Towards a new biological control approach for Photorhabdus temperata bioinsecticide production through the bioconversion of Tunisian industrial wastewater

© 2020, The Author(s). A novel bioconversion approach of Tunisian wastewater to low-cost Photorhabdus temperata bioinsecticide is presented in this study. Our results showed that when cultured on the food industry wastewater (WS4), P. temperata cells exhibited oral toxicity of about 42%, which is the same as those cultured in complex medium (CM), used as control. Moreover, variants small colony polymorphism (Vsm) of the strain K122 was completely avoided after a prolonged incubation. However, viable but non-culturable (VBNC) state was enhanced with the maximum colony-forming units (CFU) count of 9 × 106 cells/mL obtained after 48 h of incubation in the WS4. According to flow cytometry analysis, almost 100% of P. temperata cells were viable until 48 h of incubation. The appearance of propidium iodide (PI) positively stained cells was observed after a prolonged incubation with a maximum of 17% of damaged cells in WS1. In order to follow the progress of P. temperata fermentation process carried out in industrial wastewater, we established for the first time, the mathematical relationship between total cell counts, CFU counts and oral toxicity of P. temperata strain K122. Indeed, irrespective of the medium used, the relationship between CFU count and total cell count followed a power law. Additionally, when plotting CFU count, or total cell count against toxicity, a semi-log linear relationship was obtained. Our results proved the efficiency of this bioconversion approach to produce bioinsecticide based on the entomopathogenic bacterium P. temperata, with practical benefits in terms of cost production and wastewater management.[Figure not available: see fulltext.

Lessons learned from COVID-19 impact of pandemic on children with neurological disorders in Sfax, Tunisia

To record the experience of caregivers for neurologically impaired children during the lockdown periods. Data from 286 children's caregivers were collected through an administered questionnaire to record: access to care services during the lockdown periods, causes underlying loss of access to care, mitigations adopted by caregivers and patients’ outcomes. The mean age of children was 8.11 years-old and sex ratio (F/M) was 0.66. They were mainly followed-up for epilepsy or epileptic encephalopathy (53%) and cerebral palsy (21%). During the lockdown periods, caregivers reported that 45% of children had no access to healthcare majorly for neurorehabilitation (76.7%) and medicines (70.7%). Most caregivers (36%) related limitations in access to fear from catching the virus. The majority resorted to continuation of the same treatment via primary healthcare facilities (41%) and postponement of their appointments (24%). Our results show that access to healthcare for children with neurological disorders was deeply disrupted during the COVID-19 pandemic. The already precarious health systems’ infrastructures might have been the main causes for this and should be thus considered in the health policy and planing

Improvement of Photorhabdus temperata strain K122 bioinsecticide production by batch and fed-batch fermentations optimization

Optimization of a fermentation process for bioinsecticides production by Photorhabdus temperata strain K122 was investigated into fully controlled 3-L fermenter using an optimized medium (OM). Development of large-scale inocula showed that the composition of the growth medium greatly influenced the physiological state of P. temperata cells. The effect of pH, agitation and dissolved oxygen concentration (DO) on the growth, culturability and oral toxicity of P. temperata cells were also investigated. Indeed, maintaining the pH at 7 and controlling DO concentration at 50 % saturation throughout the fermentation process, improved biomass production, CFU counts and oral toxicity by 41.1, 35 and 32.1 %, respectively, as compared to cultures carried out in 500 mL shake flasks. At such conditions, 8 g/L glucose fed-batch fermentation, enhanced cell lysis and variants small colony (Vsm) polymorphism appearance. To overcome such limitations, glucose concentration should be maintained at 4 g/L. In this case, P. temperata cells were produced at high cell density and culturability reaching 4.5 and 1.2 × 10(9) cells/mL, respectively. In addition, the stability of the primary form was maintained for a long period in the stationary growth phase and Vsm polymorphism was completely avoided that can be crucial for scale-up the bioprocess of P. temperata bioinsecticide.Tunisian Ministere de l’Enseignement Superieur, de la Recherche Scientifique (MESRS

Overcoming the production limitations of Photorhabdus temperata ssp. temperata strain K122 bioinsecticides in low-cost medium

For low-cost production of Photorhabdus temperata ssp. temperata strain K122 bioinsecticide, a cheap complex medium was optimized. Diluted seawater was used as the source of micronutrients, especially sodium chloride, involved in the improvement of cell density, culturability and oral toxicity of the bacterium P. temperata against Ephestia kuehniella larvae. Thus, the new formulated medium was composed only of 10 g/l of soya bean meal, used as the carbon and nitrogen main source, mixed in sevenfold diluted seawater. At such conditions, several limitations of P. temperata bioinsecticide productions were shown to be overcome. The appearance of variants small colony polymorphism was completely avoided. Thus, the strain K122 was maintained at the primary form even after prolonged incubation. Moreover, the viable but nonculturable state was partially overcome, since the ability of P. temperata cells to form colonies on the solid medium was prolonged until 78 h of incubation. In addition, when cultured in the complex medium, P. temperata cells were produced at high cell density of 12 × 10(8) cells/ml and exhibited 81.48% improvement of oral toxicity compared to those produced in the optimized medium. With such medium, the large-scale bioinsecticides production into 3-l fully controlled fermenter improved the total cell counts, CFU counts and oral toxicity by 20, 5.81 and 16.73%, respectively. This should contribute to a significant reduction of production cost of highly potent P. temperata strain K122 cells, useful as a bioinsecticide.Tunisian Ministere de l’Enseignement Superieur, de la Recherche Scientifique (MESRS

Involvement of oxidative stress and growth at high cell density in the viable but nonculturable state of Photorhabdus temperata ssp. temperata strain K122

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 24 h 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.Tunisian Ministère de l’Enseignement Supérieur, de la Recherche Scientifique et de la Technologie (MESRS)

Review on biopesticide production by Bacillus thuringiensis subsp. kurstaki since 1990: Focus on bioprocess parameters

Irrespective use of chemical pesticides has led, over the last decades, to several problems such as soil, water and food sources pollution, and generation of a selective pressure causing the emergence of pest resistance. Consequently, researchers have been focusing more on the use of biological control as an alternative strategy. Bacillus thuringiensis (Bt) is one of the most widely studied bacteria in industrial biotechnology and commercialized as an environmentally sustainable biopesticide. Therefore, a huge interest has been allocated for research on this bacterium and several scientific studies have been published on the issue. In this review, we tried to evaluate the scientific production over the last thirty years, for the first time, in terms of number and geographical origin, focusing particularly on B. thuringiensis kurstaki (Btk). It is worth emphasizing that the Btk process engineering involving factors affecting growth, sporulation and toxin formation yields by Bt has not been fully investigated in previous reviews. To this end, the second section of this review provided an updated survey about these conditions, such as nutritional requirements, culture media and fermentation technologies. Relevant information was collected in comparative tables that could be very useful for the scientific community interested in Btk-based biopesticides

Potential of Photorhabdus temperata K122 bioinsecticide in protecting wheat flour against Ephestia kuehniella

The present study reports investigations on the insecticidal activity of the entomopathogenic bacterium Photorhabdus temperata K122 against the Mediterranean flour moth Ephestia kuehniella. Cultured in the optimized medium, P. temperata K122 cells aged 32 h exhibited 51% growth inhibition at a concentration of 9 × 108 cells/ml. However, culture must be prolonged up to 48 h incubation in the proteose peptone medium to reach only 28.6% inhibition. At the same concentration, no adult emergence was observed in the case of larvae feeding on wheat flour treated with the whole culture of P. temperata K122 after physical lysis. Interestingly, P. temperata K122 cells in the viable but non culturable (VBNC) state retained the same toxicity level as the culturable cells. At a high concentration of 12 × 108 cells/ml, 100% mortality of E. kuehniella larvae could be reached. Insect mortality is due to toxaemia as confirmed by the absence of Variants small colony (Vsm) or P. temperata colonies in E. kuehniella tissue. The investigation of the histopathological effect of P. temperata toxins on the gut of infected E. kuehniella larvae showed destruction of the gut epithelium, appearance of large cavities and cellular disintegration.Ministere de l'Enseignement Superieur et de la Recherche Scientifiqu

Development of a cost-effective medium for Photorhabdus temperata bioinsecticide production from wastewater and exploration of performance kinetic

Abstract This study investigates the optimization of the culture conditions for enhancing Photorhabdus temperata biopesticide production using wastewater (WS4) as a raw material. Box-Behnken design (BBD) was used to evaluate the effects of carbon to nitrogen ratio (C/N), sodium chloride concentration and inoculum size on P. temperata biomass production and insecticidal activity. For an enhanced biopesticide production, the optimum operating conditions were as follows: inoculum size = 4%; C/N ratio = 12.5 and [NaCl] = 4 g/L for two responses. 1.95 and 2.75 fold improvements in oral toxicity and biomass production were respectively obtained in the cost-effective medium developed in this study (WS4 I) using the three variables at their optimal values. Under the optimized conditions, WS4 I-grown cells exhibited higher membrane integrity according to flow cytometry analysis since dead cells presented only 9.2% compared to 29.2% in WS4. From batch fermentations carried out in WS4 I and WS4, P. temperata kinetic parameters in terms of biomass production and substrate consumption rates were modeled. The obtained results showed that the maximum specific growth rate in WS4 I was of 0.43 h−1 while that obtained in WS4 was of 0.14 h−1. In addition, the efficiency of P. temperata to metabolize organic carbon was enhanced by optimizing the culture conditions. It reached 72.66% instead of 46.18% in the control fermentation after 10 h of incubation. Under the optimized conditions, P. temperata cells showed the highest specific consumption rate resulting in a toxin synthesis improvement