 AB 1,955E+05 1,955E+05 0,36 0,5620 AC 1,470E+05 1,470E+05 0,27 0,6143 BC 6,628E+05 6,628E+05 1,22 0,2952 A 2 1,623E+05 1,623E+05 0,30 0,5966 B 2 1,064E+07 1,064E+07 19,59 0,0013 C , ,28 0,076 0,7882 Residual 5,433E+06 5,433E+05 Lack of Fit 5,399E+06 1,080E ,21 < Pure Error 33700, ,02 Cor Total 2,868E+07 Through the analysis of variance, we can conclude that the reaction temperature and the reaction temperature squared represent close to 91% of the total variance. However a closer analysis reveals that the linear term leads to a decrease in resistivity whereas the quadratic term leads to a resistivity increase, which means that an optimum exist between 60ºC and 120ºC. In fact, when the reaction temperature increased from 60ºC to 90ºC, taking into account the variations that occur in the other variables, the resistivity decrease from around 3000 Ohm/sq to around to 200 Ohm/sq. When the reaction temperature was increased further to 120ºC a clear increase in resistivity was observed, having the resistivity increased approximately 4 times. This behavior can be observed in fig 2 and 5. Fig. 2 also reveals the effect of the other variables studied. The model takes into account the factors and the interactions between the factors. The Model F-value of 4.75 implies the model is significant, what shows that the range of factors were well select. The model also presents a correlation coefficient (R 2 ) of , showing that 81.06% of the points obtained fit the model. The p-value Prob> F of for the lack of fit, indicates that there is no lack of adjustment of the model, as described by other authors [12]. The final equation in terms of actual factors for the electrical conductivity response is presented in table 3. Fig. 2. Effect of the reaction temperature in the resistivity Table 3. The final equation in terms of actual factors. Resistivity = +9676, ,96439 * Drying time -206,45483 * Reaction Temperature -21,13105 * Reaction time -0,86841 * Drying time * Reaction Temperature -1,12952 * Drying time * Reaction time +0,47972 * Reaction Temperature * Reaction time -2,94811 * Drying time 2 +0,95490 * Reaction Temperature 2-0,13397 * Reaction time 2 Analysing the results of the ANOVA table (Table 2) and the level of significance of the coefficients of the final equation, we have verified that the most sensitive factor is the reaction temperature, in the linear form and in the quadratic effect as well.. Some interactions between factors also have some importance, but are not significant (p-value >0.05). The reaction temperature square is in fact the most important factor in the variation of the value of the electrical conductivity because it is the variable with the highest F-value. The drying time factor is presented as insignificant or without significant influence. The influence of the interaction between variables can be more easily observed in Response Surfaces (3D), where the blue color represents the minimum value and the red color the maximum value, of resistivity in the PEDOT polymerization. In Fig 3 we can analyse the effect of the interaction between drying time and reaction temperature, for a reaction time of 40 minutes. The graph illustrates that 45

56 despite the effect of drying time, an increase on the reaction temperature causes a very significant decrease in resistivity, having the resistivity varied between 180 and 3000 Ohm/sq. Fig 3. Resistivity as a function of drying time (A) and the reaction temperature (B), keeping fixed the reaction time (C) on 40 minutes. In Fig 4 we can observe the effect of the interaction between the variables, drying time and reaction time, for a reaction temperature of 90ºC. The graph shows that the interaction between these two variables is not significant. Which is in good agreement with the table of variance analysis. Fig 5. Resistivity as a function of the reaction time (C) and the reaction temperature (B), keeping fixed the drying time (A) on 16 minutes. The effect of the oxidant concentration on resistivity was observed when the best conditions obtained in the Design Expert were tested with two different oxidant concentrations. The resistivity results obtained for oxidant concentrations of 0.2, 0.4 and 0.8M were 4562, 192 and 25 Ohm/sq, respectively. Table 4 compares the preliminary and current results for sheet resistivity. We can see that a similar low resistivity was achieved with a single layer of PEDOT against the two previously needed layers [13]. Table 4. Comparison between the preliminary and de Design Expert results. Run with two layers[13] with one layer Fig 4. Resistivity as a function of the reaction time (C) and the drying time (A), keeping fixed the reaction temperature (B) at 90ºC. In Fig 5 we can observe the effect of the interaction between the reaction temperature and the reaction time, for a drying time of 16 minutes. The graph indicates that the interaction between these two variables influence the resistivity, which is in good agreement with the table of variance analysis. According to the analysis made to the values and graphs obtained for the modeling of the resistivity in the polymerization, it can be said that the factor that most influences the result is the reaction temperature. Process VPP VPP Oxidant conc. [M] Dipping time [min] Drying time [min] Drying time [min] Reaction time [min] Reaction Temperature [ºC] Oxidant FTS FTS Monomer EDOT EDOT Sheet Resistivity [Ohm/sq] Conclusions In this work it was obtained the optimization of PEDOT polymerization using the Design Expert. We can conclude that the model takes into account the interactions between the factors and that the model is significant, what shows that the range of factors were well select. The most sensitive factor is the reaction temperature. The optimized 46

57 conditions allowed obtaining woven samples with electrical conductance characterized by sheet resistivity of 25 Ω/sq for an FTS (oxidant) concentration of 0.8M with a drying time of 16 minutes at 60ºC and 73.64minute of reaction at 90ºC. These results are comparable to those reported in the literature by our research group for oxidant solutions of FeCl3 with concentrations of 0.8M with a drying time of 3 minutes at 60ºC, a 30-minute reaction time at 60ºC and two layers [9]. Acknowledgements This work was supported by a grant, PD/BD/52636/2014 from FCT-Science and Technology Foundation. References [1] A. J. Heeger, Nobel Lecture: Semiconducting and metallic polymers: The fourth generation of polymeric materials, Reviews of Modern Physics 73 (2001) [2] T. Bashir, M. Skrifvars, N. K. Persson, Surface modification of conductive PEDOT coated textile yarns with silicone resin, Materials Technology 26:3 (2011) [3] A. G. MacDiarmid, Nobel Lecture: Synthetic metals : A novel role for organic polymers, Reviews of Modern Physics 73 (2001) [4] F. Jonas, G. Heywang, W. Schmidtberg, J. Heinze, M. Dietrich, Polythiophenes, process for their preparation and their use, United States Patent Brevet , 22 Jan [5] D. Bhattacharyya, R. M. Howden, D. C. Borrelli, K. K. Gleason, Vapor Phase Oxidative Synthesis of Conjugated Polymers and Applications, Journal of Polymer Science Part B: Polymer Physics. 50 (2012) [6] X. Yang, S. Shang, L. Li, X.-M. Tao et F. Yan, Vapor phase polymerization of 3,4-ethylenedioxythiophene on flexible substrate and its application on heat generation, Polymers Advanced Technologies. 22 (2011) [7] T. Bashira, M. Skrifvars, N.-K. Persson, Production of highly conductive textile viscose yarns by chemical vapor deposition technique: a route to continuous process, Polymers Advanced Technologies 22 (2011) [8] T. Bashir, M. Skrifvars, N.-K. Persson, Synthesis of high performance, conductive PEDOT-coated polyester yarns by OCVD technique, Polymers Advanced Technologies. 23 (2012) [9] I. G. Trindade, J. Matos, J. Lucas, R. Miguel, M. Pereira, M.S. Silva, Synthesis of poly(3, 4- ethylenedioxythiophene) coating on textiles by the vapor phase polymerization method, Textile Research Journal 85:3 (2015) [10] I. G. Trindade, F. Martins, P. Baptista, High electrical conductance poly(3,4-ethylenedioxythiophene) coatings on textile for electrocardiogram monitoring, Synthetic Metals 210 (2015) [11] R. H. Myers, D. C. Montgomery, Response Surface Methodology, Process and Product Optimisation using Designed Experiments, second ed., Wiley, New York, [12] I. C. Gouveia, J. M. Fiadeiro, J. A. Queiroz, Enzymatic removal of plant residues from wool: Application of experimental design techniques for optimization parameters, Biochemical Engineering Journal 41 (2008) [13] P. Baptista, I. G. Trindade, G. Soares, R. Simões, R. Miguel, Electrically Conductive Polymers for Wearable Electronics, Atas do I Simpósio Materiais Fibrosos e Tecnologias Ambientais (2016), ISBN

58 Industrial solid waste landfill leachate treatment by electrochemical processes R. Silva, A. Fernandes, L. Ciríaco, M.J. Pacheco, A. Lopes FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal Abstract The objective of this work was to study the elimination of the organic load from a leachate collected in an industrial solid waste landfill by electrochemical processes, namely anodic oxidation (AO) followed by electrocoagulation (EC). During AO, leachate sample was subjected to different current densities ( A m -2 ) for 8 hours, using a boron doped diamond (BDD) plate as anode material. Organic load removal increases with the current intensity, as well as energy consumption. The highest organic load removal (98%) was attained for a current intensity of 1.4 A. Despite the good results obtained in the removal of the organic load, the final concentrations of total nitrogen, TN=466 mg L -1, and ammonium, [NH 4 + ]=189 mg L -1, didn t reach the discharge limit values (TN=15 mg L -1 ; [NH 4+ ]=10 mg L -1 ). To reduce the concentration of the N-containing compounds in the final effluent obtained with the AO treatment, EC assay was carried out, using aluminum as consumable anodes, and final TN=38 mg L -1 and [NH 4+ ]=35 mg L -1 were attained. Although EC proved to be efficient in the removal of nitrogen species, this method generates sludge and introduces aluminum ions in the treated effluent. Despite this, the proposed treatment proved to be effective for the treatment of industrial landfill leachate. Introduction The increasing industrialization and the growth of the global population results in high amounts of industrial solid waste that are landfilled, causing impacts on the public health and on the environment [1]. Because of the rainwater percolation through these wastes, extracting and bringing with it several pollutant materials, a very complex wastewater is produced, usually known as landfill leachate [1,2]. Several types of pollutants can be found in the landfill leachates, such as all types of organic and inorganic compounds, some of them refractory and toxic, and heavy metals. An inadequate leachate management involves serious risks, particularly the contamination of water resources, at the surface and groundwater, and soils, and consequently, it may induce genome damage in the population that consumes the contaminated water [3]. Recently, electrochemical processes have gained notoriety in the treatment of complex effluents due to their environmental compatibility, versatility and amenability of automation, as well as the high effectiveness shown in the elimination of persistent pollutants [4-6]. Anodic oxidation (AO) is the most popular electrochemical procedure for removing organic pollutants from wastewaters [7]. It is a process based on the effluent electrolysis and, in its simplest form, consists in the oxidation of pollutants in an electrolytic cell, which is formed by two electrodes connected by an external circuit, so that electrochemical reactions can take place [7,8]. Depending on the anode material, the hydroxyl radical s mediation can promote the partial oxidation and selective oxidation of the organic pollutant and its complete mineralization [9]. Boron-doped diamond has proven to be one of the best electrodes in electrochemical processes due to the fact that it presents mechanical and chemical stability, high efficiency in the formation of hydroxyl radicals, to allow the mineralization of pollutants, and long life [10]. Electrocoagulation (EC) is a process that uses consumable electrodes to supply ions to the solution/suspension, allowing suspended, emulsified, or dissolved contaminants to form agglomerates [11]. In this process, contaminants present in the solution are treated either by chemical reactions and precipitation or by physical and chemical attachment to colloidal materials generated by the electrode corrosion [11,12]. In this study, the treatment of a leachate from an industrial landfill was performed, using combined electrochemical processes (AO+EC), using a BDD anode during AO and aluminium consumable anodes during EC. Experimental The leachate sample utilized in this study was collected in a Portuguese industrial landfill and was kept refrigerated until use. The results for the characterization of the sample are presented in Table 1. Table 1. Physicochemical characteristics of the industrial landfill leachate. Property Mean value ± SD a COD / mg L ± 47 TDC / mg L ± 52 DIC / mg L ± 12 DOC / mg L ± 44 TDN / mg L ± 72 Chloride ion / mg L ± 34 Ammonium ion / mg L ± 85 Sulfate ion / mg L ± 23 ph 8.3 ± 0.1 Conductivity / ms cm ± 0.2 a SD Standard deviation AO and EC experiments were conducted in batch mode and the experimental conditions utilized are presented in Table 2. BDD anode was purchased from Adamant Technologies. Gap between anode and cathode was

59 cm. All the electrochemical assays were performed at least in duplicate and the values presented for the parameters used to follow the assays are the mean values. Table 2. AO and EC experimental conditions. V sample / ml v stirring / rpm t / h AO EC 200 b 0 4 b Leachate treated by AO A cathode / cm 2 20 (Stainless steel) 40 (Al) A anode / cm 2 j / A m (BDD) (Al) The effluent collected in the management site and those obtained after electrochemical assays were analyzed. according to standard procedures [13]. for the following parameters: chemical oxygen demand (COD). total dissolved carbon (TDC). dissolved organic carbon (DOC). dissolved inorganic carbon (DIC). total dissolved nitrogen (TDN). COD determinations were made using the closed reflux titrimetric method. TDC. DOC. DIC and TDN were measured in a Shimadzu TOC-VCPH analyzer combined with a TNM-1 unit. Before TDC. DOC. DIC and TDN determinations. samples were filtered through 0.45 μm glass microfiber filters. Measurements of ph and conductivity were carried out with a Mettler-Toledo ph-meter and a Mettler-Toledo conductimeter. respectively. Ammonium. chloride. chlorate. perchlorate. nitrate and sulfate ions concentrations were determined by ion chromatography with a Shimadzu 20A Prominence HPLC system that was equipped with a Shimadzu CDD 10Avp conductivity detector. For the determination of the NH 4+. an IC YK-A Shodex (4.6 mm ID 100 mm) column was utilized. The mobile phase consisted of a 5.0 mm tartaric acid. 1.0 mm dipicolinic acid and 24 mm boric acid aqueous solution at a flow rate of 1.0 ml min 1. The column temperature was 40 ºC. For the determination of anions. an IC I-524A Shodex (4.6 mm ID 100 mm) anion column was employed. The mobile phase consisted of an aqueous solution of 2.5 mm of phthalic acid and 2.3 mm of tris(hydroxymethyl) aminomethane at a flow rate of 1.5 ml min 1.The column temperature was 40 ºC. All solutions for the chromatographic analysis were prepared with ultrapure water obtained with Milli-Q equipment. All eluents were HPLC grade and supplied by Sigma- Aldrich. Results and discussion The results obtained for the different evaluation parameters after the 8-h AO assays are presented in Table 3. COD decay with time for the different applied experimental conditions is presented in Figure 1. Table 3. Final values attained for the different evaluation parameters after the 8-h anodic oxidation assays. j / A m -2 COD / mg L -1 DOC / mg L -1 TDN / mg L -1 [NH4 + ]/ mg L -1 [NO3 - ]/ mg L -1 [Cl - ]/ mg L -1 [ClO3 - ]/ mg L -1 [ClO4 - ]/ mg L -1 ph Cond./ ms cm Figure 1. COD decay along time and equations adjusted to the experimental data The removal of the different organic and inorganic load evaluation parameters increases with the applied current density. as well as the final ph value of the treated effluent. Figure 1 also includes the adjusted equations that can be utilized to extrapolate the time needed to attain the wanted COD final value. for instance. the value of 150 mg L -1. which will allow the treated effluent to be dumped in water courses. In the tested cases. the times obtained were and 3.5 h. for the applied current densities of and 700 A m -2. respectively. The energetic consumptions obtained for these different situations. which increase with the applied current density. were and 169 kwh m -3 of treated effluent. Regarding color removal (Figure 2). it was faster for the tests conducted at higher current density. although after 8- h assay none of the treated samples presented color. Despite the good results obtained with AO at a BDD anode for the organic load removal. the final values for total and ammonia does not reach the discharge limit values. Also. nitrate concentration higher than the allowed discharge value. due to the oxidation of the ammonia. is observed in the treated effluent. 49

60 Figure 2. Color of the different samples collected during AO assays. performed at 300 (a) and 700 (b) A m -2. at times and 8 h. Thus. in order to further reduce the concentration of the different parameters in the treated effluent. EC tests were carried out with the samples treated by AO. The results obtained in the EC assays are presented in Table 4. The combined AO+EC treatment allowed to reach the proposed objectives: nitrate was completely removed and the concentrations of organic load and total and ammonium nitrogen were also reduced. On the other and. ph increased drastically. However. it should be noted that the application of an EC treatment with aluminum consumable electrodes. in addition to generate sludge. introduces aluminum into the final effluent. Table 4. Final values attained by the different evaluation parameters after the combined AO(8h)+EC(4h) treatment. j / COD / DOC / TDN / [NH4 + ]/ [NO3 - ]/ [Cl - ]/ [ClO3 - ]/ [ClO4 - ]/ Cond./ ph A m -2 mg L -1 mg L -1 mg L -1 mg L -1 mg L -1 mg L -1 mg L -1 mg L -1 ms cm Conclusions Electrochemical processes can be applied in the treatment of leachates from industrial waste landfill. For the experimental conditions tested. it was observed that BDD anode is very efficient in the removal of the organic load. However. this anode material enhances conversion of ammonium nitrogen to nitrates. A possible solution to remove nitrates may be the application of electrocoagulation with aluminum consumable electrodes. This technique proved to be effective not only in the removal of nitrates. but also in the removal of other forms of nitrogen and in the removal of the organic load. as well as chlorate and perchlorate. However. it has as main disadvantages the production of sludge. the introduction of aluminum in the treated effluent and the increase in ph. Depending on the operating conditions. it is possible to reduce the quantities of the different parameters to the discharge limit values. either for municipal waste water treatment plants or for water courses. The greater the desired removal. the greater the energy consumption. Application of lower current densities generally leads to lower energy consumption. However. treatment times increase considerably. Acknowledgements The authors gratefully acknowledge the financial support received from: Fundação para a Ciência e a Tecnologia. FCT. for the funding of the FibEnTech-UBI Research Unit. project UID/Multi/00195/2013. and for the grant awarded to A. Fernandes. SFRH/BPD/103615/2014; FC/Santander Totta Universidades UBI/2016. for the grant awarded to R. Silva. BID/ICI-UID. References [1] D. Bhagawan. S. Poodari. N. Chaitanya. S. Ravi. Y.M. Rani. V. Himabindu. S. Vidyavathi. Industrial solid waste landfill leachate treatment using electrocoagulation and biological methods. Desalination and Water Treatment 68 (2017) [2] A. Fernandes. M.J. Pacheco. L. Ciríaco. A. Lopes. Review on the electrochemical processes for the treatment of sanitary landfill leachates: Present and future. Appl. Catal. B Environ. 176 (2015) [3] A. Anglada. A. Urtiaga. I. Ortiz. Contributions of electrochemical oxidation to wastewater treatment: fundamentals and review of applications. J. Chem. Technol. Biotechnol. 84 (2009) [4] A. Fernandes. D. Santos. M.J. Pacheco. L. Ciríaco. A. Lopes. Electrochemical oxidation of humic acid and sanitary landfill leachate: Influence of anode material. chloride concentration and current density. Sci. Total Environ. 541 (2016) [5] A. Fernandes. M.J. Pacheco. L. Ciríaco. A. Lopes. Anodic oxidation of a biologically treated leachate on a boron-doped diamond anode. J. Hazard. Mater (2012) [6] A. Fernandes. D. Santos. M.J. Pacheco. L. Ciríaco. A. Lopes. Nitrogen and organic load removal from sanitary landfill leachates by anodic oxidation at Ti/Pt/PbO 2. Ti/Pt/SnO 2-Sb 2O 4 and Si/BDD. Appl. Catal. B Environ. 148 (2014) [7] L. Ciríaco. C. Anjo. J. Correia. M.J. Pacheco. A. Lopes. Electrochemical degradation of Ibuprofen on Ti/Pt/PbO 2 and Si/BDD electrodes. Electrochim. Acta 54 (2009)

61 [8] P. Canizares. P. Rubén. C. Sáez. M.A. Rodrigo. Costs of the electrochemical oxidation of wastewaters: a comparison with ozonation and Fenton oxidation processes. J. Environ. Manage. 90 (2009) [9] Bo Zhou. Zhiming Yua. Qiuping Wei. HangYu Long. Youneng Xie. Yijia Wang. Electrochemical oxidation of biological pretreated and membrane separated landfill leachate concentrates on boron doped diamond anode. Applied Surface Science 377 (2016) [10] F.C. Moreira. J. Soler. A. Fonseca. I. Saraiva. R.A.R. Boaventura. E. Brillas. V.J.P. Vilar. Electrochemical advanced oxidation processes for sanitary landfill leachate remediation: Evaluation of operational variables. Applied Catalysis B: Environmental 182 (2016) [11] J. Rivas. A.R. Prazeres. F. Carvalho. F. Beltrán. Treatment of cheese whey wastewater: combined coagulation-flocculation and aerobic biodegradation. J. Agr. Food Chem. 58 (2010) [12] E. Brillas. C.A. Martínez-Huitle. Decontamination of wastewaters containing synthetic organic dyes by electrochemical methods. An updated review. Appl. Catal. B Environ. 166 (2015) [13] A. Eaton. L. Clesceri. E. Rice. A. Greenberg. M.A. Franson. Standard methods for examination of water and wastewater. 21 ed.. Washington. DC. American Public Health Association

62 Theoretical Essay on the Networked Collaborative Consumption S. R. Fernandes 1, J. M. Lucas 1, M. J. Madeira 2, A. I. C. Barreiros 3, F. T. S. Gonçalves 1 1 FibEnTech-UBI, Textile Science and Technology Department, Universidade da Beira interior, Covilhã, Portugal 2 CIEO, Department of Management and Economics, Universidade da Beira Interior, Covilhã, Portugal 3 Polytechnic Institute of Castelo Branco, Castelo Branco, Portugal Abstract The present theoretical essay aims to make an outlook with various approaches related to collaborative economics. Also features several collaborative consumption platforms. Discusses the sharing of goods and services through online and offline networks. Finally, highlights five important points of collaborative economics. Keywords Collaborative Consumption, Collaborative Economics, Sharing, Sustainable Development Introduction The 20th century is enshrined as the century of excess consumption. The result of this practice generated exhaustion and unsustainability of natural resources in the development processes. To ensure life of the human being on Earth, we must reduce the extraction and waste of natural resources, as well as the generation of waste. Only in this way we can ensure the sustainability of the planet. The concept of sustainable development was established in 1987 by the World Commission on Environment and Development of the United Nations with the publication of the report "Our Common Future", through the so-called Brundtland Commission that defined it as: "a development that aims to meet the needs of the present without compromising the ability of future generations to meet their own needs "[1]. The term sustainable development is well-preserved in the international meeting on the Environment, convened by the United Nations (Eco-92), held in Rio de Janeiro, Brazil, in 1992 and constituting a strategic step for the survival of life on the planet. The conference endorses the fundamental concept of sustainable development, which combines economic progress and material use without compromising the needs of future generations. Sustainable development is a global challenge that requires local initiatives with a view to reduce the effects of environmental impacts. New economic opportunities should be able to promote a transformation of the finite resources of the planet, in order to house the next generation. The starting point for an innovation towards sustainability is trying to dissociate the business success of the relentless expansion of material consumption, seeking to minimize the depletion of resources, the pollution and the associated effects, such as climate change [2]. Sustainability requires an urgent overhaul of the current patterns of production and consumption to achieve social equity, economic efficiency and environmental preservation. Sharing Today s smart choice: Don t own. Share Walsh [3] The sharing of common assets is touted as a way for this change. It overcomes outdated as the excess consumption behaviours, which consecrated with the slogan "consume is to exist" and points to a new way of consuming and to be. Important part of this is sharing collaborative economics. In this the new established saying [4] is: "what's mine is yours". Consumption based on access to and use where we share what already exists rather than producing more. Rifkin has published a book called "The Age of Access," in which presented the future transitions of economic activities based on access to goods and services through the shared use [5]. The shared consumption will cause a real revolution of behaviours for the sake of sustainability, pointing to the replacement of products for services [6]. The shared practices are ways of optimizing the lifetime of the products, reducing the need for new products, leading to lower environmental impact [7]. This is to say that it reduces the amount of discarded products which can reduce up to 50% the environmental impact as compared with the single use [8]. In 2011, Time magazine noted the Collaborative Economy as one of the 10 ideas that will change the world [3]. Collaborative Economy is also part of the incentive for Circular Economy, which aims to find a new economic paradigm that integrates environmental concerns and opens the path to new, more sustainable business models. The Collaborative Economy refers to the optimization of the use (or even production) of a product or service through the use of sharing. Among other aspects, the Collaborative Economy questions the urgency of the needs and the requirement to satisfy them through the use of material resources. Collaborative Economy is based on five "R"s: Re-thinking, Re-designing, Re-educate, Reduce and Reuse, in order to share and preserve the social, economic and environmental dimensions [6]. In 2016, the European Commission has launched "The future of the EU collaborative economy Using scenarios to explore future implications for employment", which recognizes the potential of Collaborative Economy in creating new sources of income, offering benefits for consumers through 52

63 new business, for temporary use of goods and services, such as accommodation, sharing of cars, bicycles, televisions, lawnmowers, drills, appliances, clothes among others [9]. Companies are moving towards providing services with their products rather than selling them, and business models based on online platforms, once called collaborative economy, are becoming mainstream [10]. The term "Mesh Business", created by Gansky [11] is based on features: (1) the offer can be shared, within a value chain, community or market, including products, services and raw materials; (2) the Web and mobile data networks are used to track products and users, customers and information about goods; (3) offers, ads and recommendations are passed on through word-of-mouth and extends through network services partners. The Mesh Business and Collaborative Economy are different terms, however, very similar. Both are formed through the use of social and personal networks, communication technologies and the use of applications on mobile devices. Collaborative Economy Collaborative Economy, anyway, can be seen as a technological and economic emergent phenomenon, that develops through information and communication technologies. These generate collaborative communities on the web and create the social sharing of goods and services. It is a question of business models through decentralized networks with peer-to-peer platforms or direct contact between people. In some cases, they create their own rules and standards for new business models through the use of idle assets [12]. Collaborative consumption is divided into three systems: Product Service Systems (PSS), Redistribution Markets and Collaborative Lifestyles [4]. Product Service Systems Product Service Systems allow that products owned by a company are shared by enterprises (car sharing, solar power, automatic laundry) or that privately owned products are shared among interested people, i.e., a product of individual property, often with limited usage, is replaced by a shared service that optimizes its usefulness. Users pay for using it without the need to own it, counting with the following benefits: no need to pay the full value of the product, nor its maintenance, repair and secure costs. Redistribution Markets There are Redistribution Markets with the possession of goods and not only the access, such as in product service systems: goods of private property without use are redistributed via online social networks and physical stores. There are cases where redistribution is based entirely on free trade (e.g., Freecycle, Kashless and Around Again). Others, where the goods are sold in exchange for points (e.g. Barterquest and UISwap) or money (e.g., e- Bay, Flippid), or in which markets are a mixture of it (ex: SCoodle and Craigslist). There are even online companies that make exchanges of goods such as makeup, accessories, clothing, books, toys, etc., for similar products (e.g. Thredup, Makeup Alley; Swapstyle, Toyswap, 'N' Dig Swap) or for goods of the same value (SwapTree, SwapSimple). Exchanges are usually carried out by unknown persons while there are cases where people who know each other (e.g. Share Some Sugar, Neighbor Goods). Regardless of the type of return, the redistribution market encourages the re-use and re-sale of stranded products, keeping them in circulation. Collaborative Lifestyles The Collaborative Lifestyles refer to the shared workspace systems (Coworking Space Citzen, Hub Culture), goods (Neighborrow), tasks, time and messages (DaveZillion, Ithaca HOURs), gardens (Urban Gardenshare, Landshare), skills (Brooklyn Skillshare), food (Neighborhood Fruit), parking spaces (ParkatMyHouse) and housing (Airbnb, CouchSufing, Roomorama). People with common interests that seek to divide and share intangibles such as time, space, skills and money. These various sharing modes are reporting to a new field of Economy: Collaborative Economy. The OuiShare divides the collaborative economy in six blocks, namely: 1. Collaborative consumption is most known currently. Is based on the use of the Internet and peer-to-peer networks to reinvent the sharing through rental, exchange or trading of goods and services. 2. Collaborative production is made possible by the democratization of digital production tools. Is related with the Maker movement and Peer production, also do-ityourself (DIY), as well as the use of free software for manufacturing. Other forms are the sharing of creative spaces (FabLabs, HackerSpaces, MakerSpaces) and the exchange of knowledge and open information between manufacturers. 3. The open and collaborative knowledge comes to the democratization of information or data that can be freely used, reused and redistributed without the legal, technological or social constraints. It is the basis for creating collaborative and sustainable societies through the opening of science, technology, education, culture, economy and transparency of Governments. (Ex: Open knowledge, OuiShare Academy, Stanford, MIT, Harvard, Open Badge, and Local Motors). Open licenses (e.g. Creative Commons, Copyleft, Arduino, and Open Source Ecology). 4. Collaborative funding. Currently, many credit banking, investment, cash, bank transfer, both national and international, are also a part of this new economy. (PayPal, Kanotox, TransferWise and others.) The Transferwise has more than 20 intentional coins and allows to transfer money with a charge of 0.5% in comparison with the usual 4.5% tax of banks. There are also services offered by specialized start-ups in which collaboration and empowerment through digital tools allow people to manage financial operations. The crowdfunding is a form of 53

64 cooperation among many people whose goal is to collect a sum of money, or other non-monetary resources, which support the development of a music, handicrafts, sport, food, science and other initiatives. There are other forms of collective financing: the crowdfunding rewards (KickStarter, Indiego, Ulule and Goteo) that are known to international level, and the crowdfunding based on donations (Microdonanes.net, Migranodearena.org, WorldCoo and Teaming) and loans and investments (Funding Circle Lending Club). 5. Open Government and horizontal organizations refer to the concept of partner or state that accompanies, facilitates, discloses, and spreads out data. Governments and administrations are also open to popular contribution in participatory budgets to the co-creation of solutions or the integration of citizens as a source of information. It is important to stress that in this process of transformation of cities in collaborative cities, the role of public administration is to monitor and facilitate the development of citizen initiatives. Examples of collaborative cities are Amsterdam, Portland and Seoul, both at the level of district and town, by supporting the creation of informative web sites. 6. Exchange of value systems (Complementary and alternative currencies, banks, etc.). Complementary currencies are used, along with the currency of each country, to boost the local economy and generate employment. Alternative currencies produce synergies with communities who choose to reinvent its own currency. They create more social and environmentally friendly alternatives, the traditional currency being not necessary. An example is the Bank of time (BdT) in which the currency is generally the time [13]. Conclusions Historically, the share of products always existed. This happened through networks of trust between family members, neighbours and friends, with the purpose to supply the needs of a product, without having to own it. The structure of the traditional sharing, however, was broken due to the growth of large cities. People took one of the other unknown, thus breaking the network of interaction and trust. In the 21st century, thanks to the internet and the technological and digital evolution, social networks have emerged imitating human social life. The "online world" grows exponentially and allows to expand social networks to anywhere on the planet, linking both individuals as organizations. The links that are created using the Web end up strengthening outside of the net. In Collaborative Economy, there are five important points worth highlighting: (1) the social, in which the demand for goods and services increases along with concerns related to improve sustainability in the community; (2) the economical, focused on the continuity of the life-cycle of stranded products; (3) the technology, made possible by social networks thanks to devices and mobile platforms, as well as ease of payment systems; (4) the service of collaborative consumption that consists in the mechanism of reputation and the ability to generate trust among strangers across platforms and users; (5) the co-creation related to collective intelligence as key element for collaborative innovation processes. Acknowledgements The authors of this paper acknowledge to FCT - Fundação para a Ciência e Tecnologia and to FibEnTech (Fibres, Environment and Technology) Research and Development Unit for their technical and financial supports. References [1]Vezzilli, Carlo.Design de sistema para a Sustentabilidade:teoria, métodos eferramentas para o design sustentavel de sistemas de satisfação. Salvador: EDUFA, (2010). [2]Fletcher, K; Grose L. Moda & Sustentabilidade. De-sign para mudanc a. Sa o Paulo: SENAC, (2011). [3]Walsh, Bryan.10 ideas that will change de world. Today s smart choice: Don t own. Share. Time (17/3/2011).Available on /specials/packages/article/0,28804, _ ,00. html [Accessed on 3, August 2017]. [4] Botsman, R., Rogers, R., O que e meu e seu: como o consumo colaborativo vai mudar o nosso mundo. Porto Alegre: Bookman, (2011). [5] Rifkin, Jeremy. A era do acesso. São Paulo: Makron Books, (2001). [6] Circular Europe Network (CEN), Orientação gerais para a implementação de Estratégias Integradas de Economia Circular de Nível Regional. [Accessed on 5 July, 2017]. [7] Manzini, E., Vezzoli, C. O Desenvolvimento de Produtos Sustentáveis. Os requisitos ambientais dos produtos industriais. São Paulo: Editora da Universidade de São Paulo, (2005), (1º Ed. 2001). [8] Tukker, A. Eight types of product-service system: eight ways to sustainability? Experiences from Suspronet. Business Strategy and the Environment.v.13, p , (2004), doi: /bse.414 [10] Bock, A. K., Bontoux, L., Figueiredo do Nascimento, S. Szczepanikova A., The future of the EU collaborative economy Using scenarios to explore future implications for employment; EUR EN, (2016), doi: / [11] Gansky, Lisa.Mesh: Por que o Futuro dos Nego cios e Compartilhar. Rio de Janeiro: Atla Books, (2011). [12] Stokes, K.; Clarence, E., Anderson, L., Rinne, A. Making sense of the UK, Collaborative Economy. (2014). Collaborative Lab, p. 48. Available on: _of_the_uk_collaborative_economy_14.pdf. [Accessed on 12 July, 2017]. [13]OuiShare_COEC._) [Accessed on 15 July, 2017]. 54

65 Creative textiles design: traditional crafts as source of inspiration L.S. Ribeiro, R.A.L. Miguel FibEnTech-UBI, Textile Science and Technology Department, Universidade da Beira interior, Covilhã, Portugal Abstract This paper reflects on the importance of traditional crafts to creative textiles development, not only by the add value they incorporate on clothing or accessories items, but also as a mean for fashion design students to experiment and test creative design manually for latter industrial (machinery made) applications. The methodology applied in class and student s outcomes in the unit Creative Textiles Lab (Fashion Design course, UBI) are presented. Introduction Creativity is a complex concept which had generate some debates and an amount of definitions [1]. Some decades ago creativity was considered something divine, conducted by superior forces. Until 1970 s scientific studies around creativity were scarce, and only recently this academic unexplored theme started to be investigated by psychology, neuropsychology, and education, and lately by marketing and management academy [2]. In a human and broadly perspective, creativity can be defined as the ability to develop creative ideas [3] [4], the creative persons, but consequently creativity is transferred into creative products, creative processes, or even creative organizations [5]. Thus, creativity relies in the development of new and useful ideas, not only in art contexts as usually associated, but in any domain [6]. In the case of textiles, creativity is crucial to new product development for the fashion clothing industry to use as working material, considering market and consumer s demand. Market and consumer needs and desires are constantly changing, and in the last few years we have witness a change in human mindsets and lifestyles. There is a return to the origins and to what is more connected with nature and with the self. The search for slow fashion and sustainable products has grown, and hand work, traditional crafts and customisation are becoming source of add value to the consumers [7]. Concerning traditional crafts as embroidery, hand sewing, hand knitting or lace arts, they have always been part of the fashion industry, namely in haute couture. They have been synonymous of luxury, exclusivity and as Mirja Kälviäinen state The human-product-time relationship is also a remarkable source of authenticity. [8] (pag.8). Outside the haute couture culture, crafts are often associated with preindustrial folk culture and makers. Although, some artists ended by following a traditional approach to crafts and continuing to reproduce processes and product done previous generations [9], others following the social mindset change, have taken the knowledge of those crafted processes to develop contemporary products. Indeed, the resurgence of interest specifically in craft-based textiles is facing as Gail Kenning states ( ) a boom time. [10]. So, is important that young fashion designers realize the power that nowadays, crafts have to create an emotional and experiment relationships between the consumer and the products [8]. Textile Design Process: inspiration and experimentation crafts As stated previous, crafts have a huge potential to add value to fashion design pieces, by giving that sense of tradition, meaning and uniqueness, and consequently create a closer connection to the consumer. But explore crafts and other types of artisanal techniques, in textile design, encounters other possibilities, namely being source of inspiration and experimentation to new textile developments. The first stages of textile development are research (trends; state of the art) and concept definition. Concepts can be represented visually trough story boards, sketches and also by material samples. When developing and exploring new textiles ideas, designers manipulate and experiment with design elements, creating samples, trying to transfer creative concepts into fabric form [11]. In this stage crafts and artisanal work can be a useful tool, were the limits are the imagination and creativity of the designer. In following stages textiles can remain in a craft based production or also be used as guidelines to apply to industrial products [8]. Application of traditional crafts and outcomes The recognized importance of textiles materials creation to fashion design along with exploration of creativity, have been the driving forces to the development of a new subject in the Fashion Design course in University of Beira Interior. During the semester, students are challenged through several assignments to developed their creative potential applied to textile creations. A several of classroom workshops give them practical experimentation of traditional crafts and artisanal techniques (object printing and pattern creation; sewing textile manipulation; embroidery; felt; artisanal weaving; hand knitting; crochet and macramé). A final assignment assessed their capability to develop a creative textile mini collection. The only imposition is in the beginning concept stage where a word is attributed randomly to each one. With the word in hand, the students 55

66 develop the concept for their collection using the mind mapping technique, exploring associations related to the word (see Figure1). Those associations are the triggers to define colors, tactile sensations, patterns and any other type of visual references. Then, using processes and techniques explored in class, the process of textile experimentation and creation of a collection of samples begin. Fig.3 Textile final sample. Techniques: hand knitting; artisanal dyeing and experimental sea foam effect with shaving foam and glue. Fig.1 Mind mapping development. Student: Angélica Costa The student s creativity is the limit. Consequently, in this assignment the main purpose is exploration and the development of new ideas. Real application of the textile material in the industry is not required. The students are also encouraged to not only rely on common textiles supplies (e.g. yarns, fabrics knits) but any other kind of physical objects that went in line with their concept (e.g. leaves, twigs, paper, plastic, metal). Some examples of the student s work in the year 2016/17 is presented in figure 2, 3 and 4. Fig.4 Final collection sample. Techniques: Artisanal weaving, felt, cracking printing effect, oxidized metal, embroidery. Student: Susana Marques Conclusions Developing and creating new and unique textiles is a creative and design challenge by the plethora of elements that must be considered. Make use of crafts to explore textile developments in a unique and creative perspective, is without doubt a powerful tool for fashion design students to explore. Further Acknowledgements Fashion Design students from the 3rd year 2016/17 at University of Beira Interior References [1] S. H. Klausen, The Notion of Creativity Revisited: A Philosophical Perspective on Creativity Research, Creativity Research Journal, 22(4), (2010) [2] J. Goldenberg and D. Mazursky, Creativity in Product Innovation, Cambridge University, [3] B. A. Hennessey and T. M. Amabile, Creativity, Annual Review of Psychology, 61 (2010) Fig.2 Textile process development. Techniques: Hand drawing and painting; embroidery. Student: Marta Fernandes 56

67 [4] R. Sternberg, Handbook of creativity, Cambridge University, [5]T. Amabile, Creativity and innovation in organizations, Harvard Business School, [6] T. Amabile, R. Conti, H. Coon and J. Lazenby, Assessing the work environment for creativity, Academy of management journal, 39 (5) (1996) [7] C. Treadaway, Digital Crafting and Crafting the Digital, The Design Journal, 10(2) (2007) [8] M. Kälviäinen, The Significance of Craft Qualities in Creating Experiential Design Products, The Design Journal, 3 (3) (2000) [9] J. Turney, Here s One I Made Earlier, Journal of Design History 17(3) (2004) [10] G. Kenning, Fiddling with Threads: Craft-based Textile Activities and Positive Well-being, TEXTILE, 13 (1) (2015) [11] J. Moxey and R. Studd, Investigating Creativity in the Development of Fashion Textiles, The Journal of The Textile Institute, 91(2) (2000)

68 Flame Atomic Absorption Spectrometry: parameters optimization L.A.F. Matias 1, A. Marques 2 1 Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 2 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal Abstract Quantitative determination of iron in drinking water is part of the routine control 2, according to the Decree 306/2007 (27 th of August) of the Portuguese legislation. The method used for this determination is Flame Atomic Absorption Spectroscopy (FAAS) with an oxidant flame of airacetylene. The main purpose of this study was to determine the Fe content of water samples from the Universidade da Beira Interior s particular water supply system, using an optimized FAAS system. To increase the sensibility of the method, two system parameters were optimized, flow rate and burner height, and the working range of the calibration curve was established. These laboratory procedures must be followed for the detection and determination of analytes at low levels in natural waters. Introduction Water quality is essential to reduce waterborne pathogens and to ensure safe water supply to population, which directly affects the quality of people s life. Annually, diseases related to water shortage or to its poor quality kill millions of people in the world, especially in developing countries. Hence, the need for a more rational use of water resources and the effective control of the quality of water is very important to the populations [1,2]. Approximately twenty inorganic elements, many of them metals, are found in living systems and are essential to the proper functioning of these systems. These inorganic elements, and in particular the metals, are supplied to living organisms through their food. One of the important metals for the organism is the iron that is supplied by solid foods and also by water [3]. Iron is an essential element to life, and daily food should contain between 10 and 50 mg of this element. Iron, once absorbed by the body, will intervene in its metabolism. It is part of the hemoglobin molecule, some iron-proteins and enzymes essential to life. It is also an essential component of numerous biological oxidation systems. This metal, being one of the most abundant in the earth crust, is normally in the natural waters in concentrations that can vary from 0.5 to 50 mg L -1 [4]. The iron concentration in drinking water is not a parameter linked to the greater or lesser toxicity of this water. It is considered an undesirable element as other metals also present in water such as copper, zinc and manganese. These metals alter the appearance and taste of water, resulting in an undesired, cloudy or odorous color, because these metals provide the development of microbial flora. The different forms in which iron is found in water are highly dependent on ph and dissolved oxygen. When the ph value is in the range of 4.5 up to 9.0, iron is usually in the form of ferrous iron. Natural waters contain, in general, low iron content. When the iron content in the water treated for human consumption is very high it may be an indicator of a poorly conducted coagulation/ flocculation operation [5]. The Universidade da Beira Interior (UBI) itself has several water mines and fountains in which captures water to supply some of its facilities and at the same time is the entity that manages the system. The water thus collected is subjected to a disinfection treatment with sodium hypochlorite and stored in a water treatment plant (WTP) and after that is distributed as tap water. At this stage of the process, the Water Analysis Laboratory of the Chemistry Department at UBI performs the obligatory analyzes, according to Decree 306/2007, which defines the water quality regime for human consumption. In the mandatory analyzes defined by this law decree, the iron is one of the parameters that must be controlled in the "routine control 2". The water analysis frequency is function of the volume of water provided in the WTP. In this case, the periodicity is monthly. According to the legislation [6,7], the iron concentration is determined by flame atomic absorption spectrometry (FAAS). The spectrometer [8] used in this work is shown in Fig. 1. Fig. 1. Atomic absorption spectrophotometer. The flame atomic absorption equipment, currently commercialized, has coupled specific software where the manufacturer of the equipment introduces average analytical parameters for the determination of the selected metals. It is up to the operator of the equipment to judge whether or not to optimize the predefined instrumental parameters, which allows a lower limit of quantification (LD) and a greater sensitivity. 58

69 In this work we intend to demonstrate that there is better performance of the equipment by optimizing analytical and instrumental parameters, such as: burner height, fuel flow rate, and the adjustment of the working range analytical curve. The main purpose of this study was to analyze water samples for their Fe contents by using an optimized FAAS system. Thus, in order to improve the sensitivity of the analysis, the optimization of the instrumental parameters, burner height and the acetylene flow rate, was performed. The working range was established taking into account the linear range in the calibration curve and the "history" of known values of the iron content in this water samples collected for at least ten years. Fig. 2. Absorbance as a function of burner height. Materials and Methods From a stock solution of Fe, 1000 mg L -1, Merck, CertiPUR, diluted solutions with concentrations from 0.1 up to 1.0 mg L -1 were prepared. For the instrumental parameters optimization, it was used a 2 mg L -1 solution. Nitric acid solution, 65 %, Merck, pro-analysis, was used to acidify all the prepared solutions. All the solutions were prepared with purified water, with a resistance, R 18 MΩ, obtained by a MilliQ + Integral 3, Merck Millipore apparatus. The Atomic absorption spectrophotometer (Perkin Elmer, AAnalyst 800) is equipped with a S10 Autosampler, Perkin Elmer, S10, and a segment array of photodiodes. The analytical signal is absorbance. The 2 m L -1 solution was aspirated into the flame and the absorbance values were read in function to the burner height. The same procedure was applied in function of the acetylene flow rate. Results and Discussion The test to determine the burner height is time consuming and, therefore, the amount of solution aspirated during the test is large. It is important not to change the solution during the test so as not to disturb the system. The vertical and horizontal alignment of the burner are automatically adjusted as soon as the spectrometer is turned on. This value is considered as zero. In the manual adjustment, when positive values appear on the computer screen, it means that the burner is descending from the initial position; negative values means that the burner rises in relation to the initial position. The results obtained are presented in Fig. 2 and it can be verified that the highest absorbance signal, which indicate that the optimize burner height for the iron determination is not the referenced, as value zero, but 3.75 mm below the initial position. The second parameter to be optimized is the acetylene flow rate that was also performed with the 2 mg L -1 iron solution. The reference flow rate given by the equipment manufacturer is 1.4 L min -1 [8]. Fig. 3 shows the absorbance variation as a function of acetylene flow rate. Fig. 3. Absorbance as a function of flow rate. It can be observed that the highest analytical signal corresponds to the flow rate of 2.9 L min -1. The optimization of this instrumental parameters allows the flame conditions to be corrected in order to obtain higher method sensitivity. The "historical" knowledge of the samples allows the adjustment of the standards to evaluate the analytical method. For the normal calibration curve with external standards, the concentration range recommended by the Standard Methods for Examination of Water and Wastewater [9] for Fe is 0.3 up to 10.0 mg L -1. However, in this study the working range was adapted to the real situation. It was prepared a set of standard solutions in the concentration range of 0.18 to 1.00 mg L -1 and the absorbance was confirmed to obey to Lambert-beer law. The calibration curve is shown in Fig. 4 and can be expressed by Eq. 1, where A is the absorbance and C Fe is the Fe concentration, in mg L -1. The correlation coefficient is R 2 = [10]. A = C Fe (1) With the adjusted regression line, Eq. 1, improved, detection limit, LD, and quantification limit, LQ as well as sensitivity. 59

70 References Fig. 4. Adjusted regression line Conclusions Since the water under study presents a very low mineralization degree and an iron content close to the quantification limit (0.184 mg L -1 ) it was necessary to optimize analytical and instrumental parameters, such as iron working concentration, burner height and fuel gas flow rate. The best iron concentration range was between 0.18 and 1.00 mg L -1, and the optimized values for the flow rate and burner height are 2.9 L -1 acetylene and 3.75 mm, respectively. Acknowledgements The authors wish to thank FCT, for the funding of the FibEnTech Research Unit. [1] Worth Health Organization, WHO, Guidelines for drinking-water quality: fourth edition incorporating the first addendum, [2] A. Sousa, M. Taveira and L. Silva, Groundwater from Private Drinking Water Wells: Imminent Public Health, Water Resources, 42 (2015), [3] B. Silva and P. Faustino, An overview of molecular basis of iron metabolism regulation and the associated pathologies, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1852 (2015) [4] B. Mendes and J.F.S. Oliveira, Qualidade da água para consumo humano. LIDEL, 2004, pp [5] F.J. Millero, W. Yao, J. Aicher, The speciation of Fe(II) and Fe(III) in natural waters, Marine Chemistry, 50 (1995) [6] Decreto-Lei n.º306/2007, Diário da Republica de 27 de Agosto de 2007, pp [7] World Health Organization, WHO, Guidelines for Drinking-water Quality, vol. 1 (recommendations), 3 rd Ed., Geneva, [8] Atomic Absorption Spectrometer, User s Guide, PerkinElmer, Inc, [9] Sanard Methods for the Examination of Water and Wastewater; SMEWW, 19 th Edition; [10] Guia relacre nº 13,

71 Quality control techniques for routine analysis used in accredited laboratories: physical-chemical analysis L.Borges 1, Rita Palmeira-de-Oliveira 1, A. Marques 2 1 Labfit-HPRD: Health Products Research and Development; Ubimedical, Covilhã, Portugal 2 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal Abstract It has long been recognized the need of laboratories to define appropriate procedures which assure the quality of results and the performance of the analytical method. This paper describes the methodologies for quality control used in an accredited laboratory and is intended to promote such tools in the context of non-accredited laboratories, facilitating its implementation in routine and it is a challenge for all laboratory technicians. The implementation of a internal and external quality control program to ensure the quality of the results of total nitrogen in wastewater, collected from the daily routine, are presented. Introduction Analytical method validation is the first level to guarantee that the analytical requirements are fulfilled. The second level is to maintain performance of the analytical procedure when it is used in routine analysis [1]. Quality control includes all activities used to ensure that all quality requirements are being met. During the validation stage the method is largely applied to samples of known content. Once the method is in routine use it is used for samples of unknown content. Therefore, suitable quality control should be planned and implemented to allow ongoing monitoring of day-to-day and batch-to-batch analytical performance [2]. It remains the responsibility of the laboratory to define the statistical criteria or control rules and the number of control measurements, according to the quality required for the assay. Two different control quality approaches can be done in the laboratory: internal quality control (actions that depends only on the laboratory) and external quality control (actions carried out by the laboratory, but whose realization depends on an intervention or participation outside the laboratory). The most useful techniques for quality control are those that are coordinate by the laboratory and that provide the analyst with immediate feedback about the system s state of statistical control. Internal control procedures are related to the results' quality ensurance for a specific samples or set of samples, which includes: internal reference samples, measurement standards (quality control standards), blanks, duplicates, spiked samples, blind samples, use of control charts to monitor trends and assessment of correlation of results obtained for different characteristics of a sample, provided that a known relationship exists [1, 2]. Internal reference is a sample with known values, prepared within the laboratory and included in the analytical batch. Internal reference samples allow control of the performance of the whole analytical procedure including the sample preparation step [1]. The use of blanks will indicate that the results obtained for test samples can be suitably corrected to remove any contributions to the response which are not attributable to the analyte [2]. The most common blank consists of the analytical reagent without the target analyte introduced into de analytical procedure and carried through all subsequent steps. Duplicate analysis of routine test samples are an effective method for determining the precision of an analysis. In most cases the duplicate samples are taken from a single gross sample, although in some cases the duplicates must be independently collected gross samples [3]. Spiked samples are samples to which a known quantity of analyte has been added [3]. The spike recovery measures the effects of interferences caused by the sample matrix and reflects the accuracy of the method for that particular matrix. It is widely accepted that for routine analysis, a level of internal quality control of 5% is sufficient [4]. External quality control usually includes the use of Certified Reference Materials (CRM), or equivalent standards and participation in appropriate interlaboratory comparison [4]. The CRM establishes the traceability of the chemical measurements in the current state of the art and allows control the accuracy of the test. Different matrix samples can be purchased from international organizations. Participation in proficiency testing scheme provide an evaluation of the analytical performance of a laboratory. In that case the performance of a laboratory is evaluated objectively by comparing its results with those of other laboratories at regular time intervals [1]. In addition, these schemes can be a useful source of reference samples which can be put to good use internally by participating laboratories [5]. The use of control charts is a suitable way of documenting internal and external quality control. In control charts the variable of interest is usually plotted on y-axis versus time or sequential run number. The new control values are 61

72 collected and added to the control charts, and compared to given upper/lower control limits and upper/lower warning limits, providing a continuous record of the statistical state of the method. These limits are generally calculated from a number of sufficient stable observations. The warning and control limits could be calculated from a minimum of 15 observations [1]. It is an advantage if analytical data of routine work are recorded on control charts immediately after each completed determination. A single measured value that falls outside the upper/lower control limits normally requires that one stop the analytical process, investigate the problem and if necessary take a corrective action [6]. When a system is in statistical control, the data points should be randomly distributed about the central line [3]. The main function of the chart is to detect departure from statistical control. Deviation from the prescribed limits indicates that the method used does not meet the set requirements [1]. Given the wide range of analytical techniques routinely used in the laboratory the quality control tools are not applicable in all assays. Some methods require additional quality control, which should be seen on a case-by-case basis. The main purpose of this paper is to present a full strategy for implementing quality control procedures for physicalchemical methods, to produce good quality results. It includes consideration of calibration curves, measures of accuracy and precision, control charts and acceptance criteria. Materials and Methods The determination of total nitrogen in wastewater is routinely carried out in the laboratory by high-temperature catalytic combustion and chemiluminescense detection method (CLD). Working range of the method is between 0.5 and to 10.0 mg L -1 and the quantification limit of the method is 0.5 mg L -1. The tests were performed in a Carbon and Nitrogen Analyzer, Formacs Series Analyzer, Skalar. Quality control plan for total nitrogen are presented in detail in a quality control procedure of the laboratory: - on each working series, a seven-point curve was made: 0.5 mg L -1 ; 1.0 mg L -1 ; 2.0 mg L -1 ; 3.0 mg L -1 ; 4.0 mg L -1 ; 5.0 mg L -1 and 10.0 mg L -1.The standard solutions used in calibration curve were prepared and analyzed daily for each analytical set, and each value determined was the arithmetic mean of 3 injections. The linear regression analysis was constructed by plotting the CLD count against the standard concentration. Evaluation calibration parameters, such us: correlation coefficient, slope and linearity test according to ISO ; - quality control standard solution (glycine; 5.0 mg L -1 ), analyzed in each set of 20 or fewer samples, at the beginning and at the end of each working series. - reagent blank, every set of 20 or fewer samples; - one sample in duplicate, analyzed in each set of 20 or fewer samples; - one spiked sample analyzed in each set of 20 or fewer samples and determine a spike recovery; - verification of quantification limit. The standard solution of 0.15 mg L -1 was prepared from a stock solution which is not the same stock solution as used for the preparation of the calibration solutions; - on each working series, all the results obtained are plotted in control charts or acceptance control charts and observed if a control value is falling outside the control limits or if a certain systematic pattern is observed in the control values over a period of time: 5 consecutive points between the warning and control limit, 7 control values in consecutive order gradually increase or decrease, 10 control values are lying in the same side of the central line (7 values for range charts). These rules are not applicable to the acceptance chart control. If the method is in statistical control, the analyst can report the results. If any control value is outside control limits or if a certain trend is observed, no analytical results can be reported and the problem must be investigated - twice a year participation in a proficiency test. Results and Discussion The data collection for this study was take place in a water laboratory whose method of determination of total nitrogen used routinely, is accredited by the Portuguese Institute of Accreditation. Control quality results were collected from fifty working series and participation in two proficiency tests. Calibration/Linearity For all working series the r 2 (correlation coefficient) were obtained and all curves passed the linearity test. Figure 1 shows the example of the calibration curve obtained in one of working series performed (20 th series). The value of the r 2 obtained was 1.000, which is higher than the acceptance criteria defined by the laboratory ( 0.995). CLD count 3.0E E E E E E+06 Calibration curve 0.0E Concentration (mg L -1 ) Fig. 1. Calibration curve obtained in 20 th series. According with the linearity test, which results are shown in Table 1 Calibration function is linear chosen field [7]. 62

73 Table 1. Linearity test results obtained in 20 th series. Parameters Results If the response for any parameter varies from the acceptance criteria defined, a new calibration curve must be prepared. Slope The slope is a measure of the sensitivity of the method. The slopes of the different calibration curves must not differ significantly from the value found at the validation of the method. Fig. 2 shows the slopes obtained in the period of the study, and all results are within the control limits. 3,6E+06 3,4E+06 3,2E+06 3,0E+06 2,8E+06 2,6E+06 2,4E+06 2,2E+06 DS E+11 S 2 y F 21.2 PG Linearity YES Probabability ,0E Fig. 2. Control chart of the slope. UCL: Upper Control Limit; LCL: Lower Control Limit; UWL: Upper Warning Limit; LWL: Lower Warning Limit; CL: Central line. Quality Control Standard The mean and range control chart corresponding to the results obtained for the glycine quality control standard are shown in Figures 3 and 4, respectively. QCS (mg/l) Slope 5,40 5,30 5,20 5,10 5,00 4,90 4,80 Slope - Total Nitrogen Series QCS - Total Nitrogen 4, Series UCL UWL Fig. 3. Control chart of the mean of glycine control quality standard. UCL: Upper Control Limit; LCL: Lower Control Limit; UWL: Upper Warning Limit; LWL: Lower Warning Limit; CL: Central line. CL LWL LCL UCL UWL CL LWL LCL Range (R) 0,60 0,50 0,40 0,30 0,20 0,10 0, Fig. 4. Control chart of the range of glycine control quality standard. CL: Control Limit; WL: Warning Limit; R: mean value of the range. The mean chart evidence that there is one control result beyond lower control limit (5 th series; 4.72 mg N L -1 ), the working series was classified as out of statistical control and no samples results were reported. In this case, the laboratory having taken the fowling corrective action: two more control standard and 10% of the set samples were re-analyzed. Also, the calibration curve was verified using the minimum and maximum standard solutions of calibration curve. The new glycine standards results obtained falls within the control limits of the mean and range chart and the new samples results are located within the control limits of the range chart of duplicates. The results of the minimum and maximum standard solutions of calibration are in agreement with the criterion of accepted by the laboratory (relative error 10%). Therefore, the analytical results of 5 th analysis were reported. Duplicate Ranges from duplicate analysis are presented in figure 5. All values obtained are below the control limit (0.113), and only the value of 19 th series is up to warming limit (0.087). Range (R) 0,12 0,10 0,08 0,06 0,04 0,02 QCS - Total Nitrogen Series Duplicates - Total Nitrogen 0, Series Fig. 5. Control chart of the range of duplicates. CL: Control Limit; WL: Warning Limit; R: mean value of the range of duplicates. Spiked samples The spike recovery was calculated and represented in an acceptance control chart as shown in Fig. 6. R CL WL CL WL R 63

74 It can be observed that the results falls within control limits (80-120%), what it means that the contents of the samples do not interfere with the analytical method. Recovery (%) Spike samples - Total Nitrogen 85 LCL Series Fig. 6. Control chart of spike recovery. UCL: Upper Control Limit; LCL: Lower Control Limit; CL: Central line Blanks All the blanks results obtained during the study are below the limit of detection of the method, 0.15 mg L -1. Verification of quantification limit All results obtained for the quantification limit standard fulfilled the acceptance criteria: relative error 10%. External quality control (Proficiency testing) External quality control is also implemented by semiannual participation in proficiency testing (Aquacheck Scheme, Group 10). According with this organization, z- scores in the range +2 to 2 are satisfactory, and indicate analytical accuracy, z-scores in the range 2 to 3 indicate questionable results, and z-scores in excess of 3 are unsatisfactory results. Examples of the results obtained in two participations are given in Table 2 and demonstrate that the z-score obtained was satisfactory. Table 2. Results of the participation in proficiency testing. Distribution z-score Evaluation Satisfactory Satisfactory Results of quality control obtained demonstrate that only in a working series the method was out of statistical control but a corrective action was taken. Daily interpretation of control charts did not show a trend in quality control results and are in accordance with the rules defined by the laboratory. UCL CL Conclusions Implemented quality control procedures at accredited laboratory, demonstrates how well is the method used during routine analysis. The objective of quality control is not to produce control charts with points within of control limits, but is to identify no conform result and then to identify the cause of the error and correct it, before acceptance of results. The application of the quality procedures must be then useful for a lot of laboratories that are not accredited have the aim of obtaining good results, avoiding analytical errors and need to continually improve their methodologies. On the other hand, sample quality control analysis and interpretation of quality control data involves knowledge on the whole method, time consuming and costs. Therefore a good balance between control work and analysis of samples is essential. Acknowledgements The authors wish to thank FCT, for the funding of the FibEnTech Research Unit. References [1] P. Masson, Quality control techniques for routine analysis with liquid chromatography in laboratories, Journal of Chromatography A, 1158, , [2] V. Barwick (Ed), Eurachem/CITAC Guide: Guide to Quality in Analytical Chemistry: An Aid to Accreditation (3rd ed. 2016). ISBN Available from [3] D. Harvey, Modern Analytical Chemestry, McGraw- Hill Higher Education, ISBN , [4] OGC 002, Guia para acreditação de Laboratórios de ensaio, IPAC, [5] Guidelines for quality management in solid and plant laboratories, 9. External quality of data, FAO, [6] NMKL Procedure No3, Control charts and control materials in internal quality control in food chemical laboratories, [7] A. Valcan, Performance evaluation parameters for Merck Determination of total phosphorus in water, ProEnvironment 4, ,

75 Use of the phytoremediation for tuning cheese wastewater pretreated by chemical precipitation A. Almeida 1, A. Prazeres 2, C. Ribeiro 1, H. Chaves 1, F. Carvalho 1,3 1 Departamento de Ciências e Tecnologias Aplicadas, Escola Superior Agrária de Beja, Instituto Politécnico de Beja (IPBeja), Beja, Portugal 2 Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo (CEBAL)/ Instituto Politécnico de Beja (IPBeja), Beja, Portugal 2 Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM), Universidade de Évora, Évora, Portugal 3 FibEnTech-UBI, Universidade da Beira Interior, Covilhã, Portugal Abstract Cheese whey wastewater has a high concentration of organic matter, 0.8 to 77 g L -1 COD, which around 50% can be removed by basic precipitation. However, it still needs further extensive treatment, namely removal of organic matter to avoid environmental problems. This work deals with a post-treatment system using a vertical flow constructed wetland (VFCW). The influence of hydraulic load, depth bed as well as dissolved oxygen (DO) on organic matter removal was studied, using two VFCWs (0.24 m m; 0.24 m m ; A and B) planted with Vetiveria zizanioides in light expanded clay aggregates (Leca NR10/20). The VFCWs influent composition had COD of mg L -1, DO of 0-5 mg L -1 and redox potential (Eh) -50 to 150 mv. The VFCWs were continuously fed and operated in parallel. Hydraulic loads (H L) of 35 ± 1 Lm -2 d -1 to 187 ± 4 L m -2 d -1 were tested. Establishing 51 ± 2 g m -2 d -1 and 31 ± 3 g m -2 d -1, and 86 ± 3 % and 53 ± 3 % as maximum COD removed and removal efficiency obtained for the VFCW A and B, respectively. Introduction The cheese manufacturing industry is responsible for three main types of effluents; generally presents acidic characteristics; however basic ph's have also been reported [1]. Cheese whey wastewater has an elevated concentration of organic matter, 0.8 to 77 (COD) and 0.6 to 16 g L 1 (BOD) [1].The physicochemical pre-treatments like chemical precipitation or coagulation/flocculation could be an solution. Almost 50% of COD was removed in the process and the supernatant was highly biodegradable. This wastewater yet needs more extensive treatment, previously to its disposal. Constructed wetlands (CWs) could be a viable technology for treat various kinds wastewater, such domestic wastewater, agricultural wastewater and industrial effluent, mine drainage, landfill leachate, storm water, polluted river water, and urban runoff in the last few decades. CWs have the advantage of being low-cost and easy to operate in comparison to conventional technologies CWs are generally effective in removing organic matter, suspended solids, and N when properly designed [2]. Organic matter is decomposed in constructed wetlands by aerobic and anaerobic microbial processes as well as by sedimentation and filtration of particulate organic matter [2]. Because of the heavy organic load, anoxic/anaerobic processes prevail while aerobic processes are restricted to small zones adjacent to roots and rhizomes (radial oxygen loss). Vertical Flow Constructed Wetland (VFCW) promotes higher atmospheric oxygen diffusion inside the matrix, which can boost by organic matter removal [3]. The removal compounds are very dependent on several factors namely ph, temperature, DO, feeding mode, hydraulic load and hydraulic retention time (HRT), ammonium nitrogen concentration, organic matter, plant species and harvesting, deep bed, [2]. Some of them are interrelated one with each other. So, dissolved oxygen (DO) and his diffusion into the constructed wetland depended the plants species, type of the flow (vertical flow or horizontal flow, intermittent or continue), H L, HRT and the kind of wastewater and loading pollutant, play an important role in nitrogen removal mechanisms; [2,3,4]). Hydraulic loading and hydraulic retention time are some the most important factors, that control the performance of subsurface flow wetland systems. Greater H L promotes a quicker passage of wastewater through the substrate, thus reducing the optimum contact time. Nevertheless, greater H L, can contribute to oxygen diffusion inside the matrix, which can boost nitrification. In contrary, lack of oxygen inhibits nitrification, but predominant anoxic conditions can promotes denitrification [5, 6]. HRT is determined by flow rate, surface area of the CW, flow depth, porosity of the substrate which expresses the space available for the water to flow through the media, as well as roots plants wetland and depth of the substrate [2]. The depth of the substrate also has a significant effect on the removal efficiency of pollutants, the substrate adsorption, the quantity and activity of microorganisms, and the growth conditions for plants [7]. It has also been reported that the plants root near the top layer of the wetland substrate were more developed and denser than in the others depths [8]. The plants root play an important role in promoting the formation of an oxidized layer around the root and creating a redox gradient [3]; that than contributes to removal nitrogen compounds, as well as releasing exudates (like carbohydrates and amino acids) which serve as nutrient sources for the growth and long term survival of microorganisms [9]. In addition, can influence soil enzyme activity that interfere with nitrogen removal, namely urease and protease, and are higher in the top layer of the substrate than in the deeper layers, in wetland planted with Vetiveria zizanioides [8]. 65

76 Material and Methods The experimental work was carried out with two vertical flow constructed wetlands (VFCWs) planted with Vetiveria zizanioides in Leca NR10/20 (0.24 m m (VFCW A); 0.24 m m (VFCW B) (Fig.1), with a plant density higher than 120 plants m -2. Fig. 1 Schematic representation (not at scale) of the VFCW system, composed by two pilot VFCWs with different depth (VFCW A and VFCW B). Batches of 125 L of with pre-treated cheese whey wastewater (PCWW) obtained by basic precipitation [1], diluted with tap water, were homogenized by two submersible pumps and VFCW and VFCW were feed through a network of equidistant sprinkler, continuously in parallel mode. With this effluent drainage type and due to the substrate material used, the medium was not saturated, allowing that oxygen diffusion had occurred into the VFCWs. The flooding levels were maintained at 14% through a siphon in the outlet. This study was carried out in four successive experimental trials. The experimental conditions applied to the wetlands during each trial are presented in Table 1. Table 1. Operational condition applied to the VFCW A and VFCW B. Exp HL COD T air T soil (L m -2 d -1 ) (g m -2 d -1 ) (ºC) 1 35 ± 1 9 ± 1 10 ± 3 10 ± ± 3 24 ± 2 17 ± 3 14 ± ± 3 42 ± 3 17 ± 3 14 ± ± 4 75 ± 4 16 ± 2 13± 2 Wastewater sampling collection and analysis During the week, the samples were taken every day, in influent and effluent both VFCWs. The dissolved oxygen (DO), ph and redox potential (Eh) were immediately measured. After corresponding standard pre-treatment and reagent addition Chemical oxygen demand (COD) was analyzed using the methods as described in Standard Methods [10]. Air and soil temperature were measured daily, at same time. Soil temperature was measured using two thermometer inserted into each one VFCW and air temperature with another one suspended on the wall near the VFCWs. Rainfall was excluded by covering the VFCWs with a tunnel of transparent fine plastic. Vetiveria zizaniodes plants were visually inspected on a weekly basis for toxicity signals. Twenty plants were randomly selected from each VFCW, and the high of leaves monitored during this experience. The aboveground tissues (leaves) were cut and analyzed at the beginning and at the end of the experimental work. Three independent determinations were performed. In the aboveground plant tissue concentration of the essential elements and nutrients, namely total kjeldhal nitrogen (TKN), phosphorus (P), calcium (Ca), magnesium (Mg), sodium (Na), potassium (K), and dried matter were determined. Vetiveria zizanoides leaves were cut into small pieces, well mixed and oven dried to constant weight at 70 ºC for 48 h to moisture determination; the remaining residue was ashed in a muffle furnace at temperatures of 500 to 550 ºC for 4 to 8 hours [11,12].The ash was dissolved using 10 ml of 3 M hydrochloric acid to determine the concentration of Ca, Mg, K, Na and Pshould be presented as part of the text, with one line space above and below. Hydraulic retention time and hydraulic load calculation The hydraulic retention time (HRT) was established from the Eq. (1) [13]. HRT = V/Q i= Ayp/Q i (1) where Qi (L d 1 ) is the influent flow rate, measured every day in inlet of the VFCW, and A is the surface area of the VFCW (m 2 ), V (m 3 ) is the VFCW volume; y (m) is the flow depth; p is the porosity, which expresses the space available for the water to flow through the media, roots and other solids in the sub surface constructed wetland system [13]. Data analysis Results were statistically verified using software Statitistica 8.0 (StatSoft, Inc., USA). Differences in wastewater quality between influent and effluent of the constructed wetlands were tested using ANOVA at the significance level of p<0.05. Post-hoc (a posteriori) Tukey s test was used to determine differences between 66

77 means of specific variables. All results are presented as means ± standard deviation (S.D.), calculated for n 10. Results and discussion The plants growth were very low, about 0.5 ± 0.1 cm d -1, probably due to the time of the year in which the tests were performed (between March and May). These values were smaller than the values obtained by Almeida (2012), using swine wastewater was treatment in summer, 1.4 ± 0.1 cm d -1 plants growth were obtained. So, above ground biomass the nitrogen, potassium and calcium increasing during the trials, being removed from influent, without significant differences between the values obtained in both VFCWs. COD removal occurred in both VFCWs, so in VFCW A (0.70 m depth) was higher than that obtained in VFCW B (depth 0.35 m)) (Figure 1 a, b). In VFCW A the maximum value obtained was 51 ± 2 g m -2 d -1 and in VFCW B of the 31 ± 3 g m -2 d -1, to COD applied of 75 ± 4 g m -2 d -1 (Fig. 2, a, b). In VFCW B there were no significant differences (p<0.5) between the DO values in influent and effluent, however showed tendency to decrease in effluent, because its uptake by microbial biomass to degrade organic matter (Fig. 3). Fig.3 Dissolved oxygen: in influent and effluent in VFCW A (0.70 m depth); in influent and effluent in VFCW B (0.35 m depth), fed in parallel with PCWW for the different COD load applied. Redox potential (Eh) in the influent and effluent in each VFCW was not significantly different (p<0.5) (Fig. 4).Eh in influent it varied from -30 up to 150 mv and was founded a tendency to decrease after treatment. Fig. 2 a) COD load removed ( ) and COD removal efficiency ( ) in VFCW A (0.70 m depth); COD load removed ( ) and COD efficiency removal ( )(VFCW B) (0.35 m depth). Removal efficiency (ƞ) presented significant differences (p<0.5) between both VFCWs. In VFCW A it presented a maximum value of 86 ± 3 % (Fig. 2, a), increased slowly with the H L and COD load, until H L 126 ± 3 L m -2 d -1 and COD 42 ± 3 g m -2 d -1. The hydraulic retention times (HRT) was of 4 ± 0.2 h. In the last trial done with COD=75 ± 4 g m -2 d -1 this value decreased to 71 ± 1 % with a HRT of 3 h ± 0.3 h, due to the increased in COD applied. In VFCW B ƞ increased until H L 126 ± 3 L m -2 d -1 and COD 42 ± 3 g m -2 d -1. The highest efficiency, 53 ± 3 % (Fig. 2, b), was obtained for a HRT of 2 ± 0.1h. Decreasing to 46 ± 4 % when COD increased to 75 ± 4 g m -2 d -1, the HRT was 1.5 ± 0.1 h. The HRT was always different between the two VFCW, contributing to the differences in COD removal. It is according to referred by [14] that founded that HTR is very important to COD removal when HRT varies from 5 until 10 d. Dissolved oxygen (DO) in the influent and effluent of VFCW A, in the trials made with 24 ± 2 g m -2 d -1 COD 42 ± 3 g m -2 d -1 (second and third trials) presented significant differences (p>0.5). So, there was increased in effluent although COD applied as well as removed and have been increased. Fig. 4 Redox potential: in influent and effluent in VFCW A (0.70 m depth); in influent and effluent in VFCW B (0.35 m depth), fed in parallel with PCWW for the different COD load applied. Conclusions In this study a pilot system composed by two VFCWs, planted with Vetiveria zizanioides was applied to treat a pretreated cheese whey wastewater with heavy loads of organic matter and a wide range of H L from 35 ± 1 to 187± 4 L m - 2 d -1. COD removal efficiencies increased till 86 ± 3 % (VFCW A) and 53 ± 3 % (VFCW B), corresponding to an HRT 4 ± 0.2 h and 1.5 ± 0.1 h, in VFCW A and B respectively. The higher VFCW depth, seems to favor the creation a more extensive root system, with different oxidations conditions which allow, in VFCW A, a higher COD removal efficiency. Also, HRT associated with different contact times between plants, microorganisms and pollutants, can contribute the differences in results obtained. Due to the OD content in both the effluent and the effluent (both beds), in addition to the microbial aerobic degradation, there were others processes, through which the organic matter was removed, namely phytoremediation processes by plants and microbial enzymatic activity, Further work is needed to determine how bed depth influences these processes and how removal of organic matter occurred in the presence of smaller DO and without bad smells from the VFCW. 67

78 References [1] F. Carvalho, A. Prazeres, F. Rivas, Cheese whey wastewater: Characterization and treatment. Science of the Total Environment (2013) [2] R. H. Kadlec, S. Wallace, Treatment Wetlands 2nd ed.,crc Press, Taylor & Francis, New York (2009), 998 pp, [3] P. Cooper, The performance of vertical flow constructed wetland system with special reference to the significance of oxygen transfer and hydraulic loading rates. Wat. Sci. Tech. 51 (9), (2005) [4] H. Brix, Do macrophytes play a role in constructed wetlands. Wat. Sci. Tech. 35, (1997) [5]T. Saeed, G. Sun, A review on nitrogen and organics removal mechanisms in subsurface flow constructed wetlands: Dependency on environmental parameters, operating conditions and supporting media. J of Environ Manag 112: (2012) [6] Y. Yan, J. Xu, Improving winter performance of constructed wetlands for wastewater treatment in Northern China: a review.wetlands 34, (2014) [7] Y. X. Ren, H. Zhang, C. Wang, Y. Z. Yang, Z. Qin, Y. Ma, Effects of the substrate depth on purification performance of a hybrid constructed wetland treating domestic sewage, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 46:7 (2011) [8] L. Kong, Y.B. Wang, L. N. Zhao, Chen, Z.H. Enzyme and root activities in surface-flow constructed wetlands. Chemosphere, 76 (5) (2009) [9] D. K. Singh, S. Kumar, Nitrate reductase, arginine deaminase, urease and dehydrogenase activities in natural soil (ridges with forest) and in cotton soil after cetamiprid treatments. Chemosphere 71 (2008) [10] Standard Methods for the Examination of Water and Wastewater. 22t h American Public Health Association/American Water Works Association/Water Environment Federation, Washington DC, USA [11] C.R. Campbell, C.O. Plank, Preparation of plant tissue for laboratory analysis, in: Y.P. Kalra (ed) Handbook of Reference Method for Plant Analysis. CRC Press, Boca Raton, FL, (1998) pp [12] R.O. Miller, Determination of Dry Matter Content of Plant Tissue: Gravimetric Moisture, in: Y.P. Kalra (ed) Handbook of Reference Method for Plant Analysis. CRC Press, Boca Raton, FL, (1998) pp [13] W. Crites, E. J. Middlebrooks, S.C. Reed, Natural wastewater treatment system. 1th Edition CRC Press Taylor & Francis Group, (2006) pp [14] S. Kantawanichkul, S. Pilaila, W. Tanapiyawanich, S. Kamkrua, Wastewater treatment by plants in vertical - flow constructed wetlands. Wat. Sci. Tech., 40 (3) (1999)

79 Synthesis of carbohydrate derivatives with potential activity for the treatment of bipolar disorder M. Gomes 1, M. Lucas 1, J.A. Figueiredo 1, S. Silvestre 2, M.I. Ismael 1 1 FibEnTech-UBI, Departmento de Química, Universidade da Beira Interior, Covilhã, Portugal 2 CICS-UBI, Health Sciences Research Centre, Universidade da Beira Interior, 6200 Covilhã, Portugal Abstract The aim of this work is to design and synthesize carbohydrate derivatives, namely pseudo-c-nucleosides, with potential interest in the treatment of bipolar disorder. Bipolar disease, also known as manic-depressive illness, is a serious, incapacitating, and chronic neuropsychiatric disorder in which the patient has recurrent episodes of two opposing mood states, depression and mania. The available medical treatment for this disorder is not the most appropriate, once antipsychotic drugs are usually used to treat maniac disease and antidepressant drugs to treat depressive crisis. The synthesized pseudo-c-nucleosides with potential interest for the treatment of this manic-depressive disorder included pyrazolidine-3-ones and 5-hydroxypyrazoles. The prepared compounds were analyzed by 1 H- and 13 C- Nuclear Magnetic Resonance (NMR) and will be subject of further in vitro biological evaluation. Introduction Carbohydrates have a central role in numerous physiological events, such as in cellular communication, and are part of the basic structure of some endogenous biomolecules, which makes them usually biocompatible and good starting materials for the synthesis of new compounds with potential biological activity [1]. This class of compounds presents several chiral carbons that confers them stereogenic centres, which is an advantage for the selective interaction with the biological targets [2]. In fact, carbohydrates are considered privileged scaffolds and can be linked to several pharmacophores, such as heterocycles. Therefore, they are largely used at the therapeutic level in the treatment of several illnesses, including in central nervous system pathologies [2-4]. Among the neuropsychiatric diseases, one of the most challenging is bipolar disease also known as manicdepressive disorder. This is a disabling, chronic and severe disease, in which the patient has recurrent episodes of two opposite mood states - mania and depression [4-8]. It is known that, between other neurobiological changes involved in this disease, a GSK3β hyperactivity occurs, which leads to apoptosis of neuronal cells causing reductions of neurons and glial cells. Hence, it is important to develop a compound presenting at least the ability to inhibit the GSK3β phosphorylation and having neurotrophic effects to reduce this loss of neuronal cells [6]. With this study, we aimed to synthesize carbohydrate derivatives, namely pseudo-c-nucleosides, with potential interest for the treatment of the manic-depressive disorder, for further in vitro evaluation. Results and Discussion The pseudo-c-nucleosides were synthesized from a commercially available compound, 1,2:5,6-di-Oisopropylidene-α-D-glucofuranose (1). Of these, 5-(3-O-benzyl-1,2-O-isopropylidene-α-Dthreofuranos-4-C-yl)-pyrazolidine-3-one (12) was synthesized from compound 1. For this, a benzylation of the hydroxyl group at C3, followed by acidic hydrolysis of the isopropylidene in C5 and C6 of the furanosidic ring and further oxidative cleavage was performed (Scheme I). Scheme I- Reactional pathway to obtain the aldehyde 3-Obenzyl-1,2-O-isopropylidene-α-D-xylo-pentodialdo-1,4-furanose (4) from 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (1). Reactional conditions: a) BnBr, DMF, NaH, r.t., 20 min.; b) CH3COOH 80%, 60ºC, 1 h.; c) NaIO4, ethanol, water, r.t., 20 min. Then, a Wittig reaction allowed the preparation of an α,βunsaturated ester, which is the precursor for the final reaction, in which was obtained the pyrazolidine-3-one linked to the C4 of the furanose ring. For obtaining 5-(1,2- O-isopropylidene-α-D-threofuranos-4-C-yl)-pyrazolidine- 3-one (13) the same synthetic pathway was followed, but without the step of the benzylation of the hydroxyl group linked to C3 (Scheme II). 69

80 O O O O O OH f) O O O O 1 16 O O g) O O O OH 17 O O Scheme III- Synthetic pathway to compound 17 from compound 1. Reactional conditions: f) PCC, anhydrous dichloromethane, 40 C, 1 h.; g) Anhydrous ethanol, sodium borohydride, 0 C, 20 min. Scheme II- Synthetic pathway of the pyrazolidine-3-one derivatives and yields. Reactional conditions: d) (Carbethoxymethylene)triphenylphosphorane, Chloroform, 30 C, 30 min; e) Ethanol, Hydrazine monohydrate, 80 C, 1h. [9]. Compounds 5-(3-O-benzyl-1,2-O-isopropylidene-α-Derythrofuranos-4-C-yl)-pyrazolidin-3-one (14) and 5-(1,2- O-isopropylidene-α-D-erythrofuranos-4-C-yl)- pyrazolidin-3-one (15) were obtained from the precursor 1,2:5,6-di-O-isopropylidene-α-D-allofuranose (17), which was prepared from compound 1, by an oxidation of the free hydroxyl linked to C3 and posterior reduction to obtain (Scheme III). The further reactions were the same described for compounds 12 and 13, respectively. (Scheme II) The difference between the final yields of these compounds can be explained by the fact that the precursor 1,2:5,6-di- O-isopropylidene-α-D-allofuranose, is less reactive than the homologous glucose derivative. Compounds 3-(3-O-benzyl-1,2-O-isopropylidene-α-Dthreofuranos-4-C-yl)-5-hydroxypyrazole (21), 3-(1,2-O- isopropylidene-α-d-threofuranos-4-c-yl)-5- hydroxypyrazole (22) and 3-(3-O-benzyl-1,2-O- isopropylidene-α-d-erythrofuranos-4-c-yl)-5- hydroxypyrazole (23), were obtained by the same procedure applied to synthesize compounds 12, 13 and 14, respectively, until the α, β-unsaturated esters were prepared. After that, an addition reaction was performed to obtain a β-enaminoester, which is the precursor for the final reaction, in which was obtained the 5-hydroxypyrazole linked to the C4 of furanose ring (Scheme IV). CO 2 Et R 1 R 2 O O O 8 R 1 =OBn ; R 2 =H 9 R 1 =OH ; R 2 =H 10 R 1 =H ; R 2 =OBn h) H 2 N HN N R 1 R 2 R 1 R 2 O OH O CO 2 Et O i) O O 18 R 1 =OBn ; R 2 =H 19 R 1 =OH ; R 2 =H 20 R 1 =H ; R 2 =OBn O 21 R1 =OBn ; R2 =H ( =84%) 22 R1 =OH ; R2 =H ( =24%) 23 R1 =H ; R2 =OBn ( =83%) Scheme IV- Synthetic pathway of the 5-hydroxypyrazole derivatives and yields. Reactional conditions: h) DMF, NaN3, MW (450 W), 6 min; i) Ethanol, Hydrazine monohydrate, 80 C, 1 h. [9]. 70

81 In the case of 5-hydroxypyrazoles 21-23, the benzylated derivatives were obtained in superior yields than without any protecting group attached to C3. At this moment, in vitro cytotoxicity assays are being performed. Conclusions The pseudo-c-nucleosides 12, 13, 21 and 23 were obtained in good yields when compared with compounds 14, 15 and 22, which have been achieved with very low yield. As future work, we pretend to optimize the synthetic pathways for the pyrazolidine-3-one derivatives, whose precursor is the 1,2:5,6-di-O-isopropylidene-α-Dallofuranose, to posterior in vitro biological evaluation, in which is expected to confirm the potential interest to control the maniac-depressive disorder. If the compounds present good in vitro results in cell assays, we pretend to study the effects of these compounds in in vivo assays. References [1] H. Zhang, Y. Ma, X. Sun, Recent Developments in Carbohydrate-Decorated Targeted Drug/Gene Delivery, Med Res Rev, 30 (2009) [2] S. Hanessian, Iminosugars: From synthesis to therapeutic applications, first Ed., John Wiley&Sons Ltd, London, [3] D.F. Mccomsey, V.L. Smith-Swintosky, M.H. Parker, D.E. Brenneman, E. Malatynska, H.S. White, B.D. Klein, K.S. Wilcox, M.E. Milewski, M. Herb, M.F.A. Finley, Y. Liu, M. Lou Lubin, N. Qin, A.B. Reitz, B.E. Maryanoff, Novel, Broad-Spectrum Anticonvulsants Containing a Sulfamide Group: Pharmacological Properties of (S)-N- [(6-Chloro-2,3-dihydrobenzo[1,4]dioxin-2- yl)methyl]sulfamide, J. Med. Chem. 56 (2013) [4] S. Gupta, P. Masand, B. Frank, K.L. Lockwood, P.L. Keller, Topiramate in Bipolar and Schizoaffective Disorders: Weight Loss and Efficacy, J. Clin. Psychiatry. 2 (2000) [5] J.W. Young., D. Dulcis, Investigating the mechanism(s) underlying switching between states in bipolar disorder, Eur. J. Pharmacol. 759 (2015), [6] R. Machado-Vieira, H. Manji, C.A. Zarate Jr, The role of lithium in the treatment of bipolar disorder: convergent evidence for neurotrophic effects as a unifying hypothesis, BiPolar Disord. 11:Suppl. 2 (2009) [7] A.C. Andreazza, B.N. Frey., B. Erdtmann, M. Salvador, F. Rombaldi, A. Santin, C.A. Gonçalves, F. Kapczinski, DNA damage in bipolar disorder, Psychiatry Res. 153:1 (2007) [8] T. Kato, M. Kubota, T. Kasahara, Animal models of bipolar disorder, Neurosci. Biobehav. Rev. 31:6 (2007) [9] J.M.A. Pinheiro, Preparação de novos pseudo-cnucleósidos e sua actividade biológica, Tese de Doutoramento, Universidade da Beira Interior,

82 Phytochemical characterization and determination of biological activity of bark's extracts of Salix salviifolia M. Lucas 1, M. Gomes 1, J. A. Figueiredo 1, A. P. Duarte 2, M. I. Ismael 1 1 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal 2 CICS-UBI Health Sciences Research Centre, Universidade da Beira Interior, Covilhã, Portugal Abtract In this work, were studied the extracts of Salix salviifolia bark obtained by refluxing with ethanol/water (80:20) and with n-hexane. The aim of this study was to characterize the chemical composition of those extracts, with the purpose of determining its biological activity, by evaluating the antioxidant activity of both extracts. The determination of the antioxidant activity was made by the DPPH method. In the chemical characterization of the hydroethanolic extract, the Folin-Ciocalteu method was used for the total phenols quantification and for determination of total flavonoids, a colorimetric method was used using aluminum chloride. The antioxidant capacity of this extract was also evaluated by the β- carotene/linoleic acid system. The phytochemical identification was obtained by gas chromatography-mass spectroscopy (GC-MS). The hydroethanolic extract showed a very strong antioxidant activity, being rich in phenolic compounds. Both extracts have different chemical constituents, being present phenolic compounds, flavonoids, steroids, among others. Introduction Nowadays, compounds extracted from the plants are used for pharmacological purposes and crude extracts also show pharmacology interest. Thus, in order to know the structural components of pharmacological interest, a battery of tests is usually performed, in order to conclude what actually leads to this activity and its extension. [1] Salix salviifolia subsp. Salviifolia (Figure 1), commonly known as White Willow, among other denominations, is an endemic species of the Iberian Peninsula and in Continental Portugal it is widely distributed in the northern and central regions, being autochthonous in these places. The trees of this species grow preferentially in humid places, choosing the conditions offered by the waterways as the ideal ones. [2,3] Although there are several studies focusing on the Salix species and their medicinal properties, it is not of our knowledge the existence of studies on Salix salviifolia. Experimental The extracts were obtained from logs and branches harvested in the parish of Três Povos, near the village of Escarigo, Fundão (Coordinates: 40 14'14.78"N / 7 16'42.03"W). After collection, the barks were removed and dried at 30 C for 15 days, and then milled. The samples were collected on January [4] Figure 1 Salix salviifolia subsp. Salviifolia flowering. [2] The extraction of the active compounds was carried out in two phases. First, the non-polar compounds were collected by extraction with n-hexane in hot soxhlet. The extract obtained was concentrated under vacuum to remove the solvent. After this extraction, the barks were submitted to another extraction process, in order to collect the polar compounds, under reflux with ethanol/water (80/20) in which the ratio of the sample to the solvent was 1:20. After extraction, the filtrates were vacuum-dried and the filtrates obtained were concentrated. [4] The determination of total phenols was performed by the Folin-Ciocalteu colorimetric method. The calibration curve was prepared with different concentrations of methanolic solutions of gallic acid. Gallic acid was used as standard in the following concentrations: 50, 100, 125, 150, 200, 225, 250, 300, 325, 350, 400 and 500 mg/l. For this assay, 450 μl of distilled water was added to 50 μl of the abovementioned solutions and then 2.5 ml of 0.2 N Folin- Ciocalteu reagent was added. After addition of gallic acid, distilled water and Folin-Ciocalteu reagent, 5 minutes were allowed to react at room temperature. Then, 2 ml of sodium carbonate (75 g/l aqueous solution) were added. This mixture was allowed to incubate, in a water bath at 30 C, it for 90 minutes, with intermittent stirring. The blank was prepared as described above, replacing the 50 μl of solution with 50 μl of methanol. The assay was performed in triplicate and after incubation, the absorbances were read at 765 nm. To determine the total phenol content in the extract, the initial 50 μl was replaced by 50 μl of a solution of the extract in methanol and proceeded as described above. To calculate the amount of total phenols present in the extract, it was used the equation of the calibration curve of gallic acid in methanol (Graphic 1). [5-7] 72

83 Absorbance (765 nm) Total phenols y = x R² = Concentration of gallic acid (mg/l) To determine the antioxidant activity by the colorimetric method with DPPH, a calibration curve was constructed. A stock solution of DPPH in methanol was prepared with a concentration of mm or 85 mg/l. From this solution, different dilutions were prepared with defined concentrations, which are listed in Table 1. [5] Table1 Concentrations of the methanolic solutions of DPPH used in the construction of the calibration curve. [DPPH] mm [DPPH] mg/l Graphic 1 Gallic acid calibration curve for determination of total phenols, obtained from the average of the absorbance of triplicate assays The determination of the total flavonoids was performed using the colorimetric method of aluminum chloride. The calibration curve was prepared with different concentrations of methanolic solutions of quercetin, the reference compound, with the following concentrations: 12.5, 30, 50, 70, 100, 230, 150 and 200 μg/ml. [5,8] To 500 ml of the above solutions was added 1.5 ml of methanol, 0.1 ml of aqueous aluminum chloride solution (10%), 0.1 ml of 1M aqueous potassium acetate solution and finally 2.8 ml of distilled water. At room temperature, it was allowed to react for 30 minutes with intermittent stirring. The blank was prepared as described above, replacing the 500 μl of solution with 500 μl of methanol. The assay was performed in triplicate and after incubation time, the absorbance was read at 415 nm. For the determination of the total flavonoid content in the extract, the initial 500 μl was replaced by 500 μl of a solution of the extract in methanol and was carried out as described above. For the extract, the assay was performed in quintuplicate. To calculate the amount of total flavonoids present in the extract, the equation of the quercetin calibration curve in methanol was used (Graphic 2). [5,8] Absorbance (415 nm) y = x R² = Concentration of quercetine (µg/ml) After the preparation of those solutions, the absorbances were read at 517 nm against a blank consisting of methanol. Absorbance readings were done in triplicate. The calibration curve was constructed with the concentrations of DPPH, in mg/l, on the abscissa axis while the absorbances read were placed on the ordinate axis (Graphic 3). [5] Absorbance (517 nm) Total Flavonoids y = x R² = DPPH Concentration of DPPH (mg/l) Graphic 3 DPPH calibration curve for determination of antioxidant activity, obtained from the average of the absorbance of triplicate assays. Graphic 2 Quercetin calibration curve for determination of total flavonoids, obtained from the average of the absorbance of triplicate assays. For the determination of the antioxidant activity of the extract by this method, three solutions of DPPH in methanol, with concentration , and

84 mm, were prepared. All DPPH solutions used were prepared daily, allowed to stabilize and protected from light, due to the sensitivity of DPPH. As for the extract, dilutions of in methanol were prepared with concentrations of 25, 50, 100, 150, 200 and 250 μg/ml. [5,9] For each prepared DPPH solution and for each concentration of the extract, the following experimental procedure was performed: 3.9 ml of DPPH solution was added to 0.1 ml of the sample. It was allowed to incubate in the dark at room temperature for 90 minutes and after the absorbances were read at 517 nm. For each DPPH solution, a control consisting of 0.1 ml of methanol and 3.9 ml of DPPH solution was prepared, which was subjected to the same conditions as the samples under study. Blank contained only methanol. The assays were performed in triplicate. [5,9] Rutin, trolox and gallic acid were used as antioxidant positive controls. Methanolic solutions of these standards were prepared. To determine the antioxidant activity by the β- carotene/linoleic acid method, it was necessary to prepare an emulsion daily. For this, 500 μl of a solution of β- carotene in chloroform (20 mg/ml), 40 μl of linoleic acid, 400 μl of Tween 40 and 1 ml of chloroform were added to a round bottom flask. The mixture was homogenized and the chloroform was evaporated using the rotary evaporator at 45 ºC for 5 minutes. After evaporation under vigorous and continuous stirring, 100 ml of distilled water saturated with oxygen was added to the above mixture. [5,10,11] For the determination of the antioxidant activity of the extract, by this method, dilutions in methanol with concentrations of 5, 50, 100, 250, 500, 750 and 1000 μg/ml, were prepared. For each concentration of the extract, the following experimental procedure was performed in a test tube: to 5 ml of the previously prepared emulsion was added 300 μl of sample. For each sample a control was prepared, where 300 μl of sample was replaced with methanol. An emulsion without β-carotene was used for the blank test. Thus, the white emulsion was prepared in the same manner as above, where only 500 μl of β-carotene solution was replaced with 500 μl of chloroform. [5,10] The assay was performed in duplicate and the absorbances were read at 470 nm after one hour of reaction in a 50 C bath. The absorbance of the control was read, at time zero and after one hour of reaction, against the blank. [5] For positive control, butylated hydroxytoluene (BHT) was used as the standard. The experimental procedure used for the standard was the same as described for the sample. The antioxidant activity of the sample was compared with that of the BHT standard. [5] Results and Discussion The determination of the total phenolics, total flavonoids and the antioxidant activity by the two different methods was applied to the extract obtained by reflux of 4 hours with ethanol/water (80/20). The result of the determination of total phenols is expressed in milligrams of gallic acid equivalents per gram of drymaterial (mg GAE/g dry-material), while the determination of total flavonoids is expressed in milligrams of quercetin equivalents per gram of dry-material mg QE/g drymaterial). (Table 2) Table 2 Contents of total phenols and total flavonoids in hydroethanolic extract. Extract Total phenols (average ± standard deviation) ± 4.81 mg GAE/g dry-material Total flavonoids (average ± standard deviation) ± 1.61 mg QE/g dry-material Concerning the antioxidant activity determined by the DPPH method, the inhibitory concentration (IC 50) was calculated, that is, the antioxidant concentration required to reduce the initial concentration of DPPH by 50%. The results of the antioxidant activity are expressed according to the Antioxidant Activity Index (AAI). [5,9] The AAI was calculated with the following formula (Eq.1): AAI = DPPH in control at 90 minuts (μg/ml) IC 50 (μg/ml) According to the AAI, the antioxidant activity of the compounds are classified as poor, moderate, strong and very strong. (Table 3) [5,9] Table 3 Classification of a sample as to its antioxidant activity. Antioxidant Activity AAI Poor AAI < 0.5 Moderate 0.5 < AAI < 1.0 Strong 1.0 < AAI < 2.0 Very strong AAI > 2.0 The obtained results were compared to three positives controls with different antioxidant capacity: rutin, trolox and gallic acid. (Graphic 4) Antioxidant Activity - DPPH method Extract Rutin Trolox Gallic Acid IC50 AAI Graphic 4 Antioxidant activity for the hydroethanolic extract and for the three standards, obtained from the average of the absorbance of triplicate assays. The IC50 is presented in mg/l. (1) 74

85 The hydroethanolic extract presents an IC 50 of lower than two of the standards (rutin and trolox), thus showing greater antioxidant capacity than those. According to the AAI, the extract is classified as very strong. Antioxidant Activity (% inhibition) Antioxidant Activity - method β-carotene Concentration (µg/ml) BHT Extract Graphic 5 Antioxidant activity of hydroethanolic extract and BHT standard, obtained from the average of the absorbance of duplicate assays. The antioxidant activity determined by the β- carotene/linoleic acid method demonstrates that at the lowest concentration the extract has no activity, but subsequently increases with increasing concentration. The antioxidant activity of the extract is lower than that of the BHT standard, indicating that it has less capacity to sequester the peroxide radical of linoleic acid. As for the extract of n-hexane, it was not possible to determine the antioxidant activity by these methods, since the extract was not completely soluble in methanol, it was necessary to find another characterization technique. So, a phytochemical identification by GC-MS was carried out, which is not yet finalized, but both extracts showed very different chemical constituents, being present flavonoids, other phenolic compounds, steroids, some of which in the form of glycosides. Conclusions According to the DPPH method, the hydroethanolic extract has a very strong antioxidant activity and high phenolic content, while the flavonoid content is relatively low. By the β-carotene/linoleic acid method, the hydroethanolic extract has lower antioxidant activity than the BHT standard. The hydroethanolic and n-hexane extracts are still studied in order to obtain the photochemical identification. References [1] Proença da Cunha, A. Farmacognosia E Fitoquímica, 3rd ed.; Fundação Calouste Gulbenkian: Lisboa, [2] Flora-On: Flora de Portugal Interactiva Sociedade Portuguesa de Botânica. Salix salviifolia subsp. salviifolia (accessed May 30, 2017). [3] Lourenço, J.; Araújo, P. V.; Pereira, E. P.; Caraça, R.; Aguiar, C.; Carapeto, A.; Almeida, J. D.; Henriques, T. M. Salix salviifolia Brot. subsp. salviifolia - mapa de distribuição (accessed May 30, 2017) [4] Martins, N.V.D.F. Bioprospecção de Plantas: Obtenção de Extratos de Cascas de Salix Salviifolia, Project presented at Universidade da Beira Interior to obtain the degree of BSc in Medicinal Chemistry, [5] Luís, Â. Avaliação das actividades antioxidante e anti- Microbiana de extractos de resíduos florestais. Dissertation presented to the Universidade da Beira Interior to obtain a Master's Degree in Biochemistry, [6] Miliauskas, G.; Venskutonis, P. R.; Van Beek, T. A. Screening of Radical Scavenging Activity of Some Medicinal and Aromatic Plant Extracts. Food Chem. 2004, 85 (2), [7] Tawaha, K.; Alali, F. Q.; Gharaibeh, M.; Mohammad, M.; El-Elimat, T. Antioxidant Activity and Total Phenolic Content of Selected Jordanian Plant Species. Food Chem. 2007, 104 (4), [8] Pourmorad, F.; Hosseinimehr, S. J.; Shahabimajd, N. Antioxidant Activity, Phenol and Flavonoid Contents of Some Selected Iranian Medicinal Plants. African J. Biotechnol. 2006, 5 (June), [9] Scherer, R.; Godoy, H. T. Antioxidant Activity Index (AAI) by the 2,2-Diphenyl-1-Picrylhydrazyl Method. Food Chem. 2009, 112 (3), [10] Broinizi, P. R. B.; de Andrade-Wartha, E. R. S.; Silva, A. M. D. O.; Novoa, A. J. V.; Torres, R. P.; Azeredo, H. M. C.; Alves, R. E.; Mancini-Filho, J. Avaliação Da Atividade Antioxidante Dos Compostos Fenólicos Naturalmente Presentes Em Subprodutos Do Pseudofruto de Caju (Anacardium Occidentale L.). Ciência e Tecnol. Aliment. 2007, 27 (4), [11] Marco, G. J. A Rapid Method for Evaluation of Antioxidants. J. Am. oil Chem. Soc. 1968, 45 (9),

86 Studies on the Solubility of Methyl Orange and Acid Orange 8 in Water C.M.G. Pinto 1, M.I.A. Ferra 1, I.M.S.C. Gonçalves 1, M.J.A. Pacheco 1, A. Marques 1 1 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal Abstract The solubility of two azo dyes, methyl orange (MO) and acid orange 8 (AO8), was measured in water. The experimental data show a smooth solubility curve for MO at temperatures lower than 45 ºC. At higher temperatures, no significant solubility variation was found and the reproducibility was lower. The data obtained for AO8 indicate no solubility variation at temperatures above 40 ºC. This observation is probably related to the formation of aggregates with high number of molecules which are likely to be present in the saturated solutions. Introduction Azo dyes have been widely applied in textile, paper, cosmetics, pharmaceutical and food industries, among others, being by far the largest group of colorants [1]. They possess at least one azo bond (-N=N-) in their molecular structure which makes them quite resistant to chemical degradation. Many dyes show a high tendency to form aggregates and dimmers can be found even in quite dilute solutions [2]. Self-association generally increases as concentration and ionic strength increase or temperature decreases. Although solubility is an important property in dye characterization, data are scarce and sometimes contradictory, particularly in water, probably due to the difficulty in its experimental determination, when aggregation takes place. In this work, the solubility of two azo-dyes, methyl orange (MO) and acid orange 8 (AO8) was measured in water and the respective temperature influence is discussed. They are available as sodium salts and the respective molecular structure is shown in figure 1. Fig. 1. Molecular structure of MO (a) and AO8 (b) Experimental The MO solubility in water was determined, at 5 ºC intervals, over the temperature range 5 to 60 ºC and the AO8 solubility was measured at temperatures above 20 ºC. The temperature was controlled with a set of glass precision thermometers and read with a precision of ± 0.05 ºC. MO (85%) and AO8 (65%), purchased from Sigma- Aldrich, were used with no further purification and all solutions were prepared from purified water, with a resistivity of 18 MΩ cm, obtained by Milli-Q 185 Plus (Millipore) apparatus. The experiments were carried out in a glass bottle with a working volume of about 100 ml, with a Teflon-coated magnetic stirrer, put in a thermostatic water-circulator bath, Grant LTD 20, equipped with an auxiliary cooler, Grant C 2G. An excess amount of dye was added to water a solid - liquid equilibrium was reached [3] after the solution had been stirred for 12 h and then kept isothermal for three days. A preheated syringe was used to withdraw three samples, each one of about 3 ml, of the upper portion of the solution. They were filtered using filter paper, Whatman, SPARTAN 30/0.45 RC, injected into 100 ml flasks and weighed on a Mettler PM 200 balance. After appropriate dilutions, the samples were analyzed by UV/visible double-beam UNICAM Heλios-α spectrophotometer. The absorbance measurements were performed at the maximum absorption wavelength (λ=468 nm and λ=488 nm for MO and AO8, respectively) and the concentrations obtained from the respective calibration straight lines. Results and Discussion As illustrated in figure 2, which represents the mean of three measurements as a function of temperature, the MO solubility in water, s MO, increases with the temperature up to around 45 ºC. From that point, there is no evident change and data become more erratic. According to Kendrick and Gilkerson [4], dimmers can be formed in MO aqueous solutions at concentrations as low as 10-4 mol dm -3 and aggregates with higher number of molecules may be formed at concentrations higher than 10-3 mol dm -3. This way, aggregates are likely to be present in the saturated solutions but do not interfere in the solubility determination as they dissociate with dilution, before absorbance measurements take place. However, at higher temperatures, i.e. over 45 ºC, aggregates with very high number of molecules, or ions, may be present in the saturated solutions and they are probably adsorbed onto the solid surface. 76

87 S MO /g.kg -1 Fig. 2. Temperature dependence of methyl orange solubility At temperatures between 5 and 45 ºC, the solubility may be calculated by equation (1), with correlation coefficient R 2 = and standard error σ = 0.332, s MO = (t-25) (t-25) 2 (1) where t indicates the temperature, in ºC. Experimentally, the determination of the AO8 solubility in water is far more difficult, mainly due to the formation of a very dark coloured solution which makes the observation of a clear solid-liquid equilibrium very difficult. Even so, measurements were attempted at several temperatures, as illustrated in figure 3. S AO8 /g.kg t /ºC t / ºC Fig. 3. Temperature dependence of acid orange 8 solubility These results indicate a temperature dependence of the AO8 solubility similar to that observed for MO, with no smooth variation at temperatures higher than around 40 ºC. According to those values, AO8 is much more soluble than MO, so its concentration in the saturated solutions is higher, favouring self-association at lower temperatures. On the other hand, the amount of impurities in the purchased chemical substance is higher (35 %) which may also interfere in the final measurements, becoming less reproducible. Conclusions MO solubility in water follows a smooth curve at temperatures between 5 and 45 ºC. At higher temperatures, no evident change in the solubility was noticed, probably due to the formation of aggregates with high number of molecules or ions. Steady solubility values for AO8 were found to be more difficult to obtain, with lower reproducibility. The results indicate a temperature dependence similar to that observed for MO. As it is much more soluble, more complex aggregates are expected to be present in the saturated solutions. Reliable and reproducible data for azo dyes solubility are difficult to obtain probably due to aggregation phenomena, which may take place in saturated solutions. Acknowledgements The authors wish to thank FCT, for the funding of the FibEnTech Research Unit References [1] H. Zollinger, Color Chemistry: syntheses, properties and applications of organic dyes and pigments, third revised ed., Wiley-VCH, Weinheim, [2] D.J. Edwards, A.P. Ormerod, G.J.T. Tiddy, A.A. Jaber and A. Mahendrasingham, in A.T. Peters and H.S. Freeman, Physico-Chemical Principles of Color Chemistry, vol. 4, Blackie Academic & Professional, Chapman & Hall, [3]R. Cohen-Adad and M.T. Cohen-Adad, in G.T. Hefter and R.P.T. Tomkins, The Experimental Determination of Solubilities, John Wiley & Sons, LTD, [4]K.L. Kendrick, W.R. Gilkerson, The state of aggregation of methyl orange in water, J. Sol. Chem., 16 (1987),

88 Phytochemical composition and assessment of rheological and antioxidant properties of juices of berries Â. Luís 1, S. Sousa 2, A. P. Duarte 1, L. Pereira 3, F. Domingues 1 1 Centro de Investigação em Ciências da Saúde (CICS-UBI), Universidade da Beira Interior, Av. Infante D. Henrique, Covilhã, Portugal 2 Materiais Fibrosos e Tecnologias Ambientais (FibEnTech-UBI), Universidade da Beira Interior, Rua Marquês d Ávila e Bolama, Covilhã, Portugal 3 Centro de Matemática e Aplicações (CMA-UBI), Universidade da Beira Interior, Rua Marquês d Ávila e Bolama, Covilhã, Portugal Abstract Berries provide significant health benefits because of their high levels of bioactive compounds, namely polyphenols. The main goal of this study was to evaluate the phytochemical composition of some commerciallyavailable juices of berries and associating it with their physicochemical, rheological and antioxidant properties. It was observed that in general, the juices analyzed presented great concentrations of polyphenols and anthocyanins; and, in what concerns to their rheological properties, non-newtonian pseudoplastic fluid characteristics (n<1) were observed. The antioxidant properties of the juices were evaluated using both the DPPH free radical scavenging assay and the β-carotene bleaching test. Overall, the results of the % Inhibition of DPPH free radical by the tested juices indicate that they possess considerable capacity to scavenge free radicals, resulting in good antioxidant properties. Introduction The consumption of berries and their contribution to improving the human health is a subject of considerable interest, have already resulted in several research projects and numerous clinical trials in humans [1]. Among all the varieties of berries, the most consumed ones are blackberries, raspberries, blueberries, cranberries, strawberries, and also acai, blackcurrants, chokeberries, elderberries, mulberries and goji berries [1]. Berries provide significant health benefits because of their high levels of bioactive compounds, namely polyphenols, antioxidants, and vitamins [2]. Many polyphenols, individually or combined, possess high antioxidant properties and are of great interest for nutritionists and food technologists due to the opportunity to use them as ingredients for functional foods [3]. Berries are popularly consumed, not only in their fresh and frozen forms, but also as processed and derived products, including dried and canned fruits, yogurts, beverages or juices, jams, and jellies [2]. Juices of berries have become very popular for people due to the consumer demand for healthy nutraceutical foods that could possibly reduce some health risks and improve various health conditions, in addition to their widespread market availability and low price [4]. Thus, the objectives of this study were to evaluate the phytochemical composition of some commerciallyavailable juices of berries associating it with their physicochemical, rheological and antioxidant properties. Material and Methods Juices The juices of berries were acquired commercially in local supermarkets (Covilhã and Lisbon, Portugal) and were randomly numbered (from 1 to 25) to simplify their designation throughout the study. Physicochemical properties ph, density, and total solids A digital ph meter was used to measure the ph values of the juices of berries at 25ºC. The density of the juices was measured by weighing samples of 5 ml of each juice at 25ºC. The total solids were estimated by total evaporation (105ºC, 48 h) of aliquots of 5 ml of each juice, being the results expressed in terms of percentage of total solids relatively to the total mass. Rheological properties The rheological behaviour of the juices of berries was studied with a Haake RS150 rheometer, using a cone/plate geometry of 2º/35 mm, at 25ºC, by varying the shear rate from 0 to 1300 s -1 in 180 seconds. Apparent viscosity (η app) was determined at 1300 s -1. The shear rate versus shear stress was used to calculate the different rheological parameters (consistency coefficient - k, and flow behaviour index - n) using the Haake software, according to the Ostwald-de-Waele model, also known as the power law model, given by the equation [5], [6]: τ = k ẏ n, where τ is the shear stress (Pa); k is the consistency coefficient (Pa.s n ); ẏ is the shear rate (s -1 ), and n is the flow behaviour index. Phytochemical characterization - total reducing sugars, total phenolics, flavonoids and anthocyanins The total reducing sugars were determined by dinitrosalicylic acid (DNS) method [7]. The phenolics were determined by Folin-Ciocalteu s colorimetric method [8]. The aluminum chloride colorimetric method was used to determine the content in flavonoids [8]. Anthocyanins were measured by the ph differential method [9]. 78

89 Antioxidant properties - DPPH free radical scavenging assay and β-carotene bleaching test The DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay was used to evaluate the antioxidant activity of the juices of berries [10]. The β-carotene bleaching test was also employed to evaluate the antioxidant properties of the juices [11]. Statistical analysis The data were analyzed using the statistical program SPSS version 24. The mean values and standard deviation (SD) were calculated for all cases. One-way analysis of variance (ANOVA) was undertaken to test for significant differences among means (p<0.05 was considered significant). Results and Discussion Physicochemical properties The physicochemical properties of the juices of berries considered in this study are reported in Table 1. In general, juices are acidic solutions, presenting ph values lower than Juice 25, composed solely by cranberries, presented the lowest ph value (2.46), which is probably due to the chemical composition of cranberries. Regarding the density of the juices, it was near 1 for all the samples, which is consistent to the aqueous nature of the juices. Concerning total solids, juice 21 presented the highest value (61.25%) indicating the presence of high quantities of organic matter, which is also observed in its density (1.34 g/ml). This juice is a syrup of various berries which may explain the results obtained. Rheological properties The rheological properties of the juices of berries were also evaluated, being the results presented in Table 2. Concerning the values of apparent viscosity (η app), it was found that most of the juices presented low viscosity, with six of them (juices 4, 5, 9, 14, 15 and 21) presenting significantly higher (p<0.05) viscosity than the remaining. In general, the juices that exhibited higher viscosity presented simultaneously higher content in total solids and total reducing sugars. The consistency coefficients (k) and flow behaviour indexes (n) are adequately ascertained employing the Ostwald-de-Waele model, as showed by the regression coefficients (R 2 ) (Table 2). The juices analysed presented non-newtonian pseudoplastic fluid characteristics (n<1), except for juice 21 that had a Newtonian behaviour, since their n is equal to 1. This juice is the one with the highest total solids content (Table 1), which may explain its Newtonian behaviour, caused by the low molar mass of the solutes which constitute it [12]. Different fluid characteristics were detailed in literature for juices and nectars of berries; other researchers had previously verified that raspberry and blueberry juices had predominantly Newtonian behaviour over the studied range of temperature and total solids content [13]. Phytochemical characterization The results of the phytochemical characterization of the juices of berries are summarized in Table 3. Sugar is the general term for a group of sweet-flavored compounds used as food, and a relevant ingredient in many foods and beverages; it also occurs naturally in fruits, vegetables, milk, and honey. Juice 10 presented the lowest concentration of total reducing sugars (59.00 mg GE/100 ml), in contrast with juice 5, which presented the highest concentration ( mg GE/100 ml). However, juice 5 is only made of fresh fruit, indicating that the sugars present are only the naturally-occurring ones. Table 1. Physicochemical properties of the juices of berries studied. Juice No. ph Density (g/ml) Total solids (%) ± 0.01 q 1.01 ± 0.02 ab 1.64 ± 0.01 cd ± 0.01 s 1.02 ± 0.05 ab 2.61 ± 0.01 e ± 0.01 j 1.12 ± 0.02 bc ± 0.02 m ± 0.01 l 1.02 ± 0.04 ab ± 0.25 j ± 0.01 p 0.89 ± 0.08 a ± 0.06 k ± 0.01 f 1.02 ± 0.01 ab 4.83 ± 0.01 g ± 0.01 fg 1.03 ± 0.02 ab 5.06 ± 0.01 g ± 0.01 hj 1.03 ± 0.02 ab 3.75 ± 0.14 f ± 0.01 gh 1.04 ± 0.05 ab 9.30 ± 0.09 i ± 0.01 i 1.02 ± 0.02 ab 2.06 ± 0.01 d ± 0.01 k 1.07 ± 0.02 ab ± 0.01 l ± 0.01 i 1.05 ± 0.02 ab 9.21 ± 0.17 i ± 0.01 o 1.08 ± 0.02 ab ± 0.33 jk ± 0.01 n 1.07 ± 0.03 ab 9.34 ± 0.01 i ± 0.01 m 1.02 ± 0.07 ab ± 0.30 k ± 0.01 n 1.06 ± 0.02 ab ± 0.05 k ± 0.01 e 1.01 ± 0.02 ab 0.77 ± 0.01 a ± 0.01 c 1.02 ± 0.02 ab 4.75 ± 0.06 g ± 0.01 e 1.01 ± 0.02 ab 3.70 ± 0.04 f ± 0.01 d 1.00 ± 0.01 ab 1.19 ± 0.02 abc ± 0.01 b 1.34 ± 0.06 c ± 0.02 n ± 0.01 r 0.99 ± 0.03 ab 1.43 ± 0.01 b ± 0.01 e 1.01 ± 0.01 ab 1.05 ± 0.01 abc ± 0.01 d 1.02 ± 0.01 ab 1.23 ± 0.01 abc ± 0.01 a 1.05 ± 0.01 ab 6.86 ± 0.05 h Data expressed as means ± SD of triplicate assays; Mean values in a column with different letters are significantly different (p<0.05). Juice 12 presented simultaneously the highest concentration of total phenolics, flavonoids and anthocyanins, in strong contrast with juice 6 that had the lowest concentrations. Juice 12 is mainly composed by pomegranate, which is also a polyphenol-rich food and often consumed in combination with berries, whereas juice 6 is mostly constituted by water (88.2%), explaining the lowest values obtained for bioactive phytochemicals. It is difficult to establish a correlation between the phytochemical characterization and the composition of the juices, namely in what concerns to the type of berry, since the juices are a complex mixture of compounds, and 79

90 in many cases a mixture of several types of berries with other fruits, namely apple/pear, which are used as basis for the juices. Data expressed as means ± SD of triplicate assays; Mean values in a column with different letters are significantly different (p<0.05). Antioxidant properties The antioxidant properties of the juices under study were evaluated using both the DPPH free radical scavenging assay, and the β-carotene bleaching test; the results are presented in terms of % Inhibition and are listed on Table 4. Overall, the results of the % Inhibition of DPPH free radical by the tested juices indicate that they possess considerable capacity to scavenge free radicals, resulting in good antioxidant properties. Since berries are claimed to be functional foods, mainly due to their antioxidant bioactive compounds, and being widely used to minimize the side effects of oxidative-stress related pathologies [14], it is now demonstrated that juices of berries maintain the antioxidant properties attributed to berries. Juice 3, supplemented with resveratrol, presented the highest % Inhibition of DPPH radicals (99.54%). In contrast, juice 6 showed the poorest antioxidant activity as measured by the DPPH method (% Inhibition=23.53%), but this juice is mostly made of water, presenting low quantities of bioactive phytochemicals. Another relevant result was obtained for juice 22 (% Inhibition=96.05%), which is composed by cranberries, indicating that this type of berries possesses remarkable antioxidant properties. Table 2. Rheological properties of the juices of berries studied. Apparent Consistency Flow Regression Juice viscosity ηapp coefficient - k behavior index coefficient No. Table 3. Phytochemical characterization of the juices of berries (Pa.s) 10-3 (Pa.s n ) - n - R 2 considered in this study ± a ± a ± Total Total reducing Flavonoids Anthocyanins ± a ± a ± Juice phenolics sugars (mg (mg QE/100 (mg cy-3-glu ± a ± a ± No. (mg GAE/100 GE/100 ml) ml) Eq/100 ml) ± e ± d ± ml) ± f ± e ± ± 1.41 bc ± 8.89 f 5.28 ± 0.64 c 0.00 ± 0.00 a ± a ± a ± ± 4.24 de ± g 9.94 ± 0.41 e 0.00 ± 0.00 a ± a ± a ± ± i ± g ± 0.59 hi 0.22 ± 0.09 a ± a ± a ± ± l ± c ± 0.27 i 6.00 ± 0.69 e ± b ± a ± ± m ± c ± 0.07 gh 6.59 ± 0.35 ef ± a ± a ± ± 2.83 a ± 0.64 a 0.68 ± 0.04 a 0.88 ± 0.06 a ± a ± a ± ± 4.24 abc ± 2.08 a 1.84 ± 0.07 ab 1.35 ± 0.05 ab ± a ± a ± ± 5.66 de ± 0.16 b 6.46 ± 0.14 c 3.93 ± 0.44 d ± a ± a ± ± h ± 9.82 c ± 0.96 f 1.44 ± 0.18 ab ± c ± bc ± ± 1.41 a ± 3.69 b 7.93 ± 0.35 d 0.83 ± 0.08 a ± c ± c ± ± 1.41 e ± 6.76 d ± 0.27 g ± 0.64 g ± a ± a ± ± 1.41 f ± 9.49 h ± 0.77 i ± 0.45 g ± a ± a ± ± 7.07 j ± 8.01 e ± 0.43 gh 4.09 ± 0.06 d ± a ± a ± ± l ± 0.31 e ± 0.98 f 8.30 ± 0.60 f ± a ± a ± ± k ± 5.58 c ± 0.54 i 3.72 ± 0.61 cd ± a ± a ± ± 7.07 g ± 0.64 b 9.97 ± 0.09 e 0.00 ± 0.00 a ± d ± a ± ± 1.41 a ± 0.47 a 1.84 ± 0.06 ab 1.54 ± 0.00 ab ± a ± ab ± ± 1.41 a ± 0.60 a 2.78 ± 0.08 ab 0.70 ± 0.02 a ± a ± a ± ± c ± 1.00 a 3.72 ± 0.08 b 1.52 ± 0.08 ab ± a ± a ± ± 1.41 a ± 0.69 a 5.35 ± 0.09 c 1.98 ± 0.09 bc ± a ± a ± ± 7.07 bc ± 1.61 a 3.81 ± 0.34 b 2.99 ± 0.05 bcd ± 4.24 bc ± 0.99 a 2.48 ± 0.12 ab 0.14 ± 0.03 a ± 5.66 bc ± 0.53 a 2.03 ± 0.06 ab 0.87 ± 0.02 a ± 2.83 bc ± 1.46 a 2.02 ± 0.18 ab 0.50 ± 0.06 a ± 5.66 d ± 1.90 bc ± 0.45 e 3.50 ± 0.05 cd GE glucose equivalents; GAE gallic acid equivalents; QE quercetin equivalents; cy-3-glu Eq cyanidin-3-glucoside equivalents; Data expressed as means ± SD of triplicate assays; Mean values in a column with different letters are significantly different (p<0.05). Besides the DPPH method, the β-carotene bleaching test was also employed to evaluate the antioxidant activity of the juices. Regarding the results obtained with this method (Table 4), it was possible to verify that in general the antioxidant activity is weaker than that measured by the DPPH free radical scavenging assay. Juice 23 possesses no capacity to inhibit the oxidation of β- carotene, indicating that it is not able to inhibit the lipid peroxidation cascade reactions. In contrast, juice 2 presented 89.60% of % Inhibition, being this the highest 80

91 measured value of inhibition. In addition to several berries, this juice is also made with pomegranate and grape seeds, a mixture of known antioxidant foods. Table 4. Antioxidant properties of the juices of berries considered. Juice No. DPPH free radical scavenging assay β-carotene bleaching test % Inhibition % Inhibition ± 0.17 ghi ± 1.85 j ± 0.37 c ± 0.39 l ± 0.14 kj ± 1.00 e ± 1.04 b ± 0.33 j ± 0.13 f ± 1.22 l ± 0.43 a ± 0.31 e ± 0.08 d ± 0.36 h ± 0.57 f ± 0.66 g ± 1.67 b ± 0.19 h ± 0.07 gij 8.82 ± 1.15 b ± 0.25 f ± 0.33 e ± 0.69 fg ± 0.65 c ± 0.41 e ± 0.57 d ± 1.98 f ± 1.11 f ± 1.75 b ± 0.76 f ± 1.42 f 0.57 ± 0.05 a ± 0.89 a 5.91 ± 0.55 b ± 0.49 fgh 1.76 ± 0.22 a ± 1.19 c 0.37 ± 0.09 a ± 1.80 d ± 1.60 g ± 0.11 b 3.38 ± 0.52 b ± 1.40 ij ± 1.70 i ± 1.37 f 0.00 ± 0.00 a ± 0.76 e ± 1.34 d ± 0.87 fgh ± 0.86 c Data expressed as means ± SD of triplicate assays; Mean values in a column with different letters are significantly different (p<0.05). Conclusions This study demonstrated that the physicochemical properties of the juices influence their rheological behavior; and the phytochemical composition impacts their antioxidant properties. This study also confirmed the potential health benefits of berries and berries-derived products, namely juices, that maintain the bioactive phytochemicals and the antioxidant properties of the original berries. Acknowledgements Ângelo Luís acknowledges the Post-Doc research fellowship within the scope of the protocol signed between Universidade da Beira Interior and bank Santander/Totta with the reference BIPD/ICI-FC-BST- UBI References [1] A. Basu, M. Rhone, T. Lyons, Berries: emerging impact on cardiovascular health, Nutr. Rev. 68 (2010) [2] S. Nile, S. Park, Edible berries: Bioactive components and their effect on human health, Nutrition 30 (2014) [3] S. Skrovankova, D. Sumczynski, J. Mlcek, T. Jurikova, J. Sochor, Bioactive compounds and antioxidant activity in different types of berries, Int. J. Mol. Sci. 16 (2015) [4] P. Pinto, S. Cardoso, R. Ferreira, C. Santos, Daily polyphenol intake from fresh fruits in Portugal: contribution from berry fruits, Int. J. Food Sci. Nutr. 64 (2013) [5] M. Šimunek, A. Jambrak, S. Dobrović, Z. Herceg, T. Vukušić, Rheological properties of ultrasound treated apple, cranberry and blueberry juice and nectar, J. Food Sci. Technol. 51 (2014) [6] S. Manjunatha, P. Raju, A. Bawa, Modelling the Rheological Behaviour of Enzyme Clarified Lime (Citrus aurantifolia L.) Juice Concentrate, Czech J. Food Sci. 30 (2005) [7] K. Baskan, E. Tütem, E. Akyüz, S. Özen, R. Apak, Spectrophotometric total reducing sugars assay based on cupric reduction, Talanta 147 (2016) [8] Â. Luís, D. Neiva, H. Pereira, J. Gominho, F. Domingues, A.P. Duarte, Stumps of Eucalyptus globulus as a Source of Antioxidant and Antimicrobial Polyphenols, Molecules 19 (2014) [9] J. Lee, R. Durst, U. Trenn, J. Wightman, Determination of Total Monomeric Anthocyanin Pigment Content of Fruit Juices, Beverages, Natural Colorants, and Wines by the ph Differential Method: Collaborative Study, J. AOAC Int. 88 (2005) [10] M. Colon, C. Nerín, Synergistic, antagonistic and additive interactions of green tea polyphenols, Eur. Food Res. Technol. 242 (2016) [11] Â. Luís, A.P. Duarte, F. Domingues, Bioactive Compounds, RP-HPLC Analysis of Phenolics, and Antioxidant Activity of Some Portuguese Shrub Species Extracts, Nat. Prod. Commun. 6 (2011) [12] Z. Filho, V. Telis, E. Oliveira, J. Coimbra, J. Romero, Rheology and fluid dynamics properties of sugarcane juice, Biochem. Eng. J. 53 (2011) [13] C. Nindo, J. Tang, J. Powers, P. Singh, Viscosity of blueberry and raspberry juices for processing applications, J. Food Eng. 69 (2005) [14] C. Céspedes, M. Morales, J. Alarcón, O. López, Phytochemical profile and the antioxidant activity of Chilean wild black-berry fruits, Aristotelia chilensis (Elaeocarpaceae), Food Chem. 119 (2010)

92 Assessment of Cork Boiling Wastewater Treatment by Constructed Wetland at Horizontal Subsurface Flow Configuration A.C. Gomes 1, M.L. Silva 1, A. Stefanakis 2, R. Simões 1, A. Albuquerque 3 1 FibEnTech-UBI, Department of Chemistry, Universidade da Beira interior, Covilhã, Portugal 2 Bauer Resources GmbH 3 FibEnTech-UBI, Department of Civil Engineering and Architecture, Universidade da Beira Interior, Covilhã, Portugal Abstract Cork boiling wastewater (CBW) purification by conventional biological treatment is a challenging task due to high organic load (average COD of 1623 mg/l), intense dark color and low biodegradability (average BOD 5/COD of 0.19). Constructed wetlands (CW) with horizontal subsurface flow (HSF) using LECA as substrate material and planted with Phragmites australis were successfully used for the first time over a 4-years period and organic loading rates (OLRs) of g COD/m 2 /d corresponding to eight stepwise increments and operation periods. The average removals of the planted unit exceeded those of the control (unplanted) and were of 53.4%, 83.3% and 43.6% for COD, BOD 5 and total phenolic compounds (TPh) and corresponded to mass removals rates (in g/m 2 /d) up to 10.20±2.51, 1.69±0.24 and 0.47±0.08, respectively. The main drawback of operation was the decolourization limited to 34.8% and in some periods, had negative to small reductions, possibly because it was dependent on physical-processes rather than on biodegradation. Overall, HSFCW can be an appropriate and affordable alternative to conventional biological treatments for the cork processing industry. Introduction Cork is a unique natural material extracted from the bark of the cork oak (Quercus suber L.), mainly used to produce cork stoppers for high quality wines. The production and extraction cycles are strictly regulated to ensure a life span production over 200 years and ultimately contribute for the preservation of more than 2.2 million hectares of natural forest ecosystem typical of the Mediterranean region [1]. Portugal is the world-leading producer and exporter of cork and cork products. Portuguese industries processes over 61% of the world cork production, estimated to surpass ton per year [1]. Therefore, both activities have significant social and economic importance. However, the first stage of the industrial processing of the cork slabs is in most cases done accordingly the traditional procedure through immersion in boiling water for up to 1.5 hours. Despite production being an example of sustainability between human activity and natural resources preservation, this first stage of cork processing consumes up to 1200 L of high quality fresh water per ton of corkwood. Moreover, cork boiling wastewater (CBW) main features are the intense dark-brown color, high organic load of biorecalcitrant nature, which includes phenolic compounds some of them having high molecular size (over 100 kda) [2]. The results of CBW characterization were recently revised [3]. The reduced biodegradability of 0.19 for the biological oxygen demand after 5-days incubation (BOD 5) to chemical oxygen demand (COD) ratio, restrains the feasibility of conventional biological treatment processes to COD removals of 13-40% by activated sludge and anaerobic digestion processes [4,5]. The alternatives provide by chemical oxidation technologies, namely ozone alone or mixtures with hydrogen peroxide and associated with UV radiation, Fenton and solar photo- Fenton, electrochemical oxidation, etc., can raise the COD and colour removals up to 95% according conditions, but they are not economical feasible [3]. Hence, there is an opportunity to try an eco-friendly approach to the purification of CBW based on constructed wetland (CW) technology, which have unique advantages over the conventional biological treatments, namely low-cost, lowtechnology and simple operation/maintenance, low to nil energy requirements and environmental friendliness of being plant-based operations [6]. Over the last five decades the scope of CW application increased from domestic to include industrial wastewaters as it is the case of those releases by agro-industries, namely wineries and distilleries, olive mill and coffee processing [6]. Moreover, some of these effluents share with CBW the high concentration of phenolic compounds, namely of gallic, caffeic, ferrulic, syringic acids. The success of CWs operation relies on complex physical, chemical and biological array of processes occurring simultaneously at different locations of the bed and under diverse conditions, namely for oxygen availability. This large array of conditions also contributes to increase the diversity of the microbial activities, namely of those contributing to biological up-take, bio-transformation, cometabolism, nitrification, denitrification, etc.. Overall, the diversity of conditions and of processes contributing to wastewater purification is advantageous in the case of complex compositions, as it is the case of CBW. The aim of our research was to investigate the feasibility and efficiency of CBW depuration through biological treatment provides by CW at horizontal subsurface flow HSFCW filled with LECA and planted with Phragmites 82

93 australis. To assess the contribution of phytoremediation to the overall organic load removal we compared the performance of two microcosms: one planted and other unplanted, used as control. The acclimation and adaptation of both systems biomass to the effluent composition was carefully done and included eight increments of the organic loading rates (OLRs) corresponding to equal number of operation periods (P1 P8) over a 47-months period. The removals efficiencies of both systems were monitored for COD, BOD, TPh and colour. This investigation is an absolute novelty and intends contribute to overcome the difficulty of conventional biological treatment to fulfil the quality standards regarding discharge or reuse of cork processing wastewaters. Materials and Methods Two microcosm-scale HSFCWs made of PVC were used, one planted (CWP) and other left unplanted (CWC) allowing results comparison and assessment of plants contribution to remediation (Fig. 1). The microcosms dimensions, location and operation conditions during colonization were described elsewhere [7]. The results for CBW samples characterization are in Table 1. Table 1. CBW average characteristics. Values represent the average and standard deviation (±SD) for n=7 samples. Parameter (units) 1 Raw CBW ph ( ) 4.66±0.15 A580 ( ) 0.115±0.014 COD (mg/l) 1625±123 BOD5 (mg/l) 304±48 BOD20 (mg/l) 471±62 TPh (mg/l) 120±17 1 A580 absorbance at 580 nm for dilutions (1:10); TPh total phenolic compounds concentration expressed in terms of tannic acid concentration equivalent. The microcosms were monitored at least once a week through collection of samples from inlet, outlet and sampling points and analyzed for ph, oxidation-reduction potential (Eh), absorbance measurements at 580 nm (A 580) for 1:10 V/V dilutions and used for color quantification, COD, BOD after 5 and 20-days incubation period. These determinations were made according Standard Methods [8]. The total phenolic compounds (TPh) content quantification was made by the Folin-Ciocaulteu method and the results reported as mg/l of tannic acid equivalents determined from a linear regression correlating concentration and absorbance at 765 nm [9]. The main feature of CWs operation was the hydraulic retention times (HRT) above 5 days (average of 5.52±0.34 d) required for operation stability with low biodegradable effluents; as consequence of this the eight step-wise increment of the average organic loading rates (OLR) was implemented by consecutively feeding units with less diluted CBW. The inlet solutions ph was set to before being supplied. Additionally, during cold seasons a thermostat ensured inside temperatures in the range of 20±2ºC. During the overall operation period none intervention was done for harvest or to control plants growth because of the indoor operation, which avoid complete decay of green leaves even during cold seasons. Fig. 1. Photo of the microcosms at operation location. These samples were collected at an industry located in the Portalegre district (Portugal) at the exit of an equalization/homogenization tank used to feed a coagulation/flocculation system operated as pre-treatment before discharge into municipal collector. The CWs small size and their location, allowing close to green-house conditions, resulted in significant amounts of water losses through evaporation and evapotranspiration with the CWC and CWP operation, respectively. Thus, the removals efficiencies of COD, BOD and TPh were calculated by mass balances and expressed in percentages. The differences between inflow and outflow loads allowed the calculation of the mass removal rates (MRRs) in g/m 2 /d. To assess color removal the difference of A 580 were converted in percentage after correction of the amount of water lost. Results and discussion CBW and Inlet solutions characterization The results for CBW characterization (Table 1) were in line with results previously reported for samples collected at the same location [2,3] and from other sources [3]. The intense dark-brown color was previously correlated with 83

94 high molecular size compounds [2] and the TPh concentration of 120±17 mg/l restrained the biodegradability to 0.19±0.03 for commonly used index of BOD 5/COD. The range of COD of the inlets was from 348±9 up to 1367±51 mg/l for Phase 1 (P1) and Phase 8 (P8) operation periods, respectively. Moreover, the biodegradability ranged from 0.17±0.02 to 0.35±0.03 for P8 and P1, respectively. Overall, these values of BOD 5/COD were always minors than recommended limit of 0.40 for successful implementation of biological treatment processes [10]. Environmental and operation conditions The main operational parameters and environmental conditions average values for the overall operation were of d, L/m 2 /d and ºC for the HRT, hydraulic loading rate (HLR) and room temperature, respectively. The range of the inside temperatures was reduced to due to thermostat operation during cold seasons. Additionally, the beds location provide conditions close to greenhouse for solar exposition but without temperature control, thus, allowing the mitigation of the outside temperature variation. Anyway, the average water loss at the CWC was of 1.22±0.87 L/m 2 /d and of 3.23±1.62 L/m 2 /d, thus, justifying the removals performance evaluation through mass balances. In both cases the water balances provide results correlated with room temperature. The length of each operation phase varied according the elapsed time required to have steady results for COD, BOD 5 and TPh MRRs. The COD, BOD 5 and TPh average OLRs (in g/m 2 /d) increased from P1 to P8, being of , and , respectively. Despite the weekly control of microcosms, the variation of the OLRs in all operation periods was essentially a consequence of the use of several CBW samples to prepare the inlet solutions and variation of the flow rates provide by the pumps. The measurements of Eh in sampling points along the beds resulted in values showing that oxygen availability at the CWP was always above those at the CWC. At the middle of the CWP bed the Eh average values were of - 42±67 mv by comparison with -99±39 mv at the CWC. Taking into consideration the Eh values superior to +100 mv correspond to aerobic conditions and inferior to anaerobic conditions [6]. Thus, plants provide an additional supply of oxygen for biodegradation and increased the variety of processes. Interestingly, the Eh values at the CWP were correlated with plant activity through water losses. Removals efficiencies The removals efficiencies accomplished by the CWP exceeded those of the unplanted unit over the entire operation period (P1-P8). The values of the CWP ranged from 61-91% for COD, 86-96% for BOD 5 and 52-89% for TPh. The linear correlation between OLRs and corresponding MRRs for COD, BOD 5 and TPh with the CWP unit had correlation coefficients above 0.95 indicating the overall removal efficiencies capacity of each CW unit. Despite the small correlation with the values of the control unit, above 0.91, in mean terms the CWC was able to remove 45.7%, 76.0% and 29.5% of the COD, BOD 5 and TPh load. The performance of the CWP unit was clearly above, with average removals of 53.4%, 83.3% and 43.6%, respectively. The results for COD are presented in Fig. 2 for elucidation. Fig. 2. Correlation between the average OLRs and MRRs for COD during the overall operation with CWP. These patterns are commonly reported for biodegradation processes, including for several CW operation conditions, corresponding to a positive reaction order for the kinetics, showing that organic removals are proportional to inflow load. Regarding the comparison of the HSFCW average removal performance for COD with the alternative biological treatments previously reported the advantage of the CW is clear. For operations with HRT of 5 days the COD removal of 53.4% is above those of activated sludge-system with 13-37% and HRT of 1-4 d, and 36-40% for anaerobic digestion for operations with HRT of 15 d [4,5]. The extensive growth of plants rhizomes observed with elapsed time (Fig. 2) in addition to evapotranspiration contributed to increase the HRT and provided additional surface for interactions with pollutants and biofilm development; it is also an indication that Phragmities plants growth and physiological conditions were not negatively influenced by CBW characteristics. For operation conditions with OLR limited to 5.0 g COD/m 2 /d it was possible to have an effluent fulfilling the legal requirements for discharge, namely for COD (<150 mg/l), BOD 5 (<40 mg/l) and ph. However, descolourization had great variations, which include negative removals, and in all operation periods the effluents remained clearly visible. Consequently, colour removal was not dependent on biological processes, which performed well but on physical processes, namely adsorption, sedimentation and precipitation. This 84

95 throwback can be a consequence of the high molecular size of the compounds contributing to CBW color, with those over 100 kda accounting to 72% of color [2]. The sensitivity of these processes to operational conditions variation and their reversible nature can justify the variability of color removal reported. Fig. 2. Extensive growth and development of Phragmities plants roots after 4-years operation with CBW. CBW treatment and valorization are active fields of research at the FibEnTech, namely by studying chemical oxidation processes, membrane technologies to be sequentially associated to HSFCW and the recovery of valuable phenolic compounds having biological and antioxidant activities. Conclusions HSFCW can be a successful and affordable approach for the purification of CBW. The significant land requirement and the lack of performance under cold conditions limitation can be overcome because most of the cork processing units are located in the Iberian Peninsula, thus under warm climatic conditions in regions where large land areas are available. The extended range of time and of operational conditions allowed concluding that plants contributed to CBW purification by supplying additional oxygen and extended range of biodegradation processes. Thus, after 4-years operation and OLR ranging from g COD/m 2 /d the MRRs (in g/m 2 /d) achieved by the CWP exceeded those of the control unit and were up to 10.20, 1.69 and 0.47 for COD, BOD 5 and TPh, respectively. These performances were above those previously reported for conventional aerobic and anaerobic treatments and achieved at more interesting operation conditions. Acknowledgements The authors are grateful to COMPETE and to FEDER for the financial support of the project (PTDC/AGR- AAM/102042/2008) and to the FibEnTech Materiais Fibrosos e Tecnologias Ambientais research unit for the use of laboratories and equipment. References [1] APCOR. APCOR s Cork Yearbook Available at [2] D.C. Santos, L. Silva, A. Albuquerque, R. Simões, A.C. Gomes, Biodegradability enhancement and detoxification of cork processing wastewater molecular size fractions by ozone, Bioresource Technology 147 (2013) [3] A. Fernandes, D.C. Santos, M.J. Pacheco, L. Ciríaco, R. Simões, A.C. Gomes, A. Lopes, Electrochemical treatment of cork boiling wastewater with a boron-doped diamond anode, Environmental Technology 36 (2015) [4] F.J. Benitez, J.L. Acero, J. Garcia, A.I. Leal, Purification of cork processing wastewaters by ozone, by activated sludge and their two sequential applications, Water Research 37 (2003) [5] I.P. Marques, L. Gil, F. La Cara, Energetic and biochemical valorization of cork boiling wastewater by anaerobic digestion, Biotechnology and Biofuels 7 (2014) [6] S. Wu, S. Wallace, H. Brix, P. Kuschk, W.K. Kirui, F. Masi, R. Dong, Review: Treatment of industrial effluents in constructed wetlands: Challenges, operation strategies and overall performance. Environmental Pollution 201 (2015) [7] A.C. Gomes, L. Silva, A. Stefanakis, A. Albuquerque, R. Simões, Assessement of cork boiling wastewater treatment through horizontal subsurface flow constructed wetlands, submitted Ecological Engineering (2017). [8] APHA, Standard Methods for the Examination of Water and Waste-water, 20th ed. American Public Health Association, American Waterworks Association and Water Environmental Federation, Washington, DC, USA. [9] E.A. Ainsworth, K. Gilliespie, Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent. Nature Protocols 2 (2007) [10] Metcalf and Eddy, Wastewater Engineering: Treatment and Reuse, fourth ed. McGraw-Hill, New York, USA. The main drawback of HSFCW operation was the high variation of decolorisation achieved, with average values for the overall operation of 34±45%, which resulted in outflows clearly colored. 85

96 Development of sustainable antimalarial clothing C. I. S. Pinheiro 1(*), Nuno J. R. Belino 1, Roshan Paul 2 1 Universidade da Beira Interior, Faculty of Engineering, Department of Textile Science and Technologies, Laboratory, Covilhã, Portugal. 3 Indo-German Science and Technology Centre, New Delhi, India (*) cisp@ubi.pt Portugal Abstract Today, more than ever, the so called sub-tropical diseases exert a major influence in the quality of life and in the socioeconomic development of a large part of human population. The recent increased resistance to drugs (medicines and repellent agents) and climate changes led to important environmental changes and, consequently, to the spreading of those diseases into new geographical areas, particularly in the outskirts of Europe, never before directly affected by them. Malaria is one of such diseases. It is caused by a certain type of a plasmodium parasite and is transmitted by the female mosquito bite of the genre anopheles. This pathology has a tremendous impact in human health - three millions worldwide deaths per year and causes significant economic losses. Being aware of this situation, authors envisaged a new technological solution, completely biodegradable, to contribute for the minimization of this problem. Bearing this purpose in mind, this ongoing PhD thesis aim at the development of a new piece of cloth made with an entirely new family of PLA fibres, specifically designed. Those fibres were embedded with internally developed microcapsules containing a natural repellent agent added during the extrusion process. Based upon this new fibre, three knit structures were produced and the repellent efficacy and washing resistance were assessed. Introduction PLA fibers are increasingly penetrating markets traditionally occupied by petroleum-based synthetics. Poly(lactic acid) is a synthetic polyester derived from renewable sources such as corn. PLA has been used for many years in biomedical applications such as drug release systems, medical implants and surgical sutures because of its biodegradability and biocompatibility.. Materials Considering the necessity of having a fully biodegradable fiber which was capable to endure the thermal and rheological conditions of a melting extrusion process we selected the Biopolymer Ingeo 6201D Fiber Grade PLA from NatureWorks. The main characteristics of the aforementioned polymer are depicted in table1. Table 1 Typical PLA Ingeo 6201D Properties PHYSICAL PROPERTIES METRIC COMMENTS Specific Gravity 1,24 g/cm 3 ASTM D792 Melt Density 1,08 g/cm 3 at 230ºc ASTM D1238 Viscosity Measurement 3,1 Relative viscosity; CD Internal Viscotek method Melt Flow g/10 min at load 2,16 Kg and temperature 210 ASTM D1238 ºC THERMAL PROPERTIES METRIC COMMENTS Elongation at Break 10%-70% ASTM D2256/ASTM D3822 Tenacity 0,221-0,441 N/Tex ASTM D2256/ASTM D3822 THERMAL PROPERTIES METRIC COMMENTS Melting Point 160 ºC-170 ºC ASTM D3418 Glass Transition Temp. 55 ºC-60 ºC ASTM D3417 Shrinkage 5%-15% ASTM D32102 Boiling water Different microcapsules and nanocapsules weretested. To fill those capsules, two chemical agents were used: the first one was non-natural mosquito repellent considered to be the best option for clothing and outdoor. The second one, was the natural agent we wish to introduce and whose mosquito repellent efficacy we want to study by comparison with the non-natural standard one. Method The encapsulation of both chemical agents took place individually and were carried out through the adsorption method. Quality control tests such as DSL, TGA, FTIR and SEM, were conducted in order to characterize the obtained microcapsules. 86

97 PLA multifilament with incorporated microcapsules containing a chemical agent were extruded resorting to a 21mm twin screw extruder from Randall Technology Lta. The biopolymer pellets and the synthetized microcapsules were added through independent gravimetric feeders. The linear density, tenacity, elongation at rupture and work at rupture were determined. Conclusions This is an ongoing PhD thesis which is not completed and therefore results and conclusions are not yet possible References [1]-Diaz, J. H. (2016). Chemical and plant-based insect repellents: Efficacy, safety, and toxicity. Wilderness and Environmental Medicine, 27(1), [2]-Paul, Roshan - Ballistic and impact protection finishes for textiles. Em Functional Finishes for Textiles Hohenstein Institut für Textilinnovation ggmbh, Boennigheim Germany 1 : Elsevier, 2015 [3]-Quian L., Hinestroza, J.P. (2004). Application of nanotecnology for high performace textiles. Jorurnal of textile and apparel, technology and management. Vol.4 issue 1,summer. Figure 1. Extrusão de Multifilamentos de PLA encapsulados. Based upon those multifilament and with a Flat V-Bed Electronic Shima Seiki machine model SES 122FF three different knit structures were produced: Jersey; single piqué and French piqué. Washing fastness and mosquito repellent efficacy using the arm-in-cage method as described in the protocol WHO/CDS/NTD/WHOPES/ are to be done. [4]-WHO (World Health Organization). (2015). Who Report Geneva,27 Switzerland. Circular Jacquard Loom Figure 2. Circular Jacquard Loom. 87

98 A preliminary study on the bioactive compounds present in the leaves and stems of Urtica dioica, L. M.F.C. Tenreiro 1, M.E. Amaral 1,2 1 Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal; 2 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal Abstract Urtica dioica, L., commonly known as nettle, belongs to the Urticaceae family, encompass several genera, among which the Urtica genus, referenced by possess interesting therapeutic properties. The knowledge about the potential of bioactive compounds present in the leaves and stems of nettle was the main focus of this work. A phytochemical screening allowed to realize the qualitative bioactive compounds present in the plant under study, using staining and precipitation reactions. The main studied compounds, using a quantitative methodology, were the total phenolics and flavonoids and it was also performed the analysis of antioxidant activity. The Folin-Ciocalteu s method was used to determine total phenolics, while a colorimetric method with the aluminum chloride was used for the determination of total flavonoids. The antioxidant activity was evaluated using DPPH (2,2-diphenyl-1- picrylhydrazyl) radical scavenging assay, reported as IC 50 and antioxidant activity index (IAA). The highest concentration of total phenolics (40.57 mg of gallic acid equivalents/g dry matter) and flavonoids (48.50 milligrams of quercetin equivalents/gram dry matter) were found in leaves using the Soxhlet extraction. The samples showed a low antioxidant potential, given the values obtained for the antioxidant activity index, respectively, 0.14 for the leaves and 0.13 to the stems. This might have occurred because the plant was subject to soil and climatic factors somewhat unusual for the current period of the year. Introduction The therapeutic values of natural products have awakened worldwide attention [1,2]. Nature provides mankind with a vast therapeutic flora with a wide variety of medicinal potential [3]. In this context, the deepening of the knowledge of the physicochemical, pharmacological and nutritional properties of various vegetable species has been object of new studies by pharmaceutical companies, study centers and even confraternities in order to perpetuate its more traditional side [4]. The Urticaceae family includes several genera with traditionally known utilities, among which the Urtica genus is subdivided into species that are referenced because they have interesting therapeutic properties [5,6,7]. Native to Eurasia, Urtica dioica, L. commonly known by nettle [5] is reported by Hipocrates ( BC) in 61 remedies using this plant. Traditionally, this herb is used for the treatment of arthritis, joints and muscles rheumatism, and a study by [8] proves its anti-inflammatory activity. Used as a component of teas for diabetics [9] their hypoglycemic activity is shown later in the work [10,11]. The aqueous and ethanolic extract of nettle were able to reduce cholesterol levels in rats submitted to a hyperlipidic diet with no hepatic changes [12]. A human study using nettle root extract has proven its effect on benign prostatic hyperplasia [7,13,14]. On the other hand, an in vitro study using hydro-alcoholic extracts demonstrated an antinociceptive and anti-inflammatory activity through human abdominal pain mimicking. Of note that these extracts had a high content of flavonoids and polyphenols [14]. A strong antioxidant activity compared to α-tocopherol was demonstrated in leaf extracts using ethyl acetate as the solvent [16,17]. The presence of flavonoids, coumarins, phenolic acids, sterols, carotenoids, amino acids, vitamin K and various minerals such as calcium, copper, iron, magnesium, phosphorus, potassium and sodium [9] are reported in the chemical composition. Plants have, in addition to the primary metabolism, present in all living beings, a secondary metabolism that allows them to produce and accumulate compounds of diverse chemical nature [18]. The compounds involved in the primary metabolism have universal distribution, the secondary metabolites are characteristic of a botanical family and are divided into three main groups: terpenes, phenolic compounds and alkaloids [19]. The presence of these bioactive compounds in plants is closely related to external or abiotic factors (environmental and ecological) and internal or biotic factors (genetic, ontogenic and physiological) [18,19]. This work has as main objective to quantify some of the bioactive compounds present in the aerial parts of the nettle, as well as the analysis of its antioxidant activity. The study began with a phytochemical prospection to evaluate the qualitative composition of the different crude extracts of the leaves, using color and precipitation reactions to identify mainly the major secondary metabolites. Subsequently, crude ethanolic extracts were obtained from the stems and leaves of this plant, using two different extraction processes, in which the total phenols, flavonoids and their antioxidant activity were determined. This work was developed in scope of graduation project in Medicinal Chemistry. Why the nettle? in the municipality of Fornos de Algodres, where I am inserted, there is the Confraria da Urtiga, which has an interest to renew its use in traditional food, as well as to extend knowledge in other applications areas. Materials and Methods Plant material The aerial parts of the nettle were harvested in Serra da Esgalhada, with the following coordinates: N40º

99 'W007º32.045', after flowering season, in January The plant was dried at room temperature, in a ventilated place with no direct incidence of light. The aerial part of the plant was separated by leaves and stems. The material was pulverized and passed through a 0.25 mm screen, in order to homogenize the granulometry. After the dry matter content (TMS) was determined using an infrared (IR) scale. For this, a small amount of the powder sample was placed and by IV scanning for 30 minutes at 105 C, weight loss of the material was monitored and the TMS was visualized. This determination will serve to correct the value despite the moisture content of the samples, obtaining an absolutely dry mass (a.d.). Phytochemical analysis The general phytochemical prospection was carried out according to the scheme proposed by J.B. Harborne (1984)[20] and allowed to know in a qualitative way, using color and precipitation reactions, the bioactive compounds present in the leaves. Extract preparation Three solid-liquid extractions were performed using ethanol as the solvent. One was used to the extraction process in Soxhlet and, in the other two, to the ultrasounds for different times of extraction. The leaves and stems were placed in cellulose cartridges and fed to the Soxhlet extractor. Then, ethanol was added to the round bottom flasks and the solvent was removed until the solvent in the Soxhlet extractor became colorless. The extraction was done in duplicate for each of the samples. After extraction, the extract was poured several times into a round-bottomed flask (weighed and weighed until constant mass) and brought to the rotary evaporator, with a 40 C bath and a pressure of 175 mbar, for complete volatilization. Finally, it was placed in the oven at 30º C until a constant mass was obtained, whose value is accepted and the yield of each extraction is calculated (see table 1). To the leaf and stem samples were added 50 ml of ethanol and sonicated with a controlled temperature within the erlenmeyer at C. The ethanol was decanted and an additional 50 ml of ethanol was added under the same conditions. Two extractions with different times were made, one with two 15 minutes cycles and one with two 30 minutes cycles. After extraction, proceed as described above, evaluating the extraction yield (table 1). The methods of analysis that will be used, require the solvent to be methanol, whereby the extracts have been re-diluted in this solvent. For each extract sample in methanol its concentration was calculated. Determination of total phenolic content The total phenolic content of the extract was determined by Folin-Ciocalteu s colorimetric method and the procedure was performed according to Luís et al., Thus, 50 μl of the above solutions was added 450 μl of distilled water, 2.5 ml of 0.2 N Folin-Ciocalteu reagent (diluted in distilled water from a commercial solution with 2 N concentration), and then allowed to react (at room temperature) for 5 minutes. After this time, 2 ml of a 75 g/l aqueous sodium carbonate solution was added and allowed to react in a water bath at 30 C for 1.5 hours with intermittent stirring. The blank was carried out in the same way, but replacing the initial 50 μl by 50 μl of methanol. After the reaction time the absorbances of the mixtures were read at 765 nm using a spectrophotometer. The standard curve was constructed with a control phenolic standard, namely gallic acid. Thus, solutions of gallic acid in methanol are prepared in different concentrations ranging from 25 to 500 mg/l and absorvances were read in order to construct the standard curve (y = 0,0012.x; R 2 = 0,9942). Results were expressed as gallic acid equivalents (gallic acid in mg/g of dry mass). These assays were conducted in triplicate. Determination of total flavonoid content The total flavonoid content was evaluated by colorimetric method as described in literature [21]. Each methanol extract (500 μl) was added 1.5 ml of methanol, 0.1 ml of 10% aqueous aluminum chloride solution, 0.1 ml of 1M aqueous potassium acetate solution and 2.8 ml of distilled water. It was allowed to react at room temperature with intermittent stirring for thirty minutes. In the blank, proceed as described above, replacing the initial 500 μl by 500 μl of methanol. After this time the absorbances were read at 415 nm using a spectrophotometer. The standard curve was made using quercetin solutions at concentrations ranging from 12.5 to 200 μl / ml in methanol (y= 0,0093.x; R 2 = 0,9982). Results were expressed as quercetin equivalents (quercetin in mg/ g of dry mass). These analyses were made in triplicate. Evaluation of antioxidant activity- DPPH scavenging assay The antioxidant activity of the extracts and standards was evaluated by the radical scavenging activity method using the 2,2- diphenyl-1-picrylhydrazyl (DPPH) radical, as previously described by Scherer & Godoy (2009)[22]. This method was applied for all samples and standard solutions. The radical scavenging activity was calculated as follows: I % = [(Abs 0 - Abs 1) / Abs 0] 100, where Abs 0 was the absorbance of the blank and Abs 1 was the absorbance in the presence of the test compound at different concentrations. The IC 50 (concentration providing 50% inhibition of DPPH radicals) was calculated graphically using a calibration curve in the linear range by plotting the extract concentration vs the corresponding scavenging effect. The antioxidant activity was expressed as the antioxidant activity index (AAI), calculated according to Scherer & Godoy, (2009) [22]. In this author work s a qualitative classification is presented, namely: poor antioxidant activity when AAI < 0.5, moderate antioxidant activity when AAI between 0.5 and 1.0, strong antioxidant activity when AAI between 1.0 and 2.0, and very strong when AAI > 2.0 [22]. Assays were carried out in duplicate. Results and Discussion Phytochemical analysis Table 1 presents the phytochemical analysis showing positive results for the group of phenolic compounds. 89

100 Table 1: Summary of phytochemical prospecting for nettle leaves. group of Reagents Results compounds Alcaloids Terpenoids Phenolic compounds Dragendorff, Mayer e Bertrand Wagner Salkowski Liebermann Burchard Ferric chloride Negative Negative Negative Negative Positive Flavonoids Shinoda Positive Extraction yields Extraction yields show that Soxhlet method allows higher yields (8.5% leaves and 4.5% stems) when compared to ultrasound extractions (from 1.1% stems to 2.5 % leaves and 2x30 minutes). In the work done by Mohammad et al. (2015) using the maceration process (24 h) of the net parts of nettle in three different solvents and yields were respectively 4.4%, 20.1% and 22.9% for chloroform, methanol: water (4:1) and water [23]. It can be said that the extraction time, the method used and the type of solvent are important parameters that influence the yields obtained. Phenolic compounds Phenols have been referred to as potent antioxidants and therefore their determination is very important since a linear correlation between the content of phenolic compounds in a given sample and its antioxidant activity has been demonstrated [24,25,26]. Table 2 presents the values of the total phenolic content and flavonoids present in the extracts under study, related to the different types of extraction. From the analysis of this table it can be mentioned that the leaves are richer in phenolic compounds than the stems and that the extraction in Soxhlet allows to obtain a greater content. These results have a higher total phenolic content than those obtained by Mohammad et al. (2015) for the different solvents and the whole aerial part [23]. However, they are much lower when compared to those obtained by Luís et al. (2011) [21]. Other results presented for aqueous extracts of nettle by Valiollah et al. (2012) indicate a value of 7.82 for the root, 9.91 for the stem, and 7.62 for the leaves in mg EAG / g dry matter, which in a way corroborates some of the results obtained [8]. Table 2: Total phenolic compounds and total flavonoids for leaves and stems, using different extractions methods. Extraction method Soxhlet Ultrasounds 2 x 15 minutes Ultrasounds 2 x 30 minutes Part of the plant Total Phenolic *(mg EAG/g) mean± standard deviation Total Flavonoids **(mg EQ/g ) mean± standard deviation leaves 40,57 ± 4,42 48,50 ± 4,50 stems 23,72 ± 2,35 23,51 ± 1,27 leaves 11,81 ± 5,78 23,52 ± 3,11 stems 2,39 ± 0,94 7,38 ± 0,35 leaves 13,76 ± 3,22 28,07 ± 1,81 stems 3,93 ± 1,58 8,04 ± 1,35 * milligrams of gallic acid equivalents (EAG) per gram of dry matter. ** milligrams of quercetin equivalents (EQ) per gram of dry matter. The nettle leaves have values of total flavonoids that are much higher than those of the stem and the highest leaf content is found for Soxhlet extraction. The extract with the lowest flavonoid content corresponds to the stems, when the extraction method was ultrasound in two 15 minutes cycles. As for phenols, these results have a higher total flavonoid content than those obtained by Mohammad et al. (2015) for the different solvents and the whole shoot, respectively, 15.4 mg EQ / g for extraction with chloroform, 5.77 mg EQ / g for extraction with methanol (80%) and 7.89 mg EQ / g for aqueous extract [23]. The flavonoid content obtained is comparable to some of the results obtained for several species and presented by Luís et al. (2011) [21]. These compounds can act as antioxidants because they can donate an electron or a hydrogen atom, but this only happens for flavonoids with a certain structure, namely the position of the hydroxyl group in the molecule [21]. Antioxidant activity The ethanolic extracts from the stems and leaves of Urtica dioica, L. (Soxhlet extraction) showed a high IC 50 (382.6 ± 67.8 mg / L and ± 81.0 mg / L) with a consequent activity index antioxidant (AAI), compared to the values obtained with standard compounds (trolox, gallic acid and rutin), which have a very strong AAI [22]. On the other hand, a direct relationship between total phenol content and antioxidant activity is reported in the literature, which corroborates the results obtained in this study [21,24]. A study of aqueous extracts (Soxhlet extraction) and diethyl ether (maceration) of nettle before and after flowering showed a decrease of IC50 after flowering of 28% for aqueous extracts and of 45% for extracts in ether [ 27]. Some of the IC 50 values presented in the literature indicate a value of 1200 mg / L for an aqueous extract of nettle [28] 90

101 and values of mg / L for chloroform extract, mg /L for methanolic extract and mg/l for aqueous extract [23]. Conclusions The extraction processes used revealed different differences, with Soxhlet extraction being the one that showed greater efficiency in the extraction of bioactive compounds more than the ultrasonic extraction process. Regarding the study plant, the leaf samples presented a better overall performance than the stems with respect to both the total phenol content and the flavonoids content. In the present work, Urtica dioica, L. was found to be a species with a low antioxidant potential due to the low concentration of phenolic compounds and the presence of flavonoids that do not have the capacity to donate either a hydrogen atom or an electron to a free radical. It should be noted that these results can be related to several environmental factors, climatic, rainfall and edaphic aspects, as well as, physiological factors, related to seasonal rhythm, hormonal production, reproductive status. As is well known, the production of active substances is related to abiotic and biotic factors. A more detailed study on the chemical (presence of secondary metabolites) and environmental interface, related to the influence of the type of solvent and the extraction methods, is therefore proposed for future work; to carry out a study on the root and to evaluate the viability of the nettle stalks in the production of nanocellulose. References [1] J.S.C. Rodrigues, Contributo para o Estudo Etnobotânico das Plantas Medicinais e Aromáticas na Área Protegida da Serra do Açor, Instituto da Conservação da Natureza, [2] S.Cañigueral, Las monografias de calidad, seguridad y eficacia en el uso racional de los preparados a base de plantas medicinales, Revista de Fitoterapia, 6(S1): 25-29, [3] A.P.Cunha, A.P.Silva, O.R.Roque, Fitoterapia na atualidade, In: A.P. Cunha, (Ed.), Plantas e Produtos Vegetais em Fitoterapia, Fundação Calouste Gulbenkian, Lisboa, pp , [4] A. R. A.Carvalho, Urtica spp. Bioatividade e Cultivo, Faculdade de Ciências e Tecnologia, Dissertação de mestrado em Biotecnologia Vegetal, Universidade de Coimbra, [5] N.Virgilio, E.G.Papazoglou, Z.Jankauskienec, et al., The potential of stinging nettle (Urtica dioica, L.) as a crop with multiple uses, Ind. Crops Prod., 42 49, [6] S.Otles, B.Yalcin, Phenolic Compounds Analysis of Root, Stalk, and Leaves of Nettle, The Scientific World Journal, Vol. (2012), Article ID , (2012),12 pages. [7] S.Castroviejo, C.Aedo, M. Laínz, et al., Urticaceae, Flora Iberica, [8] V.Hajhashemi, V.Klooshani, Antinociceptive and antiinflammatory effects of Urtica dioica leaf extract in animal models, Avicenna J. Phytomedicine, Vol.3, nº2, pp , [9] N.G.P.B. Lima, A.G.S. Cabral, F.F.Furtado, et al., Urtica dioica: uma revisão dos estudos das suas propriedades farmacológicas, Rev. Bras. Farm., 89(3): pp , [10] M. Bnouham, F.Z. Merhfour, A. Ziyyat, Antihyper glycemic activity of the aqueous extract of Urtica dioica, Fitoterapia, 74(7-8), pp , [11] C.F. Daher, K.G Baroody, G.M. Baroody, Effect of Urtica dioica extract intake upon blood lipid profile in the rats, Fitoterapia, 77 (3), pp , [12] S. Bisht, S.Bhandari, N. S. Bisht, Urtica dioica (L): an undervalued, economically important plant, Agr. Sci. Res. J., Vol. 2(5); pp , [13] E.Yesilada, G. Honda, E. Sezik, et al., Traditional medicine in Turkey IV. Folk medicine in the Mediterranean subdivision, J. Ethnopharmacol., 39, pp , [14] M. Khalili, M. Rezazarandi, S. Vahidi, Anti-inflammatory effect of alcoholic Urtica dioica extractoin male NMRI rats, Basic. Clin. Pathophys, pp , [15] K. K. Ghaima, N. M. Hashim, S.A. Ali, Antibacterial and antioxidant activities of ethyl acetate extract of nettle (Urtica dioica) and dandelion (Taraxacum officinale), J. of Appl. Pharm Sci. Vol. 3 (05), pp , [16] I. Gülçin, O.I. Küfrevioglu, M.Oktay, et al., Antioxidant, antimicrobial, antiulcer and analgesic activities of nettle (Urtica dioica L.), J. Ethnopharm., 90, pp , [17] A.A. Garcia, E. P-U. Carril, Metabolismo secundário de plantas, Reduca (Biologia), serie Fisiologia Vegetal, 2 (3): pp , [18] Information on metabolismo-secundario-das-plantas/, acedido em 19/06/2016. [19] A. Cunha, F. Teixeira, A. Silva, et al.,plantas terapêuticas: Farmacologia e Ensaios clínicos, Fundação Calouste Gulbenkian, Lisboa, [20] J. B. Harborne, Methods of Plant Analysis, Springer Netherlands, pp. 1-36, [21] A. Luís, F. Domingues, A. P. Duarte, Bioactive compounds, RP-HPLC analysis of phenolics, and antioxidant activity of some Portuguese shrub species extracts, Nat. Prod. Commun., Dec. 6(12): , [22] R. Scherer, H. T. Godoy, Antioxidant activity index (AAI) by the 2,2-diphenyl-1-picrylhydrazyl method, Food Chem. 112, pp , [23] M. A. Ebrahimzadeh, M. Gharekhani, M. Ghorbani, et al., Effect of Extract of Aerial Parts of Urtica dioica (Urticaceae) on the Stability of Soybean Oil, Tropical J. of Pharmaceutical Research, 14 (1), pp , [24] C. M. M. Sousa, H. R. Silva, G. M. Vieira, et al., Total phenolics and antioxidant activity of five medicinal plants, Química nova, vol.30 nº2, pp , [25] E. M. Silva, J. N. S. Souza, H. Rogez, et al., Antioxidant activities and polyphenolic contents of fifteen selected plant species from the Amazonian region, Food Chem., 101, pp , [26] G. Miliauskas, P. R. Venskutonis, T. A. van Beek, Screening of radical scavenging activity of some medicinal and aromatic plant extracts, Food Chem., 85, pp , [27] M. Monfared, A. Kamkar, S. G. Khaligh, et al., Antioxidative effects of Iranian Urtica dioica L. extracts on the oxidation of sunflower oil, J. Med. Plants Res., Vol. 5(18), pp , [28] S. Fattahi, E. Zabihi, Z. Abedian, et al., Total Phenolic and Flavonoid Contents of Aqueous Extract of Stinging Nettle and In Vitro Antiproliferative Effect on Hela and BT-474 Cell Lines, Int. J. Mol. Cell. Med. Spring, Vol 3 No 2, pp ,

102 Anaerobic mesophilic biodegradation of raw olive pomace V. Guilherme, I.C. Gonçalves, A. Marques, A. Lopes and I.Ferra FibEnTech-UBI, Universidade da Beira Interior, Chemistry Department, R. Marquês D Avila e Bolama, Covilhã, Portugal Abstract Portugal represents only 1.7% of the total olive oil world production [INE, 2011]. Technologies used for olive oil extraction are nowadays based on centrifuging processes. It is well known the potentially harmful effects associated to the direct discharge into the environment of the residues from the olive mill processes. Therefore, both the dry solid pomace and the semi-solid olive pomace require an appropriate treatment technology for its processing to decrease the impacts in the ecosystems. In the present work, preliminary tests on anaerobic biodegradation of a semi-solid and a dry solid pomace were carried out in batch mode, at mesophilic regime. The degradation process was followed by measuring the COD removal and the biogas production. A soluble COD removal up to 92.5%was obtained for the semi-solid pomace. A decrease on both COD removal and biogas production was observed for the dry pomace. Introduction Olive oil production has been mainly confined to the Mediterranean area where it is linked to the cultures since the ancient times. Olive tree cultivation accounts for about 96% of olives used for olive oil extraction and the others 4% for olives production [INE, 2010].The worldwide olive oil production is about 2.6 million tons per year, from which 78.1% are produced in European Union (in southern Europe countries). Spain, Italy and Greece are the main producers within the European Union representing 34.6%, 22.6% and 18.5% of the world production, respectively. Portugal represent only 1.7% of the total world production [INE, 2011]. The cultivation of olive trees is still growing due to the healthy characteristics of olives and olive oil [in Awad et al., 2006]. So, it is expected an increase of the resulting process wastes (liquid and solid types). Technologies used for olive oil extraction are nowadays based on centrifuging processes. Basically, after olives washing, a milling and beating phase followed by a centrifugation (for oil and pomace separation) and a final oil washing phases are the main steps involved. These are continuous solid-liquid decanting systems with less time consuming and with a high productivity. The three phase centrifugation system appeared first and the main difference relatively to the two phase system is in the mode operation of the centrifugation system. In the three phase system it is required the addition of raw hot water in large quantities in order to separate the resulting three phases: oil, water and solid suspension. Thus, huge amounts of liquid wastewater, along with a dry solid pomace are produced as residues. In comparison with the two phase centrifugation system, the last consumes less water and energy, and there is no addition of raw water during the centrifugation process. In this case a solid residue with a high moisture content (65-75%), that is called wet or semi-solid olive pomace (or two-phase olive pomace) is generated [Albuquerque et al., 2004; Freitas, 2007]. The semi-solid olive pomace from the two phase system contains all the olive vegetation water resulting in an increase of about 50% in water relatively to the amount of water of the solid dry pomace produced in the three phase system [Borja et al., 2003]. Besides vegetation water the semi-solid pomace contains (g/kg dry weight): lignin ( ), hemicellulose ( ), cellulose ( ), fats ( ), proteins ( ), carbohydrates ( ) and phenols ( ). It is also rich in K (19.8), total N (11.4) and P (1.2) [Albuquerque et al., 2004]. The dry solid pomace has basically the same components, but almost no humidity. It is also considered a lignocellulosic residue with (in g/ kg dry weight) lignin (294.0), cellulose (266.0), K (6.5), N(11.0) and P(1.0) and recalcitrant to biodegradation [Silva, 2009]. The solid dry pomace is less environment friendly compared with the residue obtained in the two-phase system. It is used to produce an extra oil after an extraction process with an organic solvent (such as hexane). After the chemical extraction, the final solid waste can be used as fuel for energy production (Albuquerque et al., 2004). It is well known the potentially harmful effects associated to the direct discharge into the environment of the residues from the olive mill processes. Therefore, both the dry solid pomace and the semi-solid olive pomace require an appropriate treatment technology for its processing in order to decrease the impacts in the ecosystems. The aim of this work is to assess the anaerobic treatability of two types of pomaces arising from different phases of the olive oil processing, in tandem with their potential of biogas production. Materials and Methods Raw material The raw olive pomaces used in the experiments are both originated from a three-phase centrifuging system. The semi-solid olive pomace (with a high moisture content) was collected before the separation of the olive mill 92

103 wastewater in the decanter (with a horizontal centrifuging) and after the milling and beating phases. This is supposed to have a similar composition to the one produced in the two-phase centrifuging system (twophase olive pomace residue - TPOP). It was well homogenized and stored at 4ºC before being used in the experiments. The dry solid pomace was collected at the end of the process, after the drying step. Drying was performed after the centrifuging phase and separation of the olive oil and olive wastewater. This pomace was milled and sieved before to be used in the tests. The composition of pomaces is shown in Table 1. Two replicates were used for the analyses. In general the organic matter content is higher for the semi-solid pomace. Table 1 Average values and standard deviations for the total solids (TS), total volatile solids (TVS), ash and protein. Substrate Semi-solid Dry pomace pomace Parameter Total Solids (g/g pomace) ± ± Total Volatile Solids ± ± (g/g pomace) Ash (g/g pomace) ± ± Protein (mg N-NH 3/g pomace) 14.3± ±1.3 Anaerobic biomass The inocula used for the anaerobic biodegradation tests was gathered from mesophilic batch lab-scale reactors fed with a simulated effluent containing glucose as sole carbon source (~ 1.5 g/l). The anaerobic seed biomass had a suspended solid content (SS) of 20.9 g SS/L approximately. Solutions The feed solutions were based in a mineral medium containing macro and micronutrients [Brás, 2003]. The mineral medium was buffered with a phosphate solution (3 g/l Na 2HPO 4 and 0.3 g/l NaH 2PO 4). Analyses Chemical Oxygen Demand (COD), Suspended Solids (SS), Suspended Volatile Solids (SVS), ash and ph of the samples were analyzed in the soluble fraction of the samples collected [APHA, AWWA, 2005]. All the analyses were performed in duplicate. Acid precipitable lignin (APL) was analyzed in the soluble fraction after acidification with 72% H 2SO 4 (w/w) and correction for the acid insoluble ash and protein. Kjeldahl method was used to quantify the protein content using the N x 6.25 conversion factor. Total phenol was measured in Gallic Acid Equivalents (GAE) based on Folin-Ciocalteu method (Magalhães et al., 2007). Anaerobic biodegradation experiments Biodegradation tests were carried out for raw semi-solid pomace and dry pomace at mesophilic temperature (37 ± 2ºC) in batch mode operation. 10 ml of anaerobic biomass (approximately 3.48 g SS/L) was charged to each bottle containing 50 ml of mineral medium. Further 1 g of semi-solid olive pomace (TPOP) or 5 g of solid (dry) olive pomace were supplemented and bottles flushed with nitrogen for 5 minutes. Four replicates of each type of pomace were run in amber glass vials of 120 ml capacity sealed with rubber septa and equipped with magnetic stirring. The ph, COD and biogas were monitored in samples collected throughout of the entire course of the experiments. At the end of the essays protein, lignin, SS, VSS, ash and total phenol were also analyzed. The mass of pomace was selected taking into account the results obtained in leaching experiments performed with semisolid (5g) and dry (1g) olive pomaces, for which nearby values of soluble COD were achieved (Figure 1). These experiments were run in batch mode at 37 ± 2ºC at the same pomace/basal medium ratio used in the biodegradation tests. Tests were run in duplicate and samples collected from the soluble fraction were analyzed for the same parameters, to characterize the type of soluble substrates available for the anaerobic cultures. For each type of pomace the efficiency of biodegradation was evaluated by comparison of the residual value of each parameter at the end of the anaerobic tests with the corresponding maximum value in the leachates. mg CQO/L dop ssop t (d) Figure 1 Results of soluble COD vs time obtained in leaching tests for semi-solid (ssop) and dry olive pomaces (dop) Results and Discussion The anaerobic biodegradation tests were carried out with quantities of semi-solid pomace and dry pomace that produced leachates with COD, protein, soluble lignin and ammoniacal nitrogen values of the same range of magnitude, to provide enough organic matter available to 93

104 be metabolized by microorganisms and to guarantee a balanced growth of the syntrophic groups in the mixed populations. Results of leaching tests show that a huge amount of organic compounds are solubilized in the first 24h for both type of pomaces (Figure 1). However, the total phenol level is particularly higher in the leachates from the dry pomace than that produced for the semi-solid type, which can lead to different results in terms of biodegradability. The experimental results depicted in Table 2 and Figure 2, indicate, in general, a higher efficiency degradation of the soluble organic load for the semi-solid olive pomace in comparison with the values obtained for the dry one. Both the presence of phenolic compounds and the non-adapted biomass used as inoculum should explain the longer period of time required for degradation of the dry solid pomace. The residual COD value of 453 mg O 2/L obtained after 100 days of incubation time is almost two times greater than the residual COD value for the semisolid pomace in a 27 days period of reaction time (219 mgo 2 /L, approximately). Therefore, among other factors, the high residual content of phenolic compounds in the dry pomace leachate should explain the slower degradation process along with the lower efficiency treatment achieved in this case. Also, according to the literature phenolic compounds arising from hydrolysis of lignin could work as inhibitors [Chen et al., 2008]. Table 2 Average efficiency removal (%) and standard deviations of soluble organic matter for 4 replicates. Substrate Dry pomace Semi-solid pomace Parameter COD (mg O 2/L) 75.8 ± ±0.8 Protein (g/l) 77.8 ± ± 3.7 Nitrogen (mg NH 3 /L) 47.5 ± ±5.5 LS (g/l) 83.0 ± ±1.8 LPA (g/l) 72.7 ± ±1.6 Total Phenol (mggae/l) 76.4 ± ±1.1 For the semi-solid pomace a faster decrease of the residual organic load occurs, rendering a higher biogas production (Figure 2) and suggesting that both hydrolysis/ acidogenesis and methanogenesis phases proceed in balanced conditions. In this case the maximum value for the biogas production per gram of pomace within a 28- days period of time (92 ml/g pomace) is around seven times greater than the value obtained for the dry type pomace (13 ml/g pomace) in a 100 days period of time. These findings are in accordance with the results obtained for the soluble COD removal as shown in Table 2, in which a higher COD removal is shown for the semi-solid pomace (of 91.8%). a) S (mg COD/L) b) Biogas (ml/g p) t(d) ssop dop ssop dop t(d) Figure 2 Examples of time courses of the residual COD (a) and biogas production (b) for semi-solid (ssop) and dry olive pomaces (dop) Conclusions Solid residues from olive mil processes could be used as a source of biogas production hence anaerobic digestion is chosen as the appropriate technology for their treatment. The semi-solid pomace as substrate of anaerobic digestion render as a higher efficiency treatment, as a higher potential of biogas production. Furthermore in this type of pomace less process water is consumed, which decrease the amount of liquid wastewater to be processed, which is in general hardly degraded. Theoretical values of the biogas production estimated with basis on the soluble COD removal were of 106 ml biogas/g pomace for semi-solid pomace and 16 ml/ g pomace for the dry solid pomace, approximately. These values are close to the experimental results, showing that the semi-solid pomace is more prone to the anaerobic biodegradation than the dry type. Ackowledgements The authors express their gratitude to the FibEnTech and FCT for the financial support. References Albuquerque, J; Gonzalvés, J; García, D;Cegarra, J. (2004). Agrochemical characterisation of alperujo, a solid by-product of the two-phase centrifugation method for olive oil extraction. Bioresource Technology, 91:

105 Awad, A; Salman, H; Hung, Y. (2006). Olive Oil Waste Treatment. Taylor & Francis Group, LLC. APHA, AWWA (2005). Standard methods for the examination of water and wastewater.american Public Health Association, American Water Works Association, Water Environment Federation, 21sth edition, Washington, DC, USA. Borja, R; Rincón, B; Raposo, F; Alba, J; Martín. A. (2003). Kinetics of mesophilic anaerobic digestion of the two-phase olive mill solid waste Biochemical Engineering Journal, 15: Brás, R. (2003). Anaerobic Biodegradation of azo dyes by methanogenic and mixed cultures. PhD Thesis, Ubi, Covilhã. Chen, Y., Cheng, J.J., and Creamer, K.S. (2008). Inhibition of anaerobic digestion process: A review. Bioresource Technology, 99: Freitas, M. (2007). Avaliação do potencial energético dos resíduos sólidos dos lagares do Alentejo. Dissertação de Mestrado. Instituto Superior de Agronomia, Universidade Técnica de Lisboa. Guilherme, V. (2012). Anaerobic biodegradation of olive pomace. MSc Thesis, UBI, Covilhã. Instituto Nacional de Estatística, 2010, Estatísticas Agrícolas Edição ISSN Instituto Nacional de Estatística, 2011, Estatísticas Agrícolas Edição ISSN Magalhães L.M., Segundo, M.A., Reis, S., Lima, J.L.F.C., Tóth, I.V., Rangel., A.O.S.S. (2007). Automatic flow system for sequential determination of ABTS + scavenging capacity and Folin-Ciocalteu index: A comparative study in food products, Analytica Chimica Acta, 592: Silva, C. (2009). Aplicación directa de residuos de almazaras de dos fases enun olivar conriego localizado: Efectos en las propiedades edáficas y el cultivo. Tesis Doctoral. Facultad de Ciencias; Universidad de Extremadura. 95

106 Essential oils from fennel seeds collected in different regions from Portugal L. Silva 1, V. Lopes 2, E. Bettencourt 3, S. Pombal 1, J. Rodilla 1 and A. Gomes 1 1 Departamento de Química, Materiais fibrosos e Tecnologias Ambientais FibEnTech-UBI, Universidade da Beira Interior, Covilhã, Portugal 2 Banco Português de Germoplasma Vegetal lniav, I.P. Quinta de São José, S. Pedro de Merelim, Braga, Portugal. 3 Unidade Estratégica de Investigação e Serviços de Biotecnologia e Recursos Genéticos, INIAV, I.P. (retired), Portugal. Abstract The objective of this work was to compare the chemical composition of essential oils obtained from seeds of five fennel accessions collected from wild populations growing in Centre (Guarda and Santarém) and South of Portugal (Évora, Beja and Setubal). Essential oils were isolated from the seeds by hydrodistillation and analyzed by Gas Chromatography-Flame Ionization Detector (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS). The yields of the essential oils obtained vary greatly with a range of 0.5 to 1.3 % and the chemical composition changes from one region to another. A total of 16 constituents were identified. The main compounds were three: fenchone ( %), estragole ( %) and trans-anethole ( %) Percentages of these three main compounds were used to determine the relationship between the different oil samples and grouped them in five different chemotypes: Anethole; Anethole/Estragole; Fenchone; Anethole/Fenchone and Estragole. Introduction Foeniculum vulgare Mill. is a plant native to the Mediterranean region and it occur in Portugal. It is a species with several applications in the food, cosmetic and pharmaceutical industry. This plant is characterized by having as main compounds two phenylpropanoids: estragole and trans-anethole and one oxygenated monoterpenoid: fenchone. Previous studies on Portuguese fennels essential oils, have already shown the existence of chemotypes (anethole, anethole/fenchone, anethole/estragol) that deviate from the international accepted standards. As part of our continuous search for improving the knowledge about the essential oils composition of fennel in Portugal, five indigenous accessions collected from wild populations growing in the Centre and South of Portugal were studied. Materials and Methods Plant material: Five fennel populations were identified by Eng. Eliseu Bettencourt and collected in the Centre (Guarda and Santarém) and South of Portugal (Setubal, Évora and Beja). All of them are maintained in ex situ by Banco Português de Germoplasma Vegetal (BPGV). The seeds from wild populations were morphologically characterized and the progeny were analyzed. The protocol of plant material management in field was unique: no fertilizations, drip irrigation between June to August once a week, and seeds harvested in October. Essential oils isolation: The seeds essential oils were obtained from 50 g of each sample by hydrodistilation in 2 L of water for 2 hours, using a Clevenger-type apparatus. The essential oils obtained were stored under nitrogen in tightly closed dark vials between -20 ºC and -30 º C until analysis. Gas chromatography flame ionization detection: For gas chromatographic separations, an Agilent 7890 instrument coupled to an ionization flame detector (FID) was used. Volatile components were separated on a DB-5MS capillary column (5% phenylmethylpolysiloxane, 25 m, 0.25 mm i.d.; 0.25 μm film thickness) (J&W Technologies), with the following temperature program: 5 min at 60 C, subsequently 10 C/min up to 250 C, held for 20 min. Injector temperature was 250 C and detector temperature was 280ºC. Helium was used as the carrier gas, at a flow rate of 1.0 ml/min; injection volume: 1 µl; split ratio, 1:50. A mixture of aliphatic hydrocarbons (C7-C25) (Sigma, Milan, Italy) in n-hexane was directly injected into the GC injector under the above temperature programme, to calculate the retention index of each compound. Oil samples were diluted 1:100 in n-hexane and injected in a volume of 1 μl. Analysis was repeated 3 times. Gas chromatographic mass spectral analysis: The F. vulgare seeds essential oils were analyzed by GC-MS using an Agilent 7890 GC coupled with an Agilent 5975 C inert XL mass selective detector [MSD, operated in the electron impact EI mode (electron energy = 70 ev), scan range = amu, and scan rate = 3.99 scans/sec), and an Agilent ChemStation data system. The GC column was a DB-5MS fused silica capillary with a (5% phenyl)- polymethylsiloxane stationary phase, film thickness 0.25 μm, length 30 m, and an internal diameter of 0.25 mm. The carrier gas was helium with a column head pressure of 48.7 kpa and a flow rate of 1.0 ml/min. Injector temperature was 250 C. The GC oven temperature programme was used as follows: 60 C initial temperature, hold for 5 min; increased at 10 C/min to 250 C. A solution of the sample in CH 2Cl 2 was prepared and 1 μl was injected using a ratio split 1:50. Identification of components was achieved by comparison of the retention times and mass spectra with those of the 96

107 pure standard compounds. All mass spectra were also compared with those of the data system library NIST and Wiley. Results and Discussion The yields of the essential oils obtained from F. vulgare seed samples varied greatly within a range of 0.5 to 1.3% (v/w) (Table 1). Table 1: Collection data and essential oil yields of analyzed fennel samples (Foeniculum vulgare). Accessions Location Yield % Chemotype (v/w) Évora 1.2 Anethole Santarém 0.9 Anethole/Estragole Beja 0.8 Fenchone Setúbal 1.3 Anethole/Fenchone Guarda 0.5 Estragole The chemical composition of the essential oils is summarized in Table 2 according to the sequence of elution on a DB5-MS column. In spite of trans-anethole, estragole and fenchone were main constituents in essential oils, significant differences in essential oil composition between samples from different origins were noted. Thus, the accessions were distributed in five chemotypes: anethole, anethole/estragole, fenchone, anethole/fenchone and estragole (Table 1). Table 2: Relative chemical composition (%) of essential oils from six accessions of wild F. vulgare. Compound IR a) α-thujone 927-0,0 0,0 - - α Pinene 935 0,1 0,1 0,4 0,2 0,2 Camphene 953 0,0 0,0 0,0 0,0 0,1 Sabinene 975 0,0 0,0 0,0 0,0 0,1 Myrcene 990 0,1 0,1 0,1 0,1 0,1 α-phellandrene ,0 0,0 0,0 0,1 0,1 p-cymene ,0 0,0 0,0 0,0 0,0 Limonene ,7 0,6 0,8 0,9 1,3 1,8-Cineol ,2 0,2 0,1 0,1 0,3 β-ocimene ,1 0,5 0,1 0,1 0,2 -Terpinene ,3 0,1 0,1 0,1 0,1 Fenchone ,4 18,9 21,7 37,0 34,8 Camphor ,0 0,3 0,4 0,5 0,5 Terpinen-2-ol ,0 0,0 0,0 0,0 0,1 Estragole ,8 11,3 2,6 5,5 15,2 cis- Anethole ,0 0,0 0,0-0,0 trans-anethole ,2 68,3 73,4 55,1 46,9 a) IR Retention Index on DB5-MS; t-traces (less than 0.05%) Évora Santarém Beja Setúbal Guarda Conclusions The lower essential oil yield is from accession collected in Guarda and the higher is from Setubal. The central and south regions of Portugal have great chemical inter-variability. These five populations have specific chemical characteristics, all of them belong to different chemotypes. Acknowledgements The authors gratefully acknowledge the financial support received from Fundação para a Ciência e a Tecnologia, FCT, for the financing of the FibEnTech Research Unit, project Pest-OE/CTM/UI0195/2014 and for the grant awarded to Lúcia Silva SFRH/BSAB/113777/2015. References 1. Roque, O., Proença da Cunha, A. "Composição do óleo essencial de Foeniculum vulgare Miller, espontâneo do Algarve". Bol. Fac. Far. Coimbra 13(1989) Cavaleiro, C., Roque, O. and Proença da Cunha, A. "Contribution for the characterization of Portuguese fennel chemotypes". J. Essent. Oil Res. 5 (1993) Lopes, V., Barata, A., Farias, R., Mendes, M., Pedro, L., Barroso, J., Figueiredeo, A. "Morphological and Essential Oil Variability from Nine Portuguese Fennel (Foeniculum vulgare Mill.) Accessions" Acta Horticulturae 860 (2010) Hans Krüger & Karl Hammer Chemotypes of Fennel (Foeniculum vulgare Mill.) J. Essent. Oil Res.11 (1999) J. Bernáth, É. Németh, A. Kattaa & É. Héthelyi "Morphological and Chemical Evaluation of Fennel (Foeniculum vulgare Mill.) Populations of Different Origin" J. Essent. Oil Res.8 (1996) The complexity and variability of composition of essential oils from seeds of F. vulgare, we suggest they could be recommended as a potential source of pharmaceutical materials for the preparation of new antifungal and antibacterial agents. 97

108 Production of high performance filaments from nano/microfibrillated vegetal cellulose. V.L.D. Costa 1, J.W. Gomulka 1, P.D.L. Gaspar 1, D.J.M. Santos 1, A.P. Costa 1, M.E. Amaral 1, R.M.S. Simões 1 1 FibEnTech-UBI, Department of Chemistry, Universidade da Beira Interior, Covilhã, Portugal Abstract This study focuses on the production of filaments from vegetal nanofibrillated cellulose (NFC) aqueous suspensions through wet spinning and their mechanical performance. For this investigation, two NFC suspensions were produced from bleached sulphite and kraft eucalypt pulps through homogenization. The suspensions were characterized using SEM and TEM. A wet spinning system with a NaCl solution and ethanol baths was set up. Filaments were spun from the suspensions with concentrations between 2.5 and 3.22 wt.%, with flow rates from 800 to 3000 μl/min (corresponding to spinning rates from 0.45 to 1.70 m/min, respectively). The filaments were air dried at room conditions and some were afterwards submerged in a water bath and air dried once again. The filaments were mechanically tested at standard conditions. An increase in spinning rate improved the resistance parameters of the filaments, which might indicate that some level of fibril alignment is being achieved. An additional 5-minute water bath increased the mechanical performance of the filaments, mainly the tensile strength: for a 3 wt.% NFC suspension, at the same flow rate, the tensile strength increased from to MPa and the elastic modulus increased from 9.1 to 11.7 GPa. Despite some improvements of the filaments mechanical properties have been achieved, nanofibril morphology, hydrodynamic nanofibril alignment, additional treatment and drying conditions are still being optimized. Introduction Many biological materials show impressive and controllable properties determined by their micro and nanostructures. The most common building blocks found in nature are fibers, which are hierarchically organized forming strong and cohesive structures as the vegetal cell walls, for instance [1, 2]. Like nature, man has attempted to manufacture continuous fibers of several materials and for a wide range of applications, such as composites or textiles, allowing a wide flexibility in the design and manufacture, controlling the orientation and the architecture of the fibers [3]. In recent decades, natural fibrous materials, particularly nanofibrillated cellulose (NFC), have received increasing attention because of their potential to produce high performance mechanical filaments, while respecting environmental and social requirements. These filaments represent basic blocks to construct biomaterials with low environmental impact, low production cost, high mechanical performance and susceptible to nanoscale functionalization [2, 4]. The transformation of the cellulosic fibers obtained from lignocellulosic materials into cellulosic filaments with uniform characteristics, through "green" and energetically sustainable processes is a challenge. In order to provide a greater uniformity to the material, it is necessary to deconstruct the cellulosic fiber structure (obtaining NFC) and to rebuild the filament (also called macrofiber) under conditions that lead to uniformity and consistency of the property. It has been reported that the nanofibrils must be aligned and assembled in a controlled manner in order to make use of the whole potential of the NFC [2]. The objective of the present work is to investigate the influence of solid content and wet spinning operation conditions of vegetal nanofibrillated cellulose suspensions on the mechanical performance of the wet-spun filaments. Experimental part The following section describes the whole experiment setup and operating conditions. NFC production For this investigation, two types of NFC suspensions were produced: NF1 was produced from a commercial bleached sulphite eucalypt pulp and NF-AK-2 was produced from a commercial bleached kraft eucalypt pulp. The pulps were subjected to different TEMPO mediated oxidation pretreatments and had final average degrees of polymerization of 117 and 770, respectively. Both pulps were then subjected to homogenization in a high-pressure homogenizer: a first pass was done at 500 bar, followed by a second pass at 1000 bar. Morphological characterization After homogenization, the NFC suspensions were diluted to a solid content of 0.1 wt.% and were dispersed in an ultra-sound bath for 5 minutes. A drop of the diluted suspension was allowed to dry overnight at room temperature on a glass cover which was attached on a microscope sample holder with double-sided tape. Microscopy observations were performed using scanning electron microscopy (SEM) (Hitachi S-2700) operated at 20 kv. All the samples were previously gold-sputtered by cathodic spraying. For transmission electron microscopy (TEM) imaging, aqueous NFC suspensions were diluted to wt.% and sonicated. Drops of the suspensions were deposited on carbon coated electron microscope grids and negatively stained with 2 wt. % uranyl acetate. The grids were air dried and observed with a Hitachi HT 7700 TEM operated at an acceleration voltage of 80 kev. 98

109 Wet spinning set-up Results In the present investigation, NF1 and NF-AK-2 suspensions were used to produce cellulosic filaments through wet spinning. The original suspensions (which had an initial solid content of around 1 wt%) were let to evaporate at room temperature with occasional mixing until the desired solid content was reached. The NF1 was adjusted to 3.0 wt.% and the NF-AK-2 was adjusted to 2.5 and 3.22 wt.%. The wet spinning set-up consisted of a syringe filled with the NFC suspension (assembled in a syringe pump) that ejected filaments through a 2.5 cm long cone like nozzle (with internal diameters from 6 to 1.5 mm at the outlet) into a 1M NaCl coagulation bath at three different flow rates: 800, 1500 and 3000 μl/min, which correspond to spinning rates of 0.45, 0.85 and 1.7 m/min, respectively, promoting gelation by decreasing the electrostatic repulsion between the nanofibrils. A schematic representation of the wet spinning set-up is given in figure 1. The fibrils morphology was investigated through SEM and TEM imaging. Figure 2 gives an overview of the morphologic characteristics of the NF1. ② ③ a) 500 μm b) ④ ① nm nm Fig. 1. Scheme of the wet spinning set-up: ① Pump; ② Syringe with NFC suspension; ③ Coagulation bath; ④ Wet-spun filaments. Fig. 2. a) SEM image of the NF1 suspension with an amplification of 100 ; b) TEM micrograph of a NF1 negatively stained suspension. The filaments were allowed to stay in the coagulation bath for 30 minutes and were then transferred to a fixation ethanol bath for an additional 30 minutes. The produced filaments were allowed to air dry at room conditions, at fixed length, preventing them from shrinking during drying. Some of the produced filaments were afterwards submerged in a water bath for 5 minutes, in order to enable the electrolyte diffusion out of the filaments, and were air dried once again. As it can be seen in figure 2, the fibrillation of the material appears to be not homogeneous. Although, fine fibrillar structures with diameters in the nanometer range can be spotted (figure 2 b), bigger cellulose fibrils with micrometer dimensions are also clearly visible (figure 2 a). Figure 3 shows the resulting NF-AK-2 (2.5 wt.%) filaments produced with a spinning rate of 1.7 m/min and dried under tension. Mechanical characterization of the filaments The diameter of the filaments was measured through optic microscopy against a glass slide with a measuring scale, assuming a circular cross section. The produced filaments were conditioned overnight at 22ºC and 50% relative humidity, before testing. Tensile tests were carried out using a universal testing machine (Thwing-Albert Co, EJA series) in the same environment. The essays were performed at 0.2 mm/min with a 50 N load cell, at 5 cm span length. A minimum of 6 essays were performed for each condition and the arithmetic average is presented for each parameter. Fig. 3. Appearance and size of the NFC filaments produced through wet-spinning. 99

110 The produced filaments have a nearly circular cross section and are quite smooth along their whole length (figure 3), even after reimersion in a 5-minute water bath and second drying. The cross-sectional shapes of the spun filaments were not affected by the change in spinning rates. Table 1 shows the mechanical performance of the produced filaments. The obtained filaments have been evaluated regarding the maximum force, tensile strength, strain-to-failure and elastic modulus. Table 1. Wet spinning conditions and filaments mechanical properties. NFC Solid content [wt.%] Sinning rate [m/min] Diameter [μm] Linear mass [mg/cm] Additional 5- min. water bath Tensile strength [MPa] Max. force [N] Strain-tofailure [%] Elastic modulus [GPa] NF-AK No No No No Yes NF No No Yes The obtained filaments have been evaluated regarding the maximum force, tensile strength, strain-to-failure and elastic modulus. Table 1 shows that, in general, an increase in the spinning rate improved the resistance parameters of the filaments, which might indicate that some level of fibril alignment is being achieved. This result is in good agreement with what has been reported in the literature [2, 5] although the filaments have been produced at much lower spinning rates. The mechanical performance didn t improve, however, with the increase of the solid content of NF-AK-2 for the same spinning conditions: for example, for a spinning rate of 1.7 m/min the tensile strength decreased from to 84.0 MPa when the solid content increased from 2.5 to 3.22%, despite the later had a slightly higher diameter. This might be a consequence of the increase of the cross-section or could also be explained by the decrease in the freedom of motion that can impair the fibrils alignment. Even so, filaments produced from NF1 at a solid content of 3.0 wt.% had higher tensile strength than those produced from 2.5 wt.% NF-AK-2 (148.2 and MPa respectively) for a spinning rate of 0.85 m/min, for which we can hypothesize that the fibrils morphology might have an important role in the fibrils assembling in the filament. An additional water bath after the fixation with the ethanol bath led to an increase in the mechanical performance of the filaments, mainly in the tensile strength, but also in the maximum force, probably due to the removal of the residual NaCl which might weaken the fibril-fibril interaction. Håkansson et al. used a 24-hour water bath in order to enable the electrolyte to diffuse out of the produced filaments prior to transferring them to an acetone bath for fixation [2]. In this investigation, a water bath as short as 5 minutes proved to be effective in removing the NaCl from the already dried filaments, increasing the tensile strength from 84.0 to MPa for NF-AK-2 spun at 1.70 m/min and from to MPa for NF1 spun at 0.85 m/min. Additional studies revealed that increasing the water bath residence time doesn t improve the mechanical performance of the filaments (data not shown). In fact, longer residence times in the water bath (as long as 15 minutes) damaged the filaments integrity and the structures fell apart. Conclusions For this investigation, two NFC aqueous suspensions were produced through homogenization of eucalypt pulps and wet spinning was used as a method of forming filaments. The influence of solid content and wet spinning operation conditions of the NFC suspensions on the mechanical performance of the wet-spun filaments was studied. The NFC filaments were successfully spun into a NaCl 1M coagulation bath. After fixation in an ethanol bath and air drying they had smooth appearance and circular crosssectional shapes. The obtained filaments have been evaluated regarding the maximum force, tensile strength, strain-to-failure and elastic modulus. According to the results, an increase in the spinning rate improved the resistance parameters of the filaments, which might indicate that some level of fibril alignment is being achieved. The increase of solid content did not improve the mechanical performance of the filaments, probably due to the decrease in the freedom of motion that might impair the fibrils alignment. The fibrils morphology might also have an important role in the fibrils assembling in the filament. An additional water bath led to an increase in the mechanical performance of the filaments, mainly in the tensile strength probably due to the removal of the residual NaCl which might weaken the fibril-fibril interaction. Despite some improvements of the filaments mechanical properties have been achieved, nanofibril morphology, hydrodynamic nanofibril alignment, additional treatment and drying conditions are still being optimized. 100

111 Acknowledgements This research was performed under the UBI-Celtejo agreement. References [1] B. Ji, H. Gao, Mechanical properties of nanostructure of biological materials. J Mech Phys Solids 52:2004 (2004) [2] K.M.O. Håkansson, A.B. Fall, F. Lundell, S. Yu, C. Krywka, S.V. Roth, G. Santoro, M. Kvick, L.P. Wittberg, L. Wågberg, L.D. Söderberg, Hydrodynamic alignment and assembly of nanofibrils resulting in strong cellulose filaments. Nature Communications 5:4018 (2014) [3] C. Clemons, Nanocellulose in Spun Continuous Fibers: A Review and Future Outlook. J Renew Mater 4:5 (2016) [4] A. K. Bledzki, J. Gassan, Composites reinforced with cellulose based fibres. Prog Polym Sci 24 (1999) [5] S. Iwamoto, A. Isogai, T. Iwata, Structure and mechanical properties of wet-spun fibers made from natural cellulose nanofibers. Biomacromolecules 12:3 (2011)

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