Lipopeptides efficiently disperse spore crystal aggregates in Bacillus thuringiensis israelensis

The separation of Bacillus thuringiensis (Bt) spores and crystals is often compromised by dense aggregates, particularly in Bt subsp. israelensis. This study evaluates (sonication), chemical (SDS), and biological (lipopeptide) treatments to improve aggregate dissociation in BUPM98, using AR23 as a reference and Lip (Bt subsp. kurstaki) as a naturally dissociated control. Initial hexane-based purification highlighted the impact of aggregation, with significantly lower recovery rates for israelensis compared to kurstaki. Electron microscopic analysis revealed tightly integrated spore crystal complexes in Bt israelensis and independent bipyramidal crystals in Lip. While physical sonication and chemical SDS treatments improved dissociation, biological treatment with lipopeptides yielded the most significant results. This approach drastically increased viable spore counts by up to 8000-fold and enhanced crystal recovery to 60%, offering a superior and more efficient biosurfactant-based alternative to traditional methods. PCR analysis identified the kurstakin synthetase gene exclusively in the naturally dissociated kurstaki strain, suggesting a native role for these biosurfactants in preventing aggregation. To our knowledge, this is the first demonstration that lipopeptides can effectively dissociate Bacillus thuringiensis spore crystal aggregates, offering an efficient biosurfactant-based alternative to physical or chemical methods.