Screening of alginate lyase-producing bacteria and optimization of media compositions for extracellular alginate lyase production

Background: Alginate is a linear polysaccharide consisting of guluronate [polyG] and mannuronate [polyM] subunits

Methods: In the initial screening of alginate-degrading bacteria from soil, 10 isolates were able to grow on minimal medium containing alginate. The optimization of cell growth and alginate lyase [algL] production was carried out by the addition of 0.8% alginate and 0.2-0.3 M NaCl to the culture medium. Of 10 isolates, one was selected based on its fast growth rate on minimal 9 medium containing 0.4% sodium alginate. The selected bacterium, identified based on morphological and biochemical characteristics, as well as 16S rDNA sequence data, was confirmed to be an isolate belonging to the genus Bacillus and designated as Bacillus sp. TAG8

Results: The results showed the ability of Bacillus sp. TAG8 in utilizing alginate as a sole carbon source. Bacillus sp. TAG8 growth and algL production were augmented with an increase in sodium alginate concentration and also by the addition of 0.2-0.3 M NaCl. Molecular analysis of TAG8 algL gene showed 99% sequence identity with algL of Pseudomonas aeruginosa PAO1. The algL produced by Bacillus sp. TAG8 cleaved both polyM and polyG blocks in alginate molecule, as well as acetylated alginate residues, confirming the bifunctionality of the isolated lyase

Conclusion: The identification of novel algL genes from microbial communities constitutes a new approach for exploring lyases with specific activity against bacterial alginates and may thus contribute to the eradication of persistent biofilms from clinical samples

Dissolving mechanism of strain P17 on insoluble phosphorus of yellow-brown soil

Strain P17 was a bacterial strain identified as Bacillus megaterium isolated from ground accumulating phosphate rock powder. The fermentation broth of strain P17 and the yellow-brown soil from Nanjing Agricultural University garden were collected to conduct this study. The simulation of fixed insoluble phosphorous forms after applying calcium superphosphate into yellow-brown soil was performed in pots, while available P and total P of soil were extremely positive correlative with those of groundwater. Then the dissolving effect of strain P17 on insoluble P of yellow-brown soil was studied. Results showed that Bacillus megaterium strain P17 had notable solubilizing effect on insoluble phosphates formed when too much water-soluble phosphorous fertilizer used. During 100 days after inoculation, strain P17 was dominant. Until the 120th day, compared with water addition, available P of strain P17 inoculation treated soil increased by 3 times with calcium superphosphate addition. Besides available P, pH, activity of acid and alkaline phosphatase and population of P-solubilizing microbes were detected respectively. P-solubilizing mechanism of P-solubilizing bacteria strain P17 seems to be a synergetic effect of pH decrease, organic acids, phosphatase, etc.

The nematicidal effect of some bacterial biofertilizers on Meloidogyne incognita in sandy soil

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.

Production of polyhydroxyalkanoate [PHAs] and copolymer [P[HB-co-HV]] by soil bacterial isolates in batch and two-stage batch cultures

NINETY TWO local bacterial isolates, from the rhizosphere and soil around the root system of bean [Viciafaba] grown in Kalubia Governorate in Egypt, were bio-prospected for polyhydroxyalkanoate [PHA] accumulation. Three isolates accumulated >/=20% of PHAs, they were identified as Pseudomonas flu orescens S48, Bacillus megaterium 7A and B. megaterium UBFI9. The tested isolates gave the maximum PHAs content on basal medium containing glucose and ammonium sulfate at C/N ratio of 30/1 after 72 hr at 30°C using shake flask culture technique. Two-stage batch were implemented with varying loading levels of nitrogen and phosphorus, inoculated with washed cells. Nitrogen omission of 70% led to increase the PHAs content by 19%, 3% and 8.5% using washed cells of Ps. fluorescens S48, B. megaterium UBF 19 and Bacillus megaterium 7A, respectively comparing with batch production on the same medium after 72 hr. The Copolymer poly[hydroxybutyrate-co-hydroxyvalerate] [P [HB-co-HV]] content level was increased when valerie/glucose [V/G] was 0.19 mol.mo[-1] after 96 hr being 25.97% and 20.11% by Ps. fluorescens S48 and B. megaterium UBFI9, respectively and reached 23.73% by B. megaterium 7A at propionic/glucose [PIG] of 0.5 mol.mol[-1]. The corresponding highest values of valeric content of copolymer at V/G 3.08 mol.mol[-1] were 63%, 49% and 45%, respectively, comparing with other V/G ratios by using GC analysis . Replacing glucose with 2% corn oil or 1% soybean oil increased the PHAs content of Ps. fluorescens S48 cells to 54.21% and 52.12%, respectively, after 72

Production conditions of exopolysaccharide from bacillus megaterium identified by 16S rRNA gene sequencing

THE BACILLUS megaterium exopolysaccharide [EPS] producer was isolated from the Egyptian soil and identified by molecular biology technique [16S rRNA]. It was found that the exopoiysaccharide showed a high yield after 72 hr, with an initial pH of 5.0, in 250 ml Erlenmeyer flasks containing 50 ml of culture, medium having 2% sucrose as a sole carbon source. The yeast extract [0.1%] and animonium chloride [0.1%] were the best organic and inorganic nitrogen source, respectively, as they gave the maximum EPS production. Optimum incubation temperature was found to be 35°C. Pretreated molasses from sugarcane and sugarbeet were used as carbon source and they gave a considerable yield [-tow fold] in relation to sucrose. The pretreatment of molasses by sulfuric acid showed high yield of polysaccharide and maximum yield coefficient

Enterotoxigenic gene profiles of Bacillus cereus and Bacillus megaterium isolates recovered from honey / Búsqueda de factores de virulencia en cepas de Bacillus cereus y de Bacillus megaterium aisladas de miel

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.

Effect of carbon source and precursors on the production of plant growth regulators by azotobacter chroococcum [R19] and bacillus megaterium var. phosphaticum [R44]

An experiment was carried out to examine the effect of carbon source, tryptophan and adenine concentrations on the production of growth regulators by A. chroococcum [R19] and B. megaterium var. phosphaticum [R44]. Mannitol and glucose were the best carbon sources for the production of plant growth regulators [PGRs] by A. chroococcum and B. megaterium var. phosphaticum, respectively. A. chroococcum produced higher amounts of zeatin and kinetin compared to those produced by B. megaterium var. phosphaticum while B. megaterium var. phosphaticum produced higher amounts of [9R] benzyl adenine and [9G] bcnzyl adenine compared with those produced by A. chroococcum. Production of auxins, gibberellic acid [GA[3]] and cytokinins was increased with increasing tryptophan concentration. The highest amounts of PGRs produced by the two strains were obtained with tryptophan at 1000 micro M. Highest amounts of PGRs produced by A. chroococcum and B. megaterium var. phosphaticum at 10 and 100 micro M of adenine, respectively. Also, the produced amounts of gibberellic acid and cytokinin than that produced by B. megaterium var. phosphaticum. Generally, obtained data showed that the application of the optimal conditions together gave highest amounts of PGRs as compared with the other individual factors. This result is logic and was anticipated

Exploring the use of natural antimicrobial agents and pulsed electric fields to control spoilage bacteria during a beer production process / Exploración del uso de agentes antimicrobianos naturales y de campos eléctricos pulsantes para el control de bacterias contaminantes durante el proceso de elaboración de cerveza

Different natural antimicrobials affected viability of bacterial contaminants isolated at critical steps during a beer production process. In the presence of 1 mg/ml chitosan and 0.3 mg/ml hops, the viability of Escherichia coli in an all malt barley extract wort could be reduced to 0.7 and 0.1% respectively after 2 hour- incubation at 4 °C. The addition of 0.0002 mg/ml nisin, 0.1 mg/ml chitosan or 0.3 mg/ml hops, selectively inhibited growth of Pediococcus sp. in more than 10,000 times with respect to brewing yeast in a mixed culture. In the presence of 0.1mg ml chitosan in beer, no viable cells of the thermoresistant strain Bacillus megaterium were detected. Nisin, chitosan and hops increased microbiological stability during storage of a local commercial beer inoculated with Lactobacillus plantarum or Pediococcus sp. isolated from wort. Pulsed Electric Field (PEF) (8 kV/cm, 3 pulses) application enhanced antibacterial activity of nisin and hops but not that of chitosan. The results herein obtained suggest that the use of these antimicrobial compounds in isolation or in combination with PEF would be effective to control bacterial contamination during beer production and storage.

Diferentes antimicrobianos naturales disminuyeron la viabilidad de bacterias contaminantes aisladas en etapas críticas del proceso de producción de cerveza. En un extracto de malta, el agregado de 1 mg/ml de quitosano y de 0,3 mg ml de lúpulo permitió reducir la viabilidad de Escherichia coli a 0,7 y 0,1%, respectivamente, al cabo de 2 horas de incubación a 4 °C. El agregado de 0,0002 mg/ml de nisina, 0,1 mg/ml de quitosano o de 0,3 mg/ml de lúpulo inhibió selectivamente (10.000 veces más) el crecimiento de Pediococcus sp. respecto de la levadura de cerveza en un cultivo mixto. El agregado de 0,1 mg/ml de quitosano permitió disminuir la viabilidad de una cepa bacteriana termorresistente, Bacillus megaterium, hasta niveles no detectables. Por otra parte, el agregado de nisina, quitosano y lúpulo aumentó la estabilidad microbiológica durante el almacenamiento de cervezas inoculadas con Lactobacillus plantarum y Pediococcus sp. aislados de mosto de cerveza. La aplicación de campos eléctricos pulsantes (CEP) (3 pulsos de 8kV/cm) aumentó el efecto antimicrobiano de la nisina y del lúpulo, pero no el del quitosano. Los resultados obtenidos indicarían que el uso de antimicrobianos naturales en forma individual o en combinación con CEP puede constituir un procedimiento efectivo para el control de la contaminación bacteriana durante el proceso de elaboración y almacenamiento de la cerveza.

Degradation of predigested distillery effluent by isolated bacterial strains.

Batch studies were conducted on degradation of anaerobically digested distillery wastewater by three bacterial strains, viz. Xanthomonas fragariae, Bacillus megaterium and Bacillus cereus in free and immobilized form, isolated from the activated sludge of a distillery wastewater treatment plant. The removal of COD and colour with all the three strains increased with time up to 48 hr and only marginal increase in COD and colour removal efficiency was observed beyond this period up to 72 hr. After this period removal efficiency remained fairly constant up to 120 hr. The maximum COD and colour removal efficiency varied from 66 to 81% and 65 to 75%, respectively for both free and immobilized cells of all the three strains. The strain Bacillus cereus showed the maximum efficiency of COD (81%) and colour (75%) removal out of the three strains. An interrelationship between the percent COD and colour removal was carried out by correlation and regression analysis and was justified by high values of coefficient of correlation (r = 0.99) for all the cases. The first order removal rate kinetics was also applied and rate constants were evaluated for COD and colour removal efficiencies.

Isolation, screening and identification of bacterial strains for degradation of predigested distillery wastewater.

Three bacterial isolates from the activated sludge of a distillery wastewater treatment plant identified as Xanthomonas fragariae, Bacillus megaterium and Bacillus cereus were found to remove COD and colour from anaerobically digested distillery wastewater in the range of 55 to 68% and 38 to 58% respectively.

Isolamento e caracterizaçäo de bacilos alcalofílicos em água de lavagem de cana-de-açúcar / Isolation and characterization of alkalophilic bacilli in the washing water of sugar cane

Foram determinadas as características fenotípicas de 78 cepas de Bacillus alcalofílicos, isolados da água alcalina usada na lavagem da cana-de-açúcar da usina Costa Pinto de Piracicaba - SP, em pH alcalino e em pH neutro. Com base nos resultados obtidos em pH neutro, foram caracterizadas segundo o Manual de Bergey, 9 cepas de Bacillus megaterim, 17 cepas de Bacillus firmus, 34 cepas de Bacillus licheniformis, 14 cepas de Bacillus lentus, 2 cepas de Bacillus subtilis e 2 cepas de Bacillus pasteurii.

Evaluation of penicillin acylase production by two strains of Bacillus megaterium

Penicillin acylase is a key enzyme for the production of semisynthetic beta-lactam antibiotics. The intracellular enzyme from Escherichia coli has been thoroughly studied and characterized. The extracellular enzyme from Bacillus megaterium, despite its potential advantages, has received less attention in the recent scientific literature. A comparative study is presented for the production of penicillin acylase with two strains of Bacillus megaterium in batch fermentation in previously optimized complex and defined media. The enzyme produced by the selected strain has been recovered, partially purified and its kinetic behaviour determined

Enriquecimiento en mutantes de Bacillus megaterium deficientes en la síntesis de poli-ß-hidroxibutirato (PHB) / Enrichment for mutants of Bacillus megaterium deficient in the synthesis of poly-ß-hydroxybutyrate (PHB)

La centrifugación en gradientes de sacarosa permite la separación de células según su densidad boyante de acuerdo al contenido en poli-ß-hidroxibutirato (PHB). En este trabajo este método se evaluó y se adaptó para detectar mutantes deficientes en la síntesis de PHB de B. megaterium analizando un bajo porcentaje de la población mutagenizada