Bactérias diazotróficas em mudas de bananeira

Este trabalho objetivou selecionar bactérias diazotróficas isoladas de bananeira (Musa spp.) e avaliar sua influência no crescimento de mudas micropropagadas. Bactérias do tipo Herbaspirillum e relacionadas a Burkholderia cepacia foram inoculadas em plântulas de banana cv. Prata Anã e cv. Caipira. As bananeiras cv. Prata Anã, cultivadas in vitro com substrato pobre em N, apresentaram maior crescimento na presença de bactérias do tipo Herbaspirillum, ao passo que as bananeiras cv. Caipira cresceram melhor com o inóculo contendo bactérias do gênero Burkholderia. Em casa de vegetação, as bananeiras cv. Caipira crescidas em sacolas de plástico contendo areia e vermiculita (1:2), suplementada com a solução de Hoagland contendo 5 mg L-1 de N, apresentaram maior crescimento quando da inoculação simultânea dos dois gêneros de bactérias, em comparação à inoculação individual. A inoculação simultânea proporcionou um crescimento nas bananeiras equivalente ao observado nas plantas-controles adubadas com 50 mg L-1 de N no substrato, porém o teor e o acúmulo de N na parte aérea das bananeiras foram menores. A contribuição de bactérias diazotróficas no crescimento de bananeiras é demonstrada pela primeira vez.

Resposta de plantas micropropagadas de abacaxizeiro à inoculação de bactérias diazotróficas em casa de vegetação

O objetivo deste trabalho foi selecionar e avaliar bactérias diazotróficas isoladas de abacaxizeiro (Ananas comosus (L.) Merril) no desenvolvimento de cultivares micropropagadas da mesma espécie em casa de vegetação. Plantas da cultivar Perolera foram submetidas à inoculação com Asaia bogorensis (AB219) e cultivadas em tubetes, durante 145 dias, com as misturas: casca de arroz carbonizada, folha de carnaubeira triturada e vermicomposto; casca de arroz carbonizada, pó da casca do coco maduro e vermicomposto; casca de arroz carbonizada, vermiculita e vermicomposto. Plantas da cultivar Primavera receberam inóculos com o AB219 e bactérias relacionadas a Burkholderia cepacia (AB202 e AB213), enquanto plantas das cultivares Pérola e Smooth Cayenne receberam AB219 e AB213, sendo cultivadas, por 140 dias, em tubetes com a mistura de vermicomposto e vermiculita. A colonização dos abacaxizeiros pelas bactérias diazotróficas foi confirmada. As plantas da cultivar Perolera cresceram melhor em casca de arroz carbonizada, vermiculita e vermicomposto e responderam positivamente ao AB219. Já as plantas da cultivar Primavera não apresentaram resposta significativa à inoculação com AB219, AB202 e AB213. Houve incremento de 23,1% a 38,5% na matéria seca de raízes das plantas da cultivar Pérola na presença de AB213 e AB219, respectivamente. A presença de AB213 incrementou em 15,2% a matéria seca da parte aérea das plantas da cultivar Smooth Cayenne. Os resultados revelam a eficiência de bactérias diazotróficas na promoção do crescimento de abacaxizeiros.

Efeito de bactérias diazotróficas na produção de abacaxizeiro 'Cayenne Champac', sob irrigação, em dois níveis de adubação nitrogenada

Avaliou-se o efeito de bactérias diazotróficas na produção de abacaxizeiro cv. Cayenne Champac (Champaka), sob irrigação, em dois níveis de adubação nitrogenada. Os tratamentos constaram dos níveis de N de 180 kg ha-1 ano-1 e 300 kg ha-1 ano-1, nas parcelas e subparcelas de mudas micropropagadas inoculadas com bactéria relacionada a Burkholderia cepacia AB213, de bactéria Asaia bogorensis AB219 e controles sem inoculação bacteriana, utilizando-se de três repetições. A inoculação bacteriana foi realizada no laboratório (10(8) células planta-1) e a aclimatação das mudas, em casa de vegetação. Após 5 meses, as mudas foram transplantadas no campo, utilizando-se de 0,9 m entre as linhas e 0,3 m entre as plantas. A cultivar apresentou bom desempenho sob irrigação no solo arenoso de Pacajus, Estado do Ceará, quando adubado com o maior nível de N. Para cada quilograma do elemento fornecido, houve incremento de 124,3 kg ha-1 na massa fresca dos frutos com coroa, entre os níveis da adubação nitrogenada. Na dose maior de N, plantas inoculadas do AB219 produziram frutos maiores e massas frescas 19,4% e 17,3% superiores aos controles, respectivamente, para frutos sem e com coroa. O efeito do AB213 na produção foi menor (9,9%) no nível mais baixo de N. A evidência do efeito de bactérias diazotróficas na cultivar Cayenne Champac foi demonstrada.

Hydrolytic activity of bacterial lipases in amazonian vegetable oils

The data presented describe the development of an enzymatic process in vegetable oils. Six bacterial lipases were tested for their ability to hydrolyze. For each lipase assay, the p-NPP method was applied to obtain maximum enzymatic activities. The lipase from Burkholderia cepacia (lipase B-10) was the most effective in buriti oil, releasing 4840 µmol p-NP mL-1. The lipase from Klebsiella variicola (lipase B-22) was superior in passion fruit oil, releasing 4140 µmol p-NP mL-1 and also in babassu palm oil, releasing 2934 µmol p-NP mL-1. Research into the bioprocessing of oils aims to provide added value for this regional raw material.

Imobilização de lipase por encapsulação em sílica aerogel

Lipase from Burkholderia cepacia was immobilized in a silica matrix and dried in high pressure carbon dioxide media (aerogel). The protic ionic liquid (PIL) was used in the immobilization process by encapsulation. The objective of this work was to evaluate the influence of the drying technique using supercritical carbon dioxide in biocatalysts obtained through the sol-gel technique by evaluating temperature and pressure and, after selecting the best drying conditions, to investigate the application of the technique for the biocatalyst using ionic liquid as an additive in the immobilization process. The results for immobilized biocatalysts showed that the best conditions of pressure and temperature were 100 bar and 25 ºC, respectively, giving a total activity recovery yield of 37.27% without PIL (EN) and 44.23% with PIL (ENLI). The operational stability of the biocatalysts showed a half-life of 11.4 h for ENLI and 6 h for EN. Therefore, solvent extraction using supercritical CO2, besides shortening drying time, offers little resistance to the immobilization of lipases, since their macropores provide ample room for their molecules. The use of the ionic liquid as an additive in the process studied for the immobilization of enzymes produced attractive yields for immobilization and therefore has potential for industrial applications in the hydrolysis of vegetable oils.

Lipase-catalyzed Approaches towards Secondary Alcohols: Intermediates for Enantiopure Drugs

The use of enantiopure intermediates for drug synthesis is a trend in pharmaceutical industry. Different physiological effects are associated with the enantiomers of chiral molecules. Thus, the safety profile of a drug based on an enantiopure active pharmaceutical ingredient is more reliable. Biocatalysis is an important tool to access enantiopure molecules. In biocatalysis, the advantage of selectivity (chemo-, regio- and stereoselectivity) is combined with the benefits of a green synthesis strategy. Chemoenzymatic syntheses of drug molecules, obtained by combining biocatalysis with modern chemical synthesis steps usually consists of fewer reaction steps, reduced waste production and improved overall synthetic efficiency both in yields and enantio- and/or diastereoselectivities compared with classical chemical synthesis. The experimental work together with the literature review clearly indicates that lipase catalysis is highly applicable in the synthesis of enantiopure intermediates of drug molecules as the basis to infer the correct stereochemistry. By lipase catalysis, enantiopure secondary alcohols used as intermediates in the synthesis of Dorzolamide, an antiglaucoma drug, were obtained. Enantiopure _-hydroxy nitriles as potential intermediates for the synthesis of antidepressant drugs with 1-aryl-3- methylaminopropan-1-ol structure were also obtained with lipases. Kinetic resolution of racemates was the main biocatalytic approach applied. Candida Antarctica lipase B, Burkholderia cepacia lipase and Thermomyces lanuginosus lipase were applied for the acylation of alcohols and the alcoholysis of their esters in organic solvents, such as in diisopropyl ether and tert-butyl methyl ether. Candida Antarctica lipase B was used under solvent free conditions for the acylation of ethyl 3-hydroxybutanoate.

EFEITOS DA INOCULAÇÃO COM BACTÉRIAS DIAZOTRÓFICAS E DA ADUBAÇÃO NITROGENADA NO CRESCIMENTO E NA QUALIDADE DE MUDAS DE Inga laurina (SW.) Willd. (Fabaceae)1

Inga laurina é uma espécie arbórea com ampla distribuição na América do Sul, útil para sistemas agroflorestais, restauração florestal e arborização urbana. Este estudo teve como objetivo avaliar os efeitos da inoculação com bactérias fixadoras de nitrogênio (N) e da adubação nitrogenada no crescimento e qualidade de mudas de I. laurina. O experimento teve duração de 170 dias, e as mudas foram cultivadas em tubetes plásticos com 115 cm3 de capacidade, contendo uma mistura 9: 1 em volume de HS Florestal® e pó de fibra de coco como substrato. Foram analisados seis tratamentos, sendo quatro inoculações (Bradyrhizobium japonicum 1 - BJ1, Rhizobium miluonense - RM, Bradyrhizobium japonicum 2- BJ2 e Burkholderia cepacia - BC), o controle positivo - C+ (sem inoculação e com adubação nitrogenada semanal, 60 mg dm3 de N, na forma de ureia) e o controle negativo - C- (sem inoculação e sem adubação nitrogenada). Os tratamentos com inoculação foram pouco efetivos em relação ao crescimento das mudas, visto que as médias das variáveis de crescimento, da massa foliar específica (MFE) e do índice de qualidade de Dixon (IQD) foram significativamente superiores no C+, em comparação com os demais tratamentos. No entanto, os isolados RM e BJ2 foram efetivos na produção de nódulos, pois apresentaram os maiores valores médios da massa de matéria seca de nódulos (MSN). Além disso, os valores médios do índice de clorofila Falker (ICF) foram significativamente superiores nos tratamentos com inoculação em relação ao C-. Os melhores resultados entre os tratamentos com inoculação foram obtidos para RM, seguido de BJ2.

Studies on chemoenzymatic synthesis: Lipase-catalyzed acylation in multistep organic synthesis

In this thesis, biocatalysis is defined as the science of using enzymes as catalysts in organic synthesis. Environmental aspects and the continuously expanding repertoire of available enzymes have firmly established biocatalysis as a prominent means of chemo-, regio- and stereoselective synthesis. Yet, no single methodology can solve all the challenges faced by a synthetic chemist. Therefore, the knowledge and the skills to combine different synthetic methods are relevant. Lipases are highly useful enzymes in organic synthesis. In this thesis, an effort is being made to form a coherent picture of when and how can lipases be incorporated into nonenzymatic synthesis. This is attempted both in the literature review and in the discussion of the results presented in the original publications contained in the thesis. In addition to lipases, oxynitrilases were also used in the work. The experimental part of the thesis comprises of the results reported in four peer-reviewed publications and one manuscript. Selected amines, amino acids and sugar-derived cyanohydrins or their acylated derivatives were each prepared in enantio- or diastereomerically enriched form. Where applicable, attempts were made to combine the enzymatic reactions to other synthetic steps either by the application of completely separate sequential reactions with isolated intermediates (kinetic and functional kinetic resolution of amines), simultaneously occurring reactions without intermediate isolation (dynamic kinetic resolution of amino acid esters) or sequential reactions but without isolating the intermediates (hydrocyanation of sugar aldehydes with subsequent diastereoresolution). In all cases, lipase-catalyzed acylation was the key step by which stereoselectivity was achieved. Lipase from Burkholderia cepacia was a highly selective enzyme with each substrate category, but careful selection of the acyl donor and the solvent was important as well.

DGGE with genomic DNA: Suitable for detection of numerically important organisms but not for identification of the most abundant organisms

Identification of all important community members as well as of the numerically dominant members of a community are key aspects of microbial community analysis of bioreactor samples. A systematic study was conducted with artificial consortia to test whether denaturing gradient gel electrophoresis (DGCE) is a reliable technique to obtain such community data under conditions where results would not be affected by differences in DNA extraction efficiency from cells. A total of 27 consortia were established by mixing DNA extracted from Escherichia coli K12, Burkholderia cepacia and Stenotrophomonas maltophilia in different proportions. Concentrations of DNA of single organisms in the consortia were either 0.04, 0.4 or 4 ng/mu l. DGGE-PCR of genomic DNA with primer sets targeted at the V3 and V6-V8 regions of the 16S rDNA failed to detect the three community members in only 7% of consortia, but provided incorrect information about dominance or co-dominance for 85% and 89% of consortia with the primer sets for the V6-V8 and V3 regions, respectively. The high failure rate in detection of dominant B. cepacia with the primers for the V6-V8 region was attributable to a single nucleoticle primer mismatch in the target sequences of both, the forward and reverse primer. Amplification bias in PCR of E. coli and S. maltophilia for the V6-V8 region and for all three organisms for the V3 region occurred due to interference of genomic DNA in PCR-DGGE, since a nested PCR approach, where PCR-DGGE was started from mixtures of 16S rRNA genes of the organisms, provided correct information about the relative abundance of original DNA in the sample. Multiple bands were not observed in pure culture amplicons produced with the V6-V8 primer pair, but pure culture V3 DGGE profiles of E. coli, S. maltophilia and B. cepacia contained 5, 3 and 3 bands, respectively. These results demonstrate DGGE was suitable for identification of all important community members in the three-membered artificial consortium, but not for identification of the dominant organisms in this small community. Multiple DGGE bands obtained for single organisms with the V3 primer pair could greatly confound interpretation of DGGE profiles. (C) 2008 Elsevier Ltd. All rights reserved.

Avaliação, identificação e atividade enzimática de bactérias psicotróficas presentes no leite cru refrigerado

Extended storage of refrigerated milk can lead to reduced quality of raw and processed milk, which is a consequence of the growth and metabolic activities of psychrotrophic bacteria, able to grow under 7oC or lower temperatures. Although most of these microorganisms are destroyed by heat treatment, some have the potential to produce termoresistant proteolytic and lipolytic enzymes that can survive even UHT processing and reduce the processed products quality. Recently, the IN 51 determineds that milk should be refrigerated and stored at the farm what increased the importance of this group of microorganisms. In this work, psychrotrophic bacteria were isolated from 20 communitarian bulk tanks and 23 individual bulk tanks from dairy farms located at Zona da Mata region of Minas Gerais State and from southeastern Rio de Janeiro. Selected milk dilutions were plated on standard agar and after incubation for 10 days at 7oC, five colonies were isolated, firstly using nutrient agar and after using McConkey agar for 24 hours at 21oC. The isolates were identified by morphology, Gram stain method, catalase production, fermentative/oxidative metabolism and by API 20E, API 20NE, API Staph, API Coryne or API 50 CH (BioMerieux). In order to ensure reproductibility, API was repeated for 50% of the isolates. Species identification was considered when APILAB indexes reached 75% or higher. 309 strains were isolated, 250 Gram negative and 59 Gram positive. 250 Gram negative isolates were identified as: Acinetobacter spp. (39), Aeromonas spp. (07), A. Hydrophila (16), A. sobria (1), A. caviae (1), Alcaligenes feacalis (1), Burkholderia cepacia (12), Chryseomonas luteola (3), Enterobacter sp. (1), Ewingella americana(6), Hafnia alvei (7), Klebsiella sp. (1), Klebsiella oxytoca (10), Yersinia spp. (2), Methylobacterium mesophilicum (1), Moraxella spp. (4), Pantoea spp. (16), Pasteurella sp. (1), Pseudomonas spp. (10), P. fluorescens (94), P. putida (3), Serratia spp. (3), Sphigomonas paucomobilis (1). Five isolates kept unidentified. Pseudomonas was the predominant bacteria found (43%) and P. fluorescens the predominant species (37.6%), in accordance with previous reports. Qualitative analysis of proteolytic and lipolytic activity was based on halo formation using caseinate agar and tributirina agar during 72 hours at 21oC and during 10 days at 4°C, 10oC and 7°C. Among 250 Gram negative bacteria found, 104 were identified as Pseudomonas spp. and 60,57% of this group showed proteolytic and lipolytic acitivities over all four studied temperatures. 20% of Acinetobacter, Aeromonas, Alcaligenes, Burkholderia, Chryseomonas, Methylobacterium, Moraxella presented only lipolytic activity. Some isolates presented enzymatic activity in one or more studied temperatures. Among Gram positive bacteria, 30.51% were proteolytic and lipolytic at 10oC, 8.47% were proteolytic at 7oC, 10oC, and 21oC, 8.47% were proteolytic at all studied temperatures (4oC, 7oC, 10oC and 21oC) and 3.38% were proteolytic only at 21oC. At 4oC, only one isolate showed proteolytic activity and six isolates were lipolytic. In relation to Gram negative microorganisms, 4% were proteolytic and lipolytic at 7oC, 10oC and 21oC, 10% were proteolytic at 10oC and 4.4% were lipolytic at 4oC, 7oC, 10oC and 21oC, while 6.4% of all isolates were proteolytic and lipolytic at 10oC and 21oC as well as lipolytic at 4oC and 7oC. These findings are in accordance with previous researches that pointed out Pseudomonas as the predominant psycrotrophic flora in stored refrigerated raw milk

Pulmonary, microbiological and hematological changes in Crotalus durissus terrificus (Serpentes, Viperidae) parasitized by nematodes of the genus Rhabdias (Nematoda, Rhabdiasidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Produção de biofilme por bastonetes gram negativos isolados de água de hemodiálise

No Brasil, 90% de pacientes com insuficiência renal crônica ou aguda dependem dos procedimentos de hemodiálise para remover produtos de degradação metabólica, excesso de água e de sais minerais do organismo, a fim de restaurar o equilíbrio ácido-base e eletrolítico. Entretanto, o processo de osmose reversa, que visa remover todos os íons da água, também remove o cloro, que exerce efeito bacteriostático sob diversas bactérias autóctones, que passam a se multiplicar na água. Entre os principais grupos dessas bactérias, estão os bastonetes Gram negativos não fermentadores (BGN) e os mais frequentemente associados a bacteremias, em pacientes de hemodiálise são Pseudomonas aeruginosa, Stenotrophomonas maltophilia e Burkholderia cepacia. Essas bactérias podem produzir biofilmes, o que torna sua eliminação do encanamento quase impossível. Assim, o objetivo do trabalho foi a análise de água e do dialisato, da Unidade de Hemodiálise de um Hospital Universitário, na pesquisa dos três BGNs citados. Também foram utilizadas 42 cepas previamente isoladas do mesmo local. Entre as 67 amostras de água, foram isoladas 8 cepas de Pseudomonas aeruginosa e as 50 cepas foram submetidas à pesquisa do gene rpoS, um dos responsáveis pela produção de biofilme. Esse gene foi observado em 20 (40%) cepas, sendo 16 (80%) P. aeruginosa, seguida por 3 (15%) cepas de S. maltophilia e 1 (5%) de B. cepacia. Essas cepas foram submetidas à produção de biofilme a 35ºC (temperatura ótima de crescimento) e a 20ºC (temperatura média da água no encanamento da Unidade de Hemodiálise), em aço inoxidável (material de muitos instrumentos cirúrgicos), PVC (matéria prima da tubulação) e plástico de poliestireno (capacidade de produção de biofilmes em diferentes materiais). Em poliestireno, somente 1 (5%) cepa de P. aeruginosa produziu biofilme a 35ºC e a 20ºC, 2 (10%) cepas foram... (Resumo completo, clicar acesso eletrônico abaixo)

PHYSICO-CHEMICAL, SPECTROSCOPICAL AND THERMAL CHARACTERIZATION OF BIODIESEL OBTAINED BY ENZYMATIC ROUTE AS A TOOL TO SELECT THE MOST EFFICIENT IMMOBILIZED LIPASE

Two microbial lipases from Burkholderia cepacia and Pseudomonas fluorescens were evaluated as catalysts for the enzymatic transesterification of beef tallow with ethanol and the most efficient lipase source was selected by taking into account the properties of the product to be used as fuel. Both lipases were immobilized on an epoxy silica-polyvinyl alcohol composite by covalent immobilization and used to perform the reactions under the following operational conditions: beef tallow-to-ethanol molar ratio of 1:9, 45 degrees C and 400 units of enzymatic activity per gram of fat. Products, characterized using Fourier Transform Infrared spectroscopy (FTIR), viscosimetry, thermogravimetry and H-1 NMR spectroscopy, suggested that the biodiesel sample obtained in the reaction catalyzed by Burkholderia cepacia lipase has the best set of properties for fuel usage.

Microwave-assisted enzymatic synthesis of beef tallow biodiesel

Optimal conditions for the microwave-assisted enzymatic synthesis of biodiesel have been developed by a full 2(2) factorial design leading to a set of seven runs with different combinations of molar ratio and temperature. The main goal was to reduce the reaction time preliminarily established by a process of conventional heating. Reactions yielding biodiesel, in which beef tallow and ethanol used as raw materials were catalyzed by lipase from Burkholderia cepacia immobilized on silica-PVA and microwave irradiations within the range of 8-15 W were performed to reach the reaction temperature. Under optimized conditions (1:6 molar ratio of beef tallow to ethanol molar ratio at 50A degrees C) almost total conversion of the fatty acid presented in the original beef tallow was converted into ethyl esters in a reaction that required 8 h, i.e., a productivity of about 92 mg ethyl esters g(-1) h(-1). This represents an increase of sixfold for the process carried out under conventional heating. In general, the process promises low energy demand and higher biodiesel productivity. The microwave assistance speeds up the enzyme catalyzed reactions, decreases the destructive effects on the enzyme of the operational conditions such as, higher temperature, stability, and specificity to its substrate, and allows the entire reaction medium to be heated uniformly.

Physico-chemical, spectroscopical and thermal characterization of biodiesel obtained by enzymatic route as a tool to select the most efficient immobilized lipase

Two microbial lipases from Burkholderia cepacia and Pseudomonas fluorescens were evaluated as catalysts for the enzymatic transesterification of beef tallow with ethanol and the most efficient lipase source was selected by taking into account the properties of the product to be used as fuel. Both lipases were immobilized on an epoxy silica-polyvinyl alcohol composite by covalent immobilization and used to perform the reactions under the following operational conditions: beef tallow-to-ethanol molar ratio of 1:9, 45ºC and 400 units of enzymatic activity per gram of fat. Products, characterized using Fourier Transform Infrared spectroscopy (FTIR), viscosimetry, thermogravimetry and ¹H NMR spectroscopy, suggested that the biodiesel sample obtained in the reaction catalyzed by Burkholderia cepacia lipase has the best set of properties for fuel usage.