Optimization of bioinsecticides overproduction by Bacillus thuringiensis subsp. kurstaki using linear regression

A multiple linear regression analyses were performed to screen for the significant factors simultaneously influencing production of delta-endotoxin, proteolytic activities and spore formation by a Bacillus thuringiensis kurstaki strain. Investigated factors included: pH of the medium, available oxygen and inoculum size. It was observed that oxygen availability was the most influencing setting on both delta-endotoxins production and spores counts, followed by initial pH of the medium and inoculum size. On other hand, pH of medium was found to be the most significant parameter for proteolytic activity, followed by inoculum size and dissolved oxygen. Our results suggested that the first order with two-factor interaction model seemed to be more satisfactory than simple first order model for optimization of delta-endotoxin overproduction. The coefficients of determination (R2) indicated a better adequacy of the second order models to justify the obtained data. Based on results, relationships between delta-endotoxins production, proteolytic activities and spores counts were established. Our results can help to balance delta-endotoxins production and its stability.Scopu

Experimental design and Bayesian networks for enhancement of delta-endotoxin production by Bacillus thuringiensis

Bacillus thuringiensis (Bt) is a Gram-positive bacterium. The entomopathogenic activity of Bt is related to the existence of the crystal consisting of protoxins, also called delta-endotoxins. In order to optimize and explain the production of delta-endotoxins of Bacillus thuringiensis kurstaki, we studied seven medium components: soybean meal, starch, KH2PO4, K2HPO4, FeSO4, MnSO4, and MgSO4 and their relationships with the concentration of delta-endotoxins using an experimental design (Plackett—Burman design) and Bayesian networks modelling. The effects of the ingredients of the culture medium on delta-endotoxins production were estimated. The developed model showed that different medium components are important for the Bacillus thuringiensis fermentation. The most important factors influenced the production of delta-endotoxins are FeSO4, K2HPO4, starch and soybean meal. Indeed, it was found that soybean meal, K2HPO4, KH2PO4 and starch also showed positive effect on the delta-endotoxins production. However, FeSO4 and MnSO4 expressed opposite effect. The developed model, based on Bayesian techniques, can automatically learn emerging models in data to serve in the prediction of delta-endotoxins concentrations. The constructed model in the present study implies that experimental design (Plackett—Burman design) joined with Bayesian networks method could be used for identification of effect variables on delta-endotoxins variation

Modeling-based optimization approaches for the development of Anti- Agrobacterium tumefaciens activity using Streptomyces sp TN71

A new aerobic bacterium TN71 was isolated from Tunisian Saharan soil and has been selected for its antimicrobial activity against phytopathogenic bacteria. Based on cellular morphology, physiological characterization and phylogenetic analysis, this isolate has been assigned as Streptomyces sp. TN71 strain. In an attempt to increase its anti-Agrobacterium tumefaciens activity, GYM + S (glucose, yeast extract, malt extract and starch) medium was selected out of five different production media and the medium composition was optimized. Plackett-Burman design (PBD) was used to select starch, malt extract and glucose as parameters having significant effects on antibacterial activity and a Box-Behnken design was applied for further optimization. The analysis revealed that the optimum concentrations for anti-A. tumefaciens activity of the tested variables were 19.49 g/L for starch, 5.06 g/L for malt extract and 2.07 g/L for glucose. Several Artificial Neural Networks (ANN): the Multilayer perceptron (MLP) and the Radial basis function (RBF) were also constructed to predict anti-A. tumefaciens activity. The comparison between experimental with predicted outputs from ANN and Response Surface Methodology (RSM) were studied. ANN model presents an improvement of 12.36% in terms of determination coefficients of anti A. tumefaciens activity. To our knowledge, this is the first work reporting the statistical versus artificial intelligence based modeling for optimization of bioactive molecules against phytopathogen

Concomitant production of delta-endotoxins and proteases of Bacillus thuringiensis subsp. kurstaki in a low-cost medium : effect of medium components

Bacillus thuringiensis is a bacterium, commonly used as a biological pesticide, and produces entomotoxic parasporal crystals (delta-endotoxins) and photolytic enzymes involved in several biological processes. The aim of present work was to enhance the production of delta-endotoxins by an isolated Bacillus thuringiensis subsp. kurstaki strain. The adopted approach was based on studying both delta-endotoxins and proteolytic activities production. Trials were carried out based on the Plackett-Burman experimental design. The statistical analysis revealed that the soybean meal was the main significant ingredient (confidence level= 99.5%) for both delta-endotoxins and proteolytic activities productions. Starch and FeSO4 were considered as significant ingredients for only protease production, the confidence levels were 98.8% and 97% respectively. The study of the nutrients effects brought out that K2HPO4, FeSO4 and starch, exhibit an opposite effect on the synthesis of delta-endotoxins and proteolytic activity production. On the other hand, MnSO4, MgSO4 and soybean meal are considered as the ingredients having dual positive effect on proteolytic activities and delta-endotoxin productions, whereas KH2PO4 had an inhibitory effect. The optimisation strategy using mathematical methods offers an efficient technique to optimize media leading to the increase delta-endotoxin production by Bacillus thuringiensis subsp. kurstaki strains by clustering of the effects of medium components

Statistical Analysis of Cultural Parameters Influencing Delta-Endotoxins and Proteases Productions by Bacillus thuringiensis kurstaki

Bacillus thuringiensis is a spore-forming bacterium that produces delta-endotoxins, which are toxic to lepidopterans, dipterans and coleopterans. Concomitantly with production of delta-endotoxins, B. thuringiensis produces proteolytic enzymes during growth and sporulation, affecting yields of toxins production. In this work, an experimental factorial design was employed for the dual evaluation of effects of delta-endotoxins and proteolytic enzyme on activity of a B. thuringiensis subsp. kurstaki strain. Main effects and interactions of three factors (soybean meal, starch and aeration) under two conditions (0 and 7 g/l NaCl) were analysed using statistical techniques. Regression models, known as useful tool to build relationships between variables and subsequently to make predictions, were suggested to fit the experimental data. Yields ranged between 6.10–15.76 × 10−7 μg and 18.18–30.30 × 10−7 IU for delta-endotoxin and proteolytic enzymes per cell, respectively. When NaCl (7 g/l) was added to the medium, yields ranged from 15.51 to 38.06 for delta-endotoxins and from 9.56 to 48.73 for proteases. Starch, soybean meal and aeration improved the delta-endotoxins production. Interactions (starch × air) and (soybean meal × air) showed a negative effect on delta-endotoxin yield, while (starch × soybean meal) had no effect. The active function of aeration was enhanced when NaCl (7 g/l) was added, and effects of all interactions on delta-endotoxins yield had been nullified. Soybean meal and aeration had negative effects on proteolytic activity, in contrast to starch, (starch × air) and (soybean × air), which improved proteolytic activity yields. The inhibitory role of soybean meal shifted to positive effect according to the presence of NaCl (7 g/l) in the culture medium.Scopu

Nutritional Requirements to Improve Delta-Endotoxins Production of Bacillus thuringiensis var. kurstaki Using Mixed Designs Modelling

Bacillus thuringiensiskurstaki is a soil bacterium that produces insecticidal toxins called delta-endotoxins. In order to increase the toxic crystal concentrations in a low-cost culture medium and thus improve the biopesticide quality to control insect pests, the Plackett Burman screening method was applied. It was shown a tool to evaluate the significance of the selected seven factors (KH2PO4, K2HPO4, MgSO4, MnSO4, FeSO4, soybean meal, starch) which are necessary to the production of the delta-endotoxins. This was performed into two different shake flasks (250 and 500 ml). The main factors that affected the production of delta-endotoxins are shown to be soybean meal, starch, and FeSO4 in 250 ml culture flasks. In 500 ml culture flasks, soybean meal and FeSO4 are the principal factors influencing the delta-endotoxin production. The multiple linear regression, a method applied as the merging dataset of the two Plackett Burman designs, established that soybean meal and starch are the factors positively affecting the production of delta-endotoxins, in contrast to FeSO4. Furthermore, the available oxygen in culture flasks showed no significant negative effect on delta-endotoxin production. This study revealed that mixed method designs were useful to identify the significance and the effect of hidden culture parameters.Scopu

Correlation between delta-endotoxin and proteolytic activities produced by Bacillus thuringiensis var. kurstaki growing in an economic production medium

Bacillus thuringiensis is a Gram positive bacterium that produces an insecticidal crystalline protein making it one of the most important biocontrol agents for pest management. Bioinsecticides based on B. thuringiensis were produced by fermentation processes in liquid media. Cultural conditions controlling proteolytic activities in different culture media were investigated to study the possible correlations between B. thuringiensis production of proteases and delta-endotoxins in a low-cost complex medium. Aeration appeared to play an important role in delta-endotoxin production. The correlation between proteolytic activity and aeration does not seem to be reliable. A negative correlation (correlation coefficient =− 0.774) was established between protease activity and delta-endotoxin production. In order to prove this correlation, protease hypo-producing and overproducing mutants were isolated through random mutagenesis of two wild strains, BUPM13 and BUPM5, by using nitrous acid. Interestingly, delta-endotoxin production of BUPM13-1, BUPM13-2 and BUPM13-3 was markedly improved when compared to the wild strain BUPM 13, reaching 2.1-fold, 3.69-fold and 8.13-fold, respectively. Maximal protease activity (540-2468 UI) obtained by BUPM5-1 and BUPM5-2 was 2.34-fold and 10.7-fold, respectively, more than that obtained by the wild strain BUPM5 with a drastic decrease of their delta-endotoxin production. Study of delta-endotoxin production by the selected mutants confirmed that insecticidal crystal protein stability in the culture strongly depends on the level of endogenous protease activity. This was also confirmed by bioassays measuring the LC50 using larvae of Ephestia kuehniella. Determining protease activity in fermentation culture could be useful in indirectly predicting the potency of B. thuringiensis strains with high insecticidal activities. This would allow low-cost selection of overproducing wild isolates or mutants in the screening programmes for the reduction of production cost, which is important from a practical point of view.Tunisian Ministry of Higher Education, Scientific Research and Technolog