Livro de Resumos e Posters do III Workshop Biotecnologia da UA by Workshop Biotecnologia UA

9 de Maio 2012

III Workshop Biotecnologia UA

III WORKSHOP BIOTECNOLOGIA UNIVERSIDADE DE AVEIRO

LIVRO DE RESUMOS E

POSTERS

Os Primeiros Mestres em Biotecnologia pela Universidade de Aveiro

9 de Maio 2012

III Workshop Biotecnologia UA

9 de Maio 2012

III Workshop de Biotecnologia da Universidade de Aveiro

“Os Primeiros Mestres em Biotecnologia pela Universidade de Aveiro”

Departamento de Ambiente e Ordenamento da Universidade de Aveiro 9 Maio 2012

BEST Aveiro Direcção de Curso da Licenciatura e Comissão Científica do Mestrado em Biotecnologia

Contactos Professor Doutor Jorge Saraiva jorgesaraiva@ua.pt III Workshop Biotecnologia UA Gil Carraco gil.carraco@gmail.com BEST Aveiro aveiro@BEST.eu.org

http://workshopbiotecnologiaua.weebly.com/ workshopbiotecnologia.ua@gmail.com

III Workshop Biotecnologia UA

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Índice Índice ........................................................................................................................................1 Os Primeiros Mestres em Biotecnologia pela UA ...................................................................3 Resumos e Posters apresentados no Workshop ........................................................................4 1.

Rhamnolipid production by bacteria isolated from contaminated soil ...........................4

Second generation bioethanol from spent sulphite liquor using Pichia stipitis suspended and immobilized .............................................................................................................6

Yeast two hybrid system in identification of novel therapeutic targets for male infertility ........................................................................................................................................8

Lichenicidin: heterologous expression and biosynthesis comparison ............................10

Strategies to improve Eucalyptus globulus drought resistance ......................................12

Avaliação do efeito do meio de cultivo no teor e perfil lipídico das microalgas haematococcus pluvialis flotow, chlamydomonas sp. ehrenberge e oocystis sp. nageli. .............................................................................................................................14

Bioethanol Production From Bio-detoxified HSSL: Saccharomyces cerevisiae vs Scheffersomyces (Pichia) stipitis ....................................................................................16

Produção de biossurfactantes utilizando culturas microbianas isoladas da Ria de Aveiro .............................................................................................................................18

Assessment of Quercus suber Soil Microbiome using DGGE Fingerprints and Barcoded Pyrosequencing...............................................................................................20

10. Ionic-liquid-based aqueous biphasic systems using phosphate-based salts ....................22 11. Design of ionic liquids for lipase purification ................................................................24 12. Assessing the significant effects of ionic liquids towards the enzymatic activity of Candida antarctica lipase B ...........................................................................................26 13. Industrial by-products as a source of VFAs by anaerobic digestion ..............................28 14. Identification and analysis of genes acquired by horizontal gene transfer in Bursaphelenchus xylophilus ...........................................................................................30 15. Sulforaphane induces cell cycle arrest and genotoxicity in MG-63 human osteosarcoma cell line ............................................................................................................................32

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16. Enhanced of oxygen transfer in an airlift reactor using silica microparticles.................34 17. Bioethanol production by Pichia stipitis for different values of KLa ............................36 18. Evaluation of AgNP nanoparticles on osteosarcoma viability: approaches to environmental studies .....................................................................................................38 19. Structural studies of the mammalian end binding protein 3 employing high-resolution solid-state MAS NMR ...........................................................................40 20. Lipophilic composition and biological activity of different parts of Cynara cardunculus L. var. altilis (DC)...........................................................................................................42 21. Ionic-liquid-based aqueous biphasic systems for the extraction of L-tryptophan ..........44 22. Production and charecterization of polyol via liquefaction of biomass ..........................46 23. Aqueous Biphasic Systems As A Green Perspective For Treatment Of Effluents Contaminated With Ionic Liquids ...................................................................................48 24. Detoxification of Sulphide Spent Liquors for Bioethanol Fermentation Employing Ion Exchange Resins .............................................................................................................50 25. Chemical functionalization of nickel nanowires for optical bio-tagging .......................52 26. Metabolic alterations in healthy pregnancy and prenatal disorders: a nuclear magnetic resonance (NMR) spectroscopy study of maternal blood plasma ..................................54 27. Effect of temperature and high pressure treatments in starch suspensions .....................56 28. High Pressure Technological Platform of University of Aveiro for Food Preservation and Modification and Biotechnological Applications ....................................................58 29. Polysaccharide coated magnetic nanoparticles for dye removal from water..................60 30. Functionalized magnetic nanoparticles for the immobilization of biomolecules ...........62 31. Microfauna community as an indicator of effluent quality and process performance in an activated sludge system for treating industrial wastewater ..............64

â&#x20AC;&#x153;Os Primeiros Mestres em Biotecnologiaâ&#x20AC;?

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III Workshop Biotecnologia UA

“Os Primeiros Mestres em Biotecnologia pela Universidade de Aveiro” O ano letivo de 2006-2007 foi o primeiro ano de funcionamento da Licenciatura em Biotecnologia da Universidade de Aveiro. Os primeiros alunos que a frequentaram optaram por ingressar na mesma sem historial nem referências anteriores, que pudessem dar-lhes indicações da qualidade do ensino em Biotecnologia na UA, demonstrando assim confiança no nível de formação que esperavam no seu percurso académico. No final da Licenciatura a esmagadora maioria destes alunos optou por realizar o Mestrado em Biotecnologia da UA, renovando e reforçando assim a sua confiança na formação científica que a UA oferece nesta área, concluindo o Mestrado entre Julho e Dezembro de 2011. Por estas razões, o III Workshop de Biotecnologia da UA, é dedicado a estes alunos, com sentido de homenagem e reconhecida gratidão por terem sido os pioneiros na escolha da UA para a sua formação em Biotecnologia.

A Organização do III Workshop de Biotecnologia da UA,

BEST Aveiro Direcção de Curso da Licenciatura e Comissão Científica do Mestrado em Biotecnologia

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Rhamnolipid production by bacteria isolated from contaminated soil Ana C.G. Martins1, Álvaro S. Lima2, M. Rosário M. Domingues3, João A.P. Coutinho1, Luísa S. Serafim1 1

Department of Chemistry, CICECO, University of Aveiro, 3810-193Aveiro, Portugal Programa de Pós-Graduação em Engenharia de Processos, Universidade Tiradentes, Av. Murilo Dantas 300, Farolândia, Aracaju-SE, Brasil 3 Department of Chemistry, QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal

Por motivos de direitos de autor, o conteúdo do Resumo relativo ao trabalho de investigação com o título enunciado acima não está disponível online. Para mais informações contactar o autor principal através de a38485@ua.pt.

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Rhamnolipid production by bacteria isolated from contaminated soil Ana C.G. Martins1, Álvaro S. Lima2, M. Rosário M. Domingues3, João A.P. Coutinho1, Luísa S. Serafim1 1

Por motivos de direitos de autor, o conteúdo do Poster relativo ao trabalho de investigação com o título enunciado acima não está disponível online. Para mais informações contactar o autor principal através de a38485@ua.pt.

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Second generation bioethanol from spent sulphite liquor using Pichia stipitis suspended and immobilized Susana R. Pereira, Diogo J.P. Nunes, Jean D. Santos, Dmitry V. Evtuguin, Luísa S. Serafim and Ana M.R.B. Xavier CICECO, Departamento de Química, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal

Sulphite Spent Liquors (SSLs) are side products from acidic sulphite wood pulping and, after their concentration by evaporation, are normally burned for energy recovery. The great advantage of SSLs over other lignocellulosic feedstocks is that during wood pulping process, delignification and hydrolysis occurs resulting in fermentable sugars. While SSLs from softwood are well characterized1, only scarce knowledge is available on the chemical composition of Hardwood Spent Sulphide Liquors (HSSL) namely from acidic sulphite pulping of eucalypt wood. The major components of HSSLs are sulphonated lignin (lignosulphonates) and sugars, around 40-45 g.L-1, mainly xylose2,3. Xylose can be converted by Pichia stipitis to second generation bioethanol. However, the presence of high amounts of acetic acid and lignosulphonates in HSSLs is inhibitory for growth3,4. The filamentous fungus Paecilomyces variotii is able to grow in HSSLs, consuming most of the inhibitory compounds 1. This process, designated as biological detoxification step of HSSLs should be performed before the ethanol production step by P. stipitis. Hence, the aim of this study was to evaluate bioethanol production by P. stipitis from the sugars present in HSSL after detoxification by P. variotii. This evaluation included the use P. stipitis free and immobilized in calcium alginate beads. The two fermentative strategies were compared in order to select the best one. The detoxification step was studied using a Sequential Batch Reactor (SBR) with 3 cycles since a higher amount of detoxified liquor was obtained. The ethanolic fermentation of detoxified HSSL with immobilized P. stipitis was the most successful approach for producing ethanol. A maximum ethanol concentration of 0.89 g.l-1 was attained with the suspended yeast, much lower than the concentration achieved with the immobilized yeast (3.7 g.l-1). A higher ethanol yield was also reached with the immobilized culture (46.6 grams of ethanol per grams of substrate) confirming the effectiveness of the yeast immobilization when compared with the suspend culture, probably due to the protective effect of the calcium alginate beads against the toxic compounds still present in the liquor.

Acknowledgments Authors thank the Portuguese Innovation Agency for the financial support of this work carried out in the frame of FEDER/PRIME Program (R&D grand SIME nº 00/22689) and FCT for the PhD grant to S. R. Pereira (SFRH / BD / 64552 / 2009) References (1) Pereira S.R.; Ivanuša Š.; Evtuguin D.V.; Serafim L.S.; Xavier A.M.R.B. Bioresour. Technol. 2012, 103, 131. (2) Marques A.P.; Evtuguin D.V.; Magina S.; Amado F.M.L.; Prates A. J. Wood Chem. Technol. 2009 4, 322. (3) Xavier A.M.R.B.; Correia M.F.; Pereira S.R.; Evtuguin D.V. Bioresour. Technol. 2010 8, 2755. (4) Nigam J.N. J. Ind. Microbiol. Biot. 2001 3, 145.

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Yeast two hybrid system in identification of novel therapeutic targets for male infertility Maria João Freitas, Luís Korrodi-Gregório, Sara LC Esteves and Margarida Fardilha Signal Transduction Laboratory, Centre of Cell Biology, University of Aveiro, Portugal.

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Lichenicidin: heterologous expression and biosynthesis comparison Joana C. Barbosa1, R.D. Suessmuth2, Tânia Caetano1, Sónia Mendo1 1

Department of Biology and CESAM, University of Aveiro, 3810-193 Aveiro, Portugal; 2 Institut für Chemie, Technische Universität Berlin, Berlin, Germany

The dissemination of drug-resistant pathogenic bacterial strains has increased the need for new antibiotics with specifically targeted activities 1. A new type of antimicrobial peptides produced by Gram positive bacteria, the so-called lantibiotics, were found to have promising effects against some of those strains, namely methicilin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) 2. Lichenicidin is a two-peptide lantibiotic, which means that two peptides (Bliα and Bliβ) are produced and act synergistically to potentiate full antimicrobial activity 3. Lichenicidin is naturally produced by Bacillus licheniformis I89 and is active against MRSA, Enterococcus faecium, Haemophilus influenza and Listeria monocytogenes 4. Moreover, lichenicidin was the first lantibiotic to be totally produced in vivo using a Gram negative heterologous expression system, Escherichia coli 4-5. Heterologous expression of lichenicidin in E. coli allows easier manipulation procedures, which is also an important feature to take in account regarding large-scale production. Also, it allowed the incorporation of nonproteinogenic amino acids in lantibiotics using an in vivo approach 6. However, all these accomplishments were achieved using heterologous expression based on a fosmid as vector and directed by B. licheniformis genetic elements (promoters, RBS, etc.). Thus, in the present work different vectors will be tested as well as the application of E. coli transcription and translation determinants. The production levels of Bliα and Bliβ in all these expression systems will be determined by HR-ESI-MS/MS in order to identify the condition(s) of maximum lichenicidin biosynthesis. Ultimately, the selected system will be applied to produce higher amounts of Bliα and Bliβ containing noncanocical amino acids.

Acknowledgments Thanks are due to LBM (Molecular Biology laboratory), department of Biology and CESAM, University of Aveiro. References (1) Z. O. Shenkarev, E. I. Finkina, E. K. Nurmukhamedova, S. V. Balandin, K. S. Mineev, K. D. Nadezhdin, Z. A. Yakimenko, A. A. Tagaev, Y. V. Temirov, A. S. Arseniev, T. V. Ovchinnikova, Biochemistry 2010, 49, 6462-6472. (2) aG. Bierbaum, H.-G. Sahl, Current Pharmaceutical Biotechnology 2009, 10, 2-18; bD. Field, C. Hill, P. D. Cotter, R. P. Ross, Molecular Microbiology 2010, 78, 1077-1087. (3) J. M. Willey, W. A. v. d. Donk, Annu. Rev. Microbiol. 2007, 61, 477-501. (4) T. Caetano, J. M. Krawczyk, E. Mösker, R. D. Süssmuth, S. Mendo, Chemistry & Biology 2011, 18, 90100. (5) T. Caetano, J. M. Krawczyk, E. Mösker, R. D. Süssmuth, S. Mendo, Applied and Environmental Microbiology 2011, 77. (6) F. Oldach, R. Al Toma, A. Kuthning, T. Caetano, S. Mendo, N. Budisa, R. D. Süssmuth, Angewandte Chemie International Edition 2012, 51, 415-418.

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Lichenicidin: heterologous expression and biosynthesis comparison Joana C. Barbosa1, R.D. Suessmuth2, Tânia Caetano1, Sónia Mendo1 1

Department of Biology and CESAM, University of Aveiro, 3810-193 Aveiro, Portugal; 2 Institut für Chemie, Technische Universität Berlin, Berlin, Germany

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Strategies to improve Eucalyptus globulus drought resistance Joana Vale, João H. Picado, Silvia M. Moreira, Claudia de Jesus, Barbara Correia, Celeste Dias, Lucinda Neves, Clara Araujo, Glória Pinto* Department of Biology & CESAM (Centre for Environmental and Marine Studies), University of Aveiro, 3810193 Aveiro (Portugal), *gpinto@ua.pt Altri Florestal SA Quinta do Furadouro, 2510-582 Olho Marinho (Portugal)

Eucalyptus globulus growth and survival are restricted by its drought sensitivity and the development of breeding programs requires information about stress-response of breeding material selected for deployment. Also, strategies to alleviate the stress in order to prevent extensive production losses should be implemented. For example, in other species the increase of resistance to abiotic stresses has been achieved by exogenous application of various organic solutes as Salycilic Acid (SA)1 2. This research was developed at Department of Biology of the University of Aveiro. E. globulus clones were selected from the Altri Florestal breeding program. Six months-old rooted cuttings were sprayed with SA (1.5 mM). Then, four groups of plants were organized as: WS (water stress treatment-18% field capacity), WW (well watered-80% FC), WS+SA and WW+SA. Photosynthetic related parameters were assessed: net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (E) and the intercellular CO 2 concentration (Ci). Results showed that SA has a positive effect in stress alleviation: the Pn decrease was less evident in plants exposed to WS and sprayed with SA. Water stress promoted the decrease of gs, E and Ci, but no differences were observed between WS and WS+SA. Further studies are needed to investigate whether SA confers plant abiotic stress resistance to Eucalyptus seedlings and if such tolerance is correlated with changes in antioxidant system and hormones.

Acknowledgments This work is supported by FEDER through COMPETE (Programa Operacional Factores de Competitividade) and by the FCT project PTDC/AGR-CFL/112996/2009. Glória Pinto is hired under the programme Ciência 2008 (FCT, Portugal), co-funded by the Human Potential Operational Programme (National Strategic Reference Framework 2007-2013) and European Social Fund (EU). FCT supported the fellowship of M. C. Dias (SFRH/BPD/41700/2007).

References (1) Ghasemzadeh, A.; Jaafar, H. Z. E. Effect of salicylic acid application on biochemical changes in ginger (Zingiber officinale Roscoe). Journal of Medicinal Plants Research. (2012) 6:790-795 (2) Horváth, E; Janda, T.; Szalai, G. Induction of Abiotic Stress Tolerance by Salicylic Acid Signaling. J Plant Growth Regul (2007) 26:290–300

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Avaliação do efeito do meio de cultivo no teor e perfil lipídico das microalgas haematococcus pluvialis flotow, chlamydomonas sp. ehrenberge e oocystis sp. nageli Carolina Espirito Santo1 , Joana Ramos Costa1 , Paula Mota1, Kiril Bahcevandziev2 1

Microalgae - Investigação e Desenvolvimento, Lda., Bencanta, 3040-316 Coimbra, Portugal 2 Escola Superior Agrária de Coimbra, Bencanta, 3040-316 Coimbra, Portugal

As microalgas têm sido, nas últimas décadas, alvo de intensa investigação científica, resultado da inúmera variedade de espécies encontradas bem como consequência da capacidade destas em sintetizar substâncias de elevado valor comercial (ácidos gordos, βcaroteno e astaxantina, entre outros), com aplicabilidade nas indústrias farmacêutica, ambiental ou agro-alimentar1. Neste estudo, foram utilizadas as microalgas Haematococcus pluvialis Flotow, Chlamydomonas sp. Ehrenberge e Oocystis sp. Nageli, pertencentes ao Filo Chlorophyta (algas verdes)2, tendo sido nosso objetivo a avaliação do efeito de um meio de cultivo no teor e perfil lipídico das microalgas acima referidas. Foram utilizados dois meios distintos: um meio (Meio MB), criado pela empresa Microalgae e um meio de controlo (Meio M7), desenvolvido pela ACOI (Algoteca de Coimbra). A análise do teor lipídico envolveu a utilização da técnica de extracção por solvente e a cromatografia gasosa, para análise do perfil de FAME´s (Ésteres Metílicos de Ácidos Gordos). Nas três espécies estudadas, verificou-se um aumento significativo da percentagem de lípidos aquando do cultivo em Meio MB, quando comparado com o Meio M7. As percentagens para as três espécies estudadas, Haematococcus pluvialis (H1;H2), Chlamydomonas sp. (C1) e Oocystis sp. (O1), foi de 7,35%, 12,05%, 8,33% e 17% e de 23,53%, 23,82%, 14,65% e 21,5%, para os meios M7 e MB respectivamente. Relativamente ao perfil de ácidos gordos, este revelou-se variável, tanto ao nível do tamanho das cadeias hidrocarbonadas como quanto à posição do grau de insaturação, em função do meio de cultivo. Este estudo parece estabelecer uma relação entre o meio de cultivo e a acumulação de lípidos, revelando ainda que esta indução da acumulação origina uma alteração na sua composição. A eventual manipulação das microalgas, direccionando o seu crescimento e meio de cultivo em função do perfil desejado, exigirá sempre uma escolha criteriosa da estirpe envolvida.

Agradecimentos Microalgae - Investigação e Desenvolvimento, Lda.

Referências (1) Mata, T.M.; Martins, A.A.; Caetano.; N.S. Microalgae for biodiesel production and other applications: A review. Renew. Sustain. Energy.2009, 14, 217-232. (2)

Santos, Maria de Fátima.; Santos, Lília M. A. Iniciação ao estudo das Microalgas. 2002

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Bioethanol Production From Bio-detoxified HSSL: Saccharomyces cerevisiae vs Scheffersomyces (Pichia) stipitis D. J. P. Nunes1, V. Sànchez i Nogué2, M. F. Gorwa-Grauslund2, A. M. R. B. Xavier1 1 Department of Chemistry & CICECO, University of Aveiro, 3810-193 Aveiro, Portugal 2 Division of Applied Microbiology, Lund University, PO Box 124, SE-22100 Lund, Sweden

Spent sulphite liquors (SSLs) are the side product from acidic sulphite wood pulping and are usually burned for energy and chemicals regeneration [1]. CAIMA, S. A. industry produces hardwood SSL (HSSL), resulting from a magnesium-based Eucalyptus globulus cooking. This HSSL contains lignosulphonates (LS) and sugars, mainly D-xylose [2], which makes it a suitable substrate for highly efficient pentose-fermenting yeasts, such as S. stipitis. However, the presence of high amounts of acetate (9.6 g/L), traces of furfural and HMF, polyphenols and low molecular weight LS act as inhibitors of the microbial metabolism [3]. Thus, a pre-detoxification method is needed to remove and/or reduce the inhibitors content [2]. This project compares the performance of two yeast species for bioethanol production from a Pulp and Paper industry residue previously detoxified with Paecilomyces variotii [2]: TMB3500 [4], an industrial strain of S. cerevisiae and S. stipitis, that exhibits the best potential for industrial application among the yeasts that are capable to ferment xylose [2]. Batch tests using bio-detoxified HSSL in six different percentages (0, 20, 40 60, 80 and 100%), with constant temperature and agitation were carried out and maximum specific growth rate (µmax) values were calculated. With 60% of HSSL there was a good compromise between the final biomass, the µmax and the ability to successfully resist to the presence of yeast inhibitors. This percentage was used as a pre-adaptation step to enhance fermentation efficiencies of undiluted HSSL (100% HSSL). Fermentations 100% bio-detoxified HSSL were performed during 100h in 1L fermentors using an initial amount of 1.08 x 108 CFUs/mL of S. cerevisiae TMB3500 or S. stipitis. The higher ethanol yield was obtained using S. cerevisiae TMB3500 (0.34 ± 0.10 g eth/g sugars) and the CFUs were stable until the end of the assay. S. stipitis was less efficient and led to an ethanol yield of 0.1 ± 0.02 g eth/g sugars and to an increase in CFUs/mL. However, this lower ethanol yield is due to the aeration used in the fermentors that must be reduced to microaerophilic conditions.

Acknowledgments Authors thanks to TMB/Division of Applied Microbiology of Lund University for using S. cerevisiae TMB3500 and FCT for financial support to Associate Laboratory CICECO (Pest-C/CTM/LA0011/2011). CAIMA, S. A. for providing the HSSL and Aveiro University/AN PROALV for the Erasmus scholarship that allowed Diogo J.P. Nunes to be in Lund for the Autumn Semester of 2011-2012. References (1) E. Sjöström, Wood Chemistry. Fundamentals and Applications. New York, USA: Academic Press. (1993). (2) S. R. Pereira, Š. Ivanuša, D.V. Evtuguin, L.S. Serafim, A. M. R. B. Xavier, Biological treatment of eucalypt spent sulphite liquors: A way to boost the production of second generation bioethanol, Bioresource Technol. 103 (2012) 131-135. (3) C. P. Van Zyl, B. A. du Preez, James C., "Acetic Acid Inhibition Of D-xylose Fermentation By Pichia stipitis," Enzyme and Microbial Technology. 13 (1991) 82-86. (4) J.R.M. Almeida, K. Karhumaa, O. Bengtsson, M.F. Gorwa-Grauslund, Screening of Saccharomyces cerevisiae strains with respect to anaerobic growth in non-detoxified lignocellulose hydrolysate, Bioresource Technology. 100 (2009) 3674-3677. “Os Primeiros Mestres em Biotecnologia”

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Produção de biossurfactantes utilizando culturas microbianas isoladas da Ria de Aveiro S. Quintinha1, A.C.G. Martins1, J.F.B. Pereira1,A. Cunha2, L. S. Serafim1 and J.A.P. Coutinho1 1 2

CICECO, Departamento de Química, Universidade de Aveiro, 3810-193Aveiro, Portugal Departamento de Biologia e CESAM, Universidade de Aveiro, 3810-193 Aveiro, Portugal

Na atualidade os surfactantes têm grande importância na sociedade pois podem ser aplicados em diversas áreas, desde a agricultura, saúde, indústria alimentar e ainda em proteção ambiental. Os surfactantes são moléculas anfipáticas, constituídas por uma parte hidrofóbica (apolar) e outra hidrofílica (polar) e por essa razão têm capacidade de reduzir a tensão superficial e interfacial, bem como de formar emulsões em fluidos. A produção destes compostos com capacidade tensioativa por microrganismos, biossurfactantes, permite o alargamento da sua aplicação a nível industrial. Os biossurfactantes possuem vantagens de utilização em relação aos surfactantes químicos tais como a baixa toxicidade, elevada biodegradabilidade e ampla faixa de pH e temperatura.[1] Dependendo do microrganismo produtor, obtêm-se diferentes classes de biossurfactantes com propriedades distintas: ramnolípidos, produzidos por Pseudomonas aeruginosa [2] e surfactina, produzida por Bacillus subtilis [3]. No entanto os elevados custos de produção associados têm dificultado a aplicação de biossurfactantes. A seleção de estirpes microbianas isoladas de ambientes contaminados, bem como a utilização de resíduos industriais como substratos para o crescimento permitem a diminuição do preço final dos biossurfactantes [3]. Neste trabalho pretende-se analisar o potencial de estirpes bacterianas, isoladas da Ria de Aveiro, para a produção de biossurfactantes. A produção de moléculas com capacidade tensioativa, pelas estirpes isoladas, foi avaliada pela redução da tensão superficial e por medição do índice de emulsificação. Finalmente caracterizaram-se as classes de biossurfactantes produzidas.

Referências 1. Nitschke, M. and G.M. Pastore, Biosurfactants: Properties and applications. Quimica Nova, 2002. 25(5): p. 772-776. 2. Arino, S., R. Marchal, and J.P. Vandecasteele, Identification and production of a rhamnolipidic biosurfactant by a Pseudomonas species. Applied Microbiology and Biotechnology, 1996. 45(1-2): p. 162-168. 3. Nitschke, M. and G.M. Pastore, Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava wastewater. Bioresource Technology, 2006. 97(2): p. 336-341.

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Assessment of Quercus suber Soil Microbiome using DGGE Fingerprints and Barcoded Pyrosequencing Diogo Pinho1,2, Paula Gomes2, Cristina Barroso2, Isabel Henriques3, Miguel Pinheiro2, António Correia3, Mariana Telles4, Conceição Silva4, Conceição Egas2 1

Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal Advanced Services Unit, Biocant, Parque Tecnológico de Cantanhede, Nu.04, Lt03, 3060-197 Cantanhede, Portugal 3 CESAM – Centre for Environmental and Marine Studies & Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal 4 Associação de Produtores Florestais do Concelho de Coruche e Limítrofes, Rua dos Guerreiros, 54, 2100-183 Coruche, Portugal 2

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Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal Advanced Services Unit, Biocant, Parque Tecnológico de Cantanhede, Nu.04, Lt03, 3060-197 Cantanhede, Portugal 3 CESAM – Centre for Environmental and Marine Studies & Department of Biology, University of Aveiro, 3810193 Aveiro, Portugal 4 Associação de Produtores Florestais do Concelho de Coruche e Limítrofes, Rua dos Guerreiros, 54, 2100-183 Coruche, Portugal 2

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Ionic-liquid-based aqueous biphasic systems using phosphate-based salts T. Mourão, A. F.M. Cláudio, I. Boal, M. G. Freire and J. A. P. Coutinho Departamento de Química, CICECO, Universidade de Aveiro, 3180-193 Aveiro, Portugal

The development of efficient extractive and recovery techniques for common addedvalue (bio)molecules is a crucial requirement under the biotechnological field. These techniques must allow the recovery of (bio)molecules with a high level of purity, give rise to high extraction yields, and reduce the environmental hazards and economical costs associated. Therefore, some efforts have been made during the last decades to apply aqueous biphasic systems (ABS) as novel extractive techniques. Besides the low cost of these techniques, the use of volatile organic solvents is also avoided. Common ABS are formed by mixing two different polymers, one polymer and one salt, or two salts at certain concentrations in an aqueous solution. In recent years, the substitution of common polymers by ionic liquids (ILs) has leaded to novel ABS with a huge relevance.1,2 These IL-based ABS have many advantages over conventional systems, namely, their low viscosity and faster phase separation times, and the possibility of tailoring the phases’ polarities by an adequate selection of the ionic fluid. In this work, the ternary phase diagrams of several IL-based ATPS were determined at 298 K and atmospheric pressure. Three phosphate based-salts and one mixture of salts (K3PO4, K2HPO4, K2HPO4/KH2PO4, and KH2PO4) were combined with ten 1-butyl-3methylimidazolium-based ILs, and the combinations between the several salts and ILs were evaluated toward their ability to create ABS. Moreover, the ternary systems were further characterized by the determination of their tie-lines and tie-line lengths. Finally, the pH of the coexisting phases was determined to ascertain on the applicability of these systems in the biotechnological field.

Acknowledgments The authors acknowledge FCT- Fundação para a Ciência e a Tecnologia for the Pest-C/CTM/LA0011/2011 and PTDC/QUI-QUI/121520/2010 projects, and doctoral and post-doctoral grants SFRH/BD/74503/2010 and SFRH/BPD/41781/2007 of A. F. M. Claúdio and M. G. Freire, respectively.

References (1) S.P.M. Ventura, C.M.S.S. Neves, M.G. Freire, I. M. Marrucho, J. Oliveira and J.A.P. Coutinho, “Evaluation of Anion Influence on the Formation and Extraction Capacity of Ionic-Liquid-Based Aqueous Biphasic Systems”, J. Phys. Chem. B. Vol. 113, pp. 9304-9310, 2009. (2) S.P.M. Ventura, S.G. Sousa, L.S. Serafim, A.S. Lima, M.G. Freire and J.A.P. Coutinho, “Ionic-Liquid-Based Aqueous Biphasic Systems with Controlled pH: The Ionic Liquid Anion Effect.” J. Chem. Eng. Data. Vol. 57, pp. 507-512, 2012.

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“Os Primeiros Mestres em Biotecnologia”

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Design of ionic liquids for lipase purification Sónia P. M. Ventura, Sílvia G. Sousa, Mara G. Freire, Álvaro S. Lima and João A. P. Coutinho Department of Chemistry & CICECO, University of Aveiro, 3810-193 Aveiro, Portugal

A considerable number of lipase (EC 3.1.1.3) is produced by microorganisms such as yeast, molds and bacteria. 1 Microbial lipase is normally obtained by fermentation processes, which are conduced mainly by three techniques: submerged fermentation, solid-state fermentation and immobilized-cell fermentation. When produced by microorganisms, these fermentation processes generate, beyond the desired components, diverse products that frequently restrict the use of the fermented broth in industrial procedures. For the success of the commercial production of enzymes and proteins, efficient downstream processing techniques were needed. Aqueous biphasic systems (ABS) appears, in this context, as good alternative techniques aiming the enzymatic purification steps, since they could be considered as harmful to the living system, because the high water content and the possibility of maintain the neutral pH value. An alternative for ABS is the use of ionic liquids (ILs) and waterstructuring salts. The aim of this work was to study the lipase (Candida antarctica lipase B CaLB) partition using different ABS systems, varying the type of salt and also the IL. As a result, the purification factors and recoveries of each system for both phases were determined. The salt solutions used were K3PO4, K2HPO4 and a buffer solution composed by K2HPO4 and KH2PO4. The ILs belongs to different families, pyrrolidinium, piperidinium, pyridinium and imidazolium. The latest was used to study the influence of the alkyl chain length - [C2mim]Cl, [C4mim]Cl, [C6mim]Cl, [C7mim]Cl, [C8mim]Cl, [C7H7mim]Cl and the anion [C4mim][CH3SO3], [C4mim][CF3SO3], [C4mim][N(CN)2], towards the lipase activity. Accordingly to the results, was concluded that the hydrophobic ILs with higher alkyl chain length, were responsible for the major effect in the purification factor. Moreover, was proved that the aromatic nature as no significant influence in this purification process. Therefore, the higher purification factor with a higher recovery was obtained for the [C 8pyr][N(CN)2]. According to these results, is possible to prove the success of using ILs-based ABS as an alternative in the removal of the contaminating proteins from a fermentation process. 2

Acknowledgments The authors are grateful for financial support from Fundação para a Ciência e a Tecnologia for the Ph.D. grant SFRH/BD/37830/2007 of S.P.M. Ventura and from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the post-doctoral scholarship of A.S. Lima (process number 2185/09-1). The authors also thank the support from Novozymes for the supply of the enzyme and Rita Ferreira for the electrophoresis analysis.

References (1) Schomburg, D; Salzmann, M, Enzyme Handboook 3. Berlim: Springer-Verlag 1991 (2) Ventura, S. P. M.; Sousa, S. G.; Freire, M. G.; Serafim, L. S.; Lima, A. S.; Coutinho, J. A. P., J. Chromatogr. B 2011, 879, 2679.

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III Workshop Biotecnologia UA

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Assessing the significant effects of ionic liquids towards the enzymatic activity of Candida antarctica lipase B Sónia P. M. Ventura, Luísa D. F. Santos, Jorge A. Saraiva and João A. P. Coutinho Department of Chemistry & CICECO, University of Aveiro, 3810-193 Aveiro, Portugal

Enzymes have found great uses in several industries such as food, pharmaceutical, detergent, textile, pulp and paper, animal feed, leather and cosmetics.1 In nature, enzymes act as catalysts of living systems and are designed to function in aqueous solutions. However, anhydrous conditions are needed for synthetic transformations using these macromolecules. The use of organic solvents as non-aqueous environments offers the possibility of carrying out synthetic reactions by hydrolytic enzymes, increasing the solubility of molecular substrates.2 However, biocatalysts in non-aqueous media often suffer from reduction of activity, selectivity and/or stability.3 In addition, conventional organic solvents present high volatility and flammability being harmful to the environment. The use of ionic liquids (ILs) in biocatalytic processes has recently gained much attention as an environmentally attractive alternative to classical organic solvents. They have been seen as good solvents in a wide variety of biochemical processes, because they can improve enzyme stability, substrate and/or product selectivity and also are responsible for the suppression of some side reactions.4 The main idea of this work was to provide some information about the influence of several imidazolium- based ILs in the activity of Candida antarctica lipase B (CaLB). Parameters such as the IL concentration, the effect of different anions and distinct alkyl chain lengths were considered in this study. This cation core was used to study the effect of different anions and also the effect of the elongation of the alkyl chain (C 2 to C10). The studied anions were bromide Br, dicyanamide [N(CN)2], hydrogenosulphate [HSO4], methanesulfonate [CH3SO3], triflate [CF3SO3], acetate [CH3COO], trifluoracetate [CF3COO] and chloride Cl. The enzyme activity was assayed spectrophotometrically. The main conclusion of the present work is that the enzymatic activity was affect by all the parameters studied. In terms of the effects of the different anions, these results indicate [HSO4] as the responsible for the major negative effects and Br as the responsible for the smaller decrease. The influence of different concentrations was also studied being proved that this parameter has a major role in the effects of alkyl chain in the enzymatic activity.

Acknowledgments The authors are grateful for financial support from Fundação para a Ciência e a Tecnologia (FCT) for the PostDoctoral grant SFRH/BPD/37830/2007 of S.P.M. Ventura. The authors also thank the support from Novozymes company for the supply of the enzyme.

References (1) Houde, A; Kademi, A.; Leblanc, D. Appl. Biochem. Biotechnol. 2004, 118, 155. (2) Wang, L.; Chen, Y. Chin. J. Biotechnol. 2009, 25, 1789. (3) Gorman, L. A. S.; Dordick, J. S. Biotechnol. Bioeng. 1992, 39, 392. (4) Van Rantwijk, F.; Sheldon, R. A. Chem. Rev. 2007, 107, 2757.

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“Os Primeiros Mestres em Biotecnologia”

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Industrial by-products as a source of VFAs by anaerobic digestion Fátima L. Sousa, Susana R.S. Pereira, Ana M. R. B. Xavier, Dmitry V. Evtugin and Luísa S. Serafim Department of Chemistry & CICECO, University of Aveiro, 3810-193 Aveiro, Portugal

Pollution, climate changes and eminent exhaustion of natural resources (fossil fuels) are the main consequences of the fossil fuels dependency [1]. Anaerobic Digestion is the process (with four steps: hydrolysis, acidogenesis, acetogenesis and methanogenesis) whereby bacteria break down, organic material in the absence of air, yields a biogas containing methane. This process has shown great potential in using renewable resources [2]. HSSL (Hardwood Spent Sulphite Liquor) is a by-product of the paper industry, which constitutes an environmental problem. This waste is rich in lignocellulose that, with appropriate pre-treatment, provides carbon substrates as xylose, glucose and other sugars. HSSL can serve as a substrate for yeast like Scheffersomyces stipitis to produce ethanol. After the fermentation process the fermentative medium still contains a high amount of sugars. Those sugars can act as substrate in an anaerobic digestion (AD) step to produce added value products like hydrogen, ethanol or VFAs [2]. VFAs can be applied as building blocks to produce add-products as bioenergy, biomaterials (bioplastics) and chemicals (solvents) [3]. The main goal of this work is to valorise different fractions of HSSL without pretreatment (SSL), after chemical pre-treatment (SSLq) and after bioethanol production (SSLs) into VFAs. The effect of different methanogenic inhibitors as bromoethanesulfonate (BES), temperature and high pressure (HP) was also evaluated. The results of this study show that acidification of HSSL and wastes from bioethanol production were possible with the production of acetic, propionic and butyric acid. The effect of methanogenic suppression, HP (never studied before) and BES had a similar effect, an important result considering the high price of BES.

Acknowledgments Thanks are due to the CICECO Research Unit and FEDER Funds through the Programa Operacional Factores de Competitividade – COMPETE and national funds through FCT – Fundação para a Ciência e a Tecnologia under the project PEST-C/CTM/LA/0011/2011. References [1] Y-H. Zhang, “Reviving the carbohydrate economy via multi-product lignocelluloses biorrefineries,” J Ind Microbiol Biotechnol, vol. 35, pp. 367–375, 2008. [2] J.Marta-Alvarez, S. Macé, P. Llabrés, “Anaerobic digestion of organic solid wastes:na overview of reach achivements and perpectives”, Bioresour. Technol, vol. 74, pp. 3-16, 2000. [3] A.Yalini, P. Malarmagal, N. Nagamany, “Predicting VFA formation by dark fermentation of particulate substrates”, Bioresour. Technol. vol. 101, pp. 7492-17499, 2010. “Os Primeiros Mestres em Biotecnologia”

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“Os Primeiros Mestres em Biotecnologia”

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Identification and analysis of genes acquired by horizontal gene transfer in Bursaphelenchus xylophilus Daniel F. Martins1,2, Cristina Barroso2, Paula Gomes2, Miguel Pinheiro2, Felipe Santos2, Jorge Saraiva1 and Conceição Egas2 1

Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal 2 Advanced Services Unit, Biocant, 3060-197 Cantanhede, Portugal

Pine wilt disease was first identified in Japan in 1905 and its association with the nematode Bursaphelenchus xylophilus dates back to 1972. The devastating nature of this disease and the big economical losses it generates along with the ecological impact led the European Plant Protection Organization to consider its causal agent as a european quarantine pest. Until now, information regarding the nature of B. xylophilus with reference to its morphological characters, life cycle, vector association and potential host have been gathered and documented. However, little is known on the molecular mechanisms of the disease. According to current knowledge there two factors that may play a role in the disease: bacteria associated with the nematode and new nematode functions acquired through horizontal gene transfer (HGT).1-2 The transcriptome of B. xylophylus was recently sequenced and annotated at Biocant, providing a large amount of genomics information. The goal of our work was to identify new genes incorporated in the nematode genome through HGT in these transcripts. To accomplish our objective we established a data pipeline composed of several filters to discard genes of nematodal origin and select those of bacterial or fungal origin. B. xylophylus annotated transcripts were first screened for gene functions, where transcripts with annotations with Evalue ≤10-6 were discarded, for organism of origin, where only transcripts belonging to bacteria and fungi were kept and for liability of the classification, where hypothetical and predicted proteins were also discarded. A final screen was performed by removing all hits matching the microbial community surrounding the nematode, as determined by barcode pyrosequencing. The pipeline outlined 18 candidates that were submitted to a BLAST search against bacteria, fungi, nematodes and other organisms. Homologous sequences were then downloaded, aligned with the candidate and the phylogenetic relationship between them determined. This last step evidenced the presence of 2 genes resulting from HGT (1 from bacteria and 1 from fungi). This pipeline identified two HGT genes, endo-β-1,3-glucanase, already described for B. xylophilus3, and alcohol dehydrogenase, previously described for C. elegans4 thus validating our approach. The role of the identified HGT genes in nematode and disease will be studied, expecting to provide new information on the disease mechanism and in the identification of new targets eligible for disease control.

References 1.Vicente, C.; Espada, M.; Vieira, P.; Mota, M., Pine Wilt Disease: a threat to European forestry. Eur. J. Plant Pathol. 2011, 1-11. 2.Jones, J. T.; Moens, M.; Mota, M.; Hongmei, L.; Kikuchi, T., Bursaphelenchus xylophilus: opportunities in comparative genomics and molecular host–parasite interactions. Mol. Plant Pathol. 2008, 9 (3), 357-368. 3.KIKUCHI, T.; SHIBUYA, H.; JONES, J. T., Molecular and biochemical characterization of an endo-²-1,3glucanase from the pinewood nematode Bursaphelenchus xylophilus acquired by horizontal gene transfer from bacteria. 2005. 4.Parkinson, J.; Blaxter, M., SimiTri—visualizing similarity relationships for groups of sequences. Bioinformatics 2003, 19 (3), 390-395. “Os Primeiros Mestres em Biotecnologia”

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Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal 2 Advanced Services Unit, Biocant, 3060-197 Cantanhede, Portugal

“Os Primeiros Mestres em Biotecnologia”

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Sulforaphane induces cell cycle arrest and genotoxicity in MG-63 human osteosarcoma cell line Maria Costa, Catarina Remédios, Pedro Pinto, Francisco Pinho, Tiago Pedrosa, Helena Oliveira, Miguel Oliveira and Conceição Santos CESAM & Laboratory of Biotechnology and Cytomics, Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal

Sulforaphane (SFN), mainly found in Brassicaceae (e.g., broccoli), is the most extensively studied isothiocyanate for its putative anticancer properties 1,2. Research is mainly focused on its anti-tumoral activity, namely SFN ability to promote inhibition of cell viability and cell cycle arrest, as shown by several studies 3,4. However, regarding osteosarcoma cell lines, the effects of SFN on cell proliferation, cell cycle progression, and genotoxicity are still poorly understood. This work aimed to study the effects of SFN on cell cycle and cell viability, and also its genotoxic effects in human osteosarcoma cells. We exposed MG-63 cells to different concentrations of SFN (0, 5, 10 and 20 μM) for different periods (24h and 48h). Cell growth and morphology was daily observed using an inverted microscope and cell viability was determined using the MTT reduction assay. For cell cycle evaluation, we used flow cytometry and the evaluation of DNA damage was performed by the reactive oxygen species (ROS) assessment, comet assay and cytokinesis-block micronucleus cytome assay. For both periods of exposure, we observed a decrease in cell viability when compared to control; for 48h this effect was dose-dependent. Also, the highest concentrations of SFN interfered in cell cycle progression, causing an accumulation of cells in G2/M phase. Similarly, the highest SFN doses induced an increase in ROS formation with a concomitant DNA damage, as showed by comet and micronucleus assays. In conclusion, our results indicate that SFN inhibited proliferation and induced cell cycle arrest, as well as genotoxicity in human osteosarcoma cells, suggesting its potential as a therapeutic agent against cancer.

Acknowledgments FCT supported the fellowships of Helena Oliveira (SFRH/BPD/48853/2008) and Miguel Oliveira (SFRH/BPD/74868/2010).

References (1) Hayes, J. D.; Kelleher, M. O.; Eggleston; I. M. Eur. J. Nutr. 2008, 47, 73. (2) Juge, N.; Mithen, R. F.; Traka, M. Cell. Mol. Life Sci. 2007, 64, 1105. (3) Kim, J. H.; Han Kwon, K.; Jung, J. Y.; Han, H. S.; Hyun Shim, J.; Oh, S.; Choi, K. H.; Choi, E. S.; Shin, J. A.; Leem, D. H.; Soh, Y.; Cho, N. P.; Cho, S. D. J. Clin. Biochem. Nutr. 2010, 46, 60. (4) Chuang, L. T.; Moqattash, S. T.; Gretz, H. F.; Nezhat, F.; Rahaman, J.; Chiao, J. W. Acta Obstet. Gyn. Scan. 2007, 86, 1263.

“Os Primeiros Mestres em Biotecnologia”

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“Os Primeiros Mestres em Biotecnologia”

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Enhanced of oxygen transfer in an airlift reactor using silica microparticles Ivus L.O. Matos, Matheus M. Pereira, Cleide F.Soares, Eliane B. Cavalcanti and Álvaro S. Lima Universidade Tiradentes UNIT – Av. Murilo Dantas, 300. CEP: 49032-290. Aracaju-SE, Brasil

The aeration in aerobic fermentation processes is one of the most limiting factors in the successful operation of those processes1. The oxygen transfer can not be changed significantly for increasing and decreasing temperature, since the solubility of oxygen slightly altered from 25 to 35°C 2. The aim of this work is to use silica particles produced by sol-gel technique, silicon dioxide and beach sand treated by abrasion acid-base, to promote the shearing of the bubbles of oxygen and consequently to increase the volumetric oxygen transfer coefficient (kLa). It was analyzed the influence of particle using a conventional type airlift bioreactor 7 L working volume. The particles were classified by light microscopy in different sizes (10, 75, 150 e 350µm). Hydrodynamic characterization was carried out, and the particles have been introduced in aqueous solution ranging in concentration and size, using the best operating condition (6 L/m). The presence of the particles helped in increasing the volumetric coefficient of oxygen transfer (kLa) and the values were similar between the particles used (Table 1). The most significant results were obtained using 10μm particles of silica sol-gel, silicon dioxide and beach sand treated that have achieved yields of 87%, 80% and 79% respectively.

Table 1. The enhanced of volumetric oxygen transfer coefficient.

Silicon dioxide 80% 39% 16% 11%

Increment of volumetric oxygen transfer coefficient (%) Beach sand Silica sol-gel DP (μm)* treated 79% 87% 10 37% 37% 75 23% 27% 150 11% 11% 350

* Dp: Particle diameter.

Acknowledgments The authors acknowledge the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the financial support.

References (1) Amaral, P.F.F., Freire,M.G., Rocha-Leão, M. H. M.; Marrucho, I. M.; Coutinho, J. A.P.; Coelho, M. A. Z. Journal of Chemical Technology and Biotechnology, 2006, 81, 1368-1374. (2) Ntwampe, S.K.O.; Williams, C.C.; Sheldon, M.S. African Journal of Biotechnology. 2010, 9, 1106-1114.

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“Os Primeiros Mestres em Biotecnologia”

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Bioethanol production by Pichia stipitis for different values of KLa M. D. D. R. F. Moreira2, Daniel L. A. Fernandes1, Luísa S.Serafim1, Ana M. R. B. Xavier1, Inês Portugal1 1

Department of Chemistry & CICECO, University of Aveiro - Campus de Santiago, 3810-193 Aveiro, Portugal Instituto Superior de Engenharia de Coimbra, Instituto Politécnico de Coimbra, 3030-199 Coimbra, Portugal

Wood, the main renewable material on Earth, is largely used in paper-pulp manufacturing but it can also be used to produce second generation biofuels and contribute to the reduction of emissions by partially substituting fossil fuels [1]. Agro-forestry wastes can be converted to bioethanol after wood hydrolysis [2]. Spent sulfite liquor (SSL), a pulp mill effluent from the sulfite process, contains dissolved lignin and hemicelluloses that can be used to produce second generation bioethanol, single cell protein, xylitol and polyhydroxyalkanoates [3]. SSLs are currently burned for energy and chemicals recovery [4], however the conversion to bioethanol is more appealing both economically and environmentally [1-3]. The fermentation of Eucalyptus globulus SSL by P. stipitis has been studied [4]. However, the implementation of this innovative process on an industrial scale requires additional modeling and optimization studies, especially the aspects related to micro/macro aeration. It is known that oxygen transfer rate (OTR) is related to the fermentation medium properties (such as density and viscosity) but also to the reactor/stirrer geometry, stirring speed, gas distribution system and operational factors such as temperature and aeration rate [5]. The volumetric gas/liquid mass transfer coefficient (k La) is an important variable and in most aerated biological processes is considered directly related to the OTR. Typical results for KLa, oxygen uptake rate (OUR) and OTR are presented in Table 1. These parameters increased with increasing flow rate, which is consistent with the literature [5]. The results for cell growth, sugar consumption and ethanol production, at two aeration rates, are presented in Table 1. The air flow rate increment, from 8.4 to 16.8 mL/min, slightly improved cell growth. In contrast, the highest ethanol concentration (1.13 g/L), productivity rate (PE = 0.04 g L−1h−1) and yield (YP/S = 0.26 g/g) were achieved for the lower air flow rate. SSL fermentation by P. stipitis has a significant potential for ethanol production but requires the optimization of appropriate aeration conditions. Table 1 - Influence of aeration rate on ethanol fermentation of SSL by Pichia stipitis. Air flow rate (mL/min) -1

Fermentation I Fermentation II 16.8 8.4

KLa (h )

20.52

9.25

OUR (mmol O2 L-1 h-1)

0.127

0.091

OTR (mmol O2 L-1 h-1)

1.761

0.334

Y P/S (g g-1)

0.25

0.26

Y X/S (g g-1)

0.14

0.082

PE (g L−1h−1)

0.033

0.040

Acknowledgments M. D. D. R. F. Moreira (SFRH/PROTEC/68063/2010) and D. L. A. Fernandes (SFRH/BPD/65482/2009) thank FCT for their PhD and PostDoctoral grants.

References [1] S. Octave, D. Thomas, Biochimie, (2009), 91, 659-664 [2] Xavier, A. M. R. B., Correia, M. F., Pereira, S. R., Evtuguin, D. V., Bioresource Technology, (2010), 101, 2755–2761. [3] S. R. Pereira, D. L. A. Fernandes, C. M. Silva, L. S. Serafim, A. M.R.B. Xavier, D. V. Evtyugin, VIII Jornadas CICECO, (2011), March 17-18, Aveiro. [4] Correia, M., Produção de bioetanol a partir de licor de cozimento ao sulfito ácido, in Tese de Mestrado, Dep. Química Universidade de Aveiro (2008). [5] Galaction, A. I., Casacaval, D., Oniscu, C. e Turnea, M., Biochemical Engineering Journal, (2004), 20, 85-94.

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Evaluation of AgNP nanoparticles on osteosarcoma viability: approaches to environmental studies Fernanda Rosário, Helena Oliveira, Miguel Oliveira and Conceição Santos CESAM & Laboratory of Biotechnology and Cytomics, Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal

Nanoparticles (NP) are considered to be the building blocks for nanotechnology, and are referred to particles with at least one dimension <100 nm. Since their characteristic such as antimicrobial and catalytic properties and light emitting there are an increasing number of applications being developed for the use of nanoparticles in various fields 1,2. Silver NPs (AgNPs) are, due to their antimicrobial properties, the most widely used NPs in commercial products and despite that, there are few reports available 3 on the toxicity of silver NP. In this study, and in order to mimic NP as environmental contaminants, we investigated the effects of unstabilized AgNPs with different sizes (<100 nm) on osteosarcoma (MG-63) viability. For that, in vitro cultured cells were exposed to different concentrations (0, 10, 50, 100, 200 and 400µM) of AgNPs for 24h and 48h. Cell growth and morphology was daily observed using an inverted microscope and cell viability was measured by MTT reduction assay. For both exposure periods the results are not conclusive since the MTT assays reveal different profiles. From our data we concluded that though unstabilized AgNP putatively mimic environmental contaminants, the evaluation of their toxicological without stabilization and normalization is complex and leads to inconclusive results, so not being adequate to assess AgNP cytotoxicity in vitro.

Acknowledgments This work was supported by COMPET program/FCT (Fundação para a Ciência e Tecnologia) with the project PTDC/AAC_AMB/113649/2009. FCT also funded the fellows of Helena Oliveira (SFRH/BPD/48853/2008) and Miguel Oliveira (SFRH/BPD/74868/2010).

References (1) Foldbjerg, R.; Dang, D. A.; Autrup, H. Arch. Toxicol. 2011, 85,743. (2) Service, R. F. Science. 2003, 300, 243. (3) Marambio-Jones, C.; Hoek E. M. V. Nanopart. Res. 2010, 12, 1531.

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“Os Primeiros Mestres em Biotecnologia”

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Structural studies of the mammalian end binding protein 3 employing high-resolution solid-state MAS NMR Mariana Sardo1,2, Luís Mafra1, João Rocha1, Anja Böckmann3, Beat Meier2 1

CICECO –University of Aveiro, 3810-193 Aveiro, Portugal Physical Chemistry, ETH-Zurich, 8093 Zurich, Switzerland 3 IBCP, UMR 5086 CNRS/Université de Lyon 1, 7 passage du Vercors, 69367 Lyon, France 2

Solid-state nuclear magnetic resonance (NMR) spectroscopy still needs the development of procedures and protocols in order to become a generally applicable tool for 3D high-resolution protein structure determination. Crystalline model proteins play an important role in this context, as they allow further developing and optimizing the different approaches including for spectral assignments, the measurement of restraints and structure calculation. Here, we use the end binding protein 3 (EB3), a small dimeric protein, containing both an N-terminal calponin homology domain, responsible for the interaction with microtubules (MTs), and a C-terminal coiled-coil domain extending into a four-helix bundle, essential for dimer formation.1 EB3 belongs to a family of proteins that associate specifically with the plus ends of growing MTs (plus end tracking proteins, +TIPs). +TIPs form clusters at the end of growing MTs and this specific sub-cellular position makes them ideally suited for regulating MT dynamics.2 Our work focuses on the analysis of the N-terminal globular part of the EB3 protein (132 residues, 15 kDa, PDB reference3CO11) employing high-resolution solid-state MAS NMR methods. The initial steps for solid-state NMR 3D structure determination will be presented, namely sample preparation and experimental 2D/3D 13C-15N correlation spectra for assignment purposes.

Solid-state NMR studies of the mammalian EB3 protein in view of the further development of high-resolution structure determination methods. Acknowledgments Financial support was provided by the Swiss National Science Foundation, the ETH Zurich and the CNRS. MS also acknowledges the Portuguese Foundation for Science and Technology for a post-doctoral grant – SFRH/BPD/65978/2009.

References (1) Y. Komarova et. al, J. Cell Biol. 2009, 184, 691. (2) A. Akhmanova, M. O. Steinmetz, Nat. Rev. Mol. Cell Bio. 2008, 9, 309. “Os Primeiros Mestres em Biotecnologia”

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Structural studies of the mammalian end binding protein 3 employing high-resolution solid-state MAS NMR Mariana Sardo1,2, Luís Mafra1, João Rocha1, Anja Böckmann3, Beat Meier2 1

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III Workshop Biotecnologia UA

Lipophilic composition and biological activity of different parts of Cynara cardunculus L. var. altilis (DC) P. Ramos1,2, J.J. Villaverde2, A. Guerra1, O. Guerreiro1, H. Pereira3, C. S. R. Freire2, M. Duarte1, A. J. D. Silvestre2 1

CICECO and Centro de Biotecnologia Agrícola e Agro-Alimentar do Baixo Alentejo e Litoral (CEBAL) / Instituto Politécnico de Beja (IPBeja), 7801-908 Beja, Portugal 2 CICECO and Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal 3 CESAM & Department of Biology, Laboratory of Biotechnology and Cytometry, University of Aveiro, 3810 Aveiro, Portugal

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9 de Maio 2012

III Workshop Biotecnologia UA

“Os Primeiros Mestres em Biotecnologia”

9 de Maio 2012

III Workshop Biotecnologia UA

Ionic-liquid-based aqueous biphasic systems for the extraction of Ltryptophan H. Passos, A. R. Ferreira, Ana Filipa M. Cláudio, João A. P. Coutinho, Mara G. Freire Department of Chemistry, CICECO, University of Aveiro, 3810-193 Aveiro, Portugal

Aiming at avoiding the use of organic solvents in common liquid-liquid extractions, there has been a huge amount of research on the use of aqueous biphasic systems (ABS). Common ABS are constituted by two different polymers or a polymer and a salt in aqueous solutions. Since ABS are mainly constituted by water they represent more benign approaches for the extraction of several molecules. Recently, the typical polymers used in the ABS formulations have been replaced by ionic liquids (ILs). These new ABS have low viscosity and faster phase separation times, and have shown to be improved extractive techniques due to the possibility on the tailoring of the phases’ polarities. Phase diagrams of ternary systems composed of ILs, salts and water are crucial for the development, optimization, and scale-up of extraction processes using ABS. Most of the studies previously carried out used inorganic salts which can bring environmental concerns to effluent streams. In this context, organic salts, such as citrate-based, appear as potential replacements. In this work, novel phase diagrams for ABS composed of several ILs, potassium citrate and water were determined at 298 K. The effects of the IL cation core, the cation side alkyl chain length, and the anion nature were evaluated. Finally, the partitioning of L-tryptophan was also addressed to explore the applicability of the proposed systems in the biotechnology field. From the gathered results, longer aliphatic chains at the cation facilitate the formation of ABS. The IL cation core ability to form ABS follows the order: [P4444]+ > [N4444]+ >> [C4mpy]+ ≈ [C4mpip]+ > [C4mpyrr]+ > [C4mim]+. The effect of the anion nature on the ABS phase behaviour is according to: [CH3CO2]- < [PO4(CH3)2]- < [CH3SO3]- < Cl- << Br- < [CF3CO2]- << [N(CN)2]- < [SCN]- < [CF3SO3]-. The extraction efficiencies of Ltryptophan range between 72 % and 99 %. Therefore, from the results obtained, it can be anticipated the ABS composed of biodegradable salts and ionic liquids could be applied in biotechnological approaches. 100

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Acknowledgments The authors acknowledge FCT- Fundação para a Ciência e a Tecnologia for the Pest-C/CTM/LA0011/2011 and PTDC/QUI-QUI/121520/2010 projects, and post-doctoral and doctoral grants SFRH/BPD/41781/2007, SFRH/BDE/33835/2009, and SFRH/BD/74503/2010 of M. G. Freire, A. R. Ferreira, and A. F. M. Cláudio, respectively.

“Os Primeiros Mestres em Biotecnologia”

III Workshop Biotecnologia UA

“Os Primeiros Mestres em Biotecnologia”

9 de Maio 2012

III Workshop Biotecnologia UA

9 de Maio 2012

Production and charecterization of polyol via liquefaction of biomass B. Soares1, N. V. Gama1, A. Ferreira2, C. Freire1, A. Barros-Timmons1, I. Brandão3 and C. Pascoal Neto1 2 CICECO and ESTGA/ Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal 3 Sapec-Química SA, Zona Industrial de Ovar – Lote 18, 3880 Ovar, Portugal e-mail: belindasoares@ua.pt

Polyols productions from coffee grounds via acid liquefaction were investigated using Polyethylene glycol and Glycerol in the presence of sulphuric acid. We evaluated the influence upon the liquefaction yield of three operating variables, the maximal temperature (160–200ºC), the amount of added sulphuric acid (1–3% w/w) and the reaction time at maximal temperature (30–60 min) using an experimental design methodology. The obtained products were characterized using infrared spectroscopy analysis, residue content, hydroxyl number and viscosity. The results indicated that it´s possible to convert coffee ground into liquid polyols, but it´s necessary to optimize the process parameters. The urgency to find alternative raw materials to fossil ones in order to reach sustainable development is leading a number of research groups to devise synthetic paths and processes based on agro-forest residues [1]. In the present study we investigated the valorization of coffee grounds via the liquefaction for subsequent use as polyols in the production of polyurethanes for example. The main purposes of the present work were to: (i) assess the suitability of the liquefaction process using polyhydric alcohols (PEG# 400/Glycerol) to convert coffee grounds into liquid polyols, (ii) apply a statistical design of experiments to optimize the variables of the liquefaction reaction and (iii) to characterize the resulting polyols in terms of biomass residue, hydroxyl number, viscosity and by spectroscopic methods (FT-IR and 1H –NMR). Coffee grounds liquefaction was carried out at atmospheric pressure using polyethylene glycol and glycerol as solvents and sulphuric acid as a catalyst. In all experiments, the polyethylene glycol #400/Glycerol ratio was 90:10 (w/w) and the feedstock/liquefaction solvents ratio was 1:4 (w/v). A second order factorial design involving 8 experiments was developed to optimize liquefaction process. The independent variables were: (1) the maximal temperature, (2) the weight ratio of sulphuric acid to liquefying solvent and (3) the reaction time at maximal temperature. The dependent variable or system response was the biomass residue (%). We succeeded in liquefying coffee grounds however, our results showed that the process parameters had to be adjusted due to the specific characteristics of the biomass used in this project. The statistical analysis revealed that the most significant effects are the maximum temperature and the weight ratio of sulphuric acid to liquefying solvent had great influences on the residue content (90 % confidence interval). Acknowledgments The authors thank QREN/FEDER for funding the Ecopolyols project (Nº 11435). NESTLÉ Portugal S.A are gratefully acknowledged for supplying coffee grounds. CICECO acknowledges FCT for the Pest-C/CTM/LA0011/2011 project.

References [1] A. Gandini, “Polymers from Renewable: A Challenge for the future of Macromolecular Materials,” Macromolecules, vol. 41, issue 24, pp. 9491-9504, 2008. [2] S. A. Rezzoug and R. Capart, “Assessment of wood liquefaction in acidified ethylene glycol using experimental design methodology, “ Energy Conversion and Management, vol. 44, pp. 781-792, 2003. [3] R. Briones, L. Serrano, R. Llano-Ponte and J. Labidi, “Polyols obtained from solvolysis liquefaction of biodiesel production solid residues,” Chemical Engineering Journal, vol. 175, pp. 169-175, 2011.

“Os Primeiros Mestres em Biotecnologia”

9 de Maio 2012

III Workshop Biotecnologia UA

“Os Primeiros Mestres em Biotecnologia”

III Workshop Biotecnologia UA

9 de Maio 2012

Aqueous Biphasic Systems As A Green Perspective For Treatment Of Effluents Contaminated With Ionic Liquids C. F. C. Marques, C. M. S. S. Neves, M. G. Freire and J. A. P. Coutinho Departmento de Química-CICECO,Universidade de Aveiro, 3810-193 Aveiro, Portugal cfmarques@ua.pt.

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III Workshop Biotecnologia UA

9 de Maio 2012

“Os Primeiros Mestres em Biotecnologia”

III Workshop Biotecnologia UA

9 de Maio 2012

Detoxification of Sulphide Spent Liquors for Bioethanol Fermentation Employing Ion Exchange Resins D.L.A Fernandes, C.M. Silva, A.M.R.B Xavier, D.V. Evtyugin CICECO, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal e-mail: daniel.fernandes@ua.pt

Alternatives to produce fuel and chemicals from non-fossil feedstocks have attracted considerable interest around the world, to face the needs of energy supply minimizing the environmental impacts. Bioethanol is one of the attractive biofuels, since it can be easily produced in large amounts and blended with gasoline or used pure as a “green” fuel. The production of bioethanol from non-food staff (“second generation bioethanol”) is an emerging challenge in the area (1). Sulphide spent liquors (SSLs) are side products from acidic sulphide pulping of wood, and are normally burned to recover energy and the inorganic base or just sailed away after pre-concentration (2). The major components of SSLs are lignosulphonates (LS) and sugars, which are recognized perspective valuable by-products for the production of added value products (3). Regarding the bioethanol production, SSL possess advantage over common lignocellulosic materials because already contains monomeric sugars thus avoiding complex industrial processing involving hydrolysis (1). However, SSL contains several lowand high-molecular weight inhibitors of fermentation that require a previous detoxification step (4). The present study focused on purification of SSL from acidic sulphite pulping of eucalypt wood by ion exchange resins aiming to provide sugar substrates suitable for bioconversion into ethanol. The organic compounds of SSL were composed roughly by 10% of LS, 5% of sugars, 1% of acetic acid and 0.1% of polyphenolics. The high molecular weight inhibitors (main part of LS) were removed using cation exchange resin that showed also sizeexclusion performance. The monomeric-dimeric LS, sugars (mainly xylose), acetic acid and polyphenolics were separated using pre-selected anion exchange resin. This allowed a pure sugar solution that is suitable for the fermentation to ethanol with a high yield or for other technical purposes. The sugars-free monomeric-dimeric LS were suggested to be a perspective feedstock for the synthesis of flavours and adhesives.

References (1) S.I. Mussatto, G. Dragone, P.M.R. Guimaraes, J.P.A. Silva, L.M. Carneiro, I.C. Roberto, A. Vicente, L. Domingues and J.A. Teixeira, "Technological trends, global market, and challenges of bio-ethanol production," Biotechnology Advances, vol. 28, 2010. (2) J.P. Casey on Pulp and Paper, Chemistry and Chemical Technology, 3rd ed. Vol. 1. New York, USA: John Wiley & Sons, 1980. (3) J. Plank, "Applications of biopolymers and other biotechnological products in building materials," Applied Microbiology and Biotechnology, vol. 66, pp. 1-9, 2004. (4) A.M.R.B. Xavier, M.F. Correia, S.R. Pereira and D.V. Evtuguin, "Second-generation bioethanol from eucalypt sulphite spent liquor," Bioresource Technology, vol. 101, 2010.

“Os Primeiros Mestres em Biotecnologia”

III Workshop Biotecnologia UA

“Os Primeiros Mestres em Biotecnologia”

9 de Maio 2012

III Workshop Biotecnologia UA

9 de Maio 2012

Chemical functionalization of nickel nanowires for optical bio-tagging P. Pinheiroa*, C. Sousab, A. G. Guiomar c, J. P. Araújob, T. Trindadea a

Departmento de Química-CICECO,Universidade de Aveiro, 3810-193 Aveiro, Portugal Faculdade de Ciências, Departamento de Física e INIFIMUP, Universidade do Porto,4169-007 Porto, Portugal c Department of Life Sciences of the University of Coimbra *pcpinheiro@ua.pt

Since the discovery of carbon nanotubes (CNT) by Iijima in 1991, quasi-onedimensional (1D) nanostructures have attracted great attention [1]. Nanowires are 1D nanostructures that have been intensively studied due to their potential applications in diverse nanodevices. A general limitation in using and handling CNT in those devices is the ability to control their position relative to specific surroundings. Nickel nanowires present unique magnetic properties and can be easily manipulated using an external magnetic field. Additionally, these materials offer the possibility to develop new types of multifunctional biomarkers for in vitro procedures by adequate chemical functionalization of the surfaces with fluorophores. In this context, a number of issues related to the functionality and colloidal stability of the modified Ni nanowires need to be addressed from a perspective of materials design. In this work, nickel nanowires with about 40-60 nm cross section were prepared at an anodic aluminum oxide (AAO) template. The highly ordered AAO template was fabricated using aluminum and anodized electrolytes using a two-step method. Following the Ni nanowires synthesis, this research involved sequential steps of chemical modification of their surfaces in order to achieve magnetic and luminescent materials. First, modification of the Ni nanowires with polymer charged species (polyelectrolytes) was performed and the properties of the several modified samples will be discussed. For one selected system, the polyelectrolyte was labeled with a fluorescein derivative (fluorescein isothiocyanate– FITC) enabling their characterization and monitoring using fluorescence spectroscopy. The potential application of these nanomaterials as optical and magnetic markers for in vitro bioapplications will be discussed by taking in consideration relevant issues such as their toxicity, biofunctionalization and colloidal stability in physiological medium.

Acknowledgement: We thank A. V. Girão for the SEM images.

References: [1] X. T. Zhang, Z. Liu, K. M. Ip, Y. P. Leung, Quan Li, S. K. Hark, J. Appl. Phys., 10 (2004) 5752. [2] A. K.Bentley, M. Farhoud, A. B. Ellis, G. C. Lisensky, A. L. Nickel, W. C. Cron, J. Chem. Educ. , 85 (2005) 765. [3] R. M. Metzger, V. V. Konovalov, M. Sun, T. Xu, G. Zangari, B. Xu, M. Benakli, W. D. Doyle, IEEE Transactions on magnetic, 36 (2000) 30.

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III Workshop Biotecnologia UA

9 de Maio 2012

Chemical functionalization of nickel nanowires for optical bio-tagging P. Pinheiroa*, C. Sousab, A. G. Guiomar c, J. P. Araújob, T. Trindadea a

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9 de Maio 2012

III Workshop Biotecnologia UA

Metabolic alterations in healthy pregnancy and prenatal disorders: a nuclear magnetic resonance (NMR) spectroscopy study of maternal blood plasma Joana Pintoa, António S. Barrosb, Iola F. Duartea, Brian J. Goodfellow a, Eulália Galhanoc, Cristina Pitac, Isabel M. Carreirad, Maria do Céu Almeidac and Ana M. Gila a

CICECO and bQOPNAA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro Portugal; cMaternity Bissaya Barreto, Coimbra, Portugal, dCytogenetics and Genomics Laboratory, Faculty of Medicine, University of Coimbra and CENCIFOR - Forensic Science Centre & CIMAGO-Centro de Investigação Meio Ambiente, Genética e Oncobiologia, Coimbra, Portugal. Firstly, this work presents the 1st NMR metabolite profiling of maternal blood plasma for the monitoring of healthy pregnancy and, secondly, the application of the same strategy to the study of prenatal disorders (fetal malformations (FM) and chromosomal disorders (CD)). Multivariate analysis was applied to the standard 1D, T2-edited and diffusion-edited 1H-NMR spectra. Results showed markedly metabolite trajectory from the non-pregnant state throughout pregnancy (Fig. 1). Overall, plasma lipids, glucose, albumin, lactate, choline, and several amino acids showed significantly differences during pregnancy. In addition, the impact of each disorder on 2nd trimester blood plasma composition was measured, with most significant alterations found for the FM group (lipids, 3-hydroxybutyrate, acetone, betaine and methanol). Finally, Statistical Correlation Spectroscopy (STOCSY) was performed for the FM group, considering the plasma metabolite changes together with those observed in the corresponding urine and amniotic fluid, as reported previously1,2, in order to probe inter-fluid n gravidas,1T,2T,3T_8.5-0.5ppm_2_exp.M2 (PLS-DA), UVClass autofit 1 t[Comp. 1]/t[Comp. 2] correlations and hence mother/fetusClass metabolic interactions. 2 Colored according to classes in M2 LV=2 R2X=0.329 R2Y=0.437

Class 3 Class 4

Q2=0.316 100

Acknowledgments Funding is acknowledged from the European Regional Development Fund through the Competitive Factors Thematic Operational Programme and from FCT, Portugal (PTDC/QUI/66523/2006, SFRH/BD/73343/2010 and the Portuguese National NMR Network, RNRMN).

LV2

50 0 -50 -100

-100

100

LV1

Figure 1. PLS-DA R2X[2] scores scatter plot Ellipse: of T2Hotelling R2X[1] = 0.204389 = 0.12428 T2 (0.95) edited 1H NMR spectra obtained for nonpregnant women (, n=17), 1st (, n=11), 2nd (, n=15) and 3rd (, n=5) trimesters. SIMCA-P 11.5 - 20-04-2012 18:57:23

References (1) Graça, G.; Duarte, I. F.; Barros, A. S.; Goodfellow, B. J.; Diaz, S. O.; Pinto, J.; Carreira, I. M.; Galhano, E.; Pita, C.; Gil, A. M. J. Proteome Res. 2010, 9, 6016. (2) Diaz, S.O.; Pinto, J.; Graça, G.; Duarte, I. F.; Barros, A. S.; Galhano, E.; Pita, C.; Almeida, M. C.; Goodfellow, B. J.; Carreira, I. M.; Gil, A. M. J. Prote “Os Primeiros Mestres em Biotecnologia”

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9 de Maio 2012

“Os Primeiros Mestres em Biotecnologia”

III Workshop Biotecnologia UA

9 de Maio 2012

Effect of temperature and high pressure treatments in starch suspensions M. D. Santos, J. A. Saraiva and M.T. Gomes Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

Nowadays, starch is present in many food products, contributing with 50% to 70% of the energy required in the human diet (direct source of glucose) 1. Of all extracted starch, 60% is used in the food industry, while the remaining 40% is used in the pharmaceutical industry and non-food industries2. The different sources of starch lead to different functional properties that enable its use in several industries. Currently, chemically modified starches and the formation of starch-lipid complexes are used to overcome the variability of native starch. The application of thermal or/and high pressure (HP) treatment can also be used to overcome the variability of the native starch. The gelatinization of starch is generally regarded as a set of processes that occur during starch heating in the presence of water: granules swelling; amylose leaching; amylopectin melting and formation of a gel or paste-like mass3. There are also loss of crystallinity, loss of birefringence, destruction of molecular order 4-5 and viscosity increase5. The aim of this work was to study the viscoelastic properties of native and high pressure treated starch suspensions. An acoustic wave sensor was used to analyze the suspensions, and those studies were complemented with other data obtained by other techniques: differential scanning calorimetry (DSC), polarized light microscopy and scanning electron microscopy (SEM). When high pressure treated maize starch (500 MPa, 5 minutes, room temperature) was analyzed by SEM it was possible to see that the granules collapsed and became “doughnut-shaped”, with no visible break in the surface. The loss of birefringence was monitored by the loss of Maltese Cross in the granules (polarized light microscopy). Despite the HP treatment, the results were similar for both starches (native and treated maize starch), at 74,0 ºC the starch suspensions lost their birefringence (all Maltese Crosses disappeared). However, it was found that the high pressure treated maize starch suspensions presented viscoelastic properties different from native starch. The onset temperature of viscosity increase was higher for the high pressure treated maize starch (77,5 ºC) when compared with native starch suspensions (73,5 ºC). The changes in viscosity observed at temperatures above 70 ° C were smaller for high pressure treated starch. It was also possible to conclude that the heating rate of starch influences the suspension viscoelastic properties and the temperatures of viscosity changes related to the starch gelatinization process. It should be noted that in the maize starch suspension a change in the piezoelectric crystal frequency due to the breaking of the granules was detected. The influence of temperature on high pressure treatment was also studied for rice starch. Suspensions were treated at pressures of 400 MPa for 15 minutes at 5, 10, 23 and 34 ° C. It was found that the higher the processing temperature, the higher the temperature at which the viscosity increase began. At the end of this work, the HP treatment of starch suspensions showed to be a very interesting technique for starch properties modification. References (1) Copeland, L.; Blazek, J.; Salman, H.; Tang M. C. Food Hydrocolloids 2009, 23, 1527. (2) Burrell, M. M. Journal of Experimental Botany, 2003, 54, 451. (3) Brouillet-Fourmann, S.; Carrot, C.; Mignard, N. Rheol Acta, 2003, 42, 110. (4) Hoover, R. Carbohydrate Polymers, 2001, 45, 253. (5) Nelles, E. M.; Dewar, J.; Bason, M. L.; Taylor, J. R. N. Journal of Cereal Science, 2000, 31, 287. “Os Primeiros Mestres em Biotecnologia”

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9 de Maio 2012

Effect of temperature and high pressure treatments in starch suspensions M. D. Santos, J. A. Saraiva and M.T. Gomes Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

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9 de Maio 2012

High Pressure Technological Platform of University of Aveiro for Food Preservation and Modification and Biotechnological Applications L. Fidalgo, M. D. Santos, I. Delgadillo and J. A. Saraiva Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

Several scientific fields are exploring the unique effects of high pressure (HP) technology. Just like temperature, pressure is an important thermodynamic parameter that influences molecular systems1. In our biosphere, pressure extends from 0.1 MPa (atmospheric pressure at sea level) to 110 MPa in the deepest point of the ocean, however, despite this hostile condition, a biodiversity of bacteria has been observed. Piezophilic microorganisms are able to exist in the deep sea and in subsurface terrestrial habitats and are evolutionarily adapted to grow and proliferate optimally at pressures of up to 100 MPa or higher. These microorganisms are an invaluable source of HP-resistant enzymes and provide important knowledge regarding the structural adaptations that are necessary for mesophilic enzymes to withstand HP1. HP allows deep see (under pressure) living environments simulation. For instance, glucose fermentation by Saccharomyces cerevisiae occurs faster under HP and ethanol content is closer to the theoretical value 2. HP inactivation of bacteria was the main objective of the first experiments conducted under HP3. The effects of HP on proteins have been studied and HP protein refolding technology is also being studied to improve the functionality of therapeutic protein, for use in several debilitating diseases, such as Alzheimer’s disease and Parkinson’s disease 1. Since some cancers have been found to be caused by protein misfolding3, HP has also potential applications on this case. HP processing can also reduce the allergenic activity of foods, as milk and rice 3. Enzyme activity can be modulated by pressure, because at pressures lower than 200 MPa, the stability and functionality of most enzymes are not altered 3, being a notable example for food preservation, the inactivation of pectinmethylesterase and of polygalacturonase present in tomato 1. Moderate HP treatments have raised signiﬁcant interest as an alternative method for the inactivation of viruses in the context of vaccine development. Can also be applied on orthopedics concerning the disinfection of infectious or tumor-afflicted bone segments before their reimplantation1. University of Aveiro is now creating a High Pressure Based Multidisciplinary Research and Technological Platform, based on a portfolio of three HP equipments (lab, semi-pilot and pilot scales, Fig. below), aiming to create a center of international competitiveness in Portugal, to promote research and industrial applications of HP.

Acknowledgments Thanks are due to QOPNA Research Unit (Project PEst-C/QUI/UI0062/2011). References (1) Aertsen, A.; Meersman, F.; Hendrickx, M.; Vogel, R.; Michiels, C. Trends in Biotechnology 2009, 27, 434. (2) Picard, A.; Daniel, I.; Montagnac, G.; Oger, P. Extremophiles 2007, 11, 445. (3) Rivalain N.; Roquain, J.; Demazeau G. Biotechnology Advances 2010, 28, 659. “Os Primeiros Mestres em Biotecnologia”

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“Os Primeiros Mestres em Biotecnologia”

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9 de Maio 2012

Polysaccharide coated magnetic nanoparticles for dye removal from water A. M. Salgueiro, A. L. Daniel-da-Silva, T. Trindade CICECO & Department of Chemistry , University of Aveiro, 3810-193 Aveiro, Portugal

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III Workshop Biotecnologia UA

9 de Maio 2012

“Os Primeiros Mestres em Biotecnologia”

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9 de Maio 2012

Functionalized magnetic nanoparticles for the immobilization of biomolecules R. P. Silva1, R.Vitorino1,2, A.L. Daniel-da-Silva1,3 1

Department of Chemistry, 2QOPNA, 3CICECO, University of Aveiro, 3810-193 Aveiro, Portugal e-mail: a38640@ua.pt; rvitorino@ua.pt; ana.luisa@ua.pt

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III Workshop Biotecnologia UA

9 de Maio 2012

Functionalized magnetic nanoparticles for the immobilization of biomolecules R. P. Silva1, R.Vitorino1,2, A.L. Daniel-da-Silva1,3 1

Department of Chemistry, 2QOPNA, 3CICECO, University of Aveiro, 3810-193 Aveiro, Portugal e-mail: a38640@ua.pt; rvitorino@ua.pt; ana.luisa@ua.pt

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Microfauna community as an indicator of effluent quality and process performance in an activated sludge system for treating industrial wastewater Cátia S. R. Azenha1, Rita Manso2, Anabela Antunes2, Ana M. R. B. Xavier1 1

Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal Prio Energy, Terminal de Graneis Líquidos - Lote D - Porto de Aveiro, Gafanha da Nazaré, 3834-907 Aveiro, Portugal

Biological wastewater treatment is a process of increasing importance in a world with an increasing human population. Biological wastewater treatment by activated sludge systems, can be considered as artificial ecosystems subjected to extreme conditions. Thus from all heterotrophic organisms only small protozoa and metazoa, with life cycles shorter than the sludge retention time, are capable of competing in these processes. 1 As protozoa are mostly bacterivorous and graze on bacteria and particulate matter, it is commonly assumed that the main function of protozoa in wastewater treatment is the clarification of the effluent.1 Also, they are potentially important indicators of process performance and efficiency,2 since species and functional groups depend on environmental conditions and most of the protozoa found in activated sludge have an universal distribution. The structure of protozoa community can be considered as a valid indicator for the performance of the plant. This means that all major variations in the treatment performance are indicated by the dominant protozoa group. 1, 3, 4 As microfauna species identification is difficult and time consuming, it is recommended the use of functional groups in these type of studies.3 These functional groups are testate amoebae, small flagellates and ciliates. Bacterivorous ciliates can also be subdivided into three groups based on their behaviour: 1) free-swimmers, swim in the fraction of liquor; 2) crawlers, move on the surface of the sludge flocs; and 3) sessile or attached that are firmly attached by a stalk to the flocs. 1 In this work, to investigate the relation between biological and physicochemical parameters, the non-parametric correlation coefficient test of Spearman’s rho was used to obtain a correlation matrix of all physicochemical parameters versus relative abundance of functional groups. The results showed that sessile and free-swimming ciliates, testate amoebae and small flagellates can be used as bioindicators of process performance. Sessile ciliates and testate amoebae constitute a positive control group. They have a positive correlation with the efficiency of biological wastewater treatment and are indicators of high effluent quality, once they show a strong negative association with the Chemical Oxygen Demand (COD) of the effluent. On the other hand, a negative control group is constituted by free-swimming ciliates and small flagellates that increase in number when the process performances fail. This is suggested by the strong positive correlation observed between this group and the effluent COD. It has also been observed that when the plant load is above 300kgCOD/day the abundance of sessile ciliates decreases, and the opposite is observed for small flagellates and free-swimming ciliates. Thus, as an optimization parameter, the plant load must be kept below 300kg COD/day, and the feed rate must be determined daily since the influent COD is very variable. References

(1) Madoni, P. Italian Journal of Zoology 2011, 78, 3-11. (2) Dubber, D.; Gray, N. F. European Journal of Protistology 2011, 47, 274-286. (3) Zhou, K.; Xu, M.; Liu, B.; Cao, H. Journal of Environmental Sciences 2008, 20, 482-486. (4) Madoni, P. Water research 1994, 28, 67-75. “Os Primeiros Mestres em Biotecnologia”

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“Os Primeiros Mestres em Biotecnologia”

9 de Maio 2012