Rapid Estimation of Astaxanthin and the Carotenoid-to-Chlorophyll Ratio in the Green Microalga Chromochloris zofingiensis Using Flow Cytometry

Abstract

The green microalga Chromochloris zofingiensis can accumulate significant amounts of valuable carotenoids, mainly natural astaxanthin, a product with applications in functional food, cosmetics, nutraceuticals, and with potential therapeutic value in cardiovascular and neurological diseases. To optimize the production of astaxanthin, it is essential to monitor the content of astaxanthin in algal cells during cultivation. The widely used HPLC (high-performance liquid chromatography) method for quantitative astaxanthin determination is time-consuming and laborious. In the present work, we present a method using flow cytometry (FCM) for in vivo determination of the astaxanthin content and the carotenoid-to-chlorophyll ratio (Car/Chl) in mixotrophic C. zofingiensis. The method is based on the assessment of fluorescent characteristics of cellular pigments. The mean fluorescence intensity (MFI) of living cells was determined by FCM to monitor pigment formation based on the correlation between MFI detected in particular channels (FL1: 533 ± 15 nm; FL2: 585 ± 20 nm; FL3: >670 nm) and pigment content in algal cells. Through correlation and regression analysis, a linear relationship was observed between MFI in FL2 (band-pass filter, emission at 585 nm in FCM) and astaxanthin content (in HPLC) and applied for predicting astaxanthin content. With similar procedures, the relationships between MFI in different channels and Car/Chl ratio in mixotrophic C. zofingiensis were also determined. Car/Chl ratios could be estimated by the ratios of MFI (FL1/FL3, FL2/FL3). FCM is thus a highly efficient and feasible method for rapid estimation of astaxanthin content in the green microalga C. zofingiensis. The rapid FCM method is complementary to the current HPLC method, especially for rapid evaluation and prediction of astaxanthin formation as it is required during the high-throughput culture in the laboratory and mass cultivation in industry.

Keywords: Chromochloris zofingiensis; HPLC; astaxanthin; flow cytometry; fluorescence; rapid estimation.

Figure 1

Microscopic images of C. zofingiensis cells during mixotrophic growth. (a) Initial stage at 0 h; (b) Final stage at 288 h; Bars: 10 μm. The mixotrophic growth experiment was performed in modified basal medium with 30 g/L of glucose and 0.6 g/L of NaNO₃ with a high C/N ratio of 200. A light intensity at 80 μmol photon m⁻² s⁻¹ was employed for astaxanthin accumulation after inoculation of seed culture grown mixotrophically under 10 μmol photon m⁻² s⁻¹.

Figure 2

Pigment composition of the mixotrophic C. zofingiensis cells at 288 h determined by HPLC at 480 nm. Tentatively identified peaks: 1. Violaxanthin; 2. Neoxanthin; 3. Chlorophyll b; 4. Keto-lutein; 5. Lutein; 6. Astaxanthin; 7. Chlorophyll a; 8. Canthaxanthin; 9, 11. Keto-lutein mono-esters; 10. Astaxanthin mono-ester; 12. Carotene-like carotenoid; 13–18. Astaxanthin di-esters.

Figure 3

Algal biomass, pigment cell content and Car/Chl variation determined by HPLC in the culture of C. zofingiensis.

Figure 4

Percentage (%, total pigments) of pigments determined by HPLC in the mixotrophic cells of C. zofingiensis.

Figure 5

The fluorescence emission spectra of chlorophyll-rich green cells and astaxanthin-rich red cells of mixotrophic Chromochloris zofingiensis using a spectrofluorometer (488 nm excitation). (a) Chlorophyll-rich cells at 0 h; (b) Astaxanthin-rich cells at 288 h.

Figure 6

Fluorescence images of mixotrophic C. zofingiensis based on confocal laser scanning microscopy (CLSM). (a) Chlorophyll-rich cells at 0 h; (b) Astaxanthin-rich cells at 288 h (excitation at 488 nm, emission at 530 nm ± 10 nm, 580 nm ± 10 nm, 700 nm ± 30 nm). Bars: 25 μm.

Figure 7

(a) The mean fluorescence intensities (MFI) in the FL1, FL2, and FL3 channels; (b) The MFI ratios of FL1/FL3 and FL2/FL3 in mixotrophic cells of C. zofingiensis. FL1: 533 nm ± 15 nm; FL2: 585 nm ± 20 nm; FL3: >670 nm; a.u.: arbitrary units.

Figure 8

The cytograms of mixotrophic C. zofingiensis during cultivation based on FCM analysis. (a) FSC vs. FL1; (b) Cell count vs. FL1 (533 nm ± 15 nm); (c) FSC vs. FL2; and (d) Cell count vs. FL2 (585 nm ± 20 nm). Green dotted lines (or dots) represent the initial cells (the start of culture); orange solid lines (or dots) represent the final cells (the end of culture). M1 represents the population of small cells with low fluorescence; M2 corresponds to the population of large cells with high fluorescence.

Figure 8

The cytograms of mixotrophic C. zofingiensis during cultivation based on FCM analysis. (a) FSC vs. FL1; (b) Cell count vs. FL1 (533 nm ± 15 nm); (c) FSC vs. FL2; and (d) Cell count vs. FL2 (585 nm ± 20 nm). Green dotted lines (or dots) represent the initial cells (the start of culture); orange solid lines (or dots) represent the final cells (the end of culture). M1 represents the population of small cells with low fluorescence; M2 corresponds to the population of large cells with high fluorescence.

Figure 9

Linear relationships of astaxanthin contents versus mean fluorescence intensities (MFI) at FL1 (a) and FL2; (b), and linear relationships of Car/Chl ratios versus the ratios of FL1/FL3; (c) and FL2/FL3; (d) using FCM. Each dot represents the astaxanthin content (μg/g, dry weight) determined by HPLC and MFI by FCM from the same sample. The values of mean fluorescence intensity (a.u.) and the astaxanthin content (μg/g, dry weight) of algal cells were logarithmically transformed (log10) to improve the fit and to equalize variances.

Figure 9

Linear relationships of astaxanthin contents versus mean fluorescence intensities (MFI) at FL1 (a) and FL2; (b), and linear relationships of Car/Chl ratios versus the ratios of FL1/FL3; (c) and FL2/FL3; (d) using FCM. Each dot represents the astaxanthin content (μg/g, dry weight) determined by HPLC and MFI by FCM from the same sample. The values of mean fluorescence intensity (a.u.) and the astaxanthin content (μg/g, dry weight) of algal cells were logarithmically transformed (log10) to improve the fit and to equalize variances.