Bt Research 2024, Vol.15, No.1, 65-75 http://microbescipublisher.com/index.php/bt 66 2 Production Techniques of Bt Biopesticides 21 Fermentation processes Fermentation processes are critical for the production of Bacillus thuringiensis (Bt) biopesticides. Solid-state fermentation (SSF) and submerged fermentation (SmF) are the two primary methods used. SSF involves the growth of microorganisms on solid materials without free-flowing water, making it suitable for utilizing agricultural and industrial wastes. For instance, biowaste digestate has been successfully used as a substrate for Bt production in SSF, demonstrating the importance of reactor configuration and temperature control in optimizing viable cell and spore counts (Rodríguez et al., 2019). Similarly, wastewater sludge has been employed as a raw material in both SSF and SmF, showing significant improvements in oxygen transfer and protease activity at larger scales (Yezza et al., 2004; Li et al., 2011). The use of vegetable wastes and soy fiber residues has also been explored, highlighting the potential for cost-effective and environmentally friendly Bt biopesticide production (Ballardo et al., 2016; Pan et al., 2021). 2.2 Formulation and stabilization Formulation and stabilization are crucial steps to ensure the efficacy and shelf-life of Bt biopesticides. The development of compost-like materials enriched with Bt through SSF has been shown to produce stable and effective biopesticides suitable for soil amendment (Ballardo et al., 2017; Mattedi et al., 2023). The addition of specific carbon and nitrogen sources, as well as metal ions, can enhance the insecticidal activity of Bt, as demonstrated in studies using vegetable wastes (Pan et al., 2021). Moreover, the transformation of heavy metals in wastewater sludge during SSF reduces their bioavailability and environmental risks, further stabilizing the biopesticide product. These strategies not only improve the stability and effectiveness of Bt biopesticides but also contribute to sustainable waste management practices. 2.3 Scale-up and industrial production Scaling up the production of Bt biopesticides from laboratory to industrial scale involves addressing several challenges, including maintaining consistent oxygen transfer, nutrient availability, and process control. Studies have shown that scaling up from shake flasks to larger fermentors can significantly improve viable cell and spore counts, as well as protease activity, due to better oxygen transfer (Yezza et al., 2004). The use of box-type SSF equipment has enabled kg-scale production of Bt biopesticides, providing technical support for large-scale industrial production (Chen, 2009). Additionally, the optimization of fermentation conditions, such as temperature, pH, and aeration, is essential for maximizing toxin protein yield and entomotoxicity potential (Wafa et al., 2020). Future research should focus on reducing production costs, modifying fermentation processes, and developing efficient Bt strains through genetic methods to further enhance industrial-scale production. 3 Genetic Engineering for Enhanced Bt Strains 3.1 Gene cloning and expression Gene cloning and expression techniques have been pivotal in enhancing the efficacy of Bacillus thuringiensis (Bt) biopesticides. By isolating and inserting specific Bt genes into various host organisms, researchers have been able to produce strains with improved insecticidal properties (Zhou et al., 2020). For instance, the construction of Bt recombinant engineered strains through genetic engineering has become a major focus, allowing for the expression of Bt proteins that are structurally and functionally different from naturally occurring Bt prototoxins. This approach not only enhances the insecticidal activity but also addresses the environmental persistence of Bt proteins, which is crucial for sustainable agricultural practices (Li et al., 2022; Ortiz et al., 2023). 3.2 CRISPR and genome editing The advent of CRISPR/Cas9 genome editing technology has revolutionized the development of enhanced Bt strains. This precise genome editing tool allows for targeted modifications that can significantly improve the resistance management and efficacy of Bt biopesticides. For example, CRISPR/Cas9-mediated knockout of specific genes in the diamondback moth, Plutella xylostella, has demonstrated high levels of resistance to Bt Cry1Ac toxin, providing in vivo evidence for the role of these genes in Bt toxin resistance (Guo et al., 2019).
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