Optimization of submerged fermentation process for improved production of β-carotene by Exiguobacterium acetylicum S01

Abstract

Carotenoids are natural pigments with substantial applications in nutraceutical, pharmaceutical, and food industries. In this study, optimization of the fermentation process for enhancement of β-carotene and biomass production by Exiguobacterium acetylicum S01 was achieved by employing statistical designs including the Placket-Burman design (PBD) and response surface methodology (RSM). Among the seven variables investigated by two levels in PBD, glucose, peptone, pH and temperature were indicated as crucial variables (p < 0.0001) for β-carotene and biomass productivity. Response surface methodology was further applied to evaluate the optimal concentrations of these four variables for maximum β-carotene and biomass productivity. The optimized medium contained glucose 1.4 g/L, peptone 26.5 g/L, pH 8.5, and temperature 30 °C, respectively. A significant increase in β-carotene (40.32 ± 2.55 mg/L) and biomass (2.19 ± 0.10 g/L) productivities in E. acetylicum S01 were achieved by using RSM, which was 3.47-fold and 2.36-fold higher in the optimized medium compared to the un-optimized medium. Further, the optimum fermentation condition in the 5-L bioreactor was achieved a maximal β-carotene yield of 107.22 ± 5.78 mg/L within 96 h. Moreover, the expression levels of carotenoid biosynthetic genes (phytoene desaturase (CrtI) and phytoene synthase (CrtB)) were up-regulated (2.89-fold and 3.71-fold) in E. acetylicum under the optimized medium conditions. Overall, these results suggest that E. acetylicum S01 can be used as a promising microorganism for the commercial production of β-carotene.

Keywords: Biotechnology; Food science; Food technology; Microbiology.

Fig. 1

Effects of different medium components and physical factors on biomass (g/L) and β-carotene (mg/L) productivities in E. acetylicum S01. Data are shown as mean ± SD (n = 3). Bar graphs showing the effects of carbon source (A), nitrogen source (B), trace element (C), pH (D), and temperature (E).

Fig. 2

Pareto chart analysis: the effect of media component on biomass (A) and β-carotene (B) productivities were represented by t-value.

Fig. 3

Three-dimensional (3-D) response surface plots representing the interactions of the two factors with biomass (g/L) and β-carotene (mg/L) productivities in E. acetylicum S01 has grown in the RSM-CCRD optimized medium. The 3-D surface plots of pH and peptone (A), peptone and temperature (B and C), and temperature and pH (E).

Fig. 4

Gene expression level of carotenoid biosynthetic genes for the early stationary phase cultures of E. acetylicum S01 cultivated in un-optimized medium (control) and RSM optimized medium conditions for 96 h. Data represent the mean of the three independent biological repeats (n = 3). Bar graphs showing CrtI and CrtB gene expression in un-optimized medium (control) and RSM optimized medium conditions. ****p < 0.0001 indicates a significant difference between the un-optimized medium in response to RSM optimized medium conditions. Non-adjusted images provided in the supplementary material. M - DNA ladder (0.25 kb–10 kb), CTL - Un-optimized medium, RSM - RSM Optimized medium.