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

marine drugs

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

Junhui Chen 1, Dong Wei 1,* ID and Georg Pohnert 2

1 School of Food Sciences and Engineering, South China University of Technology, Guangzhou 510641, China; [email protected] 2 Institute for Inorganic and Analytical Chemistry, Bioorganic Analytics, Friedrich Schiller University Jena, Lessingstr. 8, D-07743 Jena, Germany; [email protected] * Correspondence: [email protected]; Tel./Fax: +86-208-711-3849

Received: 23 February 2017; Accepted: 11 July 2017; Published: 19 July 2017

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

1. Introduction Astaxanthin (3,30-dihydroxy-β,β-carotene-4,40-dione) is a well-known red keto-carotenoid with high commercial value in functional food, cosmetics and nutraceuticals industry because of its high anti-oxidative activities [1]. Astaxanthin is a potential therapeutic agent for cardiovascular and neurological diseases owing to its anti-oxidative, anti-inﬂammatory and anti-apoptotic effects [2,3]. Natural astaxanthin prevalently has (3S,3’S) stereochemistry, while synthetic astaxanthin is composed of (3S,3’S), (3RS,3’RS) and (3R,3’R) stereoisomers. The natural product is thus a superior source for highly deﬁned additives to food and pharmaceutics [4,5]. Both the free astaxanthin and its

Mar. Drugs 2017, 15, 231; doi:10.3390/md15070231 www.mdpi.com/journal/marinedrugs Mar. Drugs 2017, 15, 231 2 of 23 esters from Haematococcus pluvialis exhibit multiple bioactivities including anti-cancer, anti-oxidation, anti-inflammation and anti-diabetes activities and cardiovascular prevention [1]. Also, the microalgal powder of H. pluvialis has been associated with anti-aging potential [6]. The green microalga H. pluvialis is the best source for the commercial production of astaxanthin followed by the red yeast Phaffia rhodozyma [7]. Recently, the unicellular green microalga Chromochloris zofingiensis, which was renamed from Chlorella zofingiensis [8], is considered an alternative promising producer of natural astaxanthin with the potential to replace H. pluvialis. It is superior to H. pluvialis since C. zofingiensis has a high growth rate and it is easy to achieve high cell density in the presence of organic substrates [9]. It accumulates astaxanthin under stress conditions [10]. Other secondary carotenoids such as keto-lutein, adonixanthin, and canthaxanthin generally formed from primary carotenoids, also remarkably accumulate in algal cells as a strategy to reduce photo-damage and to adapt to changing light conditions along with the degradation of chlorophylls and primary carotenoids [11]. Accordingly, the carotenoid-to-chlorophyll ratio (Car/Chl), as the preferable indicator of carotenogenesis in microalgae [12], increases markedly under the combined stress of high irradiance and nitrogen deprivation due to the accumulation of secondary carotenoids. Easier cell-wall disruption and higher recovery of valuable pigments make C. zofingiensis more attractive for astaxanthin production than H. pluvialis [13]. However, the content or production of astaxanthin from C. zofingiensis is still much lower than that of H. pluvialis [10]. Strategies to enhance astaxanthin accumulation in C. zofingiensis are still under investigation [9,14]. A fast screening method for the formation of this high-value product is required to enable carotenoid evaluation and control the production of astaxanthin. Rapid and accurate methods for the relative and absolute astaxanthin quantification in living cells are thus crucial in the high-throughput screening of cultures. HPLC methods involving C18 or C30 columns for the separation of cellular pigments are commonly used, but these protocols are time-consuming and require extraction of the cells before measurement [15]. Some rapid methods including Raman spectroscopy, near-infrared spectroscopy (NIRS) and nuclear magnetic resonance spectroscopy (NMR) are feasible for the quantitative measurement of natural carotenoids [15–19]; however, these methods demand expensive and specialized equipment and often do not fulfill the requirements for high-throughput screening. The fluorescence spectroscopy-based investigation is a widely used technology that is convenient for carotenoid analysis with acceptable sensitivity and accuracy. Flow cytometry (FCM) is a powerful technology for single cell analysis through detecting forward- and side-scattered light and fluorescence signals in living cells. To date, FCM has been widely applied in the eco-physiological studies of unicellular microalgae, especially in the analysis of cellular pigments (chlorophylls, carotenoids and phycobiliproteins) based on their auto-fluorescence in real time without losing accuracy [20]. It is speculated that the auto-fluorescence of astaxanthin has the potential use to measure the content using FCM without applying additional fluorescent dyes. FCM-based methods for rapid assay and screening have been reported recently to replace the conventional methods for carotenoids and astaxanthin quantification in yeasts and the green microalga H. pluvialis [21–23]. The interferences of auto-fluorescence between carotenoids and chlorophylls are highlighted as major reasons for the limited application of FCM in pigment quantification [22]. In the present work, we present a simple FCM method to estimate the astaxanthin content in living cells of C. zofingiensis as a preferably complementary method to HPLC, especially for rapid evaluation and prediction of astaxanthin formation in living algal cells during high-throughput cultivation. The method allows for the relative quantification of the astaxanthin content and the Car/Chl ratio. Based on the correlation of the fluorescence intensity of algal cells and cellular pigment content measured by HPLC, the FCM method can potentially, after calibration, be used for the absolute quantification as well. The cells were first cultivated mixotrophically under the stress condition of a high C/N ratio to induce astaxanthin accumulation. Additionally, the fluorescence (FL1-FL3) of the stressed cells was monitored by FCM, followed by the measurement of astaxanthin content in Mar. Drugs 2017, 15, 231 3 of 23 Mar. Drugs 2017, 15, 231 3 of 22 frozencontent dried in frozen biomass dried using biomass an established using an HPLC established method. HPLC Finally, method. the FCM Finally, method, the based FCM on themethod, close correlationbased on the of fluorescenceclose correlation intensities of fluorescence with carotenoid intensities quantities with determinedcarotenoid quantities by HPLC, wasdetermined established by toHPLC, rapidly was evaluate established the to astaxanthin rapidly evaluate content the and astaxanthin Car/Chl ratiocontent in livingand Car/Chl cells. The ratio interferences in living cells. of otherThe interferences carotenoids of to astaxanthinother carotenoids quantification, to astaxanthin recovery quantification, and repeatability recovery of theand FCM repeatability method are of discussedthe FCM method in detail. are discussed in detail.

2. Results and Discussion

2.1. Cell Growth and Pigment Accumulation The green microalga C. zofingiensiszofingiensis has a high growthgrowth raterate andand showsshows substantialsubstantial astaxanthin accumulation duringduring unfavorable unfavorable growth growth conditions conditions such such as oxidative as oxidative stress, stress, high light high and light nutrient and starvationnutrient starvation [10,24]. In[10,24]. the present In the present work, thework, mixotrophic the mixotrophic cells were cells non-motilewere non-motile and spherical and spherical with differentwith different sizes (Figuresizes (Figure1). Small 1). autosporesSmall autospores were asexually were asexually produced produced from large from parental large parental cells during cells theduring culture. the culture. Cells started Cells to started accumulate to accumulate carotenoids, caro mainlytenoids, astaxanthin, mainly astaxanthin, as the response as the to unfavorableresponse to conditionsunfavorable of conditions high irradiation of high and irradiation nitrogen and starvation nitrogen for starvation survival. Therefore,for survival. the Therefore, cell color the started cell tocolor become started orange to become or red orange gradually or red from gradually green duringfrom green the surveyedduring the 288 surveyed h of culturing. 288 h of Theculturing. larger cellsThe larger were obviously cells were redder obviously than othersredder atthan the endothers of theat the cultivation end of the (288 cultivation h), where the(288 highest h), where cellular the astaxanthinhighest cellular content astaxanthin was observed conten (Figurest was observed2 and3). (Figures 2 and 3). Biomass concentrationconcentration and and pigment pigment composition composition were were analyzed analyzed daily fromdaily thefrom sixth the day sixth onwards. day Theonwards. pigments The pigments were analyzed were analyzed by HPLC by andHPLC identified and identified by comparing by comparing the retention the retention times times and and the UV-visthe UV-vis absorption absorption and and spectral spectral fine fine structures structures with with the the standards standards and and informationinformation from literature. As shownshown in Figurein Figure2, astaxanthin 2, astaxanthin and its derivativesand its de andrivatives precursors, and keto-lutein,precursors, lutein, keto-lutein, canthaxanthin lutein, andcanthaxanthin chlorophylls and were chlorophylls the main pigmentswere the inmain the pi stressedgments cells in the at thestressed end of cells the cultivationat the end (288of the h), withcultivation similar (288 chromatographic h), with similar profileschromatographic as reported profiles previously as reported [10,11 previously]. Three forms [10,11]. of Three astaxanthin forms (free,of astaxanthin mono-ester (free, and mono-ester di-esters) wereand di-esters) detected inwere the detected HPLC chromatogram in the HPLC chromatogram (Figure2), indicating (Figure that 2), astaxanthinindicating that primarily astaxanthin existed primarily as di-esters, existed while as di-esters, keto-lutein while was keto-lutein mainly present was mainly in the form present of the in mono-ester.the form of the The mono-ester. other carotenoids The other existed carotenoids predominantly existed inpredominantly the free form. in the free form.

(a) (b)

Figure 1. Microscopic imagesimages ofof C.C. zoﬁngiensiszofingiensiscells cells during during mixotrophic mixotrophic growth. growth. (a ()a Initial) Initial stage stage at at 0 h;0 (h;b )( Finalb) Final stage stage at 288 at h;288 Bars: h; 10Bars:µm. 10 The μm. mixotrophic The mixotrophic growth growth experiment experiment was performed was performed in modiﬁed 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 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 −2 −1 intensitylight intensity at 80 µ molat 80 photon μmol m photon−2 s−1 was m employed s was foremployed astaxanthin for accumulationastaxanthin accumulation after inoculation after of −2 −1 seedinoculation culture of grown seed culture mixotrophically grown mixotrophically under 10 µmol under photon 10 m μ−mol2 s− photon1. m s . Mar. Drugs 2017, 15, 231 4 of 23 Mar. Drugs 2017, 15, 231 4 of 22

60 5 50

40 9 11 15 10 12 30 7 8 16

Absorbtion (mAU) 13 3 20 4 14 17 18 10 6 1 2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 Time (min)

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

Figure 3 shows the variation of biomass, pigment content and Car/Chl ratio in mixotrophic Figure3 shows the variation of biomass, pigment content and Car/Chl ratio in mixotrophic cultures. Algal cells grew rapidly during the first 192 h; then, the specific growth rate decreased cultures. Algal cells grew rapidly during the first 192 h; then, the specific growth rate decreased gradually due to limited nitrogen supply, and the final accumulated biomass was only 3.27 g/L. The gradually due to limited nitrogen supply, and the final accumulated biomass was only 3.27 g/L. astaxanthin content began to increase remarkably when the cells entered the stationary growth The astaxanthin content began to increase remarkably when the cells entered the stationary growth phase after 192 h of the culture in response to low nitrogen and high irradiation stress. In general, phase after 192 h of the culture in response to low nitrogen and high irradiation stress. In general, chlorophylls and primary carotenoids such as neoxanthin, violaxanthin and lutein, are mainly chlorophylls and primary carotenoids such as neoxanthin, violaxanthin and lutein, are mainly located located in chloroplasts of green algae. Primary carotenoids are associated with chlorophylls in the in chloroplasts of green algae. Primary carotenoids are associated with chlorophylls in the collection collection of light energy and transfer the energy to chlorophylls for photosynthesis [25]. Nitrogen of light energy and transfer the energy to chlorophylls for photosynthesis [25]. Nitrogen deficiency deficiency and high light intensity as the stress conditions led to the accumulation of secondary andcarotenoids high light such intensity as astaxanthin as the stress and conditions keto-lutein led to theresist accumulation the excess light of secondary damage carotenoidsalong with suchthe asactive astaxanthin degradation and keto-luteinof chlorophylls to resist and theprimary excess carotenoids light damage [11]. along Astaxanthin with the and active its degradationesters were ofreported chlorophylls as the andmajor primary specific carotenoids carotenoids [ 11existing]. Astaxanthin in C. zofingiensis and its esterscells [10]. were In reportedthis work, as under the major the specificstress conditions carotenoids applied, existing the in asC.taxanthin zofingiensis contentcells increased [10]. In in this a more work, pronounced under the manner stress conditions than the applied,other pigments, the astaxanthin and reached content the increased maximum in of a more2.3 mg pronounced/g at 288 h (Figure manner 3), than which the otherwas consistent pigments, andwith reached a previous the study maximum [11]. Keto-lutein of 2.3 mg/g increased at 288 h from (Figure 0.253), to which 0.34 mg/g was in consistent C. zofingiensis with cells, a previous while studythe other [11]. pigments Keto-lutein such increased as lutein, from chlorophyll 0.25 to 0.34 a and mg/g b changed in C. zofingiensis slightly cells,during while the culture the other (Figure pigments 3). suchAs a as result lutein, of chlorophyllthe increase ainand theb cellularchanged asta slightlyxanthin during content, the the culture Car/Chl (Figure ratio3). and As atotal result pigment of the increasecontent inincreased the cellular over astaxanthintime, and finally content, reached the Car/Chl values as ratio high and as 4.81 total and pigment 3.78 mg/g, content respectively. increased overThe time,percentage and finally of astaxanthin reached values relative as high to all as 4.81pigments and 3.78 increased mg/g, respectively.from 38.1% Theat 146 percentage h to the of astaxanthinmaximum of relative 60.3% toat all288 pigments h (Figure increased 4), which fromwas 38.1%slightly at higher 146 h tothan the the maximum value of of50% 60.3% previously at 288 h (Figurereported4), for which C. zofingiensis was slightly growing higher under than theunfavorable value of 50%conditions previously [10]. Keto-lutein reported for at C.9.1% zofingiensis of total growingpigments under was found unfavorable as the conditionssecond most [10 abundant]. Keto-lutein carotenoid at 9.1% after of total astaxanthin. pigments The was percentage found as theof secondchlorophylls most abundant(a + b) relative carotenoid to all pigments after astaxanthin. decreased Thefrom percentage 31.9% at 146 of chlorophyllsh to 17.5% at (288a + hb )(Figure relative 4). to allOverall, pigments the decreasedchanges in from pigment 31.9% composition at 146 h to 17.5% of C. atzofingiensis 288 h (Figure led4 to). Overall,the shift thein color changes from in green pigment to compositionred, indicating of C.that zofingiensis the stressled of tonitrogen-depletion the shift in color and from exposure green to to red, high indicating irradiation that resulted the stress in ofa nitrogen-depletiondecrease in chlorophylls and exposure and primary to high carotenoids irradiation resultedand the inaccumulation a decrease in of chlorophylls secondary carotenoids, and primary carotenoidsespecially astaxanthin and the accumulation and keto-lutein. of secondary carotenoids, especially astaxanthin and keto-lutein. Mar. Drugs 2017, 15, 231 5 of 23 Mar. Drugs 2017, 15, 231 5 of 22

3.5 Chlorophyll a Chlorophyll b Lutein Carotene-like Canthaxanthin Ketolutein Astaxanthin Biomass 3.0 Total pigments Car/Chl 5.0

2.5 4.0

2.0 3.0

(mg/g) 1.5

2.0 Car/Chl (wt/wt) 1.0 Total pigment content (mg/g) Total 1.0 0.5 Biomass (g/L), carotenoids (mg/g), chlorophylls (mg/g), chlorophylls Biomass (g/L), carotenoids 0.0 0.0 146 168 192 216 240 264 288

Time (h) Figure 3. Algal biomass, pigment cell content and Car/Chl variation determined by HPLC in the culture of C. zofingiensis. Figure 3. Algal biomass, pigment cell content and Car/Chl variation determined by HPLC in the culture of C. zoﬁngiensis.

Mar.Mar. Drugs Drugs 20172017, 15, 15, 231, 231 6 of6 of 23 22

100% Chlorophyll a 90%

80% Chlorophyll b 70% Lutein 60%

50% Carotene-like

40% Canthaxanthin 30% Percent of pigments (%) Percent Ketolutein 20%

10% Astaxanthin 0% 146 168 192 216 240 264 288 Time (h)

FigureFigure 4. 4.Percentage Percentage (%,(%, totaltotal pigments)pigments) ofof pigmentspigments determineddetermined by HPLC in the the mixotrophic mixotrophic cells cells of of C.C. zofingiensis zoﬁngiensis. .

Mar. Drugs 2017, 15, 231 7 of 23

2.2. Fluorescence Images and Flow Cytometric Analysis Mar. Drugs 2017, 15, 231 7 of 22 The fluorescence emission spectra of the suspension of chlorophyll-rich green cells and astaxanthin-rich2.2. Fluorescence red Images cells inand pure Flow water Cytometric were Analysis measured using a spectrofluorometer (488 nm excitation) as shownThe in fluorescence Figure5. The emission mixotrophic spectra cellsof the emitted suspension green, of yellow,chlorophyll-rich and red green fluorescence cells and with differentastaxanthin-rich intensities, andred thecells fluorescence in pure water intensities were measured changed duringusing thea spectr stress-inducedofluorometer carotenogenesis. (488 nm Consideringexcitation) that as shown photosynthetic in Figure 5. pigments The mixotrophic such as cells chlorophylls emitted green, and yellow, carotenoids and red are fluorescence the important fluorescentwith different compounds intensities, in biological and the systems fluorescence [26], the intensities changes inchanged auto-fluorescence during the ofstress-induced algal cells might be correlatedcarotenogenesis. with cellular Considering pigment that photosynthetic composition. pigments In green such algal as cells, chlorophylls chlorophyll and acarotenoids, chlorophyll b are the important fluorescent compounds in biological systems [26], the changes in and lutein are the main pigments, while small amounts of other carotenoids, such as neoxanthin, auto-fluorescence of algal cells might be correlated with cellular pigment composition. In green violaxanthin, astaxanthin, ketolutein and canthaxanthin, also exist in green algal cells [11]. According algal cells, chlorophyll a, chlorophyll b and lutein are the main pigments, while small amounts of to previousother carotenoids, studies [ 26such–28 as], neoxanthin, chlorophyll violaxanthina emits maximum, astaxanthin, red ketolutein fluorescence and canthaxanthin, at about 680 also nm and low far-redexist in fluorescencegreen algal cells at [11]. 735 nm,According and these to previous fluorescence studies intensities[26–28], chlorophyll correlated a emits with maximum the content of chlorophyllred fluorescencea, which at was about in accordance680 nm and withlow far-red our study fluorescence as shown at in735 Figure nm, and5a. these The redfluorescence fluorescence at 680intensities nm could correlated be reabsorbed with the by contentin vivo of chlorophyllchlorophyll a,a whichof cells; was therefore, in accordance the weak with our red study fluorescence as mightshown be undetected, in Figure 5a. which The red could fluorescence explain at the 680 phenomenon nm could be reabsorbed that we only by in detected vivo chlorophyll the weak a of far-red fluorescencecells; therefore, of algal the cells weak in Figure red 5fluorescenceb. might be undetected, which could explain the phenomenon that we only detected the weak far-red fluorescence of algal cells in Figure 5b.

33 29 (a) 25 21 17 13 9 5 Relative Intensity (a.u.) 1 500 525 550 575 600 625 650 675 700 725 750 775 800 Wavelength (nm) 20 18 (b) 16 14 12 10 8 6 4 Relative Intensity (a.u.) 2 -1 500 525 550 575 600 625 650 675 700 725 750 775 800 Wavelength (nm) Figure 5. The fluorescence emission spectra of chlorophyll-rich green cells and astaxanthin-rich red Figure 5. The fluorescence emission spectra of chlorophyll-rich green cells and astaxanthin-rich cells of mixotrophic Chromochloris zofingiensis using a spectrofluorometer (488 nm excitation). (a) red cells of mixotrophic Chromochloris zofingiensis using a spectrofluorometer (488 nm excitation). Chlorophyll-rich cells at 0 h; (b) Astaxanthin-rich cells at 288 h. (a) Chlorophyll-rich cells at 0 h; (b) Astaxanthin-rich cells at 288 h. As for the astaxanthin-rich red cells, their pigment composition, as shown in Figures 3 and 4, wasAs for significantly the astaxanthin-rich different from red that cells, of theirgreen pigment algal cells. composition, Considering as that shown astaxanthin, in Figures accounting3 and4, was significantly different from that of green algal cells. Considering that astaxanthin, accounting for 60.3% Mar. Drugs 2017, 15, 231 8 of 23 of total pigments, was the main fluorescent compound in algal cells, the changes in auto-fluorescence of algal cells and their intensities at specific wavelengths (530 nm & 580 nm), as shown in Figures5–7, might have potential relationships with these pigments, especially astaxanthin. According to previous studies [26–28], the emission wavelength of 530 nm was probably caused by carotenoids, while the origin of the yellow fluorescence at 570 nm was not certain. In a previous study, the maximum excitation and emission wavelengths of astaxanthin in acetone by fluorescence analysis were 350 and 570 nm, respectively [22]. To our knowledge, the fluorescence emission spectrum of in vivo astaxanthin in algal cells has not been reported. In the microalga H. pluvialis, yellow fluorescence at 575 ± 15 nm (488 nm excitation) [29] and 600 ± 40 nm (546 nm excitation) [30] was used for astaxanthin analysis, although these references did not contain detailed experimental evidence of the fluorescence originating from in vivo astaxanthin. Considering that algal cells can simultaneously synthesize various carotenoids and their precursors with a similar chemical structure, it is not possible to obtain the fluorescence emission spectrum of in vivo astaxanthin due to the serious interference of auto-fluorescence between astaxanthin and other carotenoids. However, in our current work, astaxanthin, accounting for 60.3% of total pigments, was the important fluorescent compound in algal cells; thus, its fluorescence would be surely reflected in the fluorescence emission spectra of algal cells. Generally, carotenoid fluorescence is mainly attributed to the presence of the symmetrical tetraterpene skeleton formed by the conjugation of two geranylgeranyl diphosphate molecules, which is the basic structure of various carotenoids [31]. The conjugation length and chemical modification in the circuit of electrons along the conjugated bonds has been reported to affect the absorbance spectrum of carotenoids [31,32]. Thus, we assumed that the yellow fluorescence mainly originated from the series of conjugated double bonds of carotenoids and could be further affected by chemical modification of in vivo carotenoids such as esterification with different fatty acids in lipid droplets. Astaxanthin fluorescence may be also reabsorbed or disturbed by other carotenoid derivatives and precursors due to their similar structures including the symmetrical tetraterpene skeleton. Additionally, it is worth mentioning that the quantum yield of fluorescence emission depends not only on the properties of the fluorescing chromophore but also on its surroundings, which significantly influence the quenching of excitation energy [33,34]. Although there is no consensus about the origins of the green (530 nm) and yellow (570 nm) fluorescence of algal cells at present, based on our results as shown in Figures3–6, we propose that the changes in the fluorescence emission spectrum of algal cells in the present study were caused by the significant accumulation of secondary carotenoids, mainly astaxanthin during stress-induced carotenogenesis. Mar. Drugs 2017, 15, 231 9 of 23 Mar. Drugs 2017, 15, 231 9 of 22

530 nm 580 nm 700 nm Merged

(a)

(b)

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 Figurecells at 6.288Fluorescence h (excitation images at 488 nm, of mixotrophic emission at C.530 zoﬁngiensis nm ± 10 nm,based 580 onnm confocal ± 10 nm,laser 700 nm scanning ± 30 nm). microscopy Bars: 25 μ (CLSM).m. (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.

Mar. Drugs 2017, 15, 231 10 of 23 Mar. Drugs 2017, 15, 231 10 of 22

45000 1000000

40000 (a) 900000 35000 800000 30000 FL1 FL2 25000 700000 FL3 20000 600000 15000 MFI in FL3 (a.u.)

MFI in FL1 and FL2 (a.u.) 500000 10000

5000 400000 120 144 168 192 216 240 264 288 312 Time (h) 0.12 0.050 (b) 0.045 0.10 FL1/FL3 0.040 FL2/FL3 0.035 0.08 0.030 0.06 0.025 FL2/FL3 FL1/FL3 0.020 0.04 0.015 0.010 0.02 0.005 0.00 0.000 120 144 168 192 216 240 264 288 312 Time (h) Figure 7. (a) The mean fluorescence intensities (MFI) in the FL1, FL2, and FL3 channels; (b) The MFI 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 ratios of FL1/FL3 and FL2/FL3 in mixotrophic cells of C. zofingiensis. FL1: 533 nm ± 15 nm; FL2: nm ± 20 nm; FL3: >670 nm; a.u.: arbitrary units. 585 nm ± 20 nm; FL3: >670 nm; a.u.: arbitrary units. Confocal laser scanning microscopy (CLSM) was employed to visualize the cellular distributionConfocal laser of pigments scanning as microscopy shown in Figure (CLSM) 6. Fluorescence was employed images tovisualize of C. zofingiensis the cellular showed distribution that of pigmentschlorophyll-rich as shown cells in emitted Figure6 very. Fluorescence strong red fluo imagesrescence, of C. whereas zofingiensis astaxanthin-richshowed that cells chlorophyll-rich exhibited cellsboth emitted green very and strong yellow redfluorescence. fluorescence, The changes whereas in astaxanthin-rich auto-fluorescence cellswereexhibited closely related both to green the and pigment composition of algal cells at different stages of the stressed culture, which could be applied yellow fluorescence. The changes in auto-fluorescence were closely related to the pigment composition to predict pigment formation. Therefore, in the following experiments, the fluorescence at of algal cells at different stages of the stressed culture, which could be applied to predict pigment approximately 530, 580, and 700 nm was selected for FCM analysis. formation.In Therefore,vivo auto-fluorescence in the following of the experiments, mixotrophic thecells fluorescence (referred to atas approximately the mean fluorescence 530, 580, and 700 nmintensities, was selected MFI, in for three FCM channels: analysis. FL1 at 533 ± 15 nm, FL2 at 585 ± 20 nm, and FL3 > 670 nm) was recordedIn vivo auto-fluorescence by FCM. MFI in FL1 of and the mixotrophicFL2 increased cellsnoticeably (referred over to the as time the meancourse fluorescence of the experiment, intensities, MFI,while in three MFI channels: in FL3 kept FL1 on atdecreasing 533 ± 15 in nm,the stressed FL2 at 585culture± 20 (Figure nm, and7). Accordingly, FL3 > 670 nm)the MFI was ratios recorded by FCM.of FL1 MFI to FL3 in FL1(FL1/FL3) and FL2and increasedFL2 to FL3 noticeably(FL2/FL3) bo overth increased, the time from course 0.049 of to the 0.095 experiment, and 0.015 to while MFI in FL3 kept on decreasing in the stressed culture (Figure7). Accordingly, the MFI ratios of

FL1 to FL3 (FL1/FL3) and FL2 to FL3 (FL2/FL3) both increased, from 0.049 to 0.095 and 0.015 to 0.032, respectively, during the stressed cultivation. Several previous studies have demonstrated Mar. Drugs 2017, 15, 231 11 of 22 Mar. Drugs 2017, 15, 231 11 of 23 0.032, respectively, during the stressed cultivation. Several previous studies have demonstrated that the fluorescence detected by FL3 (675 ± 20 nm) could be used to monitor the content of chlorophylls thatwhen the excited fluorescence at 488 detectednm [35], byand FL3 FCM (675 could± 20 be nm) applied could to be measure used to monitorthe relative the content of chlorophyllschlorophyll a when and b excited in Dunaliella at 488 nmviridis [35 cells], and by FCM determining could be appliedthe MFI toin measureFL3 and theFL4 relative (675 ± 25 content nm), ofrespectively chlorophyll [36].a and Theb inyellowDunaliella fluorescence viridis cells at 575 by determining± 15 nm (488 the nm MFI excitation) in FL3 and [29] FL4 and (675 600± ±25 40 nm), nm respectively(546 nm excitation) [36]. The [30] yellow was applied fluorescence for the at 575analysis± 15 of nm astaxanthin (488 nm excitation) in H. pluvialis [29] and. Furthermore, 600 ± 40 nm it (546was nmreported excitation) that the [30 ]chlorophyll was applied fluorescence for the analysis ratio of of astaxanthin F684/F735 in couldH. pluvialis supply. Furthermore,useful information it was reportedfor the rapid that theestimation chlorophyll of cellular fluorescence stress ratioin the of algal F684/F735 photosynthetic could supply apparatus useful [37]. information Thus, the for MFI the rapidratios estimationof FL1/FL3 of and cellular FL2/FL3, stress which in the algalkept photosyntheticon increasing during apparatus carotenogenesis [37]. Thus, the in MFI the ratiospresent of FL1/FL3study, may and be FL2/FL3, used to investigate which kept th one cellular increasing stress during in algal carotenogenesis cells. In C. zofingiensis in the present cells, photosynthetic study, may be usedpigments to investigate such as thechlorophylls cellular stress and inastaxanthi algal cells.n InareC. the zofingiensis main fluorescentcells, photosynthetic compounds. pigments Under suchenvironmental as chlorophylls stresses, and the astaxanthin accumulation are of the secondary main fluorescent carotenoids compounds. was reported Under to co-occur environmental with the stresses,degradation the accumulationof chlorophylls of secondaryunder environmenta carotenoidsl wasstress reported [11], towhich co-occur could with further the degradation affect the ofauto-fluorescence chlorophylls under of algal environmental cells. The appropriate stress [11], in whichterpretation could further of these affect auto-fluorescences the auto-fluorescence and their of algalintensities cells. Theat specific appropriate wavelengths interpretation may ofgive these usef auto-fluorescencesul information about and theirthe intensitiespresence of at specificspecific wavelengthspigments and may the givephysiological useful information states of aboutalgal cells the presence[26]. of specific pigments and the physiological statesTwo of algal different cells [cell26]. size populations were present in the cultures as indicated by the signals of forwardTwo scatter different (FSC) cell (Figure size populations 8). These weretwo subpopu presentlations in the culturesof cells are as indicatedin accordance by the with signals the ofmicroscopic forward scatter observation (FSC) (Figureand CLSM8). Theseimages. two The subpopulations stress of nitrogen of cells deprivation are in accordance together with with high the microscopiclight irradiation observation resulted andin the CLSM increase images. in the The percentage stress of nitrogenof small deprivationcells (M1) from together ca. 22% with to high46% lightafter irradiationthe cultivation resulted (Figure in the 8a,c), increase indicating in the percentage that the ofstressed small cells conditions (M1) from resulted ca. 22% in to the 46% large after theparental cultivation cells having (Figure comparatively8a,c), indicating thin that cell the walls stressed and conditions releasing resultedmore small in the autospores. large parental This cells was havingalso consistent comparatively with a thinprevious cell walls study and which releasing reported more that small light autospores. could promote This wasalgae also proliferation consistent withand accelerate a previous cell study division which in reporteda culture that under light light could [38]. promote Moreover, algae the proliferation cytometric results and accelerate showed cellthat divisionsmall cells in a culture(M1) emitting under light weak [38 ].fluorescen Moreover,ce theexhibited cytometric no resultssignificant showed difference that small in cellsthe (M1)fluorescence emitting intensities weak fluorescence in both FL1 exhibited and FL2 no significantcompared differenceto the small in the cells fluorescence before stressing intensities of the in bothculture. FL1 Conversely, and FL2 compared the large to cells the small(M2) emitted cells before fluorescence stressing strongly of the culture. in both Conversely, FL1 and FL2 the at large the cellsend (M2)of the emitted cultivation, fluorescence although strongly the in percenta both FL1ge and of FL2 large at thecells end significantly of the cultivation, decreased although to theapproximately percentage of45%. large It cellsis known significantly that during decreased the life to approximatelycycle of C. zofingiensis 45%. It is, the known vegetative that during cells thecontain life cyclemore ofchlorophyllsC. zofingiensis and, theless vegetativeastaxanthin cells under contain favorable more conditions, chlorophylls whereas and less under astaxanthin adverse undergrowth favorable conditions, conditions, they turn whereas into large under red adverse cells growthin combination conditions, with they the turn high into accumulation large red cells inof combinationcarotenoids, mainly with the astaxanthin, high accumulation and the degradatio of carotenoids,n of chlorophylls mainly astaxanthin, [11]. Carotenoid and the degradation accumulation of chlorophyllsin large cells [ 11is ].consistent Carotenoid with accumulation the changes in in large fluorescence cells is consistent intensity with based the changeson FCM. in Accordingly, fluorescence intensitythe fluorescence based on intensities FCM. Accordingly, indicated by the FCM fluorescence could be intensities clearly applied indicated for bythe FCM relative could quantitative be clearly applieddetermination for the of relative certain quantitative cellular pigments. determination of certain cellular pigments.

(a) (b)

Figure 8. Cont.

Mar. Drugs 2017, 15, 231 12 of 23 Mar. Drugs 2017, 15, 231 12 of 22

Figure 8. The cytograms of mixotrophic C. zofingiensis during cultivation based on FCM analysis. (a) Figure 8. The cytograms of mixotrophic C. zofingiensis during cultivation based on FCM analysis. FSC vs. FL1; (b) Cell count vs. FL1 (533 nm ± 15 nm); (c) FSC vs. FL2; and (d) Cell count vs. FL2 (585 (a) FSC vs. FL1; (b) Cell count vs. FL1 (533 nm ± 15 nm); (c) FSC vs. FL2; and (d) Cell count vs. FL2 nm ± 20 nm). Green dotted lines (or dots) represent the initial cells (the start of culture); orange solid (585 nm ± 20 nm). Green dotted lines (or dots) represent the initial cells (the start of culture); orange lines (or dots) represent the final cells (the end of culture). M1 represents the population of small 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. cells with low fluorescence; M2 corresponds to the population of large cells with high fluorescence. 2.3. Linear Regression of the Astaxanthin Content versus Fluorescence Intensities 2.3. Linear Regression of the Astaxanthin Content versus Fluorescence Intensities Assuming that the auto-fluorescence of algal cells at specific wavelengths (530 nm & 580 nm) correlatedAssuming positively that the with auto-fluorescence the cellular pigments, of algal the cells relationships at specific wavelengthsbetween fluorescence (530 nm &intensities 580 nm) correlatedin FL1 and positively FL2 measured with the by cellularFCM and pigments, the cellular the relationships astaxanthin betweencontent measured fluorescence by intensitiesHPLC were in FL1explored and FL2 and measured compared by as FCM shown and in the Figure cellular 9. astaxanthinConsidering content that the measured values of by fluorescence HPLC were exploredintensity andwere compared several orders as shown of inmagnitude Figure9. Consideringhigher than thatthose the of values the pigment of fluorescence content, intensity the variables were several were orderslogarithmically of magnitude transformed higher thanto satisfy those the of homogene the pigmentity content,of variances the variablesassumption were for logarithmically the errors and transformedlinearize the tofit satisfy as much the homogeneityas possible. Then, of variances the astaxanthin assumption content for the from errors HPLC and linearizemeasurements the fit µ as(μg/g) much was as possible.plotted against Then, the astaxanthinmean fluorescence content intensity from HPLC (MFI) measurements in the channel ( g/g)FL1 wasor FL2. plotted The againstcorrelation the coefficient mean fluorescence between intensitythe astaxanthin (MFI) in content the channel and MFI FL1 in or FL2 FL2. was The obtained correlation at 0.96 coefficient (n = 21, betweenp < 0.01), thewhich astaxanthin was slightly content higher and than MFI the in value FL2 was of 0.93 obtained (n = 21, at 0.96p < 0.01) (n = between 21, p < 0.01), the astaxanthin which was slightlycontent higherand MFI than in theFL1. value Furthermore, of 0.93 (n the= 21, correlationp < 0.01) betweencoefficients the between astaxanthin Car/Chl content ratios and and MFI the in FL1.ratios Furthermore, of FL1/FL3 theor FL2/FL3 correlation were coefficients calculated between using Car/Chlsimilar procedures, ratios and theand ratios these of values FL1/FL3 both or FL2/FL3reached 0.91 were (n calculated = 21, p < 0.01). using The similar value procedures, of the correlation and these coef valuesficient both higher reached than 0.91 0.90 (n indicated= 21, p < 0.01). that Thestrong value positive of the correlation coefficientexisted between higher thanthese 0.90 variables, indicated which that strongwas consistent positive correlation with our existedresults betweenpresented these above. variables, In order which to predict was consistentthe astaxanthi withn our content results based presented on fluore above.scence In orderintensity to predictin FL1 theor FL2 astaxanthin by FCM, contentregression based analysis on fluorescence was applied intensity to find inthe FL1 best or fit FL2 of byastaxanthin FCM, regression evaluation, analysis and wasthe linear applied regression to find the was best estimated fit of astaxanthin through the evaluation, coefficient and of thedetermination linear regression (R2). The was best estimated linear 2 throughregression the curve coefficient was ofselected determination for the (Rrapid). The de besttermination linear regression of astaxant curvehin. was Furthermore, selected for the rapidrelationship determination between ofthe astaxanthin. ratios of FL1/FL3 Furthermore, or FL2/FL3 the and relationship Car/Chl ratios between was theestablished ratios of by FL1/FL3 similar orprocedures. FL2/FL3 andThe Car/ChlFCM method ratios with was the established established by similarregression procedures. curves was The then FCM used method for predicting with the establishedthe astaxanthin regression content curves and Car/Chl was then ratio used of for unkn predictingown samples the astaxanthin under specific content conditions and Car/Chl through ratio ofdirectly unknown detecting samples the underfluorescence specific intensities conditions of through unknown directly samples. detecting the fluorescence intensities of unknownAs shown samples. in Figure 9a,b, the astaxanthin content was positively correlated with MFI in both FL1 andAs shown FL2. Among in Figure these,9a,b, theMFI astaxanthin in FL2 was content closely was correlated positively with correlated the astaxanthin with MFI content in both ( FL1R2 = 2 and0.92) FL2. by Amongcomparison these, to MFI that in of FL2 FL1 was (R closely2 = 0.87). correlated This was with in the accordance astaxanthin with content the (Rfluorescence= 0.92) by 2 comparisonemission maximum to that of of FL1 algal (R =cells 0.87). (Figure This was5). Our in accordance results indicated with the that fluorescence MFI in FL2 emission was feasible maximum to of algal cells (Figure5). Our results indicated that MFI in FL2 was feasible to estimate the cellular estimate the cellular astaxanthin content by the regression equation (log10 (astaxanthin (μg/g)) = 1.53 astaxanthin content by the regression equation (log (astaxanthin (µg/g)) = 1.53 × log (MFI in FL2 × log10 (MFI in FL2 (a.u.)) + 3.07 (R2 = 0.92)). The10 coefficient of determination, as 10high as 0.92, 2 (a.u.))demonstrated + 3.07 ( Rthat= 0.92)).FCM correlated The coefficient well ofwith determination, the HPLC values as high and as 0.92,could demonstrated be directly applied that FCM to correlatedpredict the well astaxanthin with the content. HPLC values and could be directly applied to predict the astaxanthin content. In a previous study, the auto-fluorescence intensity around 675 nm based on FCM showed good correlation with the actual content of astaxanthin (0.10–0.63 mg/g) in the yeast

Mar. Drugs 2017, 15, 231 13 of 23

In a previous study, the auto-fluorescence intensity around 675 nm based on FCM showed good correlation with the actual content of astaxanthin (0.10–0.63 mg/g) in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodoxyma)[22]. A similar result was reported for β-carotene content (0.35 to 0.66 mg/g) analysis in the red yeast Rhodotorula glutinis by FCM [39]. Freitas et al. also suggested Mar. Drugs 2017, 15, 231 13 of 22 that the auto-fluorescence measured in FL3 (>670 nm, excited with 488 nm light) was best correlated with the contentXanthophyllomyces of total carotenoidsdendrorhous (Phaffia in the rhodoxyma red yeast) [22].Rhodosporidium A similar result toruloides was reported[40 ].for These β-carotene foregoing results arecontent contradictory (0.35 to 0.66 to mg/g) the existing analysis knowledgein the red yeast that Rhodotorula chlorophylls, glutinis particularly by FCM [39]. chlorophyll Freitas et al. a, not carotenoids,also suggested emit auto-fluorescence that the auto-fluorescence at the maximummeasured in wavelengthFL3 (>670 nm, ofexcited nearly with 680 488 nm light) when was excited best correlated with the content of total carotenoids in the red yeast Rhodosporidium toruloides [40]. with 488 nm light [41]. We assumed that this might by caused by the low content of carotenoids, These foregoing results are contradictory to the existing knowledge that chlorophylls, particularly and theirchlorophyll fluorescence a, not was carotenoids, seriously emit interfered auto-fluorescence with other at the pigments maximum such wavelength as carotenoid of nearly precursors, 680 proteins,nm nucleotides, when excited porphyrins, with 488 nm flavins, light [41]. etc. We [21 assume,42]. Ind that our this present might work, by caused the astaxanthinby the low content content in C. zofingiensisof carotenoids,cells reached and 2.41their mg/g,fluorescence which was was seri significantlyously interfered higher with than other that pigments of previous such as studies. Our resultscarotenoid indicated precursors, that the proteins, fluorescence nucleotides, in FL2 porphy (580rins, nm) flavins, based etc. on [21,42]. FCM In was our closelypresent work, correlated with thethe content astaxanthin of cellular contentcarotenoids, in C. zofingiensis particularly cells reached astaxanthin, 2.41 mg/g, which and was FL3 significantly (>675 nm) higher correlated than that of previous studies. Our results indicated that the fluorescence in FL2 (580 nm) based on directly with chlorophylls, which can be intuitively rationalized by the corresponding emissions of FCM was closely correlated with the content of cellular carotenoids, particularly astaxanthin, and the respectiveFL3 (>675 pigments nm) correlated [35]. Kleinegris directly with et chlorophylls, al. reported which that can the be 560 intuitively nm fluorescence rationalized ofby the the green microalgacorrespondingDunaliella salina emissionswas emittedof the respective by β-carotene pigments (excitation [35]. Kleinegris at 488 et nm). al. re InportedSymbiodinium that the 560, thenm green fluorescencefluorescence signal in of FL1 the(529 green± microalga28 nm band-pass Dunaliella filter) salina waswas generallyemitted byused β-carotene for β-carotene (excitation analysis at 488 [43]. These resultsnm). demonstratedIn Symbiodinium, thatthe green the fluorescence fluorescence intensitiessignal in FL1 at (529 the ± range 28 nm of band-pass 530–580 nmfilter) had was a close generally used for β-carotene analysis [43]. These results demonstrated that the fluorescence relationship with carotenoids, which was the foundation of the FCM method for astaxanthin analysis. intensities at the range of 530–580 nm had a close relationship with carotenoids, which was the The Car/Chlfoundation ratio of the was FCM previously method for demonstrated astaxanthin analysis. as the informative marker of cellular physiological stress in the microalgaThe Car/ChlH. pluvialisratio wasunder previously high light demonstrated irradiation as and theParietochloris informative incisamarker[44 of,45 cellular]. Moreover, the Car/Chlphysiological ratio correlated stress in the closelymicroalga with H. pluvialis the specific under high optical light irradiation density inand the Parietochloris blue-green incisa region normalized[44,45]. to the Moreover, red absorption the Car/Chl maximum ratio correlated (OD500/OD closely678), with which the could specific be appliedoptical density for the in monitoring the of carotenogenesisblue-green inregionH. pluvialis normalizedusing to opticalthe red sensorsabsorption [12 maximum]. In this work,(OD500/OD we678 explored), which thecould correlation be applied for the monitoring of carotenogenesis in H. pluvialis using optical sensors [12]. In this work, of the Car/Chl ratio with the fluorescence intensities in FL1-FL3 channels using FCM. The Car/Chl we explored the correlation of the Car/Chl ratio with the fluorescence intensities in FL1-FL3 ratio of mixotrophicchannels usingC. FCM. zofingiensis The Car/Chlincreased ratio of mixotrophic substantially C. zofingiensis during culturing increased undersubstantially stress during conditions along withculturing the accumulation under stress ofconditions astaxanthin along as with well the as theaccumulation degradation of astaxanthin of chlorophylls. as well Accordingly,as the the ratiosdegradation of FL1/FL3 of and chlorophylls. FL2/FL3 Accordingly, increased along the rati withos of the FL1/FL3 changes and of FL2/FL3 pigments increased (Figure along7b), indicatingwith a positivethe correlation changes of betweenpigments the(Figure Car/Chl 7b), indicating ratio with a positive the ratio correlation of FL1/FL3 between or FL2/FL3.the Car/Chl Figure ratio 9c,d shows thatwith the the Car/Chl ratio of FL1/FL3 ratio was or linearlyFL2/FL3. relatedFigure 9c,d with shows FL1/FL3 that the and Car/Chl FL2/FL3, ratio was though linearly the related coefficients 2 with FL1/FL32 and FL2/FL3, though the coefficients of determination (R ) were 0.82. Therefore, the of determinationfluorescence (R intensities) were 0.82. in FL1, Therefore, FL2 and the FL3 fluorescence could also be intensitiesapplied to the in assay FL1, FL2of the and Car/Chl FL3 couldratio also be appliedin toC. zofingiensis the assay cells. of the Car/Chl ratio in C. zofingiensis cells.

3.5 y = 1.9380x - 5.6681 (a) 3.4 R² = 0.8725 g/g)

μ 3.3

3.2

3.1

3.0 Log Astaxanthin ( 2.9

2.8 4.40 4.50 4.60 4.70 Log (MFI in FL1)

Figure 9. Cont.

Mar. Drugs 2017, 15, 231 14 of 23 Mar. Drugs 2017, 15, 231 14 of 22

3.5 y = 1.5325x - 3.0668 (b) 3.4 R² = 0.9209

g/g) 3.3 μ 3.2

3.1

3.0

Log Astaxnthin ( 2.9

2.8 3.85 3.95 4.05 4.15 4.25 Log (MFI in FL2)

6.0 (c) 5.0

4.0

Car/Chl 3.0

2.0 y = 50.85x - 0.2631 R² = 0.8262 1.0 0.030 0.050 0.070 0.090 0.110 FL1/FL3

4.5 (d)

3.5

2.5 Car/Chl

1.5 y = 139.19x + 0.0991 R² = 0.8267

0.5 0.008 0.013 0.018 0.023 0.028 0.033 FL2/FL3

Figure 9. FigureLinear 9.relationships Linear relationships of astaxanthin of astaxanthin contents contents versus versus mean fluorescence ﬂuorescence intensities intensities (MFI) (MFI) at at FL1 (a) and FL2; (b), and linear relationships of Car/Chl ratios versus the ratios of FL1/FL3; (c) and 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. Mar. Drugs 2017, 15, 231 15 of 23

To evaluate the determination of the astaxanthin content and Car/Chl ratio, we compared the values of the astaxanthin content and Car/Chl ratio measured by the FCM method with that measured by HPLC using correlation analysis according to the literature [46,47]. These correlation coefficients were 0.95 (n = 21, p < 0.01) and 0.91 (n = 21, p < 0.01), respectively, indicating that these different methods had a strong positive correlation with respect to measuring the astaxanthin content and Car/Chl ratio in C. zofingiensis cells. Furthermore, the FCM method can be further calibrated by HPLC through regression analysis to obtain more accurate values of the astaxanthin content and Car/Chl ratio. The regression curve for astaxanthin content determination was Y (astaxanthin content measured by FCM) = 0.8885 X (astaxanthin content measured by HPLC) + 0.1644 (R2 = 0.91). As for the Car/Chl ratio, the regression curve obtained by the similar procedures was Y (Car/Chl ratio measured by FCM) = 0.8278 X (Car/Chl ratio measured by HPLC) + 0.5383 (R2 = 0.83). However, our current work primarily aimed to examine the relationship between the fluorescence intensity and cellular astaxanthin content of algal cells and evaluate the reliability of the FCM method for predicting the astaxanthin content from the fluorescence intensity of algal cells. Thus, the FCM method without further calibration by HPLC was applied in the following method validation.

2.4. Linearity, Range, and Limits of Quantitation for the FCM Method The FCM method was validated by the evaluation of its performance characteristics, such as linearity, precision, accuracy and recovery, and proved that it complied with established criteria for chemical analysis [48,49]. Through analyzing validation samples with different astaxanthin contents, we could demonstrate that the FCM method was capable of rapidly quantifying the astaxanthin content and Car/Chl ratio in living cells of mixotrophic C. zofingiensis. The best-fit linear regression of the astaxanthin content using HPLC versus MFI in FL2 using FCM was established through the coefficient of determination (R2 = 0.92) in this work. The lower limit of astaxanthin quantification (0.82 mg/g) corresponded to the minimum fluorescence intensity of 7988 by FCM (Figure9b). The favorable range for accurate astaxanthin analysis based on FCM was between 0.82 and 2.41 mg/g dry cell weight (Figure9b). Using HPLC analysis, the astaxanthin content in stress-induced C. zofingiensis was determined mainly in the range of 0.69–1.31 mg/g dry cell weight [10]. The approach we developed allows the direct monitoring of astaxanthin in C. zofingiensis. The method is thus suitable for the real-time monitoring of carotenogenesis in C. zofingiensis cells. In the above results (Figure9c,d), the ratios of FL1/FL3 and FL2/FL3 were linearly correlated with the Car/Chl ratios, respectively, with the same coefficient of determination (R2 = 0.82). Considering the astaxanthin assay, the FL2/FL3 ratio was selected for the rapid estimation of the Car/Chl ratio in C. zofingiensis cells. The suitable range for the accurate determination of the Car/Chl ratio should be limited between 1.42 and 4.75 according to the regression.

2.5. Precision, Accuracy, and Recovery of FCM Method The precision of the FCM method is documented in Table1. The sample of C. zoﬁngiensis cells with an astaxanthin content of 2.08 mg/g and Car/Chl ratio of 3.89 (determined by HPLC) was used for this evaluation. The mean astaxanthin content of 2.03 mg/g by FCM was very close to the true value based HPLC with a relative error (ER) of 1.93% and relative standard deviation (RSD) of 2.02% (n = 5), indicating the good precision of the FCM method. Similarly, the mean Car/Chl ratio of 4.32 based on FCM was also close to the value determined by HPLC with a relative error (ER) of 11.10% and relative standard deviation (RSD) of 1.10% (n = 5), indicating that this approach is feasible for rapidly estimating the Car/Chl ratio with a suitable precision. Mar. Drugs 2017, 15, 231 16 of 23

Table 1. The precision of the FCM method for the rapid quantiﬁcation of astaxanthin and the Car/Chl ratio.

Astaxanthin Car/Chl Ratio Asta. Content Car/Chl MFI in MFI in Relative Relative No. by FCM Ratio by Mean Relative Relative FL2 (a.u.) FL3 (a.u.) Standard Mean Standard (mg/g) Value Error Error FCM Deviation Value Deviation (mg/g) (RE, %) (RE, %) (RSD, %) (RSD, %) 1 14,230 468,000 1.99 4.37 2 14,350 468,430 2.01 4.36 2.03 ± 3 14,747 489,000 2.10 4.30 1.93 2.20 4.32 ± 0.04 11.05 1.10 0.05 4 14,630 484,860 2.07 4.30 5 14,405 480,100 2.02 4.28 No. Five independent samples were taken from two independent culturing flasks. MFI: mean fluorescence intensity; FL2: 585 nm ± 20 nm; FL3: >670 nm; a.u.: arbitrary unit; Asta: astaxanthin; Car/Chl ratio: the carotenoid-to-chlorophyll ratio; a sample of C. zofingiensis cells with astaxanthin content of 2.08 mg/g and Car/Chl of 3.89 (determined by HPLC) was used for the evaluation.

In pharmaceutical analysis, recovery assays using spiked matrices and spiked samples are important tools for assessing the method accuracy [50], which is generally recognized as the difference between the measured mean values by the established method and the true values of validation samples [49]. In the current work, the accuracy and recovery of the FCM method were accordingly developed and modified according to the recovery analysis of spiked samples. In the following experiments, two sets of samples taken from the same stressed culture were measured by FCM followed by HPLC for accuracy analysis. Astaxanthin contents based on HPLC were 0.92 ± 0.12 and 1.65 ± 0.22 mg/g dry weight for these two samples, respectively. According to the results listed in Table2, the astaxanthin content determined by FCM was basically consistent with that of HPLC with less than 12% of overall variation between the two methods. The Car/Chl ratio determined by FCM was equivalent to that determined by HPLC with ca. 3% overall variation. These results demonstrated that the FCM approach is feasible to determine the astaxanthin content and Car/Chl ratio in the suitable range and fulfills the requirements of the analytical methodology without sacrificing accuracy.

Table 2. The accuracy and recovery of the FCM method for the rapid quantiﬁcation of astaxanthin and the Car/Chl ratio.

No. 1 2 3 4 5 Mean Value MFI in FL2 (a.u.) 7914 8056 8301 8233 8117 8124 ± 151 Sample 1 MFI in FL3 (a.u.) 634,600 651,000 726,080 718,860 704,560 687,000 ± 41,500 FL2/FL3 0.0125 0.0124 0.0114 0.0115 0.0115 0.0119 ± 0.0005 Asta. (mg/g) 0.81 0.83 0.87 0.86 0.84 0.84 ± 0.02 Measured value by FCM Car/Chl 1.83 1.82 1.69 1.69 1.70 1.75 ± 0.07 Asta. 87.75 90.17 94.41 93.23 91.22 91.36 ± 2.61 Accuracy analysis (%) Car/Chl 79.15 78.57 72.92 73.03 73.44 75.42 ± 3.15 MFI in FL2 (a.u.) 12,177 12,281 12,545 12,408 12,186 12,319 ± 156 Sample 2 MFI in FL3 (a.u.) 558,740 565,610 598,240 591,460 580,000 578,800 ± 16,700 FL2/FL3 0.0218 0.0217 0.0209 0.0209 0.0210 0.0213 ± 0.0005 Asta.(mg/g) 1.56 1.58 1.64 1.61 1.57 1.59 ± 0.03 Measured value by FCM Car/Chl 3.13 3.12 3.02 3.02 3.02 3.06 ± 0.06 Asta. 94.75 95.99 99.17 97.52 94.86 96.46 ± 1.88 Accuracy analysis (%) Car/Chl 103.65 103.28 99.86 99.90 100.00 101.34 ± .95 Asta. 103.58 103.33 105.16 102.93 99.45 Recovery (%) Car/Chl 78.60 78.73 80.41 80.31 80.02 Asta. 102.89 ± 2.10 Average recovery (%) Car/Chl 79.61 ± 0.88 Relative standard deviation Asta. 2.04 of recovery (RSD, %) Car/Chl 1.10 MFI: mean fluorescence intensity; FL2: 585 nm ± 20 nm; FL3: >670 nm; a.u.: arbitrary unit; Asta.: astaxanthin; Car/Chl ratio: the carotenoid-to-chlorophyll ratio; Sample 1: C. zofingiensis cells with astaxanthin content of 0.92 mg/g and Car/Chl of 2.32 (determined by HPLC); Sample 2: C. zofingiensis cells with astaxanthin content of 1.65 mg/g and Car/Chl of 3.02 (determined by HPLC). Mar. Drugs 2017, 15, 231 17 of 23

Additionally, the measured values of the samples subjected to FCM and HPLC were examined to assess the recovery analysis of the current method. As shown in Table2, the average recovery of samples subjected to FCM for astaxanthin analysis was 102.89%, within the acceptable range of 80–120%. The relative standard deviation (RSD) of samples (n = 5) was as low as 2.04%, indicating the good repeatability in the recovery analysis of the FCM method. The average recovery of the Car/Chl ratio was relatively low owing to the weak correlation of FL2/FL3 vs. Car/Chl at a low carotenoid content. A reasonable explanation might be the variation of carotenoid composition and the steady aggregation of other carotenoids at different cultivation stages of mixotrophic C. zofingiensis [12]. Thus, we concluded that FCM, as a rapid and simple method, can be applied to the analysis of the astaxanthin content and Car/Chl ratio with acceptable precision and accuracy. It is worth noting that an obvious disadvantage of this method is that it requires instrumental determination under identical conditions and the pigment concentration in the linear range of the method. When cultivated in different systems and stress conditions, microalgae have a wide range of intercellular carotenoids and variable pigment composition that could be easily surveyed by the introduced FCM approach. Variations contributed to the non-linear relationship between the estimated pigments and the fluorescence intensities when using FCM. Furthermore, algal cells sampled from the cultures had to be analyzed by FCM directly and immediately under identical conditions to avoid environmental factors affecting the determination of the auto-fluorescence of algal cells. The system settings of FCM, for example, the event limit (total events collected), fluorescence detector voltages, and compensation controls, are also crucial for the determination of samples as well as the above-mentioned factors. These parameters of system settings must be identical in each assay of FCM to avoid technique errors. Taking together all the considerations, if the culture conditions of algal cells or the settings of FCM were changed, an FCM assay with a suitable regression equation should be re-established accordingly for the robustness of this approach in rapid estimation.

3. Materials and Methods

3.1. Microalgae and Culture Conditions The green microalga Chromochloris zofingiensis (ATCC30412) was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained at 4 ◦C on agar slants or plates of modified basal medium [51]. The cells were cultivated in the same liquid medium containing 10 g/L of glucose in shaking flasks at 26 ◦C, 150 r/m under continuous illumination with white fluorescence lights (ca. 10 µmol photon m−2 s−1). The culture reached the exponential phase after 96 h; then, we transferred 10 mL of the seed culture into 100 mL of modified basal medium containing 30 g/L of glucose and 0.6 g/L of NaNO3 in 250 mL Erlenmeyer flasks to generate a high C/N ratio of 200. The continuous light intensity was increased to 80 µmol photon m−2 s−1 for astaxanthin accumulation during the following 288 h. From 146 h of the stressed culture, the cells were sampled every 24 h and harvested by centrifugation at 3800 × g for 2 min. The biomass concentration was used to monitor the cell growth. The fluorescence intensities of living cells in FL1–FL3 were determined by FCM, while the pigment content in frozen dry biomass was analyzed by HPLC.

3.2. Chemicals and Reagents HPLC-grade methanol and methyl tert-butyl ether (MTBE) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Astaxanthin, lutein, zeaxanthin, canthaxanthin, chlorophyll a and b as standards were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other chemicals and reagents of analytical grade were purchased from the local companies. Deionized water was purified by using a Milli-Q water system (Merck Millipore, Billerica, MA, USA) and filtered through a 0.22 µm membrane for HPLC analysis. Mar. Drugs 2017, 15, 231 18 of 23

3.3. Emission Spectra of Fluorescence and Imaging by CLSM The fluorescence emission spectra of C. zofingiensis cells were detected in the range from 500 nm to 800 nm using a fluorospectrophotometer (HITACHI F-7000, Hitachi, Tokyo, Japan) at an excitation wavelength of 488 nm. A Leica TCS SP5X confocal laser scanning microscopy (CLSM, Leica Microsystems, Milton Keynes, UK) was used to capture the fluorescence images of mixotrophic cells of C. zofingiensis. Intracellular distribution of pigments in cells was viewed by scanning using a 488 nm argon laser, and light emission was recorded in the green (520–540 nm), yellow (570–590 nm), and red channels (670–730 nm) to monitor fluorescence signals of C. zofingiensis cells.

3.4. Flow Cytometry Flow cytometry (model BD Accuri C6 Accuri Cytometers, Inc., Ann Arbor, MI, USA) was used for the cytometric analysis of C. zofingiensis. Two 50 mW air cooled lasers (exciting at 488 nm and 640 nm) and four interference filters (FL1 at 533 ± 15 nm, FL2 at 585 ± 20 nm, FL3 > 670 nm, and FL4 at 675 ± 25 nm) were used. The detailed procedure was modified according to a previous reference [22]. Briefly, the cells were centrifuged at 3800× g for 2 min and washed twice with potassium phosphate buffer (10 mM, pH 7.4). The cell resuspension was adjusted to 0.6–2 × 106 cells/mL as the optimal cell density and then filtered through a membrane (400 mesh) before cytometric analysis. In FCM analysis, according to the cell size of C. zofingiensis, the flow rate of injection was optimized to 35 µL/min, and 10,000 cells were count for analysis of each sample. The signals of forward scatter (FSC), side scatter (SSC), fluorescence intensities in four channels (FL1–FL4) were detected simultaneously. Data were further analyzed by Accuri® CFlow software (Accuri Cytometers, Inc., Ann Arbor, MI, USA) and Flowjo software (FlowJo, LLC., Ashland, OR, USA) for statistical analysis. All samples were performed in triplicate.

3.5. HPLC Analysis The lyophilized biomass was prepared using a freeze dryer (Modulyod-230, Thermo Scientific, Waltham, MA, USA). According to the literature [11] with a few minor modifications, the pigments were extracted by repeatedly adding methanol/dichloromethane (3:1, v/v) containing 0.1% (w/v) butylated hydroxytoluene (BHT) into the bead-beating tube until the biomass became colorless. The supernatant containing pigments was blow-dried by nitrogen gas and resolved in methanol/MTBE (1:1, v/v) containing 0.1% (w/v) BHT for HPLC analysis. The extraction process was performed under dim light, and the extract was kept at 4 ◦C. A reversed-phase HPLC system (DIONEX P680, Thermo Scientific, Waltham, MA, USA) equipped with a Waters YMC™ Carotenoid C30 column (4.6 × 150 mm, 3 µm) and a photodiode array detector (PDA-100, Thermo Scientific, Waltham, MA, USA) was employed to separate and identify the pigments. The mobile phase consisted of methanol (A), MTBE (B), and deionized water (C) at a flow rate of 0.8 mL/min. A linear gradient program was employed as follows: 0–6 min, from 95% A/5% B to 80% A/20% B; 6–12 min, to 60% A/38% B/2% C; 12–28 min, to 50% A/48% B/2% C and isocratic for 5 min. A 20 µL sample was injected into the column, and the temperature was maintained at 30 ◦C. All peaks were scanned at 300–700 nm and identified by comparison with the retention times and the UV-vis absorption and spectral fine structures of the standards and information from the literature [11] followed by quantification based on the standard curves.

3.6. Correlation and Regression Analysis of the Astaxanthin Content versus Fluorescence Intensities The correlation and regression analyses of astaxanthin content determined by HPLC and fluorescence intensities measured by FCM of algal cells were performed to explore their potential relationships. Initially, the values of fluorescence intensities (a.u.) in FL1 and FL2 and the astaxanthin content (µg/g, dry weight of algal cells) were logarithmically transformed to satisfy the homogeneity Mar. Drugs 2017, 15, 231 19 of 23 of variances assumption for the errors and to linearize the fit [21,52]. Then, Pearson’s correlation coefficients (r) between fluorescence intensities and astaxanthin contents were calculated by SPSS software (version 19.0, SPSS, Inc., Chicago, IL, USA) to measure the degree of association between these variables. If a strong correlation existed, the fluorescence intensities in FL1 and FL2 were plotted against the astaxanthin content of every sample, and regression analysis was performed to obtain the regression equations for predicting astaxanthin from fluorescence intensities based on FCM. With similar procedures, the correlations between Car/Chl ratios and FL1/FL3 or FL2/FL3 were assessed by Pearson’s correlation coefficients (r), and the regression curves for predicting the Car/Chl ratios were constructed for the samples if strong correlations existed (i.e., Pearson’s correlation coefficients were higher than 0.8). The regression curves with the highest coefficients of determination (R2) were selected for predicting the astaxanthin content and Car/Chl ratio for the unknown samples through the fluorescence intensities measured by FCM under specific conditions. In order to avoid environmental preparation errors, algal cells sampled from the cultures should be analyzed by FCM instantly and directly. All system settings of FCM should be identical in each assay of each set of samples, or the values determined from the regression curve might be invalid. If the culture conditions of algal cells or the system settings of FCM are changed, the regression curve must be re-established accordingly to avoid technique errors in sample determination. Moreover, the correlation between these two methods was determined based on the values of the astaxanthin content and Car/Chl ratio measured by FCM and HPLC according to the literature [46,47]. The correlation coefficients were calculated by SPSS 19.0 (SPSS, Inc., Chicago, IL, USA) and applied to estimate the correlation between these two methods. If a strong correlation was detected, regression analysis between the FCM and HPLC values was performed. This procedure served to explore the possibility that the FCM method can be further calibrated by HPLC to obtain more accurate values of the astaxanthin content and Car/Chl ratio.

3.7. Method Validation The FCM method validation was performed from the analysis of validation samples to evaluate its performance characteristics and prove that it fulﬁlled the requirements of the established criteria for determination.

3.7.1. Linear Range and Limits for FCM Method The fluorescent intensities in FL1 and FL2 determined by FCM were plotted against the astaxanthin content to show the best-fit correlation, while the ratios of FL1/FL3 and FL2/FL3 were correlated with Car/Chl ratios in order to investigate their relationships. Based on these strong correlations, regression analysis was performed, and the regression curves with the highest coefficients of determination (R2) were selected for predicting the astaxanthin content and Car/Chl ratio of the unknown samples. The linear range and limits for the FCM method were obtained according to the regression curves.

3.7.2. Precision The living cells of C. zoﬁngiensis in mixotrophic growth were directly analyzed in vivo immediately by FCM after sampling to avoid the potential degradation of astaxanthin. The precision of the FCM method was assessed by analysis of ﬁve technical replicates from the same culture and represented as the relative error (RE) and relative standard deviation (RSD).

3.7.3. Accuracy and Recovery Accuracy, generally recognized as the difference between the measured mean values by the established method and the true values of validation samples [49], can be estimated from recovery tests using samples spiked with a known concentration of the analyte [50]. The accuracy assessment of the FCM method in our study was accordingly developed and modiﬁed to this approach. In brief, we Mar. Drugs 2017, 15, 231 20 of 23 conducted a similar study assessing the accuracy by the use of spiked samples. Two sets of cells taken from the same stressed culture with either high or low astaxanthin content were detected by FCM and HPLC methods to ensure the accuracy, which was represented as the percentage of the mean or true value variation. The recovery was carried out from the two sets of cells similar to spiked samples. The acceptable recovery value should be between 80% and 120%.

4. Conclusions The fluorescence signals and pigment contents in mixotrophic C. zofingiensis cells during cultivation were analyzed by FCM and HPLC. A positive linear regression between the auto-fluorescence of cells based on FCM and the accurate content of astaxanthin or the Car/Chl ratio based on HPLC was established. The method introduced is suitable as a screening procedure complementary to HPLC. The FCM approach based on the auto-fluorescence has the advantages of simplicity and speed by comparison with the traditional HPLC assay. This FCM method can thus complement the traditional assay of pigments and provides a rapid and efficient approach for on-site monitoring of stress-induced carotenogenesis during the cultivation of C. zofingiensis. The FCM method developed in the current work is reliable and affordable for the rapid estimation of carotenoids, particularly astaxanthin, in C. zofingiensis cells with an acceptable precision. It is comparable and competitive to established rapid methods for carotenoid analysis. Our study demonstrates that flow cytometry with suitable algorithms, after calibration by HPLC data, is a powerful tool for studies of carotenogenesis and for physiological characterization of microalgae. The method is especially recommended in processes that require high-throughput methods for the rapid analysis and real-time monitoring of cellular pigments in C. zofingiensis under stress conditions.

Acknowledgments: This work was supported by the National Sciences Foundation of China (NSFC, Grant No. 31370383), National Hi-tech Research and Development Program (863 Project) (Grant No. 2013AA065802), Science and Technology Development Program in Marine and Fishery of Guangdong (Grant No. A201401C01), and Science and Technology Development Program of Guangdong (Grant No. 2015A020216003, 2016A010105001). Author Contributions: Junhui Chen and Dong Wei conceived and designed the experiments, analyzed the data and wrote the paper; Georg Pohnert modified the manuscript and provided valuable comments and suggestion in editing. All authors contributed to review of the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Ambati, R.R.; Phang, S.M.; Ravi, S.; Aswathanarayana, R.G. Astaxanthin: Sources, extraction, stability, biological activities and its commercial applications-a review. Mar. Drugs 2014, 12, 128–152. [CrossRef] [PubMed] 2. Fassett, R.G.; Coombes, J.S. Astaxanthin: A Potential Therapeutic Agent in Cardiovascular Disease. Mar. Drugs 2011, 9, 447–465. [CrossRef][PubMed] 3. Wu, H.; Niu, H.; Shao, A.; Wu, C.; Dixon, B.J.; Zhang, J.; Yang, S.; Wang, Y. Astaxanthin as a potential neuroprotective agent for neurological diseases. Mar. Drugs 2015, 13, 5750–5766. [CrossRef][PubMed] 4. Higuera-Ciapara, I.; Felix-Valenzuela, L.; Goycoolea, F.M. Astaxanthin: A review of its chemistry and applications. Crit. Rev. Food Sci. Nutr. 2006, 46, 185–196. [CrossRef][PubMed] 5. Schmidt, I.; Schewe, H.; Gassel, S.; Jin, C.; Buckingham, J.; Huembelin, M.; Sandmann, G.; Schrader, J. Biotechnological production of astaxanthin with Phafﬁa rhodozyma/Xanthophyllomyces dendrorhous. Appl. Microbiol. Biotechnol. 2011, 89, 555–571. [CrossRef][PubMed] 6. Huangfu, J.; Liu, J.; Sun, Z.; Wang, M.; Jiang, Y.; Chen, Z.-Y.; Chen, F. Antiaging effects of astaxanthin-rich alga Haematococcus pluvialis on fruit ﬂies under oxidative stress. J. Agric. Food Chem. 2013, 61, 7800–7804. [CrossRef][PubMed] 7. Guerin, M.; Huntley, M.E.; Olaizola, M. Haematococcus astaxanthin: Applications for human health and nutrition. Trends Biotechnol. 2003, 21, 210–216. [CrossRef] 8. Champenois, J.; Marfaing, H.; Pierre, R. Review of the taxonomic revision of Chlorella and consequences for its food uses in Europe. J. Appl. Phycol. 2015, 27, 1845–1851. [CrossRef] Mar. Drugs 2017, 15, 231 21 of 23

9. Solovchenko, A.E. Recent breakthroughs in the biology of astaxanthin accumulation by microalgal cell. Photosynthesis Res. 2015, 125, 437–449. [CrossRef][PubMed] 10. Liu, J.; Sun, Z.; Gerken, H.; Liu, Z.; Jiang, Y.; Chen, F. Chlorella zofingiensis as an Alternative Microalgal Producer of Astaxanthin: Biology and Industrial Potential. Mar. Drugs 2014, 12, 3487–3515. [CrossRef] [PubMed] 11. Mulders, K.J.M.; Weesepoel, Y.; Bodenes, P.; Lamers, P.P.; Vincken, J.-P.; Martens, D.E.; Gruppen, H.; Wijffels, R.H. Nitrogen-depleted Chlorella zofingiensis produces astaxanthin, ketolutein and their fatty acid esters: A carotenoid metabolism study. J. Appl. Phycol. 2015, 27, 125–140. [CrossRef] 12. Solovchenko, A.; Aflalo, C.; Lukyanov, A.; Boussiba, S. Nondestructive monitoring of carotenogenesis in Haematococcus pluvialis via whole-cell optical density spectra. Appl. Microbiol. Biotechnol. 2013, 97, 4533–4541. [CrossRef][PubMed] 13. Kim, D.-Y.; Vijayan, D.; Praveenkumar, R.; Han, J.-I.; Lee, K.; Park, J.-Y.; Chang, W.-S.; Lee, J.-S.; Oh, Y.-K. Cell-wall disruption and lipid/astaxanthin extraction from microalgae: Chlorella and Haematococcus. Bioresour. Technol. 2016, 199, 300–310. [CrossRef][PubMed] 14. Liu, J.; Sun, Z.; Gerken, H.; Huang, J.; Jiang, Y.; Chen, F. Genetic engineering of the green alga Chlorella zofingiensis: A modified norflurazon-resistant phytoene desaturase gene as a dominant selectable marker. Appl. Microbiol. Biotechnol. 2014, 98, 5069–5079. [CrossRef][PubMed] 15. Amorim-Carrilho, K.T.; Cepeda, A.; Fente, C.; Regal, P. Review of methods for analysis of carotenoids. Trac-Trends Anal. Chem. 2014, 56, 49–73. [CrossRef] 16. Rivera, S.M.; Canela-Garayoa, R. Analytical tools for the analysis of carotenoids in diverse materials. J. Chromatogr. 2012, 1224, 1–10. [CrossRef][PubMed] 17. Nicolaie, B.M.; Beullens, K.; Bobelyn, E.; Peirs, A.; Saeys, W.; Theron, K.I.; Lammertyn, J. Nondestructive measurement of fruit and vegetable quality by means of NIR spectroscopy: A review. Postharvest Biol. Technol. 2007, 46, 99–118. [CrossRef] 18. De Oliveira, V.E.; Neves Miranda, M.A.C.; Silva Soares, M.C.; Edwards, H.G.M.; Cappa de Oliveira, L.F. Study of carotenoids in cyanobacteria by Raman spectroscopy. Spectrochim. Acta Part A-Mol. Biomol. Spectrosc. 2015, 150, 373–380. [CrossRef][PubMed] 19. Rungpichayapichet, P.; Mahayothee, B.; Khuwijitjaru, P.; Nagle, M.; Mueller, J. Non-destructive determination of beta-carotene content in mango by near-infrared spectroscopy compared with colorimetric measurements. J. Food Compost. Anal. 2015, 38, 32–41. [CrossRef] 20. Hyka, P.; Lickova, S.; Pribyl, P.; Melzoch, K.; Kovar, K. Flow cytometry for the development of biotechnological processes with microalgae. Biotechnol. Adv. 2013, 31, 2–16. [CrossRef][PubMed] 21. An, G.H.; Suh, O.S.; Kwon, H.C.; Kim, K.; Johnson, E.A. Quantification of carotenoids in cells of Phaffia rhodozyma by autofluorescence. Biotechnol. Lett. 2000, 22, 1031–1034. [CrossRef] 22. Ukibe, K.; Katsuragi, T.; Tani, Y.; Takagi, H. Efficient screening for astaxanthin-overproducing mutants of the yeast Xanthophyllomyces dendrorhous by flow cytometry. FEMS Microbiol. Lett. 2008, 286, 241–248. [CrossRef] [PubMed] 23. An, G.H.; Bielich, J.; Auerbach, R.; Johnson, E.A. Isolation and characterization of carotenoid hyperproducing mutants of yeast by flow cytometry and cell sorting. Biotechnology 1991, 9, 70–73. [CrossRef][PubMed] 24. Cordero, B.F.; Couso, I.; Leon, R.; Rodriguez, H.; Angeles Vargas, M. Isolation and Characterization of a Lycopene epsilon-Cyclase Gene of Chlorella (Chromochloris) zofingiensis. Regulation of the Carotenogenic Pathway by Nitrogen and Light. Mar. Drugs 2012, 10, 2069–2088. [CrossRef][PubMed] 25. Demmig-Adams, B.; Gilmore, A.M.; Adams, W.W., 3rd. Carotenoids 3: In vivo function of carotenoids in higher plants. FASEB J. 1996, 10, 403–412. [PubMed] 26. Gabriela Lagorio, M.; Cordon, G.B.; Iriel, A. Reviewing the relevance of fluorescence in biological systems. Photochem. Photobiol. Sci. 2015, 14, 1538–1559. [CrossRef][PubMed] 27. Kleinegris, D.M.M.; van Es, M.A.; Janssen, M.; Brandenburg, W.A.; Wijffels, R.H. Carotenoid fluorescence in Dunaliella salina. J. Appl. Phycol. 2010, 22, 645–649. [CrossRef][PubMed] 28. Ermakov, I.V.; Sharifzadeh, M.; Ermakova, M.; Gellermann, W. Resonance Raman detection of carotenoid antioxidants in living human tissue. J. Biomed. Opt. 2005, 10, 1179–1181. [CrossRef][PubMed] 29. Rioboo, C.; Gonzalez-Barreiro, O.; Abalde, J.; Cid, A. Flow cytometric analysis of the encystment process induced by paraquat exposure in Haematococcus pluvialis (Chlorophyceae). Eur. J. Phycol. 2011, 46, 89–97. [CrossRef] Mar. Drugs 2017, 15, 231 22 of 23

30. Wayama, M.; Ota, S.; Matsuura, H.; Nango, N.; Hirata, A.; Kawano, S. Three-dimensional ultrastructural study of oil and astaxanthin accumulation during encystment in the green alga Haematococcus pluvialis. PLoS ONE 2013, 8, e53618. [CrossRef][PubMed] 31. Schoefs, B. Chlorophyll and carotenoid analysis in food products. Properties of the pigments and methods of analysis. Trends Food Sci. Technol. 2002, 13, 361–371. [CrossRef] 32. Englert, G. UV/Visible spectroscopy. In Carotenoids; Volume 1B: Spectroscopy; Britton, G., Liaaenjensen, S., Pfander, H., Eds.; Birkhäuser: Basel, Switzerland, 1995; Volume 1B, pp. 13–63. 33. Porcar-Castell, A.; Berry, J.A. Linking chlorophyll a fluorescence to photosynthesis for remote sensing applications: Mechanisms and challenges. JEXB 2014, 65, 4065. [CrossRef][PubMed] 34. Narayan, A.; Misra, M.; Singh, R. Chlorophyll Fluorescence in Plant Biology. In Biophysics; Intech: Rijeka, Croatia, 2012; pp. 171–192. 35. Hu, Z.; Li, Y.; Sommerfeld, M.; Chen, F.; Hu, Q. Enhanced protection against oxidative stress in an astaxanthin-overproduction Haematococcus mutant (Chlorophyceae). Eur. J. Phycol. 2008, 43, 365–376. [CrossRef] 36. Davis, R.W.; Carvalho, B.J.; Jones, H.D.T.; Singh, S. The role of photo-osmotic adaptation in semi-continuous culture and lipid particle release from Dunaliella viridis. J. Appl. Phycol. 2015, 27, 109–123. [CrossRef] [PubMed] 37. Eullaffroy, P.; Vernet, G. The F684/F735 chlorophyll fluorescence ratio: A potential tool for rapid detection and determination of herbicide phytotoxicity in algae. Water Res. 2003, 37, 1983–1990. [CrossRef] 38. Chen, T.; Liu, J.; Guo, B.; Ma, X.; Sun, P.; Liu, B.; Chen, F. Light attenuates lipid accumulation while enhancing cell proliferation and starch synthesis in the glucose-fed oleaginous microalga Chlorella Zofingiensis. Sci. Rep. 2015, 5, 14936. [CrossRef][PubMed] 39. Cutzu, R.; Clemente, A.; Reis, A.; Nobre, B.; Mannazzu, I.; Roseiro, J.; da Silva, T.L. Assessment of beta-carotene content, cell physiology and morphology of the yellow yeast Rhodotorula glutinis mutant 400A15 using flow cytometry. J. Ind. Microbiol. Biotechnol. 2013, 40, 865–875. [CrossRef][PubMed] 40. Freitas, C.; Nobre, B.; Gouveia, L.; Roseiro, J.; Reis, A.; da Silva, T.L. New at-line flow cytometric protocols for determining carotenoid content and cell viability during Rhodosporidium toruloides NCYC 921 batch growth. Process Biochem. 2014, 49, 554–562. [CrossRef] 41. Kula, M.; Rys, M.; Mozdzen, K.; Skoczowski, A. Metabolic activity, the chemical composition of biomass and photosynthetic activity of Chlorella vulgaris under different light spectra in photobioreactors. Eng. Life Sci. 2014, 14, 57–67. [CrossRef] 42. Mueller, S.; Galliardt, H.; Schneider, J.; Barisas, B.G.; Seidel, T. Quantification of Forster resonance energy transfer by monitoring sensitized emission in living plant cells. Front. Plant Sci. 2013, 4, 413. [CrossRef] [PubMed] 43. Lee, C.S.; Yeo, Y.S.W.; Sin, T.M. Bleaching response of Symbiodinium (zooxanthellae): Determination by flow cytometry. Cytom. Part A 2012, 81, 888–895. [CrossRef][PubMed] 44. Solovchenko, A.E.; Chivkunova, O.B.; Maslova, I.P. Pigment composition, optical properties, and resistance to photodamage of the microalga Haematococcus pluvialis cultivated under high light. Russ. J. Plant Physiol. 2011, 58, 9–17. [CrossRef] 45. Solovchenko, A.E.; Khozin-Goldberg, I.; Cohen, Z.; Merzlyak, M.N. Carotenoid-to-chlorophyll ratio as a proxy for assay of total fatty acids and arachidonic acid content in the green microalga Parietochloris incisa. J. Appl. Phycol. 2009, 21, 361–366. [CrossRef] 46. Lee, J.; Rennaker, C.; Wrolstad, R.E. Correlation of two anthocyanin quantification methods: HPLC and spectrophotometric methods. Food Chem. 2008, 110, 782–786. [CrossRef] 47. Lao, F.; Giusti, M.M. Quantification of Purple Corn (Zea mays L.) Anthocyanins Using Spectrophotometric and HPLC Approaches: Method Comparison and Correlation. Food Anal. Methods 2016, 9, 1367–1380. [CrossRef] 48. Taverniers, I.; De Loose, M.; Van Bockstaele, E. Trends in quality in the analytical laboratory. II. Analytical method validation and quality assurance. Trac-Trends Anal. Chem. 2004, 23, 535–552. [CrossRef] 49. Peters, F.T.; Drummer, O.H.; Musshoff, F. Validation of new methods. Forensic Sci. Int. 2007, 165, 216–224. [CrossRef][PubMed] 50. Gonzalez, A.G.; Herrador, M.A.; Asuero, A.G. Intra-laboratory testing of method accuracy from recovery assays. Talanta 1999, 48, 729–736. [CrossRef] Mar. Drugs 2017, 15, 231 23 of 23

51. Chen, J.; Liu, X.; Wei, D.; Chen, G. High yields of fatty acid and neutral lipid production from cassava bagasse hydrolysate (CBH) by heterotrophic Chlorella protothecoides. Bioresour. Technol. 2015, 191, 281–290. [CrossRef][PubMed] 52. Asuero, A.G.; Bueno, J.M. Fitting Straight Lines with Replicated Observations by Linear Regression. IV. Transforming Data. Crit. Rev. Anal. Chem. 2011, 41, 36–69. [CrossRef]

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).