Δ-endotoxin Production Research Articles

Optimization of Culture Conditions and Wheat Bran Class Selection in the Production of Bacillus thuringiensis-Based Biopesticides

Bacillus thuringiensis is the leading microbial-based biopesticide, thanks to its parasporal crystal proteins or δ-endotoxins, which are toxic to insect larvae upon ingestion. Once in the insect larvae midgut, the crystal is solubilized by the alkaline pH and the δ-endotoxins activated by proteolytic cleavage. Thanks to its high efficiency as a biopesticide, several efforts have been made to enhance its growth and δ-endotoxins production, in various types of culture media. In this study, a culture medium based on wheat bran (WB), the by-product of cereal grain milling, was used to grow Bacillus thuringiensis and produce δ-endotoxins. Using the response surface methodology (RSM), the effects of three variables were evaluated: WB particles granulometry, their concentration, and their agitation in a 48-h shake-flask culture at 30 °C. Three response parameters were targeted: δ-endotoxins production, final culture pH, and dry-matter consumption. According to the RSM results, the optimum would be at 3.7 g WB/50 mL, with a granulometry above 680 μm and agitation between 170 and 270 rpm. This study is key to developing natural and cheap culture media that can be used at an industrial level for Bacillus thuringiensis-based biopesticides.

Application of scanning electron microscopy and MALDI-TOF MS in the exploration of the parasporal insecticidal crystals of Bacillus thuringiensis strains isolated from Qatar soil

Bacillus thuringiensis (Bt) plays an important role in health and agriculture due to its insecticidal activities. In this study, six Bt strains isolated from Qatar soil were used to explore their crystals morphologies and δ-endotoxin profiles. Bipyramidal crystal producing strains exhibited the genes vip3Aa58, cry1Aa/Ac, cry2Ab, cry1Ba and cry1Ia, predicting toxicity against lepidopteran, dipteran and hemipteran insects. A local strain QBT474 showed a high δ-endotoxin production of 647.32 ± 8.49 × 10−6 ng/cell compared to 359.259 ± 15.02 × 10−6 ng/cell in reference HD1 strain. The scanning electron microscopy showed bipyramidal, cuboidal and spherical crystals among the strains. MALDI-TOF-MS crystal protein profile data allowed to evidence differences in Bt crystal proteins. This is a first report that MALDI-TOF could be used in differentiating Bt δ-endotoxins in the strains forming same crystal forms. This study also demonstrates that local Bt strains might be considered for the industrial production of biological insecticides.

IMPACT OF SINGLE AND TWO-PHASE DISSOLVED OXYGEN TENSION CONTROL ON Bacillus thuringiensis CULTIVATION AND δ-ENDOTOXIN PRODUCTION

Constant δ-endotoxin production is crucial in Bacillus thuringiensis cultivation with aeration strategy playing a significant role in the process. In this study, the impact of DOT control strategies on the cultivation performance of B. thuringiensis was investigated using a 5-L stirred tank bioreactor. Single-phase DOT control recorded low spore count (<5.0 × 1011 spore/mL) and low percentage of sporulation (<45%). Two-stage DOT control cultivation with DOT at 80% during the active growth phase, followed by a switch to 60% or 40% at mid-exponential growth phase contributed to enhanced sporulation (>60%), high maximum cell count (1.5 cfu/mL) with δ-endotoxin formed as early as 8 hr of cultivation. A high maximum specific growth rate, μmax (>0.1 h-1) was also essential in ensuring δ-endotoxin production. High cell count obtained in cultivation with DOT level of 80% saturation during active growth was concurrent with high cell count, high μmax, fast generation time, high OUR, high CER value, and high respiratory quotient (RQ) value. During the two-phase DOT control cultivation, low RQ values were reported. The result showed that the exhaust gas data could be used to monitor B. thuringiensis cultivation performance.

Effect of the volumetric oxygen mass transfer coefficient on producing δ-endotoxins by Bacillus thuringiensis in culture medium based on forage palm

Bacillus thuringiensis is the main commercial source of safe insecticidal toxins. On the industrial scale, oxygenation is the most important variable for enhancing the production of δ-endotoxin by B. thuringiensis and improving its toxicity. Therefore, the parameters of growth kinetics, the formation of spores, the production of δ-endotoxin, and its toxicity against Spodoptera frugiperda were evaluated in batch cultures of B. thuringiensis cultivated in a culture medium based on forage palm with different KLα values. In all conditions, diauxic growth curves were observed due to the change of carbon source. Maximum cell concentration increased when KLα values were raised from 11 to 16 h−1, but biomass productivity was enhanced only when KLa was increased from 11 to 13 h−1. Due to the diauxic growth, spore production and δ-endotoxin production were observed at two different times. The first peak was generally lower than the second peak. At both peaks, a high concentration of spores was reached using a KLa of 13 h−1, but the highest production of δ-endotoxin and the maximum toxin yield based on spores or substrate were observed in the KLa of 16 h−1. Therefore, a bioassay against S. frugiperda was performed using δ-endotoxin produced under a KLa of 16 h−1. Although more δ-endotoxin was produced in the second peak than in the first peak, the highest toxicity was observed with toxins produced in the first peak. These results suggest that the KLα affects the production of δ-endotoxin and that toxicity against S. frugiperda decreases when fermentation time is increased.

Development and evaluation of lepidopteran insect resistant jute expressing the fused Bt-Cry1Ab/Ac toxin driven by CaMV35S promoter

The global demand for jute fibres in industrial and commercial applications makes jute (Corchorus sp.) a popular cash crop. Jute is an ideal eco friendly material for packaging and product manufacturing owing to its long, shiny, non-toxic, biodegradable bast fibres and high yield of cellulosic biomass produced per acre in cultivation. In order to match the increasing global demand for jute fibres, sustainable protection of jute cultivation from insect pests is essential. Lepidopteran pest resistant Bt (Bacillus thuringiensis) jute lines were developed by expressing the fused Bt-cry1Ab/Ac gene, driven by the CaMV35S constitutive promoter in the white jute (C. capsularis) cultiver- JRC321 and named as CBT (CaMV35S Bt). Molecular analyses confirmed the single copy integration of the Bt-cry1Ab/Ac gene in the transgenic CBT lines and 0.25 ng/mg of Bt-Cry1Ab/Ac δ-endotoxin production in the leaf tissue. The study involved evaluation of the insect resistance and the level of Bt-cry1Ab/Ac gene expression in different parts (leaf, stem, seed coat, and root) of the CBT lines in comparison with the previously developed, actin1 promoter driven Bt-cry1Ab/Ac gene containing, ABT lines (actin1 Bt). The CBT and ABT (Bt jute) lines were tested against two major jute pests- hairy caterpillars (Spilarctia obliqua) and indigo caterpillars (Spodoptera exigua). The Abbott’s insect mortality was observed for hairy caterpillars and indigo caterpillars on CBT lines as 90.74 % and 76.37 % respectively and on ABT lines as 97.11 % and 80.74 % respectively. Progeny plants of both the CBT and ABT lines showed similar and significant effects of insect resistance on the test insects under bioassay. Quantification of the expression of Bt-cry1Ab/Ac gene transcripts was similar in the leaves of both CBT and ABT lines but for stems, seed coats, and roots, the expression was found to be higher in ABT lines. This study confirmed that ABT lines were superior and more promising than CBT lines for jute pest management. The progeny plants from both CBT and ABT lines were similar to WT plants in terms of agronomic parameters.

Optimization of a culture medium based on forage palm for δ-endotoxin production

An alternative culture medium based on forage palm (Cactaceae) and inexpensive nitrogen sources for δ-endotoxin production by Bacillus thuringiensis var. berliner (Btb) was optimized using response surface methodology and the influence of the nitrogen sources on biological activity and morphological properties of the δ-endotoxin has been studied. Effects of forage palm extract concentration (15–25%), ammonium sulphate (0–1 g/L), ammonium chloride (0–1 g/L) and urea concentrations (0–1 g/L) on δ-endotoxin production were studied by a 2 4 full factorial design and optimized by a 2 2 central composite design (CCD). Statistical analysis showed that forage palm and ammonium sulphate were the main factors to enhance δ-endotoxin production. Different nitrogen sources influenced on biological activity and morphology of crystals. Culture medium constituted by forage palm extract and ammonium sulphate increased δ-endotoxins production in the form of bipyramidal crystals and was more toxic to Spodoptera frugiperda . Forage palm extract and ammonium sulphate were considered to optimization of δ-endotoxin production. Maximum δ-endotoxin production (356 mg/L) took place at 30.7% (w/v) of forage palm extract concentration and 1.016 g/L of ammonium sulphate. • δ-endotoxins were produced in an inexpensive forage palm-based culture medium. • N. cochenillifera is more appropriate as a carbon source to δ-endotoxin production. • Ammonium sulphate increases the δ-endotoxin production and its toxicity. • The optimum medium had 30.7% N. cochenillifera and 1.016 g/L ammonium sulphate. • Optimum culture medium allowed the production of 356 mg/L of δ-endotoxin.

Structural approaches for the DNA binding motifs prediction in Bacillus thuringiensis sigma-E transcription factor (σETF).

The sigma-E transcription factor (σETF) can be found in most of the bacteria cells including Bacillus thuringiensis. However, the cellular regulatory mechanisms of these transcription factors in the mass production of δ-endotoxins during sporulation stage are yet to be revealed. In addition, the recognition of DNA towards σETF DNA binding motifs that led to the transcription activities is also being poorly studied. Therefore, this work studied the possible DNA binding motifs of σETF by utilising in silico approaches. The structure of σETF was first built via three different computational methods. A cognate DNA sequence was then docked to the predicted σETF DNA-binding motifs. The binding free energy calculated using molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) for triplicate 50ns simulation of σETF-DNA complex revealed favourable binding energy of DNA to σETF (average ∆Gbind = -34.57kcal/mol) mainly driven by non-polar interactions. This study revealed that σETF LYS131, ARG133, PHE138, TRP146, ARG222, LYS225 and ARG226 are most likely the key residues upon the binding and recognition of DNA prior to transcription actives. Since determination of genome-regulating protein which recognises specific DNA sequence is important to discriminate between the proteins preferences for different genes, this study might provide some understanding on the possible σETF-DNA recognition prior to transcription initiated for the δ-endotoxins production.

Substrate Feeding Strategy Integrated with a Biomass Bayesian Estimator for a Biotechnological Process

Abstract This work proposes a substrate feeding strategy for a bioprocess integrated with a biomass estimator based in nonlinear filtering techniques. The performance of the proposed estimator and the substrate strategy are illustrated for the δ-endotoxin production of Bacillus thuringiensis (Bt) in batch and fed batch cultures. Nonlinear filtering techniques constitutes an adequate option as estimation tool because of the strongly nonlinear dynamics of this bioprocess and also due to nature of the uncertainties and perturbations that cannot be supposed Gaussians distributed. Biomass estimation is performed from substrate and dissolved oxygen. Substrate feeding strategy is intended to obtain high product concentration. Simulations results along with their experimental verifications demonstrate the acceptable performance of the proposed biomass estimator and the substrate feeding strategy.

The effect of aeration conditions, characterized by the volumetric mass transfer coefficient KLa, on the fermentation kinetics of Bacillus thuringiensis kurstaki

The aeration is a key factor for Bacillus thuringiensis growth, sporulation and δ-endotoxins production. The objective of our work was to study the effect of aeration on the fermentation kinetics of Bacillus thuringiensis kurstaki (Btk), cultivated in a cereal milling byproduct (CMB) mono-component medium, in order to improve the δ-endotoxins productivity. Aeration conditions were systematically characterized by the volumetric mass transfer coefficient KLa. In the 6% CMB culture medium, different values of the maximal specific oxygen uptake rate were obtained at different values of KLa. For KLa of 7.2 h(-1), the growth was inhibited and the sporulation was defective. There was a linear increase of the average specific growth rate and faster sporulation and liberation of spores and δ-endotoxins crystals when KLa was increased between 13.3 h(-1) and 65.5 h(-1). Similar kinetic was observed in cultures performed at KLa equal to 65.5 h(-1) and 106.2 h(-1). The highest toxins productivity of 96.1 mg L(-1) (h)-1 was obtained in the 9% CMB culture medium for KLa of 102 h(-1). It was possible to track the evolution of the bacterial cells between vegetative growth, sporulation and liberation of mature spores by following the variation of the CO2 percent in the effluent gas.

Biphasic fermentation is an efficient strategy for the overproduction of δ-endotoxin from Bacillus thuringiensis.

This study illustrates a biphasic solid-state fermentation (SSF) strategy for the overproduction of δ-endotoxin from Bacillus thuringiensis subsp. kurstaki (Btk) and also purification of δ-endotoxin from the solid-fermented medium. The fermentation strategy had two phases (biphasic); i.e., the first short phase was semisolid state (12 h), and the remaining long phase was strict SSF. To achieve the biphasic SSF, after 12 h (150 rpm, 37 °C) fermentation of the medium [Luria-Bertani (LB) supplemented with 30 % (w/v) raw soybean flour (phase I)], the supernatant in it was completely centrifuged out (1,000 × g, 10 min) aseptically for harvesting the extracellular enzymes as by-product. The resultant wet solid matter without free-flowing liquid but with embedded Btk was incubated 60 h more (phase II) for enhancing δ-endotoxin production at static condition (37 °C). Coupled with this, δ-endotoxin was purified by the modified phase separation method, and its purity was physically confirmed by both staining and microscopic techniques. The maximum δ-endotoxin yield from solid medium (48 h) was 15.8 mg/mL (recovery was 55-59 %) LB-equivalent, while that of LB control (recovery was 95 %) was only 0.43 mg/mL (72 h), i.e., thus, in comparison, 36.74-fold more yield in solid medium obtained by 24 h less gestation period. The purified crystal proteins showed apparent molecular weights (MWs) of 45, 35, and 6 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, this unique study physically demonstrates how Btk δ-endotoxin is purified (95-99 % purity) from solid-fermented matter for the first time, coupled with its overproduction at the expense of only 21.5 % higher production cost.

Selection and characterization of Bacillus thuringiensis mutants over-producing δ-endotoxins

Improvement of the microbial strain by subjecting the genetic material to mutagenic agents offers an opportunity to reduce the production cost. This study aims to enhance the δ-endotoxin production of Bacillus thuringiensis strain S24 that is 2-fold more toxic than the commercialized strain HD1 against Ephestia kuehniella (Lepidoptera: Pyralidae) larvae through random mutagenesis. B. thuringiensis mutants were generated from the S24 strain by using nitrous acid, as chemical mutagenic agent. Three mutants (M29, M12 and M20) proved to be the most promising δ-endotoxin producer among 30 mutants analyzed in a screening program performed in shake flasks using a complex medium based on starch and soya bean. These mutants demonstrated an improvement of δ-endotoxin production of up to 21 and 74% compared with the parental wild strain S24 and HD1, respectively. The characterization of the over-producing mutants showed that their colony morphology, crystal shape, plasmid and protein patterns, cry gene content and toxicity are not affected by mutagenesis treatment. However, the crystal protein patterns of three under-producing mutants (M21, M14 and M11) were different to that of the parental wild strain. The high potentiality of the over-producing mutants makes them excellent candidates as biological agents for the control of pyralid stored-product pests.

Solid-State Fermentation for the Concomitant Production of δ-Endotoxin and Endospore from <i>Bacillus thuringiensis</i> subsp. <i>kurstaki</i>

Water stress and limited aeration imparted by solid-state fermentation (SSF) were reported as crucial factors for the enhancement of endospore production by Bacillus thuringiensis (Bt); and thus, more δ-endotoxin could be produced concomitantly with reduced time. Therefore, Bt subsp. kurstaki (Btk) was employed in the present study to evaluate its efficiency for the concomitant production of endospores and δ-endotoxin in LB medium supplemented with various naturally available agricultural products, i.e., flours of soybean, Bengal gram or jack seed at various concentrations (10%, 20%, 30%, 40%, 50%, 60%, 80% or 100%, all w/v). After 12 h fermentation, the supernatant in it was centrifuged off aseptically to obtain solid substrate for subsequent SSF. Of them, soybean (30%) supplemented medium was the best for the enhanced production of endospore and δ-crystals. The maximum yield of endospores during solid-state fermentation was observed 48 h, i.e., compared to submerged fermentation in LB, it was 24 h less gestation period. In control sample, the endospores achieved the maximum length (1.10 ± 0.13 μm) and diameter (0.63 ± 0.07 μm) at 72 h; while in soybean supplemented medium, the maximum length (2.10 ± 0.16 μm) and diameter (1.63 ± 0.16 μm) were at 48 h and 72 h, respectively. Upon staining, acridine orange specifically stained the endospores; malachite green-saffranin stained both δ-crystals and endospores; and coomassie brilliant blue specifically stained δ-endotoxin. Briefly, normal gestation period or harvest time for Btk is 72 h, which could be reduced to 48 h, if SSF is employed as demonstrated in this study.

Dual production of amylase and δ-endotoxin by Bacillus thuringiensis subsp. kurstaki during biphasic fermentation

This study examined the efficacy of a Bacillus thuringiensis (Bt) strain in producing amylase (EC 3.2.1.1) as a by-product without affecting its unique ability for producing δ-endotoxin, thus to establish a cultivation strategy for the dual production and recovery of both δ-endotoxin and amylase from the fermented medium with an industrial perspective. LB medium was individually supplemented (5 to 100%, wt/vol) with flour from six naturally available starchy stored foods (banana, Bengal gram, jack seed, potato, taro or tapioca); after initial fermentation (12 h), the supernatant in the medium obtained by centrifugation (1000 g, 10 min) was used for harvesting amylase and the resultant pellet was further incubated aseptically for the production of endospores and δ-endotoxin by solid-state fermentation. Maximum crude amylase activity (867 U/gram dry substrate, 12 h) was observed in potato flour-supplemented medium (10% wt/vol, 12 h), while the activity in LB control was only 4.36 U/mL. SDS-PAGE profile of the crude (supernatant), as well as partially purified (40–60% (NH4)2SO4 fractionation) amylase showed that its apparent molecular mass was 51 kDa, which was further confirmed by native PAGE. The harvest of industrially significant extracellular amylase (probably α-amylase) produced as a byproduct during early growth phase would boost the economics of the Bt-based bio-industry engaged in δ-endotoxin production.

Selection and characterisation of an HD1‐like Bacillus thuringiensis isolate with a high insecticidal activity against Spodoptera littoralis (Lepidoptera: Noctuidae)

Spodoptera littoralis (Boisduval) larvae are known by their susceptibility to Bacillus thuringiensis subsp. aizawai strains. In order to prevent the appearance of B. thuringiensis (Bt) resistance and to develop economical Bt-based biopesticides, the selection and the characterisation of a B. thuringiensis isolate toxic against S. littoralis larvae and overproducing δ-endotoxins were investigated. Among 124 Tunisian B. thuringiensis isolates assessed against S. littoralis larvae, four isolates showed toxicity similar to and higher than the toxicity of the aizawai strain HD133 and the kurstaki strain HD1 respectively. The plasmid pattern of the selected isolates was similar to that of HD1. Polymerase chain reaction (PCR) analysis using specific primers revealed that these isolates present different gene contents. The only detected gene encoding Spodoptera-specific toxin was cry9. The selected isolates were found to produce bipyramidal and cubic crystals. The assessment of δ-endotoxin production by these isolates showed that BUPM28 produced 43.71 and 80.81% more δ-endotoxin than HD1 and HD133 respectively. The application of osmotic or heat shock stress on the BUPM28 isolate made it possible to enhance δ-endotoxin production by 22 and 23% respectively. On the basis of its potent insecticidal activity and its high level of δ-endotoxin production, the BUPM28 isolate can be considered to be an effective alternative for the control of S. littoralis.

Potato flour mediated solid-state fermentation for the enhanced production of Bacillus thuringiensis-toxin

In this study, we explored the efficacy of raw potato flour (PF) as supplement to the conventional LB medium (LB control, designated as M1) for enhancing the concomitant production of endospores and δ-endotoxin from Bacillus thuringiensis subsp. kurstaki by solid-state fermentation (SSF). Of different concentrations and combinations of media tested, 10% (w/v) PF supplemented LB medium (M2) was found as the best source for the maximum yield of toxin. After 12h submerged fermentation (SmF) at 37°C and 125rpm, M2 was made into a wet-solid matter for SSF by removing the supernatant (1000 ×g, 10min); the resultant pellet subsequently incubated statically (37°C) for the production of B.thuringiensis subsp. kurstaki toxin (Btk-toxin). In comparison to M1, yield of δ-endotoxin purified by sucrose density gradient centrifugation method from M2 was about 6-fold higher (53% recovery). This maximum yield from M2 was obtained at 48h (as against 72h from M1), thus the gestation period of M2 was reduced by 24h with higher yield. In addition to the quantitative data, qualitative photomicrographs taken by image analyzer, scanning electron and fluorescent microscopes and digital camera showed physical evidences for the upper hand of SSF over conventional SmF for the enhanced production of Btk-toxin. SDS-PAGE image of the purified δ-endotoxin showed three major fractions with apparent MWs 66, 45 and 30kDa. Briefly, if low-cost agricultural products like PF is used as supplement to LB, by SSF strategy, production of Btk-toxin could be enhanced to 6-fold in short gestation time without losing its entomotoxicity efficiency.

Oxygen supply in Bacillus thuringiensis fermentations: bringing new insights on their impact on sporulation and δ-endotoxin production

The growth kinetics, sporulation, and toxicity of Bacillus thuringiensis var. israelensis were evaluated through the analysis of batch cultures with different dissolved oxygen (DO) profiles. Firstly, DO was maintained constant at 5%, 20%, or 50% throughout fermentation in order to identify the most suitable one to improve the main process parameters. Higher biomass concentration, cell productivity, and cell yield based on glucose were obtained with 50% DO. The higher aeration level also resulted in higher spore counts and markedly improved the toxic activity of the fermentation broth, which was 9-fold greater than that obtained with 5% DO (LC(50) of 39 and 329 mg/L, respectively). Subsequently, using a two-stage oxygen supply strategy, DO was kept at 50% during the vegetative and transition phases until the maximum cell concentration was achieved. Then, DO was changed to 0%, 5%, 20%, or 100% throughout sporulation and cell lysis phases. The interruption of oxygen supply strongly reduced the spore production and thoroughly repressed the toxin synthesis. On the contrary, when DO was raised to 100% of saturation, toxic activity increased approximately four times (LC(50) of 8.2 mg/L) in comparison with the mean values reached with lower DO levels, even though spore counts were lower than that from the 50% DO assay. When pure oxygen was used instead of normal air, it was possible to obtain 70% of the total biomass concentration achieved in the air assays; however, cultures did not sporulate and the toxin synthesis was consequently suppressed.

Including dissolved oxygen dynamics into the Bt δ-endotoxins production process model and its application to process control

This paper proposes a model to characterize the Dissolved Oxygen Dynamics (DO) for the Bacillus thuringiensis (Bt) δ-endotoxins production process. The objective of this work is to include this dynamics into a phenomenological model of the process in order to facilitate the biomass estimation from the knowledge of oxygen consumption; and for control purposes, by allowing the addition of a new control variable in order to favorably influence the bioprocess evolution. The mentioned DO model is based on first principles and parameter estimation and model verification are supported by real experimental data. Finally, a control strategy is designed based on this model with its corresponding asymptotic stability and robustness analysis.

Biomass estimation in batch biotechnological processes by Bayesian Gaussian process regression

This paper proposes a biomass concentration estimator for a batch biotechnological process based on Bayesian regression with Gaussian process. On the basis of experimental data, a two-stage bootstrap technique has been developed for the estimator design. In the first stage, the biomass data set was augmented with virtual filtered measurements, and in the second stage, the biomass estimator design was completed. The method provides information on the confidence level of the estimates, and the biomass estimator performances are illustrated for the Bacillus thuringiensis δ-endotoxins production process.

Influence of amino nitrogen in the culture medium enhances the production of δ-endotoxin and biomass of Bacillus thuringiensis var. israelensis for the large-scale production of the mosquito control agent

We reported here the role of amino nitrogen in the commercial production of Bacillus thuringiensis var. israelensis media design. The insect pathogen B. thuringiensis var. israelensis was cultured in different media containing varying initial levels of amino nitrogen sources obtained from three different commercial venders. The biomass, mosquito larval toxicity and spore count produced were measured during the fermentation process. The results showed that the higher level of initial amino nitrogen concentrations in the medium led to higher yield of biomass (dry weight 4.78 g l(-1)), larvicidal activity (LC(50) 18.52 ng ml(-1)) and spore count (3.24 x 10(11) CFU ml(-1)). Similarly decreasing the initial amino nitrogen concentration in the medium led to a decreased biomass (dry weight 1.64 g l(-1)), larvicidal activity (LC(50) 27.01 ng ml(-1)) and spore count (3.7 x 10(10) CFUml(-1)).

Starch processing wastewater as a new medium for production of Bacillus thuringiensis

Production of Bacillus thuringiensis (Bt) based bioinsecticide was studied by using starch processing wastewater (SPW) as a raw material. Results indicated that the nutrients contained in SPW were sufficient for growth, sporulation and δ-endotoxin production of Bacillusthuringiensis subsp. kurstaki (Btk). The final cell counts and spore counts achieved in SPW medium were 72% and 107% respectively higher than those in the soybean meal based commercial medium. Higher δ-endotoxin yield of 2.67 mg mL−1 and higher entomotoxicity of 1,050 IU μL−1 were also obtained in SPW medium as compared with the commercial medium at the end of fermentation. The morphological observations also revealed that the fermentation cycle of Btk could be shortened in this new medium. This process provides solutions for safe SPW disposal and production of high potency and low cost bioinsecticide.