Persistent organic chemicals of emerging environmental concern

The use of chemicals and chemical derivatives in agriculture and industry has contributed to their accumulation and persistence in the environment. Persistent organic pollutants (POPs) are among the environmental pollutants of most concern since, when improperly handled and disposed, they can persist in the environment, bioaccumulate through the food web, and may create serious public health and environmental problems. Development of an effective degradation process has become an area of intense research. The physical/chemical methods employed, such as volatilization, evaporation, photooxidation, adsorption, or hydrolysis, are not always effective, are very expensive, and, sometimes, lead to generation/disposal of other contaminants. Biodegradation is one of the major mechanisms by which organic contaminants are transformed, immobilized, or mineralized in the environment. A clear understanding of the major processes that affect the interactions between organic contaminants, microorganisms, and environmental matrix is, thus, important for determining persistence of the compounds, for predicting in situ transformation rates, and for developing site remediation. Information on their risks and impact and occurrence in the different environmental matrices is also important, in order to attenuate their impact and apply the appropriate remediation process. This chapter provides information on the fate of pesticides and polycyclic aromatic hydrocarbons (PAHs), their impact, bioavailability, and biodegradation. © Springer Science+Business Media Dordrecht 2014.

Chemical functionalization in bionanoparticles

Dissertação de mestrado em Biofísica e Bionanossistemas

Biotechnological production and application of fructooligosaccharides

Currently, prebiotics are all carbohydrates of relatively short chain length. An important group is the fructooligosaccharides, which are a special kind of prebiotics associated to their selective stimulation of the activity of certain groups of colonic bacteria that have a positive and beneficial effect on intestinal microbiota, reducing incidence of gastrointestinal infections, respiratory and also possessing a recognized bifidogenic effect. Traditionally, these prebiotic compounds have been obtained through extraction processes from some plants, as well as through enzymatic hydrolysis of sucrose. However, different fermentative methods have also been proposed for the production of fructooligosaccharides, such as solid-state fermentation utilizing various agroindustrial by-products. By optimizing the culture parameters, fructooligosaccharides yields and productivity can be improved. The use of immobilized enzymes and cells has also been proposed as being an effective and economic method for large-scale production of fructooligosaccharides. This paper is an overview on the results of recent studies on fructooligosacharides biosynthesis, physicochemical properties, sources, biotechnological production and applications.

Malolactic fermentation in winemaking: evaluation of operational parameters

Dissertação de mestrado em Bioengenharia

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.

Behavior of triatomines (Hemiptera: Reduviidae) vectors of Chagas' disease: I. Courtship and copulation of Panstrongylus megistus (Burm-1835) in the laboratory

A study of the courship and copulation behaviour of Panstrongylus megistus was carried out in the laboratory. fifty-five newly-fed virgin couples were used. Experiments were performed during the day (9:00 to 12:00 a.m.) and at night (7:00 to 10:00 p.m). Behaviour was recorded by direct observation and was found to consist of the following sequence of behavioral patterns: the male approached the female and jumped on her or mounted her; he took on a dorsolateral position and immobilized the female dorsally and ventrally with his three pairs of legs; the male genital was placed below those of the female; the paramers of the male immobilized the female's genitals; copulation started. The couple joined by the iniciative of the male. The female could be receptive and accept copulation, or nonreceptive and reject the male. Copulation occurred more often on the occasion of the first attempt by the male. Duration of copulation was X = 29.3 ± 9.3 min (CV = 83%). No behavioral differences were observed couples tested during the day or at night.

Decoloración de tintes por lacasa

Informe de investigación realizado a partir de una estancia en el Instituto de Biotecnología Medioambiental de la Universidad Tecnológica de Graz, Austria, entre Julio y Agosto de 2006. Se ha estudiado la decoloración de varios tintes sintéticos de estructuras químicas diferentes (Rojo Congo, Azul de Naftol, Indigo Carmín, Lanaset Gris, Azul de Nilo) por la enzima lacasa inmovilizada. La inmovilización de la enzima lacasa se llevó a cabo sobre esferas de alúmina (Al2O3) de 3 mm de diámetro debido a la resistecia mecánica de este material. La lacasa y la proteína inmovilizada se determinaron como la diferencia entre las concentraciones iniciales y residuales (obtenidas en los lavados). El porcentaje de lacasa inmovilizada fue del 68% y la cantidad de proteína inmovilizada por gramo de soporte de 5,6 mg. La enzima lacasa inmovilizada fue capaz de decolorar tintes de diferente estructura sin la necesidad de añadir mediadores redox, lo cual la hace una enzima muy adecuada para su aplicación en la decoloración de efluentes procedentes de la industria textil. De todas formas, son necesarios más estudios para optimizar la técnica de inmovilización así como el proceso de decoloración. Es interesante destacar que aunque hay muchos estudios basados en la oxidación de tintes textiles por lacasa la mayoría utilizan la enzima libre y hay muy poca información disponible sobre la aplicación de lacasas inmovilizadas, por lo que el presente estudio constituye una aportación muy interesante y novedosa.

Use of a human-like low-grade bacteremia model of experimental endocarditis to study the role of Staphylococcus aureus adhesins and platelet aggregation in early endocarditis.

Animal models of infective endocarditis (IE) induced by high-grade bacteremia revealed the pathogenic roles of Staphylococcus aureus surface adhesins and platelet aggregation in the infection process. In humans, however, S. aureus IE possibly occurs through repeated bouts of low-grade bacteremia from a colonized site or intravenous device. Here we used a rat model of IE induced by continuous low-grade bacteremia to explore further the contributions of S. aureus virulence factors to the initiation of IE. Rats with aortic vegetations were inoculated by continuous intravenous infusion (0.0017 ml/min over 10 h) with 10(6) CFU of Lactococcus lactis pIL253 or a recombinant L. lactis strain expressing an individual S. aureus surface protein (ClfA, FnbpA, BCD, or SdrE) conferring a particular adhesive or platelet aggregation property. Vegetation infection was assessed 24 h later. Plasma was collected at 0, 2, and 6 h postinoculation to quantify the expression of tumor necrosis factor (TNF), interleukin 1α (IL-1α), IL-1β, IL-6, and IL-10. The percentage of vegetation infection relative to that with strain pIL253 (11%) increased when binding to fibrinogen was conferred on L. lactis (ClfA strain) (52%; P = 0.007) and increased further with adhesion to fibronectin (FnbpA strain) (75%; P < 0.001). Expression of fibronectin binding alone was not sufficient to induce IE (BCD strain) (10% of infection). Platelet aggregation increased the risk of vegetation infection (SdrE strain) (30%). Conferring adhesion to fibrinogen and fibronectin favored IL-1β and IL-6 production. Our results, with a model of IE induced by low-grade bacteremia, resembling human disease, extend the essential role of fibrinogen binding in the initiation of S. aureus IE. Triggering of platelet aggregation or an inflammatory response may contribute to or promote the development of IE.

Absorbance enhancement in microplate wells for improved-sensitivity biosensors

A generic optical biosensing strategy was developed that relies on the absorbance enhancement phenomenon occurring in a multiple scattering matrix. Experimentally, inserts made of glass fiber membrane were placed into microplate wells in order to significantly lengthen the trajectory of the incident light through the sample and therefore increase the corresponding absorbance. Enhancement factor was calculated by comparing the absorbance values measured for a given amount of dye with and without the absorbance-enhancing inserts in the wells. Moreover, the dilution of dye in solutions with different refractive indices (RI) clearly revealed that the enhancement factor increased with the ΔRI between the membrane and the surrounding medium, reaching a maximum value (EF>25) when the membranes were dried. On this basis, two H2O2-biosensing systems were developed based on the biofunctionalization of the glass fiber inserts either with cytochrome c or horseradish peroxidase (HRP) and the analytical performances were systematically compared with the corresponding bioassay in solution. The efficiency of the absorbance-enhancement approach was particularly clear in the case of the cytochrome c-based biosensor with a sensitivity gain of 40 folds and wider dynamic range. Therefore, the developed strategy represents a promising way to convert standard colorimetric bioassays into optical biosensors with improved sensitivity.

Bacterial community structure of a pesticide-contaminated site and assessment of changes induced in community structure during bioremediation.

The introduction of culture-independent molecular screening techniques, especially based on 16S rRNA gene sequences, has allowed microbiologists to examine a facet of microbial diversity not necessarily reflected by the results of culturing studies. The bacterial community structure was studied for a pesticide-contaminated site that was subsequently remediated using an efficient degradative strain Arthrobacter protophormiae RKJ100. The efficiency of the bioremediation process was assessed by monitoring the depletion of the pollutant, and the effect of addition of an exogenous strain on the existing soil community structure was determined using molecular techniques. The 16S rRNA gene pool amplified from the soil metagenome was cloned and restriction fragment length polymorphism studies revealed 46 different phylotypes on the basis of similar banding patterns. Sequencing of representative clones of each phylotype showed that the community structure of the pesticide-contaminated soil was mainly constituted by Proteobacteria and Actinomycetes. Terminal restriction fragment length polymorphism analysis showed only nonsignificant changes in community structure during the process of bioremediation. Immobilized cells of strain RKJ100 enhanced pollutant degradation but seemed to have no detectable effects on the existing bacterial community structure.

The use of ferromagnetic dacron as solid-phase in enzyme immunoassays

Ferromagnetic dacron is proposed as an alternative solid-phase for magnetic enzyme immunoassays. Human serum albumin (HSA) was covalentlyimmobilized onto ferromagnetic dacron and as enzyme immunoassay was developed using anti-HSA rabbit sera. Peroxidase, o-phenylenediamine (OPD) and hydrogen peroxide were used anti-HSA rabbit sera. Peroxidase, o-phenylenediamine (OPD) and hydrogen peroxide were used as the enzymatic label and substrates, respectively. Best results were observed when particles of 63-100 µm (diameter) and 10 µg of immobilized antigen were used. Positive reactions were detected until dilutions of1:51200 of immune sera. Its reproducibility was similar to standard ELISA. Disruption of the immunocomplexes formed and recuperation of the immobilized antigen in other immunoassays also proved to be reliable.

Avaluació del creixement del fong ligninolític Trametes versicolor sobre diferents suports sòlids per a l'aplicació en tractaments de bioremediació

Els fongs de podridura blanca (WRF - de l'anglès: White-rot fungi) són de gran interès en l'àmbit de la bioremediació per la seva capacitat de degradar la lignina. La lacasa, un dels enzims extracel·lulars que aquests fongs excreten per degradar la lignina, pot ser utilitzada per degradar els contamiants presents en una matriu donada. Trametes versicolor ha estat estudiat per la seva capacitat de produïr aquest enzim en rersidus agrícoles com a substrats. Un primer triatge basat en la producció de CO2, la mesura de l'activitat lacasa i la quantificació de l'ergosterol han permés seleccionar els substrats on es donava un major poder oxidatiu dels cultius inoculats amb T. versicolor. Els posteriors experiments de colonització de sòls, on es monitoritzava l'activitat lacasa i l'ergosterol, han mostrat que T. vesicolor és capaç de colonitzar sòls i que té una major activitat lacasa en condicions no estèrils. També h'ha provat, mitjançant el test ND24, que T. versicolor és capaç de degradar un contaminant emergent, el naproxè, en sòls estèrils i no estèrils esmenats amb residus agrícoles.

Production of an affinity-purified antibody against an aldehyde-treated neurofilament protein for use in immunocytochemistry.

Fixation enhances cellular morphology and reduces loss of molecules during tissue processing. Antibodies against fixation-resistant epitopes are very useful, because they allow an immunocytochemical detection in tissue of better preserved morphology. However, fixatives can alter antigenicity and adversely affect the result of immunohistochemical procedures. To address this problem, this study examined the feasibility of generating antibodies to a paraformaldehyde-fixed antigen for use in immunohistochemical procedures. The large subunit of neurofilament proteins was selected for this study. Crude neurofilament proteins were isolated and separated by SDS-polyacrylamide gel electrophoresis. The large subunit of neurofilaments (NF-H) was electroeluted from the electrophoresis gel and exposed to paraformaldehyde, and used for immunization of a rabbit. The rabbit antiserum was affinity purified on CNBr-sepharose immobilized neurofilament proteins. On Western blots, the antibody reacted with the NF-H protein in a phosphorylation-dependent manner. In aldehyde-fixed cerebellum, the antibody strongly stained axons. In contrast, in alcohol-fixed cryostat sections the immunocytochemical detection was substantially reduced. The procedure presented in this study, involving a simple pretreatment of the immunogen, allows for the generation of an antibody that may be used in immunohistochemical studies where localization of the immunogen may be reduced or even lost by aldehyde fixation.

Characterization of the Aspergillus nidulans biotin biosynthetic gene cluster and use of the bioDA gene as a new transformation marker.

The genes involved in the biosynthesis of biotin were identified in the hyphal fungus Aspergillus nidulans through homology searches and complementation of Escherichia coli biotin-auxotrophic mutants. Whereas the 7,8-diaminopelargonic acid synthase and dethiobiotin synthetase are encoded by distinct genes in bacteria and the yeast Saccharomyces cerevisiae, both activities are performed in A. nidulans by a single enzyme, encoded by the bifunctional gene bioDA. Such a bifunctional bioDA gene is a genetic feature common to numerous members of the ascomycete filamentous fungi and basidiomycetes, as well as in plants and oömycota. However, unlike in other eukaryota, the three bio genes contributing to the four enzymatic steps from pimeloyl-CoA to biotin are organized in a gene cluster in pezizomycotina. The A. nidulans auxotrophic mutants biA1, biA2 and biA3 were all found to have mutations in the 7,8-diaminopelargonic acid synthase domain of the bioDA gene. Although biotin auxotrophy is an inconvenient marker in classical genetic manipulations due to cross-feeding of biotin, transformation of the biA1 mutant with the bioDA gene from either A. nidulans or Aspergillus fumigatus led to the recovery of well-defined biotin-prototrophic colonies. The usefulness of bioDA gene as a novel and robust transformation marker was demonstrated in co-transformation experiments with a green fluorescent protein reporter, and in the efficient deletion of the laccase (yA) gene via homologous recombination in a mutant lacking non-homologous end-joining activity.

Interaction of Diocleinae Lectins with Glycoproteins Based in Surface Plasmon Resonance

Interaction of glucose/mannose-binding lectins in solution with immobilized glycoproteins was followed in real time using surface plasmon resonance technology. The lectins which share many biochemical and structural features could be clearly differentiated in terms of their specificity for complex glycoconjugates. The most prominent interaction of the lectins with PHA-E comparing with soybean agglutinin, both glycoproteins exhibiting high mannose oligosaccharides, suggests that the whole structure of the glycoproteins themselves, may interfere in affinity. These findings also support the hypothesis that minor amino acid replacements in the primary sequence of the lectins might be responsible for their divergence in fine specificity and biological activities. This is the first report using surface plasmon resonance technology that evidences differences of Diocleinae lectins in respect their fine glycan-specificity.