Immobilization in alginate as a new technique for the separation of Bacillus thuringiensis var. israelensis spore crystal complex

Bacillus thuringiensis var. israelensis (Bti) spore crystal complex (SCC) produced by fermentation has to be separated before use for mosquito control in the breeding habitats. In this paper we report the development of a novel immobilization technique using sodium alginate as a matrix to separate the SCC of Bti and compared with acid precipitation method. Two strains of Bti VCRC B-17 and WHO standard strain IPS-82 were tested. Wet biomass yield of VCRC B-17 and IPS-82 separated by acid precipitation method was 215 and 224 g/L, respectively, whereas by alginate immobilization method it was 258 and 270 g/L, respectively. Spore yield of the respective strains, by acid precipitation method was 1.87 × 1014 and 2.17 × 1014 CFU/mL, whereas by alginate immobilization method 2.3 × 1015 and 3.0 x 1015 CFU/mL, respectively. Lethal concentration (LC50) of SCC of VCRC B-17 and IPS-82 by acid precipitation method was 1.18 nl/mL and 0.92 nl/mL, respectively, whereas LC50 values by alginate immobilization method was 0.94 nl/mL and 0.71 nl/mL, respectively. In the case of industrial substrate soya medium the yield of wet biomass, spore count and lethal concentration of VCRC B17 obtained by acid precipitation and alginate immobilization method were 311 and 360 g/L, 1.40 × 1014 and 2.07 × 1015 CFU/mL and 1.22 and 0.95 nl/mL, respectively. Protease activity measured after acid precipitation and alginate immobilization method was 10.8 and 103.67 U/mL, respectively. The international toxic units (ITU) of the VCRC B-17 by acid precipitation and alginate immobilization method were 13,636 and 13,888 ITU/mg, respectively. Thus the alginate immobilization method has potential application in the harvesting and formulation of biopesticides.

Graphical abstractSeparation of spore crystal complex from culture broth using sodium alginate immobilization is a new downstream process for the separation of spore crystal complex from Bacillus thuringiensis var. israelensis fermentation. Advantages of the immobilization technique are the simplicity of preparation compared to other available techniques and requires no specialized equipments. In immobilization technique the supernatant can be used for the enzyme preparation, in acid precipitation method supernatant cannot be used for enzyme preparation. This technique will be useful for B. thuringiensis var. israelensis production industry.(a) Spore crystal complex of acid precipitation method of VCRC B17 and (c) IPS-82. (b) Spore crystal complex of alginate immobilization method of VCRC B17 and (d) IPS-82.Figure optionsDownload full-size imageDownload as PowerPoint slideHighlights► Separation of spore crystal complex using immobilization is a new technique. ► Useful for the Bacillus thuringiensisisraelensis fermentation downstream process. ► Advantages of the immobilization technique are the simplicity of preparation. ► Very useful in the B. thuringiensis var. israelensis production industry.