ABSTRACT
Abstract This research has been conducted to investigate the biodegradation of polystyrene (PS) by isolated strain Bacillus megaterium from Zophobas morio's. The viability and metabolic activity of Bacillus megaterium utilizing emulsified PS in liquid carbon free basal medium as sole carbon source was confirmed using redox probe 2,3,5-triphenyltetrazolium chloride (TTC). Bacillus megaterium showed prominent result with TTC, which forms red colored insoluble TPF (triphenyl formazan) within four to seven days. The formation of TPF confirmed the metabolism activity of the cell using PS as source of carbon. The biodegradation activity of Bacillus megaterium on PS film was also confirmed by FTIR analysis, the result showed changes in functional groups due to microbial activities. FESEM analysis was carried out on the PS surface, revealing the formation of bacterial biofilm as well as occurrence of porosity and fragility of the bacterial deteriorate surface compared to control. Overall, the identification of plastic degrading bacteria (PDB) will become a fundamental platform promoting more in-depth research for optimal plastic waste management to switch from the conventional landfilling to enzymatic biodegradation. In the long run, it is to regulate the current global plastic waste accumulation on Earth which poses potent ecological threat.
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Aims: This study aimed to evaluate the antagonist effect of the bacteria Bacillus megaterium against the toxigenic fungus Aspergillus flavus using different methods. Methodology: According to the method described by Vincent et al. (1991) and Arras (1993) we have determined the effect of B. megaterium on the growth of A. flavus using fungal disc and fungus spore respectively. In order to see the effect of cell free supernatant of B. megaterium on the dry mass of A. flavus, culture bacteria of 5 days old was centrifuged; filtered and cell-free supernatant was incubated with 5 mm circular plug of A. flavus. After incubation at 28°C for 9 days. The dry mass was determined by weighting every 72 h and compared with the control. Elucidation of antagonistic mechanism of B. megaterium was examined using the following tests: Hydrogen Cyanide production, production of Ammonia (NH3) and production of extracellular enzymes such: Protease, Chitinase and Amylase. Results: The results showed that B. megaterium is an antagonistic bacterium that has been shown high effectiveness against the fungus A. flavus isolated form poultry feeds in Algeria. Results indicated an almost entirely decrease (47.56%) of mycelial growth using fungal disc and (40.75%) using fungus spore. In cell-free supernatant in vitro assay, B.megaterium showed significant inhibitory activity against A. flavus when the dry mass of mycelium decrease from 1.25 g to 0.83 g compared with the control. Conclusion: This research shows that B. megaterium is quite important and effective as biocontrol agent against the toxigenic mold A. flavus in poultry feeds. This inhibition action is probably due to the synergistic effects of the factors such as the production of antibiotics and the extracellular enzymes such as protease, cellulase, chitinase and amylase.
ABSTRACT
In our previous studies, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15 have been shown to be antagonistic to Aspergillus flavus in stored rice grains. In this study, the biocontrol activities of these strains were evaluated against Aspergillus candidus, Aspergillus fumigatus, Penicillium fellutanum, and Penicillium islandicum, which are predominant in stored rice grains. In vitro and in vivo antifungal activities of the bacterial strains were evaluated against the fungi on media and rice grains, respectively. The antifungal activities of the volatiles produced by the strains against fungal development and population were also tested using I-plates. In in vitro tests, the strains produced secondary metabolites capable of reducing conidial germination, germ-tube elongation, and mycelial growth of all the tested fungi. In in vivo tests, the strains significantly inhibited the fungal growth in rice grains. Additionally, in I-plate tests, strains KU143 and AS15 produced volatiles that significantly inhibited not only mycelial growth, sporulation, and conidial germination of the fungi on media but also fungal populations on rice grains. GC-MS analysis of the volatiles by strains KU143 and AS15 identified 12 and 17 compounds, respectively. Among these, the antifungal compound, 5-methyl-2-phenyl-1H-indole, was produced by strain KU143 and the antimicrobial compounds, 2-butyl 1-octanal, dimethyl disulfide, 2-isopropyl-5-methyl-1-heptanol, and 4-trifluoroacetoxyhexadecane, were produced by strain AS15. These results suggest that the tested strains producing extracellular metabolites and/or volatiles may have a broad spectrum of antifungal activities against the grain fungi. In particular, B. megaterium KU143 and P. protegens AS15 may be potential biocontrol agents against Aspergillus and Penicillium spp. during rice grain storage.
Subject(s)
Aspergillus flavus , Aspergillus fumigatus , Aspergillus , Bacillus megaterium , Bacillus , Fungi , Germination , In Vitro Techniques , Penicillium , PseudomonasABSTRACT
In our previous study, three bacterial strains, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15, were selected as effective biocontrol agents against Aspergillus flavus on stored rice grains. In this study, we evaluated the inhibitory effects of the volatiles produced by the strains on A. flavus growth and aflatoxin production on stored rice grains. The three strains significantly reduced mycelial growth of A. flavus in dual-culture assays compared with the negative control strain, Sphingomonas aquatilis KU408, and an untreated control. Of these tested strains, volatiles produced by B. megaterium KU143 and P. protegens AS15 markedly inhibited mycelial growth, sporulation, and conidial germination of A. flavus on agar medium and suppressed the fungal populations in rice grains. Moreover, volatiles produced by these two strains significantly reduced aflatoxin production in the rice grains by A. flavus. To our knowledge, this is the first report of the suppression of A. flavus aflatoxin production in rice grains using B. megaterium and P. protegens volatiles.
Subject(s)
Aflatoxins , Agar , Aspergillus flavus , Aspergillus , Bacillus megaterium , Bacillus , Germination , Pseudomonas , SphingomonasABSTRACT
In the study, Bacillus megaterium T04 isolated from Rach Lang stream in Vietnam was identified. The stream samples were diluted in 0.9 % NaCl broth and then spread onto the ISP4 supplemented with rice and wheat starch. The colonies showed the strongest hydrolyzing activity were picked up and identified by biochemical test and 16S rRNA sequencing analysis. The starch hydrolyzing ability of this strain was detected by starch agar plate method. For maximum α-Amylase production, including 174.7 IU/ml and 0.2 IU/ml in medium containing wheat and rice, respectively was obtained after 72 h of incubation. The enzyme still showed high activity in 60% ammonium sulfate that was necessary for study on the enzyme characteristics. As a result, Bacillus megaterium T04 could produce high yield of amylase in simple conditions.
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Bacillus megaterium T04 isolated from Rach Lang stream in Vietnam was tested for antimicrobial activities. The antimicrobial activities of Bacillus megaterium T04 (57.3x108 cfu/mL) against Candida albicans, Salmonella typhi, Pseudomonas aeruginosa, Staphylococcus sciuri, Micrococcus luteus were detected by agar well diffusion method in different cultivation conditions at three temperatures (25, 37, and 45oC) in three incubation periods (24, 48, and 72 hours). The efficacy of antimicrobial activities of this strain were determined in comparison with ceftriaxone activity against Candida albicans, Salmonella typhi, Pseudomonas aeruginosa, Staphylococcus scuiri, Micrococcus luteus. The antimicrobial activity potency was equivalent to ceftriaxone in a range ( 3.3 0.6 μg/mL to 46.5 6.2 μg/mL) for Candida albicans (0.9 0.2 μg/mL to 35.5 7.7 μg/mL) for Salmonella typhi, , (0.4 0.1 μg/mL to 28.4 4.4 μg/mL) for Pseudomonas aeruginosa, (119.8 21.2 μg/mL to 283.7 26.0 μg/mL) for Staphylococcus scuiri, (3.3 0.4 μg/mL to 64.4 7.4 μg/mL) for Micrococcus luteus.
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Bacillus megaterium is a promising host for expression of heterologous proteins. This paper reports the nutrient consumption patterns and production of metabolites for three different strains of B. megaterium, ATCC 14945, QMB 1551 and PV 361, which is QMB 1551 with seven constitutive plasmids deleted. 14 h cultivations in agitated flasks were run, for two different media: A (LB plus 10g/L glucose) and B (medium A, with the yeast extract replaced by tryptone). Strains PV361 and QMB 1551 showed higher maximum specific growth rates in medium B, reaching 0.42 h-1 and 0.48 h-1 respectively. The main by-products of the glucose overflow mechanism were acetate and lactate, for all three strains, which had preferential amino acids for substrate: Ala, Asp, Glu, Ser. No production of alcohols was detected.
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Background: Unlike petroleum-based synthetic plastics, biodegradable biopolymer generation from industrial residue is a key strategy to reduce costs in the production process, as well as in the waste management, since efficient industrial wastewater treatment could be costly. In this context, the present work describes the prospection and use of bacterial strains capable to bioconvert cassava starch by-product into biodegradable polyhydroxyalkanoates (PHAs). Results: The first step of this study was the bacterial competence screening which was conducted with 72 strains covering 21 Bacillus and related species. The microorganism growth in a medium with a starch substrate was measured by an innovative MTT assay, while the ability of the bacteria to secrete amylase and produce PHA was evaluated by the Nile Red Dye method. Based on growth and potential for PHA production, four isolates were selected and identified as Bacillus megaterium by 16S rRNA sequencing. When cultivated in hydrolyzed cassava starch by-product, maximum production reached 4.97 g dry biomass/L with 29.7 percent of Poly-(3-hydroxybutyrate) (characterized by FTIR). Conclusions: MTT assay proved to be a reliable methodology for monitoring bacterial growth in insoluble media. Selected amylolytic strains could be used as an alternative industrial process for biodegradable plastics production from starchy residues, reducing costs for biodegradable biopolymer production and wastewater treatment operations.
Subject(s)
Bacillus megaterium , Biodegradable Plastics , Polyhydroxyalkanoates , Starch , Biopolymers , Biotransformation , Tetrazolium Salts , ThiazolesABSTRACT
A highly alkaline amylase producing Bacillus megaterium was isolated from enriched soil under lab conditions. In order to enrich the soil for alkaline amylase producing Bacteria, soil was supplied with raw potatoes (substrate) and water at pH 10. Soil was left for 1month. After serial dilution five different colonies were obtained and were screened for amylase production. The culture MJS1105 showing maximum hydrolysis zone was subjected to solid state fermentation using wheat bran as substrate. The enzyme was purified using ammonium sulphate precipitation followed by dialysis. Total activity of 7.056 U was obtained at 37ºC after 96 hours of incubation and was found to have a molecular weight of 63KDa (approx.) by SDS – PAGE. The enzyme was relatively stable between pH 5 – pH 13 and at temperature ranging from 32ºC to 50ºC. Moreover, activity was enhanced by using metal ions such as Ca2+, Mg2+ and Na2+. The decline in activity was observed by adding SDS and EDTA.
ABSTRACT
In a greenhouse experiment, the nematicidal effect of some bacterial biofertilizers including the nitrogen fixing bacteria (NFB) Paenibacillus polymyxa (four strains), the phosphate solubilizing bacteria (PSB) Bacillus megaterium (three strains) and the potassium solubilizing bacteria (KSB) B. circulans (three strains) were evaluated individually on tomato plants infested with the root-knot nematode Meloidogyne incognita in potted sandy soil. Comparing with the uninoculated nematode-infested control, the inoculation with P. polymyxa NFB7, B. megaterium PSB2 and B. circulans KSB2, increased the counts of total bacteria and total bacterial spores in plants potted soil from 1.2 to 2.6 folds estimated 60 days post-inoculation. Consequently, the inoculation with P. polymyxa NFB7 increased significantly the shoot length (cm), number of leaves / plant, shoot dry weight (g) / plant and root dry weight (g) / plant by 32.6 percent, 30.8 percent, 70.3 percent and 14.2 percent, respectively. Generally, the majority treatments significantly reduced the nematode multiplication which was more obvious after 60 days of inoculation. Among the applied strains, P. polymyxa NFB7, B. megaterium PSB2 and B. circulans KSB2 inoculations resulted in the highest reduction in nematode population comparing with the uninoculated nematode-infested control. They recorded the highest reduction in numbers of hatched juveniles/root by 95.8 percent, females/root by 63.75 percent and juveniles/1kg soil by 57.8 percent. These results indicated that these bacterial biofertilizers are promising double purpose microorganisms for mobilizing of soil nutrients (nitrogen, phosphate and potassium) and for the biological control of M. incognita.
Subject(s)
Anti-Bacterial Agents/analysis , Bacillus megaterium/isolation & purification , Phosphates/analysis , Nematoda , Nitrogen , Nitrogen Fixation , Pest Control, Biological , Plants, Edible , Sandy Soils , Spores, Bacterial , Methods , Plants , MethodsABSTRACT
One hundred and thirty two Bacillus cereus and 52 Bacillus megaterium isolates from honeys were evaluated for the presence of genes encoding enterotoxin HBL, enterotoxin-T, cytotoxin K and the NHE complex, respectively. The relationship between hemolytic and coagulase activity and its correlation with the presence of the four mentioned enterotoxins was determined by principal component analysis (PCA). PCA in B. cereus revealed a positive correlation among free coagulase, hemolysis and the presence of genes hblA, hblB, hblC, hblD (HBL complex) and bceT (enterotoxin-T), but no correlation with the clumping factor (bound coagulase) and the presence of sequences of the NHE complex. On the other hand, PCA in B. megaterium showed a high positive correlation between coagulase (bound and free) and the haemolytic activity but no correlation in relation to the presence of genes of the HBL complex, cytotoxin K, enterotoxin T and the NHE complex. To our knowledge, this is the first report of the detection of cytotoxin K and of the NHE complex genes in B. megaterium. The relationship between the coagulase activity and the presence of virulence factors has not been described before in the genus Bacillus, being this work the first report of this correlation. Interestingly, the presence of the cytK gene was almost independent of the presence of the rest of virulence factors herein analyzed both in B. cereus and B. megaterium populations. Our results suggest that honey could be a possible vehicle for foodborne illness due to the presence of toxigenic B. cereus and B. megaterium strains containing different virulence factors.
Se evaluaron 132 aislamientos de Bacillus cereus y 52 de Bacillus megaterium provenientes de mieles de distintos orígenes geográficos para investigar la presencia de secuencias de ADN relacionadas con genes de virulencia y su posible correlación con la actividad hemolítica y coagulasa. Con respecto a los genes de virulencia, se analizaron por PCR secuencias de ADN de los genes nhe (A, B y C), HBL (A, B, C, D), cytK y bceT. La relación entre las variables fue evaluada mediante un análisis de componentes principales, donde se encontró que los aislamientos de B. cereus mostraron una correlación positiva entre actividad de coagulasa (coagulasa libre) y presencia de los genes del complejo HBL y bceT, mientras que en B. megaterium se halló una alta correlación positiva entre actividad de coagulasa (libre y fija) y actividad hemolítica, pero no se observó correlación significativa entre la presencia de genes de virulencia y dichas actividades. Este estudio constituye el primer registro de la presencia de los genes cyt K y NHE en cepas de B. megaterium y el primer trabajo que analiza la relación entre la actividad de coagulasa y la presencia de genes de virulencia en B. cereus y B. megaterium. La presencia del gen cytK en ambas especies resultó totalmente independiente del resto de los factores de virulencia analizados. Nuestros hallazgos sugieren que la miel podría vehiculizar enfermedades transmisibles por alimentos debido a la presencia de cepas de B. cereus y B. megaterium potencialmente tóxicas.
Subject(s)
Bacillus cereus/genetics , Bacillus megaterium/genetics , Enterotoxins/genetics , Bacillus cereus/isolation & purification , Bacillus megaterium/isolation & purification , Honey/microbiologyABSTRACT
A associação de plantas com rizobactérias pode promover o crescimento vegetal, reduzindo custos de produção. As rizobactérias benéficas podem ser uma alternativa para a produção de cebola. Neste trabalho, foi avaliado o efeito da aplicação de rizobactérias no crescimento e na produtividade da cebola da cultivar 'Bola Precoce'. A aplicação foi realizada na Estação Experimental da Epagri, em Ituporanga, Santa Catarina (SC), no ano de 2008. O delineamento experimental foi em blocos ao acaso, com cinco repetições, sendo utilizadas as rizobactérias Pseudomonas spp. W6, Bacillus megaterium W19 e Bacillus cereus UFV40, microbiolizadas isoladamente nas sementes ou em mistura, juntamente com uma testemunha não tratada. Os tratamentos com aplicação de rizobactérias proporcionaram maior altura, diâmetro de pseudocaule e número de folhas nas plantas avaliadas aos 90 dias após o transplante. Plantas que receberam os tratamentos com rizobactérias apresentaram maior rendimento de bulbos em relação à testemunha. A massa dos bulbos foi afetada positivamente pelos tratamentos, com destaque para Bacillus megaterium W19.
The association of plants with rhizobacteria can enhance plant growth, reducing production costs. Benefic rhizobacteria can stimulate plant growth being an alternative for production of onions. This research assesses the impact of applying rhizobacteria in the growth and productivity of cv. Bola Precoce onions. The study was conducted in the Experimental Station of Epagri, Ituporanga, SC, Brazil in 2008. The experimental design was in random blocks with five repetitions using Pseudomonas spp. W6, B Bacillus megaterium W19 e Bacillus cereus UFV40 rhizobacteria, microbiolized singly in seeds or in a formula together with non-treated control. The treatments involving rhizobacteria application yielded higher heights, diameter of pseudo-trunk and number of leaves in the plants assessed at 90 days after transplant. All the plants which received treatments with rhizobacteria presented better results considering the fresh weight and yield of bulb in relation to the control. The mass of bulbs were positively affected by the treatments, especially for Bacillus megaterium W19.
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Bacillus megaterium strains are commonly used in microbial fertilizer(MF) . MF products are often contaminated by other B. cereus group members,which have similar phenotype such as Bacillus cereus,B. thuringiensis,B. mycoide. For quality control and safety of MF,a rapid and accurate method is needed to distinguish the strains of Bacillus megaterium from B. cereus group. Based on specific nucleotide sequences of the spoOA genes,2 pairs of species-specific primers were designed and a multiplex-PCR(mPCR) was developed for this purpose. When the optimized mPCR was used to detect the DNAs of 24 reference strains from three genera of Bacillus,Paenibacillus,and Brevibacillus,all B. megaterium strains showed singlefragment of 443 bp and Bacillus cereus group showed a fragment of 411 bp. However,no any amplified product was from the other bacteria. The sensitivity of mPCR was 105 CFU/mL. The mPCR results of 10 isolates of B. megaterium/B. cereus group and 8 products of MF coincided with the biochemical assay. Taken together,our newly developed mPCR assay was species-specific and effective in application. It can be used to detect and identify the strains of B. megaterium and B. cereus group from microbial fertilizer products.
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Penicillin Acylase from B. megaterium was immobilized on the porous bead carriers based on methacrylate, N,N-methelene-bis-methacrymide, glycidyl methacrylate, Allyl ether copolymers (Eupergit-c) either directly or after chemical modification with 1.6-deaminohexane and L-Lysine. Directly binding with oxirane groups, the most efficient immobilization results were achieved. The immobilization yield was markedly influenced by the ratio of amount of free enzyme to the weight of the carrier. The specific activities of 89 up to 475IU/g (wet) and binding protein of 6.9 to 112 mg/g (dry) were obtained when the free enzyme added to the immobilization solution was from 323IU/g (dry) up to 6250IU/g (dry). The residual activity of immobilized PGA in a recycling system at the 20th was about 92.5% of the initial value.
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Rapidly metabolizable compounds such as glucose or glycerol were not utilized by Bacillus megaterium in the absence of manganese when grown in the supplemented nutrient broth medium. Under these conditions, growth ceased at low cell titre, 3-phosphoglyceric acid accumulated inside the cells and normal sporulation process was arrested. Addition of manganese to the medium caused disappearance of 3-phosphoglyceric acid, growth resumed and normal sporulation was observed. Synthesis of 3-phosphoglyceric acid occurred only in the mother cell compartments and it was transported for accumulation inside the forespores of Bacillus megaterium when grown in supplemented nutrient broth medium. Incubation of forespores in the presence of glucose or glycerol had no effect on 3-phosphoglyceric acid synthesis/accumulation, but it was completely utilized when forespores were incubated with manganese plus ionophore (X 537A). No other metal(s) could substitute for manganese suggesting that manganese plays crucial role in 3-phosphoglyceric acid metabolism.
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Bacillus megaterium accumulated 3-phosphoglycerate during sporulation which was utilized during spore germination. During sporulation a protein was synthesized before or at the start of 3-phosphoglycerate accumulation inside the developing spores about 1.5 h before dipicolinic acid accumulation. This protein has an affinity for Mn2+ and other divalent metal ions and inhibits phosphoglycerate mutase activity which has been shown to require Mn2+ However, the levels of the inhibitor decreased considerably (75-85%) during spore germination. No appreciable amount of the inhibitor was detected in the vegetable cell and mother cell compartment; however, the forespore compartment possesses an activity comparable to that of dormant spores. The partially purified inhibitor has a molecular weight of 11,000 and possesses both high and low affinity binding sites for Mn2+ and Ca2+ as determined by Scatchard plot analysis.