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

Abstract

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.

KEYWORDS:

• Bacillus thuringiensis israelensis
• fed batch one pot
• glycerol
• Saccharomyces cerevisiae
• biopesticide

Acknowledgments

This research work was supported by grants from CONICET (National Scientific and Technical Research Council – Argentina) and UNQ (National University of Quilmes, PUNQ 53/1030). The authors also thank Dr. Juan José García from CEPAVE (UNLP-CONICET) for larvae provision and assistance in DL₅₀ protocol assays and to Mordor Intelligence for generously providing a sample of the Global Biopesticides Market (2016 – 2021) report.

This work is dedicated to the memory of Dr. Carlos F. Mignone, RIP.

Supplementary Materials