Δ-endotoxin Production Research Articles

Enhanced Bacillus thuringiensis Production from Sewage Sludge with Alkaline and Ultrasonic Pretreatments

Sewage sludge is a cost-effective media for the production of Bacillus thuringiensis (Bt) based biopesticides. To enhance the entomotoxicity of the fermentation broth, pretreatments of sewage sludge by alkali and ultrasonic were applied in this study. Effects of alkaline and ultrasonic pretreatments on the soluble COD (SCOD) and total COD (TCOD) were evaluated by altering the alkali addition dose and the ultrasonic specific energy. Suitable pretreatment conditions were optimized with 5 g l−1 sodium hydroxide (NaOH) for alkaline treatment and 1.2 × 105 kJ kg−1 of total solid for ultrasonic treatment. Fermentations of raw and pretreated sludge for biopesticides were carried out in a bench scale fermentor. Results revealed that both pretreatments were effective for Bt growth and metabolism. Higher viable cells (VC) and viable spores (VS) counts, δ-endotoxin yields and entomotoxicity were achieved in the pretreated sludge. The enhancement was attributed to more available nutrients and better oxygen transfer. Moreover, ultrasonic pretreated sludge was superior to alkaline pretreated sludge for δ-endotoxin production and entomotoxicity owing to its higher soluble C/N ratio and finer particles.

Optimization of the Bacillus thuringiensis var. kurstaki HD-1 δ-endotoxins production by using experimental mixture design and artificial neural networks

An experimental mixture design coupled with data analysis by means of both response surface methodology (RSM) and artificial neural networks (ANNs) followed by multiple response optimization through a desirability function, was applied to the production of δ-endotoxins from Bacillus thuringiensis var. kurstaki. The composition of a culture medium was defined by testing three regional effluents: milky effluent, beer wastewater and sugar cane molasses. Both RSM and ANNs accomplished the goal pursued in this work, by predicting the optimal mixture of the effluents. ANNs provided more reliable results due to the complexity of the models to be fitted. The optimal selected blend was: 74%, 26% and 0%, respectively for each the above-mentioned effluents.

Evidence of the effect of δ-endotoxin ratio in Bacillus thuringiensis crystals on the toxicity against Ephestia kuehniella

Abstract The effect of Bacillus thuringiensis cry gene copy-number on δ-endotoxin production and on toxicity has been investigated. When a wild-type strain of B. thuringiensis kurstaki, BNS3, was transformed with the recombinant plasmids pHTcry1Aa, pHTcry1Ac, and pHTcry2Aa, δ-endotoxin synthesis was enhanced by 269, 267, and 243%, respectively. In contrast, spore counts were decreased by at least 50% which was attributed to the cloning vector effect. When B. thuringiensis Cry1Ac or Cry2Aa crystal contents were increased, the recombinant crystals were two- to threefold as toxic as the wild-type crystals against Ephestia kuehniella, demonstrating the importance of δ-endotoxin ratio in the conception of optimal B. thuringiensis bioinsecticide formulation.

Nitrogen Fertility Effects on Bt δ-Endotoxin and Nitrogen Concentrations of Maize during Early Growth

Nitrogen deficiencies interfere with protein synthesis and growth in general in maize (Zea mays L.). Transgenic maize hybrids with the Bacillus thuringiensis Berliner (Bt) gene produce proteins that are solubilized and become insecticidal in the highly alkaline midgut of certain lepidopteran insects. The effect N fertility has on Bt δ-endotoxin and how whole-plant N concentrations relate to Bt δ-endotoxin levels has yet to be documented in early-growth maize. Two Bt maize hybrids (‘Pioneer 33V08’1 with Bt event MON-810 and ‘Dekalb 626Bty’1 with Bt event DBT 418) were grown in pots in duplicate greenhouse experiments with N fertility rates of 0, 112, 224, and 336 kg ha−1 N as NH4NO3. Fertilizer was blended into a potting mixture of a 2:1:1 ratio of peat moss/sand/soil at planting. Plants were harvested at growth stage V5 and assayed for Bt δ-endotoxin using a commercial quantification plate kit. Whole-plant N concentrations were determined by semimicro-Kjeldahl. Whole-plant N concentrations were 25.8, 33.1, 35.1, and 37.7 mg g−1, and Bt δ-endotoxin concentrations were 350, 367, 486, and 534 μg kg−1 at N fertility levels of 0, 112, 224, and 336 kg ha−1, respectively. Increased available N likely increases Bt δ-endotoxin–synthesizing proteins and thus increases the Bt δ-endotoxin concentration. The response of the two Bt hybrids to increased N fertility was similar. Adequate levels of N fertility during early growth appear essential for Bt δ-endotoxin production by the plant and may affect the ability of maize plants to resist insect predation.

Stirred tank culture of Bacillus thuringiensis H-14 for production of the mosquitocidal δ-endotoxin: mathematical modelling and scaling-up studies

The effect of culture conditions on δ-endotoxin production by strain S128 of Bacillus thuringiensis H-14 (locally isolated in Egypt) was investigated using a 10l working volume fermenter. Fermentation medium formulated from the inexpensive locally available soya (as a nitrogen source) and molasses (as a carbon source) was used. Aeration, agitation, pH and initial concentration of molasses were chosen as experimental factors and their influence on toxin yield was investigated using 24 central composite experimental design. The mathematical model obtained revealed that the optimal batch cultivation conditions with respect to agitation, pH, and initial concentration of molasses were 325 rev min−1, 7.1 and 2.1% (w/v) respectively. The mathematical model obtained indicated that by increasing the aeration rate over 0.89 v/v per minute the productivity could still be increased. A simulated scaling-up study, in which the Simplex method was applied, is also presented. The results of this investigation could be of great help for large-scale production of a cheap mosquito-larvicide in developing countries where mosquito-borne diseases are still a serious health and economic problem.

Evaluation of Bt Transgenic Sweet Corn Hybrids for Resistance to Corn Earworm and Fall Armyworm (Lepidoptera: Noctuidae) using a Meridic Diet Bioassay2

A laboratory bioassay was used to evaluate Bt transgenic sweet corn hybrids for resistance against the corn earworm, Helicoverpa zea (Boddie), and the fall armyworm, Spodoptera frugiperda (J. E. Smith). Whorl leaves, silks, and kernels, either fresh or oven-dried and ground with a mill, were incorporated into a dilute pinto bean diet and bioassayed against neonate, 3-, or 6-day-old larvae. Regardless of age of the larvae, results with the diet bioassay using fresh silks, oven-dried silks or fresh kernels were highly correlated with those for the fresh silk bioassay. Differences in susceptibility between insect species to the CrylA(b) toxin produced in the transgenic plants were also readily discernable using the diet bioassay. Based on results of the bioassays, Novartis sweet corn hybrids containing a crylA(b) gene gene for δ-endotoxin production were very highly resistant to leaf, silk and kernel feeding by the corn earworm and highly resistant to leaf and silk feeding by the fall armyworm.

Nutritional requirements of a strain of bacillus thuringiensis subsp. kurstaki and use of gruel hydrolysate for the formulation of a new medium for δ-endotoxin production

Nutritional requirements of a strain ofBacillus thuringiensis (Bt) subsp.kurstaki were elucidated for δ-endotoxin production. The effect of some principal nutrients was deeply investigated, showing several nutritional and metabolite limitations in Bt growth and δ-endotoxin synthesis. This led us to formulate a new medium based on the hydrolysate of gruel, a cheap and abundant byproduct of semolina factories, supporting growth and δ-endotoxin synthesis. After hydrolysis of gruel by α-amylase, followed by proteolysis using alcalase, the resultant soluble material substituted glucose very well for Bt δ-endotoxin production. Indeed, 15 g/L total sugars coming from that hydrolysate, supplemented by 5.4 g/L ammonium sulfate as nitrogen source and either 5 g/L yeast extract or 3 g/L peptone from casein or 3 g/L casaminoacids or 0.25 g/L cysteine or aspartic acid, were the principal components of this new medium in which almost 1 g/L of δ-endotoxin in 4.5 g/L total dry biomass was produced.

Kinetic study of Bacillus thuringiensis var. israelensis in lab-scale batch process

Fermentations of Bacillus thuringiensis have long traditions for the production of δ-endotoxin as bioinsecticide in agriculture and forestry. A process analysis study of endoprotein production by batch fermentation is presented in this paper. General fermentation characteristics are given and the influence of dissolved oxygen to sporulation and toxicity is discussed. Kinetics of growth and product formation is examined and a model based on Monod with an inhibition constant is used to describe the process. The model should provide a useful framework for further process verification and optimisation in practice.

Enhancement of δ-Endotoxin Activity by Toxin-Free Spore of Bacillus thuringiensis against the Diamondback Moth, Plutella xylostella

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.

Construction of cloning vectors for Bacillus thuringiensis

The replication region of the Bacillus thuringiensis plasmid, pHT1030, was treated with hydroxylamine. Various copy-number mutants were selected and subsequently used to construct shuttle vectors with multiple cloning sites. These recombinant plasmids are very stable and allowed the cloning of a δ-endotoxin-encoding gene in B. thuringiensis. Comparison between gene expression level and vector copy-number indicated that a plateau in δ-endotoxin production is reached with a copy-number of about fifteen per equivalent chromosome.

Studies on Bacillus thuringiensis H-14 strains isolated in egypt?IV. characterization of fermentation conditions for ?-endotoxin production

Fermentation media formulated from inexpensive locally available ingredients, namely soya meal, Proflo (a partially defatted cooked cottonseed fiour) and molasses, were assessed for growth, sporulation and δ-endotoxin production, by a mosquito-toxic strain of Bacillus thuringlensis H-14 locally isolated in Egypt, using an 8 I working volume fermenter. The insecticidal activity was assayed against Culex piplens. Good growth and sporulation were obtained with all the tested medium combinations, but a medium composed of 3% (w/v) soya meal and 1% (w/v) molasses was the best for δ-endotoxin production. The optimal batch cultivation conditions with respect to aeration (0.37 to 0.62 vol/vol min(-1)), agitation (200 rev/min), pH (6.5 to 7.5), temperature (30°C) and initial concentration of carbon (1% w/v) and nitrogen (3% w/v) sources are also presented. This investigation shows that these locally available ingredients could be used for the production of low-priced mosquito larvicide in Egypt and other developing countries where these ingredients are avallable.

Studies onBacillus thuringiensis H-14 strains isolated in Egypt?III. Selection of media for ?-endotoxin production

Several medium ingredients locally available in Egypt were investigated for their ability to support δ-endotoxin production by two mosquito-toxic strains ofBacillus thuringiensis H-14 usingCulex pipiens for the bioassay of δ-endotoxin. Soya beans, black-eyed beans, common peas and lentils supported good production of toxin whereas peanuts, fodder yeast, cheese whey and corn steep liquor gave only low amounts of toxin. Molasses as the sole carbon source at 2 and 3% (w/v) with soya (or Proflo) as the sole nitrogen source at 3% gave the best yields of toxin for strain M1 and S128 respectively.

Influence of media composition on the production of δ-endotoxin by Bacillus thuringiensis var. thuringiensis

Powders of edible leguminous seeds, greengram ( Vigna radiata) or soybean ( Glycine max), were used as the major protein source with different combinations of soluble starch and/or cane sugar molasses as the major carbohydrate source for the production of δ-endotoxin by Bacillus thuringiensis var. thuringiensis serotype 1 in submerged fermentation. The primary product (lyophilized with 6 g of lactose) yield was 8.7 to 9.1 g/liter from media with dehusked greengram powder and 9.7 to 10.3 g/liter from media with defatted soybean powder in basal medium. The toxicity of primary products was assayed against fifth-instar Bombyx mori larvae by force-feeding. The primary product from the medium containing defatted soybean powder and soluble starch gave a maximum viable spore count of 91.3 × 10 6/mg, with a corresponding potency of 35,800 IU/mg, whereas the medium containing dehusked greengram powder and cane sugar molasses gave a spore count of 49.5 × 10 6/mg, with a highest potency of 38,300 IU/mg. Either legume protein in combination with cane sugar molasses yielded primary product 2.1 to 2.4 times more potent than the U.S. standard. The combined carbohydrate source consisting of soluble starch and cane sugar molasses, irrespective of the source of protein in the media, drastically reduced δ-endotoxin production, thereby reducing the potency of the primary products compared to the U.S. standard.

Comparaison des profils plasmidiques de mutants oligosporogènes chez Bacillus thuringiensis var. kurstaki HD-1 après traitement à l'ozone et à la nitrosoguanidine

Two strains of Bacillus thuringiensis were used in order to isolate sporeless and crystalliferous mutants. These strains, B. thuringiensis var. kurstaki HD-1 and var. israelensis Q-2, produce a crystalline inclusion, δ-endotoxin, which is lethal for lepidopteran and dipteran larvae, respectively. Mutants derived from strain HD-1 were easily isolated because of their characteristic morphology. By comparing different plasmidic patterns of the strains, we can confirm the relationship existing between a DNA extrachromosomic element and the production of δ-endotoxin in B. thuringiensis. However, the absence of toxic crystallin protein does not necessarily mean that all plasmids are absent.Key words: Bacillus thuringiensis, oligosporogenous, ozone, nitrosoguanidine, plasmids.

Assignment of the δ-endotoxin gene of Bacillus thuringiensis var. israelensis to a specific plasmid by curing analysis

Plasmid analysis of Bacillus thuringiensis var. israelensis revealed the presence of at least 9 plasmids. A high frequency of plasmid loss occurred when this organism was grown at elevated temperature (42°C). Analysis of over 100 isolates cured of one or more plasmids by this method revealed that loss of a 72 MDa plasmid was invariably accompanied by loss of the ability to synthesize the insecticidal δ-endotoxin protein. Deletion of any of the other plasmids had no effect on δ-endotoxin production. These results indicate the presence of a plasmid-coded copy of the structural gene for the insecticidal δ-endotoxin in B. thuringiensis var. israelensis.

Novel fermentation media for production of δ-endotoxins from Bacillus thuringiensis

Novel approaches were pursued in the development of practical media for δ-endotoxin production by Bacillus thuringiensis subspp. kurstaki and entomocidus. Several agro-industrial by-products, including cottonseed meal, fish meal, beef blood, slaughterhouse residues, corn steep liquor, and sorter liquor, were investigated for their abilities to support toxin production by these varieties. In addition, fodder yeasts and a variety of low-priced plant proteins available in Egypt, exemplified by such leguminous seeds as horse beans, kidney beans, lima beans, soybeans, chick peas, lentils, and peanuts were incorporated in fermentation media as sole sources of proteins for biosynthesis of the endotoxins. The cotton pests, Spodoptera littoralis, Spodoptera exigua, and Heliothis armigera, were used as test insects for biological assays of spore-δ-endotoxin formulations derived from the novel fermentation media. Fodder yeast, beef blood, and slaughterhouse residues were among the by-products yielding good sporulation titers and potent spore-δ-endotoxin preparations. Formulations of subsp. kurstaki produced from media containing these nutrients killed 80–100% of larvae of H. armigera when tested at 500 μg/ml diet. Most of the formulations derived from fermentations using leguminous seeds as sole sources of protein also contained high levels of spores and endotoxin. For example, LC 50 values determined against S. littoralis of spore-endotoxin preparations of subsp. entomocidus grown in media containing kidney beans, chick peas, or peanuts were 93.4, 93.4, and 110.0 μg/ml diet, respectively. The application of these findings to the practical use of B. thuringiensis is discussed.

Formation of Crystalline δ-Endotoxin or Poly-β-Hydroxybutyric Acid Granules by Asporogenous Mutants of Bacillus thuringiensis

Parental strains and asporogenous mutants of Bacillus thuringiensis subspp. kurstaki and aizawai produced high yields of delta-endotoxin on M medium, which contained 330 mug of potassium per ml, but not on ST and ST-a media, each of which contained only 11 mug of potassium per ml. On ST and ST-a media, refractile granules were formed instead. These granules had no insecticidal activity against silkworms and were isolated and identified as poly-beta-hydroxybutyric acid. Supplementation of the potassium-deficient ST-a medium with 0.1% KH(2)PO(4) (3.7 mM) led to the formation of crystalline delta-endotoxin. The replacement of KH(2)PO(4) with equimolar amounts of KCl, KNO(3), and potassium acetate or an equivalent amount of K(2)SO(4) had a similar effect, whereas the addition of an equimolar amount of NaH(2)PO(4) or NH(4)H(2)PO(4) did not cause the endotoxin to form. An asporogenous mutant, B. thuringiensis subsp. kurstaki strain 290-1, produced delta-endotoxin on ST-a medium supplemented with 3 mM or more potassium but formed only poly-beta-hydroxybutyric acid granules on the media containing </=1 mM potassium. These results clearly indicate that a certain concentration of potassium is essential for the fermentative production of delta-endotoxin by these isolates of B. thuringiensis. Manganese could not be substituted for potassium. Phosphate ions stimulated poly-beta-hydroxybutyric acid formation by strain 290-1. The sporulation of B. thuringiensis and several other Bacillus strains was suppressed on the potassium-deficient ST medium. This suggests that potassium plays an essential role not only in Bacillus cell growth and delta-endotoxin formation but also in sporulation.

Correlation between specific plasmids and δ-endotoxin production in Bacillus thuringiensis

Five strains of Bacillus thuringiensis that produce crystalline δ-endotoxin were used as parental strains in an effort to isolate acrystalliferous (Cry −) mutants: HD-2 ( B. thuringiensis var. thuringiensis, flagellar serotype 1); HD-1 and HD-73 (both var. kurstaki, serotype 3ab); HD-4 (var. alesti, serotype 3a); and HD-8 (var. galleriae, serotype 5ab). The parental strains contain complex plasmid arrays that have been previously characterized ( González and Carlton, 1980). The plasmid patterns of both Cry − and Cry + variants were analyzed and compared to the parental strains using a modified Eckhardt (1978) lysate-electrophoresis method. Most Cry − mutants derived from strain HD-2 were found to exhibit a distinctive colony morphology which facilitated their isolation. Loss of crystal production was associated with loss of a 75-Md plasmid. A 50-Md plasmid of strain HD-73 was lost in the Cry − mutants. Crystal production in strain HD-4 appears to be associated with a plasmid about 105 Md in size; in strain HD-1, a smaller plasmid (29 Md in size) seems to be involved. In strain HD-8, a large plasmid (~130 Md in size) is implicated in crystal production. Direct bioassay of several of the mutant strains has confirmed the loss of δ-endotoxin activity in the acrystalliferous isolates. The evidence obtained supports the notion of a relationship between specific extrachromosomal DNA elements and δ-endotoxin production in B. thuringiensis, and suggests that in each strain only a single plasmid is involved, although the size of the implicated plasmid varies from one strain to another.

Production of δ-Endotoxin by Bacillus thuringiensis as a Function of Glucose Concentrations

The insect pathogen Bacillus thuringiensis was cultured at different glucose concentrations. Size, protein content, and insecticidal activity of the parasporal inclusions, which are formed during the sporulation process, were measured. Increased glucose concentrations led to bigger crystalline inclusions with a higher content of protein and insecticidal activity. Maximum yields of protein and endotoxin were obtained in a semisynthetic medium that contained glucose concentrations of 6 to 8 g/liter.

Production of δ-endotoxin by eighteen isolates of Bacillus thuringiensis, serotype 3, in 3 fermentation media

The spore-δ-endotoxin complex of Bacillus thuringiensis was recovered from beers produced by 16 isolates of B. thuringiensis var. alesti (serotype 3a) and by 2 isolates of B. thuringiensis var. kurstaki (serotype 3a, 3b). The amount of endotoxin produced by the isolates varied over a wide range and depended on the isolate used and the medium on which it was grown. Thus, the activity of preparations of B. thuriengiensis could not be predicted from the serotype. Five isolates produced higher yields of the δ-endotoxin than any previously reported.