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Growth and ketocarotenoid (astaxanthin) by the fresh-water green unicellular alga Hae-
production
matococcus pluvialis has been evaluated in a 301 air-lift photobioreactor. Due to the different culture re-
quirements of the alga during the various stages of its development a two-stage batch production process
(effectively before and after addition of NaCl to the culture) was employed for (i) biomass and (ii) astaxanthin
by this alga. During the first stage, conditions within the reactor (light intensity, levels of nitrogen and phos-
phate) were maintained so as to achieve high rates of algal growth. When the algae in the reactor reached the
stationary phase of growth and levels of nitrogen and phosphate in the medium had become severely depleted
NaCl was added to stimulate the synthesis of ketocarotenoids (>95% astaxanthin, mainly in the form of mono-
and especially di-esters), partly overcoming the need to increase irradiance levels. H. pluvialis exhibited rela-
tively high rates of growth in the air-lift and accumulated up to 2.7% astaxanthin (of the dry cell weight of the
alga). This was, however, lower than could be achieved under laboratory-scale conditions (>5.5%). The use of
a hatch-production process in a air-lift reactor for the synthesis of algal carotenoids is discussed.
The occurrence of astaxanthin [(3S, 3S)-3,3-dihydro- This is in part due to several disadvantageous character-
xy-/3,/3-carotene-4,4-dione] in the freshwater microalga istics of the alga when compared to other micro-algae
Haematococcus pluvialis has been subject to increasing (e.g. the halotolerant microalga, Dunaliella spp.), includ-
experimental investigation in recent years due to great ing: (i) a relatively slow growth rate; (ii) a preference for
commercial interest in the pigmenting qualities of this low growth temperatures; (iii) a requirement of light for
carotenoid. Astaxanthin is used as a source in pigmenta- cultivation, and (iv) susceptibility to contamination and
tion of fish in aquaculture (especially salmonids; 1 and predation that may require the use of closed-growth sys-
2) and is also recognized as having superior antioxidant tem (cf. open-pond cultivation of Dunaliella spp. and
activity when compared to other carotenoids (e.g. p-caro- Spirulina spp.). Commercial cultivation of H. pluvialis
tene) and also to rw-tocopherol (3). Haematococcus spp. for the production of astaxanthin has, to date, been
accumulate astaxanthin when exposed to stress condi- limited to the growth of the alga in raceway ponds, con-
tions, generally high irradiances (4), usually in combina- taining 30,000 to one million liters of algal culture (10).
tion with nutrient deprivation (e.g. 5). The accumulation The major problem encountered when operating this
of astaxanthin in H. pluvialis (containing carotenoids open pond system is the contamination of the ponds by
typical of higher plants and the majority of green algae) bacteria, fungi and other faster growing algae, as well as
is associated with a morphological transformation from protozoan predators which have been reported to
green vegetative cells to deep-red, astaxanthin-rich, im- eliminate 90% of the algal biomass within 72 h (Spencer,
mobile aplanospores (6). These aplanospores are sur- K. G., US patent no. 4871551, 1989).
rounded by a thick cell wall composed of the bio-poly- The work reported in this study was conducted to
mer sporopollenin (7). The astaxanthin is accumulated in evaluate the use of a conventional, reliable autotrophic
lipid bodies which coalesce and eventually totally fill the system for the production of carotenoids from H. pluvia-
cell (8, 9). lis, which would minimise or eliminate the threat posed
In recent years there has been considerable interest in by potential predators and competitors. For this purpose
the synthesis of astaxanthin by Haematococcus, and a 30 I glass column, bubble airlift-photobioreactor (with
knowledge about the influence of abiotic factors such as conventional design features) was constructed (Fig. 1). A
irradiance levels, nutrient depletion and the use of ex- two-stage batch process was adopted for the production
ogenous compounds such as salt and ferrous forms of of astaxanthin from H. pluvialis designed to allow for
iron in controlling carotenogenesis in this alga is now biomass production followed by an astaxanthin accumu-
better understood. There is still, however, some debate lation stage. This involved manipulating the culture con-
as to the most suitable conditions (e.g. enrichment or ditions by the addition of NaCl at the onset of the sta-
depletion of nitrogen and phosphate in the culture medi- tionary phase of algal growth when levels of phosphate
um) which will allow for both algal growth and astaxan- and, especially nitrogen had been depleted, in order to
thin production (4, 5). Few studies have, however, consi- promote astaxanthin production in the alga.
dered the practical aspects concerned with the scale-up
of astaxanthin production in this freshwater microalga.
MATERIALS AND METHODS
TABLE 1. Carotenoid composition (expressed as a percentage of total carotenoid) of three different strains of H. pluvialis during
different stages of the life cycle as determined by reversed-phase HPLC (present study)
The vegetative cells were analysed early during the exponential phase of growth and the aplanospores towards the end of the stationary phase.
The carotenoid composition (especially esterification) of aplanospores changes with time (see Fig. 3C for additional information). The data for
the palmella stage refer to adult cells collected early in the stationary phase immediately prior to encystment (16).
thin), of which lutein was the most abundant carote- culture are substantially reduced compared to either the
noid. In contrast, highly encysted cells (aplanospores) of control (no NaCl) or the optimum value of 40 mM NaCl
W. pluvialis (harvested during the stationary phase) were (12). The effect of NaCl has been shown to be one of
primarily composed of astaxanthin (in esterified form) the most important factors in governing astaxanthin syn-
but also traces of canthaxanthin and echinenone (the
secondary carotenoids)-up to a maximum carotenoid
content of 2.7% of algal dry weight. The carotenoid
composition of these aplanospores was in agreement
with that reported previously from laboratory-scale cul-
tures of aplanospores (15) or non-motile palmella cells
(taken from early in the stationary phase-prior to
aplanospore formation) (16).
Growth and pigment levels of cells of H. pluvialis
during the course of the bioreactor batch run are shown _._
0 20 40 60 80 100
in Fig. 2. During the early stages of the batch-run the
number of cells (Fig. 2A) increased at a relatively rapid Day
rate which corresponded to high rates of chlorophyll 50
B
synthesis. However during this growth phase the level of
E
total carotenoid increased only slightly (Figs. 2A, B) 40
and represented - 1.O% of algal dry weight. The addition 30
of NaCl (between day 30-40) to the culture had a sig-
nificant effect on the growth characteristics of the culture 20
and the rate of synthesis of carotenoids. NaCl was added
10
to the reactor when the cells had reached the stationary
phase of growth and had the effect of causing an in- a- n
crease in the cell mortality rate within the culture (Fig. 0 20 40 60 80 loo-
2A). This caused a significant reduction in the levels of Day
chlorophyll but stimulated a rapid increase in carotenoid
synthesis (Figs. 2A, B). Chlorophyll levels in the bioreac- 70
tor only began to fall after the addition of NaCl on day
30 (the level on day 29 was the highest recorded and that 60
on day 35-after NaCl addition-was lower than that
measured on day 14). Previous studies on a lab-scale 50
had shown that NaCl addition was an effective way of
accelerating the process of encystment and concomitant 40
synthesis of astaxanthin in H. pluvialis (and in a number
of other fresh-water microalgae, e.g. Oocystis spp.), 30
although its addition will result in some cell death (12).
Those cells which survive the addition of 40mM NaCl Day
are capable of increasing rates of astaxanthin synthesis
FIG. 2. Growth and carotenoid accumulation of H. pluvialis cells
per cell (3-4 x greater than the rates in the absence of in the photobioreactor. Symbols: (A) 0 , cell number (S.E. C4.9?4;
NaCl; see Ref. 12). Levels in excess of 40 mM NaCl (the n=3); 0, dry cell weight (S.E.k3.3,; n=3); (B) 0, chlorophyll
concentration used in this study) will further increase the (S.E.f3.6%; n=3); 0, carotenoid (mg/l) (S.E.i4.7!4; n=3); (C)
rate of astaxanthin synthesis (on a per cell basis) but will q , chlorophyll (S.E. *3.9x; n=3); 0, carotenoid (pg per cell)
also induce higher rates of cell death in the culture and (S.E.+3.1%; n=3). Arrows indicate first and final applications of
therefore the levels of astaxanthin per unit volume of NaCl to the photobioreactor.
116 HARKER ET AL. J. FERMENT.BIOENG.,
5 a
4 100
a
._
80
.Z
.c 60
0
# 40
; 20
L
.Z 0
z 0 10 20 30 40 50 60 g
Day 0 20 40 60 80 100
FIG. 4. Effect of nitrate and phosphate concentrations on H.
pluviulis growth in the photobioreactor. (A) A, cell number
(S.E.t4.9%; n=3); (B) q , nitrate; 0, phosphate. Arrows indicate FIG. 5. Temperature and pH of the H. pluvialis culture medium
first and final applications of NaCl to the photobioreactor. in the photobioreactor. (A) A, pH; (B) 0, maximum temperature; A,
minimum temperature (C). Arrows indicate first and final applica-
tions of NaCl to the photobioreactor.
reduction and finally cessation of the growth of the
algae. The depletion of the nitrate content of the medium tion and to obtain the best light regime, high levels of
coincided with the onset of the stationary growth phase mixing were required to reach a turbulent flow of the cul-
and the increased synthesis of carotenoids (which was ture. In microalgal mass culture, productivity is affected
accelerated by the addition of NaCl-see above). Nitrate by the mixing system and when light is considered as a
levels were depleted by day 20 (down to <20x of the limiting factor of growth, the effective self shading of a
starting level-see medium composition above) by which cell has a significant effect on the growth rateicarotenoid
time the number of cells had reached the stationary production. This means that an efficient turbulent flow
phase but the total biomass continued to increase (Figs. (high liquid velocity) must be maintained within the cul-
2A and 4). The depletion of nitrogen does not specifically ture, so each cell is able to receive enough light in order
appear to be a causative agent in the reduction of total to improve the photosynthetic efficiency of the culture
chlorophyll. The level of Fe also declined during the (20). The mixing system employed in the present study
exponential phase falling from 1.79~ 10m5 M to 0.6 X had to take into account the current phase in the life
10e5 M (data not shown). cycle of the alga, so that the culture of green vegetative
During the exponential growth period the density of cells in the exponential phase of growth required a lower
the culture greatly increased restricting the amount of liquid velocity because of their fragility and the relatively
light each cell received. The bioreactor constructed for low cell concentration. The aplanospores are more
the present study was of a typical air-life design with a robust than the green vegetative cells due to their thick
relatively small ratio of surface area (available for illumi- cell wall and their culture in the stationary phase
nation): reactor volume. This is an important implica- required a higher liquid velocity to prevent cell sinking,
tion considering the importance of light for algal growth and to ensure the best light regime for carotenogenesis.
and carotenoid production (1). Green, vegetative, cells Nevertheless the high cell densities of Haematococcus
of EL pluviafis require a relatively low light intensity produced in the batch-run would affect the subsequent
[-40-50pmol rn-* SK (PAR)] to achieve high rates of ability to optimise high irradiance levels for carotenoid
growth, whilst very high light intensities [--l,SOO- synthesis in the same reactor vessel. An alternative light
2,00O/*mol m-* s-l (PAR)] significantly improve the induction system would be preferable, perhaps incor-
rate of astaxanthin synthesis in the aplanospores (17). porating a reactor design that, unlike a conventional air-
Rather than considering the total light energy impinging lift reactor, maximises light interception and is combined
on the culture surface, the most important factor is the with dilution of the culture prior to the stage of astaxan-
quantity of energy available at the cellular level. These thin accumulation.
concepts of light per cell and light regime expressed The results also pose the question of when is the best
by Richmond (18), describe the duration of each ex- time to harvest such a fermentation process? It has been
posure of the average cell in the photic zone, below the suggested that it is commercially viable to harvest Hae-
compensation point, or in darkness. Soeder (19) was matococcus once the carotenoid content of the culture
among the first to elaborate the idea of area1 density, has reached -2.0% of the dry cell weight of the culture
thereby taking into account the mutual shading of cells. (2, 18). In the present study this would have meant
Thus, an efficient growth system for large-scale cultiva- harvesting the bioreactor earlier in the batch-run.
tion of algal biomass (requiring relatively low irradiances During the batch-run the pH of the medium in the
and producing high cell densities) will generally be bioreactor did not deviate significantly from the optimal
unsuited to a process such as carotenogenesis which is pH for cell growth (pH 7.0) (Fig. SA) and no attempt
optimised at high irradiances. was therefore made to control this. Similarly the temper-
To partly overcome the phenomenon of light limita- ature of the culture medium was not subject to any
118 HARKER ET AL. J. FERMENT.BIOENG.,