Bioinsecticide Production Research Articles

Production of bio-insecticide from extracted carica papaya using NADES solvent with ultrasound-assisted extraction (UAE)

Bioinsecticide is a kind of organic pesticides that are easily degraded in nature and their residues are relatively unobstrusive to the plants and surrounding environment. The raw material of bioinsecticide production can be derived from the abundant plant wastes and has a cysteine protease compound that can damage the tissue and digestive system in plant pests, namely papaya waste. The content of cysteine protease in papaya waste in mostly in papaya skin that can be extracted using Ultrasound-assisted extraction (UAE) by varying sonication time and solid-to-liquid ratio with NADES solvent. Those variations will be comparaed using the enzyme activity test and the evication test. The results indicated that NADES solvent proved to be efficient solvents for extraction of papaya because papaya contains alkaloid that can be extracted by NADES with ChCl-oxalid acid is the type of NADES solvent.

PRODUKSI BIOINSEKTISIDA oleh Bacillus thuringiensis MENGGUNAKAN KULTIVASI MEDIA PADAT

*Penulis untuk korespondensi In bio insecticide production, the use of agricultural by products as a substrate to produce the starter has the role important on the efficient production processes, as well as the use of single isolate and production media. The bio insecticide production was carried out by the solid state cultivation, by examining the effect of substrate thick nessoncell growth and bio insecticide toxicity of bio insecticide produced. The bacterial of localisolates used were obtained from carrion worm of Attacus atlas, and identified as Bacillus thuringiens is and had important potential as bio insecticide. Tofu whey was used for starter media, while mixture of dregs sago and iles-iles as its production media. During the cultivation the media thickness affected on pH, cell growth, spores and substrate consumption. The increased of substrate thickness caused the decrease of pH, and simultaneously the decrease of cell growth, spores and substrate consumption. The product with highes toxicity capability against dipteralarvae (LC 50 of 0,24 mg/mL) and lepidoptera larvae (LC 50 of 3,3 mg/mL) was obtained under cultivation process with substrate thicknes of 1 cm. Keywords : Bacillus thuringiensis, bioinsecticide, solid-state cultivation, solid waste sago, solid waste iles-iles

Overcome of Carbon Catabolite Repression of Bioinsecticides Production by Sporeless Bacillus thuringiensis through Adequate Fermentation Technology.

The overcoming of catabolite repression, in bioinsecticides production by sporeless Bacillus thuringiensis strain S22 was investigated into fully controlled 3 L fermenter, using glucose based medium. When applying adequate oxygen profile throughout the fermentation period (75% oxygen saturation), it was possible to partially overcome the catabolite repression, normally occurring at high initial glucose concentrations (30 and 40 g/L glucose). Moreover, toxin production yield by sporeless strain S22 was markedly improved by the adoption of the fed-batch intermittent cultures technology. With 22.5 g/L glucose used into culture medium, toxin production was improved by about 36% when applying fed-batch culture compared to one batch. Consequently, the proposed fed-batch strategy was efficient for the overcome of the carbon catabolite repression. So, it was possible to overproduce insecticidal crystal proteins into highly concentrated medium.

Application of statistical experimental design for optimisation of bioinsecticides production by sporeless Bacillus thuringiensis strain on cheap medium

In order to overproduce bioinsecticides production by a sporeless Bacillus thuringiensis strain, an optimal composition of a cheap medium was defined using a response surface methodology. In a first step, a Plackett-Burman design used to evaluate the effects of eight medium components on delta-endotoxin production showed that starch, soya bean and sodium chloride exhibited significant effects on bioinsecticides production. In a second step, these parameters were selected for further optimisation by central composite design. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 30 g L(-1) starch, 30 g L(-1) soya bean and 9 g L(-1) sodium chloride. When compared to the basal production medium, an improvement in delta-endotoxin production up to 50% was noted. Moreover, relative toxin yield of sporeless Bacillus thuringiensis S22 was improved markedly by using optimised cheap medium (148.5 mg delta-endotoxins per g starch) when compared to the yield obtained in the basal medium (94.46 mg delta-endotoxins per g starch). Therefore, the use of optimised culture cheap medium appeared to be a good alternative for a low cost production of sporeless Bacillus thuringiensis bioinsecticides at industrial scale which is of great importance in practical point of view.

Overexpression of polyphosphate kinase gene (ppk) increases bioinsecticide production by Bacillus thuringiensis

Polyphosphate (polyP), synthesized by polyP kinase (PPK) using the terminal phosphate of ATP as substrate, performs important functions in every living cell. The present work reports on the relationship between polyP metabolism and bioinsecticide production in Bacillus thuringiensis subsp. israelensis (Bti). The ppk gene of Bti was cloned into vector pHT315 and the effect of its overexpression on endotoxin production was determined. Endotoxin production by the recombinant strain was found to be consistently higher than that by the wild type strain and the strain that carried the empty plasmid. The toxicity of the recombinant mutant strain (LC50 5.8±0.6ngml−1) against late 2nd instar Culex quinquefasciatus was about 7.7 times higher than that of Bti (LC50 44.9±7ngml−1). To our knowledge this is the first reported study which relates polyP metabolism with bioinsecticide biosynthesis.

Molecular Characterization and Genetic Diversity of Insecticidal Crystal Protein Genes in Native Bacillus thuringiensis Isolates

The Western Ghats of Karnataka natural ecosystem are among the most diverse and is one of the eight hottest hotspots of biological diversity in the world, that runs along the western part of India through four states including Karnataka. Bacillus thuringiensis (Bt) strains were isolated from soils of Western Ghats of Karnataka and characterized by molecular and analytical methods as a result of which 28 new Bt-like isolates were identified. Bt strains were isolated from soil samples using sodium acetate selection method. The morphology of crystals was studied using light and phase contrast microscopy. Isolates were further characterized for insecticidal cry gene by PCR, composition of toxins in bacterial crystals by SDS-PAGE cloning, sequencing and evaluation of toxicity was done. As a result 28 new Bt-like isolates were identified. Majority of the isolates showed the presence of a 55 kDa protein bands on SDS-PAGE while the rest showed 130, 73, 34, and 25 kDa bands. PCR analysis revealed predominance of Coleopteran-active cry genes in these isolates. The variations in the nucleotide sequences, crystal morphology, and mass of crystal protein(s) purified from the Bt isolates revealed genetic and molecular diversity. Three strains containing Coleopteran-active cry genes showed higher activity against larvae Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) than B. thuringiensis subsp. Morrisoni. Results indicated that Bt isolates could be utilized for bioinsecticide production, aiming to reduce the use of chemical insecticide which could be useful to use in integrated pest management to control agriculturally important pests for sustainable crop production.

Improvement of Photorhabdus temperata bioinsecticides production in low‐cost media through adequate fermentation technology

To develop a cost effective process for bioinsecticides production by Photorhabdus temperata, dissolved oxygen (DO) requirements were investigated in both the complex and the optimized media using diluted seawater as a source of micronutrients. By varying DO concentrations, tolerance to hydrogen peroxide was shown to be medium dependant. Indeed, P. temperata cells grown in the complex medium, exhibited higher tolerance than cells grown in the optimized medium (OM). Tolerance to H(2)O(2) was shown to be related to intracellular reactive oxygen species (ROS) accumulation during soya bean meal or glucose assimilation, as shown by flow cytometry analysis. To avoid oxidative stress damages in P. temperata cells cultured in the OM, DO concentration should be constant 50% saturation throughout the fermentation. However, a DO-shift control strategy was demonstrated to be beneficial for P. temperata bioinsecticide production in the complex medium. By using such a strategy biomass, culturability, and oral toxicity reached 16.5 × 10(8), 1.15 × 10(8) cells/mL and 64.2%, respectively, thus was 16.19, 26.37, and 12.2% more than in the cultures carried out at a constant 50% saturation.

Study of bioinsecticide production by Bacillus thuringiensis israelensis

Study of bioinsecticide production by Bacillus thuringiensis israelensis

Improvement of Photorhabdus temperata strain K122 bioinsecticide production by batch and fed-batch fermentations optimization

Optimization of a fermentation process for bioinsecticides production by Photorhabdus temperata strain K122 was investigated into fully controlled 3-L fermenter using an optimized medium (OM). Development of large-scale inocula showed that the composition of the growth medium greatly influenced the physiological state of P. temperata cells. The effect of pH, agitation and dissolved oxygen concentration (DO) on the growth, culturability and oral toxicity of P. temperata cells were also investigated. Indeed, maintaining the pH at 7 and controlling DO concentration at 50 % saturation throughout the fermentation process, improved biomass production, CFU counts and oral toxicity by 41.1, 35 and 32.1 %, respectively, as compared to cultures carried out in 500 mL shake flasks. At such conditions, 8 g/L glucose fed-batch fermentation, enhanced cell lysis and variants small colony (Vsm) polymorphism appearance. To overcome such limitations, glucose concentration should be maintained at 4 g/L. In this case, P. temperata cells were produced at high cell density and culturability reaching 4.5 and 1.2 × 10(9) cells/mL, respectively. In addition, the stability of the primary form was maintained for a long period in the stationary growth phase and Vsm polymorphism was completely avoided that can be crucial for scale-up the bioprocess of P. temperata bioinsecticide.

Bacteriocins from Qatari Bacillus Thuringiensis Crystal Producing Strains

Abstract Antimicrobial agents are very useful and have been the subject of intense research. Amongst these compounds, bacteriocins are defined as peptide antibiotics which do not harm the producer strain but have the ability to kill closely related bacteria. Bacillus thuringiensis is a friendly bacterium best known for the production of bioinsecticides, but is also considered as a source of bacteriocins active against the closely related pathogenic species harmful for food products, and many organisms. The major goal of this project is to evidence and characterize novel Bacillus thuringiensis bacteriocins synthesized by Qatari Bacillus thuringiensis strains. In order to obtain that, many approaches have been adopted: - Screening of delta-endotoxin producing strains of Bacillus thuringiensis. - Screening of bacteriocin producing strains of Bacillus thuringiensis. - Study of the antagonistic effects among bacteriocin producing strains. - Study of the effect of medium composition on bacteriocin synthesis. - Screening by PCR of Bacthuricin F4 like producing strains. In order to screen the bacteriocin producing strains of Bacillus thuringiensis, many techniques have been utilized such as: Direct Antagonism on Solid Media and Well Diffusion Method to check the bacteriocin activity. Bacteriocin production was studied on different growth media and against various indicator strains. The screening of 192 Bacillus thuringiensis strains isolated from Qatar, allowed the evidence of strains having different crystal forms. In fact, these strains produce parasporal crystals that have pyramidal, bipyramidal, cubical, spherical and amorphous forms. Among these Bacillus thuringiensis strains, about 70% produce spherical crystals and 30 % bipyramidal. Similar results were obtained in the laboratory by Sara Zakzok and Duha Rabah. This could be considered as a contribution to the establishment of Qatari bacterial gene bank. Screening the collection for bactericidal activities against indicator strains, revealed a variability in the size and aspect of the inhibition zones. About 76% of the collection were bacteriocin producing strains and only 24% were negative. There have been noticed many signs of novelty among our local bacteriocin producing strains. These results are very encouraging and in favour of the use of B. thuringiensis local strains for bacteriocin production.

PRODUKSI DAN FORMULASI BIOINSEKTISIDA DARI PROPAGUL AKTIF JAMUR BEAUVERIA BASSANIA

Beauveria bassiana is a well known entomopathogenic fungus that has been widely used for biocontrol of many economically important pest of crops. Production technology for massive production of bioinsecticide B. bassiana still using liquid fermentation. Product formulation is the critical stage for producing bioinsecticide B. bassiana, since its success indicator is the contain of viable propagules in the final product after long period of storage. The aim of this study is to assess the optimal production of biomass and formulation of bioinsecticide B. bassiana. Result showed that the best production technology of bioinsecticide B. bassiana is by liquid fermentation, as an innovative method aimed to reduce the production cost. Assessment of formulation of bioinsecticide B. bassiana showed that formulation using simple raw materials to make a pasta without filler is the best choice to be further developed.

Overcoming the production limitations of Photorhabdus temperata ssp. temperata strain K122 bioinsecticides in low-cost medium

For low-cost production of Photorhabdus temperata ssp. temperata strain K122 bioinsecticide, a cheap complex medium was optimized. Diluted seawater was used as the source of micronutrients, especially sodium chloride, involved in the improvement of cell density, culturability and oral toxicity of the bacterium P. temperata against Ephestia kuehniella larvae. Thus, the new formulated medium was composed only of 10 g/l of soya bean meal, used as the carbon and nitrogen main source, mixed in sevenfold diluted seawater. At such conditions, several limitations of P. temperata bioinsecticide productions were shown to be overcome. The appearance of variants small colony polymorphism was completely avoided. Thus, the strain K122 was maintained at the primary form even after prolonged incubation. Moreover, the viable but nonculturable state was partially overcome, since the ability of P. temperata cells to form colonies on the solid medium was prolonged until 78 h of incubation. In addition, when cultured in the complex medium, P. temperata cells were produced at high cell density of 12 × 10(8) cells/ml and exhibited 81.48% improvement of oral toxicity compared to those produced in the optimized medium. With such medium, the large-scale bioinsecticides production into 3-l fully controlled fermenter improved the total cell counts, CFU counts and oral toxicity by 20, 5.81 and 16.73%, respectively. This should contribute to a significant reduction of production cost of highly potent P. temperata strain K122 cells, useful as a bioinsecticide.

Improvement of Bioinsecticides Production by Sporeless Bacillus thuringiensis Strains in Response to Various Stresses in Low Cost Medium

The use of bioinsecticides, particularly those produced by sporeless Bacillus thuringiensis strains, has been shown to be a good alternative in pest management. Two types of sporeless mutants were distinguished. The asporogenic mutants which completely lack spores produce a regular bipyramidal crystal inclusion. The oligosporogenic mutants kept the ability to produce insecticidal crystal proteins. However, sporulation in such mutants was not totally blocked and very few of them could still produce spores. In order to improve bioinsecticides production, adaptation of sporeless strains to heat shock and osmotic stress was investigated. Delta-endotoxin production by 78% of sporeless mutants was significantly improved by osmotic stress with an overproduction of about 17%, compared to the wild strain BNS3. However, toxin production was improved by only 21% of mutants after heat shock, in low cost medium. The statistical analysis proved that delta-endotoxin production, cell growth, and spore formation of asporogenic and oligosporogenic mutants depended on the type of applied stress. Each strain has an important potential when applying the adequate stress. Moreover, adaptation of sporeless mutants to NaCl may allow the substitution of all minerals of the medium by diluted sea water which appeared to be a good alternative for the economic production of bioinsecticides at industrial scale which is of great importance from the practical point of view.

Improvement of Bacillus thuringiensis bioinsecticide production by sporeless and sporulating strains using response surface methodology

Statistical experimental designs, involving a Plackett-Burman design followed by a rotatable central composite design were used to optimize the culture medium constituents for Bacillus thuringiensis bioinsecticide production. This was carried out by using firstly an asporogenic strain and extrapolated to some sporeless and sporulating strains. Initial screening of production parameters was performed and the variables with statistically significant effects on delta-endotoxin production were identified: glucose, glycerol, yeast extract and MnSO(4). These variables were selected for further optimization by response surface methodology. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 22.5 g/l of glucose, 4.8g/l of glycerol, 5.8 g/l of yeast extract and 0.008 g/l of MnSO(4). Under these conditions, delta-endotoxin production was 2,130 and 2,260 mg/l into 250 and 1,000 ml flask respectively, which represent more than 38% improvement in toxin production over the basal medium (1,636 mg/l). Such medium composition was shown to be suitable for overproducing delta-endotoxins by sporeless and sporulating strains.

Biopesticide research and development: for safer agriculture, food and environment

In the frame of the protection of the environment that is continuously polluted by the massive use of chemical pesticides, we carried out a joint R&D project, on the development of biological pesticides using local bacterial strains isolated from Qatar and Tunisia. Microbial bioinsecticides were shown to be an efficient tool to control plant pests as well as human disease vectors. The Bacillus thuringiensis elta-endotoxins are the most valuable bioinsecticides currently used in commercial agriculture, forest management and mosquito control. This Gram-positive, spore-forming bacterium produces parasporal crystals composed of insecticidal elta-endotoxins. They exhibit a high specificity of insecticidal toxicity towards lepidopteran, coleopteran and dipteran insect species. From both Qatar and Tunisia, hundreds of strains of B. thuringiensis were isolated and studied and their bioinsecticides coding genes cry were cloned and characterized. Among the Tunisian strains, we evidenced the abundance of the kurstaki subspecies active on the lepidopteran olive tree pathogenic insect P. oleae, whereas from the Qatari soil samples, we found large heterogeneity among the isolated strains. Moreover Bti strains, used for the control of disease vector mosquitoes, were more abundant in the Qatari Bt strain collection than in the Tunisian one. On the other hand, very particular Qatari B. thuringiensis strains synthesizing particular crystals and harbouring different plasmid profiles and probably synthesizing novel insecticides were evidenced. Besides the genetic and molecular investigations, the development of a fermentation process for B. thuringiensis bioinsecticides production was also carried out and allowed us to do the scale-up of the production of bioinsecticides in a 430 litre fermenter. Important quantities of biological and environmentally safe insecticides were produced and applied successfully in the field.

Effects of Fetal Bovine Serum deprivation in cell cultures on the production of Anticarsia gemmatalis Multinucleopolyhedrovirus

BackgroundAnticarsia gemmatalis is a pest in South America's soybean crops, which could be controlled by the Multinucleopolyhedrovirus of A. gemmatalis (AgMNPV). Currently, its commercial production is based on infected larvae. However, the possibility of using modified baculoviruses in Integrated Pest Management programs has stimulated an interest to develop alternative multiplication processes. This study evaluated the AgMNPV production in UFL-Ag-286 cells previously deprived Fetal Bovine Serum.ResultsCulture media containing 1% FBS during the previous 48 hours achieved a synchronized condition where 90% of cells were found in G0/G1 stage, showing the presence of non-filamentous actin. All characteristics were estimated from cellular viability tests, cell actin detection trials and flow cytometer cell cycle analysis. AgMNPV production was tested by transcript studies and budded viruses (BVs) and occlusion bodies (OBs) yield quantitation. Results showed that the productivity in FBS deprived cells was 9.8 times more in BVs and 3.8 times more in OBs with respect to non-treated cells.ConclusionsUFL-Ag-286 cells previously deprived in FBS shown to be a better host for AgMNPV propagation, increasing the useful for both in vitro bioinsecticide production and applications such as recombinant protein expression or gene delivery.

Uso de bioinsecticidas para el control de plagas de hortalizas en comunidades rurales

Se evaluó el uso de bioinsecticidas elaborados a base de hongos entomopatógenos (Beauveria bassiana, Metarhizium anisopliae y Paecilomyces fumosoroseus); la producción se hizo de manera artesanal en comunidades rurales y se utilizaron para el control de plagas de hortalizas. Los productos fueron formulados con celite® y se usaron a dosis de 240g (1.2x1012 esporas/ha), para el control de plagas sobre plantaciones de lechuga Lactuca sativa L., rábano Rhapanus sativus, cebolla Allium cepa L., repollo Brassica oleracea var. capitata, papa Solanum tuberosum L. y cilantro Coriandrum sativum L. todos los aislamientos causaron mortalidades superiores al 80% a las 72 h, respecto al control, resultando efectivos para el control de la mariposa blanca de la col Pieris rapae, gusano dorso de diamante Pluxtella xylostella, gusano falso medidor Trichoplusia ni, pulgón de la col Brevycorine brassicae, Trips spp., palomilla de la papa Phthorimaea operculella, chicharrita Empoasca fabae, áfidos spp., minador Liriomyza trifolii y mosquita blanca Bemisia tabaci. Los bioinsecticidas beneficiaron a los productores de hortalizas ya que obtuvieron hortalizas libres de insecticidas químicos con mejores opciones de venta en el mercado

Development of a Cost Effective Medium for Production of Bacillus Thuringiensis Bioinsecticide Using Food Barley

Development of a Cost Effective Medium for Production ofBacillus ThuringiensisBioinsecticide Using Food BarleyGenetically manipulated food barley,Hordeum vulgareL. var.valfajr, was compared to commercial-grade starch as carbon sources in groundnut and soybean based media for supporting growth, sporulation and delta-endotoxin production byBacillus thuringiensisBerlinerB. thuringiensiskurstaki HD-1 and a promising newly isolatedB. thuringiensisstrain (referred to as BTA) were used. MgCl2, CaCl2, and MnCl2were used as trace-elements. Culture media were compared in shaken flasks and then in 5 l Fermentors. Biomass, delta-endotoxin levels and the number of spores as colony forming units (CFU) were evaluated. For each of the two strains, biomass and delta-endotoxin synthesis were not significantly different in soybean-based media after substitution starch by barley flour. There were significant differences between the two strains with respect to biomass and toxin production. Evaluated costs of media preparation showed that food barley is an economical alternative to commercial-grade starch in the production of BTA bioinsecticide.

Growth of Bacillus thuringiensis var. israelensis in glycerol derived from biodiesel industry aiming the production of bioinsecticide

Growth of Bacillus thuringiensis var. israelensis in glycerol derived from biodiesel industry aiming the production of bioinsecticide

Bioinsecticide production by the bacterium Bacillus thuringiensis. 1. Pattern of cell growth, toxin production and by-product synthesis

To develop a cost-effective process for the production of Bacillus thuringiensis-based insecticide, it is important to cultivate the bacterial strain in rich medium to obtain the highest yields of spore-crystal complexes. It was found that cultivation of the bacterium in medium with high concentrations of glucose (50–90 g l−1) resulted in much lower bacterial spores, crystal protein and lower toxicity, when tested against Spodoptera littoralis and Anagasta kuehniella larvae. The best results were obtained with glucose concentration of 20.0 g 1−1 as 7.1 × 1011 spores ml−1 and 3.4 g l−1 of crystal protein were achieved with LC50 of 40.1 and 50.2 mg kg−1 meal against S. littoralis and A. kuehniella, respectively. However, >21% of the consumed glucose was diverted into by-product synthesis at the expense of spore-crystal protein mixture. Only 78.3% of consumed glucose was converted into spores and crystal protein. Among by-products formed, acetic acid and β-hydroxybutyric acid (PHB) were produced during the phase of active growth and glutamic acid and succinic acid during the phase of active toxin production.