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To develop a cost effective process for bioinsecticides production by Photorhabdus temperata, dissolved oxygen (DO) requirements were investigated in both the complex and the optimized media using diluted seawater as a source of micronutrients. By varying DO concentrations, tolerance to hydrogen peroxide was shown to be medium dependant. Indeed, P. temperata cells grown in the complex medium, exhibited higher tolerance than cells grown in the optimized medium (OM). Tolerance to H2O2 was shown to be related to intracellular reactive oxygen species (ROS) accumulation during soya bean meal or glucose assimilation, as shown by flow cytometry analysis. To avoid oxidative stress damages in P. temperata cells cultured in the OM, DO concentration should be constant 50% saturation throughout the fermentation. However, a DO-shift control strategy was demonstrated to be beneficial for P. temperata bioinsecticide production in the complex medium. By using such a strategy biomass, culturability, and oral toxicity reached 16.5 × 108, 1.15 × 108 cells/mL and 64.2%, respectively, thus was 16.19, 26.37, and 12.2% more than in the cultures carried out at a constant 50% saturation. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012 [ABSTRACT FROM AUTHOR]

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

Abstract: Photorhabdus temperata ssp. temperata strain K122 represents a promising source of bioinsecticide. When cultured in an optimized medium, P. temperata exhibited restricted survival in terms of colony-forming ability on solid medium, which remained lower than the total cell counts. Membrane integrity assessment by flow cytometry showed that almost 100% of P. temperata cells were viable indicating that this bacterium enters in the viable but nonculturable state (VBNC). According to the double staining results, hydrogen peroxide was demonstrated to be responsible of P. temperata VBNC state. Addition of catalase or sodium pyruvate upon the inoculation of P. temperata on agar plates promoted the recovery of nonculturable cells up to 24h incubation. Further, growth at high cell density enhanced the VBNC state of this bacterium. This should evidenced extracellular signals accumulation involved in quorum sensing mechanism. Elucidation of this state is interesting for both toxicity study and production of P. temperata useful as bioinsecticide. [Copyright &y& Elsevier]

The acquisition of the erythromycin resistance by Bacillus thuringiensis kurstaki improved yields of δ-endotoxins in sporulating cells ranging from 134 to 215%. Resistance to erythromycin decreased the final spore count by at least 50%. Consequently, erythromycin resistance is an efficient tool for the improvement of bioinsecticides yields with a high ratio of δ-endotoxins to spores. [ABSTRACT FROM AUTHOR]

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

Abstract: Photorhabdus temperata ssp. temperata strain K122 is an important pathogen of the olive moth, Prays oleae. A new medium containing 5g/l glucose, 10g/l yeast extract and 1.07g/l ammonium chloride is presented that optimizes the biomass production of strain K122. We also showed for the first time that P. temperata ssp. temperata strain K122 develops variants small colony (Vsm) polymorphism after prolonged incubation. Instability and reversion from one form to another are described. Since Vsm cells reduce pathogenicity against insect larvae, conditions to avoid their appearance were determined. Medium aeration and inoculum quality are demonstrated as key factors to reduce Vsm occurrence. Maintenance of initial glucose concentration during incubation not only doubled biomass production, but also contributed to complete overcome of Vsm appearance. Following oral application, it was shown that P. temperata ssp. temperata cells produced at such conditions continued to exhibit their toxicity against Ephestia kuehniella. This study might contribute to promote the large-scale use of P. temperata ssp. temperata as a bioinsecticide. [Copyright &y& Elsevier]

Statistical experimental designs, involving a Plackett–Burman design followed by a rotatable central composite design were used to optimize the culture medium constituents for Bacillus thuringiensis bioinsecticide production. This was carried out by using firstly an asporogenic strain and extrapolated to some sporeless and sporulating strains. Initial screening of production parameters was performed and the variables with statistically significant effects on delta-endotoxin production were identified: glucose, glycerol, yeast extract and MnSO4. These variables were selected for further optimization by response surface methodology. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 22.5g/l of glucose, 4.8g/l of glycerol, 5.8g/l of yeast extract and 0.008g/l of MnSO4. Under these conditions, delta-endotoxin production was 2130 and 2260mg/l into 250 and 1000ml flask respectively, which represent more than 38% improvement in toxin production over the basal medium (1636mg/l). Such medium composition was shown to be suitable for overproducing delta-endotoxins by sporeless and sporulating strains. [Copyright &y& Elsevier]