Immobilization of an endophytic Bacillus sp. on Phragmites karka stem for lipase production in the presence of Cassia fistula seeds

Endophytic bacteria have not been reported widely for their lipolytic abilities, so they are not used for large-scale lipase production. The purpose of this study was to explore an endophytic bacterium for the production of lipase implementing cost-effective techniques, including the use of Cassia fistula seeds, as a substrate in the production medium and stem pieces of Phragmites karka as the immobilization matrix. The endophytic strain Bacillus sp. E4 was originally isolated from the halophytic plant Arthrocnemum macrostachyum. Bacillus sp. E4 produced 6.05 IU mL⁻¹ lipase in the presence of powdered seeds of Cassia fistula (golden shower tree). Initial trial experiments using a one-factor-at-a-time approach led to an improvement in lipase titers to 10.05 IU mL⁻¹. Consequently, investigations using the Plackett–Burman design suggested the influence of three significant factors – incubation period, inoculum size, and substrate concentration – on lipase production. They were optimized using the Box–Behnken design (BBD). In the response optimization experiment, strain E4 yielded 52.35 IU mL⁻¹ lipase, which was in accordance with the predicted yield and indicated an overall 8.65 fold improvement in lipase production. To investigate the use of free cells, strain E4 was immobilized on the stem pieces of a halophytic plant, Phragmites karka, which was used for the first time as an immobilization matrix. The immobilized cells retained lipase production ability for up to six cycles with the highest yield of 110 IU mL⁻¹, which corresponded to an improvement of more than eighteenfold. Scanning electron micrographs confirmed the colonization of E4 cells in the matrix and demonstrated the utilization of C. fistula seeds. Fourier transform infrared spectroscopy affirmed the utilization of components including fatty acids by the immobilized E4 cells. The study suggests that endophytic bacterial strains could be applied for the production of lipase with the utilization of nontraditional oil sources.