2nd international workshop - · pdf file2nd international workshop ... nanthi, szogi, ariel,...

2nd International Workshop

“Advances in Science and Technology of Natural Resources”

Abstract Book

October 27-29, 2010 Universidad de La Frontera, Pucón, Chile.

ORGANIZING COMMITTEE

Editor-in-Chief: María de la Luz Mora, Universidad de La Frontera, Temuco, Chile.

Managing Editors: Alejandra Jara, Universidad de La Frontera, Temuco, Chile.

Milko Jorquera, Universidad de La Frontera, Temuco, Chile. Executive Secretary: Alejandra Ribera, Universidad de La Frontera, Temuco, Chile. Coordinators: Cecilia Paredes, Universidad de La Frontera, Temuco, Chile.

Jacquelinne Acuña, Universidad de La Frontera, Temuco, Chile.

María José Flores, Universidad de La Frontera, Temuco, Chile.

Outreach Coordinator: Cristian Meriño, Universidad de La

Frontera, Temuco, Chile.

ACKNOWLEDGEMENTS

The Organizing Committee gratefully acknowledges the Doctoral Program in Sciences of Natural Resources from La Frontera University and its Director Dr. María de la Luz Mora. We would like to

thank the financial support from The Program for Improvement of Quality and Equity of Superior Education (MECESUP) and National Council of Science and Technology (CONICYT) of the

Chilean Ministry of Education through the Regular and International Cooperation Projects of the National Fund of Science and Technology (FONDECYT) and Project Ecos-CONICYT. We also

thank to La Frontera University and Students from Doctoral Program in Sciences of Natural Resources of La Frontera University.

i

CONTENTS PAGES SYMPOSIUM 1. WASTE MANAGEMENT, BIOENERGY AND CARBON SEQUESTRATION

1

ORAL PRESENTATIONS

1

ENGINEERED HEAT TREATED METHANOGENIC GRANULES, A PROMISING APPROACH FOR EXTREME THERMOPHILIC BIOHYDROGEN PRODUCTION Abreu, Ângela A.

3

BIOTECHNOLOGICAL PROCESSES FOR USING WASTE AND MICROBIAL OILS IN BIODIESEL PRODUCTION Heipieper, H.J., Azócar, L., Ciudad, G. and Navia, R.

4

CARBON SEQUESTRATION IN TOP- AND SUBSOIL HORIZONS: WHAT IS THE DIFFERENCE? Rumpel, Cornelia

6

THE MANAGEMENT OF PHOSPHORUS IN POULTRY LITTER Bolan, Nanthi, Szogi, Ariel, Seshadri, Balaji and Chuasavathi, Thammared

7

POSTER PRESENTATIONS

11

SUCESIVES CYCLES OF ENZYMATIC BIODIESEL PRODUCTION IN A TERT-BUTANOL SYSTEM Azócar, L., Ciudad, G., Heipieper, H., Muñoz, R. and Navia, R.

13

MONOMER FRACTION DURING STRONG MIX ACID PRETREATMENT OF ASPEN WOOD TO PRODUCE SUGAR Ciudad, G., Azócar, L., Janga, K. and Moe, S.

14

Candida rugose LIPASE IMMOBILIZATION ON BIOCHAR DERIVED FROM AGRICULTURAL RESIDUES Cea, M., Sangaletti, N., Gonzalez, M.E. and Navia, R.

15

CELULAR DISRUPTION EFICIENCY OF SEVERAL METHODS TO PRODUCE TOTALS OILS FROM MICROALGAE Botryococcus braunii WITH PREVIOUS EXTRACTION OF EXTRACELULAR HYDROCARBONS Concha, E., Toro, C. and Navia, R.

16

NEW SORBENT DEVELOPED FROM PETROLEUM COKE FLY ASHES AND ITS POTENTIAL APPLICATION González, A., Moreno, N., Navia, R. and Querol, X.

17

CHARACTERIZATION OF BIOCHAR FROM LIGNOCELLULOSIC WASTES González, M.E, Cea, M., González, A., Toro, C.A., Diez, M.C.and Navia, R.

18

PRODUCTION OF ELECTRICITY FROM A MIXED MICROBIAL CONSORTIUM USING A MICROBIAL FUEL CELLS Hidalgo, P. and Jeison, D.

19

CATALYTIC BEHAVIOR OF LIPASE NOVOZYME 435 IN THE FATTY ACID ETHYL ESTER (FAEE) PRODUCTION USING SOYBEAN OIL/ETHANOL EMULSION Sangaletti, N. , Briones, R., Beltran, C., Cea, M. and Navia, R.

20

BIOGENIC METHANE PRODUCTION FROM MICROALGAE BIOMASS Torres, A. and Jeison, D.

21

ii

EFFECT OF MICRO-AERATION ON BIOGAS QUALITY IN THE ANAEROBIC DIGESTION PROCESS Valdés, F., Hemmelmann, A. and Jeison, D.

22

HYPERBRANCHED POLYESTERS FROM RESIDUAL GLYCEROL FOR THE DEVELOPMENT OF POLYURETHANES. Toro, C., Valerio, O. and Navia, R.

23

SYMPOSIUM 2. ENVIRONMENTAL BIOTECHNOLOGY

25

ORAL PRESENTATIONS

TEXTILE DYES FUNGAL MECHANISMS OF BIODEGRADATION Ottoni, Cristiane A., Santos, Cledir and Lima, Nelson

27

BIODEGRADATION WITH WHITE-ROT FUNGI AND THEIR ENZYMES Rodríguez-Couto, Susana

29

ORGANOCHLORINE PESTICIDES BIODEGRADATION BY INDIGENOUS ACTINOMYCETES STRAINS Benimeli, C.S., Fuentes, M.S., Sáez, J.M., Bourguignon, N., Cuozzo, S.A. and Amoroso, M.J.

31


33

DEGRADATION OF CHLORPYRIFOS AND DIAZINON BY ACTINOMYCETES ISOLATED FROM AGRICULTURAL SOIL OF SOUTHERN CHILE

Briceño, G., Palma, G. and Diez, M.C.

35

DETERMINATION OF CRITICAL MICELLE CONCENTRATION OF SOYA LECITHIN IN KIRK MEDIUM AND EFFECT OF ADDITION OF PENTACHLOROPHENOL Bustamante, M., Rubilar, O. and Diez, M.C.

36

LIGNINOLYTIC ENZYME ACTIVITIES AND COLONIZATION BY Anthracophyllum discolor ON LIGNOCELLULOSIC SUPPORTS Elgueta, S., Lima, N. and Diez, M.C.

37

EFFECT OF LIGNOCELLULOSIC MATERIALS ON BIOLOGICAL ACTIVITIES IN BIOMIX OF BIOBED SYSTEM CONTAMINATED WITH ATRAZINE Urrutia, C., Tortella, G. and Diez, M.C.

38

METHOXYCHLOR BIOREMEDIATION BY INDIGENOUS ACTINOMYCETES USING PURE AND MIXED CULTURES Fuentes, M.S., Sáez, J.M., Bourguignon, N., Cuozzo, S.A., Benimeli, C.S.and Amoroso, M.J.

39

THESIS PROJECT: STUDY OF FACTORS RELATED TO THE EMERGING VERMIFILTRATION BIOTECHNOLOGY FOR CATTLE SLURRY TREATMENT AND VALORIZATION AS ORGANIC FERTILIZER Morales, E. and Bornhardt, C.

40

EFFECT OF BIOSTIMULATION OF AGRICULTURAL BIOBEDS ON CHLORPYRIFOS DEGRADATION. Tortella, G.R., Rubilar, O., Cea, M., Wulff, C., Martínez, O. and Diez, M.C.

41

ADSORPTION OF PENTACHLOROPHENOL (PCP) BY FUNGAL BIOMASS IN A FIXED-BED COLUMN. Rubilar, O., Tortella, G.R.., Cea, M., Cuevas, R. and Diez, M.C.

42

iii

SYMPOSIUM 3. GRADUATED DOCTORATES, DOCTORATE PROGRAM IN SCIENCES OF NATURAL RESOURCES, UNIVERSIDAD DE LA FRONTERA, CHILE

43

Dr. Leonardo Vera, Pontificia Universidad Católica De Valparaíso, Chile: “Análisis clínico de ecosistemas como base para el diseño y construcción del Parque Ecocultural Quebrada Verde”.

Dr. Howard Langer, Agrícola Nacional S.A.C. (ANASAC), Chile. “Doctores en Ciencias de Recursos Naturales, un caso de inserción a la empresa”.

SYMPOSIUM 4. SOIL MICROORGANISMS AND RHIZOSPHERE

45

ORAL PRESENTATIONS

45

MYCORRHIZAS: AN ADAPTIVE STRATEGY FOR INCREASING THE RESILIENCE OF THE SOIL-PLANT SYSTEMS IN A SCENARIO OF GLOBAL CHANGE Barea, J. M.

47

IMPACT OF ARBUSCULAR MYCORRHIZAL SYMBIOSIS ON PLANT HEALTH: AN OVERVIEW OF THE MECHANISMS INVOLVED Pozo, M., López-Ráez, J.A., Jung, S. and Azcón-Aguilar, C.

49

SOIL HEAVY METAL REMEDIATION BY ARBUSCULAR MYCORRHIZAL FUNGI Ferrol, Nuria

51

ARBUSCULAR MYCORRHIZAL FUNGAL POPULATION STRUCTURE ACROSS A REGENERATING ABANDONED COALMINE Cumming, J., Hunter-Cevera, K. and Klugh Stewart, K.

53

MICROSCALE INVESTIGATIONS IN THE INTERFACE BETWEEN RHIZOSPHERE AND DETRITUSPHERE Marschner, P., Marhan, S. and Kandeler, E.

55

BIOLOGICAL AVAILABILITY OF ORGANIC PHOSPHORUS IN SOIL Richardson, Alan E.

58


63

FLUORESCENCE DETECTION OF ALUMINIUM IN ARBUSCULAR MYCORRHIZAL FUNGAL STRUCTURES AND GLOMALIN BY USING LASER SCANNING CONFOCAL MICROSCOPY Aguilera, P., Seguel, A., Cornejo, P., Curaqueo, G., Meier, S., Medina, J., Berríos, D. and Borie, F.

65

EVALUATION OF ENZYMATIC DEGRADATION OF ORGANIC WASTE AND SEWAGE SLUDGE MIXTURES BY AUTOCHTHONOUS SAPROPHYTIC FUNGI FROM SOUTHERN CHILE Almonacid, L., Lagos, C., Cornejo, P. and Arriagada, C.

66

ARBUSCULAR MYCORRHIZA AND SOIL PROPERTIES IN AN ALFISOL OF CENTRAL-SOUTH CHILE UNDER LONG-TERM NO TILLAGE Curaqueo, G., Seguel, A., Medina, J., Aguilera, P., Rubio, R. and Borie, F.

67

EXUDATION OF ACID PHOSPHATASE AND ORGANIC ACIDS BY CLUSTER ROOTS OF Embothrium coccineum Delgado, M,, Zúñiga-Feest, A. and Borie, F.

68

EFFECTS OF ARBUSCULAR MYCORRHIZAL INOCULATION ON METALLOPHYTES AND AGRICULTURAL PLANTS GROWING ON COPPER POLLUTED SOILS Meier, S., Borie, F., Inostroza, N., Cox, R., Curaqueo, G., Seguel, A. and Cornejo, P.

69

iv

EVALUATION OF IN VITRO ANTIBACTERIAL ACTIVITY OF PLANT EXTRACTS ON BACTERIA OF IMPORTANCE PHYTOPATHOLOGICAL Concha, M., Partarrieu, A., Jofre, I., Gallardo, C., Fincheira, P., López, F. and Parada, M.

70

STUDY OF SOIL BACTERIA ISOLATED IN THE REGION OF LA ARAUCANIA, HIGHLY TOLERANT TO LEAD AND IMPLICATIONS OF THE POLYSACCHARIDE IN THIS PROCESS Gallardo, C., Godoy, K., Concha, M., Jofré, I., López F., Fincheira, P., Partarrieu, A. and Parada, M.

71

AGGREGATES AND ITS RELATION TO ORGANIC CARBON CONTENT INTO DEEP VOLCANIC SOIL OF PRISTINE TEMPERATE OLD-GROWTH RAINFORESTS IN SOUTHERN CHILE Neculman, R., Panichini, M., Matus, F. and Godoy, R.

72

IDENTIFICATION OF 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID DEAMINASE AND Β-PROPELLER PHYTASE GENES IN RHIZOBACTERIA ASSOCIATED WITH THE OLDEST LIVING PLANTS ON EARTH Jorquera, M.A., Mora, M.L. and Crowley, D.E.

73

DESIGN AND EVALUATION OF SPECIFIC PRIMER SETS FOR QUANTITATIVE PCR TO STUDY OF PHYTASE GENES IN Bacillus AND Paenibacillus Jorquera, M.A., Maruyama, F., Ulloa, P. and Mora, M.L.

74

EFFECT OF SELENIUM ADDITION ON THE COMMUNITY STRUCTURE OF NATIVE RHIZOBACTERIA, ANDISOL SOIL Padilla, E., Acuña-Sobarzo, J., Jorquera, M. and Mora, M.L.

75

ISOLATED SELENIUM-ACUMULATING RHIZOBACTERIA FROM RHIZOSPHERE OF WHEAT GROWN ON ANDISOLS SOIL Acuña-Sobarzo, J., Padilla, E., Jorquera, M. and Mora, M.L.

76

ACTIVITY CONSERVATION AND IMMOBILIZATION PATTERNS OF Rahnella sp. D75 AND E. coli ACID PHYTASES ADSORBED IN NANOCLAY SUPPORTS Menezes-Blackburn, D., Jorquera, M., Garrido, E., Gianfreda, L., Rao, M. and Mora, M.L.

77

CONTRIBUTION OF PHYSICALLY PROTECTED ORGANIC MATTER TO CARBON SEQUESTRATION IN ALLOPHANIC SOILS Panichini, M., Neculman, R., Matus, F., Godoy, R. and Rumpel, C.

78

ROLE OF ARBUSCULAR MYCORRHIZAL SYMBIOSIS IN THE ALUMINIUM TOLERANCE OF FIVE CULTIVARS OF Triticum aestivum L. GROWING IN ACID SOILS OF SOUTHERN CHILE. Seguel, A., Curaqueo, G., Meier, S., Aguilera, P., Cornejo, P. and Borie, F.

79

EARLY ARBUSCULAR MYCORRHIZAL COLONIZATION IN CEREALS GROWING IN ANDISOLS OF LA ARAUCANIA REGION Castillo, C.G. and Borie, F.

80

BIOLOGICAL AND BIOCHEMICAL SOIL INDICATORS: MONITORING TOOLS OF DIFFERENT AGRICULTURAL MANAGEMENTS Scotti, R., Sultana, S., Caceres, M., Scelza, R., Marzaioli, R., D’Ascoli, R. and Rao, M.A.

81

INFLUENCE OF NITROGEN AND PHOSPHORUS SOURCES ON MYCORRHIZAL LETTUCES UNDER ORGANIC FARMING Scotti, R., Seguel, A., Cornejo, P., Rao, M.A. and Borie, F.

82

v

SYMPOSIUM 5. SOIL, PLANT PHYSIOLOGY AND PLANT NUTRITION

83

ORAL PRESENTATIONS

83

MERCURY IN THE ENVIRONMENT: SOIL HEALTH CONCERNS AND DECONTAMINATION STRATEGIES Rao, M.A., Scelza, R. and Scotti, R.

85

ENVIRONMENTAL CHEMISTRY OF ARSENIC Violante, Antonio

86

PHYSIOLOGICAL EFFECTS OF SILICON NUTRITION ON RICE PLANTS Dettmann, K., Antunes, W., Reis, J., Araujo, W., Nunes-Nesi, A., Fernie, A. and DaMatta, F.

88

CONTRIBUTION OF MITOCHONDRIAL METABOLISM FOR PHOTOSYNTHETIC PERFORMANCE Nunes-Nesi, Adriano

89

CIRCADIAN CONTROL OF ROOT ELONGATION AND C PARTITIONING IN Arabidopsis thaliana Fisahn, Joachim

90


91

SEED PELLETIZATION WITH SELENITE AND ITS INFLUENCE ON THE ANTIOXIDANT SYSTEM OF RYEGRASS CULTIVARS Cartes, P., Yañez-Mansilla, E., Paredes, C. and Mora M. L.

92

EFFECT OF SELENIUM ON BREAD WHEAT (Triticum aestivum L.) CULTIVARS OF LA ARAUCANIA REGION Pinilla, L., Cartes, P. and Mora, M.L.

93

NITROGEN AND SULFUR FERTILIZATION AND ITS INTERACTION WITH SELENITE PELLETED SEEDS AFFECT THE OXIDATIVE STRESS IN RYEGRASS PLANTS. Yañez-Mansilla, E., Pirce, F., Vega, I., Mora, M.L. and Cartes, P.

94

ALUMINUM-SULPHUR INTERACTION EFFECTS ON TOTAL PROTEIN EXPRESSION IN Vaccinium corymbosum L. GROWING IN AN ANDISOL. Alarcón, E., Reyes-Díaz, M. and Alberdi, M.

95

PHOTOCHEMICAL EFFICIENCY OF THREE HIGHBUSH BLUEBERRY CULTIVARS UNDER ALUMINUM TOXICITY AFTER APPLICATION OF CALCIUM SULFATE UNDER CONTROLLED GREENHOUSE CONDITIONS Poblete Grant, P., Reyes-Díaz, M., Meriño-Gergichevich, C. and Alberdi, M.

96

CHANGES IN THE ANTIOXIDANT ACTIVITY IN LEAVES OF Vaccinium corymbosum L. UNDER THE EFFECT OF CALCIUM SULFATE AMENDMENT IN AN ANDISOL. Durán, M., Reyes-Díaz, M. and Alberdi, M.

97

CALCIUM SULPHATE EFFECTS ON ANTHOCYANINS AND PHENOLS IN HIGHBUSH BLUEBERRY Reyes-Díaz, M., Meriño, C., Mora, M.L. and Alberdi, M.

98

EFFECT OF MANGANESE TOXICITY AND UV-B RADIATION ON ANTIOXIDANT RESPONSE OF BLUEBERRY (Vaccinium corymbosum L.) Rojas-Lillo, Y., Reyes-Díaz, M., Alberdi, M. and Mora, M.L.

99

vi

EFFECT OF CALCIUM SULFATE APPLICATION ON LEAF SULFUR CONTENT IN HIGHBUSH BLUEBERRY (Vaccinium corymbosum L.) GROWING IN AN ANDISOL Castel , A., Meriño-Gergichevich, C., Reyes-Díaz, M. and Alberdi, M.

100

EFFECT OF MATURITY STAGES AND POSTHARVEST TREATMENTS ON PHENOLIC ACIDS, FLAVONOIDS AND ANTHOCYANINS OF HIGHBUSH BLUEBERRY (Vaccinium corymbosum L.) FRUITS Ribera, A.E., Reyes-Díaz, M., Alberdi, M., Cartes, A. and M.L. Mora

101

ACID PHOSPHATASE ACTIVITY AND PHOSPHORUS DYNAMIC IN A RHIZOSPHERE SYSTEM UNDER AMMONIUM AND NITRATE NITROGEN FERTILIZATION Paredes, C., Menezes-Blackburn, D., Cartes, P., Ribera, A.E. and Mora, M.L.

102

CHANGES IN THE CHEMICAL COMPOSITION AND ANTIOXIDANT ACTIVITY OF MORTIÑO (Vaccinium meridionale SW) FRUITS DURING GROWTH AND MATURATION Gavira, C., Lopera, Y., Hernández, J., Lobo, M., Medina, C. and Rojano, B.

103

BIOCHEMICAL ANALYSIS OF SHOOT TISSUES OF L. perenne PLANTS CULTIVATED UNDER VARYING S CONDITIONS IN THE PRESENCE OF ALUMINIUM TOXICITY Wulff-Zottele, C., Fisahn, J. and Hesse, H.

104

STANDARDIZATION OF A TECHNIQUE TO OBTAIN GOOD QUALITY DNA FROM TROUT SCALES COLLECTED AT DIFFERENT PERIODS Cerda, A., Reyes-Díaz, M. and Gajardo, G.

105

BIOGEOGRAPHIC RELATIONSHIP AGAINST TEMPERATURE AND UVB STRESS FROM BROWN ALGAE OF THE ANTARCTIC AND MAGELLAN: PHOTOCHEMICAL EFFICIENCY AND INDUCTION OF PHLOROTANNINS Cruces, E. and Gómez, I.

106

PHOTOPROTECTIVE ROLE OF PHLOROTANNINS AGAINST SHORT-TERM UVB AND TEMPERATURE STRESS IN THREE SOUTH PACIFIC KELPS Cruces, E. and Gómez, I.

107

EFFECT OF GLOBAL WARMING ON Vitis vinifera FROST-DAMAGE ON SOUTHERN CHILE Orrego, R. and E. Jorquera-Fontena, E.

108

ASSESSING THE ANTATIC OSCILLATION (OAA) AND ENSO AS PREDICTORS OF SEAZON FORECAST ON ARAUCANÍA REGION Orrego, R., Ávila, A. and Salinas, B.

109

EFFECTS OF ALUMINUM TOXICITY ON ROOT TIPS CELLS ORGANIZATION AND ANTIOXIDANT SYSTEM IN BLUEBERRY Inostroza-Blancheteau, C., Reyes-Díaz, M., Aquea, F., Mora, M.L., Arce-Johnson, P. and Alberdi, M.

110

ANTIOXIDANT ACTIVITY AND FLAVONOIDS COMPOUNDS IN FRUITS OF Vaccinium corymbosum L. TREATED WITH CALCIUM SULFATE IN AN ANDISOL Luengo, A., Reyes-Díaz, M. and Alberdi, M.

111

RELATIONSHIP BETWEEN LEAF AL-CONTENT AND CALCIUM SULFATE ADDITION ON NET PHOTOSYNTHESIS, ANTIOXIDANT CAPACITY AND TOTAL PHENOLS OF HIGHBUSH BLUEBERRY (Vaccinium corymbosum L.) UNDER AL-TOXICITY Meriño-Gergichevich, C., Reyes-Díaz, M. and Alberdi, M.

112

EFFECT OF MN EXCESS ON PHOTOSYSTEM I AND PHOTOSYSTEM II PERFORMANCE IN Arabidopsis thaliana Millaleo, R., Krol, M., Ivanov, A.G. , Huner, N.P.A., Reyes-Díaz, M. and Alberdi, M.

113

vii

CHARACTERIZATION OF ANTIOXIDANT ACTIVITY OF LEAVES AND FLOWERS OF Bougainvillea SP. Velásquez, R., Mutis, A., Alfaro, S., Scheuermann, E., Quiroz, A. and Hormazabal, E.

114

EFFECT OF AL IN OXIDATIVE STRESS AND PRODUCTION OF PHENOLIC COMPOUND IN HIGHBUSH BLUEBERRY (Vaccinium corymbosum L.) CV. LEGACY CULTIVATED IN VITRO Manquián, K., Barrientos, H., Pizarro, M., Zúñiga, G.E. and Escudey, M.

115

STRUCTURAL PROPERTIES OF SYNTHETIC ALLOPHANE NANOPARTICLES COATED WITH 6% OF IRON OXIDE AND EVALUATION OF ITS CATALYTIC PROPERTIES FOR ATRAZINE OXIDATION BY FENTON-LIKE REACTION Garrido, E.G. and Mora, M.L.

116

ARSENATE ADSORPTION IN IMOGOLITE AND FERROIMOGOLITE: A COMPARATIVE STUDY Arancibia-Miranda, N. and Escudey, M.

117

ALLOPHANIC NANOCLAYS: THEIR IMPLICATIONS ON THE POTENTIAL CARBON SEQUESTRATION Calabi-Floody, M., Jara, A., Rumpel, C. and Mora, M.L.

118

NATURAL NANOCLAYS LIKE SUPPORT MATERIALS TO IMMOBILIZE ACID PHOSPHATASE: INCREASE THE PHOSPHATE AVAILABILITY TO THE PLANTS Calabi-Floody, M., Jara, A. and Mora, M.L.

119

DESCRIPTION OF THE ADSORPTION OF CU AND ZN ON ALLOPHANIC CLAY USING THE CAPACITANCE CONSTANT MODEL Jara, A.A., Cartes, A.2 and Mora, M.L.

120

APPLICATION OF CONFOCAL MICROSCOPY AS TOOL TO EVALUATE ACID PHOSPHATASE IMMOBILIZATION ON NANOCLAYS Velásquez, G., Calabi, M. and Mora M.L.

121

ASSESSING SOIL HYDROPHOBICITY BY DIFFERENT METHODS UNDER SIMULATED WETTING-DRYING CYCLES Ramírez, J. C. and Bachmann, J.

122

SYMPOSIUM 6. CHEMICAL ECOLOGY

123

ORAL PRESENTATIONS 123 THE ROLE OF NATIVE BEES IN SUSTAINABLE CROP POLLINATION Isaacs, Rufus

125

POSTER PRESENTATIONS 127 ANTIBACTERIAL ACTIVITY FROM RED ALGA Ceramium rubrum Cortés, Y., Pardo, F., Perich, F. and Quiroz, A.

129

IDENTIFICATION OF CUTICULAR COMPOUNDS IN Hylastinus obscurus (MARSHAM) (COLEOPTERA: SCOLYTIDAE) Venthur, H., Parra, L. and Quiroz, A.

130

PRELIMINARY PHYTOCHEMICAL STUDIES OF VASODILATORS COMPOUNDS FROM AN EXTRACT OF Ruta graveolens Mansilla, K., Perich, F. and Salvatici, R.

131

viii

MORFOLOGICAL AND HISTOLOGICAL CHARACTERIZATION OF THE DIGESTIVE SYSTEM OF RASPBERRY WEEVIL Aegorhinus superciliosus (GUÉRIN-MENÉVILLE) (COLEOPTERA: CURCULIONIDAE). Medel, V., Molina, B., Seguel, J., Quiróz, A. and Rebolledo, R.

132

COMPARISON OF DIFFERENT FIBERS FOR SOLID-PHASE MICROEXTRACTION OF FUNGAL VOLATILE METABOLITES Schalchli, H., Hormazabal, E., Pardo, F., Palma, R. and Quiroz, A.

133

ROLE OF VOLATILES IN THE HETEROSPECIFIC SEXUAL COERCION BETWEEN CURCULIONIDS (COLEOPTERA) SPECIES Pacheco, B., Parra, L. and Quiroz, A.

134

OF FATTY ACIDS COMPOSITION PRESENT IN THE ROOTS OF TWO CULTIVARS AND EXPERIMENTAL LINES OF RED CLOVER (Trifolium pratense L.). Tampe, J., Quiroz, A. and Pardo, F.

135

CHEMICAL CHARACTERIZATION BY GC-MS OF CONDITIONED WATER FROM VIRGIN ADULT FEMALE OF Artemia franciscana (SAN FRANCISCO BAY, USA) Tapia, C., Gajardo, G. and Quiroz, A.

136

IDENTIFICATION OF FATTY ACIDS IN ALFALFA (Medicago sativa L.) ROOT EXTRACTS, AND BEHAVIOURAL RESPONSE OF CLOVER ROOT BORER (Hylastinus obscurus (MARSHAM)) TO THEM

Toledo, D., Pardo, F. and Quiroz, A.

137

BIOTRANSFORMATION OF ARISTOLOCHIC I AND II BY Battus polydamas archidamas (LEPIDOPTERA: PAPILIONIDAE: TROIDINII) Urzúa, A., Pinto, C. and Niemeyer, H.

138

SYMPOSIUM 4. SOIL MICROORGANISMS AND RHIZOSPHERE

139

ORAL PRESENTATION 139 SYSTEM MICROBIOLOGY TO STUDY POLYPHOSPHATE METEBOLISM IN BACTERIA Chávez, Francisco

141

SYMPOSIUM 7. AGRO-FOOD BIOTECHNOLOGY

143

BIOTECHNOLOGICAL APPLICATION OF PHYTATE-DEGRADING ENZYMES Greiner, Ralf

145

FOOD AROMA AND ESSENTIAL OILS: EXTRACTION, ANALYSIS AND APPLICATION AS BIOACTIVE COMPOUNDS Scheuermann S., Erick

148

TECHNOLOGIES APPLIED TO FOOD AND HEALTH PRODUCTS Kolodziejczyk, Paul

149

POSTER PRESENTATIONS 151 PRELIMINARY CHARACTERIZATION OF OLEOSOMES FROM PLANT SEEDS AS POSSIBLE CARRIERS FOR MICROENCAPSULATION Acevedo, F., Morales, E., Rubilar, M. and Shene, C.

153

ix

ENHANCED HYDROCARBONS RECOVERY FROM Botryococcus braunii BY AN IONIC LIQUID PRETREATMENT Medina, H. and Shene, C.

154

EXTRACTION AND CHARACTERIZATION OF OLEORRESIN FROM RED BELL PEPPER (Capsicum annum) USING SUPERCRITICAL CARBON DIOXIDE. Millao, S. and Uquiche, E.

155

EXTRACTION/FRACTIONATION OF RAPESEED CAKE OIL USING SUPERCRITICAL CARBON DIOXIDE Salazar, K. and Uquiche, E.

156

GROWTH AND EXPRESSION OF CANDIDATE GENES INVOLVED IN MUSCLE DEVELOPMENT IN ADULT MALE ZEBRAFISH (Danio rerio) FED WITH TOTAL SUBSTITUTION OF FISHMEAL BY MIXTURE OF PLANT PROTEIN SOURCES DIETS Ulloa, P., Peña A., Iturra, P. and Neira, R.

157

Botryococcus braunii CAKE AS SOURCE OF PROTEIN: EFFECT OF OIL EXTRACTION METHODS Cancino, B., Verdugo, M., Morales, E., Franco, C. and Rubilar, M.

158

RHEOLOGICAL PROPERTIES OF EDIBLE COATINGS BASED ON BLENDS OF BIOPOLYMER SOLUTIONS ENRICHES WITH POLYPHENOLS FROM Ugni molinae TURCZ. Silva-Weiss, A., Bifani, V., Ihl, M. and Gómez-Guillen, C.

159

POLYPHENOLIC COMPOUNDS FROM MURTA LEAF SEPARATED BY GEL CHROMATOGRAPHY: APPLICATIONS IN LINSEED OIL AND EMULSIONS Barra, A., Palma, B., Acevedo, F., Shene, C. and Rubilar, M.

160

EFFECT OF POLYPHENOLIC FRACTIONS OF MURTA LEAF ON OXIDATIVE STABILITY OF LINSEED OIL MICROENCAPSULATED BY SPRAY DRYER Sáez, R., Morales, E., Palma, B., Acevedo, F., Shene, C. and Rubilar, M.

161

2nd International Workshop, Universidad de La Frontera, Pucón, Chile, October 27-29, 2010.

“Advances in Sciences and Technology of Natural Resources”

1

Symposium 1

“Waste Management, Bioenergy and Carbon Sequestration”

Chairpersons:

Dr. Rodrigo Navia and Dr. María de la Luz Mora.

Oral presentations:

- Dr. Ângela Abreu, Institute for Biotechnology and Bioengineering, Centre of Biological

Engineering, University of Minho, Portugal. “Engineered heat treated methanogenic granules, a promising approach for extreme thermophilic biohydrogen production”

- Dr. Hermann Heipieper, Helmholtz, Centre for Environmental Research-UFZ:

"Biotechnological processes for using waste and microbial oils in biodiesel production”

- Dr. Cornelia Rumpel, Laboratory of Biogeochemistry and Ecology of the Earth (BIOEMCO), INRA Paris Grignon, France: “Carbon sequestration in top- and subsoil horizons: what is the difference?”

- Dr. Nanthi Bolan, University of South Australia, South Australia: “The management of

phosphorus in poultry litter”.



2



3

ENGINEERED HEAT TREATED METHANOGENIC GRANULES, A PROMISING APPROACH FOR EXTREME THERMOPHILIC BIOHYDROGEN PRODUCTION

Abreu, Ângela A.*

Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal. *Corresponding author: [email protected]

The search for new alternative fuels is a priority and hydrogen is a promising clean energy alternative to fossil fuels, since water is the only reaction product of oxidation. The most important advantage of dark fermentation is that the substrate can be supplied by several carbohydrate and/or starch-rich wastes/wastewaters, as well as cellulose-rich biomass, performing the dual function of treatment and energy production. High rate anaerobic reactors such as the expanded granular sludge bed (EGSB) reactor can be adapted for hydrogen production. The microbial selection and development of H2 producing granules from suspended sludge inocula, requires a long start-up time, which is a major drawback of this technology. Therefore, selecting the microbial communities present in matured/developed anaerobic granules towards hydrogen production, by means of environmental pressure, is a potential strategy for start-up of high rate H2 producing reactors. In the present study, it was compare two different start-up strategies for extreme thermophilic EGSB reactor systems. The first EGSB reactor was inoculated with heat treated methanogenic granules (HTG), and the second with engineered heat treated methanogenic granules (EHTG), obtained by contact of HTG with an enriched hydrogen producing culture. The two strategies were compared in terms of hydrogen production rate, process stability and bacterial community structure. Synthetic medium used in this study contained glucose and arabinose, components of various plant biopolymers (hemicellulose and pectin) and lignocellulosic hydrolysates with a significant potential for biohydrogen production. The presence of active hydrogen producers in the REHTG system during the reactor start-up resulted in the development of an efficient H2-producing bacterial community. The results showed that “engineered inocula” where known hydrogen producers are co-inoculated with HTG is an efficient way to start-up biohydrogen producing reactors.



4

BIOTECHNOLOGICAL PROCESSES FOR USING WASTE AND MICROBIAL OILS IN BIODIESEL PRODUCTION

Heipieper, H.J.1,*, Azócar, L.2, Ciudad, G.2 and Navia, R.2,3

1Department of Environmental Biotechnology, Helmholtz Centre for Environmental Research - UFZ, Permoserstrasse 15, 04318 Leipzig, Germany. 2Núcleo Científico Tecnológico en Biorrecursos, Universidad

de La Frontera, Casilla 54-D, Temuco, Chile. 3Departamento de Ingeniería Química, Universidad de La Frontera, Casilla 54-D, Temuco, Chile. *Corresponding author: [email protected]

From the beginning of the last decade there is a pressure on the European Union and other states worldwide to replace transportation fuels with those produced from renewable energy sources. Regulations regarding greenhouse gases emission included in the Kyoto protocol and dependency of the energy sector on imported crude oil forced the European Commission to setup a program to promote the use of biofuels and other renewable fuels for transport. By the year 2020 the aim of the European Commission is to replace as much as 20% of transportation fuels with alternative fuels deriving from renewable energy sources. However, due to the slow increase of the biofuel content until 2005, this aim was recently reduced to the minimum of 10% of transport fuel in each EU member state to be provided by biofuels by the year 2020 as a feasible and realistic objective. One of the first generation biofuels strongly promoted in Europe is biodiesel (Directive 2003/30/EC, 2003). It is mainly produced in the base-catalyzed transesterification process where triglyceride lipids (fatty acid glycerol esters) react with methanol yielding the corresponding fatty acid methyl esters, glycerol and water. Biodiesel in the form of fatty acid methyl esters (FAME) derived from vegetable oils is gaining more and more interest as a substitute for petroleum diesel fuel. Because of the risk of contamination with diesel oil containing significant amounts of biodiesel additive, biodegradation experiments were carried out with diesel/biodiesel blends, indicating that such blends are better degradable than pure diesel fuel. Next to these promising aspects, biodiesel has also major concerns. Biodiesel is mainly produced from edible vegetable oils. Therefore, crop soils are used for its production, increasing deforestation and producing a fuel more expensive than diesel. In order to avoid this critical socio-economic aspects of biodiesel production, the use of alternative sources for biodiesel production is an aspect for intense research. Hereby, waste lipids such as waste frying oils, waste fats and soapstock has been proposed as low-cost alternative to feedstocks. Non-edible oils such as jatropha, pongamia and rubber seed oil are also economically attractive. In addition, microalgae, bacteria, yeast and fungi with 20% or higher lipid content are oleaginous microorganisms known as single cell oil and have been proposed as feedstocks for biodiesel production. Alternative feedstocks are characterized by their elevated acid value due to the high level of free fatty acid (FFA) content, causing undesirable saponification reactions when an alkaline catalyst is used in the transesterification reaction. The production of soap consumes the conventional catalyst, diminishing FAME production yield and simultaneously preventing the effective separation of the produced FAME from the glycerin phase. These problems could be solved using biological catalysts, such as lipases or whole cell catalysts, avoiding soap production as the FFAs are esterified to FAME. In addition, by-product glycerol can be easily recovered and the purification of FAME is simplified using biological catalysts.



5

References

- Azócar L., Ciudad G., Heipieper H.J., Navia R. (2010). Biotechnological processes for biodiesel production using alternative oils. Appl. Microbiol. Biotechnol. 88:621-636.

- Azócar L., Ciudad G., Heipieper H.J., Muñoz R., Navia R. (2010). Improving fatty acid methyl esters production yield in a lipase-catalyzed process by using waste frying oils as feedstock. J. Biosci. Bioeng. 109:609-614.

- Owsianiak M., Chrzanowski L., Szulc A., Staniewski J., Olszanowski A., Olejnik-Schmidt A.K., Heipieper H.J. (2009). Biodegradation of diesel/biodiesel blends by a consortium of hydrocarbon degraders: Effect of the type of blend and the addition of biosurfactants. Bioresour. Technol. 100:1497-1500.



6

CARBON SEQUESTRATION IN TOP- AND SUBSOIL HORIZONS: WHAT IS THE DIFFERENCE?

Rumpel, Cornelia*

CNRS, BIOEMCO, Campus AgroParisTech, Thiverval-Grignon, France. *Email address: [email protected]

Despite their low carbon content, most subsoil horizons contribute to more than half of the global soil carbon stocks. Generally, carbon in deep soil horizons is characterised by a high proportion of carbon compounds with mean residence times of more than thousand years. The reasons for these long residence times are only partly understood. The aim of this presentation is to give an overview over composition, mechanisms of stabilisation and decomposability of this deep soil organic matter (SOM) and to compare it to the topsoil. More than in topsoil, the chemical composition of SOM in subsoils is soil-type specific and greatly influenced by soil inherent pedological processes. With few exceptions the radiocarbon activity and stable isotope ratios of SOM are decreasing with increasing soil depth, indicating that organic matter in deep soil horizons is highly processed and stabilised. Similar stabilisation mechanisms are operating in top- and subsoil horizons. Interaction with the mineral phase, in particular amorphous Fe and Al oxides was reported to be the main stabilisation mechanism in top- and subsoil horizons of acid soils. Similar to topsoil horizons occlusion within soil aggregates has been identified to account for a great proportion of SOM preserved in subsoils. Low C/N ratios as well as higher contributions of microbial derived carbon compounds in subsoils compared to topsoil horizons indicate that stabilised carbon in deep soil horizons is highly transformed, being depleted in energy-rich compounds. SOM precursors are different in top- and subsoil horizons. Carbon input into subsoils occurrs in form of DOC following preferential flow pathways, root exsudates and necromass along root channels and/or through bioturbation in earthworm burrows. This leads to heterogenous distribution and horizontal stratification of stabilised carbon in subsoils. In contrast to topsoil, the spatial location may be extremely important for determining the participation of subsoil carbon in soil C and N cycles. The complexity of stabilisation and destabilisation processes operating in deep soil horizons as well as the heterogeneous nature of carbon distribution suggests that modeling of carbon turnover in subsoil horizons is quite different from those of SOM in topsoil horizons.



7

THE MANAGEMENT OF PHOSPHORUS IN POULTRY LITTER

Bolan, Nanthi 1,*, Szogi, Ariel2, Seshadri, Balaji1 and Chuasavathi, Thammared1

1Centre for Environmental Risk Assessment and Remediation, University of South Australia, SA-5095;

2USDA-ARS, Coastal Plains Soil, Water and Plant Research Center, 2611 W. Lucas St., Florence, SC, 29501. *Corresponding author: [email protected]

Poultry litter provides an important source of plant nutrients including nitrogen (N), phosphorus (P), potassium (K), calcium Ca), magnesium (Mg) and sulfur (S). The potential for P surplus at the farm scale can increase when farming systems change from cropping to intensive poultry and animal production, as P inputs become dominated by poultry and animal feed rather than fertilizer. Cost-effective and innovative solutions are needed to expand the range of acceptable options in the management of nutrients, especially P in poultry manure. This will involve refining feed rations, using feed additives to increase P absorption, managing P in the manure, moving manure from surplus to deficit areas, finding alternative uses for manure, and targeting conservation practices to critical areas of P export during land application. This paper gives an overview of various strategies used in managing P in poultry manure in relation to its disposal through land application.

1 Phosphorus input in poultry feed

Phosphorus is one of the essential minerals for all animals. It plays a critical role in cellular metabolism, as a part of the energy reservoir of the cell, in cellular regulatory mechanisms, and in bone development and mineralization. Through its involvement in these metabolic and structural processes, P is essential for animals to attain their optimum genetic potential in growth and feed efficiency as well as skeletal development. Of all the poultry species, the laying hen industry feeds typically much more P relative to the requirement, largely because of concerns of inadequate mineralization of egg shells and skeletal abnormalities resulting in poor egg production, morbidity, and mortality. One third of the P is present in the forage as inorganic P which is easily digestible. The other two thirds is present as organic P especially in the form of phytic acid and phytate. The phosphate stored in this way is not available for poultry and pigs but has to be hydrolyzed first. The most P rich components in the feed include mono-calciumphosphate, dicalciumphosphate and monosodiumphosphate. Typically, less than one-third of feed P is utilized by poultry, with the remainder excreted in manure and applied to land for crop use (Patterson et al., 2005). Phytic acid P is variably available to poultry (0 to 50%), and in order to meet the P needs of the bird, inorganic P must be added to the diet. The enzyme, phytase, can liberate much of this P. 2 Phosphorus in poultry litter

Among the various nutrients in poultry litter, N and P cause some environmental concerns. Phosphorus in poultry litter is present mainly in solid-phase as organic and inorganic P. The amount of total P in poultry litter varies with the diet and bedding material, and ranges from 0.3 to 2.4 % of dry matter. Fractionation studies have shown that a large proportion of P in poultry litter is in acid soluble fraction, indicating low bioavailability. Mineral species, such as struvite (MgNH4PO4.2H2O), octocalcium phosphate (Ca4H(PO4)3.3H2O) and dicalcium phosphate (CaHPO4.2H2O) have been identified in the solid fraction of poultry manure.



8

3 Phosphorus management

3.1 Feed management

Phosphorus in manure can be reduced by feeding the birds less P or treating feed to improve phosphorus utilization efficiency. Various feed and management strategies that reduce P in poultry litter have been investigated. The first of these strategies formulates feeds closer to the birds' actual P requirements. A second feeding strategy being tested is to use phytase, an enzyme which enhances the efficiency of P recovery from phytin in grains fed to poultry. Phytase breaks down the P-phytate bonds making the P available for absorption by the bird. Another approach is to increase the quantity of P in corn that is available to poultry by reducing the amount of phytate produced by corn. The use of low-phytate corn in poultry feed can increase the availability of P and other minerals and proteins that are typically phytate-bound. A combination of these strategies is expected to result in a reduction in excreta P.

3.2 Litter management

Litter management practices include the recovery and immobilization of P in the litter. Commercially available manure amendments, such as alum, can reduce NH3 volatilization, leading to improved animal health and weight gains; they can also reduce the solubility of P in poultry litter (Moore and Miller, 1994; Nichols et al., 1997; Bolan et al., 2010). For example, the dissolved P concentration (11 mg/L) of surface runoff from fescue treated with alum-amended litter was much lower than from fescue (83 mg/L) treated with unamended litter (Shreve et al., 1995). Undoubtedly, the most direct way to resolve P surpluses at a regional or watershed level is to simply transport poultry litter to geographic areas where P is needed for crop production (Sims et al., 2005). However, increasing hauling cost remains a major limitation for economic and environmentally safe P reutilization (Keplinger and Hauck, 2006). For manure P relocation to be sustainable, it is essential some form of processing that will decrease the manure volume, increase P concentration, and produce a more valuable product with alternative use options (Greaves et al., 1999). Poultry litter management trough treatment technologies that would reduce volume and increase nutrient content can be grouped into screening, densification, biological, thermochemical and chemical processes. Thermochemical processes use high temperatures to break the bonds of organic matter and reform intermediate compounds into synthesis gas, hydrocarbons fuels, and/or a charcoal residual (Cantrell et al., 2008). Solid residues from these processes are P-dense materials amenable to be reused as fertilizer. Combustion technology of poultry litter has received major attention world wide as a method to produce heat and electricity at large centralized facilities (Kelleher et al., 2002; Turnell et al., 2007). The byproduct of combusted poultry litter is ash with high P content that can be used as fertilizer or P supplement in poultry feed. However, major environmental concern with large centralized combustion facilities is the emission of nitrogen oxides, carbon monoxide and sulfur dioxide from the combustion of poultry litter that require effective gas cleanup (Turnell et al., 2007). A chemical treatment process, called “quick wash”, was recently developed for extraction and recovery of P from poultry litter and animal manure solids (Szogi et al., 2008). The quick wash process consists of three consecutive steps: 1) P extraction, 2) P recovery, and 3) P recovery enhancement. In step 1, organically bound P is converted to soluble-P by rapid hydrolysis reactions using selected mineral or organic acids. This step also releases P from insoluble inorganic phosphate complexes. The washed litter residue is subsequently separated from the



9

liquid extract and dewatered. In step 2, P is precipitated by addition of lime to the liquid extract to form a calcium-containing P product. In step 3, an organic poly-electrolyte is added to enhance the P grade of the product. The remaining solid residue (washed litter) has a more balanced N:P ratio that is more environmentally safe for land application and use by crops.

3.3 Soil management

Soil remediation involves either chemically fixing P so it is biologically inactive or using some process to remove the compound from the soil. Aluminum- (drinking water treatment residuals (WTR) treated with alum), iron- (red mud) and Ca-based (Coal combustion products) by-products have been shown to effectively reduce P solubility and transport to surface and ground water. Several types of best management practices (BMPs) have been proposed to utilize these by-products in efforts to reduce off-site P transport. Examples include surface applying in vegetative filter strips to reduce runoff P losses and incorporating into high P soils allowing for reductions in extractable P concentrations. For example, Basta et al. (2000) have shown that that WTRs incorporated into soil with high P concentrations caused a reduction in runoff P losses between 19 to 67% compared to controls. A significant reduction in extractable P between 10 and 91% occurred after WTRs were blended with the high P soil and poultry litter.

3.4. Nutrient management

The application of poultry manure based on crop N requirements is likely to provide more of other nutrients (especially P) than is required by crops. For example, the application of 9 tons/ha of broiler litter, a rate commonly used to meet the N requirements of agronomic crops, will provide approximately 270 kg/ha of N, 100 kg/ha of P, 165 kg/ha of K and Ca/ha, 45 kg/ha of S and Mg, and 2-5 kg of Mn, Cu and Zn. Depending on the crop species grown this may result in the accumulation of some of these nutrients, especially P in soils and their subsequent contamination of surface and ground water sources. Several best management practices (BMPs) have the potential to reduce nutrients in runoff water and loading to surface waters (Sharpley et al., 2007). They can be grouped into two broad categories: (1) technologies to reduce excessive nutrient levels in the soil, and (2) technologies to reduce discharges of nutrients via runoff or sediment loss from over-application of manure. For example, growing high biomass yielding plants can remove large amounts of nutrients and may be a promising remedial strategy to export and reduce excess soil nutrients. Bermuda grass and certain warm-season annual grasses produce large dry matter yields, and thus, take up large quantities of applied nutrients.

References

- Basta, N.T., Zupancic, R.J., and Dayton, E.A. 2000. J. Environ. Qual. 29: 2007-2012. - Bolan, N. S., A. A. Szogi, T. Chuasavathi, B. Seshadri, M. J. Rothrock Jr. and P. Panneerselvam 2010.

World Poultry Science Journal 66 (in press) - Cantrell, K.B., Ducey, T., Ro, K.S., and Hunt, P.G. 2008. Bioresour. Technol. 99(17): 7941-53. - Greaves, J., Hobbs, P., Chadwick, D. and Haygarth, P. 1999. Env. Technol. 20: 697-708. - Kelleher, B.P., Leahy, J.J., Henihan, A.M., O’Dwyer, T.F., Sutton, D. and Leahy, M.J. 2002.

Bioresour. Technol. 83: 27-36. - Keplinger, K.O. and Hauck, L.M. 2006. J. Agric. Resour. Econ. 31: 424-440. - Moore, P.A., Jr., and Miller, D.M. 1994. J. Environ. Qual. 23: 325-330. - Nichols, D.J., Daniel, T.C., Moore, P.A., Jr., Edwards, D.R. and Pote, D.H. 1997. J. Environ. Qual. 26:

1002–1006.



10

- Patterson, P.H., Moore, P.A., Jr., and Angel, R. 2005. In: Phosphorus: Agriculture and the Environment. J. T. Sims and A. N. Sharpley, Eds. American Society of Agronomy, Madison, WI. Pp. 635-682.

- Sharpley, A.N., Herron, S. and Daniel, T. 2007. J. Soil Water Conserv. 58, 30-38. - Shreve, B.R., Moore, P.A., Jr., Daniel, T.C. and Edwards, D.R. 1995. J. Environ. Qual. 24, 106–111. - Sims, J.T., Bergström, L., Bowman, B.T. and Oenema, O. 2005. Soil Use Manage 21, 141-151. - Szogi, A.A., Vanotti, M.B. and Hunt, P.G. 2008. Process for removing and recovering phosphorus

from animal waste. Patent Application Serial Nº. 12/026,346, USTPO. - Turnell, J.R., Faulkner, R.D. and Hinch, G.N. 2007. World’s Poultry Sci. J. 63, 223-231.



11

Symposium 1

“Waste Management, Bioenergy and Carbon Sequestration”

Poster presentations



12



13

SUCESIVES CYCLES OF ENZYMATIC BIODIESEL PRODUCTION IN A TERT-BUTANOL SYSTEM

Azócar, L.1*, Ciudad, G.1, Heipieper, H.2, Muñoz, R1. and Navia, R.1,3

1Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Casilla 54-D, Temuco,

Chile. 2Department of Environmental Biotechnology, Helmholtz Centre for Environmental Research-UFZ, Permoserstr. 15, 04318 Leipzig, Germany. 3Departamento de Ingeniería Química, Universidad de La

Frontera, Casilla 54-D, Temuco, Chile.

*Corresponding author: [email protected] The use of an anhydrous medium for fatty acid methyl ester (FAME) production in a lipase-catalyzed process was investigated to improve the reaction yield and final product quality. FAME production catalyzed by Novozym 435 was carried out using waste frying oil (WFO) as a raw material and methanol as an acyl acceptor, as well as 3Å molecular sieves for water removal. In addition, enzyme reuse in the anhydrous medium was studied. In a preliminary study which tested raw materials with different acid values, it was established that the acid value obtained in the final product was independent from the acid value of the raw material, but may depend on the water content during the reaction. Subsequently, using an anhydrous medium which resulted from water extraction with molecular sieves, FAME yield was improved by avoiding hydrolysis and the esterification reaction, producing FAME mainly through transesterification of triglycerides (TG). The acid value of FAME was significantly diminished by TG hydrolysis reaction inhibition. Additionally, a decrease in diglycerides (DG) content was observed, because the used sieves can produce acyl migration in the reaction, improving the performance of Novozym 435, which is a lipase with sn-3 specificity. Finally, neither the use of a hydrophilic washing step with acetone nor a hydrophobic washing step with WFO did produce the expected enzyme activity recovery. However, 17 successive cycles of FAME production in an anhydrous medium using the moderate polar solvent tert-butanol as a co-solvent demonstrated that Novozym 435 can be reused in an anhydrous medium. These results indicate that the anhydrous medium would enable lipase-catalyzed processes at industrial scale to produce biodiesel mainly through the transesterification reaction, which implies that different raw materials may be used to reach properties close to the standard. Acknowledgements: This work was supported by the Chilean PBCT-CONICYT project, TPI-16; Chilean FONDECYT Project 1090382, CONICYT project 79090009; and UFZ-Centre for Environmental Research Leipzig-Halle.



14

MONOMER FRACTION DURING STRONG MIX ACID PRETREATMENT OF ASPEN WOOD TO PRODUCE SUGAR

Ciudad, G.1*, Azócar, L.1, Janga, K.2 and Moe, S.2

1Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Casilla 54-D, Temuco,

Chile. 2Department of Chemical Engineering, NTNU, Sem Sælandsvei 4, 7491 Trondheim, Norway. *Corresponding author: [email protected]

In order to produce bioethanol from wood is necessary to breakdown hemicelluloses, form oligomer and opens the remaining structure (decristalization) for subsequent hydrolysis of cellulose to produce fermentable sugar. In this work was investigated the effects of strong mix acid decristalization condition on the oligomer and monomeric fraction from the decristalization and hydrolysis steps for aspen (a hardwood). Temperature (30, 50 and 70ºC), reaction time (2, 9 and 16 h) and percent of phosphoric acid in mix with sulfuric acid (50, 75 and 100%) were evaluated using a response surface methodology (RSM). The hydrolysis step was carried on at the same condition for all the experiment (4% of sulfuric acid and 100 ºC for 3 h). The monomeric fraction was composed by glucose 52%, xylose 30% and in less quantity of cellubiose and mannose. According to the RSM results, the most important parameter was the percent of phosphoric acid in the mix with sulfuric acid, been the best condition at 70% of phosphoric acid, 50ºC and 9 h (100% glucose yield). Over 70% of phosphoric acid was found pour breakdown of the cellulose, and less than 70% was found a degradation of the glucose for sulfuric acid. The experiment showed that at 50ºC and 70% of phosphoric acid the 50% of total glucose was provided for the decristalization step. On the other hand, when was used 100% of phosphoric acid, the main monosugar was xylose (from hemicellulose), indicating the no formation of oligomer during the decristalization steps. Acknowledgements: This work was supported by CONICYT project 79090009, Chilean PBCT-CONICYT project, TPI-16; Chilean FONDECYT Project 1090382 and the Paper and Fibre Research Institute PFI, Trondheim, Norway.



15

Candida rugose LIPASE IMMOBILIZATION ON BIOCHAR DERIVED FROM AGRICULTURAL RESIDUES

Cea, M.1,2,*, Sangaletti, N.3, Gonzalez, M.E.1 and Navia, R.1,2

1Scientific and Technological Bioresources Nucleus, 2Chemical Engineering Department, University of La

Frontera, PO Box 54-D Temuco, Chile. 2Doctorate Program in Sciences: Chemistry in Agriculture and Environment Center of Nuclear Energy in Agriculture – University of São Paulo - CENA/, Brazil.

*Corresponding author: [email protected] As alternative to the conventional NaOH-catalyzed biodiesel production, the enzymes, natural biocatalysts at nanometric scale, present as main advantage due to its high selectivity associated with a high production. However, the enzymatic stability has been one of the major problems for industrial application. For this reason, the enzyme immobilization is presented as an interesting alternative to alleviate these problems. The main objective of this work was to evaluate the possibility to use biochar derived from agricultural residues as support for Candida Rugosa lipase. Two different biochar produced by pyrolysis of oats husk at 300oC (BCA 300) and 450oC (BCA 450) pretreated or not with ethanol 99% (BAC 300et and BCA 450et) were studied. A volume of 2.5 mL of enzymatic suspension of Candida Rugosa lipase (10 mg enzymes/ml buffer phosphate 0.1 M, pH 7) was added to 100 mg of support. Samples were incubated at 30oC and 150 rpm. After 24 h immobilized enzymes were separated by filtration (Whatmann 41), washed thoroughly with Buffer phosphate 0.05 M pH 7 and dried at 30oC under vacuum. The supernatant and washing solutions were assayed for protein content according to the Bradford method to obtain the amount of immobilized enzyme. The activity of immobilized and free enzyme was analyzed spectrophotometrically measuring the increment in the absorption at 410nm promoted by the hydrolysis pNPP. The lipase unit was defined as the amount of enzyme necessary to hydrolysis 1 µmol of pNPP per minute. Lipase Novozyme 435 was use as control. The results indicated that both biochar were able to immobilize a similar amount of lipase (≈ 50% of the initial protein added). The activity of free enzymes was 1.4x103 U/g and Novozyme 435 was 372.9 U/g. Biochars BCA 300, BCA 300et, BCA 450 and BCA 450et showed 334.3, 335.4, 244.4 and 315.0 U/g of enzymatic activity, respectively. Differences between enzymatic activities of the immobilized lipase on biochar were less than 10.4, 10.1, 34.5 and 15.5% compared to Novozyme 435, respectively. It was observed a difference in activity between enzymes immobilized in support BCA 300 and BCA 450 of 26.9 %. The previous pretreatment of supports with ethanol did not showed a positive significant effect over the enzyme activity in BCA 300et support. However, the immobilized lipase on BCA 450et showed 22.4 % more activity than BCA 450. In this case the ethanol improved the behavior of immobilized lipase in BCA 450. Therefore, the biochar could be considered an economic alternative to be used as support for Candida Rugosa Lipase. Acknowledgements: This work was supported by FAPESP project Nº 08/58334-4 from Brazil, CONICYT project Nº 79090009, FONDEF D07I1096 and FONDECYT Project 10902382.



16

CELULAR DISRUPTION EFICIENCY OF SEVERAL METHODS TO PRODUCE TOTALS OILS FROM MICROALGAE Botryococcus braunii WITH PREVIOUS

EXTRACTION OF EXTRACELULAR HYDROCARBONS

Concha, E.1*

, Toro, C.2 and Navia, R.

2

1Doctoral Program in Sciences of Natural Resources, University of La Frontera, Temuco, Chile.

2 Chemical Engineering Department and Scientific and Technological Bioresource Nucleus, University of La Frontera,

Temuco, Chile. *Corresponding author: [email protected]

Energy production is a huge problem nowaday because of fosil fuels are diminishing. As consequence, alternatives fuels as biodiesel are becoming important. To produce biodiesel several sources of biomass can be used: animal fats, plants oils or some unicelular microorganism as algaes and bacterias. Using microalgae has some advantages, for example, they fix CO2 helping to mitigate global warming and they do not compete for agricultural land. There are several microalgae usable for biodiesel production, but among them Botryococcus braunii is a promising alternative, due to it presents high growing rate and high content of oils. Key stages in biodiesel production from B. braunii are: cultivation, harvest, celular disruption, oil extraction and transesterification. Celular disruption is considered a critical stage because an appropiate method increase the lipid extraction efficiency. To produce biodiesel, oils from C16-18 are used, but it has been suggested that C30–C37 botryococcenes and C31–34 methylated squalenes could be converted via catalytic cracking into shorter-length fuel-type oils (C16–18). Using solvents almost all this oils can be extracted from B. braunii, but if another celular disruption methods is efficient to set free oils, solvents are not needed. In the present work, various celular disruption methods were tested to compare their eficiency. The methods used were: (1) sonication (for 10 min at nominal power of 100 watts), (2) microwaves using a microwave oven (about 100°C) for 5 min, (3) osmotic shock using a solution of NaCl (10% w/v). Subsecuently the mixture was stirring by vortex for 1 min and allowed to settle maintained for 48 h, (4) autoclaving at 125 °C with 1.5 MPa for 5 min, (5) freezing at -18ºC for 24 h and (6) violent dried at 105ºC for 24 h. To compare celular disruption methods, for each one, three samples of 3.25 g of B. Braunii (15.41% dw) was mixed by vortex with glass beads and destiled water in 1:1:2 (w:w:w) proportion. Then heptane was added in 1:10 (w/w) proportion and mixture is vortexed. In this way, extracelular oils are separated from cells without damage. After that, three phases are observed: organic, water and a solid phase. Organic phase was separated, solvent was evaporated and oils weighed. Afterwards, each disruption method were applied on the same microalgaes samples. Oil was extracted again using heptane, it was evaporated and oil was weighed. Methods eficiency to produce celular disruption was calculated as follow: (oil weight produced after celular disruption) / (dry weigth of microalgae sample). A preliminar lipids profile of B. braunii race A, used in this study, has been obtained, indicating that hexa and octadecanoic acid are the most representatives. Previous studies (Lee et al., 2010) have showed that sonication and microwaves are the most eficient celular disruption methods.

Acknowledgments: This work was supported by the CONICYT through doctorate scholarship Nº 21100412, FONDEF research grant D07I1096 and Projects CONICYT Nº 79090009 and FONDECYT Nº 3090072.

References: - Lee, J.-Y., Yoo, C., Jun, S.-Y., Ahn, C.-Y., Oh, H.-M. 2010. Comparison of several methods for effective

lipid extraction from microalgae. Bioresource Technology 101: 575-577.



17

NEW SORBENT DEVELOPED FROM PETROLEUM COKE FLY ASHES AND ITS POTENTIAL APPLICATION

González, A.1,2,*, Moreno, N.3, Navia, R.2,4 and Querol, X.3

1Ph.D. Program in Sciences of Natural Resources, University of La Frontera, P.O. Box 54-D, Temuco, Chile 2Scientific and Technological Nucleus in Bioresources, University of La Frontera, P.O. Box 54-D, Temuco,

Chile. 3Department of Geosciences, IDAEA-CSIC, C/ Jordi Girona, 18-26, E-08034 Barcelona, Spain 4Department of Chemical Engineering, University of La Frontera, P.O. Box 54-D, Temuco, Chile

*Corresponding mail: [email protected] Heavy metals and acid effluents are an important source of environmental damage. It has been estimated that mining effluents have affected about 19300 km of streams and rivers, 72000 ha of lakes and reservoirs by different pathways all over the world. Current studies of Chilean rivers have revealed an increase in the concentrations of heavy metals during the last two decades. Several authors have suggested that mining discharges into Chilean rivers (e.g., Elqui), can be important to account for high concentrations of Cu, Zn, Cd, Fe, Mn and Pb. Chemical precipitation with alkaline raw materials is one of the most common technique to reduce heavy metals concentration in acid wastewaters. Nevertheless, a high number of investigations are focused on finding the most cost-effective alternative sorbents called non-conventional sorbents (NCS) for such purpose. Two interesting residues in Chile are petroleum coke fly ashes and black liquor, which are mainly constituted by inorganic and organic material, respectively. This study treats with the use of NCS in the neutralization and heavy metals removal from acidic wastewaters. Fixed bed column tests were performed in polypropylene columns packed with 6 g of NCS. The neutralization tests consisted of feeding the acid solution at pH 2 and 4 to the columns at a flow rate of 1.5 mL/min through the NCS bed using a peristaltic pump, whereas heavy metal removal tests used initial heavy metal concentration of 100 mg L-1 at a flow rate of 1.5 mL min-1. The output solution was collected and filtered for pH measurements. Major, minor and trace elements in output samples were analyzed by ICP-MS and ICP-AES. NCS residual material was also analyzed by means of X-ray diffraction (XRD) and scanning-electron microscopy coupled with EDX (SEM-EDX). In addition, a sequential extraction procedure (SEP) was also performed to the spent material. Neutralization fixed bed column tests showed higher buffering capacity of NCS at pH 4 than at pH 2, observing that main buffering zone for both tested pHs was around pH 6. After 17 days, NCS was still able to increase the wastewater pH, allowing us to consider NCS as a suitable neutralizer of acid wastewaters, especially at pH 4. Heavy metal fixed-bed removal tests showed that NCS is also a suitable sorbent material (maximum removal capacities of 8.1 and 28.3 mg g-1 for Cu2+ and Pb2+, respectively). Tests at pH 2 and 4 indicated that heavy metal removal is favored at pH 4. At similar initial conditions, Pb2+ removal capacity was higher than Cu2+. SEM-EDX showed that the main removal mechanism was chemical precipitation, detecting lead and copper carbonates onto NCS surface. This result was corroborated with experimental data obtained from the sequential extraction, detecting Cu and Pb extraction yields of around 80% in phases associated to carbonates from NCS. Taking into account the above mentioned results, it can be concluded that NCS seems to be an interesting option to neutralize wastewaters at pH 4, simultaneously removing Cu2+ and Pb2+ from acid wastewaters.



18

CHARACTERIZATION OF BIOCHAR FROM LIGNOCELLULOSIC WASTES

González, M.E.1, Cea, M.2,3, González, A.1, Toro, C.A.2,3, Diez, M.C.2,3and Navia, R.2,3

1Doctoral Program in Sciences of Natural Resources, Universidad de La Frontera, P.O. Box 54-D, Temuco, Chile. 2Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, P.O. Box 54-D,

Temuco, Chile. 3Department of Chemical Engineering, Universidad de La Frontera, P.O. Box 54-D, Temuco, Chile. *Corresponding author: [email protected]

The production of biochar is getting increased attention all over the world. Biochar is seen as a possible bioenergy source, partially avoiding global warming simultaneously promoting agriculture in unproductive zones. Therefore, it is important to assess the physical and chemical properties of biochar, especially if for its use as a soil amendment and sorbent application. The purpose of this work was to physically and chemically characterize a biochar produced from agricultural waste at different pyrolysis temperatures and to assess its potential application as soil amendment. Biochar was produced by pyrolysis of oat hulls at 300°C and 450°C and was used in the characterization experiments. Both biochars were characterized by their physical and chemical properties. Physical properties such as moisture, bulk density and morphology by scanning electron microscopy (SEM) were determined. Among the chemical properties, the following properties were determined: available macronutrients content, pH in water, point zero charge (pHPZC) by potentiometric titration and cation exchange capacity (CEC). The results indicate that the pyrolyzed biochar (300°C and 450°C) do not significantly differ on the moisture content and bulk density. However, SEM analysis showed that the biochar obtained at 300ºC has the highest pore volume. This effect of temperature suggest that biochar created at low temperature may be suitable for controlling the release of fertilizer nutrients (Day et al., 2005), whilst high temperature biochars would be more suitable for use as adsorbent (Ogawa et al., 2006). Chemical characteristics of both biochars showed differences at 300 and 450°C. As pyrolysis temperature increased, the pH values of the biochar increased, due to the thermal decomposition of biomass. Biochar obtained at 450°C presented a major content of available nutrients (free bases), such as Na, K, Ca and Mg. Moreover, an increase in these free bases is closely related to a CEC increase. Regarding to the pHPZC, both biochars showed an acid pHPZC or also called L-carbons due to its low pyrolysis temperature production (Moreno-Castilla and Rivera-Utrilla, 2001). From an agronomic point of view, an increment in the pyrolysis temperature has a positive effect on the available nutrients content, pH and pHPZC. However, the physical properties seem to be not influenced by an increase in temperature excepting on the biochar morphology. Acknowledgements: This work was supported by the CONICYT through doctorate scholarship, FONDEF research grant D07I1096 and Projects CONICYT Nº 79090009 and FONDECYT Nº 3090072. References:

- Day, D., Evans, R.J., Lee, J.W., Reicosky, D. 2005. Economical CO2, SOx, and NOx capture from fossil-fuel utilization with combined renewable hydrogen production and large-scale carbon sequestration. Energy 30: 2558–2579.

- Ogawa, M., Okimori, Y., Takahashi, F. 2006. Carbon sequestration by carbonization of biomass and forestation: Three case studies. Mitigation and Adaptation Strategies for Global Change 11: 421–436.

- Moreno-Castilla, C. and Rivera-Utrilla, J. 2001. Carbon materials as adsorbents for the removal of pollutants from the aqueous phase. Materials Research Society: 890-894.



19

PRODUCTION OF ELECTRICITY FROM A MIXED MICROBIAL CONSORTIUM USING A MICROBIAL FUEL CELLS

Hidalgo, P.1,2,* and Jeison, D.2,3

1Doctoral Program in Sciences of Natural Resources, Universidad de La Frontera, P.O. Box 54-D, Temuco,

Chile. 2Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, P.O. Box 54-D, Temuco, Chile. 3Department of Chemical Engineering, Universidad de La Frontera, P.O. Box 54-D, Temuco,

Chile. *Corresponding author: [email protected]

During the last years the development of energy from renewable sources, as the production of the electricity from microbial fuel cells (MFC) have generated great interest, due to the reduction of the reserve of fossil fuel, that have triggering a worldwide energy crisis. Microbial fuel cells are a special kind of fuel cells which directly convert chemical energy into electrical energy through the metabolic activity of microorganisms (Pant et al., 2010). MFC is formed by an anodic chamber and a cathodic chamber separated by a proton exchange membrane (PEM) (Min et al., 2005). The anode chamber contains microorganisms that oxidize the available substrate and release both the electrons (e-) and the proton (H

+) into medium. The electrons are released on the surface of the anode and flow through an external circuit to produce electricity and H+ are transported through the cation exchange membrane to the cathode. Finally, the electrons react at the cathode with H+ and O2, thus producing H2O (Du et al., 2007). In this research was evaluated the effect of organic loading and the salinity on the coulombic efficiency and power density of an MFC. The results were as follows:

Effect of organic loading rate (OLR). The increase in ORL from 1.1 to 2.2 g COD/L/d produced an increase in both power density and Coulombic efficiency. At an ORL of 2.2 gCOD/L/d maximum values for power (69 mW/m2) and CE (2.8%) were obtained. The increase of ORL beyond 2.2 gCOD/L/d caused an important reduction of both power density and EC. Increasing the ORL may produce a reduction of CE due to the growth of methanogenic microorganisms, which actively compete with anodophilic bacteria for the substrate.

Effect of salinity. The increase in NaCl concentration resulted in an increase in both power density and CE as long as NaCl concentration remained below 2 mM. At latter value a maximum power and CE were obtained (82.1 W/m2 and 15.91% respectively).However, at high salt concentration, cations present in the solution compete with protons in their migration to the cathode, reducing the efficiency of the MFC. References:

- Du, Z., Li, H., Gu, T. 2007. A state of the art review on microbial fuel cells: A promising technology for wastewater treatment and bioenergy. Biotechnology Advances 25(5): 464-482.

- Min, B., Cheng, S., Logan, B.E. 2005. Electricity generation using membrane and salt bridge microbial fuel cells. Water Research 39(9): 1675-1686.

- Pant, D., Van Bogaert, G., Diels, L. Vanbroekhoven, K. 2010. A review of the substrates used in microbial fuel cells (MFCs) for sustainable energy production. Bioresource Technology 101(6): 1533-1543.



20

CATALYTIC BEHAVIOR OF LIPASE NOVOZYME 435 IN THE FATTY ACID ETHYL ESTER (FAEE) PRODUCTION USING SOYBEAN OIL/ETHANOL EMULSION

Sangaletti, N. 1,*, Briones, R.2, Beltran, C.2, Cea, M.2,3, Navia, R.2,3

1Doctorate Program in Sciences: Chemistry in Agriculture and Environment, Center of Nuclear Energy in Agriculture – University of São Paulo - CENA/, Brazil. 2Scientific and Technological Bioresources Nucleus,

3Chemical Engineering Department, University of La Frontera, PO Box 54-D Temuco, Chile. *Corresponding autor: [email protected]

The extraction of oil with ethanol is a viable alternative to produce esterificable oil/ethanol emulsions. From the extraction of soybean oil with ethanol we obtained two phases: The first one rich in oil (rich emulsion) and the other one rich in ethanol (poor emulsion). The rich emulsion is composed of oil (92%), ethanol (6%), unsaponifiable material (1%) and water (1%). Lipases enzymes are considered as clean alternative catalysts because they do not generate waste in the production of fatty acid esters. To avoid lipase inhibition by short chain alcohols, the stepwise addition the methanol has been proposed, however, ethanol can be less toxic than methanol in the presence of small amounts of water. The aim of this study was to evaluate the catalytic behavior of Novozyme 435 with and without stepwise addition of ethanol in a rich emulsion. The emulsion was characterized by using the methodology described in AOCS (2003). The transesterification experiments were carried out during 28 h. An ethanol/oil molar ratio of 4,5:1, Novozym 435 (9.5%), 40oC and stirring rate of 180 rpm were the operational conditions established. For the transesterification process the study was performed as follow: 1) Total addition of ethanol in one step at the beginning; 2) Ethanol added in two steps, initially one-third of the total ethanol amount and after 3 h the other two-thirds of ethanol amount 3) Ethanol added in three steps, where one-third of the total ethanol amount was added at the beginning, the second other one-third was added after 3 h and finally, the last one-third the total ethanol amount was added after 6 h . Sample aliquots were taken at 3 h, 6 h, 9 h, 24 h and 28 h and immediately stored at 4oC to stop the reaction and to later evaluate FAEE concentration. FAEE were determined using CG-MS chromatography (EN 1403). The rich emulsion presented an acid value of 0.5 mg KOH/g, oxidative stability of 14.0 h at 110ºC, iodine value of 120.9 g/100g, kinematic viscosity at 40oC of 22.46 mm2/s and specific gravity of 914.0 Kg/m3 at 20oC, thus justifying its use in enzymatic FAEE production. The studies performed with ethanol addition at different steps showed similar results, being the increment of FAEE produced similar in the three assays evaluated. For example, after 3 h of reaction FAEE (%) produced in the assays 1, 2 and 3 were 19.5, 15.5 and 9.1%, respectively. After 9 h the behavior was similar reaching for the assays1, 2 and 3 61.0, 69.2 and 49.8% of FAEE produced, respectively. Finally, after 28 h the FAEE obtained from the assays 1, 2 and 3 were 88.0, 85.7 and 84.4%, respectively. Although there are studies that show ethanol toxicity, in this case no decrease in FAEE production yield due to one-step addition of ethanol was detected. Similar FAME yields were reported by Azócar et al. (2010) using methanol and waste frying oils in one and two-step alcohol addition at the same operational conditions of our experiments, suggesting that the tested emulsion could be used as feedstock in biodiesel production using lipases as biocatalysts.

Acknowledgement. This work was supported by FAPESP project Nº 08/58334-4 from Brazil, CONICYT project Nº 79090009, FONDECYT Project 10902382

References - American Oil Chemists Society (AOCS). 2003. Official methods and recommended pratices of the American

Oil Chemists Society. 5ed. Champaign. - Azócar, L Ciudad, G., Heipieper, H. J., Muñoz, R., Navia, R. 2010. Improving fatty acid methyl ester

production yield in a lipase-catalyzed process using waste frying oils as feedstock . Journal of Bioscience and Bioengineering, 109(6), 609-614.



21

BIOGENIC METHANE PRODUCTION FROM MICROALGAE BIOMASS

Torres, A.1,3,* and Jeison, D.2,3

1Doctorate program in natural Resources Sciences, Universidad de la Frontera, Temuco, Chile. 2Department of Chemical Engineering, Universidad de La Frontera, Casilla 54-D, Temuco, Chile. 3Centre for Waste

Management and Bioenergy, Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Casilla 54-D, Temuco, Chile.*Corresponding author: [email protected]

Biogas formation from anaerobic digestion represents one of the most versatile types of bioenergy, since it can be produced from a wide variety of biomass sources, including organically polluted wastes and wastewater. In addition, the energetic yield of biogas production is notably higher than that of bioethanol and biodiesel, due to the demanding energetic processes involved in the production of liquid biofuels. Use of microalgae biomass as source of energy is generating increased worldwide interest. Such interest is based on higher microalgae productivities, when compared with land-based crops commonly used for biofuel production such as corn, sugar cane or rapeseed. Most current efforts to take advantage of microalgae as a source of bioenergy are directed to biodiesel production. However, a potentially low energetic yield is one of the main concerns. Biogas production from waste microalgae biomass, after oil extraction, is potentially feasible, and can considerably increase the energy yield from biomass. Therefore, it has been regarded as a necessary step in order to make biodiesel production from microalgae sustainable. The main objective of this study is to determine methane potential from microalgae biomass. Specific objectives are related to the evaluation of the effect of lipids extraction on methane yield and degradation kinetics. Even though lipids extraction will reduce methane yield, extraction process itself can act as a pre-treatment, facilitating the anaerobic degradation of waste microalgae biomass. A series of methane potential determinations will be carried out using batch micro-reactor systems of 250 mL, operated at 30°C, where microalgae wastes will be mixed with anaerobic biomass, responsible for methane production. Produced methane will be determined by means of liquid displacement using a NaOH solution to absorb carbon dioxide. The study includes the determination of methane potential of intact microalgae and the one after a lipid extraction procedure. Anaerobic degradation will be evaluated by means of biomass characterization pre and post digestion procedure. The results of the present research are expected to contribute towards sustainable energy production from microalgae, taking advantage of full energetic potential of this type of biomass. Acknowledgements: Conicyt scholarship N° 21100464. This proyect also will be financed support by INNOVA-CORFO / DESERT BIOENERGYconsortium.



22

EFFECT OF MICRO-AERATION ON BIOGAS QUALITY IN THE ANAEROBIC DIGESTION PROCESS

Valdés, F.1, 2,*, Hemmelmann, A.1,3, and Jeison, D.1,3,4

1Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Temuco, Chile. 2Doctorate

Program in Science of Natural Resources, Universidad de La Frontera, Temuco, Chile. 3Center of Waste Management and Bioenergy, Universidad de La Frontera, Temuco, Chile. 4Department of Chemical

Engineering, Universidad de La Frontera, Temuco, Chile. *Corresponding author: [email protected]

Introduction Renewable energies are called to play a significant role towards the mitigation of current and future energy global demand. Bioenergy represents nowadays the most important source of renewable energy. Among biofuels currently available, biogas represents an interesting alternative, since it can be produced from a wide variety of biomass sources, including wastes. Biogas can be used for different purposes, such as heat and electricity generation, household use, vehicle fuel, etc. Each of those applications requires different levels of biogas purification. Most of them require the removal of H2S, since it can cause serious corrosion problems. Biogas purification increases biogas production costs and increases process complexity. H2S content in the biogas can be controlled providing controlled levels of O2, acting as electron acceptor for H2S oxidation. Then, operation of anaerobic digesters under micro-aerating conditions should reduce H2S content of the biogas, reducing purification requirements.

General aim: To determine the feasibility of controlling the H2S-content in biogas, by means of the modification of operational digester conditions by micro-aeration.

Specific aims - To evaluate the effect of micro-aeration on H2S content of the biogas. - To develop and validate a strategy for micro-aerating anaerobic digesters in order to promote

sulfide oxidation.

Materials and methods: Two anaerobic reactors will be implemented. First reactor will be operated injecting controlled doses of oxygen in the headspace. Second reactor will be operated as control. Biogas quality and digesters performance will be evaluated and compared. Data obtained during reactors operation will be used for developing a strategy for online control of the sulfur content of the biogas, reducing the requirements for biogas purification or upgrading.

Acknowledgements: This work is part of the research "Metano biogénico para uso vehicular”. Supported by Conicyt-Fondef D08I1192. The author wants to thank to the “Comisión Nacional de Investigación Científica y Tecnológica” (CONICYT) for the doctoral scholarship.



23

HYPERBRANCHED POLYESTERS FROM RESIDUAL GLYCEROL FOR THE DEVELOPMENT OF POLYURETHANES

Toro, C.1,2,*, Valerio, O.2 and Navia, R.1,2

1Chemical Engineering Department and 2Scientific and Technological Bioresource Nucleus, University of La

Frontera, Temuco, Chile. *Corresponding author: [email protected] An alternative for diesel fuels is biodiesel, which is produced from renewable sources. The development of this type of biofuel means a great opportunity for the replacement of diesel fuels and at the same time to decrease the levels of imported oil usage in our country. Biodiesel is non-toxic, biodegradable, with a high heating value. Additionally is free of sulfides and aromatic compounds. Glycerol is the main by-product of biodiesel production. A ton of glycerol is generated for each 10 tons of biodiesel produced, so the availability of this waste has increased exponentially with the increase in biodiesel production. In consequence, a great amount of research is nowadays focused on the search of alternatives for the utilization of this waste. In the present work, glycerol has been used in the development of biomaterials, due to its characteristics as a functional monomer (B3 type monomer). Hyperbranched polyesters (HPs) synthesized from glycerol could have an important application as additive for polyurethanes, because of its similitude with dendritic polymers. In general, conventional polyurethanes have poor thermal and mechanical properties. However, HPs can improve its coating properties by including branched and/or crosslinked units, capable of modify properties such as viscosity and solubility of the polymer. Usually, HPs are synthesized by polycondensation reactions involving ABn type monomers wich need multi-step complex synthesis methodologies, increasing costs and production times. In alternative way, A2 + B3 type monomers, due to its availability and low cost, represent an excellent alternative for the synthesis process. However, they present the inconvenience of high reactivity wich can lead to gel state polymers, limiting its use as a polyurethane modifier. The aim of this research work was the fabrication of HPs presenting different structural characteristics. The effect of the synthesis conditions of HPs on its conversion degree was studied, estimating empirical models wich relates highest conversion that can be achieved in the reaction without reaching gel point and process conditions. The study was planned using the methodology of factorial design of experiments. This work showed that the most important factor influencing the conversion degree of polymer is the monomers (glycerol and adipic acid) molar ratio. In this way, in the studied range, as molar ratio of monomers moves closer to the stoichiometric cuantities, higher conversion is achieved. Addicionally a model capable of predicting the conversion degree in the range of levels studied. Finally, the highest conversion degree achieved was 81%. This work is in concordance with previous results and contributes to the establishment of a correlation that permits to synthesize hiperbranched polyesters presenting predetermined properties. Acknowledgments.This work was supported by CONICYT-Chile, through FONDECYT Nº 3090072.



24



25

Symposium 2

“Environmental Biotechnology”

Chairpersons:

Dr. María Cristina Diez and Dr. Graciela Palma

Oral presentations:

- Dr. Nelson Lima, University of Minho, Portugal: “Textile dyes fungal mechanisms of biodegradation”.

- Dr. Susana Rodriguez-Couto, Universidad de Vigo, Spain: “Bioremediation with white-rot

fungi and their enzymes”.

- Dr. Claudia Benimeli, UNSTA, Argentina: “Organochlorine pesticides biodegradation by indigenous actinomycetes strains”.

- Dr. Nelson Durán, UNICAMP, Brazil: “Nanotechnology and its biological applications”.



26



27

TEXTILE DYES FUNGAL MECHANISMS OF BIODEGRADATION

Ottoni, Cristiane A., Santos, Cledir and Lima, Nelson*

IBB – Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Campus de Gualtar, 4710-057 Braga, Portugal. *Corresponding author: [email protected]

Reactive dyes are widely used in the textile industry. Coloured effluents from dyestuff and textile industries, the major producers and users of azo dyes, not only produce visual pollution but can also be detrimental to life, as they are usually resistant to biological treatment. Additionally, fungi, mainly white rot fungi, have shown the ability to degrade numerous aromatic organopollutants, including textile dyes, via oxidative mechanisms till their complete mineralisation, avoiding the formation of anilines as intermediates. In our work, textile mono-azo dyes were synthesised using aminobenzoic and aminosulphonic acids as diazo components and bioaccessible groups such as 2-methoxyphenol (guaiacol) and 2,6-dimethoxyphenol (syringol) as coupling components (Figure 1).

Figure 1. The eight mono-azo dyes synthesised and their chemical structures. The nomenclature presented in this figure, expressing the diazo component -> coupling component, is used in textile chemistry to suggest the synthesis process. The sigla used refer the kind of acid (carboxylic––C or sulphonic––S), its position relative to the azo bond (meta–m or para––p) and also the coupling component (guaiacol––g or syringol––s). The bioaccessible groups are present in the lignin structure and seem to be access points to the ligninolytic enzymes produced by white rot fungi. The fungal biodegradation of the azo dyes were studied in order to establish the relationship between the chemical structure of the dye and the extent of biodegradation. The rule of the non-specific fungal ligninolytic enzymatic system, lignin peroxidises (LiP), manganese peroxidises (MnP) and laccases (Lcc), as well as the enzyme glyoxal oxidase wich produce H2O2 for the activities of both peroxidases were studied. The di-azo Reactive



28

Black 5 and the anthraquinone-based polymeric dye Poly R-478 have been currently used to screen the fungal biodegradation under alkaline conditions (pH ≥ 8.0). In order to adapt the fungi to this alkaline condition a chemostat is now used. To perform this work the Trametes versicolor MUM 94.04, MUM 04.100, MUM 04.101 MUM 04.104 and MUM 04.105, Pleurotus ostreatus MUM 94.08, Phanerochaete chrysosporium MUM 94.15 (ATCC 24725) and MUM 95.01, Irpex lacteus MUM 98.04, Bjerkandera adusta MUM 99.04, Fomes fomentarius MUM 04.102 and Ganoderma applanatum MUM 04.103 were used and were supplied by the culture collection Micoteca da Universidade do Minho (MUM). As a conclusion, the results show a relationship between the extent of biodegradation and the chemical structure of the dyes either to be degraded or to be used in the pre-growth medium. The identification of two hydroxylated metabolites from the degradation of the most degraded dye, allowed the proposition of a metabolic pathway either when T. versicolor was used or Phanerochaete chrysosporium. Using these fungal bioaccessible azo dyes and due to the oxidative fungal mechanisms, no hazardous anilines, resulting from reductive cleavage of the azo bond were detected. The results obtained point to the advantages of the possible use of this ‘‘green’’ bioaccessible dyes in the textile industry, thinking of a biological effluent treatment using ligninolytic fungi. Finally, T. versicolor proved to be the most suitable fungus for the degradation of the dyes studied under alkaline conditions and the current results open the possibility to use chemostat to adapt the fungus to these conditions.



29

BIODEGRADATION WITH WHITE-ROT FUNGI AND THEIR ENZYMES

Rodríguez-Couto, Susana*

CEIT, Unit of Environmental Engineering, Paseo Manuel de Lardizábal 15, 20018 San Sebastian, Spain, IKERBASQUE, Basque Foundation for Science, Alameda de Urquijo 36, 48011 Bilbao, Spain.

*Email address: [email protected] About 100-150 million tons per year of chemical products are produced world-wide and every year about 1000 new compounds are put on the market. Most of them are synthetic, i.e. extraneous for the biosphere (xenobiotics). Such feature makes them “bioresistent” with the consequent accumulation and persistence in the environment. These compounds are difficult to remove with the existing technologies which have impelled the search for new ones. In this regard, white-rot fungi are particularly suitable for the development of such technologies. White-rot fungi are a physiological group comprising fungi that are capable of biodegrading lignin and the name white-rot derives from the white appearance of the wood attacked by them, where lignin removal gives a bleached appearance (Pointing, 2001). Four main genera of white-rot fungi have shown potential for bioremediation: Phanerochaete, Trametes, Bjerkandera and Pleurotus (Hestbjerg et al., 2003). The ability of white-rot fungi to degrade lignin is related to the secretion of non-specific extracellular ligninolytic enzymes during their secondary metabolism in response to nutrient limitation, namely lignin peroxidase (ligninase, LiP, EC 1.11.1.14), manganese-dependent peroxidase (MnP, EC 1.11.1.13), versatile peroxidase (VP, EC 1.11.1.16) and laccase (benzenediol/oxygen oxidoreductase, EC 1.10.3.2) along with other accessory enzymes. The same mechanisms that give these fungi the ability to degrade lignin are also used to degrade a wide range of recalcitrant pollutants such as polycyclic aromatic hydrocarbons (PAHs), pesticides, fuels, alkanes, polychlorinated biphenyls (PCBs), explosives and synthetic dyes. Of the above-mentioned enzymes, laccases have been subject of recent research interest due to (i) laccases present a better thermo-stability than LiPs and MnPs, (ii) laccases only require the presence of oxygen from air but neither manganese nor hydrogen peroxide and (iii) in the presence of appropriate substrates (redox mediators), which act as electron shuttles, laccases can also oxidise non-phenolic compounds (Srebotnik and Hammel, 2000; Astolfi et al., 2005). Laccases represent a family of copper-containing polyphenol oxidases usually called multi-copper oxidases. They catalyse the four electron reduction of oxygen to water accompanied by the oxidation of a substrate, typically a p-dihydroxy phenol or another phenolic compound (Xu, 1996). The utilisation of laccase enzymes at industrial scale has been hampered by the cost of production and deactivation of laccase by a wide variety of environmental conditions that characterise polluted sites (pH, salts, inhibitory molecules, extreme temperature gradients). Thus, efforts have to be made in order to achieve cheap overproduction of laccase in heterologous hosts and also their modification by chemical means or protein engineering to obtain more robust and active enzymes. Another additional problem is the cost and toxicity of redox mediators. Further investigations should consider different and less polluting mediators such as the natural mediators produced by laccase in a bio-environment during lignin degradation.



30

References

- Pointing, S.B. (2001). Feasibility of bioremediation by white-rot fungi. Applied Microbiology and Biotechnology 57:20-33.

- Hestbjerg, H., Willumsen, P.A., Christensen, M., Andersen, O., Jacobsen, C.S. (2003). Bioaugmentation of tar-contaminated soils under field conditions using Pleurotus ostreatus refuse from commercial mushroom production. Environmental Toxicology and Chemistry 22:692-698.

- Srebotnik, E., Hammel, K.E.(2000). Degradation of nonphenolicn lignin by the laccase/1-hydroxybenzotriazole system. Journal of Biotechnology 81:179-188.

- Astolfi, P., Brandi, P., Galli, C., Gentili, P., Gerini, M.F., Greci, L., Lanzalunga, O. (2005). New mediators for the enzyme laccase: mechanistic features and selectivity in the oxidation of non-phenolic substrates, New Journal of Chemistry 29:1308-1317.

- Xu, F. (1996). Oxidation of phenols, anilines, and benzenethiols by fungal laccases: Correlation between activity and redox potentials ace well ace halide inhibition. Biochemistry 35:7608-7614.



31

ORGANOCHLORINE PESTICIDES BIODEGRADATION BY INDIGENOUS ACTINOMYCETES STRAINS

Benimeli, C.S. 1,3.*, Fuentes, M.S. 1, Sáez, J.M.1, Bourguignon, N.1, Cuozzo, S.A. 1 and

Amoroso, M.J. 1,2,3

1PROIMI-CONICET, 2Fac. de Bqca., Qca. y Fcia. UNT, and 3UNSTA; Tucumán, Argentina. *Email address: [email protected]

Organochlorine pesticides (OPs) have been extensively used to protect and improve quality and quantity of food commodities, building materials, clothing, animal health, and to combat certain diseases transmitted by insect vectors to man and animals. However, indiscriminate use of pesticides has caused serious concern about toxic effects by residues on non-target organisms. The γ-HCH is an organochlorine pesticide used in agriculture and medicine to world level. It has a big tendency to bioaccumulate into the environment. There are many reports indicating that hexachlorocyclohexane (γ-HCH) is present in soil, water, air, plants, agricultural products, animals, food, microbial environments etc. Considered a potential carcinogen and listed as a priority pollutant by the US EPA, γ-HCH is a lipophilic compound and therefore tends to accumulate and concentrate in the body fats of animals and humans. In Salí River, the main hydrographic system of Tucumán, Argentina, lindane was detected 10-fold in relation to the traces permitted concentrations. Hence the development of new technologies to remediate these sites using microorganisms is every time more necessary. The actinomycetes are Gram positive bacteria with great potential to bioremediate xenobiotics. One strain, Streptomyces sp M7, isolated from organochlorine pesticides contaminated sediment was selected for its capacity to grow in the presence of lindane as only carbon source. This microorganism was cultured in soil extract medium added of lindane 100 μg L-1, obtaining a maximal growth of 0.065 mg mL-1, similar to the control, with a highest lindane removal of 70.4% at 30 ºC and pH 7. When different initial pesticide concentrations (100, 150, 200 and 300 μg L-1) were added in soil medium, an increment of the microbial growth was detected in all the concentrations tested. Also a diminution of the residual lindane concentration was determined in the soil samples in relation to the abiotic controls (29.1; 78.03; 38.81 and 14.42% respectively). Besides it was determined the optimum Streptomyces sp. M7 inoculum when lindane 100 μg Kg-1soil was added to the soil sample. It was 2 g Kg-1soil for obtaining the most efficiently bioremediation process, the lindane removal in these conditions was 67.8% at 28 days of incubation. Later it was considered necessary to know the pesticide effects on maize plants seeded in lindane-contaminated soil previously inoculated with Streptomyces sp. M7. Lindane concentrations of 100, 200, and 400 mg kg-1 soil did not affect the germination and vigor index of maize plants seeded in contaminated soils without Streptomyces sp. M7. When this microorganism was inoculated at the same conditions a better vigor index was observed and 68% of lindane was removed. These results confirm the potential lindane-contaminated soil bioremediation of Streptomyces sp. M7. It is important to notice that for the first time the lindane degradation without intracellular accumulation or biotransformation products of lindane using Streptomyces sp. under aerobic conditions was reported. There are some reports regarding aerobic degradation of γ-HCH by Gram-negative bacteria like Sphingomonas and by the white-rot fungi Trametes hirsutus, Phanerochaete chrysosporium, Cyathus bulleri and Phanerochaete sordida. However, little information is available on the ability of



32

biotransformation of organochlorine pesticides by Gram positive bacteria and particularly by actinomycete species. It was demonstrated that Streptomyces sp. M7 possesses the LinA enzyme that catalyzes dehydrochlorination of γ-HCH to 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN) via γ-pentachlorocyclohexene (γ-PCCH). The increase of γ-PCCH was detected in the time by Gas Chromatography (GC-MS). On the other side two actinomycetes defined consortia with lindane biodegradation capacity, were studied. They were prepared using isolated strains from soil samples of an illegal deposit of organochlorine pesticides in the locality of Santiago del Estero, Argentina. These consortia were: Streptomyces sp. A2-A5-M7-A11 and Streptomyces A2-A5-A8. Both produced a significant increment of the specific dechlorinase activity (SDA), compared to the individual culture with 1.66 mg L-1 of lindane as the only carbon source. Therefore actinomycete consortium strains could be considered one of the most promising bacterial groups for lindane biodegradation in contaminated environment. Finally, other organochlorine pesticides (chlordane and metoxichlor) are trying to be degraded using these new isolates of actinomycete strains. Highest growth and pesticide removal were observed when chlordane was added to the culture medium as the only carbon source. Twelve out of 18 studied strains released chloride into culture supernatants, and percentages were higher with chlordane as carbon source than with lindane or methoxychlor. These results were supported by principal component analysis. It is important to notice that for the first time it was demonstrated that actinomycetes isolated from an illegal storage of organochlorine pesticide in Argentina, have capacity to growth, remove and use chlordane or metoxichlor as the only carbon source.



33

Symposium 2

“Environmental Biotechnology”




34



35

DEGRADATION OF CHLORPYRIFOS AND DIAZINON BY ACTINOMYCETES ISOLATED FROM AGRICULTURAL SOIL OF SOUTHERN CHILE

Briceño, G. 1,3, Palma, G. 2,3 and Diez, M.C. 1,3

1Department of Chemical Engineering, 2Department of Chemical Science, 3Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, P.O. Box 54-D, Temuco, Chile. Phone /Fax: 056-45-

592126. *Corresponding author: [email protected] Organophosphates pesticides such as chlorpyrifos and diazinon are a group of highly toxic agricultural chemicals widely used in soil and foliar as insecticides. They acts as inhibitor of acetylcholinestererase in insects and could affect human nervous system. In Chile, chlorpiryfos and diazinon are used in the fruit production. Considerable attention has received the potential of contamination by pesticides. Contamination of soil can result from bulk handling in the farmyard, and rising of containers and accidental release could occasionally lead to contamination of surface and groundwater. The biobed, a simple on-farm construction intended to collect and degrade spills of pesticides, can be used to minimize the risk of contamination. Studies on microbial degradation are useful in the development of bioremediation strategies. The use of actinomycetes is an attractive approach, since these microorganisms are diverse and nutritionally versatile bacteria found in a wide range of habitats, present metabolic diversity and rapid rates of growth, therefore may be well suited for bioaugmentation of decontamination systems. The goal of this work was to evaluate the degradation of chlorpyrifos and diazinon by actinomycetes isolated from agricultural soil of southern Chile. The Streptomyces sp. strains AC-5, AC-6, AC-7 and AC-9 were exposed in liquid culture with chlorpiryfos and the strains AC-5, AC1-6, ISP-4 and ISP-13 with diazinon at concentration of 25 mg L-1. Biomass and residues of pesticides were analyzed. The results of this study showed that chlorpiryfos and diazinon were rapidly degraded by actinomycetes. GC analysis showed that four strains evaluated were able to degrade between 27-97 % of chlorpirifos. Strains AC-5, AC-9 and AC-7 showed the highest degradation being able to degrade 92.0, 93.0 and 97.2 % of chlorpirifos in 96 h, respectively. Similar results were obtained for diazinon, the degradation of this compound was between 72-95 % being the strains AC1-6 and ISP-4 able to degrade 97.7 and 88.5 %, respectively. Results of biomass analysis showed an increases of all strains when were added the insecticides, the exception was the strains AC-6 exposed at chlorpirifos. The strain AC-5 and AC-6 were less efficient to degrade diazinon and chlorpirifos, respectively, compared to other strains of actinomycetes evaluated. The degradation of chlorpyrifos was favored by the culture medium containing glucose, as this insecticide is degraded by co-metabolism. Similar degradation is reported for diazinon. Future analysis will be performed with strains more efficient in the degradation of organophosphorus pesticides to be used as biotechnological tools in the bioaugmentation of biobed for the decontamination of pesticides. Acknowledgements: Postdoctoral project FONDECYT Nº 3100118 and project FONDECYT Nº 1090678.



36

DETERMINATION OF CRITICAL MICELLE CONCENTRATION OF SOYA LECITHIN IN KIRK MEDIUM AND EFFECT OF ADDITION OF PENTACHLOROPHENOL

Bustamante, M.1, Rubilar, O.2 and Diez, M.C.2

1Doctoral Program in Science of Natural Resource, University of La Frontera.

2Department of Chemical Engineering, Environmental Biotechnology Center, University of La Frontera. Francisco Salazar 01145, Casilla 54-D, Temuco, Chile.

*Corresponding author: [email protected]

Soya lecithin (SL) is a surfactant of natural origin. This surfactant has been studied in biodegradation of hydrophobic organic contaminants. In this study, critical micelle concentration (CMC) of SL, and the effect of addition of several concentration of pentachlorophenol were determinates. Besides, SL effect on pentachlorophenol degradation by Anthracophyllum discolor in liquid medium was evaluated. The CMC was determinate using modified Kirk medium (without Tween 80) with a C/N ratio of 250, at pH 5.5, and 26ºC. The concentrations of pentachlorophenol on the CMC, and biodegradation assay were 5, 10, 15, and 20 mg L-1 of PCP. The biodegradation assays were carried out using Erlenmeyer flasks with modified Kirk medium (without Tween 80) with a C/N ratio of 250, LS addition of 0.3 and 0.6 g L-1, at pH 5.5 and incubated at 26ºC in static batch cultures. The culture medium was autoclaved at 121 ± 2ºC. Results showed that the CMC of SL in modified Kirk medium was 0.61 g L-1. The CMC value decreased as the concentration of PCP increased. In both concentrations of SL evaluated (0.3 and 0.6 g L-1) PCP degradation was enhanced. Briefly, 5 mg L-1 of PCP was degraded by A. discolor in 54.2% and 57.9% when the liquid medium was supplemented with 0.3 and 0.6 g L-1 of SL, respectively. The ligninolytic enzymes produced by A. discolor (laccase, manganese peroxidase, manganese independent peroxidase, and lignin peroxidase) did not exceed 2 U L-1 after of contamination with PCP, and without SL. The fungus A. discolor increased its ligninolityc activity in presence of SL, being the maximum lignin peroxidase and manganese peroxidase activities 27.2 ± 2.5 U L-1 and 3.9 ± 0.5 U L-1 with 0.3 and 0.6 g L-1 of SL, respectively. These results showed that SL enhanced the ligninolytic activity and PCP biodegradation. Acknowledgements: This work was supported FONDECYT 1090678 and partially by DIUFRO



37

LIGNINOLYTIC ENZYME ACTIVITIES AND COLONIZATION BY Anthracophyllum discolor ON LIGNOCELLULOSIC SUPPORTS

Elgueta, S.1,2,*, Lima, N.2 and Diez, M.C.1

1Center of Environmental Biotechnology, Scientifical and Technological Bioresource Nucleus, Universidad de La Frontera, Temuco, Chile. 2IBB- Institute for Biotechnology and Bioengineering, Centre of Biological

Engineering, Universidad do Minho, Campus de Gualtar, 4710-057, Braga, Portugal. *Correspodning author: [email protected]

The increasing expansion of agro-industrial activity has led to the accumulation of a large quantity of lignocellulosic residues from wood (e.g. wood chips), herbaceous (e.g. switchgrass), agricultural (e.g. wheat straw), forestry (e.g. sawdust) and various industrial wastes all over the world. These residues results in pollution of the environment and in loss of valuable materials that can be bioconverted to several added-value products. Recently, many studies have demonstrated the importance of utilizing lignocellulose residues for enhancing production of extracellular phenoloxidase enzymes by white-rot fungi. White-rot fungus Anthracophyllum discolor native of Southern of Chile was studied for their ability to growth and produce ligninolytic enzymes on different lignocellulosic support formulated from wheat straw, chicken feed and flaxseeds in order to identify their biodegradation potential. The ligninolytic enzymes laccase (Lac), manganese peroxidase (MnP), manganese independent peroxidase (MiP), and lignin peroxidase (Lip) were measured during the experiments. The colonization of these supports was measured for 12 at the beginning of the experiment. An approximately 1 g (wet weight) sub sample of mechanically disrupted substrate-biomass composite was extracted with 5 ml of 50 mM sodium malonate (pH 4.5). The centrifuged (14,900 g) samples were stored up to 24 h at 4º C prior to analysis. All enzymes were detected. MnP gave the highest activity in all supports and lower quantity the laccase was detected. The higher activity obtained for laccase, manganese-independent peroxidase and lignin peroxidase in Flaxseed support were 38.75; 144.42 and 243.72 mmol min-1 g-1, respectively. MnP in Chicken feed was 814.16 mmol min-1 g-1. The greatest growth colonization was observed on flaxseed support more than wheat straw and chicken feed. The main enzyme detected during experiment was MnP in all supports tested. Based on the results of experimental screening flaxseed have a potential for support material for biotechnological applications with Anthracophyllum discolor.

Acknowledgments: Financed by FONDECYT Nº1090678.



38

EFFECT OF LIGNOCELLULOSIC MATERIALS ON BIOLOGICAL ACTIVITIES IN BIOMIX OF BIOBED SYSTEM CONTAMINATED WITH ATRAZINE

Urrutia, C. 1,*, Tortella, G. 2 and Diez, M.C.2

1Graduate Program in Natural Resources Science, Universidad de La Frontera, Temuco, Chile. 2Department

of Chemical Engineering, Universidad de La Frontera, Temuco, Chile. *Corresponding author: [email protected]

Among the strategies suggested to reduce the punctual contamination of pesticides the biobed system is included. This is a simple and inexpensive design, which retains and degrades pesticide spills. It consists of a biomixture of straw, peat, and topsoil (50:25:25% v v-1) with a grass layer covering the surface. Straw is the main support for pesticide degradation by microbial activity especially white rot fungi, the soil provides sorption capacity and microorganisms that promote pesticide degradation and peat that contribute to sorption capacity, pH regulation, and moisture control. The effect of lignocellulosic materials on biological activity was studied in a biomixture of biobeds contaminated with atrazine. The lignocellulosic materials used were: barley husk (BH), sawdust (SD) and straw (ST) in different volumetric proportions. The traditional Swedish biomixture composition (straw:soil:peat 2:1:1 v v-1) was used as control. All biomixtures were pre-incubated during 15 days before being contaminated with atrazine (100 mg kg-1). After pesticide application, samples were extracted at 0, 10, 20, 30 and 40 days and microbial respiration, phenoloxidase activity, fluorescein diacetate (FDA) activity, and dehydrogenase activity were measured. Biomixtures prepared with BH in the proportion of BH: ST: soil:peat (50:0:25:25 and 25: 25:25:25% v v-1) and contaminated with atrazine showed high microbial respiration and dehydrogenase activity and low phenoloxidase activity in the first 30 days of incubation compared with the other lignocellulosic materials. The samples with BH without atrazine (control) showed a high level of phenoloxidase probably due to a great number of fungi that were affected initially by atrazine addition. The values of FDA showed a similar behavior in the different biomixtures being the less production associated with the biomixture with SD:ST:soil:peat (25:25:25:25% v v-1) and the high production with the biomixture BH:soil:peat (50:25:25% v v-1). However, further studies are in progress looking for the potential of the best lignocellulosic material to activate the lignin-degrading specific enzymatic system in order to obtain the highest degradation of the pesticide.

Acknowledgements: This study was financed by Fondecyt project Nº 1090678 and partially financed by Fondecyt project Nº 3090049.



39

METHOXYCHLOR BIOREMEDIATION BY INDIGENOUS ACTINOMYCETES USING PURE AND MIXED CULTURES

Fuentes, M.S. 1*, Sáez, J.M. 1, Bourguignon, N. 1, Cuozzo, S.A. 1, Benimeli, C.S.1,3 and Amoroso, M.J. 1,2,3

1PROIMI-CONICET, 2Fac. de Bqca, Qca y Fcia. UNT. and 3UNSTA. Tucumán, Argentina.


Persistent organic pollutants (POPs) are compounds highly stable and bioaccumulative that has been known as ubiquitous environmental contaminants. Methoxychlor (MTX), like others organochlorine pesticides (OCPs), were used for purposes like insect and malaria control, sanitation and harvest optimization. Some of the OCPs were banned, but others continue to be used illegally. MTX is a developed pesticide for use as a replacement for DDT which was prohibited internationally since the 1970s. DDT and MTX accumulate in the environment due to their resistance to biodegradation and are considered toxic and estrogenic, affecting to humans and mammals. MTX exerts only slight toxicity toward microbial populations in soil under aerobic conditions. The aim of this work was to evaluate the MTX removal capacity of isolated actinomycetes, individually and as mixed cultures under controlled laboratory conditions. Streptomyces sp. M7 (M7), Streptomyces coelicolor A3 (ScA3) and four indigenous actinomycetes isolates were cultivated individually in minimal medium (MM) with MTX for acclimation. These strains, as pure and consortia of two to six microorganisms, were cultivated in MM with MTX (1.66 mg L-1). Microbial cells were used to obtain cell-free extracts for dechlorinase activity assays and the supernatants from these cultures were used to determine residual MTX by gas chromatography. The isolates were characterized by 16S rDNA amplifications and sequenced. Enzyme activities ranged between 0.00 to 159.11 μmolCl-/h/mg protein and MTX removal percentages ranged between 72 to 100%. The mixed culture A3-A12- A14 showed the best activity but not the minimal residual MTX value. Because of no linear relationship between residual MTX and enzyme activity, the ratio between these two parameters was evaluated, and seven mixed culture with the minimal relationship were selected. Actinomycetes were mostly identified as members of Streptomyces genus. These native streptomycetes present ability to grow as microbial consortia and to remove MTX. Indigenous actinomycetes consortia could be a promising tool for MTX biodegradation.



40

THESIS PROJECT: STUDY OF FACTORS RELATED TO THE EMERGING VERMIFILTRATION BIOTECHNOLOGY FOR CATTLE SLURRY TREATMENT AND

VALORIZATION AS ORGANIC FERTILIZER

Morales, E.1,* and Bornhardt, C.2

1Doctorate in Sciences of Natural Resources. Universidad de La Frontera, Av. Francisco Salazar 01145, Temuco, Chile. 2Faculty of Engineering, Sciences and Administration. Universidad de La Frontera. Av.

Francisco Salazar 01145 - Fono: (56) (45) 325955, Temuco, Chile. *Corresponding author: [email protected]

Justification problem. Traditionally, cattle slurry is used as fertilizer, applied directly to pasture in order to increase forage production. This conventional procedure involves a risk of nutrient and pathogen leaching through the soil, risk of waterlogging by excessive applications and destruction of pasture by the high weight of the machinery used for spreading slurry, especially in rainy areas. In this context, it is necessary to implement a sustainable treatment system that uses its high fertilizing potential, reducing adverse environmental impacts. A simple biotechnology is called "vermifiltration", which is a green technology using earthworms to process highly loaded wastewater. This system is used for domestic and industrial wastewater treatment such as milk industries, yeast industries, pasta industries, vegetable/plant and fruit processing industries, sausage industries, vineyards, slaughterhouses, fishery and salmon ladder, timber industries and for slurry treatment in pork farms. However, no reports have been found in literature related to the application of this system for the treatment of cattle slurry.

Hypothesis. Vermifiltration is a complex biological system, which combines physical, chemical, microbiological and macrobiological mechanisms (consortia microorganisms-earthworms), to remove organic load and nutrients from wastewater. To describe the additional beneficial effects of earthworms compared with a conventional biofilter, the following hypotheses are formulated: a) Higher removal efficiency of a vermifilter is caused by the natural habit of earthworms to dig

galleries, which favors oxygen diffusion and wastewater percolation, avoiding clogging of the filter bed.

b) Depending on the feeding strategy, using a vermifilter to treat cattle slurry will allow to obtain an environmentally sound treated effluent and a concentrated solid organic fertilizer (humus).

General goal. To study the main mechanisms and operational conditions which affect the performance of a vermifiltration system for the treatment and valorization of cattle slurry as a concentrated solid organic fertilizer (humus). Specific goals a) To evaluate the earthworms’ contribution to aeration conditions and permeability of the bed and

the removal efficiency of organic matter and nutrients (N, P, K) under controlled conditions at laboratory scale,

b) To obtain the main operating and design parameters and long-term effects of a vermifilter used for the treatment and valorization of cattle slurry as a concentrated solid organic fertilizer (humus) under field conditions at pilot scale.



41

EFFECT OF BIOSTIMULATION OF AGRICULTURAL BIOBEDS ON CHLORPYRIFOS DEGRADATION

Tortella, G.R.1*., Rubilar, O.1, Cea, M.1, Wulff, C.1., Martínez, O.1 and Diez, M.C.1,2

1Scientific and Technological Bioresource Nucleus, 2Chemical Engineering Department. Universidad de La Frontera, PO Box 54-D Temuco, Chile.


The degradation of the insecticide chlorpyrifos (160 a.i mg kg-1) using a biomix of a biobed system biostimulated with inorganic fertilizer (NPK) was investigated. Three concentrations of fertilizer (0.1, 0.5 and 1.0% ww-1) were evaluated on chlorpyrifos degradation, TCP (3, 5, 6-trichloro-2-pyrinidol) accumulation and biological activity of the biomix. The chlorpyrifos was dissipated efficiently (>75%) after 40 days of incubation and no additional dissipation was obtained with increasing concentration of NPK after 20 days of incubation. TCP formation occurred in all concentrations of NPK evaluated and its concentration increased with the increment of NPK addition raising the probability of leaching of this compound. Biological activity (FDA and ligninolytic enzyme activity) in the biomix, increased by the presence of NPK in all concentrations evaluated. The DGGE analyses showed that combined treatments with lower amounts of NPK (0% and 0.1%) and chlorpyrifos did not show significant modifications in the microbial community in the biomix. However, combined overdoses of NPK (0.5 and 1.0%) and chlorpyrifos caused significant modifications in the bacterial communities that could be associated to the reduction TCP degradation in the biomix. In conclusion, the results obtained demonstrated that the biomix prepared with Andisol and biostimulated with NPK nutrient can be recommended in biobeds as a viable alternative of chlorpyrifos dissipation avoiding soil and water contamination probability. Acknowledgments: This study was financed by Fondecyt project Nº 3090049 and partially financed by Fondecyt project Nº 1090678.



42

ADSORPTION OF PENTACHLOROPHENOL (PCP) BY FUNGAL BIOMASS IN A FIXED-BED COLUMN

Rubilar, O.1*, Tortella, G.R.1., Cea, M1., Cuevas, R1. and Diez, M.C.1,2

1Scientific and Technological Bioresource Nucleus, 2Chemical Engineering Department. Universidad de La Frontera, PO Box 54-D Temuco, Chile. *Corresponding autor: [email protected]

The PCP adsorption by Anthracophyllum discolor in a fixed-bed column at different pH and PCP concentration was investigated. In a first stage the chemical characterization of the biomass by Infrared Fourier Transform Spectroscopy (FTIR) was evaluated. In a second stage, the adsorption of PCP (20, 30 and 50 ppm) at different pH (5.0, 5.5 and 6.0) was evaluated. The Thomas model was applied and the comparison of experimental and predicted adsorption breakthrough curves of PCP was done. The results of FTIR spectra of fungal cell wall showed the presence of different characteristics peaks in agreement with the possible presence of functional groups such as C-H, O-H, COOH, NH and C-O-C. These results can evidence that different interaction such as electrostatics, hydrophobic, van der Waals and hydrogen bonds could occur between fungal mycelial and the PCP molecule. With respect to the PCP sorption at different concentration and pH values, a diminishing of pH and PCP concentration of influent gave a later breakthrough curve being the column with influent at pH 5.0 and 20 mg L-1 of PCP, the assay that demonstrated a higher breakthrough and equilibrium. The negative effect of higher pH values on adsorption is due to the biomass increase its net negative charge, and also PCP increases its anionic form. Therefore, electrostatic repulsion between the biomass surface and PCP lead to a diminishing of adsorption. As PCP concentration increase the binding sites are quickly filled resulting in a decrease in breakthrough and equilibrium time. Tomas model was able to predict breakthrough curves and good fits were obtained in all cases with correlation coefficient ranging from 0.937 to 0.986. Therefore, on the base of the preliminary experimental results of this investigation can be concluded that biomass of A. discolor could be considered a material efficient to be used as adsorbent of PCP. Acknowledments: This study was financed by FONDECYT project N° 11090255.



43

Symposium 3

“Graduated Doctorates, Doctorate Program in Sciences of Natural Resources,

Universidad de La Frontera, Chile”

Chairpersons:

Dr. Paula Cartes and Dr. Francisca Acevedo

Oral presentations:

- Dr. Leonardo Vera, Pontificia Universidad Católica De Valparaíso, Chile: “Análisis clínico

de ecosistemas como base para el diseño y construcción del Parque Ecocultural Quebrada Verde”.

- Dr. Howard Langer, Agrícola Nacional S.A.C. (ANASAC), Chile. “Doctores en Ciencias de

Recursos Naturales, un caso de inserción a la empresa”.

- Roundtable Discussion: “Experience after Graduation”.



44



45

Symposium 4

“Soil Microorganisms and Rhizosphere”

Chairpersons:

Dr. Fernando Borie and Dr. Milko Jorquera

Oral presentations:

- Dr. José Miguel Barea, Consejo Superior de Investigaciones Científicas (CSIC), Spain:

“Mycorrhizas: an adaptive strategy for helping plant performance in a scenario of global change”.

- Dr. Concepción Azcón, Consejo Superior de Investigaciones Científicas (CSIC), Spain:

“Impact of arbuscular mycorrhizal symbiosis on plant health: an overview of the mechanisms involved”.

- Dr. Nuria Ferrol, Consejo Superior de Investigaciones Científicas (CSIC), Spain: “Soil heavy

metal remediation by arbuscular mycorrhizal fungi”.

- Dr. Jonathan Cumming, West Virginia University, USA: “Arbuscular mycorrhizal fungal population structure across a regenerating abandoned coalmine”.

- Dr. Petra Marschner, The University of Adelaide, Australia: “Microscale investigations in

the interface between rhizosphere and detritusphere”.

- Dr. Alan Richardson, Australian Commonwealth Scientific and Research Organization (CSIRO), Australia: “Biological availability of organic phosphorus in soil”.



46



47

MYCORRHIZAS: AN ADAPTIVE STRATEGY FOR INCREASING THE RESILIENCE OF THE SOIL-PLANT SYSTEMS IN A SCENARIO OF GLOBAL CHANGE

Barea, J. M.*

Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Prof.

Albareda 1, 18008 Granada, Spain. *E-mail address: [email protected]

1. Mycorrhizas: concepts and significance in the soil-plant systems

Mycorrhizas are symbiotic, generally mutualistic and balanced, associations established between certain soil fungi and most vascular plants where both partners exchange nutrients and energy. Basically, the host plant receives mineral nutrients via the fungal mycelium (which colonizes and links both soil and plant roots), while the heterotrophic fungus obtains carbon compounds from the host's photosynthates. In addition, mycorrhizas improve plant health through an increased resistance against environmental stresses, whether they be biotic (pathogen attack) or abiotic (drought, salinity, heavy metal contamination, organic pollutants, etc.). Moreover, mycorrhizas improve soil structure, through the formation of water stable aggregates necessary for a good soil till and, therefore, enhance soil quality. Arbuscular mycorrhiza (AM) is the most common mycorrhizal type which is formed by more than 80 % of the terrestrial plant species including almost all of those with agronomical interest, and herbaceous and shrub species typical from natural ecosystems. The AM fungi are ubiquitous microscopic fungi, whose origin and divergence have been dated back to more than 450 million years ago, and which critically contributed to plant origin and evolution on Earth. Mycorrhizas are fundamental not only to produce healthy crops (anti-stress agents) or to ensure self-sustainability of natural ecosystems but also to contribute to prevent erosion and to minimize environmental impacts. Thus, this symbiosis could be considered as an adaptive strategy to enhance the resilience of the soil-plant systems against perturbations. In this context, resilience may be defined as the ability of an ecosystem or plant community to return to pre-disturbance conditions, while adaptive strategy refers to managements to help adaptation to perturbations. These considerations are particularly relevant in the current scenario of global change, where diverse negative impacts are affecting the productivity of both agro- and natural eco-systems. 2. A global change scenario: concepts, causes and relationships with the management of soil-plant systems

The term global change refers to the set of environmental changes which, originated by human activities (aimed at satisfying a higher demand for the increasing human population), affect key processes for the Earth´ system functioning. A primary consequence of the global change is the climatic change, a concept that, in recent usage, refers to changes in “modern” climate, which operate additionally to those climatic changes naturally happen along Earth´ history. It may be qualified as anthropogenic or induced climate change, generally known also as global warming or anthropogenic global warming. Relevant to these anthropogenic factors is the increase in CO2 levels due to emissions from fossil fuel combustion, changes in land use and deforestation. When the concentration of this and other gases responsible for the greenhouse effect (fundamental for maintaining an appropriate Earth´ temperature) increases, more solar radiation is retained by the increased size of the greenhouse gases “layer”. The effect of this is to warm the Earth´ surface causing the induced climatic change.



48

A number of anthropogenic activities acting on the soil-plant systems are know to contribute to the global change. These include deforestation, intensive agriculture, landscape fragmentation, etc., which finally induce losses in agricultural and forestry productivity, ecosystem degradation, biodiversity losses, etc. Particularly, the supra-optimal application of agrochemicals in the so-called intensive agriculture causes several types of stresses for the soil-plant system. These perturbations include an increased incidence of pathogen (pest and diseases), drought, salinity, contamination by heavy metals (HMs) and organic pollutants, plant invasions, deficit in available nutrients, soil erosion, losses in microbial and plant diversity, etc. Science and Society are generating adaptive management strategies for plant-soil systems to enhance their resilience in a scenario of global change. These strategies are based on action/concept such as: sustainability, agro-ecology, ecosystem restoration, recovery of endangered flora, inducing resistance against environmental stresses, biodiversity conservation, etc. A component of such adaptive and proactive strategies is mycorrhiza management. 3. Mycorrhiza management: a sustainable strategy to mitigate, counteract and/or avoid negative impacts of the global change on the soil-plant systems

Considering the fundamental role of mycorrhizal fungi on nutrient cycling and their involvement in increasing plant resistance against environmental stress, management of micorrizal activities was proposed as a strategy to help sustainable environmentally-friendly agricultural and forestry developments. Basic, strategic and applied research has demonstrated that some mycorrhizal activities, as those able to benefit plant fitness and soil quality, can actually be exploited as a low-input agro-biotechnology with regard to mitigate, counteract and/or avoid negative impacts of the global change by increasing the resilience of the soil-plant systems. Mycorrhizal inoculum production is an obvious pre-requisite for application purposes. In this context, multiplication of ecto- and ectendo- mycorrhizal fungi (mushrooms or truffles), and their application in reforestation, is feasible. However, the difficulty to culture the obligate AM fungal symbionts in absence of their host plant is a major obstacle to produce AM inoculants. Nevertheless, appropriate amounts of AM inocula are currently being obtained for research needs, and a number of companies worldwide are producing plant-based AM inocula which are now commercially available for field applications. Research at our Department has largely contributed to understand functioning and management of AM fungi. The main milestones can be summarized as follow: (i) it was demonstrated, under field conditions, that AM fungi, in interaction with N2-fixing bacteria, improved N-cycling, N2 fixation and N transfer to non-fixing plants; (ii) also under field conditions, AM fungi, in interaction with phosphate-solubilizing micro-organisms, improved plant use of non-available mineral or organic P sources; (iii) inoculation of native AM fungi not only improved establishment and nutrition of target plants in re-vegetation trials, but also increased soil fertility and quality (enhancement of enzymatic activities and aggregate stability), stimulating natural plant succession; (iv) it was found that AM fungi increased plant tolerance to HMs and were effective to promote bioremediation of HM-contaminated soils; (v) by using mesocosm assays it was found that AM fungi play a key role in improving water uptake and to increase plant resistance to salinity, drought, and extreme temperatures; (vi) also in mesocosms trials it was demonstrated a systematically induced resistance in mycorrhizal plants against diseases and pest; (vii) it is being investigated the impact of MA in the recovery of the endangered/endemic flora in “diversity spots”, under environmental protection.



49

IMPACT OF ARBUSCULAR MYCORRHIZAL SYMBIOSIS ON PLANT HEALTH: AN OVERVIEW OF THE MECHANISMS INVOLVED

Pozo, M., López-Ráez, J.A., Jung, S. and Azcón-Aguilar, C.*

Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, Profesor Albareda 1, 18008 Granada, España. *Corresponding author: [email protected]

Arbuscular mycorrhizal (AM) symbioses are mutualistic associations between soil fungi and most vascular plants. During AM formation, AM fungi colonize the root cortex and develop an extraradical mycelium which overgrows the soil surrounding plant roots. This mycelium expand the interface between plants and the soil environment, contributing to the plant uptake of nutrients with poor mobility or present in low concentrations in the soil solution, as it is the case of phosphorus. In return for the improved nutrient supply, the heterotrophic fungus obtains carbon compounds from the host. The benefits of the AM symbiosis on plant fitness are largely known, including a better mineral nutrition and increased ability to overcome environmental stresses. The establishment of the AM symbiosis involves the exchange of recognition signals among both symbionts and the initiation of complex genetic programs. These programs drive changes in cell organization and metabolism which facilitate plant colonization by the microsymbiont and the physical and functional accommodation of both organisms. The global reprogramming of functions in both plant and fungal symbionts involves important changes in their physiology. Concerning the plant, these changes include alterations in its hormonal balance, increases in the photosynthetic activity, changes in the partitioning of carbon compounds among different plant organs, altered primary and secondary metabolism, etc. All these changes have an impact on the interaction of the plant with the environment, modifying its responses to biotic and abiotic stresses. Increased resistance to soil-borne pathogens has been widely described in mycorrhizal plants. Although few AM isolates have been tested in this regard, some appear to be more effective than others. Furthermore, the degree of protection varies with the pathogen involved and can be modified by soil and other environmental conditions. This protection has generally been shown to be effective against soil-borne nematodes, fungi, oomycetes and certain bacteria. In addition to localized effects, split-root experimental systems allowing physical separation between AM fungi and pathogens have confirmed plant mediated systemic effects. By contrast, effects of AM symbioses on shoot diseases are more contradictory. The outcome of the interaction of AM plants with shoot attackers ranges from enhanced resistance to increased susceptibility. Despite of this variability, general trends emerge from the multiple studies dealing with mycorrhiza in diverse pathosystems, suggesting that the result of the interactions largely rely on the lifestyle and challenge strategy of the attacker. Several are the possible mechanisms involved in the bioprotection exerted by AM associations: (i) improvement of plant nutrition; (ii) compensation for pathogen damage; (iii) competition for photosynthates; (iv) competition for colonization/infection sites; (v) anatomical or morphological AM-induced changes in the root system; (vi) microbial changes in rhizosphere populations of AM plants and (vii) elicitation of plant defence mechanisms by AM fungi. Their individual contribution to the final outcome will depend on the organisms involved and the timing of the interactions. Although elicitation of plant defence reactions by AM is limited, it still may make the root more prone to react against pathogens.



50

Among the potential mechanisms involved in the induction of resistance in mycorrhizal systems, the role of plant defenses is the most controversial. During mycorrhiza formation, modulation of plant defense responses occurs, potentially through cross-talk between different signalling pathways. This modulation may impact plant responses to potential enemies by priming the tissues for a more efficient activation of defense mechanisms. In this presentation the effects of the AM symbiosis on plant susceptibility/resistance to potential deleterious organisms will be discussed. Among the deleterious organisms, root and shoot microbial pathogens, root parasitic plants and phytophagous insects will be considered, highlighting the mechanisms that may be operating in each particular interaction.



51

SOIL HEAVY METAL REMEDIATION BY ARBUSCULAR MYCORRHIZAL FUNGI

Ferrol, Nuria*

Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, C. Profesor Albareda 1, 18008 Granada, Spain. *Email address: [email protected]

High concentrations of heavy metals in the soil have detrimental effects on ecosystems and are a risk to human health as they can enter the food chain via agricultural products or contaminated drinking water. To avoid the toxicity associated with these metals, several technologies and methods have been developed to remove them from polluted soils. Traditionally, these methods have included soil removal or extraction through chemical or physical means. Unfortunately, these techniques are costly from both an economic and an environmental point of view, and could potentially have a deleterious impact on soil physical, chemical, and biological properties. As an alternative to these traditional approaches, phytoremediation, that is, the use of plants to remediate heavy metal polluted soils is a clean and cost-effective technology that is likely to be readily accepted by a concerned public. Nevertheless, metal phytoextraction can be influenced by soil microorganisms living in intimate association with plant roots. Among soil microorganisms, mycorrhizal fungi are the only ones providing a direct link between soil and roots, and can, therefore, be of great importance in heavy metal availability and toxicity to plants. Arbuscular mycorrhizal (AM) fungi belong to the phylum of the Glomeromycota and establish mutualistic symbioses with more than 80% of terrestrial plants. They occur in most ecosystems, including heavy metal-polluted soils. The extensive extraradical hyphal network produced by these fungi allows the plant to access a great volume of soil, leading to the enhancement of plant nutrient absorption and translocation. Besides promoting plant growth, AM fungi can enhance plant tolerance to environmental stresses, including heavy metals. Mycorrhizal symbiosis can affect plant growth in heavy metal polluted soils by influencing the fate of the metal in the plant and also by increasing the tolerance of the plant to this type of stress. Reductions, increases and no changes in heavy metal concentrations in plants following mycorrhizal inoculation have been observed depending on the plant-fungus-heavy metal interaction. However, the significance of AM fungi in soil remediation has been widely recognized. Buffering heavy metal-stress by AM fungi had been assigned, at least partially, to selective immobilization of the metal within those root tissues containing fungal structures or to the high metal sorption capacity of the extraradical mycelium. Despite the significant role that AM fungi play in plant interactions with soil metals and the ubiquity of AM fungi in soil environments, only recently progress has been made towards understanding the cellular and molecular mechanisms used by AM fungi to control heavy metal homeostasis and to avoid their toxicity. Research performed in our laboratory have provided evidence that these mechanisms include avoidance strategies to restrict entry of toxic levels of the heavy metal into their cytoplasm, intracellular complexation of the metal in the cytosol and compartmentalization strategies. Through the activity of specific metal transporters, the excess of the metal is translocated to subcellular compartments, mainly vacuoles, where it would cause less damage. At the level of the fungal colony, AM fungi have also evolved compartmentalization strategies based on the accumulation of the metal into specific fungal structures, such as extraradical spores and intraradical vesicles. In addition to the avoidance and compartmentalization strategies, AM fungi have also evolved mechanisms to combat the oxidative stress produced by heavy metals or to repair the induced oxidative damage.



52

Alteration of the heavy metal content in mycorrhizal plants and, consequently, an improvement in plant tolerance may be also related to changes in gene expression as well as in protein synthesis induced by the symbiosis itself. To try to understand how metal tolerance, at the whole plant level, is affected by the establishment of the AM symbiosis we have carried out transcriptional profiling using an Affimetrix Maize Oligo Microarray. A comprehensive view of the root responses induced by the development of the symbiosis in the presence of heavy metals will be presented.



53

ARBUSCULAR MYCORRHIZAL FUNGAL POPULATION STRUCTURE ACROSS A REGENERATING ABANDONED COALMINE

Cumming, J.*, Hunter-Cevera, K. and Klugh Stewart, K.

Department of Biology, West Virginia University, Morgantown, WV, USA 26506. *Corresponding author: [email protected]

Arbuscular mycorrhizal fungi (AMF) are a critical part of ecosystems. A vast majority of all plant species form symbioses with AMF, and the amount and type of AMF present at a site can play a role in determining the vegetation community. Besides increasing nutrient uptake for their plant host, some AMF have the potential to provide host plants with resistance against metal toxicity. The establishment of vegetation on a site high in toxic metals may, therefore, be partly dependent upon the mycorrhizae present for its recovery. Little is known about how mycorrhizae succession occurs on disturbed sites, or how this can affect vegetation succession. In order to better understand what role mycorrhizae may play in the re-vegetation and ecosystem development of sites historically disturbed by coalmining, the diversity and abundance of mycorrhizal fungi on an abandoned acidic coalmine site were characterized. The abandoned coalmine site is located near Morgantown, WV, USA. The site was mined in the 1950s and abandoned shortly thereafter. On this site, natural patches of re-vegetation occur within a matrix of bare, open residuum. Within the re-vegetation patches, gradients exist from sparsely vegetated edges to dense grass swards dominated by Andropogon virginicus, to areas where small trees dominate the vegetation. Soils were characterized from bare, edge, sward, and tree microsites along the re-vegetation gradients and analyzed for chemical composition. Aluminium (Al) was further characterized by its availability and speciation in the soil solution. The AMF community present in vegetation gradients was characterized by studying spore populations in field samples and fungal trap cultures established on three host plant species, Andropogon virginicus, Trifolium pratense or Solidago canadensis. Fungi were identified by spore morphology with reference to voucher specimens from the INVAM Collection at West Virginia University. A measure of infection potential and soil parameters at each microsite were also analyzed. Soils were uniformly acidic (pH ~3.0) at the site, increasing by 0.4 units across the re-vegetation gradient. Soil organic matter increased with vegetation cover, although these measures are complicated by residual coal in the samples. Extractable Al increased significantly with vegetative cover from 190 mg kg-1 in bare soils to 656 mg kg-1 in the tree plots. However, patterns of pore water Al availability differed from extractable Al: Al declined across the vegetation gradient from 1006 µM to 21 µM and monomeric Al was 690 µM in bare soils and 17 µM in sward and tree plots. Such differences in toxic Al across the system may act as a selective force on AMF communities. AMF community structure in the field changed across the re-vegetation gradient. Eight species were tabulated in field samples; six varied across vegetation gradient and six species varied among replicate sites within the study area. Spore abundance increased along the re-vegetation gradient, but declined in tree soils. Acaulospora lacunosa was found in bare soils and dominated edge microsites, A. morrowiae was most abundant in sward microsites, and the abundance of A. koskei increased to its greatest in the tree microsites. Such trade-offs in abundance of Acaulospora species may reflect niche partitioning. AMF community composition as assessed by trap cultures also exhibited distinct patterns across the re-vegetation gradient. Total spore abundance in trap cultures was greatest in edge and sward



54

microsite soils. Viable spores of fourteen species were isolated from trap cultures. These species included Archaeospora trappei, Paraglomus occultum, Entrophospora contigua, E. colombiana, Glomus clarum, G. aggregatum, Gigaspora rosea, Acaulospora koskei, A. lacunosa, A. mellea, A. morrowiae, and three undescribed species. Two of these are tentatively assigned to Acaulospora accessions WV140 and WV 141. All species, with the exception of Gi. rosea, had significantly different probabilities of being present between microsites, i.e., distribution was affected by the degree of development of vegetation or the soil chemical environment along the re-vegetation gradient. Three species, P. occultum, E. contigua, and G. clarum, were significant in abundance in bare and edge microsites. The patterns of vegetation establishment, soil chemistry, and vegetation change on these abandoned coalmine soils suggests that colonization of abandoned coalmines and ecosystem recovery is facilitated by AMF community composition. Andropogon virginicus becomes established on these sites through symbiosis with Al resistant AMF species. The distributions of G. clarum, P. occultum, A. lacunosa, and E. contigua are consistent with this role. The establishment and long-term growth of A. virginicus leads to changes in soil chemistry, including the accretion of organic matter and nutrients and reductions in available Al in the soil solution. Such altered soil chemistry facilitates microsite colonization by additional AMF species and new plant species that would otherwise not invade the low pH and high Al environment of these residual coalmine soils.



55

MICROSCALE INVESTIGATIONS IN THE INTERFACE BETWEEN RHIZOSPHERE AND DETRITUSPHERE

Marschner, P.1,*, Marhan, S.2 and Kandeler, E.2

1Soils, School of Agriculture, Food and Wine, Faculty of Sciences, University of Adelaide, Adelaide,

Australia. 2Soil Science and Land Evaluation, University of Hohenheim. *Corresponding author: [email protected]

The soil influenced by the roots (rhizosphere) and the soil surrounding plant residues (detritusphere) are characterised by high concentrations of easily available compounds and therefore are hot-spots of microbial activity. Close to the roots or residues, microbial density and activity and enzyme activity are high and decrease with increasing distance; forming distinct gradients in mm scale. Moreover, microbial community structure changes with distance from roots or residues (e.g. Kandeler et al. 2001, Poll et al. 2006). Properties of the rhizosphere and detritusphere have been studied extensively, but separately. However, in the field, roots usually grow in the vicinity of decomposing plant residues. Therefore, it is important to study the interface between rhizosphere and detritusphere on a mm scale. We used a three-compartment pot system with maize on one side and soil with wheat residues on the other, each separated from the 5 mm middle compartment by a 50 µm mesh. Soil, plant and residues differed in 13C signature. The middle compartment was sliced into 1 mm sections. In each section, enzyme activity and microbial community composition by PLFA were measured. Using the differential 13C signatures, we tracked residue or root-derived C in the PLFAs. We hypothesised that: (i) the gradients in enzyme activity and microbial community structure of rhizosphere and detritusphere overlap leading to intermediate values in mid-distance from roots and residues, or (ii) the gradient from either roots or residues dominates, diminishing the gradient from residues or roots, respectively. Methods

Experimental set up

The experiment was conducted in a three compartment pot system with two outer compartments, each separated by a 50 µm mesh from a 5 mm wide middle compartment. The soil was an agricultural top-soil (Chernozem; Corg 1.4%; pH (CaCl2) 7.0; PCAL 93 mg kg−1; Ntotal 0.06%; KCAL 137 mg kg−1; soil texture: sand 12%, silt 66%, clay 22%) from a long-term field trial in Bad Lauchstädt (Germany) with rotations including only C3 plants. Soil was sieved to 2 mm and filled in all three compartments. The mature wheat residues were obtained from a Mini-FACE experiment conducted at the University of Hohenheim (Erbs and Fangmeier 2006). The shoot residues were cut into 2 cm length and mixed thoroughly into the soil of one outer compartments at a rate of 7.5 g kg-1 dry soil. Pre-germinated maize (Zea mays, cv Amadeo) was planted in the other outer compartment. The δ13C values were: wheat -44.1‰, soil -26.5‰ and maize roots -14.1‰. There were four treatments which differed in configuration of the outer compartments: unamended soil-un-amended soil (Soil-Soil), maize-un-amended soil (Maize-Soil), maize-soil with wheat residues (Maize-Residue), soil with wheat residues-un-amended soil (Residue-Soil). Four replicates of each treatment were harvested on day 14 and 23 after planting (DAP) of the germinated maize seeds. The middle compartment was frozen at -20°C and sliced into 1 mm vertical slices using a kitchen knife.



56

Results

Plant shoot and root dry matter increased 2.5 fold from 14 DAP to 23 DAP and did not differ significantly between the treatments. On 14 DAP, maize had formed a few roots close to the mesh of the middle compartment; by the second harvest, 23 DAP, a dense root mat had formed. Moreover, root density was very high in the lower part of the pots, resulting in relatively dry soil, suggesting that although the water content of the pots was maintained by weight, the water did not penetrate sufficiently into the lower part of the pots to compensate for water uptake by the roots. The sum of PLFAs, as a measure of active microbial biomass was increased in 1-2 mm distance from the roots and the residue-amended soil with approximately 30% greater values in the detritusphere than the rhizosphere. Compared to the un-amended soil, the concentration of bacterial, Gram-negative and Gram-positive bacterial fatty acids were not increased in the rhizosphere, but were nearly two-fold increased in 1-2 mm distance from the residue-amended soil. The most distinct gradient was found for fungal fatty acids, with 5-7 fold greater concentrations in the vicinity of roots and residue-amended soil than in the un-amended soil (Figure 2). Both roots and residue-amended soil strongly increased the fungal/bacteria ratio up to 4 mm distance. Gradients were generally more distinct at 14 DAP than at 23 DAP. In the Maize-Residue treatment, there was no overlap of gradients for bacterial and fungal fatty acids; however the gradients overlapped for the fungi/bacteria ratio, resulting in increased ratios compared to the un-amended bulk soil throughout the interface.

0.0

0.5

1.0

1.5

2.0

2.5

0.0

0.5

1.0

1.5

2.0

2.5

0.0

0.5

1.0

1.5

2.0

2.5

0.0

0.5

1.0

1.5

2.0

2.5Soil ‐Soil

Maize‐ResidueResidue‐Soil

Maize ‐Soil

M 0‐1 2‐3 S4‐53‐41‐2

M 0‐1 2‐3 R4‐53‐41‐2R 0‐1 2‐3 S4‐53‐41‐2

S 0‐1 2‐3 S4‐53‐41‐2

nmolg‐1

14 DAP

23 DAP

mm mm

Figure 1. Abundance of fungal PLFA (nmol g-1) in 1 mm slices of the middle compartment and the outer compartments in Soil-Soil, Maize-Soil, Residue-Soil and Maize-Residue treatments, with S: soil, M:root compartment, R: residue compartment.



57

Maize‐Residue

Residue‐Soilδ13C

‐36

‐32

‐28

‐24

‐20

‐16

‐36

‐32

‐28

‐24

‐20

‐16

M 0‐1 2‐3 R4‐53‐41‐2

R S0‐1 2‐3 4‐53‐41‐2

14 DAP23 DAP

mm Figure 2. δ13C in the fungal fatty acids C18:2ω6/C18:1ω9 in 1 mm slices of the middle and the outer compartments in Residue-Soil and Maize-Residue treatments, with S: soil, M:root compartment, R: residue compartment. The δ13C values of the PLFAs were only determined for the plant-residue amended soil and the residue-unamended soil treatments. Of all PLFAs, only i15:0, 16:1, cy17:0/17:1/i17:0, C18:0 and C18:2ω6/C18:1ω9 showed gradients in δ13C values (Figure 3 shows C18:2ω6/C18:1ω9). The δ13C values in 1 mm distance from the residue-amended soil were depleted strongly compared to those in the soil in greater distance whereas the δ13C values in the vicinity of the root compartment were only slightly enriched, suggesting that in the 14-23 days studied here, microorganisms incorporated residue-derived C to a greater extent than root-derived C. References:

- Erbs, M. and Fangmeier, A. 2006. Atmospheric carbon dioxide enrichment effects on ecosystems - experiments and the real world. Progress in Botany. Springer-Verlag Berlin Heidelberg, pp. 441-458.

- Frostegård A, Tunlid A, Bååth E .1993. Phospholipid fatty acid composition, biomass and activity of microbial communities from two soil types experimentally exposed to different heavy metals. Applied and Environmental Microbiology 59, 3605-3617.

- Kandeler E, Marschner P, Tscherko D, Gahoonia TS, Nielsen NE. 2001. Microbial community composition and functional diversity in the rhizosphere of maize. Plant and Soil 238, 301-312

- Marx MC, Kandeler E, Wood M, Wermbter N, Jarvis SC. 2005. Exploring the enzymatic landscape: distribution and kinetics of hydrolytic enzymes in soil particle size fractions. Soil Biology and Biochemistry 37, 35-48.

- Poll C, Ingwersen J, Stemmer M, Gerzabek MH, Kandeler E. 2006. Mechanisms of solute transport influence small-scale abundance and function of soil microorganisms at the soil-litter interface. European Journal of Soil Science 57, 583-595



58

BIOLOGICAL AVAILABILITY OF ORGANIC PHOSPHORUS IN SOIL

Richardson, Alan E.*

CSIRO Plant Industry, PO Box 1600 Canberra, ACT, 2601, Australia *Email address: [email protected]

Organic forms of phosphorus (P) in soil constitute a significant component of the total soil P. Organic P is central to the operation of the soil P cycle and as such may contribute a substantial supply of phosphate for plant nutrition (Richardson et al., 2005). Organic P is particularly important in natural ecosystems, various grasslands and other low inorganic P-input and/or organic farming-based systems (Magid et al., 1996). Cycling of P in soil is intimately associated with the activity of soil microorganisms (Richardson, 2001; Obersen et al., 2005; Richardson et al., 2009a). To be available to plants, organic P substrates must first by hydrolysed (or mineralized) by phosphatase enzymes to release orthophosphate. Despite this, our understanding of processes that contribute to the accumulation of organic P in soil and factors that regulate its mineralization and the subsequent availability of released phosphate to plants remains poor. More detailed knowledge of the interactions of organic P in soil is required in order to better manage P in agricultural environments. Indeed the importance of P to the sustainability of world agriculture and possible threats of ‘P scarcity’ to global food security has recently been highlighted (Cordell et al., 2009). Our research has focused primarily on the biological interactions of inositol phosphates (inositol hexakisphosphate and its lower order derivatives) in soil, as these monoester forms of organic P constitute a significant component of the identifiable fractions (Turner et al., 2002; Turner and Richardson, 2004; Turner et al., 2007). Various studies have shown that inositol phosphates are rapidly stabilized in soil through abiotic adsorption and precipitation reactions (Celi and Barberis, 2007). As a result, and in contrast to other forms of monoester and diester P (e.g., sugar phosphates, nucleic acids, phospholipids, etc), inositol phosphates are considered to be resistant to mineralization. We have shown that plants have limited capacity to directly access P from inositol phosphates even when supplied in soluble form (Hayes et al., 2000b, Richardson et al., 2000). Low biological availability of inositol phosphates in soil can therefore be attributed to various factors that include lack of extracellular phytase activity in roots, along with poor availability of substrate and low efficacy of enzyme-substrate interactions in soil environments (Richardson et al., 2007; George et al., 2007). Furthermore the availability of inositol phosphates to plants maybe influenced by the presence of soil microorganisms and their interactions in the rhizosphere (Richardson et al, 2001b; Richardson et al., 2009b). To further investigate the biological availability of inositol phosphates in soil, and as a possible means for improving the capacity of plants to their access, we have developed transgenic plants that over-express microbial phytase genes. These plants release phytase into the rhizosphere either constitutively or in response to P-deficiency when expressed with different promoters (Richardson et al., 2001a; Mudge et al., 2003). Plants that over-express phytase clearly show improved P nutrition when supplied with phytate and grown under controlled conditions without limitation to substrate availability (Lung et al., 2005; George et al., 2004). In contrast, a more limited P nutrition response has been observed when the plants have been evaluated in soils (George et al., 2004; George et al., 2005b; George et al., 2005c; Lung and Lim, 2006). Under such conditions improved plant growth and P nutrition has been observed, but only in soils with naturally higher levels of available substrate or where the availability of substrate is amended (George et al., 2005c).



59

Nonetheless such plants are important tool for investigating the bioavailability of inositol phosphates in soil. For example, and depending on various biochemical properties of the enzymes and other soil factors, we have shown that phytases (derived from either plants or microorganisms) can be rapidly immobilised or degraded in soil which has implication for their interaction with substrate (George et al., 2005a; George et al., 2007). Additionally we have shown that once adsorbed to soil mineral or clay constituents, inositol phosphates essentially cannot be hydrolysed by phytases (Giaveno et al., 2010). In contrast, P release appears to be enhanced by the presence of organic matter, although whether this occurs by direct hydrolysis of organic P itself, or through interactions of organic matter with adsorption reactions of inositol phosphates is unclear. The presence of organic anions may also significantly enhance the enzyme-lability of soil organic P, including inositol phosphates (Hayes et al., 2000a; Tang et al. 2006) and further work to investigate the basis of such interaction is warranted. Indeed enzyme-based assays have shown that most soils do contain a significant pool of phosphatase/phytase-labile P that can potentially be mineralized (George et al. 2006; George et al., 2007) and demonstrable phosphatase activities have been shown for most soils. Various studies have also shown enhanced phosphatase (and phytase) activities in the rhizosphere which in many cases this is associated with a significant depletion of organic P around plant roots (e.g., Gahoonia and Nielsen, 1992; Chen et al. 2002; George et al. 2002; Tarafdar and Jungk 1987). However, the relative contribution of different phosphatases of either plant or microbial origin (including those from mycorrhizal fungi), to this process is poorly understood along with precise knowledge of the chemical nature of the organic P substrates that are utilized. There is need to better link methodologies used to identify organic P in soil (e.g., through fractionation and extraction procedures, or by direct analysis of extracts using NMR) with pools and forms that are biologically available. Recent studies using in vitro assays with excess concentrations of different phosphatase enzymes indicate that, depending on soil type and fertilizer history, significant amounts of orthophosphate can be released from soil extracts/suspensions (George et al., 2007; Bünemann, 2008). Understanding of factors that influence the availability of different forms of organic P and whether it can be manipulated for agricultural benefit is therefore a major challenge and is of considerable interest. Whilst variation in natural levels of various extracellular phosphatase/phytase activities can be demonstrated for different plants (e.g., Tadano et al, 1993; Li et al., 1997; Hayes et al., 1999) it remains unclear if this variation can be exploited for greater mineralisation of soil organic P, or whether availability is controlled by other root-soil interactions or abiotic soil factors. For example, George et al., (2008) reported significant variation in the activities of various intra- and extra-cellular phosphatases (measured against a range of model and actual organic P substrates) for different wheat genotypes, but this did not account for any difference in P nutrition when plant growth was assessed over a range of soils. Benefits in organic P utilization derived from root phosphatases were therefore considered to be common to all genotypes or maybe negated and/or complimented by the activities of soil microorganisms. Distinction between these possibilities is needed. In conclusion it is evident that there is value in gaining a better understanding of the mechanisms and processes that influence the biological availability of organic P in soils. Organic P is central to the soil-plant-microbial P cycle and its management or manipulation may provide opportunity for improving the efficiency of P-use in agricultural systems and, in the longer term, the sustainable use of P across the globe.



60

References

- Bünemann EK (2008) Enzyme additions as a tool to assess the potential bioavailability of organically bound nutrients. Soil Biology & Biochemistry 40:2116-2129.

- Celi L, Barberis E (2005) Abiotic stabilization of organic phosphorus in the environment. In: Organic Phosphorus in the Environment (Eds., BL Turner, E Frossard, D Baldwin) CABI Publishing, Wallingford, pp.113-132.

- Chen CR, Condron LM, Davis MR, Sherlock RR (2002) Phosphorus dynamics in the rhizosphere of perennial ryegrass (Lolium perenne L.) and radiata pine (Pinus radiata D.Don). Soil Biology & Biochemistry 34:487-499.

- Cordell D, Drangert J.-O, White S (2009) The Story of Phosphorus: Global food security and food for thought. Global Environmental Change 19:292-305.

- Gahoonia TS, Nielsen NE (1992) The effect of root induced pH changes on the depletion of inorganic and organic phosphorus in the rhizosphere. Plant and Soil 143:185-191.

- George TS, Gregory PJ, Robinson JS, Buresh RJ, Jama B (2002) Utilization of soil organic P by agroforestry and crop species in the field, western Kenya. Plant and Soil 246:53-63.

- George TS, Gregory PJ, Simpson RJ, Richardson AE (2007a) Differential interactions of Aspergillus niger and Peniophora lycii phytases with soil particles affects the hydrolysis of inositol phosphates. Soil Biology & Biochemistry 39:793-803.

- George TS, Hocking PJ, Gregory PJ, Richardson AE (2008) Variation of root-associated phosphatase in wheat cultivars explains their ability to utilise organic P substrates in-vitro, but does not effectively predict P-nutrition in a range soils. Environmental and Experimental Botany 64:239-249.

- George TS, Quiquampoix H, Simpson RJ, Richardson AE (2007) Interactions between phytase and soil constituents: implications for hydrolysis of inositol phosphates. In Inositol phosphates: Linking Agriculture and the Environment (Eds, BLTurner, AE Richardson, EJ Mullaney) CABI Publishing pp. 221-241.

- George TS, Richardson AE, Hadobas PA, Simpson RJ (2004) Characterisation of transgenic Trifolium subterraneum L. which expresses phyA and releases extracellular phytase: Growth and P nutrition in laboratory media and soil. Plant Cell and Environment. 27:1351-1361.

- George TS, Richardson AE, Simpson RJ (2005) Behaviour of plant-derived extracellular phytase upon addition to soil. Soil Biology & Biochemistry 37:977-988.

- George TS, Richardson AE, Smith JB, Hadobas PA, Simpson RJ (2005) Limitations to the potential of transgenic Trifolium subterraneum L. plants that exude phytase, when grown in soils with a range of organic P content Plant and Soil 258:263-274.

- George TS, Simpson RJ , Hadobas PA, Richardson AE (2005) Expression of a fungal phytase gene in Nicotiana tobacum improves phosphorus nutrition in plants grown in amended soil. Plant Biotechnology Journal 3:129-140.

- George TS, Simpson RJ, Hadobas PA, Marshall DJ, Richardson AE (2007b) Accumulation and phosphatase-lability of organic phosphorus in fertilized pasture soils. Australian Journal of Agricultural Research 58:47-55.

- George TS, Turner BL, Gregory P, Cade-Menun B, Richardson AE (2006) Depletion of organic phosphorus from oxisols in relation to phosphatase activities in the rhizosphere. European Journal of Soil Science 57:47-57.

- Giaveno C, Celi L, Richardson AE, Simpson RJ, Barberis E (2010) Interaction of phytases with soil minerals and availability of substrate affects the hydrolysis of inositol phosphates. Soil Biology & Biochemistry 42:491-498.

- Hayes JE, Richardson AE, Simpson RJ (1999) Phytase and acid phosphatase activities in extracts from roots of temperate pasture grass and legume seedlings. Australian Journal of Plant Physiology 26:801-809.



61

- Hayes JE, Richardson AE, Simpson RJ (2000a) Components of organic phosphorus in soil extracts that are hydrolysed by phytase and acid phosphatase. Biology and Fertility of Soils 32:279-286.

- Hayes JE, Simpson RJ, Richardson AE (2000b) The growth and utilisation of plants in sterile media when supplied with inositol hexaphosphate, glucose 1-phosphate or inorganic phosphate. Plant and Soil 220:165-174.

- Li, M., Osaki, M., Rao, I.M. and Tadano, T. (1997) Secretion of phytase from the roots of several plant species under phosphorus-deficient conditions. Plant and Soil 195:161-169.

- Lung S-C, Chan WL, Yip W, Wang L, Yeung EC, Lim BL (2005) Secretion of beta-propeller phytase from tobacco and arabidopsis roots ehnances phosphorus utilization. Plant Science 169:341-349.

- Lung S-C, Lim BL (2006) Assimilation of phytate-phosphorus by the extracellular phytase activity of tobacco (Nicotiana tabaccum) is affected by the availability of soluble phytate. Plant and Soil 279:187-199.

- Magid J, Tiessen H, Condron LM (1996) Dynamics of organic phosphorus in soils under natural and agricultural ecosystems. In ‘Humic substances in terrestrial ecosystems’. (Ed. A Piccolo) pp. 429-466. (Elsevier Science: Amsterdam).

- Mudge SR, Smith FW, Richardson AE (2003) Root-specific and phosphate regulated expression of phytase under the control of a phosphate transporter promoter enables Arabidopsis to grow on phytate as a sole P source. Plant Science 165:871-878.

- Oberson A, Joner EJ (2005) Microbial turnover of phosphorus in soil. In ‘Organic phosphorus in the environment’. (Eds BL Turner, E Frossard, DS Baldwin) pp. 133-164. (CABI: Wallingford, UK).

- Richardson AE (2001) Prospects for using soil microorganisms to improve the acquisition of phosphorus by plants. Australian Journal of Plant Physiology 28:897-906.

- Richardson AE, Barrea JM, McNeill A, Prigent-Combaret C (2009a) Acquisition of phosphorus and nitrogen in the rhizosphere and plant growth promotion by microorganisms. Plant and Soil 321:305-339.

- Richardson AE, George T.S., Jackobsen I. and Simpson R.J. (2007) Plant access to inositol phosphates in soil. In Inositol phosphates: Linking Agriculture and the Environment (Eds, BLTurner, AE Richardson, EJ Mullaney) CABI Publishing pp. 242-260

- Richardson AE, George TS, Hens M, Simpson RJ (2005) Utilisation of soil organic phosphorus by higher plants. In ‘Organic phosphorus in the environment’. (Eds BL Turner, E Frossard, DS Baldwin) pp.165-184. (CABI: Wallingford, UK).

- Richardson AE, Hadobas PA, Hayes JE (2000) Phosphomonoesterase and phytase activities of wheat (Triticum aestivum L.) roots and utilisation of organic phosphorus substrates by seedlings grown in sterile culture. Plants Cell & Environment 23:397-405.

- Richardson AE, Hadobas PA, Hayes JE (2001a) Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate. The Plant Journal 25:641-649.

- Richardson AE, Hadobas PA, Hayes JE, O’Hara CP, Simpson RJ (2001b) Utilization of phosphorus by pasture plants supplied with myo-inositol hexaphosphate is enhanced by the presence of soil microorganisms. Plant and Soil 229:47-56.

- Richardson AE, Simpson RJ, George TS, Hocking PJ (2009b) Plant mechanisms to optimize access to soil phosphorus. Crop and Pasture Science 60:124-143.



62

- Tadano T, Ozawa K, Sakai H, Osaki M, Matsui H (1993) Secretion of acid phosphatase be the roots of crop plants under phosphorus-deficient conditions and some properties of the enzyme secreted by lupin roots. Plant and Soil 155/156:95-98.

- Tang J, Leung A, Leung C, Lim BL (2006) Hydrolyis of precipiatated phytate by threee distinct families of phytases. Soil Biology & Biochemistry 38:1316-1324.

- Tarafdar JC, Jungk A (1987) Phosphatase activity in the rhizosphere and its relation to the depletion of soil organic phosphorus. Biology and Fertility of Soils 3:199-204.

- Turner BL, Papházy MJ, Haygarth PM, McKelvie, ID (2002) Inositol phosphates in the environment. Philosophical Transactions of the Royal Society, London, Series B. 357:449-469.

- Turner BL, Richardson AE (2004) Identification of scyllo-inositol phosphates in soils by solution phosphorus-31 nuclear magnetic resonance spectroscopy. Soil Science Society of America, Journal 68:802-808.

- Turner BL, Richardson AE, Mullaney EJ (2007) Inositol phosphates: Linking Agriculture and the Environment CABI Publishing 288 pp.



63

Symposium 4





64



65

FLUORESCENCE DETECTION OF ALUMINIUM IN ARBUSCULAR MYCORRHIZAL FUNGAL STRUCTURES AND GLOMALIN BY USING LASER SCANNING CONFOCAL

MICROSCOPY

Aguilera, P.1,*, Seguel, A.1, Cornejo, P.2, Curaqueo, G.1, Meier, S.1, Medina, J.2, Berríos, D.2 and Borie, F.2

1Programa de Doctorado en Ciencias de Recursos Naturales. 2Center of Amelioration and Sustainability of Volcanic Soils. BIOREN-UFRO. Universidad de La Frontera, Avenida Francisco Salazar 01145, P.O. Box

54-D, Temuco, Chile. *Corresponding author: [email protected]

Arbuscular mycorrhizal (AM) fungi are obligatory symbionts in about 80% of the terrestrial plants, some of them growing in soils with severe limitations. Under these conditions, some studies have demonstrated that the AM symbiosis can protect plants against the stress produced by toxic levels of diverse metals. The Aluminium (Al) is one of the metals more incident in the plant’s citotoxicity because of the strong effect on plant growth decrease, especially in roots, which determine a reduced capacity for water and nutrient acquisition. Among the mechanisms influencing the Al phytotoxicity it has been suggested a severe damage in specific membrane lipids. In our group, recent evidences have shown a strong relationship between exchangeable Al levels and glomalin-related soil protein (GRSP) in soil (unpublished data). Therefore the aim of this work was to study the behavior of AM fungal structures and GRSP in Al presence and its possible role in diminishing the toxic effect on plants. There are several methods and techniques to identify AM fungi structures, among which are the display by confocal laser scanning microscopy (CLSM), some methods based on the autofluorescence exhibited by several AM fungi structures, mainly hyphae, while other studies have used fluorochromes to observe in vivo hyphae and arbuscules in roots. In this study we investigated the behavior of the AM fungal native spores isolated from rhizosphere soil of Triticum aestivum plants growing at 7 and 70 % Al saturation, and GRSP from the same place. Additionally, we have included AM structures of Glomus intraradices growing under monoxenic conditions. In all cases the structures were analyzed by using CLSM. The samples (spores, hyphae and roots) were mounted on slides, one subsample with deionized water, other subsample with an AlCl3 drop, and other subsample with an AlCl3 drop and subjected to staining with hematoxilin or pyrocatechol violet. We tested them with lasers at 405, 488 and 633 nm in Fluoview FV1000 Confocal Laser Scanning Biological Microscope. The hyphae from in vitro cultures showed only autofluorescence when they were observed directly in CLSM, the hyphae showed only autofluorescence. Other sample from in vitro cultures with the addition of a few drops of solution of AlCl3 showed fluorescence in hyphae and spores. This same behavior was observed by adding the Al solution and subjected to hematoxylin or pirocatechol violet. When directly observed, the roots of D. carota showed low autofluorescence, but the colonized roots with Al addition showed intraradical hyphae, presence of vesicles and roots cell walls. GRSP from soils with 7% Al saturation showed less autofluorescence than from soil with 70% Al saturation. These results showed a high capacity for Al immobilization by mycorrhizal fungi structures, which could be a way to reduce the high Al amounts in allophanic soils. Acknowledgements. This study was supported by the project FONDECYT 1100642.



66

EVALUATION OF ENZYMATIC DEGRADATION OF ORGANIC WASTE AND SEWAGE SLUDGE MIXTURES BY AUTOCHTHONOUS SAPROPHYTIC FUNGI FROM

SOUTHERN CHILE

Almonacid, L.*1, Lagos, C.2, Cornejo, P.3 and Arriagada, C.2

1Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de la Frontera, Temuco, Chile. 2Departamento de Cs. Forestales, Facultad de Cs Agropecuarias y Forestales, Universidad de La Frontera,

Temuco, Chile. 3Departamento de Ciencias Químicas, Universidad de la Frontera, Temuco, Chile. *Corresponding author: [email protected]

The sustained increase in grain production increased the amount of crop residues generated, which are usually burned, causing a negative effect on the environment. On the other hand, the exponential installation of wastewater treatment plants has caused an increase in the amount of waste generated by them (sewage sludge), which also generates an environmental problem. Both residues contains high level of organic matter and nutrients which previously handled and processed together could be used as amendment in agroforestry systems. One alternative to crop residues burning is the use of microorganism capable of accelerating the descomposition and mineralization of organic matter and especially lignocellulosic residues, microorganisms such as saprophytic fungi that are capable to transform and enhance the residues, generating an efficient biodegradation and biofertilization. In this study we evaluated the production of enzymes generated and induced by the action of natives saprophytic fungi in the process of degradation of wheat and oat crop residue, individually or in combination with sewage sludge. Moreover, we show preliminary study of the effect of sewage sludge application on plant development in Lycopersicum esculentum Mill. For the biodegradation test the experiments were carried out in Erlenmeyer flask (250 ml). The native saprobe fungi used were Phanerochaete chrysosporium and Trametes versicolor (isolated from Rucamanque) propagated in potato dextrose broth (PDB) in stirring for 15 days at 28°C in darkness. Once multiplied they were incubated in 4 different substrates: 1. Wheat straw, 2. Mixture of wheat straw + sewage sludge; 3. Oat straw 4. Oat straw + sewage sludge, cultivated for 15 days at 25 ° C in the darkness. Extracellular enzymes laccase, beta-glucosidase, Manganese peroxidase and acid phosphatase were extracted from bioassays for analyses. Sewage sludge was applied to Tomato pots at concentrations of 10 and 20% incubated or not with P.chrysosporium and T.versicolor. Plants were harvested after 6 weeks and dry biomass of shoots were determined. The results show that the enzymatic activity in both treatments with pure lignocellulosic residues and sewage sludge presented a higher activity those incubated with both fungi compared with the control. T.versicolor showed higher values that P.chysosporium. The shoot biomass was significantly increased in all doses of Sewage Sludge treatment and this beneficial effect was more evident with the application of 20% of this residue incubated with T. versicolor. Acknowledgements: FONDECYT, project N°1100752 and Scholarship CONICYT 24080106.



67

ARBUSCULAR MYCORRHIZA AND SOIL PROPERTIES IN AN ALFISOL OF CENTRAL-SOUTH CHILE UNDER LONG-TERM NO TILLAGE

Curaqueo, G.1,2,*, Seguel, A.1,2, Medina, J.2, Aguilera, P.1,2, Rubio, R.2 and Borie, F.2

1Programa de Doctorado en Ciencias de Recursos Naturales. Universidad de La Frontera. Casilla 54-D.

Temuco, Chile. 2Center of Amelioration and Sustainability of Volcanic Soils. BIOREN-UFRO. Universidad de La Frontera, P.O. Box 54-D, Temuco, Chile.*Corresponding author: [email protected]

The no-tillage system is a management that contributes to improving the physical, chemical and biological soil properties. However, other key factor in the agroecosystems is the presence and activity of arbuscular mycorrhizal fungi (AMF) which are related to soil organic matter stabilization by means of soil particle aggregation produced by fungal mycelium activity and a glycoprotein called glomalin. Such protein is an AMF product and has been extracted from the soil and characterized like glomalin related soil protein (GRSP). The aims of this study were i) To evaluate the effect of no tillage (NT) and crop rotations on GRSP contents and humic substances; ii) To determine the contribution of GRSP to soil C pool and iii) To relate the GRSP content with organic matter and water stables aggregates. The study was carried out in an Alfisol of Central-South Chile managed during 6 (NT6), 10 (NT10) and 20 years (NT20) under no-tillage system and crop rotations in which it was included Wheat (Triticum aestivum: W), Lupin (Lupinus albus: L) and Triticale (Tritico secale: T). Soil was sampled at 0-5; 5-10 and 10-15 cm depth from W-L-T crop sequence under NT6; W-T under NT10 and W-L-T rotation under NT20 . We evaluated the content of easily extractable glomalin (EE-GRSP), and total glomalin (GRSP), content of humic and fulvic acids, water stable aggregates and water holding capacity. The results showed a decrease trend in the GRSP fractions in the soil according to more long-term of NT and soil depth, ranging from 8.9 mg g-1 to 13.4 mg g-1 at NT20 and NT6 respectively. The humic and fulvic acids showed a decreasing content under NT10 and NT20 compared with NT6 in all depths evaluated. The stable aggregate percentage was higher under NT6 (44.3%), while NT10 and NT20 reached closer percentages (27.5% and 26.6%) respectively. Results suggested an active role of GRSP on soil aggregation, and the stability of organic matter in the soil of the agroecosystem studied, being important to highlight the effect of long-term NT system, which could decrease the AMF activity and the GRSP production.



68

EXUDATION OF ACID PHOSPHATASE AND ORGANIC ACIDS BY CLUSTER ROOTS OF Embothrium coccineum

Delgado, M,1*., Zúñiga-Feest, A.2 and Borie, F.1

1Departamento de Ciencias Químicas, Universidad de la Frontera, Casilla 54-D, Temuco, Chile. 2Laboratorio de Fisiología Vegetal, Instituto de Geociencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile.

*Corresponding Author: [email protected]

Embothrium coccineum (Notro) is an endemic specie from Southern South America. In Chile it has a wide geographical distribution, from 35 º S to 56 º S. It is a specie shade intolerant and pioneer of the succession process facilitator, to able to germinate and establish in volcanic ash soils. This specie has an interesting adaptation called cluster roots (CR), which are dense clusters of fine rootlets around a main axis. These structures have been frequently reported in species that have grown in soils with a lower content of available phosphorus (P). One of the roles that have the CR is the organic anion exudation and acid phosphatase, which solubilize inorganic P (Pi) and hydrolyze organic P compounds to Pi, respectively, which is the available form to plants. Both P-raising mechanisms are important for the Chilean native species, since the volcanic soils of Southern Chile, are characterized by low levels of available phosphorous, low pH and high concentrations of aluminum. In this regard, it is noteworthy to Chilean Proteaceae species that have CR, which have been poorly studied. Because of this the aim of this study was evaluate the qualitative evidence exudation of organic acids and acid phosphatase activity in seedlings of one year old in Notro using colorimetric methods as acid-base indicators, by immersing the roots in agar with bromocresol purple and fenolftaleinfosfato, respectively. Acid phosphatase activity results shown an increased exudation of these enzymes in CR compared with normal roots. On the other hand, the exudation of acid was not detectable, which could be because it has been reported for this and other species of Proteaceae, a seasonal variation in the exudation of organic acids, which occur in autumn and winter mainly, where the plant has stopped growing. Because of this experiment was enhanced in spring time, it would confirm this seasonal variation in organic acid exudation, however, remains to quantitatively validate this situation. Therefore, in another experiment is launched, will be assessed quantitatively both; exudation of organic acids and acid phosphatase activity, which will be held under different P levels, in order to test the hypothesis that these exudates are induced in major amount under deficiency of this nutrient. These results are not available yet.



69

EFFECTS OF ARBUSCULAR MYCORRHIZAL INOCULATION ON METALLOPHYTES AND AGRICULTURAL PLANTS GROWING ON COPPER POLLUTED SOILS

Meier, S., Borie, F., Inostroza, N., Cox, R., Curaqueo, G., Seguel, A. and Cornejo, P.*

Scientifical and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, P.O. Box 54-D, Temuco, Chile. *Corresponding author: [email protected]

A pot culture experiment was carried out to study the effectiveness of different arbuscular micorhizal fungi (AMF) strains in promote plant growth and copper (Cu) concentration using two metallophytes (Oenothera affinis and Imperata condensata) and one agricultural plant (Helianthus annus) growing at increasing soil Cu levels. Plants were established in a Cu-treated soil (non Cu polluted soil -NC-, or polluted soil added with 0, 150 or 450 mg Cu kg-1), and inoculated with: i) Cu-adapted AMF (GA); ii) Glomus claroideum (GC) or iii) maintained uninoculated. Differences among AMF inoculated and uninoculated plants were found in response to higher Cu levels, and differences between the AMF strains were also observed. Although the beneficial effect of AMF in promote root biomass production was not observed, a positive and specific interaction between GA and plants increased significantly the shoot growth, especially at higher Cu concentrations. The plant Cu concentration depended not only of the soil Cu level, but also the type of mycorrhizal inoculum applied to the rhizosphere. The Cu uptake was lower, remaining a major Cu fraction in the root. However, the AMF utilization, in particular GA, produced changes in Cu distribution, increasing the translocation to the shoots. Finally, differences in mycorrhizal root colonization and AMF spore density were dependent of the plant specie analyzed, suggesting a specific compatibility between AMF and the plant. Our results indicate that under phytotoxic Cu levels in the soil metal adapted AMF is the best option to be used in order to promote phytoremediation processes. Acknowledgements: This research was supported by the National Commission for Scientific and Technological Research through Project FONDECYT 11080131 (Chile). Sebastian Meier acknowledges the support of CONICYT through Project 24091053.



70

EVALUATION OF IN VITRO ANTIBACTERIAL ACTIVITY OF PLANT EXTRACTS ON BACTERIA OF IMPORTANCE PHYTOPATHOLOGICAL

Concha, M.1, Partarrieu, A.1, Jofre, I.1, Gallardo, C.1, Fincheira, P.1, López, F.1

and Parada, M.2,*

1Estudiante de la Carrera de Biotecnología de la Facultad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera, Temuco, Chile. 2Departamento de Ciencias Agropecuarias y Recursos Naturales,

Facultad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera, Temuco, Chile. *Corresponding author: [email protected]

Phytopathogenic bacteria cause diseases in various plants and annually produce economic losses in many countries including ours, which bases part of its economy on the export of high quality fruits and must meet the requirements of purchasing countries. This is the case of Pseudomonas syringae in fruit trees and various species of Erwinia in potato crops and flowers. As a way of reducing the application of chemicals for disease control, it was considered of vital importance looking for a bactericidal product. With this objective, samples of Prunus persica with signs of gumming and samples of Solanum tuberosum with soft rot, was taken in order to isolate strains cause disease and to inhibit their growth in vitro tests. Samples of P. persica and S. tuberosum were isolated from culture media Plate Count and Müller Hilton, obtaining the strains bMBT86 and bMBT85. In vitro experiments have seen in the first instance 107 extracts obtained from various plant tissues, which were tested by agar diffusion technique, in triplicate assays and positive control compared with 0.3% gentamicin. The results indicate that both strain, bMBT86 and bMBT85, were inhibited by 4 extracts, two of which overlap. In both cases, the inhibition halos were slightly lower, equal or superior to gentamicin, which for the two strains showed a halo of 0.5 cm. On the other hand, both strains have been administered various biochemical tests and DNA extraction in order to identify and confirm the species concerned. Are currently conducting test of minimum inhibitory concentration (MIC and minimum bactericidal concentration (MBC), and then perform the test in sensitive plants to phytopathogens bacteria and assess the effect of each of the extracts and mixtures there of in search of a possible alternative to chemicals that have a high impact on environmental pollution.



71

STUDY OF SOIL BACTERIA ISOLATED IN THE REGION OF LA ARAUCANIA, HIGHLY TOLERANT TO LEAD AND IMPLICATIONS OF THE POLYSACCHARIDE IN

THIS PROCESS

Gallardo, C.1, Godoy, K.3, Concha, M.1, Jofré, I.1, López, F.1, Fincheira, P.1, Partarrieu, A.1 and Parada, M.2,*

1Estudiante de la Carrera de Biotecnología de la Facultad de Ciencias Agropecuarias y Forestales,

Universidad de La Frontera, Temuco, Chile. 2Departamento de Ciencias Agropecuarias y Recursos Naturales, Facultad de Ciencias Agropecuarias y Forestales, 3 Laboratorio Citometría de flujo, BIOREN, Universidad

de La Frontera, Temuco, Chile. *Corresponding author: [email protected]

Currently, little is known about some soil bacterial communities and how they are able to grow on different substrates and sediments. In our country major populated areas are contaminated with high levels of lead, this has motivated the search for biological tools to develop new biotechnological applications. To this end, we have the need to create a database of potentially bioremedial agencies, which began with the recovery of 91 still unidentified bacterial strains belonging to the strain collection of the Laboratory of Microbiology and rhizobiology of the Faculty of Agricultural and Forestry Science of La Frontera University. From this strain collection was isolated 36 bacteria, which were exposed to high concentrations of heavy metals (Cd, Pb, Cu, Zn, Mg). Subsequently, we selected only lead resistant bacteria bMBT23, bMBT34, bMBT35, bMBT36, bMBT42 and bMBT90, which were treated at different levels of Pb (NO3) 2 in culture medium Plate Count, obtaining 782.5 Dose resistance; 939, 1905.5 and 1252 mg of lead per kilo of medium. Some of these bacteria inoculated on plates of pure lead (on agar-water), and grown for 9 days at 28 ° C, were reisolated in culture medium Plate Count, showing surprisingly that were alive, even though its growth was not optimal. On the other hand, there was a growth curve in nutrient broth at 28 ° C, showing an exponential growth up to 6 days. In addition, tests were conducted at different temperatures (4, 28, 37, 46 and 60ºC), where strains bMBT23, bMBT34, bMBT35, bMBT36 and bMBT90 grew to 46ºC, the strain bMBT42 only increased to 28 º C bMBT90 and bMBT23 growth and was clearly increased as the temperature rose. As for the production of exopolysaccharides (EPS) in YMA medium has been observed that only strains bMBT34, bMBT36 bMBT35 and produce some kind of EPS, and could be a possible mechanism of tolerance to lead. Is currently working on isolating and sequencing their genomic DNA for identification.



72

AGGREGATES AND ITS RELATION TO ORGANIC CARBON CONTENT INTO DEEP VOLCANIC SOIL OF PRISTINE TEMPERATE OLD-GROWTH RAINFORESTS IN

SOUTHERN CHILE

Neculman, R.1,*, Panichini, M.1, Matus, F. 1, 2 and Godoy, R.3

1Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de la Frontera, Temuco-Chile. 2Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera, Temuco-Chile.

3Instituto de Botánica, Universidad Austral de Chile, Valdivia-Chile. *Corresponding author: [email protected]

Carbon (C) in the subsoil has gained importance due to its high storage capacity and means residence time of hundreds to thousands of years. However, studies of C stabilization in the subsoil are still scarce. These studies indicate that the stabilization of soil organic carbon (SOC) of the subsoil is mainly due to its chemical adsorption with aluminum (Al) and Iron (Fe). In Chile, the allophanic soils are characterized by its high content of SOC, low pH and presence of amorphous clay minerals (allophane). The temperate rainforest of southern Chile has been developed in these soils under a temperate climate, with low temperatures (4.5°C) and high precipitation (5,000-7,000 mm), where the woody vegetation produces a litter with a slow decomposition rates resulting in the SOC accumulation on the surface soil. Therefore the C study at depth soil is essential for understanding the dynamics of C. The aim study was to determine the stability of soil aggregates in the topsoil and depth, where we used the weighted average diameter (WAD) as an index of comparison and measurement of the aggregates distribution. Also, determine its C content, thus corroborating the levels of C accumulation in the subsoil. The area corresponds to the Andean mountain range located in San Pablo de Tregua (SPT), Panguipulli (39° 38 'S, 72° 05' W) and Puyehue National Park (PNP), Antillanca (40° 47 'S, 72 ° 12' W) with an elevation ranging between 550 and 1,200 meters over the sea level. The results showed that in both sites, the macro-aggregates fraction were most important in relation to meso and micro-aggregates, representing a 71% of the total soil dry weight. WAD values for SPT and PNP also showed non significant differences between the soil profiles, indicating a high aggregate stability. On the other hand, the absence of correlation between C in micro-aggregates and SOC in both sites, confirmed that fine particles (clay + silt) did not influence C accumulation, in surface and depth. However, a significant asymptotic relationship between C in the macro-aggregates and SOC showed that there is a maximum limit of C accumulation respect to Al and the physical protection, where subsoil showed a constant level of C accumulation, while the constant C input in surface could reach a level of equilibrium through the time.



73

IDENTIFICATION OF 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID DEAMINASE AND Β-PROPELLER PHYTASE GENES IN RHIZOBACTERIA

ASSOCIATED WITH THE OLDEST LIVING PLANTS ON EARTH

Jorquera, M.A.1,*, Mora, M.L.1 and Crowley, D.E.2

1Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and Technological

Bioresource Nucleus, Universidad de La Frontera, Temuco, Chile. 2Department of Environmental Sciences, University of California Riverside, Riverside, USA.


Bacterial communities in the plant rhizosphere carry out functions that are essential to plant nutrition, disease suppression and stress protection in terrestrial ecosystems. However, our knowledge on plant-bacteria interactions so far has come from studies on agricultural crops and forest tree species, and there is scarce information about bacterial communities in soil under natural vegetation. Creosote bush (Larrea tridentata) is the predominant shrub in the Mohave Desert in the USA, and they are considered as oldest living plants on Earth with some individuals having an age of more than 10,000 years. Here, we studied 1) the bacterial communities that are associated with the roots of ancient clones of the Creosote bush by using denaturing gradient gel electrophoresis (DGGE) and 2) the occurrence of acdS and BPP genes in culturable rhizobacteria, which are related to plant growth promotion by the degradation of 1-aminocyclopropane-1-carboxylic acid (ACC) and phytate, respectively. The amplified genes (16S rRNA, acdS and BPP) were sequenced and compared with those deposited in Genbank database. The DGGE analysis revealed the association of bacteria belonging to families: Chitinophagaceae, Bradyrhizobiaceae, Rhodospirillaceae, Pseudomonadaceae, Aurantimonadaceae, Desulfovibrionaceae, Thermoanaerobacteraceae, Flavobacteriaceae, Flexibacteraceae, Bacillaceae, Micrococcaceae, Enterobacteriaceae, Xanthomonadaceae and Alcaligenaceae. Moreover, our results showed that culturable Pseudomonas and Bacillus strains can harbor acdS and BBP genes. The sequence analysis revealed that acdS genes are close to genes reported for Pseudomonas and Burkholderia strains, while those BPP genes are close to reported for Bacillus pseudomycoides. It is noteworthy the isolation of a Pseudomonas strain that contain both genes. This study show that ancient clones of Creosote bush harbor a wide diversity of bacteria, which contain mechanisms that might play a relevant role in health and nutrition of this plant, contributing to the survival of plant for long time periods in extreme environment of the Mojave Desert.

Acknowledgments: The International Human Frontier Science Program, US-Israel Binational Agricultural Research and Development and Fondecyt 11080159.



74

DESIGN AND EVALUATION OF SPECIFIC PRIMER SETS FOR QUANTITATIVE PCR TO STUDY OF PHYTASE GENES IN Bacillus AND Paenibacillus

Jorquera, M.A.1,*, Maruyama, F.2, Ulloa, P.3 and Mora, M.L.1

1Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Temuco, Chile. 2Section of Bacterial Pathogenesis,

Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan. 3Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera.


Phytate is one of the most abundant sources of organic phosphorus (P) in soils but must be mineralized by phytase−producing bacteria to liberate phosphorus for plant uptake. Some members of Bacilli are recognized as phytase−producers and microbial inoculants based on Bacillus spp. have been commercially developed, but few studies have evaluated the effect of phytase−producing Bacillus (PPB), used as soil inoculants, to improve P availability to plants. In previous studies, we have isolated and identified native Bacilli strains harboring phytase genes from rhizosphere soils. Based on theses phytase gene sequences, we designed and evaluated two specific primer sets for phytase genes of Bacillus and Paenibacillus. The primer set MQHf/MQHr was designed to amplify a DNA fragment of 158 bp from Bacillus phytase genes. The primer set SPTf/SPTr was designed to amplify a DNA fragment of 233 bp from Paenibacillus phytase genes. The specificity of designed primer sets was firstly evaluated by traditional PCR using diverse Bacillus and Paenibacillus strains. The widely used primer set Bac1369F/Prok1492R, specific for 16S rRNA gene, was used as control. The electrophoresis in agarose gel did not show unspecific amplicons. Then, the designed primer sets were evaluated for quantitative PCR (qPCR) by using 7300 Real-Time PCR System (Applied Biosystems). The analysis of the dissociation curve showed one amplification peak for each primer set and confirmed the specificity of designed primer sets for each bacterial type. To evaluate the performance of each primer set, serial dilutions of target genes were analyzed and the analysis resulted in PCR efficiency of 98.4% (slope: -3.36), 104.9% (slope: -3.67) and 105.8% (slope: -3.19) for primer sets MQHf/MQHr, SPTf/SPTr and Bac1369F/Prok1492R, respectively. Our results suggested the feasibility in the use of designed primer sets for further studies on quantification and expression of phytase genes in Bacillus and Paenibacillus isolated from rhizosphere. Efficient methods to monitor the population size and activity of soil inoculants in the rhizosphere are currently needed and the primer sets designed in this study might be a useful tool to evaluate by qPCR the PPB populations in the rhizosphere and their influence on P availability for plant uptake.

Acknowledgments: Fondecyt Nº 11080159 project.



75

EFFECT OF SELENIUM ADDITION ON THE COMMUNITY STRUCTURE OF NATIVE RHIZOBACTERIA, ANDISOL SOIL

Padilla, E.1, Acuña-Sobarzo, J.1, Jorquera, M.2,* and Mora, M.L.2

1Doctorate Program in Sciences of Natural Resources, Universidad de la Frontera.

2-The Scientific and Technological Bio-resource Nucleus, Universidad de la Frontera. *Corresponding author: [email protected]

In Chile, cereal crop production like wheat is one of the most important sources of nutrition for population and crops frequently have suboptimal selenium (Se) levels, because of factors associated with plant species, soil type, fertilization and the nature of selenium compounds available. In this context, the rhizosphere harbors a wide variety of bacterial species which play an important role in the biogeochemical cycle of Se. Diverse bacterial groups, such as Bacillus, Rhodobacter, Pseudomonas, Stenotrophomonas, Aremonas and Ralstonia, have shown a high tolerance to Se and they have been suggested as biotechnological tools for bioremediation of Se-contaminated soils. However, there are no studies focused on determining the effect of Se addition applied to the plant for biofortification on the diversity of community struture of native rhizobacteria. These soils are often poor Se sources for plants, animals and human consumers by providing very low levels for optimal health. The positioning of our country as food power poses the challenge of improving optimal nutritional conditions for the production of cereal crops so as to ensure food quality and improve plant nutrition through the application of current biotechnological tools. The rhizosphere has a wide variety of bacterial species which carry out functions that are essential to plant growth, nutrition and disease control in agricultural systems. Among them, members from different phylogenetic groups have attracted considerable interest due to their great biotechnological potential for improving crop yields. In this context, our goal was to study the effect of selenite additions on bacterial community structures present in the rhizosphere in crops of wheat Triticum aestivum in two varieties: Bt and Fritz, grown in volcanic soils. For this purpose, we prepared Se-enriched samples with two different doses of Se, and after 5 weeks we took samples from the soil microcosms. After 5 weeks, the samples were analyzed using DNA extraction and PCR amplification and Denaturing Gradient Gel Electrophoresis (DGGE). The preliminary results showed no differences in the structure of community of rhizosphere in the samples neither in Bt variety nor in Fritz variety. The preliminary results indicated the Se doses did not affect the structure communities of the rhizobacteria in the soil with wheat crops, we need a deeper analysis for concluding results. Acknowledgements: This study is supported by Scholarship Conicyt and Fondecyt Grant.



76

ISOLATED SELENIUM-ACUMULATING RHIZOBACTERIA FROM RHIZOSPHERE OF WHEAT GROWN ON ANDISOLS SOIL

Acuña-Sobarzo, J.1,*, Padilla, E.1, Jorquera, M. 2and Mora, M.L.2

1Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera.

2Núcleo Científico y Biotecnológico en Biorecursos, Universidad de La Frontera. *Corresponding author: [email protected]

Selenium (Se) is an essential micronutrient with antioxidant properties. Se has received considerable attention for its important role in cancer prevention, due to biological functions related to selenoproteins and selenoenzyme (e.i. glutathione peroxidase). During the last decade, many studies have been carried out on accumulation, speciation, and biological functions of Se related to human health. In this context, Se deficiency has been related to different liver, muscles and heart pathologies. Acidic volcanic soils (Andisols) of southern Chile frequently has suboptimal levels of Se available for plants, because factors associated with soil type, fertilization and the nature of Se compounds available. In this context, analysis by the Soil and Plant Laboratory from Universidad de La Frontera showed that the soils of the IX and X regions have deficient levels of Se, with a concentration ranging from 0.02 and 0.18 mg Se kg-1 soil. Microorganisms play an important role in the transformations and availability of Se in nature. Microbiological transformations of Se includes methylation, oxidation, and reduction. They have been suggested as biotechnological tools for bioremediation of Se-contaminated soils. However, there are not studies focused on the potential application of Se-utilizing bacteria in agriculture to increase the content of Se available in the rhizosphere. In this study, our goal was to evaluate the bioaccumulation of Se by native bacteria present in the rhizosphere of wheat grown in acid soils. For this purpose, rhizosphere samples were cultured in Nutrient broth supplemented with 0.2mM of sodium selenite for 48 hrs at 30°C. Se reduction was visualized by a color change in the medium. The preliminary results showed the presence of Se-accumulating colonies with red coloration through the mechanism of reduction of Se. In this colonies, the presence of nanosphere of elemental Se (red), both extra and intracellular, was visualized by using confocal microscopy it showed. Moreover, the presence of white colonies in the culture medium suggests the existence of others mechanisms for bioaccumulation of Se by bacteria present in the wheat rhizosphere.

Acknowledgements: This study is supported by Scholarship Conicyt and Fondecyt Grant.



77

ACTIVITY CONSERVATION AND IMMOBILIZATION PATTERNS OF Rahnella sp. D75 AND E. coli ACID PHYTASES ADSORBED IN NANOCLAY SUPPORTS

Menezes-Blackburn, D.1*, Jorquera, M.2, Garrido, E.1, Gianfreda, L.3, Rao, M.3 and Mora, M.L.2

1Doctorate in Natural Resources Science, Universidad de La Frontera, Avenida Francisco Salazar

01145, Temuco, Chile. 2Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Avenida Francisco Salazar 01145, Temuco, Chile. 3Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, Università di Napoli Federico II,

Portici, Italy. *Corresponding author: [email protected]

We have studied Rhanella sp. D75 and E. coli phytase immobilization on nanoclay support in order to evaluate their activity conservation and stabilization. Cell associated acid phytase of Rhanella sp. D75 (pH of activity 3.5-5.5) was preselected among all the identified bacteria phytases for being a constitutive, low cost, easy producing phytase. Montmorillonite, allophane and iron coated allophane were used as nanoclay supports for the enzyme immobilization with contrasting surface properties. Immobilization tests were performed according to Rao et al. (1996) and phytase activity tests were performed according to Greiner (2004). Kinetics and isotherms of adsorption were also evaluated. Immobilization patterns at varying pH were greatly influenced by both enzyme and support characteristics. E. coli phytase showed a higher affinity towards montmorillonite nanoclay in comparison with other supports. Curiously, the maximum adsorption of E. coli phytase was observed to Allophane 2%Fe, and the lowest was observed to the allophane. Phytase adsorption to allophane is apparently being governed by electrostatic attraction forces, and is enhanced by acid and low ionic strength conditions. Activity conservation was significantly enhanced when phytases were adsorbed at low pHs. Uncoated allophane was considered the best support for phytase immobilization for its enhanced activity conservation after adsorption. Biotechnological applications using free and immobilized phytases will be tested in manure amendment treatments for increasing the availability organic P pool. Aknoledgements: UFRO DOCTORAL SCHOLARSHIP; CONICYT scholarship support for doctoral thesis development (2009); PROJECT FONDECYT Nº 1061262; PROJECT FONDECYT INITIATION Nº 11080159

References:

- Greiner, R. 2004. Potein J. 23, 567-576. - Rao, M, Gianfreda, L., Palmiero, F. and Violante, A 1996. Soil Sci. 161, 751.



78

CONTRIBUTION OF PHYSICALLY PROTECTED ORGANIC MATTER TO CARBON SEQUESTRATION IN ALLOPHANIC SOILS

Panichini, M.1,*, Neculman, R. 1, Matus, F. 1, 2, Godoy, R.3 and Rumpel, C.4

1Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de la Frontera, Temuco-Chile.

2Departamento de Ciencias Químicas, Universidad de La Frontera, Temuco-Chile. 3Instituto de Botánica, Universidad Austral de Chile, Valdivia-Chile. 4CNRS, BIOEMCO, Soil Organic Matter, Centre INRA Versailles-Grignon, 78850, Thiverval-Grignon, France. *Corresponding author: [email protected]

Carbon sequestration in soil is an important strategy to mitigate the increase of CO2 concentrations, since it involves the transfer of atmospheric CO2 in more stable soil organic carbon (SOC) fractions (Schimel et al., 1997). The permanence of SOC depends largely on the balance between contributions and the losses by decomposition, however there are factors that play a fundamental role in stabilizing of SOM such as mineralogy, climatic conditions and plowing. One of the most important stabilization mechanisms is the interaction of SOM with soil minerals (Rumpel et al., 2002, Six et al., 2002). But it should be bear in mind that there are other fractions of SOM such as particulate organic matter (POM) and protected organic matter in soil aggregates that may become important at time when a carbon balance should be made. The determination of the stabilization capacity of the allophanic soils under different land-use and management we conduct through density fractionation. We include ungrazed grassland, natural grassland and temperate rain forest soil. The corresponding soil depths were 0-7, 7-32 and 32-40 cm depth in Valdivia series and 0-20, 20-80 and >80 cm depth in Liquiñe series. The protocol used is a modification of the methodology described by John et al. (2005). Soil fractions were separated by density using sodium polytungstate. It resulting three fractions of different densities: fPOM, free particulate organic matter (<1.6 g cm-3), oPOM, occluded particulate organic matter (1.6-2.0 g cm-3) and MF, mineral fraction (> 2.0 g cm-3). After separation of different fractions, the samples were dried to analyze the contents of C and N by dry combustion (elemental analysis). The results showed that the MF represented over 90% of the total weight in the natural grassland and ungrazed grassland. In turn, a lower percentage was observed for oPOM and fPOM. On the other hand the MF of pristine forest fertile and infertile represent over 75% of total soil weight, but in this case the contribution of fPOM and oPOM are higher than the pasture. The concentration of C in SOM fractions isolated shown that the oPOM had the highest concentration of C in the natural grassland and ungrazed grassland. On the other hand, the highest concentration of C in the pristine forest fertile was found in thee fPOM. A different behavior was noted in the pristine forest infertile, where the oPOM average the greater concentration, especially in the deeper strata. In summary the weight distribution of fractions followed a similar pattern to those described in the literature, characterized by a higher concentration in the MF and then in the POM (John et al. 2005). The physical fractionation indicate that a large amount of carbon is present in the occluded particulate organic matter (oPOM), physically protected from microorganisms. The results indicate that a large proportion of organic carbon was found in the oPOM (physically protected) and the MF, indicating the both fractions represents a significant percentage of SOC.



79

ROLE OF ARBUSCULAR MYCORRHIZAL SYMBIOSIS IN THE ALUMINIUM TOLERANCE OF FIVE CULTIVARS OF Triticum aestivum L GROWING IN ACID SOILS

OF SOUTHERN CHILE

Seguel, A.*, Curaqueo, G., Meier, S., Aguilera, P., Cornejo, P. and Borie, F.

1Scientific and Technological Bioresource Nucleus (BIOREN-UFRO), Universidad de La Frontera. Avenida Francisco Salazar 01145, P.O. Box 54-D, Temuco, Chile. *Corresponding author: [email protected]

The main features of acid soils are the low levels of available phosphorus (P), low amounts of exchangeable bases and high percentage of aluminum (Al) saturation which represents one of the major constraints for agricultural production. In these conditions, the arbuscular mycorrhizal fungi (AMF) can play an important role protecting the roots against phytotoxic Al levels through interactions with phosphorus, promoting exudation of chelant acids by roots and also through the production of glomalin, a glycoprotein produced by AMF. The aim of this work was to study the AMF-Al interaction on the growth of five Al- tolerant cultivars of Triticum aestivum L. (wheat). Otto, Bakan, Porfiado, Invento and Crac cultivars were cultivated in an Andisol (Gorbea Series) with two levels of Al saturation, 79.8 for natural soil (NS) and 7% for soil limed with the equivalent to 4 ton lime ha-1 (LS). Harvests were made at three phenological stages of plant, tillering (60 days after sowing, DAS); anthesis (90 DAS) and physiological maturity (150 DAS). Shoot (SB) and root (RB) biomass, AMF colonization, spores number, total hyphal length and glomalin were determinate. Results of dry matter showed that Crac, Invento and Porfiado were less affected by lime addition at three harvest; however, with NS Invento cultivar did not growing, affected by the high level of Al saturation. The AMF colonization was not inhibited by high levels of Al in LS at 150 DAS and Crac, Invento and Porfiado cultivars had the highest AMF colonization (51, 52 and 56 % respectively). In LS, Porfiado and Bakan cultivars presented higher density of extraradical mycorrhizal hyphae with 38 and 36 m g-1. Similar response was observed in relation to glomalin; however, spores number increased with high levels of Al saturation. Crac cultivar growing in NS 360 spores in 100 g-1 soil were found. The study showed that Crac and Porfiado cultivars have further development to high levels of Al saturation showing a greater development of the fungal parameters assuming an important role of AMF in the possible tolerance of this cereal to high levels of Al. Acknowledgements: FONDECYT 1100642 Project, CONICYT scholarship and Campex Semillas Baer.



80

EARLY ARBUSCULAR MYCORRHIZAL COLONIZATION IN CEREALS GROWING IN ANDISOLS OF LA ARAUCANIA REGION

Castillo, C.G.1,2,* and Borie, F.1

1Center of Amelioration and Sustainability of Volcanic Soils, BIOREN-UFRO, Universidad de La Frontera,

Casilla 54-D, Temuco. 2Universidad Católica de Temuco, Facultad de Recursos Naturales, Escuela de Agronomía. Casilla 15-D, Temuco. *Corresponding author: [email protected]

The establishment of the arbuscular mycorrhizal (AM) symbiosis begins with the colonisation of a compatible plant root by the hyphae produced by AM soil propagules, be fungal mycelium, asexual spores or mycorrhizal root fragments. The aim of this study was to determine the curves of early AM colonisation in roots of cereals representatives of the region for which two bioassays were performed: I) In the greenhouse, to determine the AM colonisation rate by native fungi on roots from three different cereals, and II) In a growth chamber to study in the same cereals the colonization rate produced by the inoculation of an AM fungi strain. In greenhouse, 250 mL pots were used, containing two Andisols: Pillanlelbún, S1 (pH = 5.6, Olsen-P = 14,6 mg kg-1; OMS = 36,5 %) and Cunco, S2 (pH = 6,0, Olsen-P = 35,3 mg kg-1; OMS = 18,5 %). These soils were cropped with two cultivars of the following cereals: Triticum aestivum L. cv. “Bakan” and “Kumpa”; Avena sativa L. cv. “Pepita” and “Supernova”; Hordeum vulgare L. cv. “Sebastián” and “Barke”. To each treatment, 15 replicates in a completely randomized design with a total of 180 experimental units were realized. At 15, 30 and 45 days after sowing (DAS) root AM colonisation of wheat and barley was determined, while in oat it was measured at 15, 21, 30 and 45 DAS. For the bioassay II, a sterile substrate of sand inoculated with Glomus claroideum (native morphotype) at concentration 2,5 % v/v was used. For each cultivar, 12 replicates with a total of 144 experimental units were used. Weekly, nutrient solution was added at low P concentration, quantifying AM colonization at 15, 18 and 21 DAS. In the bioassay I, at 15 DAS, results showed scarce root colonisation by hyphae in only some roots of wheat and barley. Such colonisation was depending of Andisol, but not on the cultivar, with higher root colonisation of wheat growing in S1. On the contrary, root colonisation stage in oat was initiated at 30 DAS, where wheat colonisation significantly exceeded to the other two cereals. On the oppossite, sand inoculated with Gl. claroideum produced higher colonization in barley cv. “Barke” similar to that found at 18 DAS in oat cv. “Supernova”, and 21 DAS in both wheat cultivars, but lower 1 %. We concluded that among the three cereals, wheat growing in the Andisol with lesser available P content (S1) presented earlier colonisation by native AM fungi, regardless of cultivar, while wheat grown in the sand inoculated with G. claroideum was later colonized. Acknowledgements: FONDECYT Nº11090014.



81

BIOLOGICAL AND BIOCHEMICAL SOIL INDICATORS: MONITORING TOOLS OF DIFFERENT AGRICULTURAL MANAGEMENTS

Scotti, R.1,*, Sultana, S.2, Caceres, M.1, Scelza, R.1, Marzaioli, R.2, D’Ascoli, R.2

and Rao, M.A.1

1Department of Soil, Plant, Environmental and Animal Production Sciences, University of Naples Federico

II, via Università 100, 80055 Portici, Italy. 2Department of Environmental Sciences, Second University of Naples, via Vivaldi 43, 81100 Caserta, Italy. *Corresponding author: [email protected]

The intensive agricultural managements, increased in the last twenty years, have resulted in a decrease in fertility of soils, representing a serious threat to agricultural productivity due to both the increase in production cost, mainly for intensive use of mineral fertilizers, and the loss of the quality of crops themselves. Organic matter content is closely related to the soil fertility and its progressive reduction in cultivates soils, without a satisfactory recovery, could make agriculture untenable, resulting in a high detrimental effect on environment. But an appropriate soil management practices can improve soil quality by utilizing organic amendments as alternative to mineral fertilizers to increase soil quality and plant growth. In this context, demand of suitable indicators, whose are able to assess the impact of different agricultural managements on soil quality, has increased. It has shown that soil biological and biochemical properties are able to respond to small changes in soil conditions, thus providing information on subtle alterations in soil quality. Aim of this study was to evaluate the use of soil biological and biochemical properties as fertility indicators in agricultural soils under different agricultural managements, sited in Campania Region (Southern Italy). After a preliminary monitoring phase of soil fertility on different farms sited in five agricultural areas of Campania Region, we have selected two farms in two different study areas to assess the effect on soil quality of different organic amendments. In particular, a compost from municipal solid waste and wood from scraps of poplars pruning were supplied in different doses and ratios. Soil samplings after one month from the amendment addition and then every 4 months until a year were carried out. All collected soil samples were characterized by main physical, chemical, biochemical and biological properties. In general, the use of different organic amendments showed a positive effect on fertility of both soils under intensive farming. In general, all enzymatic activities and organic carbon content increased after 1 month, and they were still higher after 4 months from amendment application. Microbial biomass and soil potential activity (respiration) showed significantly higher values in soils added with organic amendments, for both farms and samplings, with more marked effects on respiration in the first sampling. In conclusion results showed, in general, a quick response as indicators of the assayed biological and biochemical soil properties and a good recovery in fertility of the studied agricultural soils. Acknowledgements: The project was founded by CCIIAA of Salerno.



82

INFLUENCE OF NITROGEN AND PHOSPHORUS SOURCES ON MYCORRHIZAL LETTUCES UNDER ORGANIC FARMING

Scotti, R.1,*, Seguel, A.2, Cornejo, P.2, Rao, M.A.1 and Borie, F.2

1Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali (DiSPAPA), Università degli Studi di Napoli Federico II, via Università 100, 80055 Portici, Italy. 2Departamento de

Ciencias Quimícas, Universidad de La Frontera, P.O. Box 54-D, Temuco, Chile. *Corresponding author: [email protected]

Arbuscular mycorrhizal fungi (AMF) develop symbiotic associations with plants roots. These associations are very common in the natural environment and can provide a range of benefits to the host plant. AMF improve nutrition, enhance resistance to soil-borne pests and disease, increase resistance to drought and tolerance to heavy metals, and contribute to a better soil structure. However, agricultural intensive managements, such as the use of mineral fertilizes, pesticides, mouldboard tillage, monocultures and use of non-mycorrhizal crops, are detrimental to AMF. As a consequence, agroecosystems are impoverished in AMF and may not provide the full range of benefits to the crop. Organic farming systems may be less unfavourable to AMF because they exclude the use of water-soluble fertilisers and most pesticides, and generally they plan diverse crop rotations. The AMF develop the most common type of symbiosis in nature: about 90% of the plants are mycorrhizal and many agricultural crops are mycorrhizal. One of more mycorrhizal crops is lettuce, which is very widespread in intensive agricultural under greenhouse. Therefore, cultivated lettuce is known to be responsive to mycorrhizal colonization which can reach 80% of root length and contribute to phosphorus and nitrogen absorption by this plant specie. For this work four different lettuce cultivars (Romana, Milanesa, Grande Lagos and Escarola) were used to study mycorrhization under organic agricultural system, supplying compost from agricultural waste (1 kg m-2) as background fertilization for all plots, red guano as phosphorus source (75 U ha-1 and 150 U ha-1 of P2O5), lupine flour as nitrogen source (75 and 150 U/ha of N) and a combination of both. Lettuce plants were cultivated under greenhouse and after two months of growing, plants were harvested and dried and fresh weight of lettuce roots and shoots were evaluated. The number of spores, percentage of colonization, total mycelium and glomalin content were also evaluated as mycorrhizal parameters. The results showed a different response to mycorrhization of the four lettuce Cvs. In general, mycorrhized lettuce plants had a better response to lower level of nitrogen and phosphorus sources.



83

Symposium 5

“Soil, Plant Physiology and Plant Nutrition”

Chairpersons:

Dr. Miren Alberdi and Dr. Alejandra Jara

Oral presentations:

- Dr. Maria Rao, The University of Naples Federico II, Italy: “Mercury in the environment:

soil health concerns and decontamination strategies”. - Dr. Antonio Violante, The University of Naples Federico II, Italy: “Environmental chemistry

of arsenic”. - Dr. Fabio DaMatta, Universidade Federal de Viçosa, Brazil: “Physiological effects of silicon

nutrition on rice”. - Dr. Adriano Nunes-Nesi, Universidade Federal de Vicosa, Brazil: “Contribution of

mitochondrial metabolism for photosynthetic performance”. - Dr. Joachim Fisahn, Max Planck Institute of Plant Molecular, Germany: “Circadian control

of root elongation and C partitioning in Arabidopsis thaliana”



84



85

MERCURY IN THE ENVIRONMENT: SOIL HEALTH CONCERNS AND DECONTAMINATION STRATEGIES

Rao, M.A.*, Scelza, R. and Scotti, R.

Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, Università degli Studi di Napoli Federico II, via Università 100, 80055 Portici, Italy. *Corresponding author: [email protected]

Mercury is naturally present in trace amounts in many rocks, mainly in a very resistant form (cinnabar, HgS). Anthropogenic activities are responsible for the significant impact on the global biogeochemical cycle of mercury. Following extraction and use of mercury, it escapes to the atmosphere due to its volatile nature, resulting in subsequent pollution via wet and dry deposition. Conversion of elemental mercury in mercuric salt or organic forms such as methyl mercury is one of the primary concerns regarding mercury contamination. In fact it is generally recognised that Hg toxicity and bioavailability largely depend on the chemical state of the metal rather than on the mere Hg total concentration. However, beside chemical investigations, the actual Hg toxicity and bioavailability in soil can only be assessed by studying the impact of the pollutant on soil biochemical properties and/or on selected organisms living in/on soil (e.g., bacteria, plants, earthworms, small biota, etc.). In this context, soil biological and biochemical properties are sensitive and early indicators of soil metabolic disturbance. They can be useful tools to evaluate soil quality and functionality as well as to monitor soil reclamation. Thus, soil microbial biomass carbon content, soil basal respiration, and enzymatic activities can be considered an indirect measure of the bio-availability of heavy metals in soil and a suitable indicator to study the recovery of polluted soils. Among living organisms, plants can be effective bio-indicators of soil pollution because they show a peculiar sensitivity to heavy metals. Mercury can strongly affect plant growth especially in the germination phase, the first phase of the biological cycle. Indeed, at that time seeds are sensitive, strictly depending on the capacity of metals to overcome the natural barriers of seed coats and thereby on the tolerance of each plant species according to its structural features. Plants, on the other hand, could also be adopted for their ability to extract and accumulate mercury from both the atmospheric and soil sources: Soluble and vaporous forms of Hg are readily taken in from surrounding air and soil by plants and translocated from the roots and stomata to various organs. Phytoremediation is considered to be an innovative technology that can provide a cost-effective, environmentally friendly alternative to conventional clean up methods.



86

ENVIRONMENTAL CHEMISTRY OF ARSENIC

Violante, Antonio*

Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, Università di Napoli Federico II, Portici (Napoli), Italy. *Email address: [email protected]

Arsenic is an element ubiquitous in the Earth’s crust and is extremely toxic for humans, animals and plants.. Many studies have revealed that millions of people are at serious risk in many countries around the world, especially in Southeast Asia, due to their exposure to arsenic contaminated drinking water. Tens of thousands of people already suffer from symptoms such as skin lesion, hyperkeratosis, melanosis, and liver, lung, and kidney/bladder cancer, in addition to skin cancer. Its occurrence in natural environments may be due to natural processes (weathering reactions, biological activities and volcanic emissions) as well as antropogenic activities. The mean content of arsenic in soils is about 3 mg kg-1. Arsenopyrite (FeAsS) is the most abundant arsenic-containing mineral and other minerals include realgar (AsS), orpiment (As2S3), olivenite (Cu2OHAsO4). Arsenic-bearing herbicides and pesticides have been widely used in agricultural practice until the mid-1900. In soils, surface and ground waters arsenic is found in –3, 0, +3 and +5 oxidation states, but its prevalent forms are the inorganic species, arsenate [As(V)] and arsenite [As(III)]. Arsenic also occurs as methylated forms in environmental systems. Methylation can be carried out by a variety of organisms ranging from bacteria to fungi to mammals and is believed to be part of a detoxification mechanism in living organisms. Relative to other oxyanion-forming elements, arsenic is among the most problematic in the environment because of its relative mobility over a wide range of redox conditions. Arsenite is 25-60 times more toxic than arsenate, which mainly arise from its state as H3AsO3 at pH < 9.0 as compared to the charged arsenate species which predominate in a wide pH range (H2AsO4

- between 2.5 and 7, HAsO42- between pH 7 and 12).

Arsenic mobility in soil is limited by its capacity to be sorbed on soil components, particularly metal oxides and short-range ordered aluminosilicates (allophane and imogolite) and at the edges of phyllosilicates. Many studies have demonstrated that arsenite is sorbed on Al-oxides, phyllosilicates, and calcite in a lesser extent than arsenate, whereas the opposite is true for iron oxides. Arsenite and arsenate seem to form similar surface complexes, but arsenate is more strongly held onto these soil components. Evidence for the formation of As(V) inner sphere complexes have been obtained using extended x-ray absorption fine structure spectroscopy and wide-angle x-ray scattering. It has been ascertained that on some soil components arsenate forms three different complexes: a monodentate complex, a bidentate–binuclear complex, and a bidentate–mononuclear complex, depending on surface coverage. However, As(V) forms inner sphere surface complexes on noncrystalline Al and Fe oxides, whereas As(III) forms both inner and outer sphere surface complexes on noncrystalline Fe oxide and only outer sphere surface complexes on noncrystalline Al oxide. Sorption and desorption processes control the mobility, toxicity and availability of arsenic in natural environments. Inorganic (mainly phosphate added as fertilizer) or naturally occurring organic ligands, may compete with arsenic inhibiting its sorption onto soil components or promoting its desorption from the sorbents. The sorption/desorption processes of arsenic is affected by the nature of the sorbents, pH, residence time and surface coverage.



87

Removal of arsenic from polluted soils is extremely important for remediation of these sites. The fern Pteris vittata presents an extraordinary capacity to accumulate As in its biomass (435-755 mg/kg) in uncontaminated soils and 3,520-6,800 mg/kg in contaminated soils. It has then a great capacity to translocate large amounts of As to the frond, with shoot concentrations reaching levels ~ 100-fold greater than soil concentrations. Arsenic in drinking water is much more bioavailable than arsenic in soil, because water-soluble arsenic is rapidly sorbed by humans. High concentrations of As in groundwater have been found in Argentina, Chile, Mexico, China, Hungary, West Bengal (India), Bangladesh and Vietnam. Up to around 30–35 million people in Bangladesh are estimated to be exposed to As in drinking water at concentration above 50 μg l−1.Recently, the European Union and the USA with National Priorities List (NPL) has fixed a limit of 10 µg As L-1 in drinking water. The coprecipitation of arsenic with iron and/or aluminum is a practical and effective technique to remove this toxic element from polluted waters. It has been demonstrated that arsenic promotes the formation of very poorly crystalline Al- or Fe oxides. Only, low percentages of arsenic present into the coprecipitates may be removed by phosphate (mainly) or other ligands. Arsenic removal is affected by the concentration of ligands, reaction time, nature and age of the co-precipitates. In the last decade many studies have been carried out on the possible use of layered double hydroxides (LDHs) as powerful filters for removing pollutants in anionic form from polluted waters. Only, in the last years attention has been devoted on the use of LDHs as scavengers for removal of arsenic from aqueous systems. Layered double hydroxides sorbed greater amounts of arsenate and arsenite than Fe- or Al oxides. Vice versa, higher percentages of arsenate, initially held on LDH minerals, were desorbed by phosphate (up to 80%). The presence of elevated arsenic levels in soils and irrigated waters is of considerable concern because of the possibility of plant uptake and subsequent entry into food chains. Apart from the health risk, the presence of arsenic in irrigation waters or in soils at an elevated level could hamper normal growth of plants with toxicity symptoms such as biomass reduction and yield losses. Studies have been carried out regarding the soil-to-plant transportation of arsenic and to minimize the accumulation of arsenic in plants consumed directly by humans, farm animals or wildlife. Arsenic concentrations in root, shoot and grain increases with increasing arsenic concentration in soils or irrigation water. However, phosphate fertilization minimizes the translocation of arsenic to the shoots and grain whilst enhancing phosphate status of plant. It has been demonstrated that phosphate suppresses arsenate uptake by rice, Lupinus albus, and the As-tolerant plants Holcus lanatus, Cystus striatus, and Pteris vittata. The effects of arbuscular mycorrizal inoculation and phosphorous supply on growth, arsenic and phosphorous accumulation in plants growing in polluted soils have been also investigated. The aim of this presentation is to provide the current state of knowledge on the factors (pH, surface coverage, residence time, presence of organic and inorganic ligands) which influence the mobility, toxicity and availability of arsenic in natural environments and on the integrating the existing literature on this subject with our recent findings.



88

PHYSIOLOGICAL EFFECTS OF SILICON NUTRITION ON RICE PLANTS

Dettmann, K.C.1, Antunes, W.C.1, Reis, J.V.1, Araujo, W.L.2, Nunes-Nesi, A.1, Fernie, A.R.2 and DaMatta, F.M.1,*

1Departamento de Biologia Vegetal, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, Brasil; 2Max-Planck-Institute for Molecular Plant Physiology, Am Muelenberg 1, 14476 Potsdam-Golm, Germany.


Silicon (Si) is the second most abundant element after oxygen in the earth’s crust. Since silicon dioxide comprises 50-70% of the soil solution, all plants grown in soil contain some Si in their tissues. However, Si is often assumed to be biologically unreactive and it is not considered as an essential element for higher plants. The most positive and consistent effects of Si nutrition have been found in the alleviation of biotic and abiotic stresses, as reported in many publications covering different plant species. Whilst such prophylactic effect has usually been explained by (i) a physical barrier created by Si deposition beneath the cuticle and/or (ii) by the ability of Si to act as a modulator of host resistance to pathogens (and possibly insects), Si is so far believed to cause no effect on primary metabolism in unstressed plants. In rice, a major Si accumulator, Si is actively taken up in the form of monosilicic acid; two genes encoding Si transporters (Lsi1 and Lsi2) have been recently identified in rice. Under field conditions, Si fertilization is widely used to enhance rice production. Such an enhancement has been traditionally attributed to the role of Si in alleviating abiotic and biotic stresses as well as by increasing the erectness of leaves, thus allowing better light transmittance through plant canopies that would indirectly result in improved whole plant photosynthesis. Evidence from the literature suggests that Si additions hardly affect the tiller number, root dry weight and leaf area, and evidence of direct effects on photosynthesis remains as yet inconclusive. In contrast, rice grain yield is increased by Si fertilization, as evidenced by rice mutants for Si uptake. Increased production may chiefly result from (i) lower transpiration of the spikelets (high moisture conditions play a key role for the normal development of the husk) and (ii) protection against pathogen attack. We focused our attention on the effects of Si nutrition on rice growing under unstressed conditions. By hydroponically growing two rice genotypes (‘Oochikara’ – WT, and the lsi1 mutant defective for Si uptake), we showed that Si nutrition improved rice production (up to 42% in WT as compared with its –Si counterpart) without substantially affecting both vegetative growth and leaf angle. To sustain this increased crop yield, photosynthetic rates of the flag leaf increased significantly in response to Si additions. This response was not associated with changes in stomatal conductance, maximum rate of carboxylation and the maximum rate of carboxylation limited by electron transport, but was linked to increased mesophyll conductance. Transpiration rates, in contrast to earlier reports for rice, were unaffected by Si, as assessed through both gravimetric and thermal imaging evaluations. These short-term responses were consistent with the long-term pattern of carbon isotope discrimination. Increased photosynthesis could be, at a first glance, more directly related to increased sink strength mediated by the Si nutrition, rather than by a direct effect of Si addition per se. Nonetheless, we noted that the rate of 14CO2 uptake (assessed under saturating CO2 and therefore in the absence of diffusion-mediated limitations of photosynthesis – thus reflecting the mesophyll capacity for carbon fixation) was larger in +Si plants than in –Si plants, irrespective of genotype and sink strength. Furthermore, changes in the partitioning of the recently fixed 14C were observed under the various experimental treatments between amino acids, proteins and organic acids. Metabolic profiling analysis revealed some changes in abundance of the main intermediates of respiration as well as in the pools of several amino acids, but not in the levels of sugars such as fructose, glucose, maltose and trehalose. In conclusion, we have shown that Si nutrition, in addition to improving harvestable crop in rice, might also affect photosynthesis and primary metabolism through an-as-yet unresolved mechanism.



89

CONTRIBUTION OF MITOCHONDRIAL METABOLISM FOR PHOTOSYNTHETIC PERFORMANCE

Nunes-Nesi, Adriano*

Departamento de Biología Vegetal, Universidade Federal de Viçosa, Viçosa-MG, Brazil.

*Email address: [email protected] The importance of mitochondrial function during photosynthesis has been a matter of discussion for several years. Recently transgenesis approach has been used in order to understand the roles of the tricarboxylic acid (TCA) cycle in tomato (Lycopersicum esculentum). The work started with the characterization of the spontaneous mutant Aco1 of L. pennellii which was deficient in expression and activity of aconitase displayed elevated photosynthetic performance. Subsequently transgenic plants deficient in the expression and activity of each TCA cycle enzyme were generated and characterized at physiological, molecular and metabolic levels. These transgenic plants, characterized at optimal conditions for growth and development, showed distinct phenotypes in response to the reduction in TCA cycle activity. These responses can be grouped into three classes: (i) changes related to redox poise altering photosynthetic rates [malate dehydrogenase (Nunes-Nesi et al., 2005) and aconitase (Carrari et al., 2003)]; (ii) changes in the regulation of stomatal aperture [fumarase (Nunes-Nesi et al., 2007)] and (iii) changes in nitrogen metabolism [citrate synthase (Sienkiewicz-Porzucek et al., 2008) and isocitrate dehydrogenase antisense (Sienkiewicz-Porzucek et al., 2010) plants]. In this talk I will focus on the understanding of the various physiological phenotypes observed in our transgenic plants. Additionally I will discuss the importance of mitochondrial activity for plant growth and development under abiotic stress. References

- Carrari F, Nunes-Nesi A, Gibon Y, Lytovchenko A, Ehlers-Loureiro M, Fernie AR (2003) Reduced expression of aconitase results in an enhanced rate of photosynthesis and marked shifts in carbon partitioning in illuminated leaves of wild species tomato. Plant Physiol 133: 1322–1335

- Nunes-Nesi A, Carrari F, Gibon Y, Sulpice R, Lytovchenko A, Fisahn J, Graham J, Ratcliffe RG, Sweetlove LJ, Fernie AR (2007) Deficiency of mitochondrial fumarase activity in tomato plants impairs photosynthesis via an effect on stomatal function. Plant J 50: 1093–1106

- Nunes-Nesi A, Carrari F, Lytovchenko A, Smith AMO, Loureiro M, Ratcliffe RG, Sweetlove LJ, Fernie AR (2005) Enhanced photosynthetic performance and growth as a consequence of decreasing mitochondrial malate dehydrogenase activity in transgenic tomato plants. Plant Physiol 137: 611–642

- Sienkiewicz-Porzucek A, Nunes-Nesi A, Sulpice R, Lisec J, Centeno DC, Carrilo P, Leisse A, Urbanczyk-Wochniak E, Fernie AR (2008) Mild reductions in mitochondrial citrate synthase activity result in a compromised nitrate assimilation and reduced leaf pigmentation but have no effect on photosynthetic performance or growth. Plant Physiol 147: 115-127.

- Sienkiewicz-Porzucek A, Sulpice R, Osorio S, Krahnert I, Leisse A, Urbanczyk-Wochniak, E, Hodges M, Fernie AR, Nunes-Nesi A (2010) Mild reductions in mitochondrial NAD-dependent isocitrate dehydrogenase activity result in altered nitrate assimilation and pigmentation but do not impact growth. Molecular Plant, 3: 156-173.



90

CIRCADIAN CONTROL OF ROOT ELONGATION AND C PARTITIONING IN Arabidopsis thaliana

Fisahn, Joachim*

Max Planck Institute of Molecular Plant Physiology, 14476 Potsdam/Golm, Germany, Am Mühlenberg. *Email address: [email protected]

The contribution of the carbon (C) supply and the clock to the diurnal regulation of primary root elongation was investigated in Arabidopsis seedlings using time-resolved infrared video imaging. The average rate of root extension varied almost 4-fold between wild-type seedlings growing in an 8, 12 or 16 h photoperiod, and in the presence or absence of external sucrose. The extension rate showed rapid transient changes after illumination and darkening, and a robust slow oscillation with a decline during the first 8-9 hours after dawn and a gradual recovery for the remainder of the 24 h cycle. When the night was extended, growth continued for 1-2 hours and then declined rapidly. The starchless pgm mutant and a mutant in starch degradation (sex1) showed a strong inhibition of growth at night, which was reversed by exogenous sucrose. These results show that wild-type seedlings balance root growth with C availability over the entire 24 h cycle, and that starch turnover is essential to secure the C supply at night. Two lines of evidence revealed that the 24 h oscillation is driven by the clock; it persisted in continuous light, and was strongly modified in clock mutants (cca/lhy, elf3, elf4). In elf3, endogenous starch and sugar levels were elevated at the end of the night, root growth was maximal towards the end of the day and declined during the night and this inhibition was not reversed by exogenous sucrose. Further, the inhibition of growth that occurs during the subjective day in wild-type seedlings in free-running continuous light was partly and completely reversed in elf4 and elf 3, respectively. In cca1/lhy, endogenous starch and sugars were prematurely exhausted before the end of the night, growth declined at the end of the night and the inhibition of growth was almost completely reversed by exogenous sucrose. We propose that the clock regulates root elongation growth in two ways. ELF3 and ELF4 act in a clock-dependent manner to depress root growth in the light and promote root growth in the night, while CCA1/LHY is required to set an appropriate rate of starch degradation and provide a supply of sugars to allow root growth throughout the night. Further, Arabidopsis responds to a sudden premature dusk by slowing the rate of starch degradation and simultaneously decreasing the rate of root growth, and this coordinated adjustment of C allocation and growth is impaired in the cca1/lhy mutant.



91

Symposium 5

“Soil, Plant Physiology and Plant Nutrition”




92

SEED PELLETIZATION WITH SELENITE AND ITS INFLUENCE ON THE ANTIOXIDANT SYSTEM OF RYEGRASS CULTIVARS

Cartes, P.1,*, Yañez-Mansilla, E.2, Paredes, C.2 and Mora M. L.1

1Center of Plant-Soil Interactions and Natural Resource Biotechnology, Scientific and Technological Bioresources Nucleus (BIOREN) and 2Doctoral Program in Science of Natural Resources. Universidad de La

Frontera, Temuco, Chile. *Corresponding author: [email protected] At global scale, selenium (Se) fertilization has been successfully employed to raise the Se content of pastures. In Chile there are not Se-fertilizers, and the Se deficiency affecting grazing cattle in the South region support the need to develop strategies for Se application in deficient areas. In this way, seed pelletization with Se may constitute an important tool to overcome Se deficiencies in grasslands systems. On the other hand, plant species differ widely in their ability to tolerate Se, and several reports have shown that at low concentration Se exerts an antioxidant role in plants subjected to abiotic stress (e.g. Cartes et al. 2010). Nevertheless, the impact of seed pelletization with Se on the antioxidative system at the cultivar level have no been reported yet. The aim of this study was to evaluate the effect of seed pelletization with increasing selenite supply levels on the Se uptake and the antioxidant ability of ryegrass cultivars under greenhouse conditions. Ryegrass (Lolium perenne) plants of three cultivars (Aries, Nui and Quartet) were grown on an Andisol in a greenhouse assay. Seeds were pelleted with sodium selenite at rates between 0 and 60 g ha-1. Two cuts of shoot biomass were harvested at 30 cm of plant height. Dry weight (DW) and shoot Se concentration were determined. Lipid peroxidation (TBARS) was measured as an index of oxidative stress. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were also analyzed. According with our results, DW of Aries, Nui and Quartet was not affected by the selenite addition level (0 to 60 g Se ha-1). Seed pelletization with Se at dosages up to 60 g ha-1 progressively increased the shoot Se concentration at the first and the second cut, and the pattern of Se accumulation did not differ between the three cultivars. Although no effect of the shoot Se concentration on the lipid peroxidation was detected at the first cut (except in Nui at the highest shoot Se content), dissimilar responses in TBARS accumulation occurred at the second cut. Whereas in Aries a threshold for TBARS reduction was detected at shoot Se concentration of about 200 µg kg-1 DW, the oxidative damage of membranes decreased in Nui and Quartet at increasing shoot Se content. In addition, SOD activity decreased in Quartet at increasing shoot Se concentration for the first cut. However, in Aries and Nui, SOD was inhibited at shoot concentration up to about 400 µg Se kg-1DW, and above this concentration the enzyme activity increased. For the second cut, a slight reduction of SOD activity occurred in Nui, and non differences were found in Quartet at all Se supply levels. In Aries, a decline of SOD activity was observed at shoot concentration up to 400 µg Se kg-1 DW, and the greatest Se concentrations activated it. This fact denotes increased production of superoxide free radicals in the shoots at the highest Se content. GSH-Px activity was raised in the three cultivars in both cuts as a consequence of the Se uptake, and the most noticeable changes in its activity occurred in Aries and Nui with respect to Quartet plants. Thus, ryegrass cultivars differed in their antioxidant responses to Se when it was applied directly to the seeds, and the pelletization with Se of ryegrass seeds can be used as a strategic tool to increase the Se concentration in pasture systems. Acknowledgements: FONDECYT Project 11080215 and CONICYT Scholarship 2010. References:

P. Cartes, A. Jara, L. Pinilla, A. Rosas and M.L. Mora. 2010. Ann. Appl. Biol. 156: 297-307.



93

EFFECT OF SELENIUM ON BREAD WHEAT (Triticum aestivum L.) CULTIVARS OF LA ARAUCANIA REGION

Pinilla, L.1,*, Cartes, P.2 and Mora, M.L.2

1Doctorate Program in Science of Natural Resources, Universidad de La Frontera. 2Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and Technological Bioresource Nucleus,

Universidad de La Frontera, Temuco, Chile. *Corresponding author: [email protected]

Selenium (Se) is an essential micronutrient for humans, although in plants it has not been yet confirmed as an essential element. However, nowadays, there are more biochemical evidences about the antioxidant function of Se in vascular plants. In humans, Se deficiency is related to various pathologies, and it has been reported that organic Se compounds improve the immune system and decrease the occurrence of different cancer types. Wheat is a cereal of mass consumption, and it might represent an important Se source. Besides, wheat is the main cereal grown in Chile, especially in the Southern area. The objectives of this research were: (i) evaluate the Se accumulation in grains of different cultivars of wheat grown in Andisols (Vilcún and Temuco Series of Southern Chile) never amended with Se fertilizers and, (ii) study the effect of Se on oxidative stress induced by selenite in roots of wheat cultivars differing in their potential of Se accumulation in grains. In the first stage of this research, Se concentration in bread wheat grains of 10 cultivars widely grown in La Araucanía Region was analyzed. In a second stage, four wheat cultivars presenting higher Se concentration were selected to conduct a hydroponic assay by using 6 Se doses (0 to 10 µM Se applied as sodium selenite) with 3 replicates per treatment. According to the results, we observed considerable differences in the potential for Se accumulation in wheat grains of different cultivars. The higher Se concentration in grains were observed in cv. Purpura (34 µg Se kg-1), followed by cv. Fritz (25 µg Se kg-1); cv. Invento (23 µg Se kg-1) and cv. Bakan (19 µg Se kg-1). Comparatively, the lower values were detected in cv. Porfiado and cv. Kumpa, which only reached 7 µg Se kg-1. The results of the hydroponic assay indicated that the effect of Se on the lipid peroxidation of roots, as measured by Thiobarbituric Acid Reactive Substances (TBARS), differed between the different wheat cultivars. Furthermore, a noticeable oxidative damage to lipids was observed at 10 µM Se applied to the culture media, especially in roots of wheat cv. Invento and cv. Fritz.

Acknowledgments: This work was supported by the FONDECYT projects 1100625 and 11080215. The author is grateful to the Doctoral Scholarship CONICYT for financial support. Authors are also thankful to Erik von Baer, Director of the Research Centre Semillas Baer for his collaboration with wheat seeds for the development of this research.



94

NITROGEN AND SULFUR FERTILIZATION AND ITS INTERACTION WITH SELENITE PELLETED SEEDS AFFECT THE OXIDATIVE STRESS IN RYEGRASS

PLANTS

Yañez-Mansilla, E.1,*, Pirce, F.2, Vega, I.3, Mora, M.L.4 and Cartes, P.4

1Doctoral Program in Science of Natural Resources; 2Soil and Plant Analysis Laboratory 3Faculty of Agronomy and Forestry Sciences; 4Scientific and Technological Bioresources Nucleus (BIOREN),

Universidad de La Frontera, Temuco, Chile. *Corresponding author: [email protected] From the point of view of plant nutrition, selenium (Se) deficiency in soils and forages of Southern Chile is well documented. The application of Se-fertilizers to the soil-plant system has proven to be beneficial for animal health, and in this way the seed pelletization technique can be used to raise the Se content in pasture systems (Cartes et al., 2010). Selenium can also exert positive effects in vascular plants and improve the tolerance against abiotic stress. Plants metabolize Se by the sulfur (S) assimilation pathway, and, only few reports shows the competitive effect of S on selenite in plant tissues. In addition, the metabolic pathways of Se or S and nitrogen (N) are linked in the biosynthesis of aminoacids, which can be incorporated into proteins later. The aim of this research was to evaluate the effect of seed pelletization with selenite on the oxidative damage in plants subjected at different nitrogen and sulfur fertilization rates. Plants of ryegrass (Lolium perenne) of cultivar Quartet were grown in an Andisol (Freire Series) of Southern Chile. We evaluated the combined effect N, S and Se in a randomized design with three replicates per treatment. Ryegrass seeds were pelleted with sodium selenite at doses of 0, 30 and 60 g Se ha-1, and before sowing soil samples were fertilized with N (0 or 100 mg kg-1 soil) and S (0, 50 or 100 mg kg-1 soil). In order to evaluate the oxidative damage, the level of lipid peroxidation was assessed by monitoring the thiobarbituric acid reactive substances (TBARS). The results showed that, in plants supplied with N, lipid peroxidation diminished as a consequence of Se addition at increasing rates irrespective of the S added, and a reduction of TBARS accumulation of about 53% was observed in plants fertilized with 50 mg S kg-1 and 60 g Se ha-1 in relation to plants non- Se treated. In contrast, when no N was applied, the highest rate of S application triggered an increase of the oxidative damage of membranes at a Se supply of 60 g ha-1. Such response indicated that at the greatest S and Se addition levels oxidative stress occurred probably due to a lower N availability for the synthesis of S or Se essential organic compounds. Thus, depending on the N and S fertilization rates, ryegrass seed pelletization with increasing Se dosages seems to be an efficient technique for reducing oxidative damage, and consequently to improve the nutritional quality of pastures.

Acknowledgements: FONDECYT project 11080215 and CONICYT Scholarship 2010.

References

- Cartes, P., Gianfreda, L., Paredes, C. and Mora M.L. Proceedings of the 19th World Congress of Soil Science. 4p.



95

ALUMINUM-SULFUR INTERACTION EFFECTS ON TOTAL PROTEIN EXPRESSION IN Vaccinium corymbosum L. GROWING IN AN ANDISOL

Alarcón, E.1, Reyes-Díaz, M.2,* and Alberdi, M.2

1Programa de Doctorado en Ciencias de Recursos Naturales, Facultad de Ingeniería, Ciencias y Administración,

Universidad de La Frontera, Temuco, Chile. 2Departamento de Ciencias Químicas y Recursos Naturales, Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and Technological Bioresource Nucleus

(BIOREN-UFRO), Universidad de La Frontera, Casilla 54-D. Temuco, Chile. *Corresponding author: [email protected]

In acid soils (Ultisols and Andisols) derived from volcanic ashes in southern Chile, aluminum (Al) toxicity is an important factor limiting in plants growth, development and production. Acid soils present nutritional deficiencies, such as N, Ca, Mg, K, S, Zn and B, which justify intensive management of fertilization and liming. Application of lime and gypsum amendments can reduce this effect in pasture on Andisols. On the other hand, about 70% of soils of southern Chile contain low levels (below 15 ppm) of sulfur (S), whereas normal range of S in the agricultural soils of humid and sub-humid regions is about 100 to 500 ppm. Furthermore, chemical plant analyses have revealed that S is a limiting nutrient for pastures in southern Chile. Sulfur is an essential macronutrient, being important as structural component of amino acids, regulatory and catalytic functions, vitamins, enzymes. It is also essential for chlorophyll synthesis and many other cell components such as glutathione. Sulfur deficiency limits the formation of proteins by plants, which has an important effect on their physiological performance, especially for the leaves, where photosynthesis takes place. Plants deficient on S are characterized for reducing photosynthetic activity, growth and productivity. Vaccinium corymbosum (highbush blueberry) is considered among all fruits and vegetables as one of the richest sources of antioxidants. This feature gave increasing interest to human health, making it an important crop species, which also acquired economic importance, and interest for scientific studies in our country and region, where it grows preferentially on Andisols. With exception of the effect of Al toxicity on some physiological parameter in cultivars of this species, nothing is known about the effect of Al-S interaction on the photosynthetic performance and protein expression in Blueberry growing on Andisols. This is the future aim of our project. Due to the importance that the knowledge of the elements in soil solution and their interaction has for an optimal plant growth, we began our study with the determination of the solution speciation by Geochem Program. The preliminary results indicated that by adding 2.5 mM CaSO4 + 100 µM AlCl3 and 5 mM CaSO4 + 100 µM AlCl3 the percentage of aluminium complexed with SO4

2- was 42.6 % a 52.5% respectively, causing in both cases a reduction in the Al-free. In relations to protein expression we expected to find differences with increasing SO4

2- for each treatment. Acknowledgments: Fondecyt Nº11080231, Berries San Luis, Mecesup fellowship.



96

PHOTOCHEMICAL EFFICIENCY OF THREE HIGHBUSH BLUEBERRY CULTIVARS UNDER ALUMINUM TOXICITY AFTER APPLICATION OF CALCIUM SULFATE

UNDER CONTROLLED GREENHOUSE CONDITIONS

Poblete Grant, P.1, Reyes-Díaz, M.2,*, Meriño-Gergichevich, C.3 and Alberdi, M.2

1Facultad de Ciencias Agropecuarias y Forestales, Universidad de la Frontera, Temuco, Chile. 2Departamento de Ciencias Químicas y Recursos Naturales, Center of Plant, Soil Interaction and Natural

Resources Biotechnology, Scientific and Technological Bioresource Nucleus (BIOREN-UFRO), Universidad de La Frontera, Temuco, Chile. 3Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de

La Frontera, Temuco, Chile. *Corresponding author: [email protected]

The highbush blueberry (Vaccinium corymbosum L.) is a major fruit crop for human nutrition and health. In southern Chile this species is well adapted to soil and climatic conditions of its habitat. However, it is sensitive to the presence of phytotoxic aluminum (Al3+) expressed as percentage of Al saturation (% sat Al), common in Andisols. The literature widely reported the negative effects on physiological processes such as photosynthesis that may have the Al3+ on different crops. Against this background, different calcareous amendments are used to reduce Al3+.One of these is calcium sulfate (CaSO4), whose effects on the reduction of Al3+, have been demonstrated. The aim of this study was to determine the effect of the application of CaSO4 on the photosynthetic apparatus of highbush blueberry grown in an Andisol with high Al-saturation. One year old plants of cultivars Brigitta, Legacy and Bluegold were established in pots (1 kg), containing soil fertilized with 40:30:60 NPK, from the Gorbea serie (pH 4.7 and 70% saturation of Al, a field capacity of 40%) in a greenhouse Three treatments of CaSO4 were established: 0 kg ha-1 (T1), 500 kg ha-1 (T2) and 1000 kg ha-1 (T3). Greenhouse conditions were: 300 µmol m-2 s-

1photosynthetic photon density, 80% relative humidity, and a mean temperature of 20ºC. At the beginning of the experiment and after 15 and 30 days photochemical parameters of the PSII (maximum quantum yield, Fv/Fm; effective quantum yield; ΦPSII; electron transport rate, ETR; and non- photochemical quenching, NPQ) were determined. Fv/Fm was not modified in any of the cultivars under the three treatments, achieving normal values (from 0.7 to 0.8). At 15 days of treatment in Bluegold a decrease of 80% of ΦPSII and ETR in T1, and 40% in T2 was found, whereas in T3 a 30% augment was exhibit at this time. At 30 days ΦPSII and ETR increased in all treatments. Brigitta showed a ΦPSII and ETR decreased 75% in T1, 50% in T2 and 30% in T3 at 30 days. Legacy values of ΦPSII and ETR also decreased in all treatments, both at 15 and 30 days. In NPQ a similar increase in the three cultivars at 15 days was observed in T2 and T3. However, these values remaining near the control at 30 days. In conclusion CaSO4 treatments were able to reduce the negative effects of Al3+ on photosynthetic apparatus in the three cultivars For ΦPSII and ETR of Legacy the best treatment was T2. Brigitta showed a higher tolerance to Al saturation because ΦPSII maintained their values similar to the control at 15 days. Bluegold, was the most sensitive cultivar, being T3 the best treatment for this cultivar. Then, the response to high aluminum saturation is genotype, time and dose of CaSO4 dependent.

Acknowledgments: Fondecyt Nº 11080231, Berries San Luis, Semillas Baer.



97

CHANGES IN THE ANTIOXIDANT ACTIVITY IN LEAVES OF Vaccinium corymbosum L. UNDER THE EFFECT OF CALCIUM SULFATE AMENDMENT IN AN ANDISOL

Durán, M.1, Reyes-Díaz, M.2,3,* and Alberdi, M.2,3

1Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco, Chile. 2Departamento de Ciencias Químicas y Recursos Naturales, Facultad de Ingeniería, Ciencias y

Administración, Universidad de La Frontera, Temuco, Chile. 3Center of Plant- Soil Interaction and Natural Resources Biotechnology, Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La

Frontera, Casilla 54-D, Temuco, Chile. *Corresponding author: [email protected]

Currently, the antioxidants importance in human health is well documented. Fruits and crops are rich sources in different antioxidants and therefore the demand for these products is steadily augmented. Thus, it is well known that fruits of the highbush blueberry (Vaccinium corymbosum L.) have high amounts of anthocyanins and flavonoids, which is associated with a high antioxidant activity. This highbush is well adapted to acid soils. In Chile, this crop is cultivated principally on volcanic soils (Andisols) characterized by low pH and high contents of phytotoxic aluminum (Al3+). A general effect of this toxicity on plants is the reduction of roots and plant growth, which finally results in reduction of crop yield and production. To ameliorate this effect different calcium (Ca) amendments can be used. Calcium sulphate (CaSO4) amendment application seems to be adequate because maintain the pH, reduce the Al toxicity and provide sulphur to these soils, which are deficient in this element. However, there is no information about the effect of this amendment on antioxidant activity (AA) and photosynthesis in leaves of V. corymbosum growing in Andisols of southern Chile. This study was performed in Berries San Luis Farm, Lautaro, Chile. Leaf samples were obtained from different plots with three cultivars (Brigitta, Legacy and Bluegold). They were amended with 0 (control), 500, 1000 and 2000 Kg ha-1 of CaSO4. Two sampling periods were considered (December 2009 and January 2010) for determinations of the AA and CO2 assimilation. The results indicated that Legacy cultivar and Brigitta did not have significant differences neither in CO2 assimilation nor AA. Furthermore, in Bluegold, the Al cultivar sensitive, shown an increase in AA with the CaSO4 treatment, this is concomitant with the decrease in CO2 assimilation. We can concluded that the higher calcium sulfate treatments to ameliorate the antioxidant activity in Bluegold.

Acknowledgments: Fondecyt Nº 11080231 project, Berries San Luis, UFRO fellowship.



98

CALCIUM SULFATE EFFECTS ON ANTHOCYANINS AND PHENOLS IN HIGHBUSH BLUEBERRY

Reyes-Díaz, M.1,*, Meriño, C.2, Mora, M.L.1 and Alberdi, M.1

1Departamento de Ciencias Químicas y Recursos Naturales, Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and Technological Bioresource Nucleus (BIOREN-UFRO), Universidad

de La Frontera, Casilla 54-D, Temuco, Chile. 2Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco, Chile.


Soils derived from volcanic ashes (Andisols) with an acid pH are frequent in South Central Chile. Acid soils permit the Al solubilization from silicates and oxides in phytotoxic aluminium (Al3+). Al toxicity, which induce ROS in several plants, seems to be important in limiting the plant growth and productivity in agricultural volcanic soils. To ameliorate the Al phytotoxicity of acid soils gypsum application is used for reduction of Al3+. Additionally, the gypsum application could also supply water-soluble calcium (Ca2+) without significantly pH changes. The interactions between Al3+ and Ca2+ are probably the most important factors affecting Ca2+ uptake and transport in plants grown in acid soils (pH < 5.5). These conditions seem to be very important for blueberry which need soil acidity for growth. Nonetheless, the few reports about the gypsum utilization in Andisols deal with its effects on soil, but not with the effects on leaf secondary metabolites with antioxidant capacity. In this work Brigitta, Legacy and Bluegold cultivars growing in hydroponic solution (Hoagland) were subjected to following treatments: control (Hoagland solution); 2.5 mM CaSO4; 5 mM CaSO4; 100 µM Al; 100 µM Al + 2.5 mM CaSO4; 100 µM Al + 5 mM CaSO4 during 15 days. After this time the total phenols and total anthocyanins and their composition were determined. Total phenols of Brigitta and Legacy decreased in 5mM CaSO4 treatment and in Legacy also by the interaction of CaSO4 and Al. Bluegold showed a decrease of total phenols in all treatments with exception of the Al treatment. Total anthocyanins of Legacy and Brigitta remained constant in all treatments, but increased in Bluegold (P≤0.05). In Bluegold, delphidin, cyanidin, petunidin, peonidin and malvidin decreased drastically (P≤0.05) with Al treatment. In Brigitta, malvidin and delphidin decreased significantly with the combination of CaSO4 and Al. In this cultivar cyanidin augmented with Al treatment. In Legacy, malvidin increased in all treatments, but this increase was better with Al treatment, whereas petunidin and delphinidin increased only in the 5 mM CaSO4 treatment. Peonidin increased in all treatments, being higher in 5 mM CaSO4 treatment in Legacy. In conclusion, our results showed that the effect of CaSO4 application on secondary metabolites of blueberry is cultivar specific dependent. On the other hand, an adequate management of the CaSO4 application could help to modulate the induction of these metabolites with antioxidant capacity to avoid oxidative stress in blueberry cultivars growing in Andisols.

Acknowledgments: Fondecyt Nº 11080231 project, technical assistance and Berries San Luis, Lautaro.



99

EFFECT OF MANGANESE TOXICITY AND UV-B RADIATION ON ANTIOXIDANT RESPONSE OF BLUEBERRY (Vaccinium corymbosum L.)

Rojas-Lillo, Y.1, Reyes-Díaz, M.2,3, Alberdi, M.2,3,* and Mora, M.L.2,3

1Programa Magíster en Ciencias de Recursos Naturales, Facultad de Ingeniería, Ciencias y Administración, Universidad de La Frontera, Casilla 54-D, Temuco, Chile. 2Departamento de Ciencias Químicas, Universidad

de La Frontera, Casilla 54-D, Temuco, Chile. 3Center of Plant, Soil Interaction, and Natural Resources Biotechnology, Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera,

Casilla 54-D, Temuco, Chile.*Corresponding author: [email protected]

The toxicity of manganese (Mn +2) has negative consequences for crops, limiting growth and yield, mainly in acid soils. In southern Chile, soils derived from volcanic ash (Andisols), with an acid pH are common. Besides, adverse environmental factors such as UV-B radiation (RUV-B) can synergistically potentate stress induced by Mn toxicity producing a strong oxidative damage. In this region, the cultivation of blueberries (Vaccinium corymbosum L.) has become very important, but very few or no studies about this theme are yet available Therefore, the objective of this study was to evaluate the antioxidant response (antioxidant activity (AA) and activity of the enzyme superoxide dismutase (SOD)) of roots and leaves of two cultivars of this species (cvs. Brigitta and Bluegold) against a combined action of toxic levels of Mn and UV-B radiation. As indicator of stress injury, lipid peroxidation was used. In addition, RUV-B absorbing compounds of leaves were also determined. Two year old seedlings of these cultivars were placed in hydroponic solution and the following treatments were applied: a) 2µM Mn, (control) b) 500 µM MnCl2 c) ultraviolet B radiation (RUV-B) d) 500 µM MnCl2 + RUV-B (Mn + RUV-B). Experiments were carried out in a greenhouse using UV-B lamps (Phillips Q-Panel 313), simulating a daily cycle of typical summer UV-B radiation of the Araucanía Region (peak 4 kW m-2 ), for 30 days. Mean temperature was 25ºC, relative humidity (75-80%) and PAR was 270 to 500 µmol photons m-2 s-1). We used a 2-way ANOVA and Tukey post test for statistical analysis. In Mn + RUV-B treatment, roots lipid peroxidation (LP), and the antioxidant activity (AA) and the activity of the enzyme superoxide dismutase (SOD) increased in Brigitta and Bluegold compared with the controls (p > 0.010). Under this treatment, leaves showed augmented AA, SOD and RUV-B absorbing compounds in both cultivars (P = 0.009). RUV-B treatment absorbing compounds were also significantly increased (P=0,009) when treated with RUV-B alone. A similar rise was observed in the total phenol contents of both cultivars in the RUV-B and Mn + RUV-B treatment. However, in the RUV-B treatment, the anthocyanin content increased only 24% in Brigitta compared with the control, whereas no difference was found in Bluegold. It can be concluded that the antioxidant defense systems of these cultivars were activated to a greater extent during the combined treatment of Mn+ RUV-B, despite the higher lipid peroxidation exhibited by roots. Acknowledgments: Project Fondecyt Nº 1080372, Scholarship UFRO and Bicentenary PSD-26 Project



100

EFFECT OF CALCIUM SULFATE APPLICATION ON LEAF SULFUR CONTENT IN HIGHBUSH BLUEBERRY (Vaccinium corymbosum L.) GROWING IN AN ANDISOL

Castel , A.1, Meriño-Gergichevich, C.2, Reyes-Díaz, M.3,4,* and Alberdi, M.3,4

1Facultad Ciencias Agropecuarias y Forestales, Universidad de La Frontera, Temuco, Chile.

2Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Casilla 54-D, Temuco, Chile. 3Departamento de Ciencias Químicas y Recursos Naturales, Facultad de Ingeniería, Ciencias y Administración, Universidad de La Frontera, Temuco, Chile. 4Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and Technological Bioresource Nucleus (BIOREN-UFRO), Universidad

de La Frontera, Casilla 54-D, Temuco, Chile. *Corresponding author: [email protected]

Highbush blueberry (Vaccinium corymbosum L.) has been developed in a main fruit crop of the La Araucanía region, Chile. This species is well adapted to soil acidity conditions (pH < 5.5), and lower levels of sulfur (S), among others, typical of Andisols. These features are probably due to environmental and agricultural conditions. Sulfur is an essential element of enzymes, proteins, and chlorophylls between others. Different S sources are employed in agricultural management of this fruit crop, however little is known about of suitable doses for this species. A good choice is the use of calcium sulfate (CaSO4), which contains 17% S, without alter the pH level required for and optimal growth of blueberry. The aim of this study was to know the effect of the application of increasing doses of CaSO4 on the leaf S content of V. corymbosum cultivars growing on an Andisol. Four treatments of CaSO4 were applied: 0 (G0), 500 (G1), 1000 (G2) and 2000 (G3) kg ha-1, on plots of Brigitta, Bluegold and Legacy cultivars, cultivated in a two years orchard placed in San Luis, Lautaro La Araucanía region, Chile, where the soil series is Lautaro. We evaluated foliar S content (%) in December 2009 and January 2010. The leaf material was analyzed by turbidimetric method described in Sadzawka et al. (2004). The results indicated that Legacy presented the lowest levels of S in the four treatments in December and January. However, these contents were higher (~ 10%) than the control in January, but in December only the G1 treatment over passed the values of control. In the three cultivars, the treatments G1, G2 and G3 increased (~10%, ~10% y ~5%, respectively) the leaf S contents in January with respect to G0. In December, Legacy and Bluegold responded with a slight increase to G1 (0.081% y 0.089%), while Brigitta did it with G2 (0.095%). G3 had no increase from G0 for all three cultivars. The effect of the application of CaSO4 on foliar S content was genotypically and seasonally dependent. Furthermore, the higher dose of CaSO4 was not correlated with an increase in S content as occurred for the lower doses.

Acknowledgments: FONDECYT Nº 11080231, Berries San Luis, Lautaro.



101

EFFECT OF MATURITY STAGES AND POSTHARVEST TREATMENTS ON PHENOLIC ACIDS, FLAVONOIDS AND ANTHOCYANINS OF HIGHBUSH BLUEBERRY (Vaccinium

corymbosum L.) FRUITS

Ribera, A.E.1*, Reyes-Díaz, M.1,2, Alberdi, M.1,2, Cartes, A.3 and M.L. Mora1

1Center of Plant-Soil Interaction and Natural Resources Biotechnology, Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Casilla 54-D, Temuco, Chile. 2Departamento de Ciencias Químicas y Recursos Naturales; Facultad de Ingeniería, Ciencias y Administración; Universidad

de La Frontera, Casilla 54-D, Temuco, Chile. 3Soil and Plant Analysis Laboratory. *Corresponding author: [email protected]

We evaluated the genotype and maturity effects on phenolic compounds of whole, skin and pulp fruits from three highbush blueberry cultivars (cv. Brigitta, cv. Bluegold and cv. Legacy) grown in southern Chile. We found that total phenolic content in unripe green and fully ripe fruits was high and similar between them, whereas the lowest levels were found in intermediate ripe fruits. The same trend was observed for fruit total antioxidant activity. This could be attributed to the higher concentrations of phenolic acids (mainly chlorogenic acid) and flavonols (mainly rutin) at immature fruit stages; whereas the high antioxidant activity in mature fruits could be explained by the elevated amounts of anthocyanin. All antioxidant compounds were mostly located in the skin. As with almost all parameters, we found significant variations in anthocyanidin concentrations and compositions among the cultivars and during the fruit maturation process. Chlorogenic acid and rutin were the most abundant cinnamic derivative and flavonol in highbush blueberry fruits. Moreover, our results showed that, despite the genotype and maturity effects, delphinidin (Dp) was the main anthocyanidin detected in all cultivars and at all fruit maturity stages. Delphinidin concentrations in blue ripe fruits significantly varied among the cultivars in the order Legacy > Brigitta > Bluegold. The relative distribution of each aglycone also varied among genotypes in the order Dp > Malvidin (Mv) > Cyanidin (Cy) > Petunidin (Pet) for Bluegold; Dp > Cy > Mv > Pet for Brigitta; and Dp > Mv > Pet > Cy for Legacy. Moreover, total amounts of anthocyanidins (Dp + Cy + Mv + Pet) in all three blueberry cultivars were significantly different. It is noticeable that highbush blueberry fruits grown in southern Chile have exceptionally higher antioxidant activity and anthocyanins contents compared with those cultivated in the northern hemisphere. We suggest that all the findings obtained in this work should be considered in processing technologies with aimed to maximize the antioxidant extraction for food, nutraceutical or pharmaceutical industry as well as for further blueberry breeding programs. In addition, we study the effect of different postharvest treatments (storage at 4ºC and UV-C radiation) on the antioxidant activity and total phenolic and total anthocyanins contents. Interestingly, our results showed that both treatments increased the antioxidant metabolites contents in all blueberry cultivars.

Acknowledgements: This work was supported by the Postdoctoral FONDECYT Nº 3090074 and Regular FONDECYT 1080372 Grants. We thank to “Berries San Luis” farm, Lautaro, La Araucanía Region, Chile. We also thank to the Agronomist Water Lobos (Fruticulture specialist) by his collaboration.



102

ACID PHOSPHATASE ACTIVITY AND PHOSPHORUS DYNAMIC IN A RHIZOSPHERE SYSTEM UNDER AMMONIUM AND NITRATE NITROGEN FERTILIZATION

Paredes, C.1,*, Menezes-Blackburn, D. 1, Cartes, P. 2, Ribera, A.E.2 and Mora, M.L2

1Programa Doctorado en Ciencias de Recursos Naturales, Facultad de Ingeniería, Universidad de La

Frontera, Temuco, Chile. 2Center of Plant - Soil Interaction and Natural Resources Biotechnology, Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Temuco, Chile.

*Corresponding author: [email protected] A short-term greenhouse experiment was conducted to evaluate the nitrogen (N) fertilization effect on phosphatase (Pase) activity and phosphorus (P) dynamics in the soil-plant system. Plants of ryegrass and tall fescue were cultivated in an acidic Andisol in the presence or absence of P, ammonium (NH4

+) and nitrate (NO3-). Pase activity, pH and Olsen P were determined in

rhizospheric soil. Plant biomass, P uptake and root P-ase were assayed for each plant species. The results show that, P uptake by tall fescue was about 67% higher than that of ryegrass at low soil P availability, which suggests that tall fescue was less sensitive to P deficiency than ryegrass. For both plant species, P-ase did not vary as a consequence of P addition. On the other hand, fertilization with the highest N-NH4

+ dose strongly decreased soil pH and shoot P content as well as it increased root Pase activity. This fact denotes that root Pase behaved as a strategic response parameter to P stress with insufficient impact on plant P nutrition in both plant species.

Acknowledgements: This work was supported by the FONDECYT Nº 1061262 project grants.



103

CHANGES IN THE CHEMICAL COMPOSITION AND ANTIOXIDANT ACTIVITY OF MORTIÑO (Vaccinium meridionale SW.) FRUITS DURING GROWTH AND

MATURATION

Gavira, C.1, Lopera, Y.1, Hernández, J.2, Lobo, M.2, Medina, C.2 and Rojano, B.1,*

1Universidad Nacional de Colombia sede Medellín, Medellín - Antioquia, Colombia. 2CORPOICA “La Selva”, Rionegro, Antioquia, Colombia.


The fruits of plants of the genus Vaccinium are characterized by high contents of phenolic acids, flavonoids, anthocyanins and high antioxidant activity. V. meridionale (mortiño) belongs to the Ericaceae family and is native to the high Andean zone, being distributed from Venezuela to Bolivia at an altitude between 2600 to 4000 m a.s.l. In Colombia, the mortiño is consumed as fresh fruit or jams, desserts and handmade liqueur. The fruits are obtained in the field from wild shrubs, due to the absence of crops. For some Vaccinium species it is reported that the process of growth and fruit maturation are associated with physical and chemical changes, such as antioxidant activity and polyphenol contents. No studies are reported yet for mortiño. The objective of this research was to determine the change in chemical composition and antioxidant activity of mortiño wild fruits during their growth and maturity, with the purpose to establish the optimal pre-harvest fruit condition. In the present study, the changes in the content of phenols and anthocyanins, chlorogenic, caffeic and ellagic acids and antioxidant activity by TEAC-ABTS, TEAC-DPPH, FRAP and ORAC methods have been performed. The results showed that the antioxidant activity and content of total phenolics and chlorogenic acids exhibited two peaks: the first at 36 days (4804 eq mg de galic acid/ 100 g FW and 646 mg chlorogenic acids 100 g-1 FW) and the second at 105 days (3140 eq mg de galic acid 100 g-1 FW and 757 mg chlorogenic acids 100 g-1 FW). Contents of galic and chlorogenic acid (1373 eq mg de galic acid 100 g-1W and 64 mg chlorogenic acids 100 g-1 FW) approximately constant were obtained before the day 134. Caffeic acid decreased from its initial value to the end of the evaluation. Ellagic acid presented variations throughout the study. The anthocyanins appears quantifiable from the 134 day (4.2 mg c3g 100 g-1 FW) and reach its maximum (272 mg c3g 100 g-1

FW) at 171 days. The maxima antioxidant activity of the fruits of V. meridionale were found in the green stages, which can be due to the ascorbate and glutathione presence in this stage as indicated for other berries. Thereafter, the antioxidant activity decreased with the fruit maturity. Polyphenols were directly related with the antioxidant activity (rFRAP = 0.9934; rORAC= 0.8976). At the end of maturity chlorogenic and ellagic acids were directly related with antioxidant activity. Based in the ORAC value and total polyphenol contents, the best period for the beginning of harvest of mortiño fruit is around the day 143, where these parameters remained constant. In relation to anthocyanins, should be noted that despite harvesting these fruit pigments can increase their value, which is very important for the fruit quality. Acknowledgements: This work was supported by MADR (agriculture Ministry and rural development of Colombia). Project “Categorization of atmospheres with relationship to the production of anti-rust of agraz or mortiño (southern Vaccinium Sw)”, 051-2008L363-33333 and DIME, Universidad Nacional de Colombia, sede Medellín.



104

BIOCHEMICAL ANALYSIS OF SHOOT TISSUES OF L. perenne PLANTS CULTIVATED UNDER VARYING S CONDITIONS IN THE PRESENCE OF ALUMINIUM TOXICITY

Wulff-Zottele, C.1,2*, Fisahn, J.2 and Hesse, H.2

1Center of Plant, Soil Interaction, and Natural Resources Biotechnology, Scientific and Technological

Bioresource Nucleus (BIOREN), Universidad de La Frontera, Casilla 54-D, Temuco, Chile. 2Max Planck Institute of Molecular Plant Physiology, 14476 Potsdam-Golm, Germany.


Aluminium toxicity is an important growth-limiting factor in acid soils. Acid injury of root tissues is mainly caused by aluminium (Al) toxicity that it is finally reflected in alterations of plant physiological functions, like the uptake of macronutrients by root tissues. In order to produce a better crop production on acid soils, farmers are recommended to apply alkaline material such as gypsum (Calcium sulfate) to increase the soil pH, reduce the amount of exchangeable Al in soil solution, and enhance the availability of plant macronutrients. In this study, we decided to approach the questions about the concerning effects of holistic physiological and biochemical changes of sulfate homeostasis, such as those caused by increased doses of sulfate, on the regulation of genes and accumulation of metabolites implicated in plant response mechanisms against Al toxicity in shoot tissues of perennial ryegrass (L. perenne). Perennial ryegrass plants (cv. Jumbo) were tested in a factorial design with four ammonium sulfate fertilization conditions (120 μM, 240 μM, 360 μM, and 480 μM) and Al-stress (200 μM) included in Taylor Foy´s nutrient solution. At day 21 of the experiment, modifications in the photosynthetic parameters of leaves of Al-treated plants, and their controls, were evaluated by a non-invasive chlorophyll fluorescence imaging technique. In parallel to chlorophyll fluorescence measurements, shoot tissues of ryegrass plants were harvested and frozen until their use for biochemical studies of changes in the metabolite composition using GC-TOF (Gas Chromatography coupled to Time of Flight Mass Spectrometer) metabolite profiling. Furthermore, qRT-PCR analysis was used in order to determine differences in the expression of genes associated in metabolic pathways involved in Al stress, that were also modified by the different sulfate supply conditions studied. The results of the methods described above were compiled, organized, and sorted using informatics and data mining tools, such as Hierarchical Clustering Analysis (HCA) and Principal Component Analysis (PCA). Chlorophyll fluorescence Imaging measurements enable the detection of Al toxicity symptoms in shoot tissues of ryegrass plants, that were also dependent of the amount of sulfate included in the nutrient solution. PCA analysis allowed the identification of significant differences in the metabolic phenotypes of shoot tissues of control and Al-treated ryegrass plants, and they were dependent of the amount of sulfate added to the hydroponic solution. On the other hand, HCA analysis determined that the photosynthesis, photorespiration, polyamine biosynthesis, nitrogen assimilation, amino acid metabolism, and tricarboxylic acid biosynthesis displayed significant modifications during the physiological response of leaf tissues against Al toxicity. Furthermore, the compiled information demonstrated that sulfate fertilization less than 360 μM in nutrient solution improves Al-tolerance in ryegrass shoot tissues. Acknowledgements: The research was founded by the Internal Fund of the Universidad de la Frontera (DIUFRO), and the International Scientific Cooperation Program between Comisión Nacional de Investigación y Tecnología (CONICYT, Chile) and the Deutsche Forschung Gemeinshaft (DFG, Germany).



105

STANDARDIZATION OF A TECHNIQUE TO OBTAIN GOOD QUALITY DNA FROM TROUT SCALES COLLECTED AT DIFFERENT PERIODS

Cerda, A.1, Reyes-Díaz, M.2 and Gajardo, G.3*

1Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco, Chile. 2Departamento de Ciencias Químicas y Recursos Naturales, Center of Plant, Soil Interaction and Natural

Resources Biotechnology, Scientific and Technological Bioresource Nucleus (BIOREN-UFRO), Universidad de La Frontera, Casilla 54-D, Temuco, Chile. 3Laboratory of Genetics, Aquaculture & Biodiversity.

Universidad de Los Lagos. Av. Fuschlocher 1305. Osorno, Chile. *Corresponding author: [email protected]

The rainbow trout (Onchorynchus mykiss) and the brown trout (Salmo trutta fario) are exotic genetic resources that became naturalized in inland waters of southern Chile after being introduced in the early twentieth century (1905). Both species are scientifically relevant since they represent an ongoing evolutionary experiment in the receiving ecosystem. Several aspects are interesting to investigate in these species such as the genetic changes experienced in about 100 years as a consequence of the adaptation to a new environment. As this process has not been investigated systematically over spatial and temporal scales, it is relevant to understand the genetic changes associated with adaptation in trouts, and their likely impact on native species hence on ecosystem stability and resilience. This is particularly relevant with rainbow trout which is a highly invasive species, but it has a great value for fly fishing an approximately US 10 million recreational business. Since exotic fishes are the second cause of biodiversity loss after habitat degradation, it is important to understand what genetic characteristics have changed over time in order to infer what makes trouts, particularly rainbow trout, highly invasive. Another event affecting freshwater ecosystem is the explosive development of aquaculture with its toll of millions of escapees likely to interbreed with naturalized trouts and native species. In this work, we are interested in monitoring invasive species at different geographical and temporal scales. In relation to this, scales collected at different periods by anglers provide an opportunity to look backwards the evolutionary process on trouts. The objective of this work was to standardize a DNA extraction technique to obtain good quality DNA from trout scales of different ages collected by anglers. The DNA isolation will be performed by means of two techniques: E.Z.N.A. TMInsect DNA Isolation Kit and a phenol/chloroform method with ethanol precipitation as described by Taggart et al. 1992. Five to ten samples will be taken from O. mykiss and S. trutta scales, with fresh salmon muscles used as positive control. The DNA obtained is expected to be partially degraded considering the small amount of DNA content in scales, particularly in older ones. Different and standardized extraction protocols will be assayed. This will allow us to compare different periods in the evolution of trouts to obtain a minimum amount of DNA of good quality, prior to and after the development of aquaculture. This study will give light to ways for dealing with the management of both species in a more systematic manner.

Acknowledgments: UFRO fellowships and ULA project N°0209F.



106

PHOTOCHEMICAL EFFICIENCY AND INDUCTION OF PHLOROTANNINS IN ANTARCTIC BROWN ALGAE SUBJECTED TO UV-B RADIATION AND

TEMPERATURE STRESS

Cruces, E.1,2,* and Gómez, I.2

1Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco, Chile. 2Instituto de Biología Marina, UACh, Valdivia, Chile. *Corresponding author: [email protected]

Due to global climate change the stratospheric ozone levels are being depleted with the consequent increase in the amounts of solar UV-B radiation reaching the Earth's surface. UV radiation and temperature increases (effect of global warming) are two major factors that affect physiological performance of algae in their habitat, especially those inhabiting the intertidal zone. Macroalgae according to their adaptation have evolved different physiological strategies to resist environmental stress (dynamic photoinhibition, synthesis of compounds with capacity to absorb different wavelengths of UV). The synthesis of phlorotannins (polymers of floroglucinol) has been claimed as a potential photoprotective strategy of brown algae against UV radiation. Apparently, these compounds can act not only as efficient UV-sunscreen, but also as strong antioxidants, minimizing the effects of UV and other stressors. The objectives of the present study were to evaluate the combined action of different doses of UV-B and temperature on different physiological responses (photochemical reactions and synthesis of phlorotannins) through a latitudinal gradient in species of brown algae from Punta Arenas and Antarctic. Results showed that the temperature along with UVB radiation produces a synergistic effect in the production of phlorotannins in Antarctic algae. Therefore, the results indicate that survival strategies are in overall differential and species-specific. Acknowledgements: FONDECYT N°1090494 and INACH N°T 20-09 Projects.



107

PHOTOPROTECTIVE ROLE OF PHLOROTANNINS AGAINST SHORT-TERM UVB AND TEMPERATURE STRESS IN THREE SOUTH PACIFIC KELPS

Cruces, E.1,2 and Gómez, I.2

1Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco, Chile.

2Instituto de Biología Marina, UACh ,Valdivia, Chile. *Corresponding author: [email protected]

Along with UV-B radiation (UV-B) the temperature is a factor that could increase the harmful effects on seaweeds. The syntheses of phlorotannins, polyphenols with high antioxidant capacity, are among the main ecophysiological strategies of brown algae to protect against the action of environmental stress. Therefore, the following work aims to describe the induction of phlorotannins in three species of brown algae of southern Chile (Lessonia nigrescens, Durvillaea antarctica, Macrocystis pyrifera) against thermal and UV-B stress, assessing the antioxidant capacity of these algae and peroxidative damage. We used 3 temperatures (10°, 20° and 28°C) in combination with UV-B. Phlorotannins synthesis was measured by the modified method of Folin-Ciocalteu and peroxidative damage is through of the accumulation of malondialdehyde. Also, the antioxidant capacity was evaluated by the DPPH method. The results of this study show that increased synthesis of phlorotannins occurred at 10°C, causing reduction of these compounds by the action of temperature. On the other hand, the highest antioxidant capacity was observed in the combined treatments of temperature and UV-B, and increased peroxidative damage in the treatment of 28°C UV-B. Therefore, the action of UV-B and the temperature would have an antagonistic effect on the synthesis of phlorotannins, in contrast to the antioxidant capacity, which would increase by synergistic effect of both stresses.

Acknowledgements: Project FONDECYT N°1090494.

+



108

EFFECT OF GLOBAL WARMING ON Vitis vinifera FROST-DAMAGE ON SOUTHERN CHILE

Orrego, R.* and E. Jorquera-Fontena, E.

Programa Doctorado en Cs. de Recursos Naturales,Universidad de La Frontera, Temuco, Chile.


The current global warming (GW) accelerates the phenological states (PS) in plants (Peñuelas and Filella 2001). Although under GW it is predicted a decrease of frost-day account (Liu et al., 2008), a fast phenological grow process might produce an overlap between sensitive periods and adverse weather conditions (mainly frost). This work aims to measurement the frost risk of some grape critical PS cultivated on southern Chile under GW conditions, based on the hyphotesis the plant phenology is mainly controled by temperature. Both global warming, and current condition are obtained of the dataset generated by Fuenzalida et al., (2006), thus, we choose the maximun and minimun temperatute (MMT) grids for simulated IPCC scenarious A2 (2070-2100) and “base line” (BL) (1961-1991). These data were entered to stochastic weather generator LARS-WG to generate time series of 50 years of daily MMT and to calculate degree-days (base 10ºC, DD). PS states were calculated based on the PS/DD relationship (Jorquera y Orrego, 2010). Crop data were obtained from two trial of var. Gewürtztraminer (GE) (14 plants) and var. Sauvignon Blanc (SB) (14 plants), planted in 2001. The measurements were done from August to the harvest, during 2003-04 and 2004-05. We accounted the frosts on dormancy, budburst, blooming and ripening, and calculated the frost likehood by the rate between the number of frost and period size. The model show that GW shorten the PS cycle in 26 days for GE and 27 days for SB. The frost ocurrence decrease from 13 to 2 frosts per year due to GW. However, this is only significant in dormancy. In budburst there is a small increment of frost likehood on GW condition (from 0 to 0.09%). In flowering does not show changes. Frost in ripening happened after the harvest in GW and BL conditions. There results is based on empirical PS/DD relationships so it is necessary to see ones with caution. Zang et al. (2008) propose that this relationship can be modified on the time, e.i. it is not stationary relationship. This implies that phenology might not changes so much because the plant is adapting to new condition. Althought GW decrease the frost ocurrency, the frost damaege likehood is increase due to the early burdburst. For impruving this prediction, we have a challenge of added the non stacionary relationship between degree-days and phenolgy. References:

- Peñuelas J., Filella I. 2001. Science 294: 793-794. - Jorquera E., Orrego, R. 2010. Agrociencia. In Press. - Fuenzalida H., Falvey M., Rojas M., Aceituno P., Garreaud R., 2006. Informe CONAMA. 71 p. - Zhang T., Zhu J., Yang X. 2008. Agricultural and forest meteorology 148:1412-1418.



109

ASSESSING THE ANTATIC OSCILLATION (OAA) AND ENSO AS PREDICTORS OF SEAZON FORECAST ON ARAUCANÍA REGION

Orrego, R.1,2,*, Ávila, A.2,3 and Salinas, B.2

1Doctorado en Cs. de los Recursos Naturales, Universidad de La Frontera, Temuco, Chile. 2Centro Agronómico de Investigación y Desarrollo (CAID), Universidad de La Frontera, temuco, Chile. 3Centro de

Excelencia de Modelación y Computación Científica (CEMCC), Universidad de La Frontera, Temuco, Chile. *Corresponding author: [email protected]

Climate is one of the main factors on crops systems and other issues related with natural resources. Currently, researches has developed important efforts for forecasting events which bring high wastes such as, frost, drought, floods and anomalous rains. In this way, season forecast is one of the main goals. Chile climate has high variability. At global scale, Pacific Anticyclone is the main factor which drive the meteorological conditions. The Pacific Anticyclone is a high preassure center which is a barrier to fronts. Thus, when Pacific Anticyclone are onto the southern aestival driest are poduced, and when the Pacific Anticyclone are onto the northern winter rains are produced. Althouhg the Pacific anticyclone determinates the typical annual climate cycle, this is high influenced by ENSO (El Niño Southern Oscillation) and OAA (Antartic Oscillation). In this work, we asses the ENSO and OAA as predictors of medium-term climate forecast, for will develop a medium term climate model. We obtained the ENSO3.4 index (http://iridl.ldeo.columbia.edu/), the OAA-index (http://www.cpc.noaa.gov/products/precip/CWlink/daily_ao_index/aao/aao_index.htm), and meteorological records of the stations “Quellen” and “Quinta Normal”. The last was used for comparing the region (boundary region of Pacific Anticyclone) with the central zone. We clustered the dataset by IRI criteria for ENSO and one standard deviation for OAA. Based on this clustering, we computed mean, standard deviation, and climatology for each class, and each station. In addition, we check the independence of ENSO and OAA (by correlation index), and the montly correlation between both index and the rainfall considering each indexes and both indexes together by a multiple correlation model, for each station. We observed that ENSO and OAA are independents. Also, we observed that there is an increase on the rainfall when ENSO condition are produced, but, this is changes by the OAA. In fact, under el Niño when the OAA is in warm phase there are an less decrease of 6% and when the OAA is in cold face the increase is 105% in Santiago. In Quellen under el Niño when the OAA is in warm phase there are an less decrease of 0.9% and when the OAA is in cold face this decrease is 18%. In the other hand, the effect of ENSO changed during the annual cycle. In this way, the ENSO effect is higher at summer month, while the OAA effect is similar in all year. The ENSO and the OAA correlation have an spatial pattern. In fact, in the northern station the ENSO correlation are higher than southern station, in opposite of the OAA correlation. The last is in agree with the decrease of the coefficient of ENSO and the increase on the OAA coefficient when we compared the southern station with the northern station.



110

EFFECTS OF ALUMINUM TOXICITY ON ROOT TIPS CELLS ORGANIZATION AND ANTIOXIDANT SYSTEM IN BLUEBERRY

Inostroza-Blancheteau, C.1,2, Reyes-Díaz, M.3,4, Aquea, F.2, Mora, M.L.3,4, Arce-Johnson, P.2

and Alberdi, M.3,4*.

1Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Casilla 54-D, Temuco, Chile. 2Laboratorio de Bioquímica, Departamento de Genética Molecular y Microbiología, Facultad

de Ciencias Biológicas, Pontificia, Universidad Católica de Chile, Casilla 114-D, Santiago, Chile. 3Departamento de Ciencias Químicas y Recursos Naturales, Facultad de Ingeniería, Ciencias y

Administración, Universidad de La Frontera, Temuco, Chile. 4Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and Tecnological Bioresource Nucleus (BIOREN-UFRO), Universidad

de La Frontera, PO Box 54-D, Temuco, Chile. *Corresponding author: [email protected] Blueberry plants (Vaccinium corymbosum L.) require acid soils for optimum growth. In Chile, the species are cultivated on acid soils, being the aluminum (Al) toxicity the major limiting factor for crop productivity in these soils. The main symptom of Al toxicity is a rapid inhibition of root growth. However, little is known about the aluminum (Al) effects at root level and the antioxidant system in acid soils. We used two blueberry genotypes (Brigitta, Al3+-resistant y Bluegold, Al3+-sensitive), which were evaluated in Hoagland’s solution during seven days for the homogenization of the nutritional conditions and follow by CaCl2 solution supplemented with Al (0 y 100 µM Al3+). Roots and shoots samples were taked at different times (0, 2, 6, 24 and 48 hours). We studied the Al effects on the cellular structure of root tips stained with toluidin blue and examined through optical microscopy. The damage (loss of the integrity of root tip cells) was observed in peripheral cells of genotype Bluegold (Al-sensitive), compared with the control (without Al). Furthermore, we also analyzed the changes in the antioxidant system of leaves and roots through the superoxide dismutase (SOD), catalase (CAT) and antioxidant activity (AA). SOD activity in roots increased 1.7-fold at 6 h in both genotypes while in the leaves of Brigitta genotype the activity increased 4-fold at the same time. CAT activity increased 2.2-fold in roots at 24 hours while in the leaves was 2-fold at 2 hours. In AA there are no significant differences between cultivars and tissues analyzed. Nevertheless, the AA in leaves is twice than in roots. In summary, these results suggest that the Al-toxicity affects the ultrastructure of root tip cells of blueberry and that the antioxidant activity is lower in this tissue. However, in the shoot there is a higher activity of the antioxidant system in the Al-resistance genotypes under Al stress, allowing better performance to the plant. Acknowledgements: This research was supported by Millennium Nucleus for Plant Functional Genomics, Fondecyt-1080372, Doctoral Supported Fellowship and Support CONICYT Ph.D. Fellowship.



111

ANTIOXIDANT ACTIVITY AND FLAVONOIDS COMPOUNDS IN FRUITS OF Vaccinium corymbosum L. TREATED WITH CALCIUM SULFATE IN AN ANDISOL

Luengo, A.1, Reyes-Díaz, M.2,3, and Alberdi, M.2,3*.


2Departamento de Ciencias Químicas y Recursos Naturales, Facultad de Ingeniería, Ciencias y Administración, Universidad de La Frontera, Temuco, Chile. 3Center of Plant- Soil Interaction and Natural

Resources Biotechnology, Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Casilla 54-D, Temuco, Chile.


In southern Chile, soils derived from volcanic ashes (Andisols) are frequent. The acidity of these soils permits the Al solubilization from silicates and oxides in form of phytoxic aluminium (Al3+). Highbush blueberry (Vaccinium corymbosum L.), an important fruit crop established in Chile, is adapted to acid soils, but it is very sensitive to Al3+, which constitutes a problem for their productivity and fruit quality. It is known, that Al stress induces an increment of reactive oxygen species (ROS) in plants, activating different antioxidant defense strategies against oxidative stress, which could be of enzymatic and non-enzymatic nature. Non enzymatic antioxidants are products from the secondary metabolism of plants such as phenols and flavonoids, carotenoids and anthocyanins. To avoid the toxic effect of Al3+ under the acid soil conditions that demands V. corymbosum for an optimal growth and development, calcium (Ca) addition is a common practice used to reduce Al-toxicity. The Ca applications are carried out as lime, gypsum or phosphogypsum. There is no information regarding the effect of Ca-amendments (CaSO4) in soils on the antioxidant features of fruits of blueberry growing on volcanic soils from Chile or other countries. In the present study, we will inform about the effect of gypsum amendments on the antioxidant activity (AA) and phenols contents of fruits of V. corymbosum growing in an Andisol. Additionally, fruit quality parameters were determined. The study was performed in Berries San Luis Farm (Lautaro, Chile). Following concentrations of CaSO4 was applied as amendment: 0 (control), 500, 1000 and 2000 Kg ha-1. The sampling of mature fruits was done in January 2010. Preliminary results indicated that Bluegold, the Al sensitive cultivar, shows a tendency to increase the AA in higher concentrations of CaSO4, improving the response of this cultivar to Al toxicity. Skin extracts showed significant increments in AA levels compared to pulp extracts. This is the first report, where differences between these two parts of fruit with respect to the effect of CaSO4 amendment are informed. Acknowledgments: FONDECYT Nº 11080231, Berries San Luis, CONICYT fellowship.



112

RELATIONSHIP BETWEEN LEAF AL-CONTENT AND CALCIUM SULFATE ADDITION ON NET PHOTOSYNTHESIS, ANTIOXIDANT CAPACITY AND TOTAL

PHENOLS OF HIGHBUSH BLUEBERRY (Vaccinium corymbosum L.) UNDER AL-TOXICITY

Meriño-Gergichevich, C.1, Reyes-Díaz, M.2,3,* and Alberdi, M.2,3


2Departamento de Ciencias Químicas y Recursos Naturales, Facultad de Ingeniería, Ciencias y Administración, Universidad de La Frontera, Temuco, Chile. 3Center of Plant, Soil Interaction and Natural

Resources Biotechnology, Scientific and Technological Bioresource Nucleus (BIOREN-UFRO), Universidad de La Frontera, Temuco, Chile. *Corresponding author: [email protected]

In Chile, highbush blueberry (Vaccinium corymbosum L.) has been developed in acid soils (pH ≤ 5.5). This species is good adapted to these soil types, but is sensitive to phytotoxic aluminium (Al3+). On the other hand, different calcareous amendments are used to improve blueberry production, being essential for a good functioning of physiological and biochemical processes. Little is known about their effects in this species. The aim of this work was to study the Al-toxicity responses under calcium sulfate (CaSO4) application. The study consider the following determinations: leaf aluminium content (LAC, mg kg-1 DW), net photosynthesis (Amax, μmol CO2 m-2 s-1), antioxidant capacity (AA, μg TE g-1 FW) and phenol contents (PC, chlorogenic acid μg g-1 FW). Legacy, Brigitta and Bluegold cultivars (cv) were grown in hydroponic solution containing 2.5, 5.0 and 10 mM CaSO4 and 25, 50, 100 and 200 µM AlCl3 for 15 days, under greenhouse conditions. In the three cv LAC was reduced (~50%) respect to control in all treatments. Bluegold showed higher Amax (10%) with CaSO4 treatments (2.5 and 5 mM), besides LAC correlated negatively with Amax (R2= 0.75 and 0.48 in Bluegold and Legacy, respectively). Antioxidant activity and PC was enhanced with 2.5 and 5 mM CaSO4 plus Al treatments in three cv respect to controls. The CaSO4 addition in 2.5 and 5 mM treatment showed better results in Amax, AA and PC in all cultivars, meanwhile 10 mM CaSO4 exhibited a higher reduction of LAC, principally in Brigitta and Bluegold. Acknowledgments: FONDECYT N º 11080231, MECESUP FRO 0601 Scholarship, Doctoral thesis support AT-24100159-2010, Berries San Luis.



113

EFFECT OF MN EXCESS ON PHOTOSYSTEM I AND PHOTOSYSTEM II PERFORMANCE IN Arabidopsis thaliana

Millaleo, R.1, Krol, M.2, Ivanov, A.G. 2, Huner, N.P.A. 2, Reyes-Díaz, M.3 and Alberdi, M.3,*

1Programa Doctorado en Ciencias de Recursos Naturales, Facultad de Ingeniería, Ciencias y Administración,

Universidad de La Frontera, Casilla 54-D, Temuco, Chile. 2Department of Biology and the Biotron, University of Western Ontario, N6A 5B7, London, ON, Canada. 3Center of Plant, Soil Interaction, and

Natural Resources Biotechnology, Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Casilla 54-D, Temuco, Chile. *Corresponding author: [email protected]

Manganese (Mn) is an essential micronutrient for higher plants. However, it can be toxic for them when it is present in excess. Toxicity of Mn is considered the second most important growth limiting factor in acid soils, after to Al toxicity. Because of its lower pH, acid soils permit a major availability of Mn in its reducing form (Mn2+). This can cause considerable damages to physiological level of plants, specifically in the photosynthetic performance. The objective of this research was to evaluate the effects of increasing Mn concentrations (18, 50, 500, 1000 and 1500 µM) on the structure (polypeptide composition) and functional performance (photochemical activities) of photosystem II (PSII) and photosystem I (PSI) in the photosynthetic apparatus of Arabidopsis thaliana. Control (18 µM) and Mn-treated plants were grown in a mixture of vermiculite perlite in a growth chamber for 21 days. SDS-PAGE and immunoblot analyses did not exhibit any significant changes in the relative abundance of PSII- associated polypeptides: PSII reaction center protein D1, Lhcb1 (major light harvesting protein of LHCII complex) and PsbO (OEC33 -33kDa protein of the oxygen-evolving complex). In addition, the abundance of Rubisco also did not change with Mn treatments. In contrast, the abundance of PSI reaction center polypeptides (PsaA and PsaB) in plants treated with the highest Mn concentration (1500 µM) was significantly lower compared to the control (18 µM Mn). In vivo measurements of the redox transients of PSI reaction centre (P700) showed a considerable decrease in the extent of P700 photooxidation (P700+) under the same conditions. This was accompanied by a faster rate of P700+ re-reduction indicating an up-regulation of the PSI-dependent cyclic electron flow in Mn-treated plants. The results presented here suggest that PSI is more susceptible to Mn toxicity than PSII. Acknowledgments: CONICYT Internship “Pasantias Doctorales en el Extranjero 2010”, Department of Biology and the Biotron, University of Western Ontario, London, Ontario, Canada. Fondecyt Project Nº 1080372.



114

CHARACTERIZATION OF ANTIOXIDANT ACTIVITY OF LEAVES AND FLOWERS OF Bougainvillea SP.

Velásquez, R1., Mutis, A1,2., Alfaro, S4., Scheuermann, E3., Quiroz, A1,2., Hormazabal, E1,2,*.

1Laboratorio de Ecología Química, Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera, Temuco, Chile. 2Center of Chemical Ecology of Terrestrial and Aquatic Systems, BIOREN-UFRO. 3Center of Food Biotechnology and Bioseparations, BIOREN-UFRO y Departamento de Ingeniería

Química, Universidad de La Frontera, Temuco, Chile. 4Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco, Chile. *Corresponding author: [email protected]

Bougainvillea is a genus plant originated from South America. Within its medicinal properties, Bougainvillea is used for controlling sand flies that cause leishmaniasis, and for curing ulcer, diarrhea, diabetes and having anti-microbial activities. The most of the reports have been focused on leaf and stem medicinal properties, but floral compounds activity has not been reported. The aim of this study was to characterize acetonic, ethanolic and aqueous extracts from leaves and flowers of Bougainvillea sp. for evaluating their total phenolic compounds (TPC) and antioxidant potential. The TPC in extracts was evaluated using Folin-Ciocalteu reagent, whereas antioxidant activity (AA) was analyzed using 2,2-diphenyl-1-picrylhydrazyl (DPPH). The extract showing the highest TPC, expressed as gallic acid equivalents (GAE) per 100 grams of dry weight (DW), corresponded to 3,857 mg present in the leaves extracted with water. AA expressed as trolox equivalents (TE) per 100 grams of dry weight (DW), ranged from 37 mg for flowers and leaves acetonic extract up to 338 mg for flowers aqueous extract. Although the flowers aqueous extract showed a lower TP in relation to leaves aqueous extract, the highest AA presented by the flowers aqueous extract, could be attributed to polyphenols chemical differences. In flowers could be predominate aglycone polyphenols to which literature has been attributed greater antioxidant effect. The mechanism of reduction of the DPPH molecule is based on a scavenging activity. These results suggested that the structure (both planar and spatial) of the antioxidant compounds present in the extract, are important for its capacity of donating hydrogen ions. Future research will be carried out to determine the polyphenol chemical compositions, allowing to explain the differences in the antioxidant activity between flowers and leaves of Bougainvillea sp.



115

EFFECT OF AL IN OXIDATIVE STRESS AND PRODUCTION OF PHENOLIC COMPOUND IN HIGHBUSH BLUEBERRY (Vaccinium corymbosum L.) CV. LEGACY

CULTIVATED IN VITRO

Manquián, K.1, Barrientos, H.2, Pizarro, M.2, Zúñiga, G.E.2 and Escudey, M.2

1Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, casilla 54-D, Chile.

2Universidad de Santiago de Chile, Facultad de Química y Biología, correo 40, casilla 33, código postal 7254758, Chile. *Corresponding author: [email protected]

Acid soils are found throughout the world. The biggest problem of acid soils is the active Aluminum (Al) content, resulting toxic to plant. The present study was undertaken to assess the effects of Al in the antioxidant activity and production of phenolic compound. Blueberry (Vaccinium corymbosum L.) cultivated in vitro were treated with Al 100 and 200 μM at pH 5.2. The effects to the concentration of Al were analyzed. The production of malondialdehyde (MDA) was measured to evaluate the level of lipid peroxidation; the antioxidant activity was measured as radical scavenging activity and ferric reducing activity; the total phenolics content were determined by colorimetric methods; profile phenolic compound were evaluated by HPLC-DAD. The MDA content, antioxidant activity and phenolics compound were estimated in blueberry after 7, 14 and 21 days supply of the aluminium. Analysis of the contents of MDA accumulated in the tissues of the blueberry seedlings indicated that Al concentrations applied increased the damage caused by lipid peroxidation within two treatments after 14 days. The antioxidant activity was high in the extracts obtained with 200 µM Al after 14 days to treatment. In these extracts were identified the majority presence of chlorogenic acid and elagic acid. These phenolic compound synthesized by blueberry exposed to Al may provide a defense mechanism against oxidative damage induced by Al.



116

STRUCTURAL PROPERTIES OF SYNTHETIC ALLOPHANE NANOPARTICLES COATED WITH 6% OF IRON OXIDE AND EVALUATION OF ITS CATALYTIC

PROPERTIES FOR ATRAZINE OXIDATION BY FENTON-LIKE REACTION

Garrido, E.G.1,* and Mora, M.L.2

1Natural Resource Science Doctorate Program and 2Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Avenida Francisco Salazar 01145, Temuco, Chile.


Allophane nanoparticles (Al-Si) with Si/Al molar ratio 2.0 were synthesized and coated with 6% of iron oxide (Al-Si-Fe6). The structural properties were evaluated by Transmission Microscopy Electronic (TEM) and by N2 adsorption-desorption isotherms at 77ºK and the application of BET equation for surface area, BJH method for mesoporosity and De Boer method (t-plot) for micropore volume. TEM showed aggregates of spherical nanoparticles with similar morphology to natural and synthetic allophane. The BET surface areas were 199 m2g-1 and 348 m2g-1 for Al-Si and Al-Si-Fe6, respectively. The coating with 6% of iron oxide increased the micropore volume and decreased the mesopore volume. The catalytic properties of 1 mg/mL of Al-Si-Fe6 were studied by atrazine (ATZ) oxidation at different pH (3.0, 4.0 and 6.0) and hydrogen peroxide concentration (0.2 M and 0.3 M). No oxidation was achieved when only Al-Si-Fe6 was added into the solution. A 77% of ATZ oxidation was achieved at pH 3.0 in 48 hours of the reaction with 0.2 M hydrogen peroxide. The ATZ oxidation decreased according the pH increased to more neutral values (48% ATZ oxidation at pH 6.0 with 0.2 M hydrogen peroxide). The use of a higher hydrogen peroxide concentration (0.3 M) increased the ATZ oxidation for all pH values. A 100 % of ATZ oxidation was achieved at pH 3.0 in 24 hours of reaction. ATZ oxidation of 82% and 66 % were obtained at 4.0 and 6.0 pH values, respectively with 0.3 M hydrogen peroxide. The Fe dissolved during the Fenton-like reaction was study by Atomic Absorption Spectrometry in order to evaluate the stability of the AL-Si-Fe6, It was observed that according the pH was more acid the Fe concentration increased into the solution. However, in all cases the Fe dissolution was less than 1% of the total Fe present on the surface of the clays. It is concluded that due the properties Al-Si-Fe6, such as, size in the nano-scale range, high surface area and microporosity, synthetic allophane nanoparticles are catalytic by the Fenton-like reaction for ATZ oxidation. These results indicated that ATZ oxidation on Al-Si-Fe6 was mediated by a heterogeneous process occurs on the surface of the catalyst.

Acknowledgements: Scholarship D-21070269 CONICYT and Nº AT-24100139 CONICYT supported this work



117

ARSENATE ADSORPTION IN IMOGOLITE AND FERROIMOGOLITE: A COMPARATIVE STUDY

Arancibia-Miranda, N.1, 2,* and Escudey, M.2, 3

1Depto. Ciencias Químicas, Universidad de La Frontera, Av. Francisco Salazar 01145, Casilla 54-D, Temuco, Chile. Nicolás Arancibia-Miranda is corresponding author. 2Center for the Development of Nanoscience and Nanotechnology, CEDENNA, 9170124, Santiago, Chile. 3Facultad de Química y Biología, Universidad de

Santiago de Chile, Av. B. O’Higgins, 3363, Santiago, Chile. *Corresponding author: [email protected]

The contamination of water with arsenic (As) is the global problem, due significant negative effect on the environmental an human health. We investigated arsenate (As(V)) adsorption on nanotubular aluminosilicates mineral (synthetic Imogolite and Ferro-imogolite) (Yoshinaga, 1968; Cradwick et al., 1972; Gutiérrez et al., 2010). The sample were characterization through powder X-ray diffraction (XRD), transmission electronic microscopy (TEM), Mössbauer spectroscopy, isoelectric point (IEP) and vibrant sample magnetometer (VSM). The characterization of the imogolite and Ferro-imogolite by XRD, TEM, Mössbauer spectroscopy, VSM and IEP, shows a significant difference. The hysteresis curves were measured for the magnetite nanoparticles, the mixture of nanotubes with magnetite (Ferro-imogolite) and the synthesis of magnetite with the nanotubes (Ferro-imogolite) and the results are shown in figure 4. It can be observed that the saturation magnetization of the magnetite nanoparticles is typical of Fe oxide samples (around 90 emu g-1). This is an indication that the Ferro-imogolite synthesis results in nanoparticles that are smaller. The hysteresis curve of the Ferro-imogolite system is compatible with superparamagnetic nanoparticles as the ones observed in figure 2, with 10 nm in diameter. The binary system (Ferro-imogolite) showed a higher adsorption of arsenate compared to Imogolite, due to a higher affinity of Fe this anion. According with the results, we can see that removal efficiencies of the Ferro-imogolite for As after 30 minutes were 60%. Since there is a higher of stripping at that time. The sorption of arsenate is characterized by initial fast adsorption of arsenate it might be due to the nanoscaled particle size of adsorbents, as fine particles were favorable for the diffusion of arsenic molecules from bulk solution onto the active sites of the adsorbents.

Acknowledgements: Financial support of this work by FONDECYT under Project 1070116, the Centro para el Desarrollo de la Nanociencia y la Nanotecnología (CEDENNA). N. Arancibia-Miranda acknowledges a scholarship from CONICYT (Chile). References:

- N. Yoshinaga, Soil Science and Plant Nutrition, 1968, 14, 8. - P.D. Cradwick, V.C.Farmer,J.D.Russell, C.R.Masson, K.Wada and N. Yoshinaga, Nature (London), Phys.

Sci., 1972, 24, 187. - M. Gutiérrez, M. Escudey, J. Escrig, J.C. Denardin, D. Altbir, J.D. Fabris, L.C. D. Cavalcante and M.T.

García-González Clays and Clay Miners. (2010, in press).



118

ALLOPHANIC NANOCLAYS: THEIR IMPLICATIONS ON THE POTENTIAL CARBON SEQUESTRATION

Calabi-Floody, M.1,4,*, Jara, A.3,4, Rumpel, C.2 and Mora, M.L.3,4

1Programa de Doctorado en Ciencias de Recursos Naturales Universidad de La Frontera, Temuco, Chile. 2Laboratoire de Biogéochimie et Ecologie des Milieux Continentaux (BIOEMCO, UMR Université Paris VI et XII-CNRS-INRA-IRD), Campus AgroParisTech, Thiverval-Grignon, France. 3Departamento de Ciencias

Químicas y Recursos Naturales, Universidad de La Frontera, 4Scientific and Technological Bioresource Nucleus (BIOREN-UFRO), Universidad de La Frontera, Temuco, Chile.

*Corresponding author: [email protected] Negative effects from the global warming on the life are generating the necessity to study alternative to mitigate these problems. Recent studies would indicate that global warming is partially caused by the increased emission into the atmosphere of greenhouse gases from the burning of fossil fuels, decomposition of carbon-rich materials and intensive deforestation. Carbon stabilization and sequestration is one of the ways to mitigate the greenhouse effect. It has been suggested that nanoparticles, with their large surface-to-volume ratio, could be highly effective in carbon sequestration. Since many nanoparticles occur widely in the natural environment, especially in soil, we focus attention on extracting nanoparticles from soil to assess their potential for carbon sequestration. Soils contain about three times more carbon than the above-ground vegetation, approximately 75% of terrestrical C pool, and hence play a key role in the global C cycle. The clay fraction of soils derived from volcanic ash (Andisols) contains different inorganic nanoparticles, among which allophane is the most abundant. Allophane is a non-crystalline aluminosilicate, it morphology is like a hollow spherule with an outer diameter of 3.5–5.0 nm. The 0.7–1.0 nm thick spherule wall is composed of an outer Al octahedral (gibbsitic) sheet and an inner Si sheet. Defects in the wall structure give rise to perforations of ~ 0.3 nm in diameter. We found that the extracted aggregates of nanoparticles retain a significant amount of carbon (11.8 %) against intensive peroxide treatment. Also we showed by pyrolysis-GC/MS that the organic compounds from organic matter stabilized persist on allophanic nanoclays after treatments to remove organic matter. As the nanoclay fraction to contain strongly bound and protected organic matter that can persist for several centuries or millennia, the results of this investigation will also provide insight into organic matter stabilization in soil.

Acknowledgements: We gratefully acknowledge CONICYT (National foundation for Science and Technology), Chile for financial support under FONDECYT Grant Numbers 1061262 and 11070241. We also thank colleagues at the Laboratoire de Biogéochimie et Ecologie des Milieux Continentaux (BIOEMCO, UMR Université Paris VI et XII-CNRS-INRA-IRD), Campus AgroParisTech, Thiverval-Grignon, France for their assistance and input.



119

NATURAL NANOCLAYS LIKE SUPPORT MATERIALS TO IMMOBILIZE ACID PHOSPHATASE: INCREASE THE PHOSPHATE AVAILABILITY TO THE PLANTS

Calabi-Floody, M.1,3, Jara, A. 2,3 and Mora, M.L.2,3

1Programa de Doctorado en Ciencias de Recursos Naturales Universidad de La Frontera 2Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera 3Scientific and Technological

Bioresource Nucleus (BIOREN-UFRO), Universidad de La Frontera. Av. Francisco Salazar 01145, P.O. Box 54-D, Temuco, Chile. *Corresponding author: [email protected]

The P-fertilizer constitutes a problem due to high phosphorus retention in acid Andisols. Te phosphate catalysis by enzymes is a suitable alternative to improve nutrient uptake. Nanomaterials like nanoclays occur widely in nature. The use of nanomaterials as immobilization support improves enzymatic stability and catalytic activity against other materials. The aims of this study were: i) to evaluate allophanic and montmorillonite clays and nanoclays as support to immobilize acid phosphatase and ii) to evaluate the organic phosphorus mineralization by means nanoclay-phosphatase complexes and aerobic degraded cattle dung in order to increase the phosphate availability from Andisols of the Southern Chile. Clays and nanoclays extracted from an Andisol and montmorillonite were characterized by TEM and AFM. The enzymatic activities were measured with ρ-nitrophenylphosphate (ρ-NPP) as substrate. The kinetics parameters (Vmax and Km) were calculated according to Michaelis–Menten equation. The phosphorous mineralization was determinate by Ion chromatography. The microscopy analysis showed the presence of nanoclay aggregates with high proportions of mesoporous suitable to enzymatic immobilization. We demonstrated that the immobilized phosphatase has a significant increasing in both, catalytic efficiency (Vmax/Km) and substrate affinity (1/Km) when allophanic and montmorillonite nanoclays were used as material support. Finally, we observed that the phosphatase acid immobilized on allophanic and montmorillonite nanoclyas increased the phosphorus mineralization from aerobic degraded cattle dung. Acknowledgements We gratefully acknowledge CONICYT (National foundation for Science and Technology), Chile for financial support under FONDECYT Grant Numbers 1061262 and 11070241.



120

DESCRIPTION OF THE ADSORPTION OF CU AND ZN ON ALLOPHANIC CLAY USING THE CAPACITANCE CONSTANT MODEL

Jara, A.A.1,2,*, Cartes, A.2 and Mora, M.L.1,3

1Departamento de Ciencias Químicas y Recursos Naturales, 2Soil and Plant Analysis Laboratory, and

3Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Temuco, Chile. *Corresponding author: [email protected]

Surface Mechanistic Models (SMMs) are a useful tool to predict the availability of the metals on soil solution and describe the adsorption mechanisms that can occur on the surface (Sposito, 1984; Goldberg, 1992). The capacitance constant model (CCM; Hohl and Stumm, 1976) and Double Layer Model (DLM; Sposito, 1984) have been used to predict the adsorptive affinities of divalent metal ions on different surfaces. In adsorption studies of Cu2+ and Zn2+ on montmorillonite (Gu et al., 2010) or ferrihidrite (Travedi et al., 2004) have been proposed that these metals form monodentate (SOM+) and bidetante (SO2M) complexes. The aim of this work was to model by CCM the Cu and Zn adsorption on allophanic clay extracted from an Andisol to determine minerals. The experiments were carried out in batch system for 24 h at pH (5.0), ionic strength (KCl 0.1 M) and temperature (25ºC) constants, with clay fraction/solution ratio 0.1 g 10 Ml-1. The metals in solution were determined by atomic absorption spectrophotometer (AAS). Data were fitted by the CCM assuming the formation of monodentate and bidentate inner sphere complexes for Cu2+ and Zn2+ using FITEQL 3.5 program. The intrinsic surface complexation constants (Kint) were determined using the adsorptive data at constant pH, ionic strength and temperature. Result from adsorption isotherms on allophanic clay showed that the affinity of Cu was slightly higher than Zn. The fit values for Zn and Cu (M) were log Kint(OS)2M -3.718 and -2.784 for bidentade complex and log KintSOM+ -2.752 and -1.215 for monodentate complex, respectively. Similar results were reported by Xu et al. (2006), who find that the adsorption mechanisms of Zn and Cu were alike, and both ions can form monodentate and bidetante complexes on the goethite surface. Acknowledgments: Financial support from Fondo Nacional de Desarrollo Científico y Tecnológico (11070241) is acknowledged. References:

- Goldberg, S. 1992. Use of surface complexation models in soils chemical systems. Adv. Agrom. 47, 233-329. - Gu, X., Evans, L.J. and Barabash, S.J. 2010. Modeling the adsorption of Cd(II), Cu(II), Ni(II), Pb(II) and Zn(II)

onto montmorillonite. Geochim. Cosmochim. Acta, 74, 5718-528. - Hohl, H. and Stumm, W. 1976. Interaction of Pb2+ with hydrous γ-Al2O3. J. Colloid Interface Sci. 55, 281-288. - Sposito, G. 1984. The surface chemistry of soils. Oxford University Press, New York. - Trivedi, P., Dyer, J.A., Sparks, D.L. and Pandya, K. 2004. Mechanistic and thermodynamic interpretations od zinc

sorption onto ferrihydrite. J. Colloid Interface Sci., 270, 77-85. - Xu, Y., Axe, L., Yee, N. and Dyer, J.A. 2006. Bidentate complexation modeling of heavy metal adsorption and

competition on goethite. Environ. Sci. Technol. 40, 2213-2218.



121

APPLICATION OF CONFOCAL MICROSCOPY AS TOOL TO EVALUATE ACID PHOSPHATASE IMMOBILIZATION ON NANOCLAYS

Velásquez, G.1,*, Calabi, M.1,3 and Mora M.L.2,3

1Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de la Frontera, Temuco

2Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera 3Scientific and Technological Bioresource Nucleus (BIOREN-UFRO), Universidad de La Frontera. Av. Francisco Salazar

01145, P.O. Box 54-D, Temuco, Chile. *Corresponding author: [email protected] In Chile, soils derived from volcanic ashes represent about 60% of agricultural soils. These soils have characteristics such as high levels of organic matter, high absorption capacity of phosphorus (P) and high concentrations of phototoxic elements such as aluminum, among others. These properties can restrict the growth of plants and crops. In general, techniques commonly applied to improve P availability to crops, such as fertilization, are not an effective response. About 90% of applied P is not captured in the first year, being absorbed by soil colloids. According to these antecedents one main research goal of our group was to increase the availability of P in crops. Therefore, there have been several studies of enzymes immobilized on soil colloids obtained as a result of acid phosphates immobilized in nanoclay which improve their catalytic activity by 33% compared with the free enzyme (Rosas et al., 2008). It is important to study the immobilization of the enzyme in nanoclay to understand the possible mechanisms of interaction, so that various techniques could be developed. Confocal microscopy is a technique that has been widely used in the study of dynamic processes in living cells and protein transport. This allows you to track changes in the spatial distribution of molecules in terms of time and disruption in the cellular environment, as well as specific interaction with certain cell structures. The aim of this study was to use confocal microscopy as a tool to evaluate the immobilization of acid phosphates on clay and soil from nanoclay Andisol in southern Chile. Preliminary results show that allophonic soil with organic matter presents higher fluorescence at 453 nm than soils treated with hydrogen peroxide at 30% where organic matter was removed. Based on these results we can conclude that confocal microscopy is a good tool for the study of soil samples. References:

Rosas, A. et al. 2008. Geoderma 145, 77-83.



122

ASSESSING SOIL HYDROPHOBICITY BY DIFFERENT METHODS UNDER SIMULATED WETTING-DRYING CYCLES

Ramírez, J.C.1 and Bachmann, J.2

1Departamento de Ciencias Químicas y Recursos Naturales, Universidad de la Frontera, Temuco. Av. Francisco Salazar 01145, Casilla 54-D. Temuco, Chile. 2Institut für Bodenkunde, Leibniz Universität

Hannover. Herrenhäuser Str. 2 – 30419 Hannover. Germany. *Corresponding author: [email protected]

Wettability can be defined as the ability of a fluid to wet a solid, being directly related to the balance of surface free energies or surface tensions (σ, mN m-1) between both interacting surfaces. In soil science, wettability is expressed as hydrophobicity, being a recurrent property observed all over the world. Soil hydrophobicity affects directly different processes, like infiltration rate, water distribution and erodibility. Typically, minerals are highly hydrophilic due their natural high σ, however, in natural soils these particles are usually covered by highly hydrophobic molecules of organic origin, inducing different levels of water repellency along the soil profile given their low σ in relation to water. Hydrophobicity is calculated by assessing the contact angle (CA) between the wetting front (liquid surface) and the test sample (solid surface). Higher CA indicates increasing hydrophobicity, with CA=90° considered as the theoretical limit defining a sample as hydrophilic or hydrophobic. Given that in natural systems hydrophobicity is not constant, but a rather variable property, with and different methods leading usually to different results, the correct assessment of CA is still a matter of research. In this context, the present work compares the results of different methods assessing CA in natural soil samples, submitted to simulated wetting-drying cycles. The goal is to determine which laboratory method is more suitable in describing the wetting behavior of the test soils. The samples used correspond to two different natural soils located in Niedersachsen (Germany) (sample A: sandy loam soil, sample B: loam soil). Before measurements, the samples were air-dried. CA was measured by using the a) Capillary Rise Method (CRM), b) Wilhelmy Plate Method- advancing CA (WPM-ACA), c) Wilhelmy Plate Method-equilibrium CA (WPM-ECA) and d) CRM in long columns (LC). After measurements, the same samples were re-wetted to saturation level (pF=0), air-dried and CA re-calculated. The cycles were repeated three times and CA compared. Initially, CRM and WPM estimate high repellency for both samples (CRM >90º, WPM-ACA=114° and 121°, for sandy loam and loam samples, respectively), while WPM-ECA estimated values in the subcritical range (72° and 86°, respectively). On the other side, LC demonstrated that liquid uptake is possible (up to 18 cm) into these soils. The re-measurement of CA (air-dried samples) showed a significant increment of water repellency, especially for the sandy-loam sample, for all methods. While CRM and WPM-ACA continue estimating CA>90°, WPM-ECA still estimate CA in the subcritical range (~86°). The comparison with LC showed that liquid uptake into the segmented columns was almost absent during the first 72 hours (H <2 cm), then increasing and reaching the equilibrium height after approximately one week. These results lead to concluded that WPM-ECA is a suitable method in characterizing water repellency in comparison to CRM and WPM-ACA. At the same time, due the total amount of organic components into the samples is expected to be relatively constant along the experiments, it is concluded that qualitative factors play a major role in determining the hydrophobicity level of natural soil samples.



123

Symposium 6

“Chemical Ecology”

Chairpersons:

Dr. Andrés Quiroz and Dr. Christian Figueroa

Oral presentations:

- Dr. Christian Figueroa, Universidad Austral de Chile, Chile: “Ecological and evolutionary genomics of pest aphids”.

- Dr. Rufus Isaacs, Michigan State University, USA: “The role of native bees in sustainable

crop pollination”.



124



125

THE ROLE OF NATIVE BEES IN SUSTAINABLE CROP POLLINATION

Isaacs, Rufus*

Department of Entomology, Michigan State University, East Lansing, Michigan 48824, USA. *Email address: [email protected]

Bees are the primary pollinators of many of the world’s fruit, vegetable, and nut crops. Without bees visiting flowers, there would be much lower production of crops such as blueberry, apple, cherry, squash, tomato, almonds, and coffee. Recent global estimates have valued the annual contribution of pollination by bees to human food at approximately US$220 billion, highlighting the importance of maintaining pollination systems. Honey bees (Apis mellifera) are the primary pollinators of many intensively-managed fruit, vegetable, and nut crops because these insects can be raised in hives by beekeepers and are flexible for delivery to farms when they are needed. However, in many regions of the world there has been a significant decline in the number of honey bee colonies, raising concerns about potential shortages of honey bees that may then limit food production. This decline in honey bees reflects a combination of maladies affecting this insect including parasites, diseases, and loss of habitat for foraging. Recent experiences with Colony Collapse Disorder have also highlighted how different factors may combine to challenge the health of honey bee colonies. With ongoing concerns about the long-term health of this primary crop pollinator and increasing demand for food projected from the growing global human population, there is a need to examine sustainable approaches to crop pollination. There are approximately 20,000 species of bees in the world and management systems have been developed for a small number of these insects. Bumble bees (Bombus spp.) nest in cavities on or in the ground in nature, and colonies can be reared in small containers. These are available commercially for distribution to farmers, and are used widely for pollination in greenhouses (e.g. tomato) and also in open field settings. Bombus bees are well adapted to pollinate some crops such as blueberry because of their ability to sonicate flowers and release pollen. Their hairy bodies and flower-visiting behaviors also make them highly efficient at transfer of pollen. In our recent research in blueberry fields in Michigan, USA, Bombus impatiens bumble bees were able to provide equivalent pollination to honey bees. Other species of Bombus are currently in use in Chile for pollination of fruit crops, including Bombus terrestris and Bombus ruderatus in blueberry fields. Osmia bees are another type of managed bee that are being adapted for use in fruit crops. These solitary bees nest in cavities in trees in nature, but they can be encouraged to nest in tubes that allow their movement to fields where they are needed. Osmia are currently being used to pollinate raspberry, cherry, apple, and almonds. There is great diversity of Osmia in temperate northern hemisphere regions, and this diversity has yet to be fully explored for identification of other potential crop pollinators. While managed bees provide an opportunity for rapid manipulation of the pollination force during the period of crop flowering, a long-term strategy for sustainable crop pollination is to encourage native wild bees to be abundant around crop fields. Most landscapes support a diverse community of native bees that can provide free pollination to farms. Indeed, our research has identified over 160 species of wild bees in Michigan blueberry fields. Some other recent studies have found that wild bees can provide all the pollination required to pollinate squash crops that bloom during the summer. While these bee communities may be diverse, many agricultural regions also have low populations of native bees because of intensive farm management techniques. Soil tillage, removal of wild habitat, and use of pesticides can all contribute to lower populations of wild bees.



126

Additionally, the high density of flowers present for a short time during bloom of some crops can make it challenging for native bee communities to provide sufficient pollination services. Development of strategies that can support wild bee populations is an active area of research currently, including exploration of pesticide effects on native bees, development and testing of habitat conservation strategies, and analysis of landscape factors that support these insects. Each of these components is expected to contribute to building native bee populations, and a combined approach is expected to provide the greatest levels of population increase and stability. To ensure that crops gain maximal pollination and can produce to their potential, sustainable crop pollination strategies are needed. These will require the integration of honey bees, other managed bees, improved farm habitat, and native bees as appropriate to the different systems and their context. This will help ensure that land is used efficiently for production of bee-dependent crops which will be important for the growing global human population. It will also help farmers reduce risk by reducing the annual fluctuations in yield due to poor pollination. Ongoing research and education programs can help develop and transfer the knowledge required for farmers to consider which components are most useful on their farms.



127

Symposium 6

“Chemical Ecology”




128



129

ANTIBACTERIAL ACTIVITY FROM RED ALGA Ceramium rubrum

Cortés, Y.1, Pardo, F.2, Perich, F.2 and Quiroz, A.2*

1Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco 4811230, Chile. 2Laboratorio de Ecología Química, Departamento de Ciencias Químicas y Recursos Naturales,

Universidad de La Frontera, Temuco 4811230, Chile. *Corresponding author: [email protected]

Because of the evolving resistance of microorganisms to existing antibiotics, there is an increasing need for new antibiotics not only in human but also in veterinary medicine. Competition for space and nutrients led to the evolution of antimicrobial defense strategies in the aquatic environment. Therefore, aquatic organisms, such as seaweeds, offer a particularly rich source of potential new drugs. In the genus Ceramium, especially in Ceramium rubrum, several authors have emphasized in their antibacterial, antifungal and antiviral properties. Because this background, the present study focused on the antibacterial activity from C. rubrum. The aim of our study was to identify chemical fractions from C. rubrum, with activity against human pathogenic bacteria. The alga was extracted exhaustively with ethanol. Subsequently the extract was partitioned with solvents with increasing polarity for obtaining dichloromethane, ethyl acetate, n-butyl alcohol and aqueous fractions. All materials were screened for their antibacterial activities against four human pathogenic bacteria strains (Escherichia coli and Enterobacter aerogenes Gram negative strains, and Staphylococcus aureus and Staphylococcus epidermidis Gram positive strains) using agar diffusion test. The involved bioassays will allow comparing the active concentration of the extract and fractions with an antibiotic control (ciprofloxacin). Therefore, it will expect that the C. rubrum affect the growth of common human pathogens. The results could suggest the use of this seaweed as a source of antimicrobial compounds or for being used as an additive in functional food.



130

IDENTIFICATION OF CUTICULAR COMPOUNDS IN Hylastinus obscurus (MARSHAM) (COLEOPTERA: SCOLYTIDAE)

Venthur, H.1; Parra, L.1,2 and Quiroz, A.1,2,*

1Laboratorio de Ecología Química, Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera, Temuco, Chile. 2Center of Chemical Ecology of Terrestrial and Aquatic System (CETAS),

BIOREN, Universidad de La Frontera, Temuco, Chile. *Corresponding author: [email protected]

The red clover root borer, Hylastinus obscurus, is a one of the most serious pests of red clover, Trifolium pratense L., throughout the world. Both larvae and adults of this scolytid bore feeds into the roots causing a significant reduction in both production levels and persistence of red clover stands after two years sowing. At the present there is no control for clover root borer. Pesticides have not been successful in controlling borer infestations. Manipulation of population with semiochemicals is another possible method for pest management. Numerous studies of insect species have shown that a subset of female cuticular hydrocarbons is used as short-range or contact pheromones. Here, we studied the chemical differentiation of the cuticle of females and males of H. obscurus, which could influence the mating behavior of this insect. Cuticular chemicals were extracted from 150 females and males H. obscurus for immersing in 500 μL of n-pentane for 1 min each sex. Extract were analyzed by coupled gas chromatography-mass spectrometry (GC-MS). The components of the cuticular extracts were identified for analysis of the resulting mass spectra and compared with a library database by a reverse search technique. Analyses of female and male cuticular extract by GC-MS indicated that H. obscurus share several long-chain aliphatic hydrocarbons, which are the major extractable cuticular components. Some sex-specific compounds were identified in the extracts, such as tetracosane which was specific to females and pentacosane which was specific to males. This information 1) will provide an understanding of the role of cuticular hydrocarbons in the mating behavior of H. obscurus and 2) the information generated in this study provides key baseline information for a detailed study of the semiochemistry of H. obscurus.

Acknowledgements: This work is part of FONDECYT Nº 1100812.



131

PRELIMINARY PHYTOCHEMICAL STUDIES OF VASODILATORS COMPOUNDS FROM AN EXTRACT OF Ruta graveolens

Mansilla, K.1,*, Perich, F.2 and Salvatici, R.3

1Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco, Chile. 2Programa de Doctorado en Ciencias de Recursos Naturales, Departamento de Ciencias Químicas y Recursos

Naturales, Universidad de La Frontera, Temuco, Chile. 3Laboratorio de Neurociencias, Universidad de La Frontera, Facultad de Medicina CEBIOR, Temuco, Chile.


Ruta graveolens L. is a member of Rutaceae family. It is a hardy, evergreen shrub of up to one metre tall, with a characteristic greyish green colour and a sharp unpleasant odour. The leaves are small, oblong, deeply divided, pinnate, glandular dotted. The stems are much ramified. Its flowers are small, yellow and in clusters during spring and summer. R. graveolens is a native of Southern Europe. The leaves are the main part used medicinally. It has been reported that R. graveolens leaves contain as active ingredients and essential oils, flavonoid glycosides, as well as capric acid, and caprylic plagónico. In addition, contains coumarins (coumarin, herniarin, gravelliferon, rutaretin), furanocoumarins (bergapten, psoralen, rutamarin), furanoquinoline alkaloids (dictamnine, skimmianine, rutacridone) and the flavonoids rutin and quercetin. Methyl nonyl ketone is a major component of the volatile oil and is used in perfumery and flavourings. A large number of plants have been used by indigenous people as part of folk medicine, including some that are used to control human fertility. Ruta graveolens L. and Ruta chalepensis are alleged to be anti-inflammatory, antipyretic, antiparasitic, antihelmintic, and antinoniceptive. In rats, the aqueous extract of rue has also shown a hypotensive effect that could be explained by a direct effect on the cardiovascular system. The fractionation bio-conducted study was carried to the detection of vasodilatory active fractions from Ruta graveolens grown in southern Chile. After the extract fractionation, using solvents at increasing polarity, we investigated the vasodilatory activity of the fractions. This analysis yielded a fraction apolar with a high vasodilator response against isolated rat aortic rings in vitro bioassays using an isolated organ team. After isolating the active fraction in the preliminary analysis of thin-layer chromatography, through a study using gas chromatography mass spectrometry, we investigated the presence of two compounds of furanocoumarins nature. These compounds when compared with the library NIST Mass Spectral Datebase team, showed a high probability that corresponds to the compounds Psoralem and Bergapten. The structural determination of the compounds will be confirmed by the methods of nuclear magnetic resonance (NMR). The expected results will be active principles responsible for vasodilator effects, which give rise to specific protocols and then are used in the development of therapeutic products for marketing.

Acknowledgments: FONDEF Nº D07I1113 project.



132

MORFOLOGICAL AND HISTOLOGICAL CHARACTERIZATION OF THE DIGESTIVE SYSTEM OF RASPBERRY WEEVIL Aegorhinus superciliosus (GUÉRIN-MENÉVILLE)

(COLEOPTERA: CURCULIONIDAE).

Medel, V.1*, Molina, B.2, Seguel, J.2, Quiróz, A.3 and Rebolledo, R.4

1Doctoral Program in Natural Resources Sciences, University of La Frontera. Temuco, Chile. 2Basic Sciences Department, Faculty of Medicine, University of La Frontera. 3Chemistry Sciences Department, Faculty of Engineering and Management, University of La Frontera. 4Agronomic Sciencies and Natural Resources

Department, Faculty Agricultural and Forestry Sciences, University of La Frontera. *Corresponding author: [email protected]

The raspberry weevil is a native insect whose larvae have powerful jaws with which they cut and grind roots and rootlets of plants, burrowing the main root near the neck where they build a pupal chamber. This larval activity causes deterioration of the plant, which can lead to its death. Research has been focused on the management and control of this species with no morphological nor histological background of the digestive system of this weevil. The aim of this paper is to describe the adult insect's digestive tract morphologically and histologically as this canal represents an area of contact between the insect and the environment, being the focus of most methods for controlling this pest. With this purpose, the adult insect's digestive system was extracted, being a long tube of variable diameter, divided into three regions: foregut or stomodeum, midgut and hindgut or proctodeum. Each region is in close relationship with numerous tracheae that run throughout the body facilitating gas exchange. The foregut consists of the pharynx, esophagus and crop, an organ attached to the proventriculus, which ends at the cardia. The midgut consists of the anterior and posterior ventricular; it measures 1.03 mm. ± 0.205 mm. in males and 1.16 mm. ± 0.367 mm. in females. The midgut is separated from the pyloric valve proctodeum, near which three Malphigian tubes are born; they are linked in the region of the midgut and proctodeum. Internally, the ventricle has a columnar epithelium and an edge with the presence of microvilli. The proctodeum is smaller than the ventricle in diameter and it consists of the ileum, which continues in a tubular portion called the colon, this has a wall of muscle cells in its outer part/side and ripples and folds in the presence of villi in the inner part. The rectum is lined with muscle cells with contractile ability and is a short tube that ends in the anus, which is attached to the reproductive system. As conclusion, the digestive system of this insect is the same as the described for other species of weevils, showing similarities and differences, with a total larger length in females than in males.

Acknowledgments: DIDUFRO DI08-0027 project.



133

COMPARISON OF DIFFERENT FIBERS FOR SOLID-PHASE MICROEXTRACTION OF FUNGAL VOLATILE METABOLITES

Schalchli, H., Hormazabal, E., Pardo, F., Palma, R. and Quiroz, A.*

Center of Chemical Ecology of Terrestrial and Aquatic Systems (CETAS), Universidad de La Frontera. Casilla 54-D. Temuco, Chile. *Corresponding author: [email protected]

A suitable technique for the capture and analysis of fungal volatile metabolites is headspace solid phase microextraction (SPME) used in combination with gas chromatography–mass spectrometry (GC-MS), allowing a fast detection of volatile compounds without the use of organic solvents. The use of this technique for trapping volatiles from fungi is increasing. Different kinds of volatile compounds can be captured depending on the chemical composition of the SPME fiber. The aim of this study was to compare different SPME fibers used for extracting volatile compounds from a saprophytic fungus. Four SPME fiber types Polydimethylsiloxane (PDMS, 100 μm), Polyacrylate (PA, 85 μm), Carboxen/PDMS (CAR/PDMS, 75 μm), and Polydimethyl-siloxane/Divinylbenzene (PDMS/DVB, 65 μm) were evaluated in terms of their efficiency in extracting volatile metabolites emitted by Schizophyllum commune grown in potato dextrose agar (PDA). Preliminary results have shown that S. commune release inhibitory volatile metabolites against some plant pathogen fungi, such as Botrytis cinerea (not published data). Different classes of volatile compounds were extracted, such as alcohols, esters, thio-esters and sulfur-derived compounds. Esters were the most abundant compounds extracted by the tested fibers excepting PA. PDMS/DVB fiber favored the extraction of esters (62.3%) and thio-esters (25.6%), whereas the profile of volatile compounds extracted using PA fiber showed the highest proportions of alcohols (75.7%). Relative area of esters diminished to 27.7% in CAR/PDMS, while thio-esters reached 64.9%. The relative area of esters and thio-esters extracted by PDMS fiber were 38.4% and 21.6%, respectively. Volatile esters have been reported by their high inhibitory activity against a wide range of pathogenic microorganisms [1]. Our results showed that fibers containing PDMS were suitable to collect this kind of compounds, contrasting PA fiber which was suitable to extract mainly alcohols.

Acknowledgements: The authors would like to thank the Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) project AT 24090207 for financially supporting this study.



134

ROLE OF VOLATILES IN THE HETEROSPECIFIC SEXUAL COERCION BETWEEN CURCULIONIDS (COLEOPTERA) SPECIES

Pacheco, B.1, Parra, L.1,2 and Quiroz, A.1,2,*

1Laboratorio de Ecología Química, Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera, Temuco, Chile. 2Center of Chemical Ecology of Terrestrial and Aquatic System (CETAS),

BIOREN, Universidad de La Frontera, Temuco, Chile. *Corresponding author: [email protected]

The sexual coercion is a third form of sexual selection along with female choice and intermale competition. Sexual coercion prevents or constrains the free exercise of mate choice by females and entails forced copulation, sexual harassment, and intimidation through punishment of uncooperative females. For example, sexual harassment entails a variety of costs for females such as greater energy expenditure and loss of feeding time associated with rejecting persistently males significantly reduce fecundity. This type of interaction has been described in different species. Thus males of order Hemiptera (family Coreidae and Lygaeidae) has been observed attempting to secure and couple with beetles, other true bugs (Hemiptera), and bees. Chemicals cues are important in mate selection in many kinds of organisms. Typically, research on insect sex pheromones has emphasized their role as sex attractants, often assuming that they allow males to make simple discriminations, for example, to determine whether another individual is a male or a female, or either a conspecific or a heterospecific. In field observations, we have observed effective copulation between females Aegorhinus nodipennis and males A. superciliosus (Coleoptera: Curculionidae) in birch trees (Betula pendula). For this reason, the objective of this study was to evaluate the role of the volatiles in the hererospecific relationship between both species. Group of female and male of both A. superciliosus and A. nodipennis were confined in a glass container. The volatiles were collected from container using purified air passed through a charcoal trap onto a glass volatile collection device containing 100 mg of Porapak Q adsorbent. The olfactometric response of males A. superciliosus towards volatiles from females A. nodipennis was significant (P<0.05). On the contrary, when tested A. superciliosus females volatiles did not elicit a significant response from males A. nodipennis (P>0.05). Our results suggest that male A. superciliosus have involved the ability to use female-produced chemical cues from A. nodipennis as an indicator of female’s sexual status. This information will provide an understanding of the role of volatiles in heterospecific sexual harassment of both species. Future work should aim to (1) identify the specific compounds that elicit attraction of males A. superciliosus toward females A. nodipennis and (2) the cost of persistent courtship in both species in terms of reduced fecundity.

Acknowledgments: We thanks to FONDECYT Nº 1100812 project.



135

OF FATTY ACIDS COMPOSITION PRESENT IN THE ROOTS OF TWO CULTIVARS AND EXPERIMENTAL LINES OF RED CLOVER (Trifolium pratense L.).

Tampe, J.1,*, Quiroz, A.1,2,3 and Pardo, F.1,2


2Laboratorio de Química Ecológica, Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera, Temuco, Chile. 3Núcleo BIOREN-UFRO, Universidad de La Frontera, Casilla 54-D,

Temuco, Chile. *Corresponding author: [email protected]

Red clover (Trifolium pratense L.) is a short-lived perennial forage legume, widely used in temperate regions of the world. It is renowned for its ability to produce high quality forage. The persistence of this forage is affected by the infestation of Hylastinus obscurus (Marsham) (Coleoptera: Scolytidae). Adults and larvae of H. obscurus are wood decay and feed on clover roots, digging deep galleries that can reach the vascular cambium. Despite the considerable damage made by this insect, there is no adequate chemical and biological control capable of controlling this pest. This is due to the long flight and location H. obscurus within the roots of the plant. To date, there have been several studies on the chemical interaction between H. obscurus and T. pratense. These have indicated the existence of a relationship between the volatile compounds emitted by the red clover and infestation by H. obscurus. Of the identified compounds, methyl benzoate (E)-2-hexenal, attracted the insect T. pratense plants, while limonene was identified as a repellent. It was recently reported that fatty acids present in bean root Quiñequeli could be involved in the selection of host. Based on this background, the objective of this study was to compare semi-quantitatively the chemical composition of fatty acids present in roots of two cultivars, Quiñequeli and Redqueli and seven lines of red clover related to population levels of H. obscurus. Treatments were three replications for each line and cultivar. Plants were collected from the Agricultural Research Institute (INIA) Carillanca. Then, they were transported to the laboratory of the Department of Chemical Ecology, Cs. Chemical and Natural Resources, University of La Frontera, where we proceeded to evaluate the presence of H. obscurus in its larval and adult stage. Subsequently, the roots were subjected to a process of washing and drying at room temperature until a constant mass. Each sample was subjected to an extraction process with a solution of dichloromethane, to be subsequently analyzed in gas chromatograph coupled to a mass spectrometer. Preliminary results of identification of the chemical composition of fatty acids present in red clover roots will establish their relationship with the arrival of the host insect, thus validating the use of fatty acids to select genotypes resistant to infestation in clover plants.

Acknowledgments: We thanks to FONDECYT Nº 1100812 project.



136

CHEMICAL CHARACTERIZATION BY GC-MS OF CONDITIONED WATER FROM VIRGIN ADULT FEMALE OF Artemia franciscana (SAN FRANCISCO BAY, USA)

Tapia, C.1,3,*, Gajardo, G.2 and Quiroz, A.3

1Doctorado en Ciencias de Recursos Naturales, Laboratorio de Ecología Química, Universidad de La

Frontera. Temuco, Chile. 2Laboratorio de Genética & Acuicultura, Universidad de los Lagos. Osorno. Chile. 3Laboratorio de Ecología Química, Universidad de La Frontera.

*Corresponding author: [email protected] Microcrustaceous Artemia is of great importance in terms of food for aquaculture. The genus is composed by di-, tri-, tetra- and pentaploid parthenogenetic population and seven bisexual species. However, the existence and characterization of new populations is still questioned. The study of crustacean semiochemicals was initiated in 1960, but the species studied are few and there is no knowledge of the existence of pheromones in Artemia genus. This information can be important for several areas, in fisheries for being used as baits and in aquaculture for the manipulation of the mating behavior, especially it can be a powerful tool to compare and contrast genetic and evolutionary aspects intra-and interspecific of the genus, considering the mating behavior and the specific-mate recognition systems. This work focused on the characterization of the less polar components present in conditioned water from virgin adult females of Artemia franciscana, in order to compare this spectrum with other antecedents for sex pheromones present in the phylum. The conditioned water was provided by living adults virgin females placed in a flask for two days without food. Then, this conditioned water was subjected to a solid phase extraction for obtaining a total ethanolic extract from conditioned water of virgin adult females of A. franciscana. Subsequently, the ethanolic extract was subjected to a vacuum distillation for separating the less polar fraction from the polar fraction. Chemical profiles of the less polar fraction were obtained by gas chromatography coupled with mass spectrometry (GC-MS). This is the first report showing chemical compounds released from living A. franciscana to the aqueous environment.



137

IDENTIFICATION OF FATTY ACIDS IN ALFALFA (Medicago sativa L.) ROOT EXTRACTS, AND BEHAVIOURAL RESPONSE OF CLOVER ROOT BORER (Hylastinus

obscurus (MARSHAM)) TO THEM

Toledo, D. 1, Pardo, F. 2 and Quiroz, A.2

1Programa de Doctorado en Ciencias de Recursos Naturales. Universidad de La Frontera. Temuco, Chile. 2Laboratorio de Ecología Química, Departamento de Ciencias Químicas. Universidad de La Frontera.

Temuco, Chile.*Corresponding author: [email protected] In Chile, the root borer, Hylastinus obscurus (Marsham) (Coleoptera: Scolytidae) is observed only in red clover Trifolium pratense L., suggesting that the insect has become a specialist on this legume. Previous reports have suggested that the settling behavior could be related to the presence of fatty acids in the roots of T. pratense. This fact is supported by reports showing that fatty acids could act as indicators of susceptibility to infestation by insect pest. Recently, the fatty acid components present in root extracts from 9-month-old red clover have been characterized and their effects on both olfactory and contact behavioural responses of the clover root bore were investigated. Individual synthetic fatty acids such as lauric, palmitic and oleic acid elicited an attractant behavioural response only from females. Contact bioassays, using wood dummies coated with root extract, four fatty acids, or respective specific blend elicited a preference from H. obscurus females. The results suggest that the long-chain free fatty acids present in red clover roots may play a role as close-range olfactory and/or tactile cues in the host-finding process of H. obscurus. Several species of legumes, including alfalfa (Medicago sativa) have been reported as host plants for clover root borer. As in Chile, alfalfa has become a non-host plant of H. obscurus, we hypothesize that the qualitative and quantitative composition of fatty acids in alfalfa roots must be different in comparison with red clover roots. In a first step, low polar compounds present in alfalfa roots were extracted two times with CH2Cl2 for 24 h each one at room temperature. The extracts were combined and filtered. The filtered extract was evaporated to dryness in a rotary evaporator. The residue was reconstituted with 5 ml of CH2Cl2. One microliter of the reconstituted mixture was injected in the GC-MS, for the identification of the free fatty acid. Afterwards, this mixture was submitted for sterification with methanol/HCl/CHCl3 at 90°C, with the aim of obtaining the sterified fatty acid (trans-esterification). This sterified mixture was injected in the GC-MS. In this work we report: a) both a qualitative and quantitative comparison of the fatty acid profile between alfalfa and red clover, and b) the olfactometric behavioural response of H. obscurus to alfalfa root extracts. The results were compared with previous reports related to the relationship between H. obscurus and red clover. This report is the first approach for understanding the role of fatty acids as key compounds in the suitability of a host to a non-host plant for H. obscurus.



138

BIOTRANSFORMATION OF ARISTOLOCHIC I AND II BY Battus polydamas archidamas (LEPIDOPTERA: PAPILIONIDAE: TROIDINII)

Urzúa, A.1,*, Pinto, C.2 and Niemeyer, H.2

1Laboratorio de Química Ecológica, Facultad de Química y Biología, Universidad de Santiago de Chile

2 Laboratorio de Química Ecológica, Universidad de Chile, Casilla 653, Santiago, Chile. *Corresponding author: [email protected]

Larvae of Battus polydamas archidamas (Papilionidae, Troidini) are able to sequester from Aristolochia chilensis their host-plant aristolochic acids that remain in their body during their lifetime. We explored the patterns of sequestration of AAs in tissues (body extracts, integument extracts and osmeterial secretions) of B. polydamas archidamas larvae along its development through the use of two meridic diets containing AAs (AAI and AAII) in concentrations higher and lower, respectively, than the mean natural concentration in the plant. Accumulation of AAs in body and integument extracts was proportional to the weight of larvae and was more pronounced in the diet containing higher concentration of AAs. Phenolic AAs (AAIa and AAIVa) not present in the diets were found in all tissues examined. Integument and body extracts showed a high AAI/AAII ratio with respect to that in the diet, suggesting a preferred AAII to AAIa transformation in agreement with a relatively high AAIa/AAIVa ratio in those two tissues. In osmeterial secretions, the value of the AAI/AAII ratio similar to that in the diets and the value of the AAIa/AAIVa ratio close to 1 suggest that hydroxylations of AAI to AAIVa and of AAII to AAIa occur to similar extents.

O

O COOH

NO2

R R1

AA-I R=H R1=OCH3

AA-II R=R1=H

AA-Ia R=H R1=OH

AA-IVa R=OH R1=OCH3



139

Symposium 4


Chairpersons:

Dr. Fernando Borie and Dr. Milko Jorquera. Oral presentations: - Dr. Francisco Chavez, Universidad de Chile, Chile: “System microbiology to study poly-

phosphate metabolism in bacteria”. - Dr. David Crowley, University of California, USA: “Soil inoculation: opportunities, challenges,

and technology for use of beneficial microorganisms”



140



141

SYSTEM MICROBIOLOGY TO STUDY POLYPHOSPHATE METEBOLISM IN BACTERIA

Chávez, Francisco*

Biology Department, Milennium Institute ICDB, Faculty of Science, University of Chile.

*Email address: [email protected] Polyphosphate (polyP) is a ubiquitous linear polymer of hundreds of orthophosphate residues (Pi) linked by phosphoanhydride bonds. PolyP has been found in the tree domains of life (Archaea, Bacteria and Eukarya) but been studied mainly in prokaryotes. In bacteria, polyP and its processing enzymes play important roles in cellular metabolism as well as in pathogenesis. PolyP is synthesized in prokaryotic cells from ATP by polyP kinases (PPKs) of which two families, PPK1 and PPK2, have been identified. PolyP was primary described as a reservoir of phosphate and, as in ATP, a source of high-energy phosphate bonds. Further genetic and biochemical experiments with ppk1 mutants in many bacteria have indicated additional roles for polyP. These include inhibition of RNA degradation, activation of the Lon protease during stringent response, involvement in membrane channel structure, and contribution to the resistance to stress generated by heat, oxidants, osmotic challenge, antibiotics and UV. Many bacterial pathogens knockout of ppk1 result in cellular defects, particularly in the context of virulence toward the host they invade. For example, ppk1 mutants of Salmonella sp. showed a drastic reduction in acid tolerance and invasiveness in epithelial cells, and bacterial survival in the macrophage cells was also severely compromised. In Shigella flexneri, the etiologicalagent of bacillary dysentery, ppk1 mutant leads to a decrease in several factors that affect virulence, including invasiveness for epithelial cells. Mutants of V. cholerae were also acid sensitive and stationary-phase defective while ppk1 mutants of N. meningitidis gene were highly sensitive to killing by human sera. Attenuated synthesis of ppk1 gene in Mycobacterium tuberculosis, by expression of antisense ppk1 results in a decrease of its ability to colonize and survive in macrophages. Similar results have been reported in H. pylori, where the lack of ppk1 gene results in the bacterial inability to colonize mice. The link between genotypes and phenotypes observed during polyP deficiency can be the result of complex networks of interaction that can be elucidated by using omics technologies. We have studied the global transcriptome and proteome changes in E. coli polyP mutants deficient in ppk1, ppx and both genes. To complement these studies we performed Phenotypic Microarrays™ studies in the same polyP mutant strains. This system biology approach enabled us to derive a hypothesis in which we proposed a link between Central Metabolism and particularly the TCA cycle and polyP metabolism. Also we have provided a potential mechanistic explanation for the link between polyP and alternative Sigma factors during polyP deficiency. Future bionformatic analysis of these omics results will allow us to make interaction networks that generate novel biological hypothesis that explain the structural and functional changes that occur during variation of polyP metabolism in bacteria.



142



143

Symposium 7

“Agro-food Biotechnology”

Chairpersons:

Dr. Carolina Shene and Dr. Berta Schnettler

Oral presentations:

- Dr. Ralf Greiner, Max Rubner-Institute, Germany:"Biotechnological application of phytate-degrading enzymes".

- Dr. Erick Scheuermann, Universidad de La Frontera, Chile: “Food aroma and essential oils:

extraction, analysis and application as bioactive compounds”.

- Dr. Paul Kolodziejczyk, University of Alberta, Canada: “Technologies applied to food and health products”.



144



145

BIOTECHNOLOGICAL APPLICATION OF PHYTATE-DEGRADING ENZYMES

Greiner, Ralf*

Department of Food Technology and Bioprocess Engineering, Max Rubner-Institute, Federal Research Institute of Nutrition and Food, Haid-und-Neu-Strasse 9, D-76131 Karlsruhe, Germany.

*Email address: [email protected] Phytases [myo-inositol(1,2,3,4,5,6)hexakisphosphate phosphohydrolases] have been identified in plants, microorganisms, and in some animal tissues (Konietzny and Greiner, 2002). They represent a subgroup of phosphatases which are capable of initiating the stepwise dephosphorylation of phytate [myo-inositol(1,2,3,4,5,6)hexakisphosphate], the most abundant inositol phosphate in nature. Phytases are a diverse group of enzymes that encompass a range of sizes, structures and catalytic mechanisms. Based on the catalytic mechanism, phytases can be referred to as histidine acid phytases (HAPhy), ß-propeller phytases (BPPhy), cysteine phytases (CPhy) or purple acid phytases (PAPhy) (Greiner, 2006; Mullaney and Ullah, 2003). Depending on their pH optimum, phytases have been divided into acid and alkaline phytases and based on the carbon in the myo-inositol ring of phytate at which dephosphorylation is initiated into 3-phytases [E.C. 3.1.3.8], 6-phytases [E.C. 3.1.3.26] and 5-phytases [E.C. 3.1.3.72]. Up to now, phytases have been mainly, if not solely, used as animal feed additive in diets largely for swine (Selle and Ravindran, 2008) and poultry (Selle and Ravindran, 2007), and to some extent for fish (Debnath et al., 2005). The first commercial phytase products were launched into market in 1991. Meanwhile the market volume is in the range of 150 million euro (Haefner et al., 2005). The small intestine of monogaster has only a very limited ability to hydrolyse phytate (Iqbal et al., 1994) due to the lack of endogenous phytases and the low microbial population in the upper part of the digestive tract. This fact explains also why phytate phosphorus is poorly available to single-stomached living beings (Walz and Pallauf, 2002). Phosphorus is absorbed as orthophosphate and thus utilisation of phytate phosphorus by single-stomached living beings will largely depend on their capability to hydrolyse phytate. Numerous animal studies have shown the effectiveness of supplemental microbial phytase in improving the utilisation of phosphate from phytate (Adeola et al., 2006; Augspurger et al., 2003; Esteve-Garcia et al., 2005; Simons et al., 1990). Therefore, including adequate amounts of phytase in the diets for simple-stomached animals reduces the need for orthophosphate supplementation of the feed. As a result, the environment is protected from pollution with excessive manure phosphorus runoffs because the faecal phosphate excretion of the animals is reduced by up to 50%. Enzyme preparations with the phytases from Aspergillus niger, Peniophora lycii, Schizosaccharomyces pombe and Escherichia coli are available commercially. In general, their large scale production is based on the use of recombinant strains of filamentous fungi and yeast. In addition, not always the wild-type phytases are produced, but mutant forms exhibiting more favourable properties regarding their application as feed supplements. Today, all phytases used for animal feed application belong to the class of histidine acid phytases. ß-Propeller phytases have been advocated for several applications. However, no commercial applications of ß-propeller phytases are currently available. Furthermore, neither a cysteine phytase nor a purple acid phytase are currently being marketed, although they have been subjected to several studies. More recently, phytases have been found increasingly interesting for processing of food for human consumption, particularly because the decline in food phytate results in an enhancement of mineral bioavailability (Greiner and Konietzny, 2006). This is seen as a way to reduce the risk of running into mineral deficiency in vulnerable groups such as child-bearing women, strictly vegetarians and inhabitants of developing countries. Different strategies could be applied to optimise phytate



146

degradation during food processing and digestion in the human alimentary tract such as adjustment of more favourable conditions during food processing for the phytases naturally occurring in the raw material, addition of isolated phytases to the production process, use of raw material with a high intrinsic phytase activity either naturally present or introduced by genetic engineering and the use of recombinant food-grade microorganisms as carriers for phytase activity in the human gastrointestinal tract. Phytate hydrolysis during germination, soaking, cooking, and fermentation is a result of the phytase activity naturally present in plants and microorganisms. The capability to dephosphorylate phytate differs greatly between different plant and microbial species due to differences in their intrinsic phytase activities and properties. To optimise food processing in respect to phytate degradation, it is essential to know the properties of the natural occurring phytases and the properties of exogenous phytases added during food processing or heterologously expressed in plants and microorganisms. Furthermore, phytases may find application in the production of functional foods or food supplements with health benefits. Individual myo-inositol phosphates have been shown to have important physiological functions in man, such as prevention of diabetes complications, anti-inflammatory, antiangiogenic and antitumour effects. Phytases render production of defined myo-inositol phosphates in pure form and sufficient quantities possible. Phytases degrade phytate by sequentially removing phosphate from the myo-inositol ring in a regio- and stereospecific manner and the majority of phytases generates only one single myo-inositolpentakis-, tetrakis-, tris-, and bisphosphate isomer. Purification of the individual isomers from the reaction mixture could be achieved by ion-exchange-chromatography and different phytases pave the way for the production of different partially phosphorylated myo-inositol phosphates. Last but not least, technological improvements are expected to occur due to phytate degradation during processing as shown for breadmaking, production of plant protein isolates, corn wet milling and the fractionation of cereal bran. However, but up to now, no phytase product for a relevant food application has found its way to the market. One ideal phytase for all food and feed applications does not exist. Thus, screening nature for phytases with more favourable properties for certain applications and engineering phytases in order to optimise their catalytic and stability features are suitable approaches to make a proper phytase available for a specific application. References

- Adeola, O., Olukosi, O.A., Jendza, J.A., Dilger, R.N. and Bedford, M.R. (2006) Response of growing pigs to Peniophora lycii- and Escherichia coli-derived phytases or varying ratios of calcium to total phosphorus. Animal Science 82, 637-644.

- Augspurger, N.R., Webel, D.M., Lei, X.G. and Baker, D.H. (2003) Efficacy of an E. coli phytase expressed in yeast for releasing phytate-bound phosphorus in young chicks and pigs. Journal of Animal Science 81, 474-483.

- Debnath, D., Sahu, N.P., Pal, A.K., Baruah, K., Yengkokpam, S. and Mukherjee, S.C. (2005) Present scenario and future prospects of phytase in aquafeed – review. Asian-Australian Journal of Animal Science 18, 1800-1812.

- Esteve-Garcia, E., Perez-Vendrell, A.M. and Broz, J. (2005) Phosphorus equivalence of a consensus phytase produced by Hansenula polymorpha in diets for young turkeys. Archives of Animal Nutrition 59, 53-59.

- Greiner, R. (2006) Phytate-degrading enzymes: Regulation of synthesis in microorganisms and plants. In: Turner, B.L., Richardson, A.E. and Mullaney, E.J. (eds.) Inositol Phosphates: Linking Agriculture and Environment, CAB International, London, England, pp. 78-96.



147

- Greiner, R. and Konietzny, U. (2006) Phytase for food applications. Food Technology and Biotechnology 44, 125-140.

- Haefner, S., Knietsch, A., Scholten, E., Braun, J., Lohscheidt, M. and Zelder, O. (2005) Biotechnological production and applications of phytases. Applied Microbiology and Biotechnology 68, 588-597.

- Iqbal, T.H., Lewis, K.O. and Cooper, B.T. (1994) Phytase activity in the human and rat small intestine. Gut 35, 1233-1236.

- Konietzny, U. and Greiner, R. (2002) Molecular and catalytic properties of phytase-degrading enzymes (phytases). International Journal of Food Science and Technology 37, 791-812.

- Mullaney, E.J. and Ullah, A.H.J. (2003) The term phytase comprises several different classes of enzymes. Biochemical and Biophysical Research Communications 312, 179-184.

- Selle, P.H. and Ravindran, V. (2007) Microbial phytase in poultry nutrition. Animal Feed Science and Technology 135, 1-41.

- Selle, P.H. and Ravindran, V. (2008) Phytate-degrading enzymes in pig nutrition. Livestock Science 115, 99-122.

- Simons, P.C.M., Versteegh, H.A.J., Jongbloed, A.W., Kemme, P.A., Slump, P., Bos, K.D, Wolters, M.G.E., Beudeker, R.F. and Verschoor, G.J. (1990) Improvement of phosphorus availability by microbial phytase in broilers and pigs. British Journal of Nutrition 64, 525-540.

- Walz, O.P. and Pallauf, J. (2002) Microbial phytase combined with amino acid supplementation reduces P and N excretion of growing and finishing pigs without loss of performance. International Journal of Food Science and Technology 37, 835-848.



148

FOOD AROMA AND ESSENTIAL OILS: EXTRACTION, ANALYSIS AND APPLICATION AS BIOACTIVE COMPOUNDS

Scheuermann S., Erick*

Chemical Engineering Department, Engineering, Science and Administration Faculty and Scientifical and

Technological Bioresources Nucleus (BIOREN), Universidad de La Frontera, Temuco, Chile. *Email address: [email protected]

Aroma is an odour with a pleasant connotation. “Odour” has been defined by the British Standards Institute as the quality of sensation perceived, via olfactory organ situated in the nasal cavity, from certain volatile substances (Mottram, 1993). Then, the aroma corresponds to the volatile compounds release from the food. However, the term “flavor or flavour” is often used rather than aroma. According Reineccius (2006) the task of identifying volatile flavor components (aroma compounds) particularly in a food matrix is one of the most formidable tasks faced by an analytical chemist. The range of chemical classes which contributes to food flavor is diverse in both chemical composition and physical properties, and includes aliphatic, alicyclic, aromatic and heterocyclic compounds (Mottram, 1993). For food aroma or food flavor analysis, traditional techniques for chemical isolation, concentration, separation and identification are used (Mottram, 1993; Reineccius 2006). New methods as the solid phase microextraction (SPME) and stir bar sorptive extraction (SBSE) have recently begun to be used by researchers for aroma volatile compounds isolation and concentration (Diez et al., 2004; Guerrero et al., 2006; Reineccius, 2006). Hall and Merwin (1988) suggest that the term “flavor” is use for a particular category of substance which causes the olfactory and taste stimulus. Sources of flavor range from the traditional fruits, vegetables, spices and herbs to contemporary synthetics. From a technological point of view, the flavor can be classified into seven groups: condiments, spices, concentrated fruits and juices, process flavor, oleoresins and solid extracts, essential oils and aromatic chemicals. Essential oils are volatile, natural, complex compounds characterized by a strong odour and are formed by aromatic plants as secondary metabolites (Bakkali et al., 2008). These flavors, composed of only the volatile fraction obtained from the distillation of spices and similar plant materials, contain aroma compounds that are present in high concentration (Lindsay, 1984). Their chemical composition include mainly terpenes, aromatics compounds and terpenoides (Bakkali et al., 2008). Essential oils (EOs) normally are obtained by distillation, hydrodifussion, expression, enfleurage and adsorption, solvent and supercritical fluid extraction (Tyrrell, 1990; Wildwood, 1996). The EOs are widely proposed and used for food preservation for their bactericidal, virucidal, fungicidal, antiparasitical and insecticidal properties (Bevilacqua et al., 2010). Also they are used as analgesic, sedative, anti-inflammatory, spasmolytic and locally anesthesic remedies (Bakkali et al., 2008). The EOs antioxidant activity has been extensively researched because they are widely used in the meat derivate product preservation. In the Food Science Laboratory at Chemical Engineering Department, Universidad de La Frontera several studies has been carry to obtain and analyze aroma and essential oil from murtilla (Ugni molinae Turcz), oregano (Origanum vulgare L.) and boldo (Peumus boldus) and to evaluate their application as bioactive compounds in other food products.



149

TECHNOLOGIES APPLIED TO FOOD AND HEALTH PRODUCTS

Kolodziejczyk, Paul*

Department of Agricultural, Food and Nutritional Science, Faculty of Agricultural, Life and Environmental Sciences. University of Alberta, Edmonton, Alberta, Canada T6G 2P5. *Email address: [email protected]

Food and medicine production and related technologies were around from the beginning of human civilization. Initially, those activities were limited to collection of plants and hunting. With the acquirement of fire, real processing was started, which included cooking, baking, roasting, and smoking meat or fish for conservation purposes. The industrial revolution and subsequent high rate of urbanization created a great demand for food products. Novel crops and new more efficient processing prompted the development of food industry, providing the mass food production to a rapidly growing population. New components in human diet and excess of food proved to cause some detrimental effects and are now often linked to obesity and diseases (cardiovascular, diabetes and some cancers). In the last couple of decades, we have experienced a dramatic change in the perception of the role food plays in human nutrition. Now food is considered as an important factor influencing human health. For example, the relationships between the consumption of polyunsaturated fatty acids and cardiovascular diseases or brain development, the effect of inulin on gut bacterial flora, or the protective role of antioxidants are presently well documented. There is evidently an increasing demand by consumers for foods that has fewer synthetic additives, or have undergone fewer changes during processing. These foods more closely resemble the original raw materials and have a “healthy” or “natural” image. Correspondingly, demand for organic foods has significantly increased in developed countries. These consumer pressures are an important influence on changes that are taking place in the food processing industry. New products that are supplemented with vitamins, minerals and probiotic cultures have appeared in recent years. Consumer pressure for more “natural” products has also stimulated development of novel “minimal” processes that reduce the change to sensory characteristic or nutritional value of foods. The new categories of food products called “functional foods”, along with “nutraceuticals” are nowadays recognised by legislation in most countries. Legislation is now increasingly international in its focus and application, and international standards for both specific products and also for methods of achieving quality assurance are in force. The processed food, functional food or nutrition products need to be protected from the damage caused by microorganisms or from enzymatic processes occurring during their storage. Traditional methods include pasteurization or irradiation (ionizing particles, microwaves or electric fields). The elevated temperature treatment or high energy irradiation often is responsible for undesired changes in flavour, texture or in composition of bioactive factors present in food. Relatively new technology is based on high pressure processing. In 1990 the first commercial products processed using this technology appeared in Japanese market. Initially, the production was limited to jams and fruit juices in flexible sealed plastic packs. Currently yogurts, and other milk products, eggs products, fruit jellies, salad dressings are produced using high pressure processing. There are advantages, mainly high quality, retained flavour, texture and preservation of vitamins and other beneficial bioactive components, which are justifying the 3-4 fold higher prices in comparison to traditional products.



150

Interestingly, several enzymes that are related to food quality vary in their barosensitivity. This is a promising avenue for further exploratory research in novel methods for the extension of shelf-life of food products. The industry is in constant need of novel products and processing methods. Standard operation units: extractions, separation (distillation, filtration) are often combined with biotechnological methods for production of new foods, functional foods, nutraceuticals or medicines. The presentation will illustrate how modern processing methods are used for processing and development of novel foods and nutritional products. The examples will present the application of polyunsaturated fatty acids and polyphenolics in human nutrition and health and review methods for processing and analysis of natural products.



151

Symposium 7

“Agro-food Biotechnology”




152



153

PRELIMINARY CHARACTERIZATION OF OLEOSOMES FROM PLANT SEEDS AS POSSIBLE CARRIERS FOR MICROENCAPSULATION

Acevedo, F.1*, Morales, E.1, Rubilar, M.1,2 and Shene, C.1,2

1Center of Food Biotechnology and Bioseparations, BIOREN, Universidad de La Frontera, Casilla 54-D,

Temuco, Chile. 2Technology and Processes Unit, CGNA, Universidad de La Frontera, Casilla 54-D, Temuco, Chile. *Corresponding author: [email protected]

Oleosomes (OL) from plant seeds are oil bodies that act as energy stores for post-germinative growth. These OL consist of an oil core with a matrix of triacylglycerol bounded by a phospholipid monolayer embedded with proteins known as oleosins. Oleosomes could offer an excellent alternative as natural biocapsules. In fact, the structure and topological orientation of oleosins make OL useful in biotechnological applications. Oleosins possess three domains: central hydrophobic domain and amphipathic N and C termini. The central hydrophobic domain penetrates through the phospholipid monolayer into the oil body matrix. N and C termini of oleosin are located on the OL surface. Their negatively charged residues exposed to the cytoplasm provide steric hindrance and electrostatic repulsion, thus maintaining the OL as separate and stable entities. In addition, oleosins have natural self-emulsifying properties which may increase the surface area for release of active biomolecules from the OL. Oleosomes and oleosins have been purified and characterized in many plant species. Chilean Amerindians have used seeds of Gevuina avellana as oil sources since pre-Columbian times and only few scientific reports about their bioactive molecules have been already found. The high content of oil (49%) from Gevuina avellana seeds suggests that it would be a potential source of oleosomes. However, to date, no purification and characterization of OL and oleosins from Gevuina avellana have been performed. Furthermore, the main goal of this study was to isolate and characterize oleosomes and oleosins from Gevuina avellana as possible encapsulation carriers. In this study, the oleosomes from Gevuina avellana were purified using an aqueous-based flotation-centrifugation method and characterized by means a proximate composition and particle size by light and confocal microscopy. Lipid content was determined by diethylether extraction. The protein content of the defatted dried OL was determined by the Bradford method. Proteins were separated using SDS-PAGE electrophoresis. Extraction and characterization of oleosomes and oleosins of linseeds and rapeseeds were run in parallel. This work has shown that oleosomes can be extracted from Gevuina avellana seed, linseed and rapeseed with an extraction yield of 5.6%, 5.8% and 5.0%, respectively. The final washed oleosomes preparation contained 85.9% lipid and 1.8% protein for Gevuina avellana seeds, 78.4% lipid and 6.7% protein for linseed and 86.9% lipid and 4.4% protein for rapeseed. The identified oleosins showed molecular mass values between 14 and 25 kDa, depending on the seed species. The spherical oleosomes presented a diameter of about 2 µm. Our findings support a potential functionality for native Chilean seeds as sources of oleosomes for possible biotechnological applications.

Acknowledgements: The authors are grateful for financial support provided by Fondecyt Project 1090516.



154

ENHANCED HYDROCARBONS RECOVERY FROM Botryococcus braunii BY AN IONIC LIQUID PRETREATMENT

Medina, H.1,* and Shene, C.2

1Doctorate Program in Science of Natural Resuorces and 2Departament of Chemical Engineering,

Universidad de La Frontera, Temuco, Chile. *Corresponding author: [email protected]

Production of biofuels based on renewable sources is currently more than ever object of study; among the different sources for sustained biofuel production most promising are microalgae. Under suitable cultivation conditions several microalgae strains are able to accumulate high contents hydrocarbons and lipids, products that can be converted into diesel through cracking and transesterification. Nevertheless, to date some factors impede the commercial applications of algae-biofuel technologyamong which is the high energy input required for the extraction and recovery of hydrocarbons. Different technologies for cell wall disruption have been suggested and evaluated, for instance, high-pressure homogenization, autoclaving, chemical and enzymatic hydrolysis, osmotic shock, among others. Ionic liquids (ILs)are molten salts that have melting point below than 100°C; these salts composed by a positive organic cation and negative inorganic/organic anion, have negligible vapor pressure and havebeen defined as “green solvents”. Recently, the use of ILs having the property to revert their polarity was tested for this process. The use of ILs could also assist the cell disruption process for releasing hydrocarbons, approach that has been used for recovering soy lipids, and as a medium for lipase assisted biodiesel production. Many reports indicate that the solubility of polysaccharides, with structures similar to those found in microalgae cell wall, is enhanced by the use of ILs. In addition stability, selectivity and activity of disrupting enzymes are improved by using ILs as solvents creating the possibility to use them in pretreatments for decreasing cell wall resistance. From data reported in literature polymerization degree (DP) and crystallinity of cellulose treated with ILs is reduced by 52% and 75%, respectively. Que es el modulo?Themodule for mechanical resistance was reduced by 0.0011 kPa/DPand enzymatic module by -0.0027 mg TRS (mg dw DP)-1 (TRS, total sugar release). The mechanism through which ILs act on polysaccharide dissolution and lipid recovery is based on the formation of anelectron donor-acceptor complex; this is controlled by hydrogen bond capacity of anion/cation in ILs and by π interaction with cation. The objective of this work is to analyze the effect of different ILs on mechanic and enzymatic resistance of B. brauniicell wall and to optimize the high pressure homogenizer and enzymatic disruption processes. At the time, preliminaryextraction experiments have been carried out with organic solvents andwet/dry biomass; chloroform extraction of wet biomass (18.4%) showeda better performance thansoxlet (petroleum ether/dry biomass 13.1%) and hexane/dry biomassmethod (12.5%). Lipid content in the dry biomass was 17.4%. Because chloroform has three Cl atoms with high hydrogen bond capacity, it has best performance in permeabilizationof the cell wall. On the other hand, hexane and petroleum ether interact by dipole-dipole and would not coordinate with polysaccharide OH-group.



155

EXTRACTION AND CHARACTERIZATION OF OLEORRESIN FROM RED BELL PEPPER (Capsicum annum) USING SUPERCRITICAL CARBON DIOXIDE

Millao, S. and Uquiche, E.*

Center of Food Biotechnology and Bioseparations, BIOREN. Universidad de La Frontera (UFRO), Temuco,

Chile. *Corresponding author: [email protected]

The commercial value of red bell pepper (Capsicum annuum) for paprika and oleorresin, it only depends of the coloring capacity, so that the quantification of the content of pigments carotenoids is very important, also its antioxidant activity. This work had as objective to study the extraction of oleorresins from flakes and pellets of red bell pepper by using supercritical carbon dioxide. Was evaluated effect of temperature and pressure on characteristics of extracts, such as color ASTA, concentration of carotenoids and antioxidant activity (TEAC). Supercritical fluid extraction (SFE) of oleorresin was carried out in a Spe-ed SFE unit (Applied Separations, Allentown, PA). In all experiments the sample was charged into the extractor vessel (50 mL) and treated with 3 L min-1 of CO2. An attempt was made to optimize three parameters of SFE's: temperature (40 y 60 ºC), pressure (300 and 500 bar) and extraction time (180 min) to obtain high extraction yield of oleoresin. During extraction, extract aliquots were periodically collected in pre-weighed glass vials (15 mL capacity), and dried in a dessicator with silica gel to remove co-extracted water, and the recovered extract was assessed gravimetrically by difference with cleaned and dried vials. The yield of oleoresin was calculated based on the cumulative mass of extract and was expressed in units of water-free solute mass per unit of dry substrate mass. The higher concentration of carotenoids and units of color ASTA were obtained in flakes at 40 ºC and 500 bar with 48.53 (mg carotenoids/kg oleorresin) and 1.393 units ASTA. In pellet was lower at same conditions, resulting in 41.07 mg carotenoids/kg oleorresin and 913 units ASTA. The pelletization pre-treatment diminished the antioxidant capacity of susbstrate, this effect was reflected in the results of analysis of p-anisidine index and TEAC values.

Acknowledgements: This work was funded by Chilean agency Fondecyt (Project 1080211).



156

EXTRACTION/FRACTIONATION OF RAPESEED CAKE OIL USING SUPERCRITICAL CARBON DIOXIDE

Salazar, K. and Uquiche, E.*

Center of Food Biotechnology and Bioseparations, BIOREN. Universidad de La Frontera (UFRO), Temuco, Chile. *Corresponding author: [email protected]

Rapeseed press cake is rich in oils with a high content of linolenic fatty acid (omega 3). However, there are other components of great functional value, known as minor lipids in this substrate such as carotenoids, tocopherols and phytosterols. The objective of this study was to identify the effect of pressure on the oil extraction yield and fatty acid composition and minor lipid concentration of oil fractions obtained in the course of extraction from press rapeseed cake using supercritical CO2. In addition, the extraction efficiency of SC CO2 was compared with traditional extraction methods, namely the Soxhlet. Supercritical fluid extraction (SFE) of oleorresin was carried out in a Spe-ed SFE unit (Applied Separations, Allentown, PA), using CO2 at 80 °C and pressures of 350, 400 and 450, and a flow rate of 4 L/min. The extraction the oil was collected for 60 minutes, as subsequent two fractions depending on a period of time extraction (30 and 60 min). The yield of extraction and fractionation of oil by using SC-CO2 was calculated as percentage regarding the extraction oil yield in a Soxhlet apparatus with hexane. The concentration of carotenoids, tocopherols and sterols was determined. It was observed that the total extraction oil yield increased when the extraction pressure also increased (350-450 bar). A higher oil mass was extracted in the first stage of supercritical extraction. On the contrary, the fractions obtained in the final stage of the extraction resulted with a higher concentration of minor lipids. So, at 80 ºC and 450 bar, the second fraction had a concentration of carotenoids, tocopherols and sterols of 71.2, 14.180 and 31.575 mg/kg oil, respectively. Fatty acids composition and antioxidant activity were analyzed in two successive oil fractions. According to our results, the fractionation of oil using supercritical CO2 was possible under the conditions studied to obtain extracts enriched with minor lipids and with a higher concentration of tocopherols, sterols and antioxidant activity.

Acknowledgements: This work was funded by Chilean agency Fondecyt (Project 11080213).



157

GROWTH AND EXPRESSION OF CANDIDATE GENES INVOLVED IN MUSCLE DEVELOPMENT IN ADULT MALE ZEBRAFISH (Danio rerio) FED WITH TOTAL

SUBSTITUTION OF FISHMEAL BY MIXTURE OF PLANT PROTEIN SOURCES DIETS

Ulloa, P.1,*, Peña A.2, Iturra, P.3 and Neira, R.4,5

1Programa de Doctorado en Ciencias de Recursos Naturales Universidad de La Frontera. 2Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Pontificia Universidad Católica de Valparaíso 3Facultad de Medicina Universidad de Chile. 4Departamento de Producción Animal, Facultad de Ciencias Agronómicas

Universidad de Chile.5Aquainnovo S.A. *Corresponding author: [email protected]

Zebrafish (Danio rerio) is a model organism widely used in studies of developmental biology, physiology, molecular genetics, and toxicology. Nowadays, zebrafish has been postulated as a model organism for nutrition and growth studies in fish. Phenotypic expression of growth is both under genetic control and influenced by environmental factors, as nutrition. All these, and zebrafish’s extensive genetic information, makes it an ideal model for nutritional genomics studies, where results can be applied to important aquaculture fish. There is little research focused on studying how different sources of animal or plant protein in fish feed affect the expression of genes involved in metabolic pathways that regulate growth and muscle development. The aim of this study is to assess growth performance and expression of genes involved in growth and muscle development in adult male zebrafish feeding diets with total substitution of fishmeal by a mixture of plant protein sources. This study characterizes the expression of eight candidate genes such as IGF-I and II, mTOR, PLD, involved in the synthesis and degradation of proteins. Myostatin (Mstn1b) is a negative key regulator of growth and muscle development. Myogenina, Myod and myf4 are involved in myogenesis in the differentiation of myoblasts and myofibers. Only gene expression in males is informed in this report. We used two wild-type strains of D. rerio to generate a base population of 24 experimental families. After the larval period (from 8 to 28 days post fertilization, dpf), each of the 24 families were divided into two to create replicates of each family (40 fish per family replicate). One sub-population or set of family replicates (24 families) was fed to satiation three times a day for 13 weeks with a diet containing fishmeal as the primary protein sources (FM diet, control diet) and the other set of family replicates (24 families) was fed with a diet with total substitution of fishmeal by mixture of plant protein sources (PP100%, experimental diet). Diets were formulated to represent those used in the salmon industry and be iso-nitrogenous and iso-energetic and with addition in indispensable amino acid. Fish were sampled before the nutritional assay (28 dpf), at 70 dpf, and at the end nutritional assay (98 dpf) by sub-population and family, for growth (mg) and length (mm). Growth curve of zebrafish fed with FM diet showed a faster growth rate than fish fed with experimental diet. Average growth for FM diet was 180.73 ± 57.83 and 135.51 ± 55.39 for experimental diet. In each sub-population, individuals were selected according to the phenotypic value for growth rate. Three families with average growth rate within each sub-population were selected. The same families were selected in both cases in order to have the same genetic background, representing growth rate of each sub-population. For gene expression analysis, three fish were evaluated in each family (3 fish x 3 family = 9 fish in each case). We used quantitative real-time PCR (qRT-PCR) analysis. Results indicate that total substitution of fish meal by plant protein sources significantly increased (x 1.6) IGF-I; (x 0.54) Mst1b; (x 4.0) Myogenin, and (x 3.4) Mrf4 expression in muscle. Expression of MyoD, mTOR, PLD and IGF-II was not modified by dietary treatment in muscle. Thus, this study demonstrates that the decrease in growth of zebrafish fed a substitution total of fishmeal by mixture of plant protein sources is linked to changes in the dynamics muscle growth and correlated with changes in expression of a gene involved in anabolic processes such as cell division, tissue repair and protein synthesis; also in myogenesis and structural growth of fish muscle.

Acknowledgements: Aquainnovo SA- INNOVA 206-5047; Beca Conicyt AT-24091052.



158

Botryococcus braunii CAKE AS SOURCE OF PROTEIN: EFFECT OF OIL EXTRACTION METHODS

Cancino, B.2, Verdugo, M.1,2,*, Morales, E.2, Franco, C.2 and Rubilar, M.2,3.

1Ph.D. student of Program in Sciences of Natural Resources, Universidad de La Frontera, casilla 54-D,

Temuco, Chile. 2Center of Food Biotechnology and Bioseparations, BIOREN, Universidad de La Frontera, casilla 54-D, Temuco, Chile. 3Technology and Processes Unit, CGNA, Universidad de La Frontera, casilla

54-D, Temuco, Chile. *Corresponding author: [email protected]

Microalgae are well known natural producers of fatty acids and hydrocarbons and additionally it is possible to extract other different kind of bioproducts. For the biodiesel production from microalgae the hydrocarbons and oil are extracted. This process left behind a big volume of residue called microalgae cake which until now is poorly utilized and it is destined mainly to animal feeding. There are a large variety of protein resource from microalgae; however in the literature there is no reports about Botyrococcus braunii as a source of proteins and there is no known report on protein derived from the B. braunii cake. The main goal of this study was evaluate the impact of microwave pre-treatment on B. braunii oil extraction and on protein content in microalgae cake. Additionally, the effect of pH, cake/solvent ratio, and contact time were studied on the soluble protein as preliminary studies to establishment the work levels for subsequent protein extraction from microalgae cake. The oil from microalgae was extracted according to AOAC standard method, the sample was extracted for 5 h with petroleum ether in a Soxhlet extractor. Furthermore, microalgae were pretreatment with microwave at medium potency during 10 min and after that the solvent extraction was made. The protein solubility from B.braunii cake was determined by lowry´s method at pH 2, 4, 6, 8 10 and 12. In the same way the effect of contac time on soluble protein was studied at 15, 30, 45, 60, 90, 120 180 and 240 min. Also the cake/solvent ratio ⅟9, ⅟19, ⅟29, ⅟39, ⅟49 were evaluated on the protein extraction from microalgae cake. The preliminary assay for the extraction oil from B. braunii showed 15.0 % and 11.9% with pretreatment and without microwave pretreatment respectively. In spite of the microwave pretreatment was more effective in the oil extraction, percentage of soluble protein was about 18,3%, which it is lower in compare to 23,7% found in the sample without pretreatment. These results are concordant with the similar studies found in literature on oilseed samples; protein quality and content strongly depend on processing conditions. The follow preliminary experiments to relate to protein extraction were performed with defatted cake without microwave pretreatment. Respect these experiments lead to the protein extraction from B. braunii cake, the pH effect on soluble protein showed higher yield on the extraction at pH 12. On the other hand the contact time had no influence on soluble protein and respect to cake/solvent ratio the higher soluble protein 43%, was found for the ⅟49 ratio. Finally this work showed that microwave pretreatment it is more effective in the oil extraction; however it produce more damage on microalga cake proteins in compare to cake obtained without pretreatment.

Acknowledgements: Author acknowledges the financial support given by Conicyt through Conicyt scholarship 21070302, Consortium Desert Bioenergy S.A. for research and development of biofuel industry from microalgae, INNOVA Chile-CORFO 09CTEI-6860, Program “Microalgae biomass for production of bio-compounds”, GAP. Dirección de Investigación at Universidad de La Frontera and DI10-2016.



159

RHEOLOGICAL PROPERTIES OF EDIBLE COATINGS BASED ON BLENDS OF BIOPOLYMER SOLUTIONS ENRICHES WITH POLYPHENOLS FROM Ugni molinae

TURCZ.

Silva-Weiss, A.1,*, Bifani, V.2, Ihl, M.2 and Gómez-Guillen, C.3

1Ph.D. Program in Natural Resources Science, Universidad de La Frontera, Temuco, Chile.

2Chemical Engineering Department., Universidad de La Frontera, Temuco, Chile 3Instituto del Frío (CSIC), Ciudad Universitaria, 28040 Madrid, Spain

*Corresponding author: [email protected] In food preservation, edible films and coatings have been particularly considered because of their capability in improving global food quality and safety. Edible films and coatings can carrier active natural compounds from plants that act as antioxidant, antimicrobial and/or anti-browning agents as well as cross-linking agents. Aqueous murta leaves extracts (Ugni molinae Turcz.) have been shown to have high antioxidant activity in vitro (1). Among the main compounds found in aqueous murta leaves extract are phenolic acids as gallic acid as well as glycosides of quercetin, myricetin and kaempferol (1, 2). In food engineering, rheology helps to understand how food material structure responds to applied force and deformation. Rheologycal characterization can relate structure and stability of food materials. Beside, these properties are required for calculation of any process involving fluid flow and play an important role in process design, quality control and sensory assessment of food materials. In this work, the effect of polyphenols from Ugni molinae Turcz. and blend type of biopolymer solution on the rheological properties of edible coatings were studied at 25ºC. The biopolymers used in the formulations of film-forming soltions were carboxymethylcellulose (CMC), unmodified corn starch (CS), modified corn starch (MS) and chitosan (CH). Biopolymer blend solutions (CH-CS, CS-CMC, MS-CMC, and CH-CMC) were prepared by mixing the film-forming solutions without and with added murta extract. The concentration of total hydrocolloids in solution (HT) was maintained at a constant 2 % w/v value. A cone-plate geometry was used for both steady shear and viscoelastic measurements at 25ºC. All film-forming solutions behaved as pseudoplastic fluids (n < 1) with shear rate above 0.4 s-1. Although a dramatic change in apparent viscosity was observed in CH-CS blend solution and CH solutions when murta extract was incorporated. The flow behavior and viscoleastic properties of film-forming solutions were affected by biopolymer blend type as well as presence or absence of polyphenols from Ugni molinae Turcz. Blend solution based on CS-CMC and MS-CMC and CMC, with and without added polyphenols from Ugni molinae Turcz.. show suitable viscosity range (2.75 to 8.49 Pas at 2 s-1 and 25 °C) for the casting process. They providing stable solutions in the wide frequency range studied. CMC was the main factor influencing both apparent viscosity and viscoelastic properties of these coatings. References: (1) Bifani V. et al. (2007). LWT- Food Science and Technology, 40, 1473-1481. (2) Rubilar M. et al. (2006). Journal of Agricultural and Food Chemistry, 54(1), 59–64.

Acknowledgement: CONICYT-CHILE AT-24090134, 29090088 and 21070302, INNOVA-CORFO Project 06N12PAT-57, and Postgraduate Program in Natural Resources Sciences, Universidad de La Frontera, Temuco, Chile.



160

POLYPHENOLIC COMPOUNDS FROM MURTA LEAF SEPARATED BY GEL CHROMATOGRAPHY: APPLICATIONS IN LINSEED OIL AND EMULSIONS

Barra, A.1, Palma, B.1, Acevedo, F.1, Shene, C.1,2 and Rubilar, M.1,2*



The murta is a shrub that grows on the coast and in the Andes Mountains of southern Chile. Both the fruit and leaf have shown a high antioxidant potential, but its effect on oil has not been evaluated yet. The objective of this study was to evaluate the antioxidant effect of murta leaf fractions on the oxidative stability of linseed oil and oil-water emulsions (o/w). Polyphenolic fractions of murta leaf were separated by gel chromatography (Sephadex LH-20) and incorporated into the oil (directly) and into emulsions o/w, comparing its effect with butylated hydroxytoluene (BHT) and trolox. As evaluation criterion, the peroxide index (PI) and the formation of conjugated diene (CD) during accelerated storage at 50 °C for 24 days were used. Eight fractions (from F1 to F8) showed different polyphenolic content and antioxidant activity. The fraction F6 was the most active for both forms of incorporation reaching a PI of 13.43 meq/kg and CD of 0,216 nm directly into the oil and a PI of 15,9 meq/kg and CD 0,203 nm into emulsions o/w. These results are slightly higher than those obtained for BHT and Trolox. The statistical analysis showed that the incorporation of antioxidants and storage time were significantly different (p≤0,05) in the formation of peroxides. As conclusion, the chromatographic method used in this study was effective for separating compounds with antioxidant activity comparable with BHT, where the fraction F6 was able to increase the resistance to the oxidation of linseed oil for both forms of incorporation. Acknowledgements: The authors are grateful for financial support provided by Fondecyt Project 1090516 and GAP, DI10-2016 of the Research Office at Universidad de La Frontera.



161

EFFECT OF POLYPHENOLIC FRACTIONS OF MURTA LEAF ON OXIDATIVE STABILITY OF LINSEED OIL MICROENCAPSULATED BY SPRAY DRYER

Sáez, R.1, Morales, E.1, Palma, B.1, Acevedo, F.1, Shene, C.1,2 and Rubilar, M.1,2*



Linseed oil is a valuable source of omega-3 fatty acids, and lipid oxidation is the main cause of deterioration that affects this type of food. The process of microencapsulation by spray drying has beneficial effects on protection of polyunsaturated fatty acids against oxidation. The objective of this study was to evaluate the effect of the incorporation of antioxidants polyphenolic fractions of murta leaf on the oxidative stability of linseed oil microencapsulated by spray drying. For this purpose, eight polyphenolic fractions were separated by gel chromatography (Sephadex LH-20), of which fractions 6 and 8 reached the highest and lowest antioxidant activity in vitro (using DPPH method) in the oil, respectively. Both fractions were incorporated into linseed oil emulsions in an aqueous solution of arabic gum (15% w/w) in a ratio of 1:4. Additionally, emulsions were elaborated with commercial antioxidants such as butylated hydroxytoluene (BHT), α-tocopherol and trolox. The conditions of microencapsulation process by spray drying were: inlet temperature (Ti) at 140°C, outlet (To) to the dryer at 95°C, feed temperature (Tf) at 20 °C and average feed rate (Rf) of 5,4 g/min. The peroxide index (PI), formation of conjugated diene (CD) and p-anisidine values (PAv) during storage at 25°C for 40 days were used as evaluation criterion. As results, the microencapsulated linseed oil (MLO) with the fraction 6 showed PI values of 15,26 meq/kg, CD of 0,409 nm and PAv of 2,55 at 40 days of storage. The similarity percentage of these results respect to those obtained for MLO with Trolox and BHT were 93,6% and 62,6%, respectively. The MLO with the incorporation of the fraction 8 showed PI values of 30,99 meq/kg, CD of 0,618 nm and PAv of 2,90. With α-tocopherol, MLO reached PI values of 205,87 meq/kg, CD 2,97 nm and PAv of 25,45. Both MLO with fraction 8 as MLO with α-tocopherol showed a prooxidant behavior with values higher than the control. Statistical analysis showed that factors such as storage time and antioxidants were significantly different (p≤0,05) in the increase of PI, CD and PAv values. The MLO with α-tocopherol showed a significantly higher oxidative process compared with other antioxidants. As conclusion, it was found that the fraction 6 of murta leaf reached an increase in the oxidative stability of MLO with comparable values to those reached with Trolox and BHT.

Acknowledgements: The authors are grateful for financial support provided by Fondecyt Project 1090516 and GAP, DI10-2016 of the Research Office at Universidad de La Frontera.

2nd international workshop - · pdf file2nd international workshop ... nanthi, szogi, ariel,...

Documents