Produção de enzimas quitosanolíticas utilizando Paenibacillus ehimensis e Paenibacillus chitinolyticus para obtenção de quitoologossacarídeos

The acquisition of oligosaccharides from chitosan has been the subject of several studies in the pharmaceutical, biochemical, food and medical due to functional properties of these compounds. This study aimed to boost its production of chitooligosaccharides (COS) through the optimization of production and characterization of chitosanolytic enzymes secreted by microorganisms Paenibacillus chitinolyticus and Paenibacillus ehimensis, and evaluating the antioxidant potential of the products obtained. In the process of optimizing the production of chitosanase were employed strategies Fractional Factorial Experimental Design and Central Composite Rotatable Design. The results identified the chitosan, peptone and yeast extract as the components that influenced the production of chitosanase by these microorganisms. With the optimization of the culture media was possible to obtain an increase of approximately 8.1 times (from 0.043 to 0.35 U.mL U.mL-1) and 7.6 times (from 0.08 U.mL-1 to 0.61 U.mL-1) in the enzymatic activity of chitosanase produced by P. chitinolyticus and P. ehimensis respectively. Enzyme complexes showed high stability in temperature ranges between 30º and 55º C and pH between 5.0 and 9.0. Has seen the share of organic solvents, divalent ions and other chemical agents on the activity of these enzymes, demonstrating high stability of these crude complexes and dependence of Mn2+. The COS generated showed the ability of DPPH radical scavenging activity, reaching a maximum rate of scavenging of 61% and 39% when they were produced with enzymes of P. ehimensis and P. chitinolyticus respectively. The use of these enzymes in raw form might facilitate its use for industrial applications

Microbial Aspects of Environmentally Benign Iron Ore

Many types of micro-organisms inhabit iron ore deposits contributing to biogenic formation and conversion of iron oxides and associated minerals. Bacteria such as Paenibacillus polymyxa arc capable of significantly altering the surface chemical behaviour of iron ore minerals such as haematite, alumina, calcite and silica. Differing mineral surface affinities of bacterial cells and metabolic products such as proteins and polysaccharides can be utilised to induce their flotation or flocculation. Mineral-specific bioreagents such as proteins are generated when bacteria are grown in the presence of haematite, alumina, calcite and silica. Alumina-grown bacterial cells and proteins separated from such cells were found to be capable of separating alumina from haematite. Biodegradation of iron ore flotation collectors such as amines and oleates can be effectively utilised to achieve environmental control in iron ore processing mills.

Microbially-induced separation of chalcopyrite and galena

Cells of Paenibacillus polymyxa and their metabolic products such as bioproteins and exopolysaccharides could be effectively used in the separation of galena from chalcopyrite. While interaction with bacterial cells resulted in significant flocculation of both chalcopyrite and galena, treatment with bioproteins selectively flocculated only chalcopyrite, dispersing galena. Microbially-induced selective flocculation after conditioning with cells, bioproteins or exopolysaccharides resulted in efficient separation of chalcopyrite and galena from their mixtures. Prior interaction with bioproteins facilitated enhanced flotation of galena from chalcopyrite. The role of bacterial cells and bioreagents such as proteins and polysaccharides in mineral beneficiation is demonstrated.

Developments in Biotechnology for Environmentally Benign Iron Ore Beneficiation

Biomineralization and biogenesis of iron ore deposits are illustrated in relation to indigenous microorganisms inhabiting iron ore mines. Aerobic and anaerobic microorganisms indigenous to iron oxide mineralization are analyzed. Microbially-induced flotation and flocculation of iron ore minerals such as hematite, alumina, calcite and quartz are discussed with respect to use of four types of microorganisms, namely, Paenibacillus polymyxa, Bacillus subtilis, Saccharomyces cerevisiae and Desulfovibrio desulfuricans. The role of the above organisms in the removal of silica, alumina, clays and apatite from hematite is illustrated with respect to mineral-specific bioreagents, surface chemical changes and microbe-mineral interaction mechanisms. Silica and alumina removal from real iron ores through biobeneficiation is outlined. Environmental benefits of biobeneficiation are demonstrated with respect to biodegradation of toxic reagents and environmentally-safe waste disposal and processing.

Prediction and validation of gold nanoparticles (GNPs) on plant growth promoting rhizobacteria (PGPR): a step toward development of nano-biofertilizers

Several soil microbes are present in the rhizosphere zone, especially plant growth promoting rhizobacteria (PGPR), which are best known for their plant growth promoting activities. The present study reflects the effect of gold nanoparticles (GNPs) at various concentrations on the growth of PGPR. GNPs were synthesized chemically, by reduction of HAuCl 4, and further characterized by UV-Vis spectroscopy, X-ray diffraction technique (XRD), and transmission electron microscopy (TEM), etc. The impact of GNPs on PGPR was investigated by Clinical Laboratory Standards Institute (CLSI) recommended Broth-Microdilution technique against four selected PGPR viz., Pseudomonas fluorescens, Bacillus subtilis, Paenibacillus elgii, and Pseudomonas putida. Neither accelerating nor reducing impact was observed in P. putida due to GNPs. On the contrary, significant increase was observed in the case of P. fluorescens, P. elgii, and B. subtilis, and hence, GNPs can be exploited as nano-biofertilizers.

Biomineralization and Biobeneficiation of Bauxite

Role of indigenous microbes in the formation and conversion of bauxite minerals is illustrated. Many types of microorganisms such as fungi, heterotrophic and autotrophic bacteria and yeasts inhabit bauxite ore deposits bringing about biogenesis and biomineraliztion. Organisms capable of iron oxidation and reduction and solubilising calcium carbonate and silica can be isolated from bauxite deposits and are used to bring about selective mineral beneficiation to remove iron, calcium and silica. Use of Paenibacillus polymyxa in the efficient removal of calcium from low grade bauxites is demonstrated through bioreactor technology. Similarly, for iron removal from bauxite, iron-reducing bacteria can be used. Silicate bacteria aid in selective silica solubilisation to control alumina: silica ratios. Microorganisms can also be used to bring about environmental control with respect to red mud disposal through bioremediation technology.

Exploiting the bioactive potential of marine sponge microbiota

Endospore-forming bacteria are often isolated from different marine sponges, but their abundance varies, and they are frequently missed by culture-independent studies. Within endospore-formers, Bacillus are renowned for the production of antimicrobials and other compounds of medical and industrial importance. Although this group has been well studied in many different environments, very little is known about the actual diversity and properties of sporeformers associated with marine sponges. Identification of the endospore-forming bacteria associated with the marine sponges; Haliclona simulans, Amphilectus fucorum and Cliona celata, has uncovered an abundant and diverse microbial population composed of Bacillus, Paenibacillus, Solibacillus, Halobacillus and Viridibacillus species. This diversity appears to be overlooked by other non-targeted approaches where spore-formers are masked by more dominant species within the ecosystem. In addition to the identification of two antibiotic resistant plasmids, this bank of sporeformers produce a range of bioactive compounds. New antimicrobial compounds are urgently needed to combat the spread of multidrug resistant pathogens, as few new options are entering the drug discovery pipelines for clinical trials. Based on the results of this project, endospore-formers associated with marine sponges may hold the answer. The power of coupling functional based assays with genomic approaches has enabled us to identify a novel class 1 lantibiotic, subtilomycin, which is active against several clinically relevant pathogens. Subtilomycin is encoded in the genomes of all the marine sponge B. subtilis isolates analysed. They cluster together phylogenetically and form a distinct group from other sequenced B. subtilis strains. Regardless of its potential clinical relevance, subtilomycin may be providing these strains with a specific competitive advantage(s) within the stringent confines of the marine sponge environment. This work has outlined the industrial and biotechnological potential of marine sponge endospore-formers which appear to produce a cocktail of bioactive compounds. Genome sequencing of specific marine sponge isolates highlighted the importance of mining extreme environments and habitats for new lead compounds with potential therapeutic applications.

Molecular characterization of the nitrifying bacterial consortia employed for the activation of bioreactors used in brackish and marine aquaculture systems

The addition of commercial nitrifying bacterial products has resulted in significant improvement of nitrification efficiency in recirculating aquaculture systems (RAS). We developed two nitrifying bacterial consortia (NBC) from marine and brackish water as start up cultures for immobilizing commercialized nitrifying bioreactors for RAS. In the present study, the community compositions of the NBC were analyzed by universal 16S rRNA gene and bacterial amoA gene sequencing and fluorescence in situ hybridization (FISH). This study demonstrated that both the consortia involved autotrophic nitrifiers, denitrifiers as well as heterotrophs. Abundant taxa of the brackish water heterotrophic bacterial isolates were Paenibacillus and Beijerinckia spp. whereas in the marine consortia they were Flavobacterium, Cytophaga and Gramella species. The bacterial amoA clones were clustered together with high similarity to Nitrosomonas sp. and uncultured beta Proteobacteria. FISH analysis detected ammonia oxidizers belonging to b subclass of proteobacteria and Nitrosospira sp. in both the consortia, and Nitrosococcus mobilis lineage only in the brackish water consortium and the halophilic Nitrosomonas sp. only in the marine consortium. However, nitrite oxidizers, Nitrobacter sp. and phylum Nitrospira were detected in both the consortia. The metabolites from nitrifiers might have been used by heterotrophs as carbon and energy sources making the consortia a stable biofilm.

Wheat seed embryo excision enables the creation of axenic seedlings and Koch’s postulates testing of putative bacterial endophytes

Early establishment of endophytes can play a role in pathogen suppression and improve seedling development. One route for establishment of endophytes in seedlings is transmission of bacteria from the parent plant to the seedling via the seed. In wheat seeds, it is not clear whether this transmission route exists, and the identities and location of bacteria within wheat seeds are unknown. We identified bacteria in the wheat (Triticum aestivum) cv. Hereward seed environment using embryo excision to determine the location of the bacterial load. Axenic wheat seedlings obtained with this method were subsequently used to screen a putative endophyte bacterial isolate library for endophytic competency. This absence of bacteria recovered from seeds indicated low bacterial abundance and/or the presence of inhibitors. Diversity of readily culturable bacteria in seeds was low with 8 genera identified, dominated by Erwinia and Paenibacillus. We propose that anatomical restrictions in wheat limit embryo associated vertical transmission, and that bacterial load is carried in the seed coat, crease tissue and endosperm. This finding facilitates the creation of axenic wheat plants to test competency of putative endophytes and also provides a platform for endophyte competition, plant growth, and gene expression studies without an indigenous bacterial background.

Interação de microorganismos na solubilização de fósforo e potássio de rochas para produção de biofertilizantes

he present model of agriculture is based on intensive use of industrial inputs, due to its rapid response, but it brings harmful consequences to the environment, and it is necessary the use of modern inputs. And an alternative is the use of rock biofertilizers in agriculture, a product easy to use, with higher residual effect and does not harm the environment. The objective of study was to evaluate the inoculation and co-inoculation of different microorganisms in the solubilization of rock phosphate and potash ground microbial evaluating the best performance in the production of biofertilizers comparing with rocks pure in soil chemical properties and, verify effect of inoculation of the bacterium Paenibacillus polymyxa in the absorption of minerals dissolved in the development of cowpea (Vigna unguiculata [L.] Walp.). The first bioassay was conducted in Laboratory (UFRN) for 72 days in Petri dishes, where the rock powder was increased by 10% and sulfur co-inoculated and inoculated with bacterial suspension of Paenibacillus polymyxa grown in medium tryptone soy broth, Ralstonia solanacearum in medium Kelman, Cromobacterium violaceum in medium Luria-Bertani and Acidithiobacillus thiooxidans in medium Tuovinen and Kelly,and fungi Trichoderma humatum and Penicillium fellutanum in malt extract. Every 12 days, samples were removed in order to build up the release curve of minerals. The second bioassay was conducted in a greenhouse of the Agricultural Research Corporation of Rio Grande do Norte in experimental delineation in randomized block designs, was used 10 kg of an Yellow Argissolo Dystrophic per pot with the addition of treatments super phosphate simple (SS), potassium chloride (KCl), pure rock, biofertilizers in doses 40, 70, 100 and 200% of the recommendation for SS and KCl, and a control, or not inoculated with bacteria P. polymyxa. Were used seeds of cowpea BRS Potiguar and co-inoculated with the bacterial suspension of Bradyrhizobium japonicum and P. polymyxa. The first crop was harvested 45 days after planting, were evaluated in the dry matter (ADM), macronutrients (N, P, K, Ca, Mg) and micronutrients (Zn, Fe, Mn) in ADM. And the second at 75 days assessing levels of macro end micronutrients in plants and soil, and the maximum adsorption capacity of P in soil. The results showed synergism in co-inoculations with P. polymyxa+R. solanacearum and, P. polymyxa+C. violaceum solubilizations providing higher P and K, respectively, and better solubilization time at 36 days. The pH was lower in biofertilizers higher doses, but there was better with their addition to P at the highest dose. Significant reduction of maximum adsorption capacity of phosphorus with increasing dose of biofertilizer. For K and Ca was better with SS+KCl, and Mg to pure rock. There was an effect of fertilization on the absorption, with better results for P, K and ADM with SS+KCL, and N, Ca and Mg for biofertilizers. Generally, the P. polymyxa not influence the absorption of the elements in the plant. In treatments with the uninoculated P. polymyxa chemical fertilizer had an average significantly higher for weight and number of grains. And in the presence of the bacteria, biofertilizers and chemical fertilizers had positive values in relation to rock and control. The data show that the rocks and biofertilizers could meet the need of nutrients the plants revealed as potential for sustainable agriculture

Recuperação e purificação de quitosanases usando adsorção em leito expandido com streamline DEAE com modelagem e simulação usando redes neurais

Expanded Bed Adsorption (EBA) is an integrative process that combines concepts of chromatography and fluidization of solids. The many parameters involved and their synergistic effects complicate the optimization of the process. Fortunately, some mathematical tools have been developed in order to guide the investigation of the EBA system. In this work the application of experimental design, phenomenological modeling and artificial neural networks (ANN) in understanding chitosanases adsorption on ion exchange resin Streamline® DEAE have been investigated. The strain Paenibacillus ehimensis NRRL B-23118 was used for chitosanase production. EBA experiments were carried out using a column of 2.6 cm inner diameter with 30.0 cm in height that was coupled to a peristaltic pump. At the bottom of the column there was a distributor of glass beads having a height of 3.0 cm. Assays for residence time distribution (RTD) revelead a high degree of mixing, however, the Richardson-Zaki coefficients showed that the column was on the threshold of stability. Isotherm models fitted the adsorption equilibrium data in the presence of lyotropic salts. The results of experiment design indicated that the ionic strength and superficial velocity are important to the recovery and purity of chitosanases. The molecular mass of the two chitosanases were approximately 23 kDa and 52 kDa as estimated by SDS-PAGE. The phenomenological modeling was aimed to describe the operations in batch and column chromatography. The simulations were performed in Microsoft Visual Studio. The kinetic rate constant model set to kinetic curves efficiently under conditions of initial enzyme activity 0.232, 0.142 e 0.079 UA/mL. The simulated breakthrough curves showed some differences with experimental data, especially regarding the slope. Sensitivity tests of the model on the surface velocity, axial dispersion and initial concentration showed agreement with the literature. The neural network was constructed in MATLAB and Neural Network Toolbox. The cross-validation was used to improve the ability of generalization. The parameters of ANN were improved to obtain the settings 6-6 (enzyme activity) and 9-6 (total protein), as well as tansig transfer function and Levenberg-Marquardt training algorithm. The neural Carlos Eduardo de Araújo Padilha dezembro/2013 9 networks simulations, including all the steps of cycle, showed good agreement with experimental data, with a correlation coefficient of approximately 0.974. The effects of input variables on profiles of the stages of loading, washing and elution were consistent with the literature

Estudo do mecanismo de produção de oligossacarídeos com atividades nutracêuticas a partir da quitosana por hidrólise enzimática com processo fermentativo simultâneo

The obtaining of the oligosaccharides from chitosanase, has showed interest of the pharmaceutical area in the last years due their countless functional properties. Although, the great challenge founded out is how to keep a constant and efficient production. The alternative proposed by this present work was to study the viability to develop an integrated technology, with reduced costs. The strategy used was the obtaining of the oligomers through enzymatic hydrolysis using chitosanolitic enzymes obtained straight from the fermented broth, eliminating this way the phases involved in the enzymes purification. The two chitosanases producing strains chosen for the work, Paenibacillus chitinolyticus and Paenibacillus ehimensis, were evaluated according to the behavior in the culture medium with simple sugar and in relation to the pH medium variations. The culture medium for the chitosanases induction and production was developed through addition of soluble chitosan as carbon source. The soluble chitosan was obtained using hydrochloric acid solution 0.1 M and afterwards neutralization with NaOH 10 M. The enzymatic complexes were obtained from induction process in culture medium with 0.2% of soluble chitosan. The enzymes production was verified soon after the consumption of the simple sugars by the microorganisms and the maximum chitosanolitic activity obtained in the fermented broth by Paenibacillus chitinolyticus was 249 U.L-1 and by Paenibacillus ehimensis was 495U.L-1. These two enzymatic complexes showed stability when stored at 20°C for about 91 days. The enzymes in the fermented broth by Paenibacillus chitinolyticus, when exposed at temperature of 55°C and pH 6.0, where the activity is maximum, showed 50% lost of activity after 3 hours Meanwhile, for the complex produced by Paenibacillus ehimensis, after 6 days of exposure, it was detected 100% of the activity. The chito-oligosaccharides obtained by the hydrolysis of a 1% chitosan solution, using the enzymatic complex produced by Paenibacillus chitinolyticus showed larger quantity after 9 hours hydrolysis and using the complex produced by Paenibacillus ehimensis after 20 minutes was observed the chito-ligosacharides with polymerization degree between 3 and 6 units. Evaluating these results, it was verified that the production of chitosan-oligosaccharides is possible, using a simultaneous process

Efeito de bactérias endofíticas do cafeeiro no controle da ferrugem

Pós-graduação em Agronomia (Proteção de Plantas) - FCA

Bioprospecção de bactérias endofíticas como agentes de biocontrole da mancha de Exserohilum turcicum e como promotoras do crescimento de plantas de milho (Zea mays L.)

Pós-graduação em Agronomia (Proteção de Plantas) - FCA

Produção e obtenção de ciclodextrinas produzidas por CGTases bacterianas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)