A selected bacterial strain for the self-healing process in cementitious specimens without cell immobilization steps.

The use of microorganisms capable of mediating the bioprecipitation process can be an important application in the self-healing processes of cement specimens. Thus, the present study identified and evaluated five Bacillus strains for potential application in the protocol of self-healing via bioprecipitation. Cell growth, enzyme production, and kinetic parameters conditions were evaluated during the fermentation process. Based on the analysis of 16S rDNA in conjunction with biochemical testing, results demonstrate that the strains are either Bacillus cereus or Bacillus thuringiensis. Strategically it was found that the addition of glycerol to fermentative medium was essential to increase the bacterial concentration (≈ 4.2 × 107 cells mL-1) and production of the enzyme urease (≈ 3.623,2 U.mL-1). The addition of this medium after 40 days of fermentation promoted the self-healing of cracks and increased compressive strength in ≈ 14.2% of the cementitious specimens; therefore, increasing the sustainability and engineering properties of cement-based materials.

2-Methylcitrate cycle: a well-regulated controller of Bacillus sporulation.

Bacillus thuringiensis is the most widely used eco-friendly biopesticide, containing two primary determinants of biocontrol, endospore and insecticidal crystal proteins (ICPs). The 2-methylcitrate cycle is a widespread carbon metabolic pathway playing a crucial role in channelling propionyl-CoA, but with poorly understood metabolic regulatory mechanisms. Here, we dissect the transcriptional regulation of the 2-methylcitrate cycle operon prpCDB and report its unprecedented role in controlling the sporulation process of B. thuringiensis. We found that the transcriptional activity of the prp operon encoding the three critical enzymes PrpC, PrpD, and PrpB in the 2-methylcitrate cycle was negatively regulated by the two global transcription factors CcpA and AbrB, while positively regulated by the LysR family regulator CcpC, which jointly account for the fact that the 2-methylcitrate cycle is specifically and highly active in the stationary phase of growth. We also found that the prpD mutant accumulated 2-methylcitrate, the intermediate metabolite of the 2-methylcitrate cycle, which delayed and inhibited sporulation at the early stage. Thus, our results not only revealed sophisticated transcriptional regulatory mechanisms for the metabolic 2-methylcitrate cycle but also identified 2-methylcitrate as a novel regulator of sporulation in B. thuringiensis.

Rapid Assessment of Water Toxicity by Plasmonic Nanomechanical Sensing.

The ability to rapidly and accurately detect water toxicity is crucial for monitoring water quality and assessing toxic risk, but such detection remains a great challenge. Here, we present a plasmonic nanomechanical sensing (PNMS) system for the rapid assessment of water toxicity. This technique is based on the plasmonic sensing of the nanomechanical movement of single bacterial cells, which could be inhibited upon exposure to potential toxicants. By correlating the amplitude of nanomechanical movement with bacterial activity, we detected a variety of toxic substances in water. The direct readout of bacterial activity via PNMS allowed for a high sensitivity to toxicants in water, thereby enabling us to evaluate the acute toxicological effect of chemical compounds rapidly. The PNMS method is promising for online alerts of water quality safety and for assessing chemical hazards. We anticipate that PNMS is also suitable for a wide range of other applications, including bacterial detection and high-throughput screening of antibacterial materials.

The signaling peptide PapR is required for the activity of the quorum-sensor PlcRa in Bacillus thuringiensis.

The transcriptional regulator PlcR, its cognate cell-cell signaling heptapeptide PapR7, and the oligopeptide permease OppABCDF, required for PapR7 import, form a quorum-sensing system that controls the expression of virulence factors in Bacillus cereus and Bacillus thuringiensis species. In B. cereus strain ATCC 14579, the transcriptional regulator PlcRa activates the expression of abrB2 gene, which encodes an AbrB-like transcriptional regulator involved in cysteine biosynthesis. PlcRa is a structural homolog of PlcR: in particular, its C-terminal TPR peptide-binding domain could be similarly arranged as in PlcR. The signaling peptide of PlcRa is not known. As PlcRa is a PlcR-like protein, the cognate PapR7 peptide (ADLPFEF) is a relevant candidate to act as a signaling peptide for PlcRa activation. Also, the putative PapRa7 peptide (CSIPYEY), encoded by the papRa gene adjacent to the plcRa gene, is a relevant candidate as addition of synthetic PapRa7 induces a dose-dependent increase of abrB2 expression. To address the issue of peptide selectivity of PlcRa, the role of PapR and PapRa peptides in PlcRa activity was investigated in B. thuringiensis 407 strain, by genetic and functional complementation analyses. A transcriptional fusion between the promoter of abrB2 and lacZ was used to monitor the PlcRa activity in various genetic backgrounds. We demonstrated that PapR was necessary and sufficient for PlcRa activity. We showed that synthetic PapRs from pherogroups II, III and IV and synthetic PapRa7 were able to trigger abrB2 expression, suggesting that PlcRa is less selective than PlcR. Lastly, the mode of binding of PlcRa was addressed using an in silico approach. Overall, we report a new role for PapR as a signaling peptide for PlcRa activity and show a functional link between PlcR and PlcRa regulons in B. thuringiensis.

Interpretation of the bacterial growth process based on the analysis of the speckle field generated by calibrated scattering media.

The speckle imaging technique has been proven to be a reliable and effective method for real-time monitoring of the growth kinetics of any bacterium in suspension. To understand the interaction between the light and the bacterial density, a simulation of the bacterial growth of Bacillus thuringiensis was performed using calibrated microspheres of different concentrations and sizes. Results show that the decrease of speckle grain size with the increase of the medium scattering coefficient reveals the two essential phases of the bacterial growth: the exponential phase where the number of the bacteria increases and the stationary phase where sporulation and cell lysis occur.

Effect of the spoIIID mutation on mother cell lysis in Bacillus thuringiensis.

SpoIIID is a small, sequence-specific DNA-binding protein which can direct many genes' transcription and has an effect on spore formation in Bacillus subtilis. We investigated the role of SpoIIID in mother cell lysis in Bacillus thuringiensis. A ß-galactosidase assay based on the promoter fusions with lacZ indicated that the sigK gene was positively regulated by SpoIIID and σK negatively regulated the expression of sigE. The spoIIID mutant strain exhibited no mother cell lysis in Schaeffer's sporulation medium (SSM) but did in ½ Luria-Bertani (LB) medium. cwlC is an essential hydrolase gene for mother cell lysis. Moreover, the expression of a PcwlC-lacZ fusion in spoIIID mutant was proved to be higher in ½ LB medium than in SSM. HD (ΔspoIIID)(ΔcwlC) mutant was obtained by knocking out the cwlC gene in HD(ΔspoIIID) and displayed no mother cell lysis in both SSM and ½ LB mediums. The deletion of spoIIID decreased the crystal protein production in HD73. The expression of Porf1cry8E and P5014 promoter fusions with lacZ gene in the acrystalliferous HD-(ΔspoIIID) mutant showed similar activity to that in the acrystalliferous HD73- strain before T7 and slightly higher than that in the acrystalliferous HD73- after T7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that Cry1Ac production in HD-(ΔspoIIID) directed by the Porf1cry8E and P5014 promoters was at a similar level as that in HD73 wild strain. Altogether, these results suggested that the spoIIID mutant with Porf1cry8E or P5014 promoters could be an alternative delivery system for cry gene expression with no mature spore formation and medium-dependent mother cell lysis.

Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism.

Bacillus thuringiensis is a widely used bacterial entomopathogen producing insecticidal toxins, some of which are expressed in insect-resistant transgenic crops. Surprisingly, the killing mechanism of B. thuringiensis remains controversial. In particular, the importance of the septicemia induced by the host midgut microbiota is still debated as a result of the lack of experimental evidence obtained without drastic manipulation of the midgut and its content. Here this key issue is addressed by RNAi-mediated silencing of an immune gene in a lepidopteran host Spodoptera littoralis, leaving the midgut microbiota unaltered. The resulting cellular immunosuppression was characterized by a reduced nodulation response, which was associated with a significant enhancement of host larvae mortality triggered by B. thuringiensis and a Cry toxin. This was determined by an uncontrolled proliferation of midgut bacteria, after entering the body cavity through toxin-induced epithelial lesions. Consequently, the hemolymphatic microbiota dramatically changed upon treatment with Cry1Ca toxin, showing a remarkable predominance of Serratia and Clostridium species, which switched from asymptomatic gut symbionts to hemocoelic pathogens. These experimental results demonstrate the important contribution of host enteric flora in B. thuringiensis-killing activity and provide a sound foundation for developing new insect control strategies aimed at enhancing the impact of biocontrol agents by reducing the immunocompetence of the host.

Solid-state fermentation of Bacillus thuringiensis var kurstaki HD-73 maintains higher biomass and spore yields as compared to submerged fermentation using the same media.

To determine the growth kinetic parameters, substrate consumption and spore yields for Bacillus thuringiensis, liquid fermentation (SmF) and solid-state fermentation (SSF), on polyurethane foam (PUF), were analysed comprising strictly the same media. The analysis included three medium concentrations, maintaining the same C/N ratio, with initial glucose at 12.5, 25, and 50 g L-1 (1X, 2X and 4X, respectively). SSF at 2X and 4X produced higher amounts of total biomass, vegetative growth and even early sporulation. Notably, at all glucose concentrations, sporulation was not inhibited in SSF as seen partially in SmF at 2X, and totally at 4X. Micrographs from PUF cultures showed thin layers of bacteria forming large horizontal aggregates, associated with the higher biomass yields and the early cell differentiation. This is the first work showing that SSF improves spore yields of B. thuringiensis in media with high substrate concentrations, using PUF as a research tool for comparative analysis with application in new production systems including biofilm-forming microorganisms.

Dracaena sanderiana endophytic bacteria interactions: Effect of endophyte inoculation on bisphenol A removal.

Bisphenol A (BPA) is one of the most abundant endocrine-disrupting compounds which is found in the aquatic environment. However, actual knowledge regarding the effect of plant-bacteria interactions on enhancing BPA removal is still lacking. In the present study, Dracaena sanderiana endophytic bacteria interactions were investigated to evaluate the effect of bacterial inoculation on BPA removal under hydroponic conditions. Two plant growth-promoting (PGP) bacterial strains, Bacillus thuringiensis and Pantoea dispersa, which have high BPA tolerance and can utilize BPA for growth, were used as plant inocula. P. dispersa-inoculated plants showed the highest BPA removal efficiency at 92.32 ±â€¯1.23% compared to other inoculated and non-inoculated plants. This was due to a higher population of the endophytic inoculum within the plant tissues which resulted in maintained levels of indole-3-acetic acid (IAA) for the plant's physiological needs and lower levels of reactive oxygen species (ROS). In contrast, B. thuringiensis-inoculated plants had a lower BPA removal efficiency. However, individual B. thuringiensis possessed a significantly higher BPA removal efficiency compared to P. dispersa. This study provides convincing evidence that not all PGP endophytic bacteria-plant interactions could improve the BPA removal efficiency. Different inocula and inoculation times should be investigated before using plant inoculation to enhance phytoremediation.

A soft sensor based on online biomass measurements for the glucose estimation and control of fed-batch cultures of Bacillus thuringiensis.

On bioprocess engineering, experimental measurements are always a costly part of the modeling effort; therefore, there is a constant need to develop cheaper, simpler, and more efficient methodologies to exploit the information available. The aim of the present work was to develop a soft sensor with the capacity to perform reliable substrate predictions and control in microbial cultures of the fed-batch type, using mainly microbial growth data. This objective was achieved using dielectric spectroscopy technology for online monitoring of microbial growth and hybrid neural networks for online prediction of substrate concentration. The glucose estimator was integrated to a fuzzy logic controller to control the substrate concentration in a fed-batch experiment. Dielectric spectroscopy is a technology sensitive to the air volume fraction in the culture media and the turbulence generated by the agitation; however, the introduction of a polynomial function for the calibration of the permittivity signal allowed biomass estimations with an approximation error of 2%. The methodology presented in this work was successfully implemented for the glucose prediction and control of a fed-batch culture of Bacillus thuringiensis with an approximation error of 6%.

Alternative low-cost process for large-scale production of Bacillus thuringiensis in a simple and novel culture system.

An industrial-scale, profitable method for production of the most widely used bioinsecticide, Bacillus thuringiensis (Bt), is challenging because of its widespread application. The aim of this study is to present a strategy to develop a low-cost, large-scale bioprocess to produce Bt H14. This study was first focused on the design of a culture medium composed of economical and available components, such as glycerol and lysed Saccharomyces cerevisiae. The production goal of 1200 ITU was achieved using a medium composed of 20:20 g L-1of glycerol:lysed yeast in batch cultures. Efforts were subsequently focused on the design of an appropriate culture system, and an original two-stage culture system was proposed. First, yeast (the primary component of the culture medium) are cultivated using a minimal mineral medium and lysed, and in the second stage, Bt is cultivated in the same bioreactor using the lysed yeasts as culture medium (supplemented with a feeding pulse of 10 g L-1 glycerol). This system was called fed batch one pot (FOP). A new inoculation strategy is also presented in this study, since these Bt cultures were inoculated directly with heat pre-treated spores instead of vegetative bacteria to facilitate the bioprocess. This study was developed from the laboratory to production-scale bioreactors (measuring from 500 mL to 2500 L), and the efficiency of the proposed strategy was evident in LD50 tests results, achieving 1796 ITU in large-scale processes. Both the use of non-conventional sources and the process development for biomass production are important for cost-effective production of Bt-based insecticides in mosquito control projects.

[The functions of yhcZ gene during Bacillus thuringiensis growth].

yhcZ and yhcY genes constitute a two-component system in Bacillus subtilis and B. cereus that regulates bacterial growth. However, the exact biological function of yhcZ gene in B. thuringiensis has not been fully elucidated. In this study, we proved that HD73_5824 is an yhcZ gene in B. thuringiensis subsp. kurstaki HD73 strain by combining gene functional annotation, analysis of upstream and downstream genes arrangement, and amino acid sequence alignment. This yhcZ gene may co-regulate bacterial growth with HD73_5825 gene (yhcY gene) by constituting a two-component system. Homologous recombination technology was employed to knock out yhcZ gene of HD73, resulting in a mutant strain HD (ΔyhcZ). The HD (ΔyhcZ) strain grew slower than wild-type strain HD73 in both LB and SSM medium. Re-introduction of yhcZ gene in HD (ΔyhcZ) strain can partially restore the growth, indicating that the deletion of yhcZ gene impacts the cell growth of HD73 strain. HD (ΔyhcZ) strain grew faster than HD73 strain in M9 medium with 0.4% glucose as the sole carbon source, implying that the yhcZ gene plays an important role in glucose utilization by HD73 strain. The results of biolog assay showed that HD (ΔyhcZ) exhibits a lower average well color development compared to HD73. HD(ΔyhcZ) cells also demonstrated a decreased capacity for absorbing and utilizing D/L-serine, formic acid, D-gluconic acid, L-histamine, D-methyl lactate, and citric acid, indicating that yhcZ gene could dramatically influence carbon source utilization of HD73 strains. Additionally, HD (ΔyhcZ) was less resistant to 8% NaCl, suggesting that yhcZ gene may be involved in the expression and regulation of genes related to high-salt stress response in bacterial cells. The results above show that the yhcZ gene significantly promotes glucose and other carbon sources utilization of HD73 strain during growth. Our findings will lay a foundation not only for analyzing the regulatory mechanisms of glucose and carbon sources utilization by yhcZ gene, but also providing a reference for the further research on bacterial growth and fermentation.

The Change in the Entomopathogenic Properties in Streptomycin Resistant Bacillus thuringiensis.

Streptomycin-resistant strains (StrR) of the entomopathogenic bacteria Bacillus thuringiensis ssp. galleriae (Btg) have been obtained. Assessment of growth rate of Btg 69-6 colonies revealed significant difference between the initial strain StrS sensitive to antibiotics and StrR. Decrease in susceptibility of instar IV larvae of Galleria mellonella to Btg 69-6 StrR by a factor of eight compared to Btg 69-6 StrS has also been recorded. In Btg 190 StrR, the insecticidal activity decreased by a factor of five. In StrR, the biochemical properties changed after acquisition of resistance compared to the initial strain.

Construction of Bacillus thuringiensis Simulant Strains Suitable for Environmental Release.

For a surrogate bacterium to be used in outdoor studies, it is important to consider environmental and human safety and ease of detection. Recently, Bacillus thuringiensis, a popular bioinsecticide bacterium, has been gaining attention as a surrogate bacterium for use in biodefense. In this study, we constructed simulant strains of B. thuringiensis with enhanced characteristics for environmental studies. Through transposon mutagenesis, pigment genes were inserted into the chromosome, producing yellow-colored colonies for easy detection. To prevent persistence of spores in the environment, a genetic circuit was designed to produce a spore without sporulation capability. Two loxP sites were inserted, one on each side of the spo0A gene, which encodes a sporulation master regulator, and a sporulation-dependent Cre expression cassette was inserted into the chromosome. This genetic circuit successfully deleted spo0A during sporulation, producing spores that lacked the spo0A gene. In addition, two major α/ß-type small acid-soluble spore protein (SASP) genes, predicted by synteny analysis, were deleted. The spores of the mutant strain showed increased UV-C sensitivity and quickly lost viability when tested in a solar simulator. When the spores of the mutant strain were administered to the lungs of BALB/c mice, cells were quickly removed from the body, suggesting enhanced in vivo safety. All strains constructed in this study contain no antibiotic resistance markers and all heterologous genes were inserted into the chromosome, which are useful features for simulants to be released into the environment.IMPORTANCEB. thuringiensis has recently been receiving increasing attention as a good spore simulant in biodefense research. However, few studies were done to properly address many important features of B. thuringiensis as a simulant in environmental studies. Since spores can persist in the environment for years after release, environmental contamination is a big problem, especially when genetically engineered strains are used. To solve these problems, we report here the development of B. thuringiensis simulant strains that are capable of forming yellow colonies for easy detection, incapable of forming spores more than once due to a genetic circuit, and lacking in two major SASP genes. The genetic circuit to produce a spore without sporulation capability, together with the deletion of SASP genes, ensures the environmental and human safety of the simulant strains developed in this study. All of these features will allow wider use of B. thuringiensis as a simulant for Bacillus anthracis in environmental release studies.

Comparison of sampling methods to recover germinated Bacillus anthracis and Bacillus thuringiensis endospores from surface coupons.

AIMS: In an attempt to devise decontamination methods that are both effective and minimally detrimental to the environment, we evaluated germination induction as an enhancement to strategies for Bacillus anthracis spore decontamination. To determine an optimal method for the recovery of germinating spores from different matrices, it was critical to ensure that the sampling procedures did not negatively impact the viability of the germinating spores possibly confounding the results and downstream analyses of field trial data. METHODS AND RESULTS: Therefore, the two main objectives of this study were the following: (i) development of an effective processing protocol capable of recovering the maximum number of viable germinating or germinated spores from different surface materials; and (ii) using a model system of spore contamination, employ this protocol to evaluate the potential applicability of germination induction to wide-area decontamination of B. anthracis spores. We examined parameters affecting the sampling efficiencies of B. anthracis and the surrogate species Bacillus thuringiensis on nonporous and porous materials. CONCLUSIONS: The most efficient extraction from all matrices was observed using PBS with 0·01% Tween 80 extraction buffer. The addition of a sonication and/or extended vortex treatment did not yield significant increases in spore or germinated spore recovery. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data demonstrate that previous germination-induction experiments performed in suspension can be reproduced when Bacillus spores are deposited onto reference surfaces materials. Our proof of concept experiment illustrated that a germination pretreatment step significantly improves conventional secondary decontamination strategies and remediation plans.

Antimicrobial effects of zero-valent iron nanoparticles on gram-positive Bacillus strains and gram-negative Escherichia coli strains.

BACKGROUND: Zero-valent iron nanoparticles (ZVI NPs) have been used extensively for the remediation of contaminated soil and groundwater. Owing to their large active surface area, they serve as strong and effective reductants. However, the ecotoxicity and bioavailability of ZVI NPs in diverse ecological media have not been evaluated in detail and most studies have focused on non-nano ZVI or Fe0. In addition, the antimicrobial properties of ZVI NPs have rarely been investigated, and the underlying mechanism of their toxicity remains unknown. RESULTS: In the present study, we demonstrate that ZVI NPs exhibited significant toxicity at 1000 ppm against two distinct gram-positive bacterial strains (Bacillus subtilis 3610 and Bacillus thuringiensis 407) but not against two gram-negative strains (Escherichia coli K12 and ATCC11634). Specifically, ZVI NPs caused at least a 4-log and 1-log reductions in cell numbers, respectively, in the two Bacillus strains, whereas no change was detected in the two E. coli strains. X-ray photoelectron spectroscopy, X-ray absorption near-edge, and extended X-ray absorption fine structure spectra confirmed that Bacillus cells exposed to ZVI NPs contained mostly Fe2O3 with some detectable FeS. This finding indicated that Fe0 nanoparticles penetrated the bacterial cells, where they were subsequently oxidized to Fe2O3 and FeS. RedoxSensor analysis and propidium iodide (PI) staining showed decreased reductase activity and increased PI in both Bacillus strains treated with a high (1000 ppm) concentration of ZVI NPs. CONCLUSION: Taken together, these data show that the toxicity of ZVI NPs was derived from their oxidative properties, which may increase the levels of reactive oxygen species and lead to cell death.

Biodegradation of anthracene by a newly isolated bacterial strain, Bacillus thuringiensis AT.ISM.1, isolated from a fly ash deposition site.

The current study is aimed to evaluate the mechanism of anthracene degradation by a bacterial strain isolated from fly ash deposition site near Jamadoba Coal Preparation Plant, Jharkhand, India. The Bushnell-Haas media cultured (containing anthracene as sole carbon source) bacterial isolate was identified by 16S rRNA gene sequence coding as the Bacillus thuringiensis strain, which showed the efficiency to degrade anthracene. The degradation efficiency of the strain has been estimated to be around 91% (for 40 mg l-1 of anthracene concentration) after 2 weeks of incubation at 33-36°C and initial pH of 6·8-7. The growth kinetics of the isolated strain has been described well by the Haldane-Andrews model of microbial growth pattern for inhibitory substrate, with a correlation factor (R2 value) of 0·9790. The maximum specific growth rate (µmax ) was 0·01053 h-1 and the value of inhibition coefficient for Haldane model was specified as 18·2448 mg l-1 . In the present study, some diphenol metabolites were identified besides the known possible biodegradation products. SIGNIFICANCE AND IMPACT OF THE STUDY: Polycyclic aromatic hydrocarbons (PAHs) are recognized as significant health risks and consequently listed as priority pollutants by environmental protection agencies across the globe. The aim of the present study was to degrade one of the important PAHs, anthracene, by a newly isolated Bacillus thuringiensis strain. This is the first report of anthracene degradation by B. thuringiensis. This is also the very first growth kinetic study of a bacteria in an anthracene-containing medium. Some diphenol metabolites were found for the first time as anthracene biodegradation by-products, which can be an indication towards a new pathway.

Effects of Bacillus thuringiensis strains virulent to Varroa destructor on larvae and adults of Apis mellifera.

The sublethal effects of two strains of Bacillus thuringiensis, which were virulent in vitro to Varroa destructor, were measured on Apis mellifera. The effects of five concentrations of total protein (1, 5, 25, 50 and 100µg/mL) from the EA3 and EA26.1 strains on larval and adult honey bees were evaluated for two and seven days under laboratory conditions. Based on the concentrations evaluated, total protein from the two strains did not affect the development of larvae, the syrup consumption, locomotor activity or proboscis extension response of adults. These same parameters were also tested for the effects of three concentrations (1, 10 and 15µg/kg) of cypermethrin as a positive control. Although no significant differences were observed after two days of treatment with cypermethrin, a dose-response relationship in syrup consumption and locomotor activity was observed. A significant reduction in the proboscis extension response of the bees treated with cypermethrin was also observed. Therefore, in contrast to cypermethrin, our results indicate that the EA3 and EA26.1 strains of B. thuringiensis can be used in beehives to control V. destructor and reduce the negative effects of this mite on colonies without adverse effects on the larvae and adults of A. mellifera. Additionally, the overuse of synthetic miticides, which produce both lethal and sublethal effects on bees, can be reduced.

Influence of Ephestia kuehniella stage larvae on the potency of Bacillus thuringiensis Cry1Aa delta-endotoxin.

The economically important crop pest Ephestia kuehniella was tested at two stages of larval development for susceptibility to Bacillus thuringiensis Cry1Aa toxin. Bioassays showed that toxicity decreased during the development of larvae stage. In fact, Cry1Aa toxins from BNS3-Cry- (pHT-cry1Aa) showed low toxicity against the first-instar larvae (L1) with a LC50 value of about 421.02µg/g of diet and was not toxic against the fifth-instar (L5), comparing to the BLB1 toxins used as positive control which represent a LC50 value of about 56.96 and 84.21µg/g of diet against L1 and L5 instars larvae, respectively. Effects of Cry1Aa toxins were reflected in histopathological observations by the weak destruction of midgut epithelium, slight hypertrophy of epithelial cells, and minor alteration of brush border membrane (BBM) detected mainly in L1 larvae stage comparing to the more extensive damage caused by BLB1 toxins. Interestingly, in vitro proteolysis of Cry1Aa toxins was found to correlate with the difference of toxicity during larval stage development. In fact, the weak proteinase activity detected inside the L1 midgut has led to the persistence of the Cry1Aa active forms (65 and 58kDa) during prolonged incubations, causing the alterations described previously. Three subfamilies of aminopeptidase (APN) receptors were detected in both larvae instars with different intensities and molecular weights (150kDa and 55kDa for APN1, and 90kDa for APN2 and APN4). Remarkably, binding assay using Cry1Aa toxin seems to have no direct correlation with larval stages toxicity differences, since same putative receptors were detected. Understanding the reasons for the clear differences in the effectiveness of Cry1Aa toxins during larval development stages of E. kuehniella is very important for the design of future improvement insecticidal approaches and for the accomplishment of resistance prevention strategies.

Pilot-scale biopesticide production by Bacillus thuringiensis subsp. kurstaki using starch industry wastewater as raw material.

Pilot-scale Bacillus thuringiensis based biopesticide production (2000 L bioreactor) was conducted using starch industry wastewater (SIW) as a raw material using optimized operational parameters obtained in 15 L and 150 L fermenters. In pilot scale fermentation process the oxygen transfer rate is a major limiting factor for high product yield. Thus, the volumetric mass transfer coefficient (KLa) remains a tool to determine the oxygen transfer capacity [oxygen utilization rate (OUR) and oxygen transfer rate (OTR)] to obtain better bacterial growth rate and entomotoxicity in new bioreactor process optimization and scale-up. This study results demonstrated that the oxygen transfer rate in 2000 L bioreactor was better than 15 L and 150 L fermenters. The better oxygen transfer in 2000 L bioreactor augmented the bacterial growth [total cell (TC) and viable spore count (SC)] and delta-endotoxin yield. Prepared a stable biopesticide formulation for field use and its entomotoxicity was also evaluated. This study result corroborates the feasibility of industrial scale operation of biopesticide production using starch industry wastewater as raw material.