Communications Agricultural Applied Biological ...

Vol 71(1) 1-330 (2006)

Communications in

Agricultural and

Applied Biological Sciences

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Vol 71(1) 1-330 (2006)

COMMUNICATIONS IN AGRICULTURAL AND APPLIED BIOLOGICAL SCIENCES

formerly known as

MEDEDELINGEN FACULTEIT LANDBOUWKUNDIGE EN TOEGEPASTE BIOLOGISCHE WETENSCHAPPEN

PUBLISHERS Professors

Pascal Boeckx Peter Bossier Guy Smagghe Walter Steurbaut Els Van Damme Erick Vandamme Niko Verhoest

Editorial address Coupure links 653 9000 Gent (Belgium) Website http://www.fbw.ugent.be ISSN 1379-1176

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Proceedings

12th SYMPOSIUM ON APPLIED BIOLOGICAL SCIENCES

Building E Faculty of Bioscience Engineering, Gent 21 September 2006

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MEMBERS OF THE ORGANIZING COMMITTEE Universiteit Gent Bruno De Meulenaer Veerle Fievez Peter Goethals, Katleen Raes Hans Saveyn Olivier Thas, Tom Vande Wiele Sofie Van Maercke Eveline Volcke Katholieke Universiteit Leuven Abram Aertsen Katrien Beullens Jeroen De Temmerman Bert Lagrain Jeroen Lammertyn Rob Lavigne Sofie Saerens Bert Sels Nele Van Beneden Boris Van Berlo Steven Vermeir

SYMPOSIUM ORGANIZED BY

Ghent University Faculty of Bioscience Engineering

Catholic University of Leuven Faculty of Bioscience Engineering

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SPONSORING

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PREFACE This year the PhD symposium on Applied Biological Sciences is organized for the 12th time. Since 5 years it is a joint organization of Ghent University and the Catholic University of Leuven. In the 11 previous editions, the format has always been the same: a one-day symposium with about 10-12 oral contributions, a large poster section, and 2 invited speakers. Since one of the primary objectives of the symposium is to create a forum where PhD students from different research areas can learn about the research activities at other departments and at their (sister) faculty, all oral presentations were organized in plenary sessions. In practice, however, we noted that some topics were too distinct regarding the field of research. For instance, forestry versus food technology, or microbiology versus agro-marketing. Yet another reality is that some PhD students (and also professors) have problems in finding or making the time to spend a whole day at the symposium. For these reasons, we have changed the format slightly. For this year’s edition, the oral contributions were clustered into 3 parallel sessions, while the two invited talks are scheduled as a plenary session. In this way, we have 17 oral presentations concentrated in only one single afternoon. Despite the few weaknesses that we mentioned before, we feel that there is still a large interest in the symposium. This is clearly illustrated by the number of submissions we received this year. We never received more proposals before. In selecting 15 from this list, we tried to find a good mix between young and more experienced PhD students. As usual, the selection process was hard, particularly because most submissions are based on high quality research. We hope that this is reflected in the abstracts presented in these proceedings and that the attendees enjoy the symposium. The organizing committee

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SYMPOSIUM PROGRAM From this year on the PhD Symposium is organized as a half-day symposium, with both plenary and parallel sessions. Below you find the structure of the program, and the session details on the next pages. Hour 13.30 Registration 14.00 Opening 14.10

KEYNOTE SPEAKER 1: Niceas Schamp (Koninklijke Vlaamse Academie van België voor Wetenschappen en Kunsten) Session 1 - CELL AND GENE BIOTECHNOLOGY

Session 2 - FOOD SCIENCE AND NUTRITION

14.50 Criel Bram (KUL) Mehdikhanloo Khosro 15.10 (ILVO)

Baert Katleen (UGent) Van Houtegem Nancy (UGent) Andjelkovic Mirjana 15.30 Deckers Daphne (KUL) (UGent) 15.50 Coffee break + poster session Session 4 - CHEMISTRY Session 5 - SOIL, FORAND BIOPROCESS TECH- EST, NATURE AND WANOLOGY TER MANAGEMENT 16.40 Mertens Pascal (KUL) Verbist Bruno (KUL) 17.00 Mangelinckx Sven (UGent)

Leroy Ben (UGent)

17.25 KEYNOTE SPEAKER 2: Erwin Lamot (Flanders’ Food) 17.55 Reception and poster price 19.30 End

Session 3 - AGRICULTURAL SCIENCES De Vos Yann (UGent) Wauters Erwin (KUL) Lourenço Marta (UGent) Session 6 - ENVIRONMENTAL TECHNOLOGY Lesage Els (UGent) Aelterman Peter (UGent)

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SESSION DETAILS Session 1 - CELL AND GENE BIOTECHNOLOGY (Chair: Rob Lavigne) B. Criel, J-F. Hausman, M. Oufir, R. Swennen, B. Panis & J. Renaut (14h30-14h50) Proteome and sugar analysis of abiotic stress underlying cryopreservation in potato

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K. Mehdikhanloo & P. Van Bockstaele (14h50-15h10) AFLP analysis of genetic diversity wihtin and between white clover varieties D. Deckers, A. Aertsen, L. Callewaert & C.W. Michiels (15h1015h30) Role of lysozyme inhibitors in bacterial colinization of egg albumen

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Session 2 - FOOD SCIENCE AND NUTRITION (Chair: Steven Vermeir) K. Baert, B. De Meulenaer, F. Verdonck, I. Huybrechts, S. De Henauw, P.A. Vanrolleghem & F. Devlieghere (14h30-14h50) Probabilistic exposure assessment of patulin in apple juice for preschool children in Flanders

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N. Van Houteghem, F. Devlieghere, S.M. Oses Gomez, J. Debevere & M. Uyttendale (14h50-15h10) Effect of CO2 on the resuscitation of Listeria monocytogenes injured by mild inactivation techniques

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M. Andjelkovic, J. Van Camp, C. Socaciu & R. Verhe (15h1015h30) Evaluation of the content and bioactivity of phenolic compounds in olive oil

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Session 3 - AGRICULTURAL SCIENCES (Chair: Veerle Fievez) Y. Devos, D. Reheul, O. Thas, E.M. De Clercq & K. Cordemans (14h30-14h50) Spatial impact of isolation distances between parcels of GM and non-GM maize

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E. Wauters (14h50-15h10) The adoption of soil conservation measures in Belgium. An application of the theory of planned behaviour

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M. Lourenço, K. Raes, S. De Smet & V. Fievez (15h10-15h30) Influence of the botanical composition of forages on rumen, milk and tissue fatty acid metabolism

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Session 4 - CHEMISTRY AND BIOPROCESS TECHNOLOGY (Chair: Bruno De Meulenaer) P. Mertens, I. Vankelecom & D. De Vos (16h40-17h00) Metal nanocolloids as innovative pseudohomogenous catalysts

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S. Mangelinckx & N. De Kimpe (17h00-17h20) β-azidocyclopropanediesters: useful building blocks with potential application in agricultural and pharmaceutical chemistry

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Session 5 - SOIL, FOREST, NATURE AND WATER MANAGEMENT (Chair: Peter Goethals) B. Verbist, R.H. Widodo, S. Susanto, M. Van Noordwijk, J. Poesen & S. Deckers (16h40-17h00) Assessment of flows and sediment loads in a catchment under conflict in Sumberjaya, Lampung, Sumatra

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B. Leroy, S. De Neve, D. Reheul & M. Moens (17h00-17h20) The quality of exogenous organic matter: influence on earthworm abundance

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Session 6 - ENVIRONMENTAL TECHNOLOGY (Chair: Eveline Volcke) E. Lesage, F.M.G. Tack, N. De Pauw & M.G. Verloo (16h40-17h00) Removal of heavy metals in constructed wetland microcosmos: effect of sorption, sulphate reduction and Phragmites australis

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P. Aelterman, K. Rabaey, H. The Pham, N. Boon & W. Verstraete (17h00-17h20) Continuous electricity generation at high voltages and currents using stacked microbial fuel cells

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CONTENT Platform presentations

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B. Criel, J-F. Hausman, M. Oufir, R. Swennen, B. Panis & J. Renaut Proteome and sugar analysis of abiotic stress underlying cryopreservation in potato

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D. Deckers, A. Aertsen, L. Callewaert & C.W. Michiels Role of lysozyme inhibitors in bacterial colinization of egg albumen

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K. Baert, B. De Meulenaer, F. Verdonck, I. Huybrechts, S. De Henauw, P.A. Vanrolleghem & F. Devlieghere Probabilistic exposure assessment of patulin in apple juice for preschool children in Flanders

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N. Van Houteghem, F. Devlieghere, S.M. Oses Gomez, J. Debevere & M. Uyttendale Effect of CO2 on the resuscitation of Listeria monocytogenes injured by mild inactivation techniques

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M. Andjelkovic, J. Van Camp, C. Socaciu & R. Verhe Evaluation of the content and bioactivity of phenolic compounds in olive oil

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Y. Devos, D. Reheul, O. Thas, E.M. De Clercq & K. Cordemans Spatial impact of isolation distances between parcels of GM and non-GM maize

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E. Wauters The adoption of soil conservation measures in Belgium. An application of the theory of planned behaviour

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M. Lourenço, K. Raes, S. De Smet & V. Fievez Influence of the botanical composition of forages on rumen, milk and tissue fatty acid metabolism

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P. Mertens, I. Vankelecom & D. De Vos Metal nanocolloids as innovative pseudohomogenous catalysts

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S. Mangelinckx & N. De Kimpe β-azidocyclopropanediesters: useful building blocks with potential application in agricultural and pharmaceutical chemistry

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B. Verbist, R.H. Widodo, S. Susanto, M. Van Noordwijk, J. Poesen & S. Deckers Assessment of flows and sediment loads in a catchment under conflict in Sumberjaya, Lampung, Sumatra

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B. Leroy, S. De Neve, D. Reheul & M. Moens The quality of exogenous organic matter: influence on earthworm abundance

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E. Lesage, F.M.G. Tack, N. De Pauw & M.G. Verloo Removal of heavy metals in constructed wetland microcosmos: effect of sorption, sulphate reduction and Phragmites australis

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P. Aelterman, K. Rabaey, H. The Pham, N. Boon & W. Verstraete Continuous electricity generation at high voltages and currents using stacked microbial fuel cells

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Poster Presentations

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D. Adriaens, O. Honnay & M. Hermy No evidence of a plant extinction debt in highly fragmented calcareous grassland in Belgium

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M. Asad RNA interference (RNAi) as a tool to engineer high nutritional value in chicory (Chicorium intybus)

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F. Asnake, F. Lambein & G. Gheysen A life-saving food plant producing more neurotoxin under envionmental stress

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C. Boeckaert, N. Boon, I.Z. Abdulsudi, W. Verstraete & V. Fievez Accumulation of biohydrogenation intermediates and changes in the rumen protozoal population after micro algae feeding to dairy catle

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L. Callewaert, A. Aertsen, D. Deckers & C.W. Michiels Searching for bacterial lysozyme inhibitors

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M. Campioli, R. Samson & R. Lemeur Seasonal growth dynamics of the moss Tomenthypnum nitens in a subarctic heath ecosystem

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P.C. Ceyssens, R. Lavigne, K. Hertveldt & G. Volckaert Isolation of lytic Pseudomonas aerugnisoa bacteriophages from worldwide collected water samples

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M. Craninx, B. Vlaeminck & V. Fievez Artificial neural networks to model the rumen fermentation pattern in dairy cattle

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E. De Backer, R. Samson & W. Steurbaut Determination of pesticide emission fluxes from canopy using micrometeorological methods

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M. De Schampheleire, P. Spanoghe, D. Nuyttens, K. Baetens, W. Cornelis & D. Gabriels The efficiency of drift-reducing measures to protect the Flemisch environment

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V. De Schepper, T. Jiang, I. Nopens & P. Vanrolleghem Modelling the performance of a membrane bioreactor controlled by air sparging

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L. De Vetter & J. Van Acker Silicon-based hydrophobation agents for wood

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K. D’haene, A. Van den Bossche, S. De Neve, D. Gabriels & G. Hofman The influence of 10 years reduced tillage on the potential carbon miniralization of silt loam soils under a temperate climate

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E. Dhooghe, M.C. Van Labeke & D. Reheul In vitro polyploidisation in the family of the Ranuculaceae

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M. Dingemans, J. Dewulf, W. Van Hecke & H. Van Langenhove Solubility of ozone in polymers: experimental determination

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A.M. Mouton, M. Schneider, P.L.M. Goethals & N. De Pauw Nature knows: heuristic search algorithms optimizing a fuzzy rulebased fish habitat suitability model for rivers

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A. Erfani & G. Wyseure The use of low-quality water on the transport and transformation of waste water in irrigation soils

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E. Fouquaert, W.J. Peumans & E.J.M. Van Damme Confocal microscopy confirms the presumed cytoplasmic/nuclear location of plant, fish and fungal orthologs of the Galanthus nivalis agglutinin

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I.M. François, E. Marien & M.De Proft Inlfuence of harvest date on the yield and quality of chicory (Cichorium intybus L.)

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W. Franssens, C. Bravo, H. Ramon & J. Anthonis Site spefici herbicide treatment of weeds in sugar beet crop

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V.M. Gomez-Lopez, P. Ragaert, F. Devlieghere & J. Debevere Prolongation of the self-life of minimally processed vegetables by mild treatments

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C. Grootaert, W. Verstraete & T. Van de Wielde Arabinoxylan oligosaccharides with different structures exert a bifidogenic effect in mixed intestinal community

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P.J. Haest, S. Ruymen, D. Springael & E. Smolders Reductive dechlorination in high aqueous TCE concentrations

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S.J. Husson & L. Schoofs Characterization of a key neuropeptide processing enzyme in C. elegans by mass spectrometry

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L.S.S. Constantine Early childhood development project in Uganda as a strategy for reducing children’s poor growth indicators: an explanatory study

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A. Maes, P.L.M. Goethals & N. De Pauw Implementation and optimisation of the Water Framework Directive Explorer for river management in Flanders

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K. Mahdian, L. Tirry & P. De Clercq Temperature alters the functional response of the predatory insect Picromerus bidens

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N. Mahna, R. Dreesen, B. Baghban Kohneh Rouz, B. Ghareyazie, M. Valizadehy, V. Grigorian & J. Keulemans Isolation and characterization of a MADS-box type gene from apple (Malux x domestica)

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M.L. Oey, E. Vanstreels, M.C. Alamar, J. De Baerdemaeker, O. Schlüter & B.M. Nicolai Inlfuence of turgor on micromechanical properties of apple tissue

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M. Reza Maleki, A. Mounem Mouazen, H. Raman & J. De Baerdemaeker Development of a variable rate fertilisation system using soil vis-nir sensor for phosphorus application

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H.K. Mebatsion, P. Verboven, Q.T. Ho, F. Mendoza, B.E. Verlinden, T.A. Nguyen & B.M. Nicolaï Modelling fruit microstructure

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M.S. Medina-Martinez, M. Uyttendaele, S. Meireman & J. Debevere J. Screening of N-acyl-L-homoserine lactone production by bacteria isolated from fresh foods

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M. Messiaen, P.L.M. Goethals & N. De Pauw Integrated benchmarking for selection of wastewater treatment technologies in Ho Chi Minh City, Vietnam

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F. Mestdagh, B. De Meulenaere, T. Cucu & C. Van Peteghem The role of water of the formation of acrylamide in a potato model system

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J. Michiels, P. Maene, J. Missotten, N. Dierick, D. Fremaut & S. De Smet Gas-chromatographic method for quantifying carvacrol, thymol, terpinen-4-ol, trans-anethole, eugenol and trans-cinnamaldehyde in media simulating pig gut conditions

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J. Missotten, J. Michiels, J. Goris, L. Herman, M. Heyndrickx, S. De Smet & N. Dierick Fermented liquid feed for weaned piglets

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K. Zendehdel, W. De Keyser & G. Van Huylenbroeck A novel approach in environmental valuation methods

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A. Van den Bossche, K. Dhaene, S. De Neve & G. Hofman Decomposition of sugar beet residues from three tillage systems in a temperate climate

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A.M.K. Van de Moortel, H. Six, E. Lesage, J. Van Acker, F.M.G. Tack & N. De Pauw Mobility of nutrients and heavy metals in the sediment of a retention basin for combined sewer overflows

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V.I.R. Unamuno, E. Meers & F.M.G. Tack The solid-solution partitioning of heavy metals (Cd and Zn) in soil and dredged sediments for environmental management purposes

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L. Anh Tuan, N. Kim Uyen & G. Wyseure Effects of common reed (Phragmites spp.) in constructed wetland for removing phosphorous and nitrogen from domestic wastewater

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P. Taheri & M. Höfte Ribovlavin induces resistance in rice against Rhizoctonia sheath diseases by acvtivating signal transduction pathways leading to upregulation of rice cationic peroxidase and formation of lignin as a structural barrier

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B. Slabbinck, P. Dawyndt, B. De Baets & P. De Vos Fame-based Bacillus species indentification using artificial neural networks

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I. Sioen, J. Van Camp, F. Verdonck, F. Vanhonacker, W. Verbeke & S. De Henauw Nutritional toxicological conflict of fish consumption: a tool for combined intake assessment

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B. Ryckaert, P. Spanoghe, S. Isebaert, B. Heremans, G. Haesaert & W. Steurbaut Quantitative determination of the influence of adjuvants on fungicides residues

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V. Rouhi, R. Samson, R. Lemeur & P. Van Damme Stomatal resistance under drought stress conditions induced by PEG 6000 on wild almond

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R. Pedreschi, E. Vanstreels, S. Carpentier, J. Robben, J-P. Noben & B. Nicolaï A proteomic approach to study flesh browning in stored conference pears

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N. Ntawubizi, K. Raes, K. Smet, G. Huyghebaert, S. Arnouts & S. De Smet Effect of antioxidant supplementation indiets of broilers on the oxidative status of plasma

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S. Shahidi Noghabi, E. Van Damme & G. Smagghe Bioassays for insecticidal activity of iris ribosome-inactiviating proteins expressed in tobacco plants

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M.T. Nguyen, I. Oey, M. Hendrickx & A. Van Loey Effect of pressure and temperature combination on the stability of (6R,S) 5-methyl and (6R,S) 5-formyltetrahydrofolic acid in model system

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P. Moons, W. Werckx, R. Van Houdt, A. Aertsen & C.W. Michiels Resistance development of bacterial biofilms against bacteriophage attack

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A. Molan & X. Gellynck Supply chain relationships and satisfaction in the agribusiness sector

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G. Van Ranst, V. Fievez, J. De Riek & E. Van Bockstaele Effect of components in red clover on plant lipase activity with possible consequences on PUFA-content of dairy products

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E. Van Twembeke & D. Geelen Micropropagation of the medicinal plant Maesa lanceolata Forssk.

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I. Vandekinderen, P. Ragaert, J. De Bruyne, J. Debevere & F. Devlieghere Antimicrobial activity of gaseous chlorine dioxide: interference by food components

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K. Vandersypen, D. Raes & J-Y. Jamin Tools for participatory water management: the modeling approach

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A. Vermeulen, N. Smigic, K.Gysemans, K. Bernaerts, A. Geeraerd, J. Van Impe, J. Debevere & F. Devlieghere Microbial safety of mayonnaise based salads: a growth/no growth model for Listeria monocytogenes as a predictive tool

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E. Wevers, P. Moons, R. Van Houdt, A. Aertsen & C.W. Michiels Role of quorum sensing in resistance of Serrata plymuthica against Acanthamoeba castellanii

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Comm. Appl. Biol. Sci, Ghent University, 71/1, 2006

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PLATFORM PRESENTATIONS

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PROTEOME AND SUGAR ANALYSIS OF ABIOTIC STRESS UNDERLYING CRYOPRESERVATION IN POTATO

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B. CRIEL1,2, J.-F. HAUSMAN2, M. OUFIR2, R. SWENNEN1, B. PANIS1 & J. RENAUT2

Laboratory of Tropical Crop Improvement, Division of Crop Biotechnics, K.U.Leuven, Kasteelpark Arenberg 13, BE-3001 Heverlee, Belgium 2 EVA, Centre de Recherche Public – Gabriel Lippmann 41, rue du Brill, LU-4422 Belvaux, GD Luxembourg

INTRODUCTION Cryopreservation complements classical conservation methods, which are carried out in the field or in vitro. It involves the storage of biological material in liquid nitrogen (-196°C). At this temperature, all chemical and physical processes are stopped, allowing a safe storage over an unlimited period of time. However, at this moment the development of improved cryopreservation protocols for potato is mainly based on a trial-and-error approach. Drought acclimation is known to improve recovery after cryopreservation in potato and other species. In this study, drought acclimation is used to analyze the mechanism underlying tolerance towards cryopreservation. MATERIAL AND METHODS Plant material Plants (Solanum tuberosum, cv. Désirée by Gaspard Bauhin (1591)) were precultured for 21 days on regular MS and MS supplemented with 0.055M, 0.11M and 0.22M sorbitol. Immediately, after pre-treatment, leaf and shoot tip samples were collected and stored at –80°C for subsequent proteomic and carbohydrate analyses. Cryopreservation Cryopreservation was carried out after 21 days of pre-treatment on the media discribed above. Potato shoot tips were cut from pre-treated plants and incubated for 15 minutes in Loading Solution, containing 9% glycerol (v/v), and 0.4M sucrose. Afterwards, shoot tips were incubated for 50 minutes on ice in Plant Vitrification Solution 2 (PVS2), containing 30% glycerol, 15% ethylene glycol, 15% DMSO (all v/v) and 0.4M sucrose. Subsequently, shoot tips were placed on an aluminum foil strip in droplets of PVS2 and plunged into liquid nitrogen (Panis et al., 2005). Thawing was done at room temperature in a highly osmotic Recovery Solution, containing 1.2M sucrose, to prevent osmotic shock. After cryopreservation, shoot tips were transferred into the dark for 1 week (Panis et al., 2005). During the first day of post-culture, shoot tips were maintained on MS media, containing 0.3M sucrose. Afterwards, regular MS media were used. After 30 days, recovery was calculated as the percentage of newly formed shoots.

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Proteomics Proteins from shoot tip and leaf samples were extracted, using a TCA/acetone extraction method. After quantification, 40 µg protein was labelled with three different fluorescent dyes (Cy2, Cy3 and Cy5). As such, 2 samples and one internal standard - containing a mix of all the samples – can be loaded on the same IPG strip (pI 4-7) for the first dimension (Unlu et al., 1997). Isoelectric focusing was carried out using the Amersham Ettan IPGphor. The second dimension was run in the Amersham Ettan Dalt Six electrophoresis system. Gels were scanned using the Typhoon 9400 scanner. Gels were analysed with the Amersham DeCyder program and EDA module. After quantification, differences in protein expression were determined and differentially expressed spots picked by the Amersham Ettan Spot Handling Workstation. In a following step, spots were digested, using Trypsin and spotted on a MALDI plate. Protein identification was executed, using the Applied Biosystems MALDI 4800 TOF/TOF analyzer. Analysis of carbohydrates and polyols 100 mg leaf samples was used for carbohydrate and polyol extractions (Van Huylenbroeck et al., 1996). The following carbohydrates and polyols were quantified: sucrose, glucose, fructose, galactose, stachyose, arabinose, melibiose, maltose, trehalose, inositol, mannitol, galactinol and sorbitol. Carbohydrates were analyzed on a Dionex HPLC ICS2500-Bio LC, using a Dionex Carbopac PA-20 column and polyol analyses were conducted on a Dionex DX-500 chromatograph, using a Dionex Carbopac MA1 column. Concentrations were assessed using seven points calibration-curves with custom-made external standard solutions (Wilson et al., 1995). RESULTS Cryopreservation Recovery rates without sorbitol were around 50% but increased with increasing concentration of sorbitol pre-treatment up to a recovery rate around 80% (Figure 1).


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Figure 1. Recovery of potato shoot tips after cryopreservation and pre-treatement under 4 different sorbitol concentrations. Two repetitions: left: n = 40 and right: n = 100.

Proteome analysis Preliminary analysis of the gels shows differences in protein patterns, when plants were precultured on different sorbitol media (Figure 2). Further analysis is in progress. Based on the observed differences, interesting spots are now picked and identified through mass spectrometry. pI 4

pI 7

High MW

Low MW Figure 2: Example of a 2D DiGE gel, run on shoot tip samples from pretreated plants (Red: Cy3, 0M sorbitol; Green: Cy5, 0.055M sorbitol; Yellow: Overlay Cy3 and Cy5; Blue: Cy2, Internal standard). Analysis of carbohydrates and polyols We observe a strong influence of the sorbitol pre-treatment on the polyol and carbohydrate metabolism. Inositol, galactinol, glucose, fructose, melibiose, stachyose and sucrose increased with increasing sorbitol concentrations up to 0.11M sorbitol. At 0.22M sorbitol their concentrations decreased. For galactose a similar trend was observed, but concentrations reached a pla-

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teau at 0.22M sorbitol pre-treatment. Trehalose and sorbitol (Figure 3) concentrations increased exponentially with an increasing molarity of sorbitol pre-treatment. Mannitol and arabinose concentrations increased up to a 0.11M sorbitol pre-treatment, but could not be measured after a 0.22M sorbitol pre-treatment . Maltose was only observed when plantlets were treated with 0.22M sorbitol. 2000

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Figure 3. Sorbitol (A) and trehalose (B) concentrations in leaf tissue, during pretreatement for 21 days on increasing sorbitol concentrations (n = 6).

DISCUSSION When plants were treated with up to 0.11M sorbitol during 21 days, carbohydrates and polyols concentrations increased. When 0.22M sorbitol was applied as pre-treatment, most sugar concentrations decreased. Since high intracellular osmolyte concentrations are needed to allow successful recovery after cryopreservation, the results from carbohydrates and polyols analyses may explain the higher recovery rate after cryopreservation observed after sorbitol pre-treatment. Extra cryopreservation experiments are needed to confirm these results. Besides the sugar analysis, changes have been observed at the protein level. Identification of these proteins will lead to a better understanding of the physiological status of the tissue during sorbitol pre-treatment. REFERENCES Panis B., Piette B. & Swennen R. (2005). Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Sci., 168, 45-55. Unlu M., Morgan M.E. & Minden J.S. (1997). Difference gel electrophoresis: a single method for detecting changes in protein extracts. Electrophoresis, 18, 2071-2077. Van Huylenbroeck J.M. & Debergh P.C. (1996). Impact of sugar concentration on photosynthesis and carbon metabolism during ex vitro acclimatisation of Statyphyllum plantlets. Physiol. Plant., 96, 298-304. Wilson L., Amy G., Gerba C., Gordon H., Johnson B. & Miller J. (1995). Water quality changes during soil aquifer treatment of tertiary effluent. Water Environ. Res., 67, 371-376.


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ROLE OF LYSOZYME INHBITORS IN BACTERIAL COLONIZATION OF EGG ALBUMEN D. DECKERS, A. AERTSEN, L. CALLEWAERT A C.W. MICHIELS Laboratory of Food Microbiology, Kasteelpark Arenberg 22, BE-3001 Leuven E-mail: [email protected]

INTRODUCTION Lysozymes are natural antimicrobial compounds that have been described from vertebrate and invertebrate origin. Based on differences in three dimensional structure and amino acid sequence, they are further classified in distinct subfamilies. Hen egg white lysozyme (HEWL) is the representative of the c-type lysozyme family and occurs naturally in high concentration in egg white (3.2 mg/ml) (Proctor and Cunningham, 1988; Conner, 1993). The substrate of lysozyme is the peptidoglycan, which is a unique component of the bacterial cell wall that determines cellular shape and provides protection against the cellular turgor pressure. Splitting of the β(1,4)-glycosidic bounds between the alternating N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) residues that form the backbone of the peptidoglycan, causes lysis of the bacteria. The enzymatic action of lysozyme is, however, limited to Gram-positive bacteria. Contrary to Gram-positive bacteria the peptidoglycan of Gram-negative bacteria is markedly thinner but is further surrounded by an outer membrane that forms a barrier for the amphipilic lysozyme molecule. However, the efficiency of lysozyme against Gram-negative bacteria can be improved by disrupting the barrier function of the outer membrane. This can be achieved by substances that complex or displace the stabilizing divalent cations from the outer leaflet of the outer membrane, such as EDTA or lactoferrin (Ellison and Giehl, 1991; Facon and Skura, 1996; Vaara, 1992), or by some physical treatments such as high hydrostatic pressure (Hauben et al., 1996). Besides the barrier function of the outer membrane, however, the contribution of lysozyme inhibitors to lysozyme resistance of Gram-negative bacteria should be strongly considered. Although the structure and enzymatic action of lysozyme has been extensively studied for several decades and the existence of lysozyme inhibitors has been regularly suggested in literature, the first known proteinaceous specific lysozyme inhibitor, Ivy (Inhibitor of Vertebrate lYsozyme) was only recently discovered in the periplasm of Escherichia coli (Monchois et al., 2001). Besides its strong inhibitory activity against HEWL in vitro, Ivy plays an important role in the lysozyme resistance of the bacteria, since the inactivation of the ivy gene increases the lysozyme sensitivity of E. coli when the stability and permeability of the outer membrane was compromised by lactoferrin or by high hydrostatic pressure treatment (Deckers et al., 2004). High hydrostatic pressure is proposed as an alternative to the thermal pasteurization process of liquid eggs. Since, opposed to heating, it can improve the microbial safety of egg white with only negligible effects on product quality. Based on the protective role of Ivy and regarding the importance of E. coli strains as food pathogens, we investigated in this paper the role of Ivy in the survival of E. coli in egg white during a high hydrostatic pressure treatment.

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MATERIAL AND METHODS Isolation of egg albumen Biological eggs were purchased from Colruyt. To avoid contamination of bacteria present on the egg shell to the egg white, the egg shell was first disinfected with 70% ethanol. The egg white was aseptically separated from the yolk and homogenized for 30s at 230 rpm (stomacher, Led Techno, Eksel, Belgium).

Escherichia coli strains Escherichia coli MG1655, the corresponding ivy knockout mutant MG1655 ivy::Kan and MG1655 pAA410, which contains the ivy gene under the transcriptional control of the arabinose inducible PBAD promoter were used throughout this study (Deckers et al., 2004). LB agar master plates were inoculated from -80°C stocks and contained ampicillin (100 µg/ml) for E. coli pAA410 or kanamycin (50 µg/ml) for E. coli ivy::Kan (Sigma-Aldrich, Bornem, Belgium). All liquid cultures were grown overnight at 37°C in Luria Bertani (LB) broth (10 g/l trypton, 5 g/l yeast extract, 5 g/l NaCl) with the appropiate antibiotics untill stationary phase. Sensitivity of E. coli strains to egg white under high hydrostatic pressure Stationary cultures of the strains described above were diluted 100-fold in fresh LB media and incubated at 37°C. At an optical density (OD600) of 0.4, 0.2% arabinose was added and growth was continued until an OD600 around 0.9 (early stationary cells, ± 109 CFU/ml). Although arabinose served only to induce ivy expression from the plasmid pAA410, it was also added to the cultures of the strains not harboring this plasmid to ensure identical growth conditions for all strains in one experiment. The cells were harvested by centrifugation and resuspended in the same volume of egg white solution (75% egg white – 25% potassium phosphate buffer 10 mM, pH 7.0). 300 µl of these suspensions were sealed in a sterile polyethylene bag after exclusion of air bubbles and subjected to a high hydrostatic pressure treatment at 250 MPa (15 min, 20°C). The experiments were done in equipment with eight parallel 8-ml vessels, which can simultaneously be pressurized and are thermostatically controlled with a water jacket (Resato, The Netherlands). The high-pressure transmission fluid was TP1, a mixture of glycols (Van Meeuwen, Weesp, The Netherlands). After decompression, the samples were aseptically removed from the bags. To assess the loss of viability untreated and treated samples were serially diluted in sterile potassium phosphate buffer (10 mM, pH 7.0) and plated on LB agar plates. Colonies were allowed to develop for 24 h at 37°C. Inactivation was expressed as a logarithmic viability reduction factor, log10 No/N, where No and N are the colony counts of untreated and treated samples, respectively. All data shown are averages of three independent experiments with standard deviations.


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RESULTS Suspensions of early stationary phase cells of E. coli MG1655 (wild-type), its corresponding ivy knock-out and ivy overexpression strain that had been induced with arabinose were pressurized at 250 MPa for 15 min at 20°C and survivors were enumerated. Besides lysozyme, egg white contains other antimicrobial compounds such as ovotransferrin that can destabilise the outer membrane and thus assist the penetration of lysozyme through the outer membrane, however, no inactivation of the strains was seen at ambient pressure (3 h at 20°C) by the egg white during the time of the experiments (data not shown). At 250 MPa, we observed a higher inactivation of the ivy knockout strain (2.1 log units), compared to 1.5 log units of the wild-type and 1.1 log units of the overexpression strain by egg white (Figure 1). Corresponding to previous high pressure experiments performed with commercial HEWL in potassium phosphate buffer, these results show that the sensitivity of E. coli to egg white was inversely related to the periplasmic Ivy content of the cells. 3

log N0/N

2

1

0 MG1655 ivy::Kan

MG1655

MG1655 pAA410

Figure 1. Inactivation (log10N0/N) of E. coli MG1655 ivy::Kan, E. coli MG1655 and E. coli MG1655 (pAA410) under high pressure (250 MPa, 15 min) in egg white. Mean values ± SD (error bars) are shown.

DISCUSSION Although the effect of Ivy on the lysozyme resistance of Gram-negative bacteria was recently revealed, its biological impact remains largely unknown (Deckers et al., 2004). However, regarding the important antimicrobial role of lysozymes in the innate immune system of vertebrate and invertebrate organisms, these inhibitors may contribute to the host colonization capacity of commensal and pathogenic bacteria. In this work we investigated the role of the lysozyme inhibitor Ivy in the survival of E. coli in egg albumen, which is a major source of c-type lysozyme. Under high hydrostatic pressure we observe that the survival of E. coli in egg white is indeed enhanced by the overproduction of the lysozyme inhibitor and that the disruption of the ivy gene resulted in an increased inactivation of the bacteria compared to the wild-

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type strain. These results indicate that the destruction of E. coli in egg white is not only dependent on applied pressure and temperature but is indeed also mediated by the antimicrobial activity of lysozyme and warn us for the role of Ivy, or bacterial lysozyme inhibitors in general, as potential virulence factors of pathogenic bacteria that protect against host lysozymes. Recently, Callewaert et al., discovered a new lysozyme inhibitor in S. Enteritidis, which is a long recognized pathogen in a wide range of foods. Especially eggs and egg containing products are responsible for a high percentage of human salmonellosis (see contribution of Callewaert et al. to this symposiumOur results regarding Ivy, do suggest that this lysozyme inhibitor is maybe an important protein for the resistance of S. Enteritidis to egg albumen by providing an extra line of defense against host lysozymes, when the outer membrane becomes permeabilized by ovotransferrin. REFERENCES Conner, D.E. (1993). Naturally occurring compunds. In: P.M. Davindson and A.L. Branen (Eds), Antimicrobials in Foods, Marcel Dekker Inc., New York, pp. 441468. Deckers, D., Masschalck, B., Aertsen, A., Callewaert, L., Van Tiggelen, C.G.M., Atanassova, M., Michiels, C.W. (2004). Periplasmic lysozyme inhibitor contributes to lysozyme resistance in Escherichia coli. CMLS, 61, 1229-1237. Ellison R.T. and Giehl T.J. (1991) Killing of gram-negative bacteria by lactoferrin and lysozyme. J. Clin. Invest., 88, 1080-1091 Facon M.J. and Skura B.J. (1996) Antibacterial activity of lactoferricin, lysozyme and EDTA against Salmonella enteritidis. Int. Diary J., 6, 303-313 Forsythe, R.D. (1970). Egg processing technology progress and sanitation programs. J. Milk Food Technol., 33, 64-73. Hauben K., Wuytack E., Soontjens C., and Michiels C. (1996) High pressure transient sensitization of Escherichia coli to lysozyme and nisin by disruption of outermembrane permeability. J. Food Prot.., 59, 350-355 Monchois, V., Abergel, C., Sturgis, J., Jeudy, S., Claverie, J-M. (2001). Escherichia coli ykfE ORFan gene encodes a potent inhibitor of C-type lysozyme. J. Biol. Chem., 276, 18437-18441. Proctor V.A. and Cunningham F.E. (1988) The chemistry of lysozyme and its use as a food preservative and a pharmaceutical. Crit. Rev. Food Sci. Nutr. 26, 359-395. Vaara M. (1992) Agents that increase the permeability of the outer membrane. Microbiol. Rev., 56, 395-411


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PROBABILISTIC EXPOSURE ASSESSMENT OF PATULIN IN APPLE JUICE FOR PRESCHOOL CHILDREN IN FLANDERS Katleen BAERT1, Bruno DE MEULENAER1, Frederik VERDONCK2, Inge HUYBRECHTS3, Stefaan DE HENAUW3, Peter A. VANROLLEGHEM2 AND Frank DEVLIEGHERE1 1

Department of Food Safety and Food Quality, Ghent University Coupure links 653, BE-9000 Ghent, Belgium 2 Department of Applied Mathematics, Biometrics and Process Control, Ghent University Coupure links 653, BE-9000 Ghent, Belgium 3 Department of Public Health, Ghent University De Pintelaan 185, BE-9000 Ghent, Belgium

INTRODUCTION The mycotoxin patulin (4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one) is produced by a large number of fungi including Penicillium expansum. This mould is commonly identified as the ‘blue mould rot’, a serious post-harvest disease of apples. As a consequence, patulin is frequently occurring in apples and apple products, like apple juice (Moake et al., 2005). The aim of the present study was to estimate the consumers’ exposure to patulin through organic, handcrafted and conventional apple juice, based on a probabilistic method. The findings were then compared with the Tolerable Daily Intake (TDI) aiming to help risk managers in the regulatory decision making process. Since, a Scientific Cooperation task (SCOOP task) has shown that small children have a higher patulin intake compared to other population groups and because apple juice and apple nectar are the main sources of patulin intake in most countries, the focus of this probabilistic exposure assessment is on this particular population group and this specific matrix (Directorate – General Health and Consumer Protection, 2002). Secondly, the study aimed to evaluate the effectiveness of a reduction of the regulatory limit for patulin in apple juice. MATERIALS AND METHODS Data on the apple juice consumption were obtained from a large-scale epidemiological study investigating nutrition habits of preschool children in Flanders (2.5-6.5 years old), using 3-day estimated diet records and parents as a proxy (Huybrechts et al., 2006). The contamination data used in the present study were derived from a previous study (Baert et al., 2006) in which 177 apple juice samples were analyzed on their patulin content (65 organic, 90 conventional and 22 handcrafted apple juices). The exposure to patulin was modeled by multiplying consumption data with contamination data of organic, conventional and handcrafted apple juice (patulin intake (µg/kg bw/day) = concentration of patulin in apple juice (µg/kg) x apple juice consumption (g/kg bw/day) x 1 (kg) / 1000 (g)). In order to characterize the variability of interindividual consumption and patulin concentrations, a non-parametric approach was used. This means that a distribution is used whose mathematics is defined by the required

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shape (Vose, 2000). In this study, a discrete uniform distribution (RiskDuniform) was used for both the observed consumption and contamination values. A discrete uniform distribution is a special case of the Discrete distribution where all possible values have the same probability of occurrence (Vose, 2000). Uncertainty characterization was performed using non-parametric bootstrap. The bootstrap theory assumes that the distribution F (of e.g. patulin concentration in apple juice) can be reasonably approximated by the distribution F’ of n observed values. This is of course a more reasonable assumption, when more data are collected. For a sufficiently large number of times, n random samples are taken with replacement from the distribution F’ and each time a statistic of interest is calculated from the sample (Vose, 2000). Contamination data below the Limit Of Detection (LOD) were replaced by random samples from a uniform distribution with α as a minimum (α = uniform distribution between zero and the LOD) and β as a maximum (β = a uniform distribution between zero and the LOD) (RiskUniform (RiskUniform (0; LOD); RiskUniform (0; LOD))). Propagation of the characterized variability and uncertainty was then performed using Monte Carlo simulation. It was executed with @RISK, a risk analysis software (@RISK 4.5 for excel professional edition, Palisade, UK). Latin Hypercube sampling was used to randomly sample the probability distribution functions of input parameters (consumption and contamination data) and the samples were used to calculate the intake by the described model. One thousand simulations were carried out to describe the consumption and contamination variability in the population and 1000 bootstrap iterations were carried out to estimate the uncertainty (expressed as a confidence interval). In the study it was assumed that children consume a certain type of apple juice (e.g. organic) and do not consume different types. It was also assumed that the consumption data were the same for the different consumers (organic, handcrafted and conventional consumers). RESULTS AND DISCUSSION In a previous study, higher patulin concentrations were found in organic apple juice (8.8 µg/L) compared to conventional (4.1 µg/L) and handcrafted apple juice (4.4 µg/L) (Baert et al., 2006). It was one of the aims of the present study to evaluate the implications of these higher concentrations with regard to public health. The estimated exposures to patulin together with the 90% CI are summarized in Table 1 for the 3 types of apple juice tested. For the three types of apple juice, the 83rd percentile shows an intake of 0 µg/kg bw/day, indicating that 83% of the children have no intake of patulin. For organic apple juice the best estimate for the 99.5th percentile is higher than the TDI (of 0.43 µg/kg bw/day). The lower limit of the 90% CI of the 99.9th percentile is also higher than the TDI. Also for conventional apple juice, the best estimate shows that the TDI will be exceeded in less than 0.1% of the cases. The best estimate of the exposure through handcrafted apple juice is below the TDI. Although higher average exposures are observed for organic apple juice compared to the other two practices, their CI overlap, which indi-


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cates that the average exposure to patulin of children will be roughly the same for the 3 kinds of apple juice tested. Table 1. Statistics on exposure (µg/kg bw/day) for different apple juices (AJ) (best estimation [90% confidence interval])

P0 P50* P83 P85 P90 P95 P97.5 P99 P99.5 P99.9 Mean Probability to exceed the TDI * 50th percentile

Organic AJ 0 [0-0] 0 [0-0] 0 [0-0] 0.0001 [0-0.024] 0.039 [0.014-0.069] 0.072 [0.027-0.117] 0.135 [0.053-0.229] 0.350 [0.143-0.822] 0.615 [0.249-1.472] 1.471 [0.526-3.066] 0.019 [0.010-0.032] 0.009 [0.003-0.018]

Conventional AJ 0 [0-0] 0 [0-0] 0 [0-0] 0 [0-0.017] 0.030 [0.011-0.049] 0.059 [0.031-0.085] 0.095 [0.057-0.133] 0.156 [0.106-0.206] 0.202 [0.141-0.287] 0.328 [0.210-0.548] 0.009 [0.006-0.013] 0.001 [0-0.003]

Handrafted AJ 0 [0-0] 0 [0-0] 0 [0-0] 0.0001 [0-0.022] 0.037[0.013-0.066] 0.065 [0.027-0.102] 0.102 [0.047-0.151] 0.150 [0.084-0.229] 0.195 [0.109-0.290] 0.298 [0.156-0.460] 0.010 [0.005-0.015] 0 [0-0.002]

Comparison of the percentage of children exceeding the TDI for the 3 types of apple juice (Table 1) indicates that the probability of exceeding the TDI is higher for organic apple juice than for conventional and handcrafted apple juice. With 90% certainty, it can be stated that between 0.3 and 1.8% of the children consuming organic apple juice exceed the TDI while for conventional and handcrafted apple juice this is between 0 and 0.3% and between 0 and 0.2% respectively. In Europe, the maximum limit for patulin is 50 µg/kg (European Commission, 2003). However during the study on the occurrence of patulin in apple juice in Flanders, two organic apple juices were found with patulin contents above 50 µg/kg (Baert et al., 2006). Therefore, the influence of a strict implementation of the current legislation on patulin exposure of young children, was tested by removing these two samples from the dataset. In the regulation 1425/2003 it was also foreseen that the commission would review the maximum levels for patulin by 30 June 2005 at the latest in order to reduce them (European Commission, 2003). However, until now no new legislation is in preparation or has been published. In order to test the effect of lowering the Maximum Limit (ML), the exposure was simulated with a limit reduced by 50%, resulting in a new limit of 25 µg/kg which is also the current ML for solid apple products. For this test, all samples above 25 µg/kg were removed from the dataset. Since concentrations above 25 µg/kg were only found for organic apple juice, both techniques were just tested for organic apple juice alone. Making a comparison of the percentage of the population exceeding the TDI (Table 2), shows that a strict implementation of the limit of 50 µg/kg reduces the percentage of children exceeding the TDI, (between 0.1% and 1.2%). A restriction to 25 µg/kg reduces the exposure higher than the TDI further, towards 0 to 0.3% of the population under study. Therefore it can be concluded that in order to reduce the exposure to patulin, a reduction of the legal limit to 25 µg/kg is necessary and causes a six fold reduction of the probability to exceed the TDI. However, it needs to be stressed that the implementation of a ML is only effective when this limit

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is followed and as a consequence control on the implementation is necessary. Table 2. Comparison of the statistics on exposure for organic apple juice for different hypotheses to reduce the level of contamination of apple juice (best estimation [90% confidence interval])

Probability to exceed the TDI

No reduction 0.009 [0.003-0.018]

Contamination 100

1.00

>100

B

0.025

CAH's [mmol]

CAH's [mmol]

A

0.0125 0 0

20

40

0.1 0.05

60

0 0

20

40

60

Time [d]

Time [d]

Figure 1. (A) Reductive dechlorination of 0.020 mmol TCE by HP10 (0.02 mmol equals an aqueous concentration of 0.2 mM TCE in the batch); (B) Reductive dechlorination of 0.083 mmol TCE by KB-1TM (0.083 mmol equals an aqueous concentration of 0.9 mM TCE in the batch).

Legend: ♦ TCE; ▲ cDCE; ■ VC; * Ethene

Tested cultures Dehalococcoides ethenogenes

Positive controls Dehalococcoides CBDB-1

Mixed culture

KB-1TM HP10

A

B

C

KB-1TM

Dehalococcoides ethenogenes strain 195

Figure 2. 16S rDNA PCR-DGGE analysis of 3 of the tested cultures. Positive controls: (A) KB1TM incubated on DCA; (B) KB1TM incubated on TCE [0.1 mM]; (C) KB-1TM incubated on VC.

A

Dehalococcoides

specific

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PCR-DGGE was performed to analyze the species composition of KB-1TM [TCE: 1 mM], HP10 [TCE: 0.1 mM] and Dehalococcoides ethenogenes mixed culture [TCE: 0.1 mM]. Figure 2 illustrates that the 3 cultures indeed contain Dehalococcoides spp. but a difference in species composition is present. HP10 contains species that are similar to Dehalococcoides ethenogenes strain 195 and Dehalococcoides strain CBDB-1. KB-1TM contains different Dehalococcoides spp. and the PCR-DGGE analysis shows that, depending on the chlorinated ethenes present and their concentration, different bands appear. This observed shifting of bands suggests a sequence of species is active in the complete dechlorination of TCE to ethene as was observed by others. The results of the abiotic column test show that the measured TCE concentration in the effluent is near the TCE water saturation of 8.4 mM (data not given). This indicates that TCE dissolves rather well out of the DNAPL droplets at ambient groundwater velocities. Unfortunately, no dechlorination was observed in batch tests with 2 mM TCE concentrations or higher present in the batch. As such, the high TCE concentrations near a TCE DNAPL will strongly inhibit the reductive dechlorination by the tested cultures, limiting the possibilities of bioremediation in a TCE source zone. Results were obtained in a homogeneous column medium with an evenly distributed DNAPL pollution. Effects of bypass flow, dilution etc. are not taken into account and can be important in situ. CONCLUSION In batch tests a toxicity of TCE on TCE degradation (‘self-inhibition’) was found above 1 mM TCE. This concentration is 8-fold below its water solubility and below the aqueous concentrations observed in the abiotic column containing TCE residual DNAPL. This suggests that self-inhibition is an important factor in a TCE source zone where elevated aqueous TCE concentrations are to be found. The extent of the reductive dechlorination is determined largely by the Dehalococcoides spp. present and results so far suggest that different Dehalococcoides spp. work in series in the complete degradation of TCE to ethene. The competitive inhibition of the higher chlorinated ethenes on the degradation of the lower chlorinated products can result in an accumulation of the more toxic VC. Future research will examine whether the above mentioned observations play a significant role in a more heterogeneous medium. If so, we postulate that bioremediation of a residual TCE source zone may serve as an efficient screen for downstream users but will not result in a substantial decrease of the source zone lifetime. ACKNOWLEDGEMENT Sincere thanks go to Dr. R. Richardson of the Cornell University (USA), to the SiREM company and to Dr. R. Lookman of the VITO (Belgium) for providing their cultures. Many thanks also to Dr. J. Dijk for his advice and kind assistance. This research is funded by a Ph.D grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen)


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REFERENCES Christ, J. A., C. A. Ramsburg, L. M. Abriola, K. D. Pennell, & F. E. Loffler. (2005). Coupling Aggressive Mass Removal With Microbial Reductive Dechlorination for Remediation of Dnapl Source Zones: a Review and Assessment. Environ. Health Persp.,113:465-477. Cupples A.M., A.M. Spormann & P.L.McCarty (2003). Comparative Evaluation of Chloroethene Dechlorination to Ethene by Dehalococcoides-Like Microorganisms. Appl. Environ. Microbiol. 69: 953-959 Harkness, M. R., A. A. Bracco, M. J. Brennan, K. A. Deweerd, and J. L. Spivack. 1999. Use of Bioaugmentation to Stimulate Complete Reductive Dechlorination of Trichloroethene in Dover Soil Columns. Environ. Sci. Technol.,33:1100-1109. He J, Ritalahti KM, Aiello MR and Löffler FE. (2003). Complete Detoxification of Vinyl Chloride by an Anaerobic Enrichment Culture and Identification of the Reductively Dechlorinating Population as a Dehalococcoides Species Appl. Environ. Microbiol.,69: 996-1003 McGuire, T.M., J.M. McDade and C.J. Newell (2006). Performance of DNAPL source depletion technologies at 59 chlorinated solvent-impacted sites. Ground Water Monit.,26:73-84. Ruymen, S. (2006). Mogelijkheden van bioremediatie voor een TCE-bronzone. Stagerapport, Katholieke Hogeschool Limburg, Belgium Soga, K., J. W. E. Page, and T. H. Illangasekare. 2004. A Review of Napl Source Zone Remediation Efficiency and the Mass Flux Approach. Journal of Hazardous Materials 110:13-27. Yu, S. and L. Semprini (2004). Kinetics and Modeling of Reductive Dechlorination at High Pce and Tce Concentrations. Biotechnol. Bioeng.,88:451-464.

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CHARACTERIZATION OF A KEY NEUROPEPTIDE PROCESSING ENZYME IN C. ELEGANS BY MASS SPECTROMETRY S.J. HUSSON & L. SCHOOFS Laboratory for Developmental Physiology, Genomics and Proteomics, Katholieke Universiteit Leuven, Naamsestraat 59, BE-3000 Leuven, Belgium E-mail: [email protected]

INTRODUCTION Neuropeptides are signaling molecules that regulate most biological processes in animals by the interaction with cell surface receptors to trigger an intracellular transduction pathway. These endogenous, bioactive peptides are derived from large proprotein peptide precursors that require several highly regulated post-translational events to yield the neuropeptides (Figure 1). A typical proprotein peptide precursor contains an aminoterminal signal peptide, which is cleaved off upon entrance into the secretory pathway by a signal peptidase. Subsequently, proprotein convertases (PCs) cleave the remaining part of the precursor at specific dibasic (KR, RR, RK and KK) or monobasic (K and R) sites. After cleavage from the proprotein peptide precursor, the resulting intermediate peptides contain carboxyterminal basic amino acids which are removed by a specific carboxypeptidase. Finally, the carboxyterminal glycine residue, if present, is transformed into an amide, a common feature of secreted, bioactive peptides. In mammals, PC2 and PC1/3 specifically recognize substrates composed of paired basic residues, reflecting their role in the processing of neuropeptide precursors. The egl-3 gene in C. elegans encodes a PC2-like convertase (Kass et al., 2001) and was first identified in a mutant screen for deficiency in egg laying. Biochemical evidence of EGL-3 being the major peptide precursor processing enzyme in C. elegans has, however, never been delivered.

Figure 1. Schematic representation of the (neuro)peptide processing pathway

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A few years ago, we introduced the concept of peptidomics, which aims at the simultaneous visualization and identification of endogenous peptides in a cell, tissue or organism by mass spectrometry (Clynen et al., 2001; Baggerman et al., 2004). This way, we analyzed the peptidome of C. elegans (Husson et al., 2005) and were able to sequence sixty naturally occurring (neuro)peptides by using an on line 2D-nanoLC-Q-TOF MS/MS setup. In the present study, we combined HPLC separation with mass spectrometric detection of naturally occurring peptides using a MALDI-TOF instrument. This off line approach allows a straightforward comparison of peptide profiles in various C. elegans strains having mutations in the presumed peptide processing enzymes (Husson et al., 2006). MATERIAL AND METHODS Strains and sample preparation - The C. elegans strains used in this work are: wild-type N2, egl-3(n729), egl-3(gk238), egl-3(n588), egl-3(n150) and egl3(ok979) which were all kindly provided by the Caenorhabditis Genetics Centre (CGC). They were all propagated on conventional nematode growth media (NGM) plates containing E. coli OP-50 bacterial lawns, at 20°C. All strains were treated separately using exactly the same extraction protocol as described earlier (Husson et al., 2005). High-performance liquid chromatography (HPLC) - HPLC analysis of the six peptide extracts was carried out on a Waters Symmetry C18 (5µm, 4.6x 250mm) column using a linear gradient over 60 minutes from 2% CH3CN to 50% CH3CN in 0.1% aqueous TFA. 1mL fractions were collected automatically every minute starting from the beginning of the gradient. Matrix assisted laser desorption ionisation – time of flight mass spectrometry (MALDI-TOF MS) - One fourth of each HPLC fraction was lyophilized and resuspended in 1µL water/CH3CN/TFA (50/49.9/0.1) and subsequently transferred to a stainless steel target plate and mixed with 1µL of a saturated α-cyano-4-hydroxy cinnamic acid in acetone solution and dried. MALDI-TOF mass spectrometry was performed on a Reflex IV instrument (Bruker Daltonic). Spectra were recorded in positive reflectron mode and were the result of 5 x 20 laser shots. RESULTS Identification of 75 neuropeptides by MALDI-TOF MS – An analytical HPLC analysis of the wild type C. elegans extract on a Symmetry C18 column resulted in a satisfying reduction of sample complexity. Screening of all 60 HPLC fractions with MALDI-TOF MS and comparing the masses of the ions with the calculated masses of predicted FLP and NLP peptides, resulted in the identification of 75 C. elegans neuropeptides; 37 of which were also detected in the previous peptidomic study of C. elegans using 2D-nanoLC-QTOF MS/MS (Figure 2A). Differential peptidomic analysis of egl-3 mutant strains - The same HPLC-MS setup was used to compare the neuropeptide profile of C. elegans strains having mutations in egl-3. Most peptide ion peaks had a fair intensity


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in the wild type strain and were completely absent in the five independent strains with mutated egl-3 alleles (Figure 2). Not a single peptide ion could be detected in egl-3(n729) and egl-3(gk238). To support our findings, we performed immunocytochemistry experiments (as adapted from Li and Chalfie, 1990) on wild type C. elegans and egl-3 mutants using anti-RFamide antibodies. A strong decrease of immunoreactivity in the mutants, when compared to wild type C. elegans, could be observed (Figure 3). This is in agreement with the mass spectrometry data, which provide biochemical evidence about FLP and NLP precursors being substrates for EGL-3.

Figure 2. Comparison of MALDI-TOF MS spectra from wild type C. elegans (A) and strains having mutated egl-3 genes (B). Peptide extracts were subjected to an HPLC analysis to generate 60 fractions, which were all analyzed by MALDI-TOF MS. As an example, zoom regions from m/z 900 to m/z 2000 of fraction 35 is shown here, indicating a severe depletion of neuropeptides in the mutant egl-3 strains

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Figure 3. -RFamide immunoreactivity in wild type C. elegans and egl-3 mutant Immunoreactivity in the C. elegans strains with mutated egl-3 alleles is strongly reduced when compared to the wild type strain which displays a very intense immunoreactivity in the nerve ring (NR) and the ventral nerve cord (VNC). This is in agreement with a depletion of FLP peptides due to the mutations in egl-3.

DISCUSSION To identify substrates for PC2 or PC1/3 in mammals, antibodies against a wide variety of endogenous peptides had to be generated and radioimmuno assays (RIAs) had to be performed, which was a laborious job. Using mass spectrometry, however, peptides in crude tissue extracts can be systematically analyzed with a greater resolution and specificity than traditional techniques like e.g. SDS-PAGE, Western blots, and RIA, which often suffers from cross-reactivity. Doing so, we were able to sequence 29 peptides derived from NLP precursors in addition to 31 FLP peptides (Husson et al., 2005). We now expand this list of peptides by applying another approach, which resulted in the identification of additional peptides. This off line HLPC-MALDI-TOF MS setup allowed a straightforward comparison of peptide profiles from various C. elegans strains having mutations in the key neuropeptide processing enzyme. A severe depletion of endogenous peptides in C. elegans with mutated egl-3 genes was observed. We hereby provide evidence about EGL-3 being the major neuropeptide processing enzyme in the nematode C. elegans. REFERENCES Baggerman G., Verleyen P., Clynen E., Huybrechts J., De Loof A. & Schoofs L. (2004) Peptidomics., J. Chromatogr. B, 803, 3-16. Clynen E., Baggerman G., Veelaert D., Cerstiaens A., Van Der Horst D., Harthoorn L., Derua R., Waelkens E., De Loof A. & Schoofs L. (2001) Peptidomics of the pars intercerebralis-corpus cardiacum complex of the migratory locust, Locusta migratoria. Eur. J. Biochem., 268, 1929-1939. Husson S.J., Clynen E., Baggerman G., De Loof A. & Schoofs L. (2005) Discovering neuropeptides in Caenorhabditis elegans by two dimensional liquid chromatography and mass spectrometry. Biochem. Biophys. Res. Comm., 335, 76-86 Husson S.J., Clynen E., Baggerman G., Janssen T. & Schoofs L (2006) Defective processing of neuropeptide precursors in Caenorhabditis elegans lacking proprotein convertase 2 (KPC-2/EGL-3): mutant analysis by mass spectrometry. J. Neurochem., accepted for publication Kass J., Jacob T.C., Kim P. & Kaplan J.M. (2001) The EGL-3 proprotein convertase regulates mechanosensory responses of Caenorhabditis elegans. J. Neurosci., 21, 9265-9272. Li C. & Chalfie M. (1990) Organogenesis in C. elegans: positioning of neurons and muscles in the egg-laying system. Neuron, 4, 681-695.


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EARLY CHILDHOOD DEVELOPMENT PROJECT IN UGANDA AS A STRATEGY FOR REDUCING CHILDREN’S POOR GROWTH INDICATORS: AN EXPLANATORY STUDY Loum L.S. CONSTANTINE ABSTRACT This study shall explore the activities of the Uganda nutrition and early childhood development project using explanatory program effects, qualitative research methods to determine the role/interplay of project factors and community dynamics in relation to poor growth and social indicators in Ugandan children. This project aimed to improve the quality of life for children age less than 6 years by improving their nutrition, health, cognitive and psychosocial development. The key concern remains how successful and sustainable are the outcomes? Data shall be collected using interviews, focus groups and document reviews. Data analysis shall employ the thematic networks analytical tool, which is aided by the ATLAS.ti software. The key hypotheses and recommendations from the analysis shall then be tested in a suitable community location and period in line with the research questions.

AIMS AND OBJECTIVES OF STUDY/MOTIVATION Overall aim: Evaluate the key factors/project approaches including community dynamics that were responsible for the successes or failures of project implementing districts; thus generate findings and recommendations that can be replicated and used in similar future projects with greatly improved outcomes. Specific objectives: Assess to what extent the project objectives have been achieved from the beneficiaries’ point of view. • • • •

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Assess the role of institutions in the project implementation Determine the current project status with respect to sustainability. Assess the level of community empowerment as a result of the project. Assess levels of project integration and capacity building of local government social service departments that are stakeholders in early childhood care and development. Assess the effectiveness of project implementation strategies with respect to project continuity and community participation and project ownership.

INTRODUCTION AND DESCRIPTION OF THE UGANDA NECDP The overall goal of the Project is to improve the quality of life for Ugandan children less than six years of age. The purpose is to improve the nutrition, health cognitive and psychosocial development of the children covered in the project areas. The Nutrition and Early Childhood Development Project (NECDP) Uganda chapter has just gone through its first phase. It is a robust programme initiated in 25 districts of Uganda with massive fund inflow for its implementation. The initial phase is 5 years after which more districts shall be added to

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increase the coverage nation wide. As its name suggests the NECD Project has been in the agenda of many international development organisations especially, UNICEF, UNESCO, and the WORLD BANK which indeed funded part of the project through the IDA project grants. Uganda is still faced with poor social indicators. This is reflected by the Infant Mortality Rate (IMR) of 97 deaths per 1000 live births, and the Maternal Mortality rate (MMR) of 506 per 100,000 live births. Chronic malnutrition (stunting) is high at 38% and the prevalence of acute malnutrition (wasting) is also high 5%. NB: The 2003 estimate from UNICEF country information show that the figures have not changed much in any case: IMR, 81; MMR, 510; Stunting, 39% and acute malnutrition is still at 5%. The NECD project of Uganda most certainly traces its conception from the Early Childhood Development (ECD) paradigm which has gained a strong foothold in poverty alleviation and development speaks of international organisations. The chronology of ECD in Africa is well illustrated in; all these developments show the need to improve on child well fare in order to prepare them for better life as adult and consequently participate meaningfully in poverty reduction in society. ECD is emerging as an independent development concept in its own right; thus policy formulations are in progress in many countries both developed and developing. The importance of ECD is further seen in the sustained spending that the World Bank has given to it. RESEARCH PROBLEM Significant number of development projects in developing countries with noble intentions to improve the lives of poor people especially children, women, the disadvantaged etc are faced with daunting tasks to prove that the projects have indeed achieved their goals and objectives. The Uganda NECDP is not any different. Poor link of project goals and objectives to the local situation and inappropriate approaches to underlying causes of the problems being tackled is a major obstacle to project success. These have been enumerated in a number of project literatures (A: 1, 11, & 12). Therefore: Many current approaches and paradigm in promoting community nutrition, health and other child development features are not producing convincing outcome. RESEARCH QUESTIONS • • • • • •

How effective have been the approaches used to implement the project? What are the critical local conditions (enabling characteristics) that stand to promote the success and sustainability of the project? How have project objectives and inputs strengthened local capacity to ensure continuity? How have project features been integrated in the local setting to promote sustainability? What are the strengths of local institutional establishments, and what level of participations have they been involved or played? How committed are the local communities to the project; in other words, what is the level of project ownership? What determines this ownership?


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JUSTIFICATION OF THE STUDY •

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•

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Early childhood projects are very important contemporary development activity; this study is set to lay emphasis on what works and what doesn’t; i.e. what is the role of the community versus project approaches in eradicating the root causes of poor children’s growth indicators. A qualitative explanatory, and programme effect case study of this nature is bound to bring out a rich, ‘thick description’ of the project and increase knowledge on previous evaluation studies. The study shall also help in justifying the use of huge funds in such projects; a detail study is the only way that can bring this to the fore. This research shall do nothing less in shedding light on what made other districts fair better than others. The research shall highlight key factors that are responsible for empowering communities to own development projects and promote continuity and sustainability.

LITERATURE REVIEW Literature search shall be guided by knowledge assumption (epistemology) of the study; in this case an interpretive philosophy in which phenomena are understood through the meaning people assign them, i.e. the social constructions such as language, consciousness and shared meaning. Thus literature that supports the use of the study design and the underlying principles of the study shall be sourced. THE RESEARCH METHOD AND DESIGN: This study shall be conducted using the Programme effects, explanatory qualitative case study method/approach, Sampling: Purposeful selection of study areas; and the target respondents are the beneficiaries and key informants. Methods of collecting empirical materials (data) shall be the followings: • • • • •

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Interviews with key informants. Focus group discussions with project beneficiaries in all the selected districts. Focus group discussions with the district project monitoring team. Field observation reports by the researchers direct field assessments of project activities. Data from project documents i.e. publication and reports from implementing agencies, district authorities, project steering committee, World Bank, and relevant government ministries. Archival records on the project and other related fields.

Data management of all field reports, interviews etc shall be profile thematically and chronologically by topic using Microsoft word programme. Analysis of empirical materials (data) is based on ‘thematic network analysis aided by Atlas.ti computer software for qualitative data analysis. Maintaining

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validity of the study under Qualitative research, the trustworthiness model of Lincoln and Guba’s shall be implemented. Finally the last phase of the study, shall be to test the developed emerging themes and or hypotheses i.e. in form of recommendations in selected location and outcomes evaluated Ethical Considerations: all the required procedures to ensure the integrity of the study shall be implemented. RESEARCH SCHEME/TIMED OBJECTIVES SCHEDULE: First Year: Completion and confirmation of the research concept protocol (protocol); a detail plan and an intensive literature review plus preparation of instrumentations for the fieldwork. Completion of a draft introduction to the thesis structure and the beginnings of a chapter of research procedures. Initial phase of data collection. Second year: Completion of data collection and analysis of Phase I field work with established methodologies for doing so. Draft at least two of literature chapters including an additional on research methodology. Plan for Phase II of the project is set in motion and a longitudinal study is effected in the implementation of field recommendations. Third year: Completion of Phase II of the field research by the end of third year and organising of the draft field report. Completion of data and field report analysis of Phase II of the project and satisfactory internalisation of relevant literature. Completed chapter on research procedures and methodology; establishment of emergent themes and detailed plan for data analysis chapters. This should culminate to the production of the first draft of the thesis; this already is within fourth year. Fourth year: Last corrections and tying of loose ends and production of the final thesis; planning for the thesis defence and then graduation.


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IMPLEMENTATION AND OPTIMISATION OF THE WATER FRAMEWORK DIRECTIVE EXPLORER FOR RIVER MANAGEMENT IN FLANDERS Annelies MAES, Peter L.M. GOETHALS & Niels DE PAUW Laboratory of Environmental Toxicology and Aquatic Ecology, Ghent University Jozef Plateaustraat 22, BE-9000 Gent, Belgium

INTRODUCTION The main objective of the WFD-Explorer is to support water managers with the development of river basin management plans; in particular to support them with respect to the determination of cost-effective strategies for the realization of ecological objectives (Van Der Most et al., 2006). Implementation of this Dutch tool on a Flemish sub basin is part of an optimisation and validation study in an international context. MATERIAL AND METHODS The WFD-Explorer The challenge for the development of the WFD-Explorer was to account for the ‘simplicity’ and transparency required in the policy process while at the same time acknowledging the complexity and fuzziness of ecological processes. The required swift response of the WFD-Explorer excludes the use of complex models. Hence the WFD-Explorer includes a simplified description of the hydromorphology and water quality of the water bodies within a river basin. In most cases the impact of measures on the ecological quality of water bodies is determined using intermediate ‘steering variables’. These steering variables make up the habitat for a particular ecological quality. The most important components of the WFD-Explorer are the knowledge rules for the relationships between measures and steering variables as well as for the relationships between steering variables and ecological quality (Figure 1). The knowledge rules for ecological quality describe how the ‘scores’ for the four different biological quality elements (phytoplankton, macro phytes, macro fauna and fishes) depend on the values of the steering variables. These steering variables are described in classes. The ecological quality is assessed through comparison of the habitat variation of the steering variables with the habitat requirements of the indicator species in reference situations. Knowledge rules have also been derived for a number of different types of measures. These knowledge rules describe how characteristics of the water system or steering variables will change because of the execution of particular measures (Van Der Most et al., 2006).

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Figure 1. Conceptual model of the WFD-Explorer (Van Der Most et al., 2006)

The WFD-Explorer is meant for analyses on the level of a river (sub) basin. Such a river (sub) basin consists of a number of connected water bodies and their associated drainage areas. For each of these water bodies simple balances of water and substances are determined as well as some hydromorphological characteristics. Application of the WFD-Explorer in the Flemish sub basin ‘De Burggravenstroom’ The sub basin De Burggravenstroom is located in the North of the region East-Flanders. It belongs to the basin ‘Canals of Ghent’ and is called after the central watercourse De Burggravenstroom. The study-area is strongly influenced by the drinking water-collection activity in Kluizen. Next to this, there is also a general occurrence of agriculture. Many stakeholders have to be taken in account and the impacts of certain activities and restoration actions are not always clear. This makes it an interesting region to test the possibilities of the WFD-Explorer. The application of this tool on a new sub basin needs various inputs. First of all the water bodies and their associated drainage areas must be defined. The co-ordinating committee ‘Integrated Water Policy’ (CIW) is authorized with the marking of the Flemish water bodies (catchment area > 50 km2). Information concerning the local water bodies of the first (catchment area: 10 and 50 km2) and second order (catchment area < 10 km2) has been obtained from the Flemish Environmental Agency (VMM) and local water managers of East-Flanders. Then, hydrological scenarios have to be determined. In this area, with a strongly influence of drinking water captation, has been chosen to make at least a difference between the period of captation and the period of no captation. Characteristics of the water bodies and drainage areas for these scenarios are necessary the modelling of the water balance (and substances) in the


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WFD-explorer. Fixed characteristics will not differ between the scenarios and include the length, the area, the bank profiles, the presence of weirs, etc. These data were mainly obtained from the department water of the Administration Environment, Nature, Land and water management (Aminal) and the provincial management of East-Flanders. Also variable characteristics like rainfall, vaporisation and drain data can be delivered by the former instance. The presence of wastewater treatment plants and sources of pollution in the area are also included in these balances based on information provided by Aquafin and the Flemish Environmental Agency. RESULTS AND DISCUSSION Data is being collected from the different instances and the digitalisation of the area is proceeding. Ten water bodies are identified in the study area: seven Flemish water bodies and three local water bodies of first order. The local water bodies of the second order are considered as drainage areas, since the catchments are too small to identify them as water bodies in the WFD-explorer. The most important measures for the study area are sorted out the sub basin management plan. An overview of the general solutions is given in Table 1. The last column shows the similarity with the measures implemented in the prototype of the WFD-Explorer. The ecological effects of these measures will be tested for different water bodies with the WFD-Explorer in the context of this pilot study in Flanders. Different intensities (and related investments) will be analysed to select the most convenient set of management options. Table 1. Overview of the general solutions

CONCLUSION The WFD-Explorer offers to water managers and stakeholders the opportunity to learn about the relationships between the objectives pursued, the measures that might be taken and the impacts of such measures (including costs). It will give a joint frame of reference with respect to effectiveness and attractiveness of measures in realizing ecological objectives (Van Der Most et al., 2006). In this context this decision support system opens interesting

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perspectives for the complex water management in Flanders related to meeting the WFD objectives by 2015. ACKNOWLEDGEMENTS The WFD-Explorer is being developed in the Netherlands by a consortium of water management organizations, research institutes and consultancy firms. The software tool development is co-ordinated by WL Delft Hydraulics. This optimization and validation project is co-ordinated by RIZA and sponsored by the research program ‘Living with water’.

REFERENCES Van Der Most, H.; Jeuken, A. & Van Schijndel, S. (2006). WFD-Explorer: a planning kit to support implementation of the Water Framework Directive. Paper presented at HIC-conference: 7th International Conference on Hydro informatics, Nice, France.


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TEMPERATURE ALTERS THE FUNCTIONAL RESPONSE OF THE PREDATORY INSECT PICROMERUS BIDENS 1

K. MAHDIAN1,2, L. TIRRY1 & P. DE CLERCQ1 Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium 2 Department of Crop Protection, Faculty of Agriculture, Vali-E-Asr University of Rafsanjan, Rafsanjan, Iran

INTRODUCTION Biological control of arthropods in agriculture often involves the use of exotic rather than indigenous natural enemies, either because the target pest (either native or exotic) lacks effective local natural enemies, or because exotic natural enemies are known to be effective. Although a wide range of arthropod biocontrol agents are commercially available for augmentative releases in numerous crops in Europe, about half of the marketed species are not native to the area of release. The introduction of an exotic natural enemy into an agro-ecosystem may however entail environmental risks. The exotic natural enemy may cause negative effects on non-target species and the ecosystems in which these species function, especially when the exotic natural enemy is a generalist. Therefore, investigation of the potential of the native natural enemy species may yield alternative solutions preventing negative effects associated with the release exotic natural enemies. Members of the pentatomid subfamily Asopinae are are predators attacking mainly larvae of Lepidoptera and Coleoptera, plus many other insect species, in various agricultural and forest ecosystems (Thomas, 1992; De Clercq, 2000). The spined soldier bug, Podisus maculiventris (Say) is the best known species of this subfamily. This predator is indigenous throughout North America. There have been multiple introductions of this species in Europe since the 1930s for classical biological control of the Colorado potato beetle, Leptinotarsa decemlineata (Say) but the predator never established. In the late 1990s, interest in the species was renewed for augmentative biocontrol of caterpillar pests in greenhouses (De Clercq, 2000). The main risks associated with the use of P. maculiventris are related to its wide host range and potential of establishment. As a result, several European countries have decided to no longer allow this species for augmentative release. Therefore, the availability of alternatives for P. maculiventris may be important for effective biocontrol of several insect pests including lepidopteran, hymenopteran and coleopteran defoliators. Picromerus bidens L. is a predatory pentatomid that is widely spread in the western Palearctic region and that also occurs in North America. It is a highly polyphagous bug that feeds mainly on leaffeeding larvae of the Lepidoptera, Coleoptera and Hymenoptera. This stinkbug has received some attention in the literature, but information regarding its predation capacity is still scarce. Arguably, successful field performance of a biocontrol agent also relies, in part, on a better understanding of its predation ability. This study investigates functional response of P. bidens to densities of noctuid pest, Spodoptera exigua (Hübner), as affected by temperature. The beet armyworm, S. exigua, is an economically important pest in numerous crops worldwide. In Northern Europe, the beet armyworm spo-

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radically infests glasshouse cultures and has been reported to cause considerable damage to a variety of vegetable and ornamental crops (De Clercq & Degheele, 1994; van der Linden, 1996). MATERIAL AND METHODS A culture of P. bidens was started with eggs originating from a laboratory colony at the Department of Entomology and Biological Control, All-Russian Research Institute for Plant Quarantine, Moscow, Russia. P. bidens used in this study was in the 2nd generation of laboratory rearing at Ghent University. The food of stock colonies of both predators consisted mainly of larvae of the greater wax moth, Galleria mellonella L. and larvae of the cotton leafworm Spodoptera littoralis (Boisduval). The functional response of female adults of P. bidens to the density of fourth instars of S. exigua was measured in the laboratory at three temperatures (18, 23 and 27 ± 1°C). The experiments were performed in plexiglas cylinders (25 cm high, 17 cm diameter), the top of which was covered with fine mesh screen. Each cylinder contained a single potted green bean plant, Phaseolus vulgaris cv. Prelude (ca. 20-25 cm tall). Six densities of fourth-instar beet armyworms were used, ranging from 1 to 24 individuals per plant arena. Prey were distributed over the bean plants to obtain the required density and a single starved female predator (3 to 4 weeks old) was introduced into each cylinder. The experiments were conducted in growth chambers set at the respective temperatures, a relative humidity of 65 ± 5 % and a photoperiod of 12:12 (L:D) h. The experiments were replicated 9 times simultaneously and controls (consisting of arenas without a predator) were used for each density. Killed prey were not replaced during the experimental period. After 24 h, the total number of live and dead prey was counted. Statistical analyses were performed using the SAS statistical package (SAS Institute 1989). A logistic regression of the proportion of prey killed and prey density was used to determine the shape of functional responses. A nonlinear least squares regression of number of prey eaten versus number offered was used to estimate and compare parameters of functional responses. Functional response data were fitted to the random predator equation

N e = N 0 {1 − exp [a (Th N e − T )]} where Ne = number of prey eaten, N0 is the initial number of prey, a = attack constant, T = total time available, and Th = handling time per prey (Rogers, 1972; De Clercq et al., 2000). RESULTS AND DISCUSSION On bean plat arenas, P. bidens females displayed high predation rates on fourth instars of S. exigua. Logistic regression analyses indicated that the type of functional response shown by P. bidens females changed as a function of temperature and suggested a type II functional response at 18 and 23 °C. A type III functional response is suggested by the outcome of the logistic regression at 27 °C. Likewise, P. maculiventris and the Neotropical species


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Podisus nigrispinus (Dallas) had a type II response at 18°C and a type III response at 27°C (Mohaghegh et al., 2001). The relationships between temperatures, handling times and attack rates show that the functional responses of female adults of P. bidens were greatly affected by temperature. Based on asymptotic 95% confidence intervals, the attack rates of P. bidens were similar at 18 and 23 °C, but handling times differed significantly among temperatures. The coefficients of determination (r²) indicated greater variation in predation at 18 °C than at 23 and 27 °C. Nonlinear regression yielded a reduced type III model at 27 °C, in which attack rate was a linear function of initial prey density. In a laboratory environment, P. bidens has demonstrated high predation capacities against noctuid caterpillars within a wide range of temperatures (18-27 °C). The performance of this Palearctic species compares well to that of the Nearctic pentatomid P. maculiventris, which is a species with a widely recognized biocontrol potential (De Clercq, 2000). Picromerus bidens may thus have potential for augmentative release in biological control programmes targeting lepidopteran defoliators. REFERENCES De Clercq, P. (2000). Predaceous stinkbugs (Pentatomidae: Asopinae). In: C.W. Schaefer and A.R. Panizzi (eds.). Heteroptera of economic importance. Boca Raton, FL, CRC Press, pp. 737-789. De Clercq, P. (2002). Dark clouds and their silver linings: exotic generalist predators in augmentative biological control. Neotrop. Entomol., 31, 169-176. De Clercq, P. & Degheele, D. (1994). Laboratory measurement of predation by Podisus maculiventris and P. sagitta (Hemiptera: Pentatomidae) on beet armyworm (Lepidoptera: Noctuidae). J. Econ. Entomol., 87, 76-87. De Clercq P., J. Mohaghegh and L. Tirry, 2000. Effect of host plant on the functional response of the predator Podisus nigrispinus (Heteroptera: Pentatomidae). Biol. Control 18: 65-70. Mohaghegh, J., De Clercq, P. & Tirry, L. (2001). Functional response of the predators Podisus maculiventris (Say) and Podisus nigrispinus (Dallas) (Het., Pentatomidae) to the beet armyworm, Spodoptera exigua (Hübner) (Lep., Noctuidae): effect of temperature. J. Appl. Entomol., 125, 131-134. Rogers, D.J., 1972. Random search and insect population models. J. Anim. Ecol. 41: 369-383. Thomas , D.B. (1992). Taxonomic synopsis of the asopine Pentatomidae (Heteroptera) of the Western Hemispher. Thomas Say foundation Monographs, Vol. 15. Entomological Society of America, Lanham, Maryland, U.S.A. 156 pp. van der Linden, A. (1996). Control of caterpillars in integrated pest management. IOBC WPRS Bull., 19, 91-94. van Lenteren, J., Babendreier, D., Bigler, F., Burgio, Hokkanen, G. H.M.T., Kuske, S., Loomans, A.J.M., Menzler-Hokkanen, I. van Rijn, P.C.J., Thomas, M.B., Tommasini, M.G. & Zeng, Q.Q. (2003). Environmental risk assessment of exotic natural enemies used in inundative biological control. BioControl, 48, 3-38.

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ISOLATION AND CHARACTERIZATION OF A MADS-BOX TYPE GENE FROM APPLE (MALUS X DOMESTICA) N. MAHNA1, 2, 3, R. DREESEN1, B. BAGHBAN KOHNEH ROUZ2, B. GHAREYAZIE3, M. VALIZADEH2, V. GRIGORIAN2 & J. KEULEMANS1 1 Lab for Fruit Breeding and Biotechnology, Center for fruit Culture (R&D Division, KULeuven), W. de Croylaan 42, Heverlee, B-3001, Belgium 2 Department of Agronomy and Plant Breeding, Faculty of Agriculture University of Tabriz, 51664, Tabriz, Iran 3 Department of Genomics, Agricultural Biotechnology Research Institute of Iran (ABRII) Seed and Plant Improvement Institutes Campus P.O. Box 31535-1897, Mahdasht Road, Karaj, Iran

INTRODUCTION Apple tree (Malus x domestica) has an extended juvenile phase of several years, during which vegetative growth is maintained. This characteristic is recognized as a disadvantage in breeding and stable annual production. Therefore it is necessary to gain an understanding of the genetic mechanisms underlying transition from vegetative to reproductive phase. Unraveling the molecular mechanisms of apple flowering would open the way for understanding and manipulating juvenility, which in turn can be applied in other fruit trees as well, especially those belonging to the Rosaceae family. Different flowering genes have already been identified and characterized in apple. Some are as shown in table 1. Table 1. A brief summary of flowering genes already isolated and characterized from apple. Apple gene MdMADS1 MdMADS2 MdMADS3 MdMADS4 MdMADS5 MdMADS10 MdCOL1 and MdCOL2 MdPI MdTFL

Arabidopsis homologue SEP SQUAMOSA SEP SEP AP1 AG CO PI TFL1

Reference Sung and An, 1997 Sung et al. 1999 Sung et al. 2000 Sung et al. 2000 Yao et al., 1999 Yao et al., 1999 Jeong et al., 1999 Yao et al., 2001 Kotoda et al., 2003

In this paper, we report the discovery of an additional MADS-box type gene (Malus x domestica SOC1, MdSOC1), which is a member of the relatively poorly characterized SOC1/TM3 class of MADS-box genes. MATERIALS AND METHODS Apple EST sequence data were downloaded from the Genome Database for Rosaceae (http://www.mainlab.clemson.edu/gdr/) and the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST/). Apple ESTs similar to the SOC1 gene (GenBank accession numbers: CV998019; CV880272; CV657904; CO722859) were identified and assem-

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bled to make a contig. An ORF translation was made which showed a strong homology with the SOC1 protein sequence. Translation and protein alignments were performed with ClustalX (Thompson et al., 1994) and other software. Primers (sense: 5’-AAGAGAGTGGGCATCCACAA-3’; antisense: 5’GAATGTTGACTTTAGACTGC-3’) designed from the MdSOC1 contig so that the amplified fragment would include the ORF. Young leaves of apple trees growing in the greenhouse were collected, quickly frozen in liquid nitrogen and stored at -80°C until further use. RNA was extracted according to the LiCl-protocol (Sambrook et al., 1989). In this protocol a DNase-treatment is included to eliminate possible contaminating DNA in preparation for the Reverse Transcription (RT) reactions. The reverse transcription reaction was carried out in 20 µl reaction volume following the protocol supplied with SUPERSCRIPT II reverse transcriptase kit (Invitrogen). The resulting cDNAs were used directly as templates for PCR. PCR conditions were as follows: 2’ 94°C, followed by 32 cycles of 94°C (30”), 55°C (30”) and 72°C (1’), ending with final extension at 72°C for 10’ and using Platinum® Taq DNA Polymerase (Invitrogen). The resulting fragment was cloned into the pGEM® -T Easy vector (Promega, Madison, WI) and was transformed into DH5α-type competent cells. The fragment sequenced using the M13 universal primers. The resulting sequence data for the coding region have been deposited in the GenBank database under accession number DQ846833. RESULTS AND DISCUSSION MADS-box proteins are transcription factors that control a diverse range of developmental processes in plants (Becker and Theissen, 2003). They are characterized by a highly conserved N-terminal DNA-binding domain termed the “MADS-box”. The MADS-box gene family contains more than 100 members in Arabidopsis and comprises of five major clades, of which only one, the so-called “MIKC” class, has been the subject of significant functional analysis (Becker and Theissen, 2003). SOC1 is a member of this “MIKC class” that integrates flowering signals from the photoperiod, vernalization, and gibberellin pathways (Borner et al.2000; Lee et al., 2000) (Figure 1).


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Figure 1. Arabidopsis genes regulating flowering time and development (Lemmetyinen, 2003)

Therefore it could be of importance to be able to manipulate the expression of this gene to change the time of flowering and growth habit. Here, we report the isolation of a Malus x domestica homolog of the SOC1 (MdSOC1) gene. The resulting sequence contains a part of 5’ UTR, a coding sequence and a part of 3’ UTR. BLAST homology searches of the coding region of this gene revealed a high homology with the SOC1 protein from Arabidopsis. The coding region contains a K-box named so because of its structural similarity to the coiled-coil domain of Keratin (Ma et al. 1991, Theissen et al. 1995). The K-box domain mediates protein-protein interactions of the MADS-box proteins to form homo- or heterodimers facilitating DNA-binding to other regulatory factors (Ma et al. 1991, Pnueli et al. 1991, Theissen et al. 1995). Furthermore, a conserved domain of SOC1, the so-called “SOC1 motif” (Nakamura et al. 2005, Vandenbussche et al. 2003) has been identified in the C-terminal region. The similarity between the MADS-box of MdSOC1 assembled contig and SOC1 protein from Arabidopsis is 85.7% while that of K-box is 71.8% and C-terminal SOC1 motif is 75%.The resulting sequence had a gap at 5’ of the coding region comparing to the contig, and consequently, it had just one quarter of MADS box (Figure 3). This gene is probably a 5’-truncated form of the complete gene which could be the result of an alternative splicing event as a way to regulate functionality. Expression analysis of this gene showed that it is expressed in young leaves but not in the terminal buds (Figure 2).

Figure 2. Expression pattern of MdSOC1 in terminal buds and young leaves. Actin was used as an internal control.

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More than one-fifth of plant genes are estimated to be alternatively spliced (Wang and Brendel, 2006). However, how to distinguish erroneous splicing from functional events is yet to be resolved. The coding region of the isolated (possibly exposed to alternative splicing) MdSOC1 has still an ATG start codon in its ORF and could be translated as a protein containing K-box and C-terminal motif. Nevertheless, its functionality is not known and it could be concluded that this probable alternative splicing event could exert a special functional role for silencing of the gene or for assigning a new function to it. Further experiments are yet to be carried out to isolate the complete gene as well as to elucidate its possible function.

Figure 3. Alignment of the amino acid sequence of the isolated MdSOC1 against the assembled contig of EST sequences and the SOC1 protein of Arabidopsis. As it is clear, there is a high homology between these sequences. A region at the 5’ terminus of the coding sequence, which is a part of the MADS-box consensus, is lacking as the result of an alternative splicing event.

ACKNOWLEDGEMENTS The authors wish to thank the Iranian Ministry of Science, Research and technology for granting a visiting scholarship to N.M.

REFERENCES Becker A, Theissen G (2003) The major clades of MADS-box genes and their role in the development and evolution of flowering plants. Mol Phyl Evol 29:464-489. Borner R, Kampmann G, Chandler J, Gleissner R, Wisman E, Apel K, Melzer S (2000) A MADS domain gene involved in the transition to flowering in Arabidopsis. Plant J 24:591–599. Jeong DH, Sung SK, An G (1999) Molecular Cloning and Characterization of CONSTANS-Like cDNA Clones of the Fuji Apple. Journal of Plant Biology 42(1): 23-31.


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Kotoda N, Wada M, Masuda T, Soejima J (2003) The break-through in the reduction of juvenile phase in apple using transgenic approaches. Acta Horticulturae 625: 337343. Lee H, Suh S, Park E, Cho E, Ahn JH, Kim S, Lee JS, Kwon YM, Lee I (2000) The AGAMOUS-LIKE20 MADS domain protein integrates floral inductive pathways in Arabidopsis. Genes Dev 14:2366–2376. Lemmetyinen J (2003) The birch genes BpMADS1 and BpMADS6 and their use in the modification of flowering. PhD Dissertation in Biology, University of Joensuu, 85 pp. Ma H, Yanofsky MF, Meyerowitz EM (1991) AGL1-AGL6, an Arabidopsis gene family with similarity to floral homeotic and transcription factor genes. Genes Dev 5: 484495. Moon J, Suh SS, Lee H, Choi KR, Hong CB, Paek NC, Kim SG, Lee I (2003) The SOC1 MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis. Plant J 35:613–623. Nakamura T, Song I, Fukuda T, Yokoyama J, Maki M, Ochiai T, Kameya T, Kanno A (2005) Characterization of TrcMADS1 gene of Trillium camtschatcense (Trilliaceae) reveals functional evolution of the SOC1/TM3-like gene family. J Plant Res. 118(3): 229-234. Pnueli L, Abu-Abeid M, Zamir D, Nacken W, Schwarz-Sommer Z, Lifschitz E (1991) The MADS box gene family in tomato: temporal expression during floral development, conserved secondary structures and homology with homeotic genes from Antirrhinum and Arabidopsis. Plant J 1(2): 255–266. Sambrook J, Fritsch EF, and Maniatis T (1989) Molecular Cloning, a Laboratory Manual. 2nd Edition, Cold Spring Harbor, Cold Spring Harbor Laboratory Press Sung SK, An G (1997) Molecular cloning and characterization of a MADS-box cDNA clone of the Fuji apple. Plant Cell Physiol 38(4): 484-9. Sung SK, Yu GH, An G (1999) Characterization of MdMADS2, a Member of the SQUAMOSA Subfamily of Genes, in Apple. Plant Physiol. 120: 969-978. Sung SK, Yu GH, Nam J, Jeong DH, An G (2000) Developmentally regulated expression of two MADS-box genes, MdMADS3 and MdMADS4, in the morphogenesis of flower buds and fruits in apple. Planta 210(4): 519– 528. Theissen G, Strater T, Fischer A, Saedler H (1995) Structural characterization, chromosomal localization and phylogenetic evaluation of two pairs of AGAMOUS-like MADS-box genes from maize. Gene 156:155-166 Thompson JD, Higgins DG, Gibson TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionsspecific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. Vandenbussche M, Theissen G, Van de Peer Y, Gerats T (2003) Structural diversification and neo-functionalization during floral MADS-box gene evolution by Cterminal frameshift mutations. Nucleic Acids Res 31:4401–4409. Wang BB and Brendel V (2006) Genomewide comparative analysis of alternative splicing in plants. PNAS 103: 7175 - 7180. Yao JL, Dong YH, Karnheden A, Morris B (1999) Seven MADS-box genes in apple are expressed in different parts of the fruit. J Amer Hort Sci 124: 8-13. Yao J, Dong Y, Morris BA (2001) Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADS-box transcription factor. PNAS 98(3):1306-11. Abbreviations: AG, AGAMOUS; AP1, APETALA1; CO, CONSTANS; EST, Expressed Sequence Tag; MdMADS1, Malus domestica MADS 1; PI, PISTILLATA; MdPI, Malus domestica PI; PTM5, Populus tremuloides MADS-box 5; RT, Reverse Transcription; SEP, SEPALLATA; SOC1, SUPPRESSOR OF OVEREXPRESSION OF CO 1; TM3, TOMATO MADS 3; Malus domestica TFL (MdTFL); TFL1, TERMINAL FLOWER 1.

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INFLUENCE OF TURGOR ON MICROMECHANICAL PROPERTIES OF APPLE TISSUE

1

M.L. OEY1, E. VANSTREELS1, M.C. ALAMAR2, J. DE BAERDEMAEKER1, O. SCHLÜTER3 & B.M. NICOLAI1

Laboratory/Flanders Centre of Postharvest Technology, BIOSYST-MeBioS, Catholic University of Leuven, W. De Croylaan 42, B-3001 Leuven, Belgium 2 Instituto Valenciano de Investigaciones Agrarias (IVIA), Ctra.Moncada-Náquera, Km.4.5, 46113 Moncada (Valencia), Spain 3 Department of Horticultural Engineering, Leibniz-Institute for Agricultural Engineering Potsdam-Bornim, Max-Eyth-Allee 100, D-14459 Potsdam, Germany

INTRODUCTION Texture, one of the primary quality attributes of fruits and vegetables, is affected by ripening stage as well as mechanical damage during harvest, transport and storage, which leads to a loss of commercial value. For evaluation of mechanical properties fruits are typically seen as a continuous material, but fruit tissue is in fact a complex conglomerate of cells, whose structural features (cell wall properties and middle lamella, intercellular spaces, internal turgor pressure) determine its mechanical strength. A micromechanical approach is thus preferable to understand the relative importance of these features on the overall mechanical behaviour of fruits. Tissue strength is thought to be largely influenced by cell turgor. It can be manipulated by soaking fruit discs in solutions of different osmotic concentrations. In general, turgor pressure increases by water uptake after incubation in solutions that are less concentrated than the cell sap, which can be measured by changes in weight and volume (Lin and Pitt, 1986). Influence of turgor on mechanical parameters of fruit or vegetable tissue has been investigated by mainly uniaxial compression tests. For potato a decrease in failure force and stress was found with increasing turgor (Lin and Pitt, 1986; Konstankiewicz and Zdunek, 2000), which was related to higher turgidity pre-stressing and weakening of the cell walls, making the tissue more brittle. In studies on apples, it has been shown that increased turgor resulted in tissue failure at lower stress and strain, while the initial modulus increased (Lin and Pitt, 1986; Scanlon et al., 1996). Reduced failure force, deformation and firmness with increasing turgor were also reported on tomato tissue and attributed to cell rupture as predominant mechanism of failure (Jackman et al., 1992). In tensile tests on pear specimen turgor did not affect force at failure in soft tissue, where cell-to-cell debonding at the middle lamella is the predominant failure mode. In firm pears the force necessary for tissue rupture increased with higher turgor suggesting stress-hardening of the cell wall (De Belie et al., 2000). The objective of this study was to investigate the influence of turgor pressure on micromechanical behaviour of soft and firm apple tissue in tensile and compression tests. In addition cell structural parameters and deformations were followed and recorded during mechanical testing.

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MATERIAL AND METHODS Samples Experiments were performed on fresh Jonagored apples and fruit stored under shelf life conditions (12 days at 18°C), selected according to their uniformity in size and firmness (average firmness of 79 N and 67 N respectively). Rectangular specimen (11 mm length x 5 mm width x 2 mm height for tensile tests; 3 mm x 11 mm x 5 mm for compression tests) were cut radially from the parenchyma using parallel razor blades. The isotonic point of fresh and shelf life apples was determined as described elsewhere (Lin and Pitt, 1986). To manipulate cell turgor samples were soaked overnight in isotonic (0,6 M), hypotonic (0,4 M) and hypertonic (0,8 M) mannitol solutions. Samples were stained 2 min with methylene blue (7,5 mg/100 ml) directly before testing to allow visualisation of the cells. Mechanical tests and image analysis Samples were tested in a miniature tensile stage (Deben Microtest, Suffolk, U.K.) mounted underneath a stereomicroscope (SMZ1000, Nikon, Japan) which was equipped with a CCD camera (type TK-1360B colour ½ inch CCD, JVC). This allowed to monitor the deformation of individual cells and to record simultaneously the corresponding force-displacement curves. In total 20 (fresh) or 15 (shelf life) samples coming from 3 respectively 2 apples were analysed. Tensile and compression tests were performed with a testing speed of 0,5 mm/min and 0,2 mm/min, respectively. For tensile tests samples needed to be glued to the grips of the miniature tensile stage by cyanocrilate adhesive. Beyond a certain travel 3 cyclic tests were carried out, and then samples were further elongated or compressed until rupture. Cell structural parameters (area, aspect, roundness, perimeter, length and width) were extracted from the recorded images, after a digitisation step in a specific Matlab program (MATLAB 6.5, The MathWorks, Inc., Natick, MA, USA), using Image-Pro Plus (Image-Pro Plus 4.5, Media cybernetics, Silver Spring, USA). Data Analysis Force-deformation data were converted to stress-strain data, from which the micromechanical parameters maximum stress (σmax), strain at failure (εmax) as well as the initial modulus (E1) and Young’s modulus at approx. 80 % stress (E2) were calculated. Statistical analysis was carried out using the general linear model procedure in SAS (SAS version 8, SAS Institute Inc., Cary, NC, USA). RESULTS AND DISCUSSION Typical stress-strain curves of apple tissue under tensile and compressive loading are displayed in Figure 1 and micromechanical parameters extracted from the curves are shown in Table 1.


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Compression Tests Stress-strain curves produced by compression tests are S-shaped, and stiffness changes with strain. For soft and firm apples the “lag-Phase” of the curve becomes longer with decreasing turgor, such that the S-shape is more pronounced, and curves are shifted to the right. This behaviour is reflected in the micromechanical parameters extracted from the curves.

firm 0.4M

soft 0.4M

firm 0.6M

soft 0.6M

firm 0.8M

soft 0.8M

Figure 1. Typical stress-strain curves of firm (fresh) and soft (stored) apple tissue with different turgor pressures

Turgor has significant influence on Young’s modulus for small strains (E1) in both firmness classes. Less turgid cells (soaked in 0.8M mannitol solution) have a smaller initial modulus than cells under full turgor, indicating that, at small strains, tissue stiffness increases with turgor. At higher strains no clear effect of turgor on Young’s modulus (E2) was found. Maximum strain values increase significantly with decreasing turgor. Soaking in hypertonic (0.8M) solutions causes the cells to release water and thus the tissue to lose its rigidity. Soaking in hypotonic (0.4M) solutions, however, provokes a shorter maximum strain as the cells expand by water uptake and the tissue becomes less compliant. Although it was reported that failure force decreases when turgor increases (Lin and Pitt, 1986), in this study no significant effect of turgor on maximum stress was found when compressing firm samples. In agreement with previous experiments carried out in our lab (M.C. Alamar, unpublished results), lower stress and strain values were found for stored apples. These differences were more pronounced for samples soaked in non-isotonic solutions. Tensile Tests Stress-strain curves of tensile tests show a similar behaviour of increasing lag-phase with decreasing turgor. Under high turgidity (0.4M), however, an almost linear relation with no lag-phase was found, indicating linear-elastic behaviour before tissue failure. Still, curves are shifted to the right when turgor decreases. Tensile stress and strain values were considerably lower than compressive stress and strain (note different scales in Figure 1).

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Effect of turgor on micromechanical parameters of fresh and stored apple tissue corresponds to its effect found in compression tests. With decreasing turgor initial modulus decreases and strain increases significantly. Also in tensile tests no significant effect of turgor on maximum stress was found. But in contrast with previous studies on pears (De Belie et al., 2000), failure force shows a slight trend to decrease with increasing turgor (0.8M). This could be explained by pre-stressing of cells by higher turgidity and thus enhancement of brittleness rather than stress-hardening. As in compression tests, no clear effect of turgor on Young’s modulus at higher strains (E2) could be observed. Unexpectedly, stored apples were found to have higher stress and strain values than in fresh apples, although penetrometer measurements indicated that stored apples were less firm. For samples soaked in hypotonic solution also stiffness increased after storage, while stiffness of samples from iso- and hypertonic solutions decreased. However, verification in additional experiments is necessary as samples were taken from only few apples and statistical analysis has shown that the apple itself has a significant influence on all measured mechanical parameters. Table 3. Means and standard deviations of micromechanical parameters of tensile and compression tests for apple tissue soaked in osmotic solutions

Compression

firm

soft

Tensile

firm

soft

c [M] 0,4 0,6 0,8 0,4 0,6 0,8 0,4 0,6 0,8 0,4 0,6 0,8

σmax [MPa] 0,532±0,05 0,524±0,06 0,551±0,087 0,305±0,07 0,488±0,08 0,316±0,02 0,175±0,06 0,189±0,07 0,223±0,06 0,180±0,07 0,228±0,05 0,247±0,06

εmax [%] 35,91±2,52 36,17±2,58 50,89±6,33 14,69±1,89 21,13±1,71 28,99±2,06 9,21±3,65 12,42±2,02 17,69±4,05 3,88±1,12 10,62±2,78 17,09±1,37

E1[MPa] 0,50±0,2 0,28±0,2 0,04±0,04 0,87±0,5 0,59±0,2 0,10±0,04 2,09±1,0 1,04±0,3 0,44±0,2 6,00±1,5 1,13±0,8 0,32±0,1

E2[MPa] 2,33±0,2 2,74±0,3 3,01±0,6 2,44±0,5 3,55±0,6 2,86±0,3 2,61±0,5 2,57±0,6 2,73±0,5 4,76±1,2 3,56±1,0 3,25±0,6

Further work comprising similar experiments on more apples will be undertaken in order to confirm the results obtained in this study. Image analysis is currently carried out and the influence of cell structural parameters on cell behaviour under deformation will be investigated with respect to different turgor pressures. ACKNOWLEDGEMENT The authors would like to acknowledge the Fund for Scientific Research Flanders (F.W.O. Vlaanderen), project G.0200.02 and the European Union (Training Site Marie Curie Grant to M. Oey, project G056).

REFERENCES De Belie N., Hallet I.C., Harker F.R., De Baerdemaeker J. (2000). Influence of ripening and turgor on the tensile properties of pears: A microscopic study of cellular and tissue changes. J. Amer. Soc. Hort. Sci., 125 (3), 350-356.


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Jackman R.L., Marangoni A.G., Stanley D.W. (1992). The effects of turgor pressure on puncture and viscoelastic properties of tomato tissue. J. Texture Stud., 23, 491505. Konstankiewicz K., Zdunek A. (2001). Influence of turgor and cell size on the cracking of potato tissue. Int. Agrophysics, 15, 27-30. Lin T.T., Pitt R.E. (1986). Rheology of apple and potato tissue as affected by cell turgor pressure. J. Texture Stud., 17, 291-313. Scanlon M.G., Pang C.H., Biliaderis C.G. (1996). The effect of osmotic adjustment on the mechanical properties of potato parenchyma. Food Res. Int., 29, 481-488.

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DEVELOPMENT OF A VARIABLE RATE FERTILISATION SYSTEM USING SOIL VIS-NIR SENSOR FOR PHOSPHORUS APPLICATION Mohammad REZA MALEKI, Abdul MOUNEM MOUAZEN, Herman RAMON & Josse DE BAERDEMAEKER Catholic University of Leuven, Department of Biosystems, Division of Mechatronics Biostatistics and Sensors (MeBioS) Kasteelpark Arenberg 30, BE-3001,Leuevn, Belgium E-mail: [email protected]

INTRODUCTION Variable rate (VR) phosphorous (P) fertilisation has economical, ecological and agronomical benefits. There are studies showing that VR fertilisation increases yield compared to uniform rate (UR) (Wittry and Mallarino, 2004; Yang et al., 2001). Two approaches exist for variable rate fertilisation application, namely map and sensor-based methods. The map-based approach includes sampling, laboratory reference analysis and development of maps to be used as input for VR fertilisation applicator. However, this method is of high cost and time required, since it is based on the traditional method of soil sampling and analysis. Furthermore, the use of a positioning system with error risks during data acquisition is the most critical feature of this method (Morgan and Ess, 1997). On the other hand, the lack of a robust onthe-go soil sensor for soil properties and calibration models valid for a wide geographical area has however, postponed the development of sensor-based VR method. The on-the-go visible (VIS) and near infrared (NIR) soil sensor developed and tested by Mouazen et al. (2005) showed promising results for the measurement of some soil properties (Mouazen et al., 2006). As a follow up of the on-the-go sensor development, research on development of VR application system of elemental P using the sensor-based VR approach is undertaken. This on-the-go soil sensor was able to acquire information about the soil fertility status over small distance intervals, which leads to a large amount of information available as input to vary the rate of fertilisers on VR applicators. The objectives of this study were to develop a VIS-NIR soil sensor based VR applicator to vary the rate of P application in a given field and compare the system to traditional UR application rate. MATERIALS AND METHODS A soil VIS-NIR sensor-based variable rate applicator was developed for P variable rate application. A fiber type Zeiss Corona spectrophotometer collected soil spectra through a soil sensor attached to the back of a subsoiler which was mounted at the front of a 3 m width row crop planter (AMAZONE; ED302) with a 4-75 fertilisation compartment. An electrical actuator changed the rate of application and could be controlled by the LabVIEW (National Instrument ®) program. A soil P model that already developed (Mouazen et al., 2006) was used to predict the ammonium lactate soil P (Pal) content while travelling across the field. A total of 186 soil samples were used to develop this model, which were divided into calibration (146 samples) and

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validation (30 samples) sets. These samples were collected from different fields in Belgium and northern France, representing a wide range of soil textures and colours. Each sample was a mixture of 25 soil cores taken randomly from each field. Collecting the samples during a relatively long period of time (5 months) resulted in samples with different moisture content and a wide range of soil Pal (min = 4.00, max = 108.00, mean = 38.75 and standard deviation = 22.37 mg/100g). The partial least squares (PLS) regression with full cross-validation was used to built the calibration model for Pal. This model predicts Pal with accuracy of R2 = 0.73 and 11.52 for root mean square error. Because, sometimes high variation in predicted P was observed for successive spots, a moving average of 10 samples was used to moderate the fluctuation. It should be noted that the process of averaging on successive samples within a window (an area of 3 m width and a length equals to the number of successive Pal being averaged) is updated with time so that the first spectrum is dropped out of the moving average and a new spectrum is added for the next calculation. Therefore, the average of some successive predicted P could be used instead of a single predicted one. The number of ten successive samples was considered as this was already optimised by Maleki et al., (2006). A model based on available recommendation table was developed to estimate the elemental P. Having predicted P and averaging 10 successive points, the elemental P was calculated. Each value was then classified in 40 categories in 5 kg/ha intervals from 0-200 kg/ha (Maleki et. al., 2006). For instance, for value 37 kg elemental P /ha, it was rounded as 35. This value was designated as applied elemental P. The penetration unit and soil sensor (Mouazen et al., 2006) was installed at 0.91 m ahead of the fertiliser applicator pipes (Figure 1). This distance was determined based on the delay time between triggering the applicator till having the proper rate at the end of the fertiliser delivery pipes. The field was divided to some strips and every other strips was used for variable rate (VR) and the rest for uniform P application.

Figure 1. The soil sensor-based variable rate applicator used in the research; the optical soil sensor attached at the front of the fertilizer applicator unit of the planter

A total number of 35 samples were taken and mixed based on traditional grid soil sampling for recommendation of UR elemental P application. The soil test revealed that 30 kg elemental P/ha had to be applied as UR application.


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A custom built LabVIEW program was used for data processing including filtering the noise and bad spectra, prediction soil P, estimation of the elemental P, controlling the rate of fertiliser application and recording the coordinates of the sample point using a DGPS (Trimble®). The planter was calibrated for maize sowing as 105000 seeds/ha. The applicator applied elemental P during planting based on the soil P variability. The application rate value of every spot was recorded and then the average of the total fertiliser consumption was compared with UR. The crop density and crop leaves were counted at three weeks after maize planting. For both VR and UR treatments 26 segment on each planted row with a length of 20 m were randomly selected and the maize plants and number of plant leaves were counted and subjected to statistically analysis. RESULTS AND DISCUSSION The total number of spectra was about 2200 in 1.2 ha. A total number of 150 soil spectra was approximately collected for each field travelling run resulting about one soil spectrum for each 6 m2. So for every 6 m2 the application rate could be adopted. The high variation of the P measured on-the-go could be observed in almost all rows in the field. Figure 2 and 3 show the predicted P normalized by the moving average of 10 and application rate maps for experimental field, respectively. The application rate for elemental P were 26.47 kg/ha. This shows about 3.5 kg/ha less application for variable rate P application.

Figure 2. Predicted phosphorus normalized by an moving average of 10 samples

Figure 3. Applied elemental P based on the 40 categories intervals from 0 to 200 kg/ha

The P variation in application varied from 0 to 95 kg/ha over all rows which shows the capability of the soil sensor and the prediction model of P. The average of VR application was less than UR application in 50% of the planting rows. It means that at least 50% of a field is being over applied using common method of fertilisers application. Although, the results of the plant density analysis was not significantly different (p=0.59) between two method

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of VR and UR however, the average of plant population was 3300 plants /ha more for VR approach. Results also showed that the application rate is less when a moving average of 10 is used compared to determination of application rate without using averaging scheme. CONCLUSION The measurement of soil P and application of elemental P simultaneously can play an important role in site specific management. Furthermore, the variable rate (VR) system can diminish the environmental impacts particularly for those countries which suffer from high application of phosphorus. As the soil P varies within a short field travelling, only a sensor-based VR can distribute proper application rate in different field spots. The application of P on field spots with lower P can increase the yield while less application for those spots with high level of P can reserve more fertilisers. As, the improper application of P contaminate environment year by year using sensorbased VR application can overcome the problem. REFERENCES Maleki, M.R., Van Holm, L., Merckx, R., Ramon, H., De Baerdemaeker, J. & Mouazen, A. M. (2006). Phosphorus sensing for fresh soils using visible and near infrared spectroscopy. Biosystems Engineering. in revision. Morgan, M.T. & Ess., D.R., 1997. The Precision Farming Guide for Agriculturalists. John Deere Publishing, Moline, IL Mouazen, A.M., De Baerdemaeker, J. & Ramon, H., 2005. Towards development of onthe-go soil moisture content sensor using a fibre-type NIR spectrophotometer. Soil & Tillage Research 80, 171-183. Mouazen, A.M., Maleki, M.R., De Baerdemaeker, J. & Ramon, H., (2006). On-the-go measurement of some selected soil properties using a VIS-NIR Sensor. Soil & Tillage Research, doi, 10.1016/j.still.2006.03.009 Wittery, D.J. & Mallarino, A.P., (2004). Comparison of uniform and variable rate phosphorus fertilization for corn-soybean rotations. Am. Soc. Agron. J., 96, 26-33. Yang, C., Everitt, J.H. & Bradford, J.M. (2001). Comparison of uniform and variable rate nitrogen and phosphorus fertilizer application for grain sorghum. Transaction of the ASAE 44(2), 201-209.


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MODELLING FRUIT MICROSTRUCTURE H.K. MEBATSION1, P. VERBOVEN1, Q.T. HO1, F. MENDOZA1, B.E. VERLINDEN2, T.A. NGUYEN1 & B.M. NICOLAÏ1,2 1 BIOSYST- MeBioS, Katholieke Universiteit Leuven W. De Croylaan 42, BE-3001 Leuven, Belgium 2 Flanders Center of Postharvest Technology W. De Croylaan 42, BE-3001 Leuven, Belgium

INTRODUCTION Materials with complex heterogeneous microstructure arise in a wide area of applications. They range from engineering materials such as ceramics, polymers and composite materials to biological materials including fruits and vegetables. The degree of the physical and transport properties of materials depend on the size, shape, and properties of the microstructure as well as their spatial distribution within the material matrix (Ghosh et al., 1996). Material stability, transport property, structural integrity or nutritional quality of fruits are the reflections of the assemblies of microstructures (cells, pores, cell walls) when embedded in, give rise to an engineered structure, which we call macrostructure (tissue and organ). Hence, defining and understanding microstructures is the key step forward to have a clear sight of the global material properties. In this respect, modelling materials structure and simulation of their behaviour is becoming a reliable tool to underpin scientific investigations and to complement traditional theoretical and experimental approaches (Ghoniem et al., 2003). Spatial geometrical models can treat a fruit as a continuum called macroscale or as an arrangement of discrete components termed as microscale. As a continuum, a geometric model of a material represents the heterogeneous properties of the tissue and the complex cellular structure as a black box. The continuum modelling approach is the classical engineering approach of modelling media with a complex microscopic geometry based on phenomenological approach in which transfer properties that appear in the macroscopic balances are determined experimentally. Although popular for their relative simplicity in representing physical complexities, these models lack critical information on the microstructural morphology. In microscopic modelling, components, which depend on the level of tissue organization, which usually go as deep as the material microstructures (cells, intercellular space, and cell wall in fruit tissues), are treated separately. The continuum mechanics principles at the microscopic level are employed to predict homogenized constitutive responses at the macroscopic level (Lee and Ghosh, 1999). The merits of this approach are that as we go down the scale, despite the difficulty of getting representative microstructural geometries, the physics of a process becomes easier and more understandable. Figure1 represents the two contrary factors, the geometry and the physics, in applying microscale modelling. The Figure explains the difficulty of constructing a representative geometry while the physics of the process becomes understandable and more meaningful and vice versa. Moreover, the homogeneous macrostructure described by the apparent continuum mechanics and the heterogeneous microstructures, the morphology consisting of distinguished cellular components, is defined.

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Figure 1. Schematic representation of complexity of macroscale vs. microscale modelling. a) macroscale model; b) microscopic image; c) TEM image of apple cells; d) TEM image of apple cell walls.

MATERIAL AND METHODS The study was based on images of fruit cellular structures obtained from light microscopy. Sample preparation and image acquisition procedures outlined by Mebatsion et al. (2006a) were followed. Samples from Conference pear parenchyma tissues were used. The geometrical characteristics of cells were estimated using moment calculations and moment based ellipse-fitting algorithms outlined by Mebatsion et al. (2006a). Voronoi tessellations were constructed from the geometric centres of cells, which we call them as Centroid based Voronoi diagrams (CVDs) (Mebatsion et al., 2006a). Voronoi tesselation algorithm divides spaces in to regions, called Voronoi cells by constructing the planes bisecting the line connecting two neighbour generating points resulting in a set of convex polygons in 2D embedding the points and their domain of attraction, completely filling up the underlying space. Microscopic fruit cell sections are elliptical (aspect ratio greater than one) (Mebatsion et al., 2006a; Schotsmans, 2003). The conic sections fitting to scattered data procedure (Fritzgibbon et al., 1999) generates the best-fit ellipse for the given set of points on the natural boundary of microscopic images. The ellipse tessellation geometry was generated from the ellipses, which were truncated when neighbouring areas overlap. By doing so, as many truncated ellipses as there are cellular images were generated filling the entire cellular space (for details refer to Mebatsion et al., 2006b). The spatial analysis of the microscopic and the virtual tissues were performed by expressing spatial arrangements in terms of their location with respect to one another by means of a “weighting function” (Brown, 1982; Odnald, 1988). In this study, the weighting function was defined as the inverse of the distance between the geometric centres (centroids) of two cellular regions. Local indicators of spatial association (LISA) tests were used to test the spatial variability of the microscopic image and its virtual counterparts.


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For details of the spatial tests of the microscopic and the virtual tissues, refer to Mebatsion et al. (2006b). From the individual Moran’s I values and that of the white random field a hypothesis of spatial significance can also be tested Mebatsion et al. (2006b). RESULTS AND DISCUSSION The microscopic and virtual parachymatic tissue of Conference pear is presented in Figure 2. The geometrical and spatial properties of the digitized fruit microstructure (Figure 2b) is more or less equal to that of the microscopic images as points were taken on the natural boundary of the cells. Nevertheless, the presence of sharp edges at the intersection of two neighbouring cells make the generation of finer meshes for finite element simulations difficult. On the other hand, Centroid based Voronoi diagram (CVD)(Figure 2c) could not produce a virtual microstructure that has similar geometrical properties and spatial characterstics. The ellipse tessellations (Figure 2d) produced virtual tissues that had similar geometrical parameters (area, aspect ratio and orientation) and spatial distribution as the microscopic image. The statistical Kolmogorov-Smirnov test proved that area, orientation and aspect ratio distributions of the micrograph and the ellipse tesselations were equal at 5% level of significance with p-values 0.99, 0.99 and 0.93 respectively . Figure 3 shows the area and the spatial distributions of the microscopic and the virtual tissues..

(a)

(c)

(b)

(d)

Figure 2. Pear fruit microstructure and its equivalent Voronoi tessellation. (a) Fruit microstructure; (b) digitized fruit microstructure; (c) Voronoi tesselation; (d) Ellipse tesselation.

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The area distribution of the microscopic image and the ellipse resselated virtual tissue are similar where as that of the Voronoi tesselation is far from the microscopic image (Figure3a). This trend is repeated for the local indicators of spatial association (LISA) distributions (Figure 3b).

Figure 3. Area and spatial distributions of microscopic and virtual tissues. a) area distribution; b) LISA distribution

CONCLUSION Virtual microstructures that could be used in finite element simulations were generated. The ellipse tesselation algorithm generates geometrically and spatially equivalent virtual tissue to the microscopic image. Such a tissue can be exported to finite element environment (such as Femlab) via interfacing Matlab code to perform in silico experiments in multiscale modelling of gas and moisture transport in fruits. Such microstructural model avoids a tiresome classical continuum modelling approach where transfer properties that appear in the macroscopic transport equation are determined experimentally. REFERENCE Brown, M. A.(1982). Modelling the spatial distribution of suburban crime. Economic Geography, 58(3), 247-261. Fitzgibbon, A.; Pilu, M.; Fisher, R. B.(1999). Direct least square fitting of ellipses. IEEE Trans. Pattern Anal. Mach. Intel., 21(5), 476-480. Ghoniem, N.M., Busso,E.P., Kioussis, N., Haung, H. (2003). Multiscale modelling of nanomechanics and micromechanics. Philosophical magazine, 83(31-34), 34753528. Ghosh, S., Lee, K., Moorthy, S. (1996). Two scale analysis of heterogeneous elasticplastic materials with asymptotic homogenization and Voronoi cell finite element model. Comput. Meth. Appl.Mech.Eng.132, 63-116. Lee, K., Ghosh, S. (1999). A microstructure based numerical method for constitutive modeling of composite and porous materials. Mater. Sci. Eng. A 272, 120-133. Mebatsion, H. K.; Verboven, P.; Verlinden, B. E.; Ho, Q.T.; Nguyen, T.A.; Nicolaï, B. M. (2006a). Microscale modelling of fruit tissue using Voronoi tessellations. Comput. Electron. Agric. 52, 36-48. Mebatsion, H.K., Verboven, P., Ho, Q.T., Mendoza, F., Verlinden, B., Nguyen, T.A., Nicolaï, B.M.(2006b). Modelling fruit microstructure using noble ellipse tessellation algorithm. CMES-Comp. Model. Eng. Sci. (in press).


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Odland, J.(1988). Spatial autocorrelation. Scientific geography series. Sage publication, USA Schotsmans, W.(2003). Gas diffusion properties of pome fruit in relation to storage potential. PhD thesis, Katholieke Universiteit Leuven, Leuven, Belgium, 186 p.

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SCREENING OF N-ACYL-L-HOMOSERINE LACTONE PRODUCTION BY BACTERIA ISOLATED FROM FRESH FOODS M.S. MEDINA-MARTINEZ1,2, M. UYTTENDAELE1, S. MEIREMAN1 & J. DEBEVERE1 1

Laboratory of Food Microbiology and Food Preservation Faculty of Bioscience Engineering, Ghent University 2 Cátedra de Microbiología de Alimentos, Facultad de Farmacia Universidad Central de Venezuela, Apartado 40109, Caracas 1040 A, Venezuela.

INTRODUCTION In members of Enterobacteriaceae family quorum sensing mediated by Nacyl-L-homoserine lactones (AHL) is involved in the regulation of some functions. In foods most of the studies about quorum sensing have been performed in processed foods (Gram et al., 1999; Bruhn et al., 2004); however, information about production of AHL in fresh foods is limited. The objective of the present study was to deduce the relevance of quorum sensing bacteria in refrigerated fresh foods with a limited shelf life due to development of Gram-negative spoilage bacteria MATERIALS AND METHODS Screenig of AHL production by Enterobacteriaceae strains The production of AHL by six Enterobacteriaceae strains was screening by induction and fluorescence assays, following the methodologies described by Ravn et al. (2001) and Medina-Martínez et al. (2006). A. tumefaciens NT1, C. violaceum CV026 and E. coli JB523 were used as sensor strains (Ravn et al., 2001, Andersen et al., 2001). The inhibition of the pigment production by C. violaceum was used to detect long chain AHL producers (Ravn et al. 2001). Screening of potential AHL producer bacteria by replica assay Fish, pork, beef, cucumber, soya, mexican salad and lettuce salad were screened for the presence of potential AHL producers. From each food sample ten-fold serial dilutions in Physiological Peptone Solution (PPS) (peptone 1 g l-1, NaCl 8.5 g l-1) were prepared. 0.1 ml of appropriate dilutions was plated on the surface of TSA plates in duplicate. The plates were incubated at 30°C for 24-48 h before enumeration of the colonies. The screening of potential AHL producer bacteria was performed by the replica method described by Bruhn et al. (2004). Colonies from the TSA plates were copied using a Whatman n° 41 filter (Maidstone, England) in plates containing an AHL monitor system bacterium. A. tumefaciens NT1 and C. violaceum CV026 were used as sensor strains The plates were incubated at 30°C for 24h before AHL positive colonies were visualized by colour change (violet or blue) in the surrounding agar. Tentative positive and negative colonies were confirmed by induction and fluorescence assays. Additionally, some isolates were checked for the production of long chain AHLs using the inhibition of

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the pigment production in C. violaceum. The preliminary identification of these isolates was performed by traditional biochemical tests and the complete identification of some of them was performed using the BBL Crystal ID system (Becton Dickinson, Sparks, MD USA). RESULTS AND DISCUSSION All the test strains, except for the E. cloacae and E. agglomeran strains, were positive in the induction assay with A. tumefaciens and C. violaceum as well as in the fluorescence assay with E. coli JB523 (Table 1). It can be concluded from these results that these strains are able to produce short chain AHLs with or without substitutions. In the inhibition of the induced C. violaceum, complementary to results described above, only E. agglomerans and E. cloacae produced long chain AHLs. These results confirm the production of the signal communication molecules AHL in Enterobacteriaceae associated with foods. Table 1. Screening of AHL production by some Enterobacteriaceae members by induction assay on solid media and fluorescence assay in broth. Strain H. alvei 253 H. alvei 267 H. alvei 259 S. liquefaciens 389 E. cloacae 015 E. agglomerans 397

Induction assay C. violaceum A. tumefaciens ++ ++ ++ ++ -

++ ++ ++ + -

Inhibition of pigment production in C. violaceum + +

Flurescence assay E.coli JB523 ++ ++ ++ ++ -

++ Strong positive, + weak positive, - negative

Results of the replica assay in the food samples are shown in Table 2. No potential AHL producers were detected with the reporter C. violaceum. Although initial results of the replica assays using A. tumefaciens as the sensor strain showed the presence of AHL producers in different food samples, it was only in mexican salad and lettuce salad that the initial positive results were confirmed. In the other food samples the initial potential positive isolates for the production of AHL were later confirmed as no AHL producers. Two potential AHL producer strains in mexican salad (Figure 1) were identified as Rahnella aquatilis and Flavimonas oryzihabitans.

Figure 1. Replica assay of Mexican salad


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Table 2. Replica assay for the screening of potential AHL producers N° colonies Potential TSA colony count Reporter strain Confirmed (CFU/g) AHL producers C. violaceum A. AHL producers ** (CFU/g)* tumefaciens Fish 1.3 x 105 3.0 x104 0/13 + Mexican salad 1.7 x 106 3.0 x 105 2/11 + 1.0 x 105 1/8 + Lettuce slad 1.2 x 106 6.0 x 104 0/12 + Cucumber 1.3 x 105 6.0 x 106 0/12 + Soya 1.8 x 107 3.0 x 104 0/8 + Minced Pork 7.0 x 104 0 0/13 Minced Beef 1.7 x 105 * The results reported are based on the number of AHL positive isolates found by replica assay. ** Confirmation of AHL producers is based in the induction assay in solid media and fluorescence assay. Food sample

In lettuce salad one isolate was detected as an AHL producer and was identified as R. aquatilis. The production of AHL by these isolates was confirmed by induction on solid media and the fluorescence assay. Figure 2 shows the results of the fluorescence assay for a strain of R. aquatilis isolated from mexican salad. According to information revised by Nguyen-the and Carlin (1994), R. aquatilis has been described as one of the most frequent Enterobacteriaceae in minimally processed vegetables. The identification of some isolates showed to Pseudomonas member and Enterobacteriaceae as principal flora in vegetables. In fish Shewanella putrefaciens was the major microorganism isolated and in meats Gram-positive bacteria was the most detected flora. Ten of 16 isolates tested caused the inhibition of the induced reporter strain, showing that these strains can produce long chain AHL molecules. Two of these AHL positive isolates were identified as E. cloacae and six as members of Pseudomonas genus 25000

Figure 2. Fluorescence assay of test strain Rahnella aquatilis isolated from mexican salad: (■) R. aquatillis, (▲) C6-HSL synthetic standard, (□) 3-oxo-C6HSL synthetic standard, (●) LB medium.

20000

RFU

15000

10000

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CONCLUSIONS The production of AHLs by several Enterobacteriaceae under in vitro conditions was demonstrated. Fresh foods or minimally processed foods may carry AHL producing bacteria which could by the quorum sensing contribute to

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spoilage in these products; however, it is not the dominant flora present in these foods. REFERENCES Andersen, J. B., Heydorn, A., Hentzer, M., Eberl, L., Geisenberger, O., Christensen, B., B., Molin, S. & Givskov, M. (2001). gfp-Based N-acyl homoserine-lactone sensor systems for detection of bacterial communication. Appl. Environ. Microbiol., 67, 575-585. Bruhn, J. B., Christensen, A. B., Flodgaard, L. R., Nielsen, K., F. N., Larsen, T., O., Givskov M. & Gram, L. (2004). Presence of acylated homoserine lactones (AHLs) and AHL-producing bacteria in meat and potencial role of AHL in spoilage of meat. Appl. Environ. Microbiol., 70, 4293-4302.


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INTEGRATED BENCHMARKING FOR SELECTION OF WASTEWATER TREATMENT TECHNOLOGIES IN HO CHI MINH CITY – VIETNAM Marjolein MESSIAEN, Peter L.M. GOETHALS & Niels DE PAUW Laboratory of Environmental Toxicology and Aquatic Ecology, Ghent University Jozef Plateaustraat 22, BE-9000 Gent, Belgium

INTRODUCTION Urban population raised from 5 million (1997) to almost 10 million (2005) in HCMC. The increasing water pollution, entails an urgent need to invest in wastewater treatment technologies. However, for the choice of effective and cost-efficient methods, both technological and social-economical information is crucial. The aim of this study is to investigate what technologies are convenient to treat wastewaters in HCMC, the capital city of Vietnam. For this purpose, expert knowledge based on literature and interviews in combination with a local pilot study were used for an integrated benchmarking of different wastewater treatment options. Feedback during a workshop allowed to gain insight in the opinion of the involved stakeholders and urban managers. MATERIALS AND METHODS The first step of this study was based on analyzing scientific studies. The resulting insights were discussed with local water managers, ward and district authorities, wastewater treatment experts and scientists. The results of this literature study and interviews were combined with an additional questionnaire to allow a broad scan of the expectations and preferences regarding wastewater treatment in HCMC. In the mean time, also results from a local aerated lagoon system, described by De Pauw et al. (1999) and Goethals et al. (2000), could be compared with data from an activated sludge and UASB system in other cities in Vietnam. The results of all these studies were presented during a workshop organized by the Vietnamese government and the Belgian Technical Cooperation (BTC) attended by about two hundred participants involved in water management. RESULTS AND DISCUSSION According to the SWOT-analysis, based on an extensive literature study, high-tech technologies, such as activated sludge systems seem highly appropriate for the treatment of wastewater in urban areas. One of the main advantages of those systems, compared with aerated lagoon systems, are the high removal efficiencies and the low land area requirements. Other weaknesses of aerated lagoons are the possible production of odors and noise. But, physico-chemical measurements of the pilot system, show that waste stabilization ponds seem to be good alternatives. The treatment plant can operate at minimized costs with a high removal efficiency, so that all the Vietnamese standards are met (Table1). Moreover, the disadvantage of the

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high land requirements is compensated by the development of a greenish area. Table 2. Comparison of the treatment results with the Vietnamese standards. For all parameters the Vietnamese standards were met. At present 6.65-7.88 28.7-31.2 43-74 7.0-22.0 7.0-20.0 16-35 12-33 13.3 – 16.0 0.9, the acrylamide levels tend to decrease with increasing aw. However, this decreasing effect is much more pronounced in potato mixtures obtained during the water desorption experiment. So it is obvious that, depending upon the adsorption/desorption status of the potato powder, the acrylamide generation upon heating is different despite the fact that the aw of the powder is the same. When evaluating these data against the sorption isotherms (Figure 1), it becomes clear that the powders experiencing water desorption have a much higher moisture content compared to the powders going through a water adsorption phenomenon, for similar aw. This hysteresis effect is much more pronounced at aw > 0.9. This leads to the hypothesis that moisture content, rather than aw, plays a role in acrylamide formation in the current experimental setup. This hypothesis was confirmed in the experiments, presented in Figure 3. Here, different amounts of distilled water were added manually to the dry potato powder, in order to obtain moisture contents above 100% (expressed on dry matter). From these results, it can be concluded that only high moisture contents (> 100% on dry matter) give rise to decreased acrylamide levels in the potato powder model system, and that aw is not the decisive factor. 7 Conc. acrylamide (mg/kg)

0.943 6

0.966 0.940 0.964

5

0.967 0.977

4

0.980

3 2 1 0 50

100

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Figure 3. Effect of moisture content (> 50%) on the acrylamide levels in homogeneous potato powder mixtures, containing different amounts of water. Mixtures were heated for 3 (∆), 5 (◊), and 7 (□) min in a closed tubular reactor. Data from the adsorption experiment are plotted as well for 3 (▲), 5 (♦), and 7 (■) min heating. Desorption experiment data, with aw values of 0.940 and 0.967, are also included, for 3 (o), 5 (●), and 7 (x) min. The labels show aw levels corresponding to the moisture content of each mixture

REFERENCES Ames, J.M. (1990). Control of Maillard reaction in food systems. Trends Food Sci. Technol., 1, 150-154. Kaymak-Ertekin, F. & Gedik, A. (2004). Sorption isotherms and isosteric heat of sorption for grapes, apricots, apples and potatoes. Food Sci. Technol., 37, 429-438. Martins, S.I.F.S. & van Boekel, M.A.J.S. (2003). Melanoidins extinction coefficient in the glucose/glycine Maillard reaction. Food Chem., 83, 135-142. Mottram, D.S., Wedzicha, B.L. & Dodson, A.T. (2002). Acrylamide is formed in the Maillard reaction. Nature, 419, 448-449.


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Pedreschi, F., Hernandez, P., Figueroa, C. & Moyano, P. (2005). Modeling water loss during frying of potato slices. Int. J. Food Prop., 8, 289-299. Yaylayan, V.A., Wnorowski, A. & Locas, C.P. (2003). Why asparagine needs carbohydrates to generate acrylamide. J. Agric. Food Chem., 51, 1753-1757.

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GAS-CHROMATOGRAPHIC METHOD FOR QUANTIFYING CARVACROL, THYMOL, TERPINEN-4-OL, TRANSANETHOLE, EUGENOL AND TRANS-CINNAMALDEHYDE IN MEDIA SIMULATING PIG GUT CONDITIONS Joris MICHIELS1,2, Peter MAENE1, Joris MISSOTTEN2, Noël DIERICK2, Dirk FREMAUT1 & Stefaan DE SMET2 1

2

Departement Biotechnologische Wetenschappen, Landschapsbeheer en Landbouw Hogeschool Gent, Voskenslaan 270, 9000 Ghent, Belgium Laboratory for Animal Nutrition and Animal Product Quality, Faculty of Bioscience Engineering, Ghent University, Proefhoevestraat 10, 9090 Melle, Belgium

INTRODUCTION Michiels et al. (2005) demonstrated that certain phytochemicals have clear and interesting in vitro antimicrobial activities against the main components of the pig gut flora. Consequently these phytochemicals have potential as an alternative for the antimicrobial growth promoters in animal feed which have been banned in the EU from January 2006. However, no data are yet available about their stability and biodegradability in gut conditions, which are necessary for an appropriate dosage of these chemicals in pig feed. Therefore an accurate method to quantify these phytochemicals is needed. The paper discusses the methodology and the validation of a gas-chromatographic (GC) method with 2-isopropyl-phenol as internal standard, to quantify these phytochemicals in media simulating pig gut conditions. MATERIAL AND METHODS Chemicals. Carvacrol, thymol, trans-anethole and eugenol were obtained from Sigma-Aldrich, Bornem, Belgium. Trans-cinnamaldehyde was delivered by Fluka, Bornem, Belgium as well as 2-isopropyl-phenol, the internal standard. Across Organics (Geel, Belgium) supplied terpinen-4-ol, which was a 50:50 racemic mixture of the (+) and (-) enantiomer. Preparation of standard matrices and extraction. Incubation media for simulation of the gastric, small intestinal and caecal conditions of the pig gut (artificial substrate, buffer solution and inoculum) were prepared in 100 ml vessels and allowed to ferment according to the procedure described by Michiels et al. (2005). After fermentation, the contents of the vessels were acidified to pH 2 with 2% of a 12 M H2SO4 solution to stop further fermentation. Thereafter, the phytochemicals were mixed into the different vessels to obtain the appropriate final concentration. These were the standard matrices used for the validation of the method. They were stored at -20°C for a short period of time. Each analysis (extraction and GC-analysis) was performed in duplicate. This procedure was adapted and modified from Friedman et al. (2000). The sample size for extraction was 2 ml. Each 2 ml sample was transferred to a 10 ml vial and spiked with 100 µl of 2-isopropyl-phenol internal standard solution (2.5 mg/ml of 2-isopropyl-phenol in ethyl acetate). The samples were extracted twice; each extraction was carried out by vigorous shaking for 2 min with 4 ml of ethyl acetate. The samples were then

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centrifuged (1400 g, 3 min) prior to transfer of the less dense ethyl acetate layer. The combined ethyl acetate extracts were reduced to dryness with nitrogen at room temperature. The residue of each was dissolved in ethyl acetate (500 µl). Aliquots (1 µl) were used for GC-analysis. Quantification by GC. GC was performed using a capillary column 30 m x 0.25 mm ID x 0.25 µm film thickness fused silica. The phase composition was 2,3-di-O-methyl-6-O-tert-butyl dimethyl silyl beta cyclodextrin doped into 14% cyanopropylphenyl/86% dimethyl polysiloxane (Restek RtβDEXsm; chiral separation column). The average helium carrier gas flow was set to 1.90 ml/min. The split ratio of the column was 60:1. The injector temperature was set at 250°C. The column oven temperature was held at 110°C for 2 min, then programmed to 170°C at 4°C/min and then to 230°C at 40°C/min, and maintained at 230°C for 4 min. The FID detector temperature was set at 280°C. Validation of the method. To validate the analytical procedure the following criteria were put forward. The GC-analysis should give a linear response in the concentration range 5 to 1000 mg/l (R2 >0.99). Standard matrices with the following phytochemical concentrations were prepared; 5, 10, 50, 100, 500 and 1000 mg/l. Six samples were taken from each standard matrix and these were further processed as described above. Response factors (RF) were calculated according to the following equation: RF = peak area of phytochemical / peak area of internal standard. These RF were used as dependent variable, and the ratio of the concentration of the phytochemical to the concentration of the internal standard as independent variable in linear regression analysis. No peak signal interference should be observed. Therefore the signal-noise ratio (S/N) of all peaks in the area ‘retention time of analyte ± 2.5%’ must be lower than 3. For each of the three types of simulation, 10 blank chromatograms were analysed. Repeatibility, expressed as coefficient of variation (CV)(residual standard deviation relative to the mean) should be lower than 5% and has been determined for three concentrations (low, medium and high) for each phytochemical and type of simulation. Therefore, six subsamples were taken from each standard matrix and the RF of the phytochemical determined. The percentage of recovery for extraction/drying should be higher than 50%. Recovery has been measured for three concentrations (low, medium and high) for each phytochemical and type of simulation. Therefore the RF of a phytochemical out of a standard matrix was compared to the RF of the respective phytochemical added to an equal concentration after extraction and drying of a blank matrix (n=4). The limit of detection (LOD) is defined as “the concentration which gives an instrument signal significantly different from the blank or background signal”. In practice it is given by the following equation : ylimit of detection = yB + 3 sB, whereas yB is the blank signal and sB the standard deviation of the blank. It is appropriate to use the sy/x (the standard deviation of random errors in the Y-direction) in place of yB. RESULTS AND DISCUSSION Linearity. A first linear regression was carried out and the 95% confidence level curves calculated. If the origin was within these 95% confidence level curves it was concluded that the Y-intercept was not different from zero. The regression was therefore forced to go through the origin. This was the case


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for all but one regression. The intercept for the regression of transcinnamaldehyde in caecal media was highly negative (-0.197). The observed RF for the lower concentrations (5, 10, 50 and 100 mg/l) were remarkably low, causing this negative intercept. According to this regression line, transcinnamaldehyde in media simulating the caecum should give only a detectable signal above a certain concentration. This could indicate that a considerable amount of this chemical is rapidly (partially) degraded or absorption to/interaction with other particles of the caecum matrix is taking place once it is added to the acidified standard caecum matrix. Further experiments showed that a standard concentration series of trans-cinnamaldehyde in ethyl acetate did give a good linear response with high RF, even for the lowest concentrations. Trans-cinnamaldehyde contains an oxidizable α,βunsaturated aldehyde function. Haarmann & Reimer (2001) demonstrated the readily biodegradability of trans-cinnamaldehyde. However, biochemical transformation of trans-cinnamaldehyde in the standard caecum matrix of our study can be excluded due to the fact that before the addition of transcinnamaldehyde, the caecum matrix was acidified to pH 2 hampering all enzymatic activity. No hydrolysis is possible for trans-cinnamaldehyde, so it is expected to be stable in the aqueous incubation medium, although it may be slowly oxidized to cinnamic acid (The Aromatic Consortium, 2005). Yuan et al. (1992) found no oxidation of trans-cinnamaldehyde to cinnamic acid in water solutions. In our study peak signals for cinnamic acid were found on the chromatograms but the peak areas were far too low to explain all losses of trans-cinnamaldehyde; certainly for concentrations > 50 mg/l. Temperature-dependent and non-enzymatic oxidation and further decomposition to benzaldehyde and glyoxal has been reported by Friedman et al. (2000), but this occurred for pure trans-cinnamaldehyde and at temperatures ≥ 60°C. Through reaction with amino groups trans-cinnamaldehyde can form Schiff base intermediates (Yuan et al., 1992). However addition of 1 or 10% formol to the standard caecal matrix to prevent formation of Schiff bases (competitive suppression) did not result in higher RF for trans-cinnamaldehyde. The exact reason for this analytical problem is not yet fully understood. Moreover, the slope of the calculated regression equation for trans-cinnamaldehyde in caecal media (0.70) was significantly (P