Enhanced Productivity of Fragrance Compounds: Biotransformation of d-limonene Using Whole Cell Immobilization of Pseudomonas putida and Rhodococcus erythropolis

Aromas and fragrances have high demand in the modern world, and most of the fragrance compounds find applications in food, cosmetics, chemicals and pharmaceutical industries. The microbial transformation has emerged as a novel approach for carrying out specific conversions using commercially low-value substrates to produce natural fragrances of high quality. However, for many microbial species, these substrates are highly toxic, consequently inhibiting their metabolism. To compare the effect of whole cell immobilization on substrate toxicity and fragrance synthesis. The transformation of d-limonene was carried out using Pseudomonas putida and Rhodococcus erythropolis to fragrant intermediates. Rhodococcus erythropolis converts d-limonene into l-carveol and l-carvone, whereas Pseudomonas putida forms in S-(−)-perillyl alcohol and S-(−)-perillic acid. This study revealed that in free cells, biomass yield dropped continuously with significant increase in limonene concentration from 5 to 10 mM, whereas immobilization on Siran® SIKUG41 glass beads showed significant tolerance up to 30–35 mM, thereby alleviating the toxic effect of the substrate. The maximum immobilization efficiency obtained using glass beads was up to 63%. The whole cell immobilization resulted in threefold rise in specific activity and that of tenfold in volumetric activity. The maximum specific and volumetric activity obtained at limonene concentration of 3 mM with P. Putida was 41 U/gCDW, and 218 U/Laq and 2 mM with R. erythropolis was 85 U/gCDW and 478 U/Laq, respectively. The experimental data were statistically analysed using two-tailed Student t test and p values were found to be < 0.05. Immobilization significantly improved productivity of fragrance compounds S-(−)-perillyl alcohol/l-carveol and S-(−)-perillic acid/l-carvone by two- and threefold, respectively.