Journal of Applied Phycology 13: 19–24, 2001.

© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

19

Evaluation of different cell disruption processes on encysted cells of

Haematococcus pluvialis: effects on astaxanthin recovery and implications
for bio-availability

M.M. Mendes-Pinto, M.F.J. Raposo, J. Bowen1 , A.J. Young1 & R. Morais∗

Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida,
4200-072 Porto, Portugal
1 Carotenoid Research Group, School of Biological & Earth Sciences, Liverpool John Moores University, Byrom

St., Liverpool L3 3AF, UK

(∗ Author for correspondence; fax +351-225090351; e-mail [email protected])

Received 26 March 2000; revised 24 July 2000; accepted 7 August 2000

Key words: astaxanthin, bio-availability, Haematococcus, microalgae, processing

Abstract
Although Haematococcus pluvialis is one of the most important natural sources of the carotenoid astaxanthin as a
pigmentor for the aquaculture industry, the thick sporopollenin cell wall in the cysts hinders astaxanthin extraction
and its subsequent bio-availability to fish. A range of physical and chemical processes were tested to promote the
disruption of the encysted cells. The efficacy of these processes was evaluated in terms of astaxanthin recovery,
which was assessed by determining the extent of leaching of astaxanthin into an organic solvent. The processes
tested were: autoclave 30 min, 121 ◦ C, 1 atm; HCl 0.1 M, 15 min and 30 min; NaOH 0.1 M, 15 min and 30 min;
enzymatic treatment with a mixture of 0.1% protease K and 0.5% driselase in a phosphate buffer, pH 5.8, 30 ◦ C, for
one hour; spray drying, inlet 180 ◦ C, outlet 115 ◦ C; and mechanical disruption, with a cell homogeniser developed
for this purpose. The mechanical (homogenisation) and autoclave treatments were the most effective in terms of
extraction and availability.

Introduction Haematococcus can accumulate up to 8% dry

weight of astaxanthin, in the form of 3S,30 S enan-
Haematococcus pluvialis Flotow (Chlorophyceae) has tiometer, as a mixture of mono and di-esters (Harker
a complex life-cycle involving several stages from et al., 1996b). The composition of the aplanospores
motile flagellated zooids through to palmella and en- is somewhat dependent upon the age of the culture so
cysted stages (Elliot, 1934). This alga is one of the that the ratio of mono-esters: di-esters decreases with
most important natural sources of the carotenoid pig- time (Harker et al., 1996b).
ment astaxanthin (3,30 -dihydroxy-β,β’-carotene-4,40- Astaxanthin biosynthesis in this alga is gener-
dione) and is cultivated commercially by several com- ally, but not exclusively (A. Hartley & A.J. Young,
panies. Under optimum conditions, astaxanthin is unpublished data), linked to the generation of apla-
completely absent from the cells. Upon exposure of nospores and the associated formation of a thick
the cells to growth-limiting conditions (typically as a sporopollenin wall around the cell. The subsequent
result of nutrient – especially nitrogen – limitation or bio-availability of the astaxanthin is limited due to this
exposure to very high irradiance or high temperature: wall in the astaxanthin-rich cysts (Good & Chapman,
Droop, 1954; Fan et al., 1994; Harker et al., 1996a), 1979). Intact astaxanthin-rich cysts of Heamatococ-
astaxanthin is synthesised and accumulated within oil cus do not cause pigmentation in salmonids (Sommer
droplets.
20

et al., 1991). Thus there is a need to extract or rup- during the biomass mechanical processing in order to
ture these cysts prior to use in order to achieve a prevent or minimise oxidation reactions that might oc-
desired level of pigmentation in the target organism. cur. All the treatments were carried out in triplicate.
Whilst it is possible to obtain a ‘cell-free’ extract of Following treatment, the astaxanthin content and com-
carotenoids, this is not practical on a large scale. Nor position of the processed biomass was determined by
is it feasible to extract and purify ‘free’ astaxanthin HPLC (see below).
from Haematococcus, in part due to the sensitivity of
astaxanthin to oxidation. A range of alternative pro- Astaxanthin determination
cesses have been suggested for processing algal cells.
For example, Bubrick (1991) described a process in- After the addition of 2 mL ethanol and glass beads
corporating cryogenic (–170 ◦ C) grinding of dried to the test tubes, triplicates (30 mg) were submit-
Haematococcus biomass in the presence of butylated ted to a combination of vortex mixing (1 min) and
hydroxytoluene. ultrasound treatment (15 min). Samples were centri-
The main aim of this study was to evaluate a range fuged (5 min at 1000 × g), the supernatant removed
of different physical and chemical treatments on the and the pellet re-suspended in 2 ml diethyl ether /
disruption of encysted cells of H. pluvialis, in terms of ethanol (1:1, v/v). After repeating all the steps the
astaxanthin recovery and the effect on subsequent ‘po- pellet was extracted with 2 ml diethyl ether and finally
tential bioavailability’ of pigment from the processed with 3 ml n-hexane. All supernatants were pooled and
biomass. the sample was evaporated to dryness under a steady
stream of oxygen-free nitrogen.

Materials and methods Extraction into acetone

Haematococcus pluvialis (34/7 strain of CCAP – Pigment availability in processed algal biomass was
Culture Collection of Algae and Protozoa, Win- assessed by extraction of astaxanthin into acetone
dermere,UK) was cultivated in modified Bold’s Basal (2 mL), from 30 mg cells (centrifuged as above).
Medium (Nichols & Bold, 1964). The culture was The material was gently mixed using a magnetic stir-
grown at 21 ± 1 ◦ C for 14 days in plastic sleeves rer and left for 16 hours at room temperature. The
of 10-L volume into which ambient air was used sample was filtered prior to analysis to remove any
to agitate the culture. Illumination was provided by cell debris. These conditions were previously found
cool white fluorescent lamps (L36 W/21 Hellweiss to permit maximum extraction of cellular astaxanthin
Luminux Coolwhite, Osram, Lisbon, Portugal) at an from astaxanthin-rich materials, without any degrada-
irradiance of 46 µmol photon m−2 s−1 . After 14 days, tion of carotenoid. This assay was used to compare the
the irradiance was increased to 80 µmol photon m−2 effects of different processing methods on subsequent
s−1 in order to stimulate astaxanthin biosynthesis in astaxanthin availability from cells of Haematococcus.
the alga.
Spectrophotometry
Processing of algal biomass
Astaxanthin content was determined using a double
After sedimentation and centrifugation (10 min, beam UV-VIS spectrophotometer (Shimadzu 1601)
1000 × g) of the encysted cells, the de-watered within a wavelength range 400–700 nm. For astax-
astaxanthin-rich biomass was divided into aliquots and anthin quantification, the extracts originated from the
submitted to the following processes: (i) autoclaved 30 solvent mixture extraction were re-suspended in n-
min, 121 ◦ C, 1 atm; (ii) HCl 0.1 M, 15 min and, (iii) hexane; those obtained from the contact with acetone
30 min; (iv) NaOH 0.1 M, 15 min and, (v) 30 min; were analysed directly in acetone. The extinction coef-
(vi) enzymatic treatment for 1 hour with a mixture of ficient of astaxanthin in both solvents is 2100 and the
0.1% protease K and 0.5% driselase in a phosphate λmax is 470 nm in n-hexane and 476 nm in acetone
buffer, pH 5.8, 30 ◦ C; (vii) spray drying, inlet 180 ◦ C, (Britton, 1995).
outlet 115 ◦ C; and, (viii) mechanical disruption using
a cell homogeniser developed for this purpose (patent
pending). A steady stream of nitrogen was applied
 21

Thin-Layer Chromatography (TLC) – one

dimensional separation of carotenoids

Pre-coated silica gel H60 thin layer chromatography

plates 20 × 20 cm (Merck, Darmstadt, FRG) were
used for the separation of carotenoids. Acetone, n-
hexane, methanol (all from Merck, Darmstadt, FRG)
and citric acid (Sigma) were laboratory grade. The
carotenoid standards, β-carotene and astaxanthin (pur-
chased from Sigma) were dissolved in a small volume
of n-hexane at 1mg/ml; 2.5 µg was applied to the
TLC plate. Samples of 30 mg from all treatments were
evaporated to dryness and reconstituted individually in
500µl n-hexane, from which 1.2 mg was applied to the
TLC plate. All samples and standards were kept dried,
under oxygen-free nitrogen, in the dark at –4 ◦ C until
used.
All separations were carried out in developing
chambers presaturated with the appropriate solvent Figure 1. Total carotenoid content of Haematococcus cysts follow-
system, at room temperature. Carotenoids were sep- ing processing: 1. Control (intact); 2. Autoclave; 3. HCl 15 min;
4. HCl 30 min; 5. NaOH 15 min; 6. NaOH 30 min; 7. Enzyme; 8.
arated using a solvent system of acetone: n-hexane, Mechanical disruption; 9. Spray drying. See Materials and Methods
3:7 (v/v) (Kobayashi et al., 1991). Before immersing for details.
the plate into the solvent system, the plate was pre-
run in a 2.5% (w/v) solution of citric acid in methanol.
This prevented the tailing of carotenoids that posses a Scanning Electron Microscopy (SEM)
3-hydroxy, 4-keto end-group such as astaxanthin and
The Haematococcus processed biomass was examined
astacene. The TLC plates were then dried at 40 ◦ C for
by scanning electron microscopy using a JEOL JSM-
10 min.
840 microscope at an accelerating voltage of 1–
Analysis of chromatograms was made using an
15 KV. The samples were lyophilised (CHRIS AL-
imaging densitometer (Model GS-700, Bio-Rad) op-
PHA 1–4,B-Braun Biotech International) at –52 ◦ C,
erating with a reflectance resolution of 56 µm (450
and coated with a thin gold-palladium film (10–15nm)
dpi), with a blue filter at a pixel depth of 8.
using a Polaron E5000 sputter unit.

High Performance Liquid Chromatography (HPLC)

Results
The HPLC analyses were carried out using a mod-
ified method based on that described by Vecchi et The carotenoid composition of the Haematococcus
al. (1987). Samples were analysed by normal phase biomass used in this study was 70.5% astaxanthin
HPLC using a diode array detector (HP1100, Hewlett mono-esters, 24.7% astaxanthin di-esters and 4.8%
Packard). A Lichrosorb column (12.5 × 4.6 cm, 5µm lutein. Traces of β-carotene were observed but were
particles) was pre-coated with 1% H3 PO4 in methanol not quantified. The astaxanthin content of the algal
(v/v) before used, to prevent tailing of astaxanthin. biomass was approximately 2% (w/dw) which is com-
The mobile phase was n-hexane: acetone, 86:14 (v/v; parable to the commercial source of this material
Merck, Darmstadt FRG) operating at a flow rate of 1.2 currently available (NatuRose, Cyanotech Corp.). This
ml/min. In order to improve separation efficiency, the pigment composition is typical of encysted cells of
polarity of the mobile phase was changed during the this alga, with high levels of astaxanthin mono-esters
run (run time 25 min); the gradient elution was 1:1 predominating (Grung et al., 1992; Harker et al.,
(v/v) n-hexane: acetone. The sample volume applied 1996b).
was 20 µl following solubilisation of the carotenoid The effects of processing of the algal biomass
extract in 100 µl n-hexane. on the carotenoid content of the cells is shown in
22

gastro-intestinal tract of salmonids. The leaching of

carotenoids, under gentle conditions, from biological
materials into an organic solvent provides a simple,
rapid estimation of the extent to which the cell wall
is disrupted. Intact cysts of Haematococcus only re-
lease ∼20% of astaxanthin into acetone over a 16 hour
period (Figure 2). Similar low yields were observed
for cells that were subject to enzymatic treatment,
spray-drying or exposure to acid or alkali. High re-
coveries of carotenoid (>85%) were only observed
for cells that had been autoclaved or mechanically
processed. This suggests that these two methods of
processing algal biomass may be most effective in
terms of pigmentation as the cell contents are readily
accessible.
In general, the leaching of carotenoids into acet-
one did not result in a high recovery of lutein and
β-carotene (data not shown). This may be due to
Figure 2. Extraction of total carotenoids into acetone from pro- their binding to the various pigment-protein com-
cessed Haematococcus biomass. 1. Control (intact); 2. Autoclave; plexes within the chloroplast, whilst the astaxanthin
3. HCl 15 min; 4. HCl 30 min; 5. NaOH 15 min; 6. NaOH 30 min; 7. esters are accumulated in extra-chloroplastic globules.
Enzyme; 8. Mechanical disruption; 9. Spray drying. See Materials
and Methods for details. In addition to using solvent extractability as a
means of assessing the effectiveness of processing
on the algal biomass the direct effect that processing
Figure 1. This shows that, compared to the control, had on the cell wall of the alga can be observed by
the highest recovery of astaxanthin was obtained in scanning electron microscopy (Figure 3 a-d). The un-
the autoclaved, spray-dried and mechanical disrupted processed cysts of Haematococcus (Figure 3a) are
cells. None of these processes has a detrimental effect fully intact with no signs of pitting or damage to the
on the carotenoid content and composition of the algal cell wall. In contrast, the cells subjected to mechanical
biomass. However, enzymatic treatment of Haemato- processing (Figure 3b) and autoclaving (Figure 3d) are
coccus cells or exposure to alkali or acid, resulted clearly disrupted. A noticeable effect of spray-drying
in a significant loss of carotenoid (20–35% of total the algal biomass (Figure 3c) was the clumping of
carotenoid) as a direct result of processing. Caroten- cells, although the cells are not damaged by this pro-
oids such as astaxanthin which possess the 3-hydroxy, cess. Exposure to NaOH did result in some distortion
4-keto end-group are unstable, especially in the pres- and collapse of some cells, especially those exposed to
ence of alkali. However, the presence of oxidative alkali for a longer period. Some pitting of the cell wall
products of astaxanthin, namely astacene and semi- was observed with treatment by HCl, whilst enzymatic
astacene, could not be confirmed by HPLC analysis. treatment had no visible effect on cell morphology
Chromatographic analysis of these processed materi- (data not shown).
als revealed that selective degradation of carotenoids
did not occur and that losses were seen for all caroten- Discussion
oids. The high yield of carotenoid from the biomass
obtained by spray-drying may be explained by the re- The bio-absorption or bio-availability of carotenoids
duced water content facilitating better extraction by in animals and humans is a critical aspect concern-
the organic solvents. ing the use of these natural pigments / anti-oxidants
The use of algal astaxanthin as a pigmentor in in the food and feedstuff industries (Castenmiller &
the aquaculture industry is dependent upon the bio- West, 1997). In aquaculture, the relatively poor re-
availability of the carotenoid from the algal biomass. tention (typically only a few percent; Torrissen et al.,
The physical barrier of the sporopollenin cell wall in 1989) of astaxanthin and canthaxanthin in salmonids
the cysts of Haematococcus is thought to be a key has significant implications in terms of cost. Nat-
factor in limiting carotenoid bio-availability in the ural sources of astaxanthin include krill waste, the
 23

Figure 3. Scanning electron micrographs of Haematococcus pluvialis (× 2000), bar size 10 µm: (a) control (intact) cells, (b) mechanical
disrupted cells, (c) spray-dried cells, and (d) cells autoclaved.

yeast Phaffia rhodozyma, flowers of Adonis annua as mono-esters, whilst the synthetic products are free
and a number of microalgae, notably Haematococcus carotenoid. A study by Foss et al. (1987) demonstrated
pluvialis (Bernhard, 1990). that racemic astaxanthin di-palmitate did not pigment
Salmonids appear to lack the digestive enzymes salmonids as effectively as free astaxanthin. A more
necessary to break down the sporopollenin wall found recent study in rainbow trout has however demon-
in cysts of Haematococcus. A number of factors strated that both the natural mono- and di-esters of
may be responsible for limiting the bio-availability astaxanthin from Haematococcus, when administered
of astaxanthin from Haematococcus cells. The use of to fish in an isolated form, pigment the muscle tis-
astaxanthin-rich cells of this alga in pigmentation tri- sues as effectively as synthetic astaxanthin (J. Bowen
als on rainbow trout has shown that intact cysts do J, S. Davies, R. Serwata, & A.J. Young, unpublished
not permit pigmentation and that cellular astaxanthin data). Another factor that distinguishes algal astax-
only becomes bio-available once the cyst is broken anthin from the synthetic form is the configuration of
or disrupted (Johnson & An, 1991; Sommer et al., the carotenoid. In algae, astaxanthin occurs as the (3S,
1991; J. Bowen, S. Davies, S. Lagocki, A.J. Young, 30 S) form (Renstrøm et al., 1981) whilst the synthetic
unpublished). In addition, astaxanthin accumulated in product is a racemic mixture of the (3R, 30 R), (3S, 30 S)
algae such as Haematococcus is esterified, principally and the meso form (3R, 30 S) (Bernhard, 1990). Foss
24

et al., (1987) demonstrated that all three epimers of Castenmiller JJM, West CE (1997) Bioavailability of carotenoids.
astaxanthin are equally utilised by salmonids, which Pure appl. Chem. 69: 2145–1250.
Choubert G, Heinrich O (1993) Carotenoid pigments of the green
suggests that the algal form of the carotenoid would alga Haematococcus pluvialis: assay on rainbow trout, Onco-
not be discriminated against by the fish. rhynchus mykiss, pigmentation in comparison with synthetic
The cracking or disruption of the algal cell there- astaxanthin and canthaxanthin. Aquaculture 112: 217–226.
fore appears to be the single most important factor in Droop MR (1954) Conditions governing haematochrome formation
and loss in the alga Haematococcus pluvialis. Arch. Mikrobiol.
utilisation of algal astaxanthin. It is, however, import- 20: 391–397.
ant to note that astaxanthin accumulation in Haemato- Elliot AM (1934) Morphology and life history of Haematococcus
coccus is not restricted to the formation of cysts and pluvialis. Arch. Protistenk. 82: 250–272.
that this carotenoid can be biosynthesised at a number Fan L, Vonshak A, Boussiba S (1994) Effect of temperature and irra-
diance on growth of Haematococcus pluvialis (Chlorophyceae).
of stages of the alga’s complex life cycle (see Intro- J. Phycol. 30: 829–833.
duction). Similarly the formation of the sporopollenin Farrow WM, Tabenkin B (1966) Process for the preparation of
cell wall, and hence the apparent ‘toughness’ of the lutein. U.S. Patent n◦ 3 280 502.
cell, alters with the age of the cyst (Burczyk, 1987). Foss P, Storebakken T, Austreng E, Liaaen-Jensen S (1987) Caroten-
oids in diets for salmonids V. Pigmentation of rainbow trout
Several methods have been proposed to disrupt and sea trout with astaxanthin and astaxanthin di-palmitate in
algal cells (Farrow & Tabenkin, 1966; Ruane, 1977; comparison to canthaxanthin. Aquaculture 65: 293–305.
Nonomura, 1987) although most methods are not Good BH, Chapman RL (1979) A comparison between the walls of
Haematococcus cysts and the loricas of Dysmorphococcus and
very efficient for disrupting the sporopollenin wall
the possible presence of sporopollenin. J. Phycol. 15: 17.
of Haematococcus cysts. These cysts are similar to Grung M, D’Souza MCD, Borowitzka M, Liaaen-Jensen S (1992)
those found in many microalgae and in pollen from Algal carotenoids 51. Secondary carotenoids 2. Haematococcus
higher plants (VanWinkle, Swift & Rickoll, 1997). pluvialis aplanospores as a source of (3S,30 S) astaxanthin esters.
J. appl. Phycol. 4: 165–168.
They are particularly resistant to chemical attack, in- Harker M, Tsavalos AJ, Young AJ (1996a) Factors responsible
cluding KOH and acetolysis but can be attacked by for astaxanthin formation in the chlorophyte Haematococcus
chromic acid. Indeed, resistance to acetolysis is used pluvialis. Biores. Technol. 55: 207–214.
in part to characterise such cell walls. According to Harker M, Tsavalos AJ,Young AJ (1996b) Autotrophic growth and
carotenoid production of Haematococcus pluvialis in a 30 liter
the results obtained from this study it can be observed air-lift photobioreactor. J. Ferment. Bioengng 82: 113–118.
that both mechanical disruption and autoclaving of Johnson EA, An G-H (1991) Astaxanthin from microbial sources.
the astaxanthin-rich algal biomass are effective treat- Crit. Rev. Biotechnol. 11: 297–326.
ments. Both processes result in a very high yield of Kobayashi M, Kakizono T, Nagai S (1991) Astaxanthin production
by a green alga, Haematococcus pluvialis accompanied with
astaxanthin (Figure 1; i.e. no detrimental effects were morphological changes in acetate media. J. Ferment. Bioengng
observed due to processing per se). In addition, the 71: 335–339.
extractability of the astaxanthin (Figure 2) from these Nichols HW, Bold HC (1964) Trichsarcina polymorpha gen. et nov.
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processed cells was very efficient suggesting effective
Nonomura AM (1987) Process for producing a naturally-derived
cracking of the cyst wall. This was confirmed by SEM carotene/oil composition by direct extraction from algae. U.S.
for both products (Figure 3). Patent no. 4 680 314.
Renstrøm B, Borch G, Skulberg OM, Liaansen-Jensen S (1981)
Optical purity of (3S,30 S)-astaxanthin from Haematococcus plu-
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Ruane M (1977) Extraction of caroteniferous materials from algae.
This work was supported by a Framework IV Project Australian Patent n◦ 72 395 74.
FAIR CT-97-1518. Sommer TR, Potts WT, Morrisey NM (1991) Utilization of mi-
croalgae astaxanthin by rainbow trout (Oncorhynchus mykiss).
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