Industrial fermentation and formulation of entomopathogenic bacteria

Couch, T. L.

doi:10.1007/978-94-017-1429-7_16

Cited by 32 publications

(31 citation statements)

References 10 publications

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“…Furthermore, no resistance has been detected on populations submitted to long-term exposure to this pathogen (Regis & Nielsen-LeRoux 2000). B.t.i formulations have been continuously improved to optimise their potency, stability (shelf life), ease of field application and residual activity (Couch 2000). Trials under simulated field conditions have been considered a reliable methodology to evaluate the efficacy and persistence of B.t.i based products because, under these conditions, many factors involved in residual activity can be better controlled and investigated than under real field conditions (Thiéry et al 1999.…”

Section: Abstract: Bacillus Thuringiensis Israelensis -Aedes Aegyptimentioning

confidence: 99%

Evaluation of a new tablet formulation based on Bacillus thuringiensis sorovar. israelensis for larvicidal control of Aedes aegypti

Melo-Santos

Sanches

et al. 2001

Mem. Inst. Oswaldo Cruz

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The effect of sunlight on the efficacy and persistence of an experimental tablet formulation based on Bacillus thuringiensis sorovar. israelensis (C4P1) was evaluated against Aedes aegypti larvae under simulated field conditions. The initial mortality ranged from 93 to 100%, and the residual activity (≥ 70% mortality) recorded in containers exposed to sunlight or shade were, respectively, 13-35 days and 40-54 days. The results suggest that C4P1 can provide long-term larvicidal effect and operational advantages. Key words: Bacillus thuringiensis israelensis -Aedes aegypti -tablet formulationBacillus thuringiensis sorovar. israelensis (B.t.i) has been used since the 80's in several programs for controlling black fly and mosquito species worldwide. It is considered highly toxic to the target organisms and environmentally safe. Furthermore, no resistance has been detected on populations submitted to long-term exposure to this pathogen (Regis & Nielsen-LeRoux 2000). B.t.i formulations have been continuously improved to optimise their potency, stability (shelf life), ease of field application and residual activity (Couch 2000). Trials under simulated field conditions have been considered a reliable methodology to evaluate the efficacy and persistence of B.t.i based products because, under these conditions, many factors involved in residual activity can be better controlled and investigated than under real field conditions (Thiéry et al. 1999.In the present work, the effect of sunlight on the efficacy and residual activity of a new tablet formulation based on B.t.i, C4P1 against Aedes aegypti larvae (Recife-Lab strain), was investigated under simulated field conditions. C4P1 is an experimental slow release solid formulation, developed by Far-Manguinhos-Fiocruz, Rio de Janeiro, Brazil. Initially, the larvicidal activity of the technical powder was determined by bioassay according to the standard protocol (de Barjac & LargetThiéry 1984). The LC 50 and the LC 90 were titrated against the B.t.i reference powder (IPS82), from Institut Pasteur. The technical powder showed LC 50 = 0.30 ± 0.01 mg/l and LC 90 = 0.44 ± 0.01 mg/l, resulting in potency of 1,146 ± 52 ITU/mg (International Toxic Units).The tests under simulated conditions were carried out in March (1st set) and May (2nd set) 2000 to evaluate the residual activity of C4P1 related to the incidence of sunlight. The containers, placed in an outdoor area, were submitted to direct sunlight and shade. The tests were conducted in plastic containers (38 x 42 x 30 cm) filled with 20 l of tap water where the temperature ranged from 27 to 29°C in the shade and from 27 to 32°C, in direct sunlight. Each container was treated with 1 tablet/ 10 l. Fifty early fourth-instar larvae (L4) were introduced into each container and the mortality rate was recorded 48 h after, by counting the surviving larvae. This procedure was always used to determine the initial mortality. Thereafter, the evaluation of residual activity was carried out using 50 L4 or 300 first instar (L1) larvae per conta...

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Section: Abstract: Bacillus Thuringiensis Israelensis -Aedes Aegyptimentioning

confidence: 99%

Evaluation of a new tablet formulation based on Bacillus thuringiensis sorovar. israelensis for larvicidal control of Aedes aegypti

Melo-Santos

Sanches

et al. 2001

Mem. Inst. Oswaldo Cruz

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“…In fact, these metabolic limitations hamper likely industrial developments with this species due to the high costs of the proteinaceous media used for toxin production compared to the costs of alternative media based on starch or molasses (5,18,26). Consequently, studies disclosing the true metabolic potential of B. sphaericus are essential for genetic-improvement programs.…”

mentioning

confidence: 99%

Existence of a True Phosphofructokinase in Bacillus sphaericus : Cloning and Sequencing of the pfk Gene

Alice

Pérez-Martı́nez

Sánchez-Rivas

2002

Appl Environ Microbiol

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Some strains of Bacillus sphaericus are entomopathogenic to mosquito larvae, which transmit diseases, such as filariasis and malaria, affecting millions of people worldwide. This species is unable to use hexoses and pentoses as unique carbon sources, which was proposed to be due to the lack of glycolytic enzymes, such as 6-phosphofructokinase (PFK). In this study, PFK activity was detected and the pfk gene was cloned and sequenced. Furthermore, this gene was shown to be present in strains belonging to all the homology groups of this heterogeneous species, in which PFK activity was also detected. A careful sequence analysis revealed the conservation of different catalytic and regulatory residues, as well as the enzyme's phylogenetic affiliation with the family of allosteric ATP-PFK enzymes.Bacillus sphaericus strains are strictly aerobic and mesophilic bacilli which form spherical endospores (15). On the basis of DNA homology, this species has been divided in five homology groups (11,17). In turn, group II was divided in two subgroups, IIA and IIB (11). Some strains in group IIA are biotechnologically very interesting, because they can produce insecticidal proteins which are active against mosquito larvae, in particular against various species of the genera Culex and Anopheles, which are known to be regular transmission vectors of filariasis and malaria (3,4,13,14,16).These bacteria can metabolize a wide variety of organic compounds and amino acids (1), but they are unable to use hexoses and pentoses as unique carbon sources (18). In fact, these metabolic limitations hamper likely industrial developments with this species due to the high costs of the proteinaceous media used for toxin production compared to the costs of alternative media based on starch or molasses (5,18,26). Consequently, studies disclosing the true metabolic potential of B. sphaericus are essential for genetic-improvement programs.The inability to metabolize carbohydrates in this species has been attributed to the absence of key enzyme activities in both the Embden-Meyerhof-Parnas pathway (phosphoglucoisomerase [EC 5.3 PFK activity in B. sphaericus. B. sphaericus and Bacillus subtilis strains were grown aerobically in Luria-Bertani (LB) medium at 30 and 37°C, respectively. At mid-log phase (optical density at 600 nm, 0.6 to 0.8), cells were harvested by centrifugation, washed twice with TDP buffer (20 mM Tris-HCl [pH 7.5], 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride), and resuspended in the same buffer at 4°C. Crude extracts were prepared by disrupting cells twice by 20-s sonication in the presence of glass beads and maintained refrigerated in an acetone-ice bath. Samples were centrifuged in a microcentrifuge at 17,400 ϫ g for 15 min, and the supernatants were used for the enzymatic assays. Reactions were performed in a 500-l final volume containing 50 mM Tris-HCl (pH 7.5), 5 mM Cl 2 Mg, 2 U each of aldolase (Sigma Chemical Co.) and triose phosphate isomerase-glyceraldehyde-3-phosphate dehydrogenase (Sigma Chemical Co.), 1 mM ...

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“…Due to the large consumption of oxygen during the cultivation, control of this parameter is very important and should not reach values below 20% (Couch, 2000). Controversy exists regarding the need for pH control during fermentation, and in some cases are used buffered media for not having an adequate system of control.…”

Section: Wwwintechopencommentioning

confidence: 99%

Bacillus sphaericus and Bacillus thuringiensis to Insect Control: Process Development of Small Scale Production to Pilot-Plant-Fermenters

Luna-Finkler¹,

Finkler²

2012

Insecticides - Advances in Integrated Pest Management

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Industrial fermentation and formulation of entomopathogenic bacteria

Cited by 32 publications

References 10 publications

Evaluation of a new tablet formulation based on Bacillus thuringiensis sorovar. israelensis for larvicidal control of Aedes aegypti

Evaluation of a new tablet formulation based on Bacillus thuringiensis sorovar. israelensis for larvicidal control of Aedes aegypti

Existence of a True Phosphofructokinase in Bacillus sphaericus : Cloning and Sequencing of the pfk Gene

Bacillus sphaericus and Bacillus thuringiensis to Insect Control: Process Development of Small Scale Production to Pilot-Plant-Fermenters

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