Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico / Identificação por técnicas moleculares de bactérias halofílicas que produzem enzimas importantes em área intocada de Campeche, México

Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Isla Arena está localizada na coordenada 20°70’N - 90°45’W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.

Optimization of Bacillus subtilis cell growth effecting jiean-peptide production in fed batch fermentation using central composite design

Background Optimization of nutrient feeding was developed to improve the growth of Bacillus subtilis in fed batch fermentation to increase the production of jiean-peptide (JAA). A central composite design (CCD) was used to obtain a model describing the relationship between glucose, total nitrogen, and the maximum cell dry weight in the culture broth with fed batch fermentation in a 5 L fermentor. Results The results were analyzed using response surface methodology (RSM), and the optimized values of glucose and total nitrogen concentration were 30.70 g/L and 1.68 g/L in the culture, respectively. The highest cell dry weight was improved to 77.50 g/L in fed batch fermentation, which is 280% higher than the batch fermentation concentration (20.37 g/L). This led to a 44% increase of JAA production in fed batch fermentation as compared to the production of batch fermentation. Conclusion The results of this work improve the present production of JAA and may be adopted for other objective products' production.

Enhanced production of mosquitocidal cyclic lipopeptide from Bacillus subtilis subsp. subtilis.

Background & objectives: A cyclic lipopeptide, surfactin produced by a strain of Bacillus subtilis subsp. subtilis (VCRC B471) was found to exhibit activity against both the larval and pupal stages of mosquitoes. The present study was aimed at increasing the production of the mosquitocidal metabolite by modifying the conventional medium. Methods: Enhancement of mosquitocidal metabolite production was attempted by replacing the existing micronutrients of the conventional NYSM and supplementing the medium with additional amounts of glucose. The LC50 value of culture supernatant (CS) against the larval and pupal stages of Anopheles stephensi was determined. Crude mosquitocidal metabolite (CMM) was separated from the CS, identified by MALDI-TOF analysis and its LC50 dosage requirement for the pupal stage of the above mosquito species determined. Results: The medium containing a new composition of micronutrients and glucose up to 1 per cent resulted in increased metabolite production. The LC50 value of the CS obtained in the improved medium against larvae and pupae of An. stephensi was 5.57 and 0.71 μl/ml, respectively. The yield of CMM was doubled in the improved medium. MALDI-TOF analysis revealed that the CMM was surfactin. Interpretation & conclusions: The new improved medium enhanced the production of mosquitocidal metabolite as the dosage required for inciting 50 per cent mortality among the pupal stages of mosquitoes was only half of that required when the metabolite was produced in the conventional medium. The mosquitocidal metabolite was identified as surfactin, a cyclic lipopeptide and biosurfactant.

Production of peptide antifungal antibiotic and biocontrol activity of Bacillus subtilis.

Among different bacterial cultures, a potent Bacillus subtilis MTCC-8114 was isolated from garden soil samples which showed 16 and 14 mm inhibition zones by spot inoculation method and 24 and 22 mm inhibition zones by well agar diffusion method against test fungi i.e. Microsporum fulvum and Trichophyton species. Among four media tested, the maximum growth and antibiotic production was found in trypticase soya broth (TSB) medium at 37 degrees C, pH-7 and 48 h of incubation. The Rf value (0.64) by Thin Layer Chromatography (TLC) technique and UV and FTIR spectral data of the active antifungal compound, indicated that the isolated compound belongs to peptide antifungal antibiotic group. MIC value of antifungal antibiotic was 135 and 145 microg/ml.

Comparison of plant growth-promotion with Pseudomonas aeruginosa and Bacillus subtilis in three vegetables / Comparação da promoção de crescimento de plantas por Pseudomonas aeruginosa e Bacillus subtilis em três vegetais

Our objective was to compare some plant growth promoting rhizobacteria (PGPR) properties of Bacillus subtilis and Pseudomonas aeruginosa as representatives of their two genera. Solanum lycopersicum L. (tomato), Abelmoschus esculentus (okra), and Amaranthus sp. (African spinach) were inoculated with the bacterial cultures. At 60 days after planting, dry biomass for plants treated with B. subtilis and P. aeruginosa increased 31 percent for tomato, 36 percent and 29 percent for okra, and 83 percent and 40 percent for African spinach respectively over the non-bacterized control. Considering all the parameters tested, there were similarities but no significant difference at P < 0.05 between the overall performances of the two organisms.

Nosso objetivo foi comparar as propriedades PGPR (rizobactérias promotoras de crescimento de plantas) de Bacillus subtilis e Pseudomonas aeruginosa. Solanum licopersicum (tomate), Asbelmoschus esculentus (ocra) e Amaranthus sp (espinafre africano) foram inoculados com as culturas bacterianas. Após 60 dias de plantio, a biomassa seca das plantas tratadas com B.subtilis e P. aeruginosa aumentou 31 por cento para o tomate, 36 por cento e 29 por cento para ocra, e 83 por cento e 40 por cento para espinafre africano, respectivamente, em comparação com o controle não inoculado. Considerando os parâmetros testados, o desempenho dos dois microrganismos foi similar, sem diferença estatisticamente significativa (p< 0,05).

Greenhouse evaluation of Bacillus subtilis AP-01 and Trichoderma harzianum AP-001 in controlling tobacco diseases / Avaliação em estufa de dois agentes biológicos de controle de doenças do tabaco

Two biological control agents, Bacillus subtilis AP-01 (LarminarTM) and Trichoderma harzianum AP-001 (TrisanTM) alone or/in combination were investigated in controlling three tobacco diseases, including bacterial wilt (Ralstonia solanacearum), damping-off (Pythium aphanidermatum), and frogeye leaf spot (Cercospora nicotiana). Tests were performed in greenhouse by soil sterilization prior to inoculation of the pathogens. Bacterial-wilt and damping off pathogens were drenched first and followed with the biological control agents and for comparison purposes, two chemical fungicides. But for frogeye leaf spot, which is an airborne fungus, a spraying procedure for every treatment including a chemical fungicide was applied instead of drenching. Results showed that neither B. subtilis AP-01 nor T. harzianum AP-001 alone could control the bacterial wilt, but when combined, their controlling capabilities were as effective as a chemical treatment. These results were also similar for damping-off disease when used in combination. In addition, the combined B. subtilis AP-01 and T. harzianum AP-001 resulted in a good frogeye leaf spot control, which was not significantly different from the chemical treatment.

Dois agentes de controle biológico, Bacillus subtilis AP-01 (Larminar®) e Trichoderma harzianum AP-001 (Trisan®) foram avaliados separadamente ou em combinação quanto à capacidade de controlar três doenças do tabaco: murcha bacteriana (bacterial wilt, Ralstonia solanacearum), tombamento de mudas (damping-off, Pythium aphanidermatum), e mancha olho-de-rã (frogeye leaf spot, Cercospora nicotiana). Os testes foram realizados em estufa, esterilizando-se o solo antes da inoculação dos patógenos. Os patógenos causadores da murcha bacteriana e tombamento de mudas foram inicialmente encharcados e acompanhados com os agentes de controle biológico e, para comparação, com um fungicida químico. Para a mancha olho-de-rã, causada por um fungo anemófilo, utilizou-se um processo de spray ao invés do encharcamento. Os resultados indicaram que nenhum dos dois agentes de controle biológico, aplicado isoladamente, foi capaz de controlar a murcha bacteriana, mas quando em combinação a capacidade de controle foi similar ao do tratamento químico. Resultados semelhantes foram obtidos para o tombamento de mudas. Além disso, a combinação de Bacillus subtilis AP-01 e Trichoderma harzianum AP-001 resultou em um controle muito eficiente da mancha olho-de-rã, que não diferiu significativamente daquele obtido com o tratamento químico.

Effect of some herbicides used in Nigeria on Rhizobium phaseoli, Azotobacter vinelandii and Bacillus subtilis.

The effect of three herbicides namely Agroxone, Atranex 50SC and 2,4-Damine on Azotobacter vinelandii, Rhizobium phaseoli and Bacillus subtilis were studied. These bacteria were isolated from a bean-garden in Lagos. The results revealed that 2, 4-Damine was the most toxic of the three herbicides studied and Azotobacter vinelandii was found to be most sensitive to the herbicides. There was a reduction in LC50 of herbicides with increased number of days. The percentage survival decreased with increased concentration of herbicides and days for Rhizobium phaseoli and Azotobacter vinelandii while an initial reduction in population was followed by increased percentage survival of organisms for Bacillus subtilis.

Effect of pH and temperature on Bacillus subtilis ATCC 601 alfa-amylase production. Some properties of the crude enzyme

Neste trabalho foram estudadas a influência do pH inicial do meio de cultura e da temperatura de crescimento sobre a produçäo de alfa-amilase por Bacillus subtilis ATCC 601, bem como algumas propriedades da enzima bruta. Houve maior produçäo de enzima quando a fermentaçäo foi realizada a 37oC. A atividade enzimática específica global, Emax/Xmax' mostrou que a maior concentraçäo enzimática no caldo de cultura fermentado a 37oC näo foi devida a um maior crescimento celular e sim a uma maior síntese de enzima por unidade de massa celular. O estudo do efeito do pH inicial do meio de cultura sobre a produçäo da enzima mostrou que ela ocorreu preferencialmente a pH 7,0, também devido a um estímulo da sítese por unidade de massa celular. A temperatura ótima da enzima está ao redor de 50oC e o seu pH ótimo está entre 5,4 e 6,4. A alfa-amilase de Bacillus subtilis ATCC 601 mostrou-se pouco estável a 60oC foi completamente inativada quando mantida durante 10 minutos a 80oC. Ions Ca +2, nas concentraçöes de 10mM e 20mM, estabilizaram sensivelmente a enzima, enquanto que a adiçäo de EDTA 10mM a desestabilizou de tal modo que a enzima apresentou a 50oC um comportamento semelhante ao observado a 80oC. Näo houve formaçäo de glicose quando o amido foi hidrolisado por esta alfa-amilase. Os açúcares formados variaram de maltose à maltoheptaose

Evaluación de promotores del crecimiento para pollos de engorda / Evaluation of growth promotores in broiler chicks

Se evaluó el efecto promotor del crecimiento de probióticos y antibióticos en pollos de engorda de 1 a 49 días de edad. Se emplearon dietas sorgo + soya e iniciación y finalización sin suplementación testigo, o con la adición de 100 ppm de Bacillus subtilis (10 esporas viables por gramo), Bacitracina zinc 100 ppm y 1000 ppm de un producto a base de cultivos vivos de Lactobacillus acidophilus, Streptococcus faecium, Sacharomyces cerevisiae y enzimas (amilasas, proteasas y celulasas). Los datos obtenidos de ganancia de peso (1904, 1933 y 1948g) fueron similares (P> 0.05) entre tratamientos. Sin embargo, numéricamente fue mayor el aumento de peso con el antibiótico respecto a los probióticos. Para conversión alimenticia (1.96, 1.94, 1.90 y 1.93) existió respuesta más favorable a la adicción del antibiótico y la mezcla de lactobacilos levaduras y enzimas. Los datos obtenidos en este estudio sugieren un efecto benéfico mayor en pollos de engorda del antibiótico respecto a los probióticos usados en el estudio

Production of alpha amylase by bacillus subtilis 1

Of 24 bacterial isolates, B. subtilis 1 was the most potent for the production of alpha-amylase. The highest yield of alpha-amylase was achieved by incubation of B. subtilis 1 for 40 hours at 45C. Addition of wheat bran, maltose, glucose or lactose to the culture medium had a stimulating effect on the production of alpha-amylase. The effect of various carbon and nitrogen sources on the production of the enzyme was also studied. The use of dipotassium hydrogen phosphate at 0.1-0.15% in the culture medium also favored enzyme production. Zn 2+ or Cu 2+ ions had adverse effect, but Ca 2+ ions and Tween-80 favored enzyme production. An initial pH of the cultural medium of 5.0 effected highest enzyme production [26.0 U/ml]

Purification of alpha-amylase from bacillus subtilis line No 1024 with ATCC 465

Alpha-amylase E.C.3.2.1.1. [1,4 glucan, 4 glucanohydrolase] can be obtained from salivary glands, pancreas and microorganisms such as Pseudomonas and Asperigllus, as well as muscles and ovarian tubes. Alpha-amylase from Bacillus subtilis #1024[ATCC 465] was purified with a highest degree of purity in our laboratory [31.59 U/mg]. The extracellular alpha-amylase was subjected to different purification techniques such as anionic and cationic exchange chromatography and preparative electrophoresis. The final fold of purification was equivalent to 38, which was higher than the previous reported values, polyacrylamide gel electrophoresis with sodium dodecyl sulphate [PAGESDS], Laemmeli method, gave a molecular weight of 82000 Daltons

Atividade antibacteriana de extratos de Bidens pilosa L / Antibacterial activity of Bidens pilosa L. extracts

Foram preparados extratos de plantas de B. pilosa provenientes de diferentes regiöes as quais mostraram atividade contra S. aureus. Extratos preparados a 80ºC näo apresentaram atividade. Foi testada a atividade antibacteriana do extrato bruto de B. pilosa contra S. aureus, S. lutea e B. subtilis cultivados sob cinco diferentes valores de pH e, para isto, mediu-se o crescimento através da turvaçäo e da atividade catalásica. A S. lutea apresentou a maior sensibilidade. Dos diferentes solventes orgânicos utilizados no fracionamento do extrato bruto, o clorofórmico foi o melhor. Na avaliaçäo da presença de princípio ativo em diferentes fases do desenvolvimento de B. pilosa, a fase cotiledonar apresentou menor concentraçäo do princípio ativo e a fase de flor a maior concentraçäo