y and Ecology 353 (2007) 191–197www.elsevier.com/locate/jembe

Journal of Experimental Marine Biolog

A simple method for the extraction and quantification ofphotopigments from Symbiodinium spp.

John E. Rogers ⁎, Dragoslav Marcovich

United States Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects ResearchLaboratory, Gulf Ecology Division, 1 Sabine Island Dr., Gulf Breeze, FL 32561, United States

Received 7 October 2006; received in revised form 28 March 2007; accepted 25 August 2007

Abstract

We have developed a simple, mild extraction procedure using methanol which, when coupled with HPLC analysis and diodearray detection (DAD), can be used to quantify the major photopigments found in cultured Symbiodinium spp. Extracts wereprepared by suspending, fresh or frozen (−70 °C), wet cell pellets in methanol and sonicating or not sonicating the cell suspensionsbefore soaking the cells for 2 h in an ice bath. To assist the soaking process, cell suspensions were vortex mixed at 30 min intervals.After soaking, 0.5 M ammonium acetate buffer was added (1 part buffer to 9 parts methanol) before suspensions were stored overnight at −20 °C. Greater than 92% the recoverable pigment was obtained in the initial extraction of the four major photopigments,chlorophyll c, peridinin, diadinoxanthin, and chlorophyll a. Neither sonication nor freezing substantially increased the recovery ofphotopigments extracted with methanol. Extraction by other commonly used solvents such as acetone or acetone:water with orwithout freezing and sonication were less effective.Published by Elsevier B.V.

Keywords: Symbiodinium; Photopigments; Methanol extraction; Coral

1. Introduction

The initial step in quantifying photopigments by highperformance liquid chromatography (HPLC) is extrac-tion into a suitable solvent that is both efficient andcompatible with the method of analysis. Efficient sol-vents penetrate the algal cell wall, have good solvationproperties, and require minimal mechanical disruption.Solvents compatible with high performance liquidchromatography (HPLC) lead to tight loading bands

⁎ Corresponding author. USEPA, Gulf Ecology Division, 1 SabineIsland Dr., Gulf Breeze, FL 32561, United States. Tel.: +1 850 9349326; fax: +1 850 934 2401.

E-mail address: Rogers.johne@epa.gov (J.E. Rogers).

0022-0981/$ - see front matter. Published by Elsevier B.V.doi:10.1016/j.jembe.2007.08.022

that increase resolution and produce sharper peaks. Theextraction of photopigments from algae in general hasbeen extensively reviewed by Wright et al. (1996).

Acetone and acetone/water (90:10) have generallybeen the solvents of choice for extracting photopigmentsfrom Symbiodinium sp. since the dichromatic methodfor quantifying chlorophylls a and c2 was introduced byJeffrey and Humphrey (1975). The spectrophotometricmethod of Jeffrey and Humphrey (1975), which is anupdated version of the original method of Jeffrey andHaxo (1968), is one of the most commonly used meth-ods for photopigment analysis in coral-related studies.Dichromatic equations are used to estimate concentra-tions of chlorophylls a and c2 from absorption spectra ofacetone or acetone:water (90:10, v/v) extracts of coral

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tissues and cultured Symbiodinium spp. Wet or drysamples of coral tissue or cultured cells are extractedrepeatedly until the residue has no visible green color.Extraction is facilitated by grinding or sonicating thetissue or cell material in solvent. Solvent extracts arecombined and the concentrations of chlorophylls a andc2 are determined from absorption values at 630 nm and663 nm and extinction coefficients fro these solvents.Iglesias-Prieto et al. (1992) modified this method byusing acetone:dimethylsulfoxide (90:10, v/v) but unfor-tunately retained the equations of Jeffrey and Humphrey(1975). Kinzie (1993) used methanol:tetrahydrofitran(80120, v/v) as the extracting solvent and a modifieddichromatic equation to calculate the concentration ofchlorophyll a. However useful these methods have beenfor quantifying chlorophylls a and c2, they are laborintensive and do not provide information on importantcarotenoids, such peridinin, diadinoxanthin anddiatoxanthin.

The more recent HPLC methods, which have seenlimited use in coral studies, detect and quantify a widerange of chlorophylls and carotenoids. Dunlap andChalker (1986) detected but did not quantify, chlor-ophylls a and c and peridinin, in methanol:tetrahydro-furan extracts of Acropora spp. Ambarsari et al. (1997)quantified chlorophylls and a number of carotenoids inacetone:water (90:10) extracts of Goniastrea asperatissues, Brown et al. (1999) quantified diadinoxanthinand diatoxanthin in acetone:water (90:10) extracts ofG. aspera, and Kufmer (2002) quantified chlorophyllsand carotenoids in methanol extracts of Poritescompressa tissues.

In the course of examining the responses of Sym-biodinium isolates to ultraviolet radiation and temper-ature we required a procedure to adequately extract andquantify the antenna photopigments, chlorophyll a,chlorophyll c2 and peridinin as well as the xanthophyllcomponent diadioxanthin. Methanol was selected for agreater extraction efficiency (Wright et al., 1996) andcompatibility with the HPLC solvent system (Wrightet al., 1991). In this manuscript we describe a mildextraction procedure compatible with HPLC that uses asingle extraction with methanol to achieve this goal.

2. Materials and procedures

2.1. Cell cultures

Symbiodinium spp. clones isolated from blastates ofPorites porites (JROZFl) and Madracis mirabilis(RDO2) were maintained and cultured in Ll medium(Guillard and Hargraves, 1993) prepared with filter

sterilized sea water (salinity 34) collected off the coastof Pensacola Beach in northwest Florida. Cultures(2 L) were grown to late log phase in 2.8 L Fernbachflasks in an incubator shaker (100 rpm) with overheadfluorescent lighting. Photosynthetically active radiation(PAR, λ 400–700 nm) was about 100 μmole quantam−2 s−1. Late log cultures were transferred to 50 mLpolypropylene tubes which were centrifuged at 2000 ×gand 4 °C in an RT6000b Sorval Centrifuge® for 15 minto pellet cells. The supernate was removed by aspirationto the 5 mL mark, celIs were resuspended in the re-maining growth media and the suspensions were trans-ferred to 15 mL polypropylene tubes, centrifuged again,and the supemate was completely removed by aspira-tion. Cell pellets were used immediately or stored at−70 °C.

2.2. Extraction of photopigments

Three extraction solvents were investigated: metha-nol/ammonium acetate, acetone, acetone/water (90:10).All solvents were HPLC grade. Methanol/ammoniumacetate extractions were initiated by adding 4.5 mlmethanol to cell pellets in 13×100 mm glass tubes withpolypropylene caps. The resulting mixtures werehomogenized by vortex mixing or sonication for 120 swith a Vibra Cell® sonicator equipped with a microtipand set to 50% on/off impulse mode. Tubes were thenplaced in crushed ice in the dark for 2 h, mixed at 30 minintervals and, after 2 h, 0.5 mL of 0.5 M ammoniumacetate, pH 7.2, was added to each tube. Samples weremixed again and then stored overnight at −20 °C beforeanalysis. Acetone and acetone/water (90:10) extractionsfollowed a similar pattern. Cell suspensions were ho-mogenized by vortex mixing or sonication as. above andthen placed in crushed ice for 2 h in the dark. Sampleswere vortex mixed at 30 min intervals. After two hours,all samples were placed in a −20 °C freezer overnight.Ammonium acetate (0.5 M) was not added to the ace-tone based extractions. The following morning allsamples were mixed and centrifuged to pellet residualcell material. A portion (1 to 2 mL) of each supernatewas filtered through an Acrodisc® 0.2 pm PTFEmembrane into an amber HPLC sample vial. Extractswere stable when stored for several days at −20 °C orfor several months −70 °C. Because of the stability ofthese samples at −70 °C we currently use extractsprepared from isolate JR02Fl as our continuingcalibration check (ccc) for all pigment analyses. Theccc controls were originally prepared by subdividing alarge volume of extract and storing the samples at−70 °C for up to 6 months.

Table 1Recovery of chlorophyll a, chlorophyll c2, peridinin, and diadinoxanthin,from fresh and frozen wet cell pellets extracted with methanol ormethanol plus sonication

Extract Fresh cell pellets Frozen cell pellets

Methanolplussonication

Methanol Methanolplussonication

Methanol

μg/ml a μg/ml μg/ml μg/ml

Mean (SD) Mean (SD) Mean (SD) Mean (SD)

Chlorophyll a1st 0.80 (0.06) 0.77 (0.08) 0.81 (0.08) 0.84 (0.06)2nd 0.05 (0.01) 0.05 (0.01) 0.05 (0.01) 0.06 (0.01)3rd 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.01 (0.02)Total 0.85 (0.07) 0.82 (0.09) 0.86 (0.09) 0.90 (0.07)% in

1st b0.94 (0.01) 0.94 (0.01) 0.94 (0.01) 0.93 (0.02)

Chlorophyll c1st 0.31 (0.02) 0.31 (0.04) 0.28 (0.03) 0.31 (0.03)2nd 0.03 (0.03) 0.03 (0.03) 0.01 (0.02) 0.00 (0.00)3rd 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.01 (0.02)Total 0.34 (0.05) 0.34 (0.06) 0.29 (0.04) 0.32 (0.03)% in 1st 0.92 (0.08) 0.92 (0.07) 0.97 (0.07) 0.98 (0.05)

Peridinin1st 0.83 (0.06) 0.82 (0.09) 0.82 (0.07) 0.84 (0.05)2nd 0.02 (0.01) 0.02 (0.01) 0.01 (0.01) 0.02 (0.01)3rd 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)Total 0.85 (0.06) 0.84 (0.09) 0.83 (0.08) 0.86 (0.05)% in 1st 0.97 (0.01) 0.97 (0.01) 0.99 (0.01) 0.9% (0.01)

Diadinoxanthin1st 0.16 (0.01) 0.14 (0.02) 0.15 (0.01) 0.15 (0.01)2nd 0.01 (0.01) 0.01 (0.01) 0.01 (0.01) 0.01 (0.02)3rd 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)Total 0.17 (0.02) 0.15 (0.03) 0.16 (0.01) 0.17 (0.02)% in 1st 0.97 (0.07) 0.97 (0.07) 0.96 (0.08) 0.94 (0.09)a μg/ml of pigment in each sequential 5.0 ml extraction.b % in first equals μg/ml recovered in first extract divided by the

total μg/ml recovered in the three sequential extracts.

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2.3. HPLC and spectrophotometric analyses

Photopigments were chromatographically separated bya modification of the Wright et al. (1991) method.Instrumentation included the following Hewlett-Packard(HP) components: 1090 liquid chromatograph with aDAD, Kayak XA personal computer with chemstation andHPuv/vis library software, 3D cooling auto sampler (4 °C),premixing autoinjector and an HP1046 A FluorescenceDetector. Reverse phase colunmswere aWaters spherisorbS5 ODS2 4×250 mm analytical column and a Watersspherisorb S% ODS2 4.6×10 mm guard column.Pigments were detected and quantified by their absorptionat 436 nm. Fluorescence excitation at 424 nm withemission monitored at 640 nm was used to identify chlo-rophylls and chlorophyll break down products. The HPLCgradient used three solvents:A.80:20 (v/v)methanol:0.5Mammonium acetate (pH 7.2); B. 80:20 (v/v) acetonitrile:water; and C. ethyl acetate. The flow rate was 0.76 mL perminute. Photopigments were separated by a series of lineargradients: from 0 to 3 min 100%A to 100%B; 3 to 21 min100%B to 35%B/65%C; 21 to 23 min 35%B/65%C to100%C; 23 to 26 min 100%C to 100%B; and 26 to 30 min100%B to 100%Awhich was followed by a 10 min washwith 100%A before the next injection. Injection volumewas 50 μL. Quantitation was based on authentic standardspurchased from DHI Water and Environment.

2.4. Determination of extraction efficiency

Photopigment extraction efficiencies were examinedusing a single solvent to biomass ratio or by varying thesolvent to biomass ratio. Extraction efficiencies weredetermined using 5 replicate fresh and frozen cell pellets(−70 °C) prepared from late log cultures of Symbiodi-nium 144 sp. (JR02Fl). Each cell pellet was extractedthree times. The combined recoveries form three ex-tractions with methanol/ammonium acetate were as-sumed to yield 100% extraction efficiency.

The effect of cell biomass on pigment recovery wasexamined using cell pellets prepared from 8,5,2.5, 1.0,0.5and 0.25 mL samples from frozen cells (−70 °C) from alate log culture of isolate JR02Fl resuspended in Llmedium or using cell pellets prepared from 50,40,30,20,and 10 mL of a late log culture of isolate RD02.

3. Results

3.1. Comparison of methanol extraction approaches

Methanol recoveries and extraction efficiencies arelisted in Table 1. In all cases the initial methanol extraction

recovered from 92 to 99% of the total from threeconsecutive extractions for the three antenna pigment(chlorophyll a, chlorophyll c2 and peridinin) and thexanthophyll cycle component, diadinoxanthin. The dif-ferences between sonication versus no sonication andfresh versus frozen cell pellets were not significant whenexamined by single factor ANOVA analysis of totalrecovery and percent in first extract for a p value of 0.05.

3.2. Comparison of methanol, acetone and acetone:water extraction efficiencies

With few exceptions, total recoveries of photopig-ments from three consecutive extractions were greater for

Fig. 1. Relative extraction efficiency of chlorophyll a, chlorophyll c2, peridinin, and diadinoxanthin, Ii-om fkesh and frozen wet cell pellets extractedwith methanol, acetone, acetone:water with or without sonication. Bars identified by NS were not significantly different by pair wise two-tailedStudent t-test comparisons with the corresponding methanol extracts at p=0.05. Error bars represent one standard deviation.

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methanol than for acetone or acetone:water (Fig. 1).Whencompared to methanol extraction of either fresh or frozencell pellets, acetone extraction recovered less than 20% ornon-detectable levels of the photopigments and acetone:water extraction recovered from 10–80% of the photo-pigments. Although sonication increased recovery, ex-traction with acetone or acetone:water fell short ofmethanol extraction efficiencies. Extraction efficienciesfor chlorophyll c2 and diadinoxanthin were not signifi-

Fig. 2. Recovery of photopigment from methanol extraction of cell pellets pculture. Values represent a single extraction.

cantly different (p=0.05) for sonicated acetone:waterwhen compared to methanol extracts, however meanefficiencies for acetone:water were less than methanol.

3.3. Ratio of methanol volume to cell biomass

The ratio of methanol to cell biomass was examinedto determine the robustness of the methanol extraction(Fig. 2). Cell pellets prepared from increasing volumes

repared with increasing volumes of a Madracis mjrabilis (RD02) cell

Fig. 3. Comparison of photopigment recoveries fi-om methanol extracts (♦) and acetone:water (90110, v/v) plus sonication extracts (■) cell pelletsprepared with increasing volumes of a Porites porites (JR02Fl) cell culture. Values represent a single extraction.

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of a late log culture of a Symbiodinium isolate RD02were extracted with 5 ml methanol-ammonium acetate.Recovery of chlorophyll c2, peridinin, and diadinox-anthin from wet cell pellets was not affected by thequantity of cell biomass examined herein (Fig. 2); in-creased biomass yielded a proportionate increase in thequantity of each of the three photopigments. Recoveryof chlorophyll a, however, was reduced for cell biomassderived from culture volumes greater than 20 mL(approximately 2.3×107 cells). At higher biomass chlo-rophyll a levels in extracts were lower than the extra-polated values (Fig. 2). In a parallel experiment (Fig. 3)both the methanol-ammonium acetate and the acetone:water plus sonication extraction methods were testedagainst increasing quantities of cell biomass (JR02Fl).Extraction by acetone:water plus sonication was lesseffective at all biomass levels investigated.

3.4. Compatibility of solvents with HPLC analysis

Solvents compatible with the initial HPLC solventgenerally result in better chromatographic resolution.The methanol: 0.5 M ammonium acetate (90:10 v/v)extraction solvent used herein, compares favorably to aninitial HPLC solvent of methanol:0.5 M ammoniumacetate (80:20 v/v). Methanol extracts (50 μL) producedchromatograms with sharp, well separated peaks(Fig. 4A). In contrast, analysis of acetone:water extractsresulted, in ill defined peaks with less separation(Fig. 4B). In addition, the chlorophyll c2 peak wasactually split into two peaks and peridinin was mergedwith two closely associated peaks. Reducing the

injection volume from 50 μL to 10 μL resulted in betterpeak separation and definition, however at the cost of afive fold reduction in sensitivity.

4. Discussion

A methanol extraction procedure compatible withHPLC-DAD analysis was evaluated for quantifyingphotopigments found in cultured Symbiodinium spp.Methanol extraction has been recommended for extrac-tion of mixed algal samples obtained in the field (Wrightet al., 1996). Freezing cell pellets or sonicating cells insolvent did not appear to increase total photopigmentrecovery. The combined recovery of three consecutiveextractions was considered to yield complete recovery ofthe four photopigments examined herein. A single ex-traction recovered greater than 92–98% of the pho-topigments chlorophyll a, chlorophyll c2, peridinin anddiadinoxanthin from cell pellets containing less thanapproximately 2.3×107 cells. HPLC analysis of photo-pigments yielded chromatograms with sharp, symmetri-cal, and well-separated peaks, indicative of compatibilitybetween the extraction solvent and the initial HPLCrunning solvent. Themethod is simple, requiring a limitedamount of mixing (vortex) coupled with soakingovernight. Cell pellets containing as little as 1.8 μgchlorophyll a (approximately 5×106 cells) were easilyextracted and quantified by thismethod. The sensitivity ofthe method could be increased 5-fold (approximately1×106 cells) by reducing the volume of methanolammonium acetate mixture to 1.0 ml. Acetone-basedsolvents on the other hand were far less effective than

Fig. 4. HPLC chromatograms of 50 μL injections of extracts frommethanol (A) and acetone:water (90/10, v/v) plus sonication (B) extractions.

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methanol. Furthermore, analysis of HPLC chromato-grams of acetone extracts indicated marked reduction inresolution of the pigments of interest probably due toincompatibility between the extracting solvent and theinitial HPLC solvent.

4.1. Reduced photopigment recovery at low ratios ofsolvent to cell biomass

It is noted, however, that extraction of cell pelletscontaining greater than 2.3×107 cells can lead to un-derestimations of chlorophyll a concentrations. Reducedrecovery at higher biomass levels (Fig. 1) presumablyoccurred in part because chlorophyll a is sparingly solublein cold methanol (Merck Index, 2001) and aqueous- thanmethanol solutions (Latasa et al., 2001).

Allomerization (Schaber et al., 1984) of chlorophylla was also observed in a few cases at higher biomasslevels, although this has not been observed at the lowerbiomass levels. Allomerization products chromatographnear the chlorophyll a peak and begin to appear as thechlorophyll a, peak decreases. Chalker and Dunlap(1981) observed numerous small peaks nested withchlorophyll a in an HPLC separation they identified as atypical chromatogram in their study. They collectivelylabeled the peaks as chlorophyll a and isomers; howeverit is more likely that the smaller peaks were allomeriza-tion products. Other unwanted pigment transformationssuch as chlorophyllase action (Barret and Jeffery, 1971),de-metallation (Porra et al., 1996), and epimerization(Struck and Sheer, 1990) of chlorophylls, and degrada-tion or isomerization of carotenoids (Liaaen-Jensen,1990) were not detected.

4.2. Compatibility of extracting solvents with HPLCchromatography

In addition to providing better recovery of photopig-ments, the methanol-ammonium acetate extracts weremore compatible with the chromatographic solvent thanthe acetone:water extracts (Fig. 3). In previous studies,Zapata and Garrido (1991) found that methanol extractsproduced sharper HPLC peaks and better separation forthe more polar pigments than did acetone. In an extensionof this work Wright et al. (1991) and Kraay et al. (1992)found that the addition of ammonium acetate (2%, 0.5 M)to methanol produced extracts with improved resolutionof the more polar peaks when compared to methanolalone. Although ammonium acetate buffer (0.5 M, pH7.1) was added primarily to enhance chromatographiccompatibility, the buffer may also have stabilized theextracts from allomerization reactions and other unwantedpigment transformations. Jeffrey and Wright (1994)found, in a survey of prymnesiophyte pigments, thatless than 1.5% of the chlorophyll a could be attributed tochlorophyllides, allomers and epimers when extracts wereprepared with methanol plus 2% ammonium acetate,0.5 M, pH 7.1. Methanol without added buffer has beenshown to promote the formation of allomers (Diehn andSeely, 1968; Schaber et al., 1984). The observation ofallomerization products herein at high biomass levelsmayhave resulted from instability of the ammonium acetatebuffering capacity at higher biomass levels.

5. Conclusions and recommendations

In summary, a simple and reliable analytical methodfor quantifying photopigments in Symbiodinium spp.

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was presented. The method efficiently extracted themajor photopigments without production of alterationproducts. The time consuming and labor intensivemethods of sonication or grinding of frozen cells insolvent was not required; resuspension of cells period-ically during the extraction process, however, enhancedextraction. Very little cell material was requiredallowing the use of smaller culture volumes and smallercoral tissue samples to increased replication to creategreater statistical power. The method is a valuable toolfor both coral and Symbiodinium spp. studies.

Acknowledgements

Peter Chapman, William Fisher, David Millie, LeahOliver, and Richard Greene made helpful comments onthe earlier drafts of the manuscript. Contribution No.1192 US EPA, Gulf Breeze, FL, USA. Mention of alltrade names or commercial products does not constituteendorsement by the US EPA. [SS]

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