Overproduction of Xanthophyll Pigment in Flavobacterium sp. JSWR-1 under Optimized Culture Conditions

Abstract

Flavobacterium can synthesize xanthophyll, particularly the pigment zeaxanthin, which has significant economic value in nutrition and pharmaceuticals. Recently, the use of carotenoid biosynthesis by bacteria and yeast fermentation technology has shown to be very efficient and offers significant advantages in large-scale production, cost-effectiveness, and safety. In the present study, JSWR-1 strain capable of producing xanthophyll pigment was isolated from a freshwater reservoir in Wanju-gun, Republic of Korea. Based on the morphological, physiological, and molecular characteristics, JSWR-1 classified as belonging to the Flavobacterium species. The bacterium is strictly aerobic, Gram-negative, rod-shaped, and psychrophilic. The completed genome sequence of the strain Flavobacterium sp. JSWR-1 is predicted to be a single circular 3,425,829-bp chromosome with a G+C content of 35.2% and 2,941 protein-coding genes. The optimization of carotenoid production was achieved by small-scale cultivation, resulting in zeaxanthin being identified as the predominant carotenoid pigment. The enhancement of zeaxanthin biosynthesis by applying different light-irradiation, variations in pH and temperature, and adding carbon and nitrogen supplies to the growth medium. A significant increase in intracellular zeaxanthin concentrations was also recorded during fed-batch fermentation achieving a maximum of 16.69 ± 0.71 mg/l, corresponding to a product yield of 4.05 ± 0.15 mg zeaxanthin per gram cell dry weight. Batch and fed-batch culture extracts exhibit significant antioxidant activity. The results demonstrated that the JSWR-1 strain can potentially serve as a source for zeaxanthin biosynthesis.

Keywords: Carotenoid; Flavobacterium; HPLC; antioxidant; pigments; zeaxanthin.

Fig. 1. Isolation and identification of Flavobacterium species JSWR-1.

(A) A red-marked region on the map indicates Wanju-gun, South Korea (Source from Wikipedia); (B) Isolates JSWR-1 showed colony morphology on R2A agar and pigmentation. (C) JSWR-1 displayed road-shaped colony morphology on scanning electron microscopy (SEM, x 10,000). (c) TEM images of JSWR-1, the arrow indicated the region of zeaxanthin accumulation.

Fig. 2. Effect of pH on growth and zeaxanthin production of JSWR-1.

(A) Specific growth in two different time intervals at dark incubation. (B) Specific growth in two time intervals at white illumination. (C) Zeaxanthin production on different pH and incubation conditions.

Fig. 3. Effect of C: N ration on bacterial growth and zeaxanthin production of JSWR-1.

(A) Specific growth in two different time intervals at white illumination. (B) Production of zeaxanthin from different C: N ratios. (C) Bacterial growth on selected C: N concentrations from different time intervals. (D) Maximum zeaxanthin production from selected conditions.

Fig. 4. Fed-batch fermentation of Flavobacterium sp. JSWR-1. Glucose (10 g l^-1) and yeast extract (5 g l^-1).

Zeaxanthin concentration represented in mean value ± SD (n = 2) respectively, different alphabetic letters indicate significant difference p < 0.05 by Duncan's multiple range test. Dry weight of the cell pellet was determined by difference after freeze drying (g l^-1). The fed-batch fermenter vessel picture was capture at 120 h.

Fig. 5. Chromatogram of yellow carotenoids from JSWR-1.

(A) UV-VIS spectra of samples compared with zeaxanthin standard (dashed line), absorbance maximum of 450, 478 nm (B) Thin-layer chromatography of standard and samples (a, β-carotene; b, lutein; c, zeaxanthin; d, BC; e, FBC), the dashed arrow indicates the BC and FBC spots on TLC; (C) HPLC chromatogram of standards zeaxanthin, the retention time 13.729 min.; (D) HPLC chromatogram of β-carotene, the retention time 29.529 min.; (E & F) HPLC profile of BC and FBC.

Fig. 6. Results from identification of yellow carotenoids by liquid chromatography –mass spectrometer data.

(A) BC, (B) FBC.

Fig. 7. Antioxidant activity of FBC and BC from JSWR-1.

(A & B) Radical scavenging activity in the DPPH assay of BC and FBC extracts; (C) Antioxidant capacity was measured using the FRAP assay. Error bars represent ± SD of the mean value, n = 3 independent experiment in which each sample was analyzed in duplicate.