Thermo-resistant enzyme-producing microorganisms isolated from composting / Microrganismos produtores de enzimas termo-resistentes isolados de compostagem

Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

Os fertilizantes organo-minerais suplementados com aditivos biológicos são uma alternativa aos adubos químicos. Neste estudo, microrganismos termoresistentes foram isolados de compostagem por procedimentos de duas etapas. Inicialmente, as amostras tomadas em diferentes períodos e temperaturas (33 dias a 52 ºC, 60 dias a 63 ºC e mais de 365 dias a 26 ºC) foram pré-incubadas a 80 oC por 30 minutos. Posteriormente, a seleção microbiana foi conduzida por métodos baseados em cultura in vitro e choque térmico a 60 oC e 100 oC por 2h e 4h. Quarenta e um isolados foram capazes de crescer a 60 °C por 4h; vinte e sete a 100 °C por 2h e dois a 100 °C por 4h. A identificação molecular por sequenciamento parcial do gene ribossomal 16S usando primers universais revelou que trinta e cinco isolados eram de oito espécies de Bacillus, um Brevibacillus borstelensis, três Streptomyces thermogriseus e dois fungos (Thermomyces lanuginosus e T. dupontii). Os dados dos ensaios de atividade de amilase, fitase e celulase e o índice enzimático (IE) mostraram que 38 dos 41 isolados termorresistentes produziram pelo menos uma enzima. Para a atividade da amilase, o maior valor de IE foi observado em Bacillus licheniformis (isolado 21C2, IE = 4,11), seguido por Brevibacillus borstelensis (isolado 6C2, IE = 3,66), Bacillus cereus (isolado 18C2, IE = 3,52) e Bacillus paralicheniformis (isolado 20C2, IE = 3,34). Para a fitase, os maiores valores de IE foram observados para B. cereus (isolado 18C2, IE = 2,30) e B. licheniformis (isolado 3C1, IE = 2,15). Em relação à produção de celulose, B. altitudinis (isolado 6C1) foi o mais eficiente (IE = 6,40), seguido por três Bacillus subtilis (isolados 9C1, 16C2 e 19C2) com valores de IE de 5,66, 5,84 e 5,88, respectivamente, e um B. pumilus (isolado 27C2, IE = 5,78). Pode-se inferir que os microrganismos selecionados são potencialmente úteis como aditivos biológicos em fertilizantes organo-minerais e outros processos biotecnológicos.

Biological control in the germination of seeds from two species native of the Cerrado region / Controle biológico na germinação de sementes de duas espécies nativas da região do Cerrado

Abstract Microorganisms have been efficiently used for the biological control of phytopathogens through the production of antimicrobial substances. However, the objectives of this work were: to study the germination of Butia purpurascens Glassman and Butia archeri Glassman seeds in different substrates, to select and identify the endophytic and rhizospheric bacterial isolates of B. purpurascens and B. archeri, and to perform antibiosis tests based on the isolated microorganisms of these tree species. No difference was found between the cultivation substrates for the percentages of germination, hard seeds, and fungal contamination in the B. purpurascens seeds. The Bacillus subtilis isolated showed the best capacity for suppressing the growth of the two deteriorative fungi tested in B. purpurascens seeds. No difference was found for inhibition of the growth of Aspergillus niger fungus (deteriorative fungus of B. archeri seeds) between the microorganisms with Bacillus sp. and Brevibacillus brevis compared to the control. In the microbiolization of B. purpurascens and B. archeri seeds performed with microbiological solutions produced from the endophytic and rhizospheric strains of Bacillus sp., no differences were observed in the percentages of germination and contamination by fungi. For B. archeri seeds, there was contamination by fungi and bacteria after one day of cultivation, primarily in the regions with lesions caused by the extraction and scarification process.

Resumo Microorganismos específicos tem sido usados eficientemente para o controle biológico de fitopatógenos através da produção de substâncias antimicrobianas. Portanto, os objetivos deste estudo foram: estudar a germinação de sementes de Butia purpurascens Glassman e Butia archeri Glassman em diferentes substratos; selecionar e identificar isolados bacterianos rizosféricos e endofíticos de B. purpurascens e B. archeri, e realizar testes de antibiose a partir de microorganismos isolados dessas espécies arbóreas. Não houve diferença entre os substratos de cultivo para as porcentagens de germinação, de sementes duras e de contaminação por fungos nas sementes de B. purpurascens. O isolado bacteriano Bacillus subtilis apresentou melhor capacidade de supressão do crescimento dos dois fungos deterioradores testados nas sementes de B. purpurascens. Não foi encontrada diferença para a inibição do crescimento do fungo Aspergillus niger (fungo deteriorador de sementes de B. archeri) entre os microorganismos com Bacillus sp. e Brevibacillus brevis em comparação ao controle. Na microbiolização das sementes de B. purpurascens e B. archeri realizada com soluções microbiológicas produzidas a partir de cepas endofíticas e rizosféricas de Bacillus sp. não foram observadas diferenças na porcentagem de germinação e contaminação por fungos. Para B. archeri houve contaminação por fungos e bactérias após um dia de cultivo, principalmente nas regiões de lesão proporcionadas pelo processo de extração e escarificação.

Enhanced production and organic solvent stability of a protease fromBrevibacillus laterosporus strain PAP04

A bacterial strain (PAP04) isolated from cattle farm soil was shown to produce an extracellular, solvent-stable protease. Sequence analysis using 16S rRNA showed that this strain was highly homologous (99%) to Brevibacillus laterosporus. Growth conditions that optimize protease production in this strain were determined as maltose (carbon source), skim milk (nitrogen source), pH 7.0, 40°C temperature, and 48 h incubation. Overall, conditions were optimized to yield a 5.91-fold higher production of protease compared to standard conditions. Furthermore, the stability of the enzyme in organic solvents was assessed by incubation for 2 weeks in solutions containing 50% concentration of various organic solvents. The enzyme retained activity in all tested solvents except ethanol; however, the protease activity was stimulated in benzene (74%) followed by acetone (63%) and chloroform (54.8%). In addition, the plate assay and zymography results also confirmed the stability of the PAP04 protease in various organic solvents. The organic solvent stability of this protease at high (50%) concentrations of solvents makes it an alternative catalyst for peptide synthesis in non-aqueous media.

Preliminary screening of the larvicidal effect of Brevibacillus laterosporus strains against the blowfly Chrysomya megacephala(Fabricius, 1794) (Diptera: Calliphoridae)

: This study evaluated whether different strains of Brevibacillus laterosporus could be used to control larvae of the blowfly Chrysomya megacephala , a pest that affects both human and animal health.

Bioprospecção de bactérias entomopatogênicas sobre o desenvolvimento pós-embrionßrio de Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae), em laboratório / Bioprospecting entomopathogenic bacteria on the postembryonic development of chrysomya megacephala (fabricius, 1794) (diptera: calliphoridae) in laboratory

Chrysomya megacephala, conhecida como mosca-varejeira, tem importânciapara a saúde pública devido ao fato de os adultos serem potenciais vetores depatógenos entéricos, e causadores de miíase, tanto em humanos quanto emanimais. Além disso, trata-se de uma espécie exótica invasora. Os poucos estudosque abordam o controle de C. megacephala empregam os inseticidas químicos,como os piretroides e os organofosfatos e o uso de parasitoides e metabolitossecundßrios de plantas. Bactérias entomopatogênicas são uma alternativainteligente para a redução do uso de agroquímicos, pois possuem maior eficßcia,menor custo e benefícios ambientais, como segurança para os humanos e outrosorganismos não alvo. Nesse sentido, este trabalho avaliou a bioatividade deBrevibacillus laterosporus e Bacillus thuringiensis sobre o desenvolvimento pósembrionßriode Chrysomya megacephala. Foram testadas (bioensaios preliminares)15 cepas da espécie Brevibacillus laterosporus e 3 cepas de Bacillus thuringiensisvar. israelensis contra larvas de primeiro instar de C. megacephala. Anßlisesestatísticas indicaram maior atividade larvicida das cepas Bon 707, IGM 16-92 e Shi3 de B. laterosporus, causando mortalidade significativa e superior a 50%. Foramrealizados bioensaios completos analisando os efeitos letais (mortalidade larval,pupal e total) e subletais (período de desenvolvimento pós-embrionßrio em dias emédia de peso das larvas), das cepas Bon 707, Shi 3 e IGM 16-92. Suspensões deesporos das cepas Shi 3 e Bon 707 apresentaram taxas de mortalidade superiores.A anßlise por microscopia eletrônica de transmissão revelou alterações intestinaisdecorrentes da ingestão pelas larvas de esporos das cepas Bon 707 e IGM 16-92...

Chrysomya megacephala, known as a blowfly species, is important for publichealth due to the fact that adults are potential vectors of enteric pathogens, andcause myiasis, both in humans and in animals. Furthermore, it is an exotic invasivespecies. The few studies that address the control of C. megacephala employchemical insecticides, such as pyrethroids and organophosphates, the use ofparasitoids and secondary plant metabolites. Entomopathogenic bacteria are a smartalternative for reducing the use of pesticides because they have greater efficiency,lower cost and environmental benefits such as safety for humans and other nontargetorganisms. Therefore, this study evaluated the bioactivity of Brevibacilluslaterosporus and Bacillus thuringiensis strains on the post-embryonic developmentChrysomya megacephala. Fifteen strains of Brevibacillus laterosporus species and 3strains of Bacillus thuringiensis var. israelensis were tested (preliminary bioassays)against first instar larvae of C. megacephala. Statistical analysis indicated higherlarvicidal activity of the strains Bon 707, IGM 16-92 and Shi 3 of B. laterosporus,causing significant mortality, higher than 50%. Complete bioassays analyzing thelethal effects (larval, pupal and total mortality) and sublethal effects (period in days ofpost-embryonic development and mean weight of larvae) were performed with strainsBon 707, Shi 3 and IGM 16-92. Spore suspensions of strains Bon 707 and Shi 3presented higher mortality rates. Analysis by transmission electron microscopyrevealed intestinal alterations resulting from the intake of spores of Bon 707 and IGM16-92 strains by the larvae...

Characterization of Bacteriocin like inhibitory substance produced by a new Strain Brevibacillus borstelensis AG1 Isolated from 'Marcha'

In the present study, a bacterium isolated from Marcha- a herbal cake used as traditional starter culture to ferment local wine in North East India, was evaluated for bacteriocin like inhibitory substance production and was tested against six food borne/spoilage causing pathogens viz. Listeria monocytogenes MTCC 839, Bacillus subtilis MTCC 121, Clostridium perfringens MTCC 450, Staphylococcus aureus, Lactobacillus plantarum and Leuconostoc mesenteroides MTCC 107 by using bit/disc method followed by well diffusion method. The bacterial isolate was identified as Brevibacillus borstelensis on the basis of phenotypic, biochemical and molecular characteristics using 16Sr RNA gene technique. Bacteriocin like inhibitory substance produced by Brevibacillus borstelensis AG1 was purified by gel exclusion chromatography. The molecular mass of the Brevibacillus borstelensis AG1 was found to be 12 kDa. Purified bacteriocin like inhibitory substance of Brevibacillus borstelensis was further characterized by studying the effect of temperature, pH, proteolytic enzyme and stability. Bacteriocin like inhibitory substance was found to be thermostable upto 100 °C, active at neutral pH, sensitive to trypsin, and partially stable till third week of storage thus showing a bright prospective to be used as a potential food biopreservative.

Analysis of microbial diversity of nitrifying bacteria by terminal restriction fragment length polymorphism / 生物工程学报

We analyzed the microbial diversity and quantity of nitrifying bacteria in the enrichment reactor by Terminal Restriction Fragment Length Polymorphism (T_RFLP), a cultured-independent molecular technique. The result indicated that nitrobacteria enriched the best, and the diversity index decreased 62.80% compared with the initial data. Nitrobacteria were predominant in the reactor. Meanwhile, we studied the microbial diversity before and after adding Nitrobacteria into shrimp ponds, and analyzed several major bacterial species that existed stably in the pond. According to the analysis by T_RFLP program, species including Brevibacillus brevis, Microbacterium lactium, Azoarcus indigens and Bordetella holmesii were the dominant bacteria in the ponds.