Downstream processing of mosquitocidal toxins from solid state fermentation of Lysinibacillus sphaericus and Bacillus thuringiensis israelensis

Aims: The aim of this study was downstream processing of moquitocidal toxins produced by Lysinibacillus sphaericus (L. sphaericus) and Bacillus thuringiensis israelensis (Bti) under solid state fermentation. Methodology and results: Two mosquitocidal strains (L. sphaericus and Bti) were grown separately in trays under solid state fermentation for toxin production. The best conditions for extraction of crude toxins from fermented solids of both cultures were tap water at 5-50 °C, for 10 min under static conditions. Also, concentrated mosquitocidal toxins were efficiently extracted from fermented solids by 4 constitutive additions of 500 mL tap water to 1 kg of fermented culture at room temperature (25 °C) for 5 min each under static conditions. Both extracted toxins were formulated with talcum powder and they were stable for 8 months at room temperature. Conclusion, significance and impact of study: It is very important to study the operating conditions for mosquitocidal toxins extraction from solid state fermentation (SSF) and its formulation in cost effective manner.

Adsorption of Toxic Metals and Control of Mosquitos-borne Disease by Lysinibacillus sphaericus: Dual Benefits for Health and Environment / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Assessment of the bacterium L. sphaericus as a dual-action candidate for biological control of mosquito-borne diseases and bioremediation of toxic metals.</p><p><b>METHODS</b>Larvae of the mosquito, C. quinquefasciatus, were first evaluated for metal tolerance and then exposed to 5 ppm cadmium, chromium, arsenic, and lead in assays together with seven strains of L. sphaericus. A probit regression analysis was used to estimate the LC(50) of Cd, Cr, As, and Pb to C. quinquefasciatus. An analysis of covariance and multifactorial ANOVA examined the metal biosorption and larvicidal properties of the seven strains of L. sphaericus.</p><p><b>RESULTS</b>We found that L. sphaericus adsorbed the toxic metal ions and was toxic against mosquito larvae. The L. sphaericus strain III(3)7 resulted in a larvae mortality of over 80% for all the tested metals. This strain also exhibited the capacity to adsorb 76% of arsenic, 32% of lead, 25% of chromium, and 7% of cadmium.</p><p><b>CONCLUSION</b>This study found combined metal adsorption and larval toxicity associated with three strains of L. sphaericus [III(3)7, OT4b.31, and CBAM5]. This suggests that a combination of these strains shows strong dual potential for biological control of mosquitos in heavy metal-contaminated areas and remediate the heavy metal contamination as well.</p>

Lipolytic Activity and Molecular Identification of Pseudomonas aeruginosa and Lysinibacillus sphaericus Isolated from Domestic Oil Rich Wastewater.

Microbial lipases have been heightened in bioremediation and various industries. Place and Duration of Study: Department of Microbiology, Ekiti State University, Ado-Ekiti, Ekiti State, Nigeria, between September 2010 and August 2011. To identify the lipolytic enzyme producing microbial strains in domestic oil rich wastewater, the 16S rRNA gene was amplified and sequenced. The sequences were used to identify the strains by comparing with related sequences in database using BLAST analysis. The enzyme activity was quantified by HPLC analysis. All the lipolytic bacteria showed appreciable growth rates in the wastewater (between 0.67 and 1.67 mg/day) within 5 days. The most effective lipolytic bacteria isolates in the oil-rich wastewater were two species of the genus Pseudomonas and one of Bacillus. Comparing the weights on the first day to the twelfth day values when lipolytic organisms were grown in palm oil, some appreciable increases in weight difference were recorded in some isolates: 28.3%, 7.84%, 4.44% and 6.98% for Pseudomonas, Staphylococcus, Bacillus and Klebsiella, respectively. The weight increase of each of the microbial cells in palm oil culture was usually lesser than what was obtained in the oil-rich wastewater culture. Two isolates showed high similar sequence (99%) to that of Pseudomonas aeruginosa and Lysinibacillus sphaericus, respectively. From palm oil, Lysinibacillus sp. produced various forms of fatty acids in the medium, including myristic acid (2.61%), palmitic acid (6.22%), stearic acid (5.18%) and arachidic (3.66%). These strains are versatile in utilizing the limited nutrient and had the ability to grow appreciably in the toxic condition (soap solution), suggesting that they may serve as candidates in treating dietary oil-rich wastewater.

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification / Nueva esterasa tolerante a los solventes orgánicos aislada por metagenómica: ideas sobre la clasificación de las esterasas/lipasas

In order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 a/ß hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.

Con el fin de aislar nuevas variantes de lipasas tolerantes a solventes organicos (OST), se construyo una libreria metagenomica a partir de ADN obtenido de una muestra de suelo de bosque templado. A traves de un monitoreo en dos etapas, basado en la deteccion de actividades, se aislo un clon con actividad lipolitica en presencia de solventes organicos. La secuenciacion del plasmido pRBest recuperado del clon positivo revelo la presencia de un gen codificante de una hipotetica lipasa/esterasa. La secuencia deducida de amino acidos (RBest1) contiene los motivos conservados de enzimas lipoliticas y esta relacionada con la lipasa OST previamente descrita de Lysinibacillus sphaericus 205y, que es la unica enzima procariota estudiada perteneciente al subgrupo 4.4 de a/ß hidrolasas (abH4.04). Estudios in vivo e in vitro sobre la especificidad de sustratos de RBest1, utilizando triacil-gliceroles o p-nitrofenil-esteres, respectivamente, revelaron que la enzima es altamente especifica para compuestos butiricos (C4), comportandose como una esterasa y no como una lipasa. La esterasa RBest1 fue purificada y caracterizada bioquimicamente. La actividad optima de esterasa fue observada a pH 6,5 y las temperaturas optimas fueron entre 38 y 45 °C. Se establecio que la actividad enzimatica, determinada por hidrolisis de p-nitrofenil esteres, es afectada en presencia de diferentes solventes organicos miscibles y no miscibles, y tambien sales. Notoriamente, RBest1 permanece significativamente activa a elevadas fuerzas ionicas. Estos hallazgos sugieren que RBest1 posee la capacidad de las enzimas OST de la adaptacion molecular en presencia de compuestos organicos, asi como la resistencia de las proteinas halofilas.

Effect of culture medium on biocalcification by Pseudomonas Putida, Lysinibacillus Sphaericus and Bacillus Subtilis

The objective of this study is to investigate the efficiency of calcium carbonate bioprecipitation by Lysinibacillus sphaericus, Bacillus subtilis and Pseudomonas putida, obtained from the Coleção de Culturas do Instituto Nacional de Controle de Qualidade em Saúde (INCQS), as a first step in determining their potential to protect building materials against water uptake. Two culture media were studied: modified B4 containing calcium acetate and 295 with calcium chloride. Calcium consumption in the two media after incubation with and without the bacterial inoculum was determined by atomic absorption analysis. Modified B4 gave the best results and in this medium Pseudomonas putida INQCS 113 produced the highest calcium carbonate precipitation, followed by Lysinibacillus sphaericus INQCS 414; the lowest precipitation was produced by Bacillus subtilis INQCS 328. In this culture medium XRD analysis showed that Pseudomonas putida and Bacillus subtilis precipitated calcite and vaterite polymorphs while Lysinibacillus sphaericus produced only vaterite. The shape and size of the crystals were affected by culture medium, bacterial strain and culture conditions, static or shaken. In conclusion, of the three strains Pseudomonas putida INQCS 113 in modified B4 medium gave the best results precipitating 96 percent of the calcium, this strain thus has good potential for use on building materials.