Aproximación al diseño de sistemas de bio-reactores para la delignificación de materiales lignocelulósicos y para la decontaminación de suelos y efluentes. Approximation to the design of bio-reactors systems for the delignificación of lignocellulosic materials and for the decontamination of soil and effluents

Los materiales lignocelulósicos residuales de las actividades agroindustriales pueden ser aprovechados como fuente de lignina, hemicelulosa y celulosa. El tratamiento químico del material lignocelulósico se debe enfrentar al hecho de que dicho material es bastante recalcitrante a tal ataque, fundamentalmente debido a la presencia del polímero lignina. Esto se puede lograr también utilizando hongos de la podredumbre blanca de la madera. Estos producen enzimas lignolíticas extracelulares fundamentalmente Lacasa, que oxida la lignina a CO2. Tambien oxida un amplio rango de sustratos ( fenoles, polifenoles, anilinas, aril-diaminas, fenoles metoxi-sustituídos, y otros), lo cual es una buena razón de su atracción para aplicaciones biotecnológicas. La enzima tiene potencial aplicación en procesos tales como en la delignificación de materiales lignocelulósicos y en el bioblanqueado de pulpas para papel, en el tratamiento de aguas residuales de plantas industriales, en la modificación de fibras y decoloración en industrias textiles y de colorantes, en el mejoramiento de alimentos para animales, en la detoxificación de polutantes y en bioremediación de suelos contaminados. También se la ha utilizado en Q.Orgánica para la oxidación de grupos funcionales, en la formación de enlaces carbono- nitrógeno y en la síntesis de productos naturales complejos. HIPOTESIS: Los hongos de podredumbre blanca, y en condiciones óptimas de cultivo producen distintos tipos de enzimas oxidasas, siendo las lacasas las más adecuadas para explorarlas como catalizadores en los siguientes procesos:  Delignificación de residuos de la industria forestal con el fin de aprovechar tales desechos en la alimentación animal.  Decontaminación/remediación de suelos y/o efluentes industriales. Se realizarán los estudios para el diseño de bio-reactores que permitan responder a las dos cuestiones planteadas en la hipótesis. Para el proceso de delignificación de material lignocelulósico se proponen dos estrategias: 1- tratar el material con el micelio del hongo adecuando la provisión de nutrientes para un desarrollo sostenido y favorecer la liberación de la enzima. 2- Utilizar la enzima lacasa parcialmente purificada acoplada a un sistema mediador para oxidar los compuestos polifenólicos. Para el proceso de decontaminación/remediación de suelos y/o efluentes industriales se trabajará también en dos frentes: 3) por un lado, se ha descripto que existe una correlación positiva entre la actividad de algunas enzimas presentes en el suelo y la fertilidad. En este sentido se conoce que un sistema enzimático, tentativamente identificado como una lacasa de origen microbiano es responsable de la transformación de compuestos orgánicos en el suelo. La enzima protege al suelo de la acumulación de compuestos orgánicos peligrosos catalizando reacciones que involucran degradación, polimerización e incorporación a complejos del ácido húmico. Se utilizarán suelos incorporados con distintos polutantes(por ej. policlorofenoles ó cloroanilinas.) 4) Se trabajará con efluentes industriales contaminantes (alpechínes y/o el efluente líquido del proceso de desamargado de las aceitunas). The lignocellulosic raw materials of the agroindustrial activities can be taken advantage as source of lignin, hemicellulose and cellulose. The chemical treatment of this material is not easy because the above mentioned material is recalcitrant enough to such an assault, due to the presence of the lignin. This can be achieved also using the white-rot fungi of the wood. It produces extracellular ligninolitic enzymes, fundamentally Laccase, which oxidizes the lignin to CO2. The enzyme has application in such processes as in the delignification of lignocellulosic materials and in the biobleaching of fibers for paper industry, in the treatment of waste water of industrial plants, in the discoloration in textile industries, in the improvement of food for ruminants, in the detoxification of polutants and in bioremediation of contaminated soils. HYPOTHESIS: The white-rot fungi produce different types of enzymes, being the laccases the most adapted to explore them as catalysts in the following processes:  Delignification of residues of the forest industry in order to take advantage of such waste in the animal feed.  Decontamination of soils and / or waste waters. The studies will be conducted for the design of bio reactors that allow to answer to both questions raised in the hypothesis. For the delignification process of lignocellulosic material they propose two strategies: 1- to treat the material with the fungi 2-to use the partially purified enzyme to oxidize the polyphenolic compounds. For the soil and/or waste water decontamination process, we have: 3- Is know that the enzyme protects to the soil of the accumulation of organic dangerous compounds catalyzing reactions that involve degradation, polymerization and incorporation to complexes of the humic acid. There will be use soils incorporated into different pollutants. 4- We will work with waste waters (alpechins or the green olive debittering effluents.

Procesos oxidativos en matrices alimentarias complejas. Oxidative processes in complex food matrices.

Las reacciones bioquímicas que ocurren como consecuencia del tratamiento y almacenamiento de los alimentos, mejoran la seguridad alimentaria, las propiedades sensoriales y la vida útil. Sin embargo, el tratamiento térmico, la exposición a la luz y el oxígeno pueden causar daño oxidativo a los lípidos y proteínas. Los procesos oxidativos de matrices complejas tienen características distintivas que no se manifiestan cuando los componentes son sometidos a oxidación individualmente. La hipótesis de trabajo es que la oxidación de proteínas en matrices alimentarias complejas altera la estructura y las propiedades funcionales de las proteínas y, que las modificaciones que se producen varían según las condiciones de procesamiento y de la composición química del alimento. Nuestros estudios intentan demostrar que el estado oxidativo de las proteínas de un alimento es un parámetro importante para la evaluación de las propiedades funcionales, sensoriales y nutricionales de un producto lácteo. El objetivo general del proyecto es el estudio de los procesos de oxidación de matrices alimentarias complejas (la leche, miel) y su relación con distintos procesos y materiales utilizados en la industria. Es decir, nos proponemos estudiar las consecuencias funcionales y biológicas (calidad nutricional, coagulación) de la oxidación proteica en modelos experimentales “in vitro” y en productos comerciales. 1. Estudiar los fenómenos de peroxidación proteica en leche entera y descremada sometida a los distintos procesos tecnológicos de la producción de leche y queso a escala laboratorio. Se realizarán las mismas experiencias con albúmina sérica y con proteínas aisladas de suero de leche para comparar diferencias entre una matriz compleja y una simple. 2. Determinar la relación entre oxidación y composición proteica de la leche, y los cambios en las fracciones proteicas aisladas (caseínas y beta-lactoglobulina). 3. Analizar el impacto de los procesos tecnológicos a nivel de producción primaria (composición proteica y estado de oxidación) en los indicadores de inflamación (contenido de células somáticas y proteína C Reactiva) y de estado redox (capacidad antioxidante de los productos lácteos y nivel de carbonilos de proteinas). 4. Comparar las características de composición química y el estado de oxidación de leche provenientes de las tres regiones (Buenos Aires, Santa Fe y Córdoba) que conforman la cuenca láctea Argentina. Este objetivo se realizará conjuntamente con los integrantes de nuestro grupo de investigación que trabajan en el Laboratorio de Control de Calidad de la Escuela Superior de Lechería. 5. Determinar los metabolitos secundarios de mieles uniflorales propuestos como responsables de la capacidad antioxidante de estas (polifenoles) y como indicadores de su origen botánico. 6. Valorar la capacidad antioxidante total de mieles uniflorales. 7. Validar los métodos analíticos y semicuantitativos utilizados y a utilizar en el presente proyecto teniendo en cuenta lo efectos de matrices típico de los fluidos biológicos y las mezclas. El estudio de las modificaciones oxidativas de matrices complejas es un tema que es importante tanto desde el punto de vista del conocimiento básico como del aplicado. Nosotros creemos que el presente proyecto aportará conocimiento sobre las características de las vías oxidativas de proteínas en matrices complejas y que podrá ser utilizado para diseñar estrategias productivas tendientes a disminuir el deterioro de la calidad de la leche debido a la exposición a energía radiante. Parte de la experiencia ganada por el grupo ha sido ya volcada a subsanar dificultades y problemas de oxidación y deterioro de la calidad de alimentos. Además, se contribuirá a discernir la paradoja que existe en el área sobre las propiedades oxidantes/antioxidantes de los polifenoles y la relación entre estas y el estado oxidativo de un alimento. The biochemical reactions that occur as a result of food treatment and storage, improve food security, sensory properties and shelf life. Heat treatment, exposure to light and oxygen can cause oxidative damage to lipids and proteins. Oxidative processes in complex matrices display distinctive features that do not appear when the components are individually subjected to oxidation. The hypothesis is that protein oxidation in complex food matrices alters the structure and functional properties of proteins and that the modifications vary according to process conditions and food composition. The main goal is to study oxidation of complex food matrices (milk, honey) with different processes and materials used in the industry. The specific aims are: 1. To study protein oxidation in whole milk and skim subject to various technological processes. The same experiences will be done with serum albumin and isolated whey proteins to compare complex and simple matrices. 2. To determine the relationship between oxidation and milk protein composition, and changes in casein and beta-lactoglobulin. 3. Analyze the impact of technological processes at the level of primary production on markers of inflammation and redox (antioxidant capacity and protein carbonyls). 4. Compare characteristics of chemical composition and oxidation state of milk. 5. Determine secondary metabolites of honey responsible for the antioxidant capacity of these. 6. To evaluate the total antioxidant capacity unifloral honey. This project will provide knowledge about characteristics of oxidative pathways of proteins in complex matrices that can be used to design production strategies aimed at reduce the deterioration of milk quality. Also, it would help to discern the paradox that exists on the oxidants/antioxidants properties of polyphenols and the relationship between these and the oxidative status of a food.

Defining habitat characteristics influencing the distribution, density and growth of Plaice (Pleuronectes Platessa) and Dab (Limanda Limanda) on west of Ireland nursery grounds

Plaice (Pleuronectes platessa, L.) and dab (Limanda limanda, L.) are among the most abundant flatfishes in the north-eastern Atlantic region and the dominant species in shallow coastal nursery grounds. With increasing pressures on commercial flatfish stocks in combination with changing coastal environments, better knowledge of population dynamics during all life stages is needed to evaluate variability in year-class strength and recruitment to the fishery. The aim of this research was to investigate the complex interplay of biotic and abiotic habitat components influencing the distribution, density and growth of plaice and dab during the vulnerable juvenile life stage and to gain insight in spatial and temporal differences in nursery habitat quality along the west coast of Ireland. Intraspecific variability in plaice diet was observed at different spatial scales and showed a link with condition, recent growth and morphology. This highlights the effect of food availability on habitat quality and the need to consider small scale variation when attempting to link habitat quality to feeding, growth and condition of juvenile flatfish. There was evidence of trophic, spatial and temporal resource partitioning between juvenile plaice and dab allowing the co-existence of morphologically similar species in nursery grounds. In the limited survey years there was no evidence that the carrying capacity of the studied nursery grounds was reached but spatial and interannual variations in fish growth indicated fluctuating environments in terms of food availability, predator densities, sediment features and physico-chemical conditions. Predation was the most important factor affecting habitat quality for juvenile plaice and dab with crab densities negatively correlated to fish condition whereas shrimp densities were negatively associated with densities of small-sized juveniles in spring. A comparison of proxies for fish growth showed the advantage of Fulton’s K for routine use whereas RNA:DNA ratios proved less powerful when short-term environmental fluctuations are lacking. This study illustrated how distinct sets of habitat features can drive spatial variation in density and condition of juvenile flatfish highlighting the value of studying both variables when modeling habitat requirements. The habitat models generated in this study also provide a powerful tool to predict potential climate and anthropogenic impacts on the distribution and condition of juveniles in flatfish nurseries. The need for effective coastal zone management was emphasized to ensure a sustainable use of coastal resources and successful flatfish recruitment to the fishery.

Estrutura floral das angiospermas usadas por Heliconius erato phyllis (Lepidoptera, Nymphalidae) no Rio Grande do Sul, Brasil

A field survey of flowering plants used as food resource by the adults of Heliconius erato phyllis (Fabricius, 1775) was carried out in four sites located in Rio Grande do Sul state, Brazil. Samples were taken in preserved areas of the Atlantic Rain and Myrtaceae forests, an Eucalyptus plantation, and an urban park. Adult feeding frequencies on flowers were registered monthly from December 1996 to May 1997, on plants located on previously marked 200 m long transects. Flowers on which H. erato phyllis fed in the field were collected, drawn and morphometrically characterized. Feeding was registered on flowers of twenty-three species, of which seventeen are new records for H. erato in Brazil . The use of a given plant varied among localities, as a function of its corresponding abundance. The most visited flowers were those of Lantana camara L. and Stachytarpheta cayennensis (Rich.) Vahl, followed by Dahliapinnata Voss in the urban site. The data suggest the existence of size and shape convergence between the proboscis and the small, tubular flowers upon which H. eratophyllis feeds. They also indicate that H. eratophyllis adults have an opportunistic nectar feeding / pollen gathering habit, using several of those flowers available in a given time and locality that fit such a morphometrical pattern. Since plant species of both primitive and derived families are used, there is no indication that phylogenetic constraints play a major role in this association, nor that color of flowers, growth pattern or size of the plants are relevant in determining their use by H. erato phyllis.

The aggregation of Chauliognathus species (Coleoptera, Cantharidae) and its possible role for coexistence and mimicry

The relative population sizes of a species complex of Chauliognathus are reported, as well as their spatial distribution associated with different patches of food plants. Field work was done at Fazenda Santa Isabel, municipality of Guaíba, State of Rio Grande do Sul, Brazil. The results suggest that two mechanisms account for the reduction in food competition among the species involved: one is asynchrony in the appearance of the species in the area, and the other is aggregation in different patches of food plants. Since the species here reported show a similar colour pattern (yellow-black) the possibility of the occurrence of serial mimicry in this complex of species is dicussed.

Feeding habits of six anuran (Amphibia: Anura) species in a rainforest fragment in Northeastern Brazil

Although Brazil encompasses one of the most abundant anuran faunas in the world, quantitative information on anuran ecology and diet are limited, especially in the Northeastern region. We analyzed the diet of six species: Hyla albomarginata, Hyla cf. branneri, Hyla minuta, Phyllomedusa aff. hypochondrialis (Hylidae), Leptodactylus natalensis, and Physalaemus cuvieri (Leptodactylidae) in a temporary pond in a rainforest remnant in Pernambuco, between 1999-2000. We analyzed diet composition, degree of food preference, and seasonal variations in diet. Leptodactylus natalensis and P. cuvieri showed higher diet diversity, whereas H. minuta consumed fewer food items. Insecta, Arachnida, and plants were preferential items for most species. Acari were consumed by all species; Hymenoptera, Odonata, and Coleoptera were also often consumed. A slight increase in diet diversity occurred in the rainy season. The species showed a generalist feeding behaviour, although P. cuvieri consumed Formicidae as major prey item.

Population dynamic of Sesarma rectum (Crustacea, Brachyura, Sesarmidae) from a muddy flat under human impact, Paraty, Rio de Janeiro, Brazil

A population of Sesarma rectum Randall, 1840 under the influence of human impact was studied. Monthly sampling (CPUE, two people during 30 min) took place from August/2001 to July/2002 at an impacted muddy flat in Paraty city, State of Rio de Janeiro (23º13'S, 44º42'W). At the laboratory, specimens were classified by sex and measured with a vernier caliper (0.01 mm). The size at the beginning of the sexual maturity was obtained by means of different techniques: in the case of males it was used the allometric procedure and the macroscopic analysis of gonads wile for females, the size of the smallest ovigerous female was also considered. The population structure was evaluated by means the analysis of the variations in the modes of the size frequency distribution. The fecundity was assessed using sub samples of the egg mass. For males, the macroscopic analyses of gonads revealed larger values of carapace width than those obtained with morphometric analysis. Males larger than 18.5 mm of carapace width can be considered as mature. For females, such size was 17.4 mm CW. Despite of the human impact in the habitat, the population presented to be stable, as indicated by a single mode on the size frequency distribution. The second mode that appeared in some months is probably related to the entrance of juveniles in the population. The sex ratio of this population is closely approximating to 1:1 until crabs reach a carapace width of about 28 mm; after that, males outnumbered females. Comparing the fecundity of the present population with a previous study from Ubatuba, it can be verified a difference in the number of eggs. The fecundity of Paraty's population is significantly lower than the Ubatuba's population. This is probably related to the scarcity of food resource in Paraty, once no vascular plant can be found in that place. The continuity of reproductive processes and the juvenile recruitment suggest this species is able to live in the area with human impact. The ability to obtaining nutrients from different source of food is probably a feature that allows S. rectum to occupy such impacted ecosystem.

Diet of the ichthyofauna associated with marginal vegetation of a mangrove forest in southeastern Brazil

The objective of this study was to analyze the diet of fish species that use the mangrove vegetation for shelter and feeding in a river southeastern Brazil. The fieldwork, including collecting and underwater observations, was carried out in the dry (July and August 2004) and in the rainy season (February and March 2005) in order to assess the existence of seasonal variation in the diets. Seven kinds of food items were consumed, two of plant origin and five of animal origin. Crustaceans predominated in the diet of most species, either in the form of unidentified fragments or discriminated in eight groups. The predominance of species using mainly a single food source (crustaceans, principally Ostracoda and Tanaidacea) and the existence of seasonal variation in the diets of some species became very evident in the analysis food niche breadth, with a predominance of dietary specialists. In the Rio da Fazenda mangrove, the submersed marginal vegetation was used by the ichthyofauna as a locale for foraging, and principally as cover by bottom-feeding species. These species may be using the vegetation for protection from aerial and aquatic predators, or even from the pull of the current during the turn of the tide. In the study area, the great diversity of crustaceans constitutes an important food source for most fish species which adjusted their diet according to seasonal changes in food availability and to interactions with other species.

Differences on allocation of available resources, in growth, reproduction, and survival, in an exotic gastropod of Physidae compared to an endemic one

Physa acuta Draparnaud, 1805 is an invasive gastropod that can affect local species. In Argentina, it is widespread and abundant, even in environments inhabited by the native species Stenophysa marmorata Guilding, 1828. Its predominance raises the question whether this could be explained by a more successful energy allocation in functional requirements (growth, reproduction and survival) compared to S. marmorata. This study was aimed at comparing growth rates, as well as survival and fecundity, between both species under laboratory conditions. Individuals born on the same day were grouped in four per aquaria and kept under controlled conditions of food, light, and temperature. Snails were weekly measured (maximum shell length), and growth rates were calculated using the Von Bertalanffy's equation. The number of eggs and survivors were grouped by week. Stenophysa marmorata was larger at birth than Physa acuta and invested more energy in growth, delaying sexual maturity. This resulted in a disadvantage in fecundity and survival compared to P. acuta, which had a lower growth rate but matured earlier and survived longer. Furthermore, the growth of P. acuta was not affected by reproduction, its reproductive period was longer, consequently with more eggs laid than S. marmorata.

Comparative dietary analysis of Eurycheilichthys pantherinus and Pareiorhaphis hystrix: two Loricariidae species (Ostariophysi, Siluriformes) from Campos Sulinos biome, southern Brazil

This work was carried out in Marco river, São José dos Ausentes municipality, state of Rio Grande do Sul, southern Brazil. This region is located within the Campos Sulinos biome, an area of great biological importance due to its high diversity and endemism of fish. The feeding habits and food overlap between Eurycheilichthys pantherinus (Reis & Schaefer, 1992) (n=108) and Pareiorhaphis hystrix Pereira & Reis, 2002 (n=60) are described. Monthly samples were obtained between September 2000 and July 2001 with dipnets using the kick sampling technique. Stomach contents were analyzed based on frequency of occurrence, volumetric frequency and index of alimentary importance. The level of dietary specialization and food overlap were determined by the Levins measure and the Morisita index, respectively. Eurycheilichthys pantherinus fed mainly on immature aquatic insects, such as Dipterans and Ephemeropterans, and was classified as insectivore. Pareiorhaphis hystrix fed mainly on detritus associated with small amounts of aquatic insects, and was classified as detritivore. Species are clearly segregated by the use of food resources in Marco River. Their diets did not overlap and the differences observed in their feeding habits probably contribute for their coexistence.

Impact of fish cage-culture on the community structure of zooplankton in a tropical reservoir

We investigated the impact of fish cage culture on the zooplankton community structure in a tropical reservoir. We hypothesized that community abundance is greater near cages and increases over time due to the increase in food availability. Samplings were performed near, upstream and downstream from net cages, and before and after net cage installation. The abundance of zooplankton increased 15 days after the experiment was set up, followed by a reduction and finally increased. Rotifer abundance showed significant differences among sites (p<0.05) and sampling periods (p<0.001). Significant differences were also observed in total zooplankton and cladoceran abundance (p<0.001). The spatial and temporal variation of the physical and chemical variables were indirectly correlated with the structure and dynamic of the zooplankton community, as they indicated the primary production in the environment. Our hypothesis was rejected, since the zooplankton was abundant at the reference site. Only rotifers showed higher abundance near cages, due to the influence of food availability. Community dynamics during the experiment was also correlated to food availability. Our results suggest an impact of fish farming on the zooplankton community.

Buccal apparatus and gastrointestinal tract dimensions associated to the diet of early life stages of Centropomus undecimalis (Centropomidae, Actinopterygii)

The present study aims to compare the buccal apparatus and gastrointestinal tract of early life stages of Centropomus undecimalis (Bloch, 1792), and relate them to its diet. A total of 190 individuals collected with a channel net in the Catuama estuary (07º40'9.9''S, 34º50'36.7''W), northern coast of the state of Pernambuco, were examined. Morphometrical and meristic data were analyzed for the two initial developmental periods (larval and juvenile). Their digestive tube was morphologically characterized and its content identified. The longest transverse axis of food items was measured, and compared to the standard length (SL) and mouth gape size (D) of the individuals. Body measurement regressions differed significantly (p<0.001) between larvae and juveniles. The stomachs with food content (n=118 individuals) presented a proportion of 62% full and 30% empty (being 8% damaged). They differed in relation to the fullness level and presented a coiled shape when empty. The number of food items in relation to SL and D did not present an evident correlation. Larvae (SL<10 mm) feed on small copepods, while juveniles (SL=11.1 to 64.7 mm) ingest larvae of various decapod species, showing a distinct diet between these initial developmental stages.

Trophic ecology and foraging behavior of Tropidurus hispidus and Tropidurus semitaeniatus (Squamata, Tropiduridae) in a caatinga area of northeastern Brazil

This study aimed to analyze the seasonal variation in diet composition and foraging behavior of Tropidurus hispidus (Spix, 1825) and T. semitaeniatus (Spix, 1825), as well as measurement of the foraging intensity (number of moves, time spent stationary, distance traveled and number of attacks on prey items) in a caatinga patch on the state of Rio Grande do Norte, Brazil. Hymenoptera/Formicidae and Isoptera predominated in the diet of both species during the dry season. Opportunistic predation on lepidopteran larvae, coleopteran larvae and adults, and orthopteran nymphs and adults occurred in the wet season; however, hymenopterans/Formicidae were the most important prey items. The number of food items was similar between lizard species in both seasons; however the overlap for number of prey was smaller in the wet season. Preys ingested by T. hispidus during the wet season were also larger than those consumed by T. semitaeniatus. Seasonal comparisons of foraging intensity between the two species differed, mainly in the wet season, when T. hispidus exhibited less movement and fewer attacks on prey, and more time spent stationary if compared to T. semitaeniatus. Although both lizards are sit-and-wait foragers, T. semitaeniatus is more active than T. hispidus. The diet and foraging behavior of T. hispidus and T. semitaeniatus overlap under limiting conditions during the dry season, and are segregative factors that may contribute to the coexistence of these species in the wet season.

Influence of seasonal changes in daily activity and annual life cycle of Geotrigona mombuca (Hymenoptera, Apidae) in a Cerrado habitat, São Paulo, Brazil

The foraging activity of Geotrigona mombuca Smith, 1863 was studied under natural conditions aiming to verify the influence of seasonal changes on daily flight activity and annual cycle of the colony. Daily flight activity was monitored for a year based on the observation and counting of foragers leaving and entering the hive, as well as the kind of material transported and meteorological factors such as day time, temperature and relative humidity. The influence of seasonal changes was evidenced by alterations on daily rhythm of flight activity and by differences on transportation of food resources, building material and garbage. These data indicate that forager behavior is related to daily microclimate conditions and it is synchronized with the requirements of colony annual cycle, which determines an intense pollen collection in the summer. Thus, the recomposition of the intranidal population in spring and summer can be ensured, which is characterized both for a higher intensity of flight activity and increase in garbage and resin transport, as well as the swarming process in the spring. In this way, an action targeting the preservation or management of the species in a natural environment should consider that survival and reproduction of the colony depends greatly on the amount of available pollen in late winter.