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Autotrophic growth and carotenoid production of Haematococcus pluvialis in a 30 liter air-lift photobioreactor
JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 82, No. 2, 113-118. 1996
Autotrophic Growth and Carotenoid Production of Haematococcus pluvialis in a 30 Liter Air-Lift Photobioreactor MARK HARKER,§
ALEX J. TSAVALOS,
ANDREW
AND
J. YOUNG*
School of Biological and Earth Sciences, John Moores University, Byrom Street, Liverpool, Received
28 August
1995/Accepted
UK
21 May 1996
by the fresh-water green unicellular alga HaeDue to the different culture requirements 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 phosphate) 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 monoand especially di-esters), partly overcoming the need to increase irradiance levels. H. pluvialis exhibited relatively 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. Growth
and ketocarotenoid
(astaxanthin)
production
matococcus pluvialis has been evaluated in a 301 air-lift photobioreactor.
[Key words:
astaxanthin,
carotenoid,
Haematococcus] This is in part due to several disadvantageous characteristics of the alga when compared to other micro-algae (e.g. the halotolerant microalga, Dunaliella spp.), including: (i) a relatively slow growth rate; (ii) a preference for low growth temperatures; (iii) a requirement of light for cultivation, and (iv) susceptibility to contamination and predation that may require the use of closed-growth system (cf. open-pond cultivation of Dunaliella spp. and Spirulina spp.). Commercial cultivation of H. pluvialis for the production of astaxanthin has, to date, been limited to the growth of the alga in raceway ponds, containing 30,000 to one million liters of algal culture (10). The major problem encountered when operating this open pond system is the contamination of the ponds by bacteria, fungi and other faster growing algae, as well as protozoan predators which have been reported to eliminate 90% of the algal biomass within 72 h (Spencer, K. G., US patent no. 4871551, 1989). The work reported in this study was conducted to evaluate the use of a conventional, reliable autotrophic system for the production of carotenoids from H. pluvialis, which would minimise or eliminate the threat posed by potential predators and competitors. For this purpose a 30 I glass column, bubble airlift-photobioreactor (with conventional design features) was constructed (Fig. 1). A two-stage batch process was adopted for the production of astaxanthin from H. pluvialis designed to allow for biomass production followed by an astaxanthin accumulation stage. This involved manipulating the culture conditions by the addition of NaCl at the onset of the stationary phase of algal growth when levels of phosphate and, especially nitrogen had been depleted, in order to promote astaxanthin production in the alga.
The occurrence of astaxanthin [(3S, 3’S)-3,3’-dihydroxy-/3,/3-carotene-4,4’-dione] in the freshwater microalga Haematococcus pluvialis has been subject to increasing experimental investigation in recent years due to great commercial interest in the pigmenting qualities of this carotenoid. Astaxanthin is used as a source in pigmentation of fish in aquaculture (especially salmonids; 1 and 2) and is also recognized as having superior antioxidant activity when compared to other carotenoids (e.g. p-carotene) and also to rw-tocopherol (3). Haematococcus spp. accumulate astaxanthin when exposed to stress conditions, generally high irradiances (4), usually in combination with nutrient deprivation (e.g. 5). The accumulation of astaxanthin in H. pluvialis (containing carotenoids typical of higher plants and the majority of green algae) is associated with a morphological transformation from green vegetative cells to deep-red, astaxanthin-rich, immobile aplanospores (6). These aplanospores are surrounded by a thick cell wall composed of the bio-polymer sporopollenin (7). The astaxanthin is accumulated in lipid bodies which coalesce and eventually totally fill the cell (8, 9). In recent years there has been considerable interest in the synthesis of astaxanthin by Haematococcus, and knowledge about the influence of abiotic factors such as irradiance levels, nutrient depletion and the use of exogenous compounds such as salt and ferrous forms of iron in controlling carotenogenesis in this alga is now better understood. There is still, however, some debate as to the most suitable conditions (e.g. enrichment or depletion of nitrogen and phosphate in the culture medium) which will allow for both algal growth and astaxanthin production (4, 5). Few studies have, however, considered the practical aspects concerned with the scale-up of astaxanthin production in this freshwater microalga.
MATERIALS
author. 5 Present address: Dept. of Genetics, The Hebrew University Jerusalem, Givat Ram Campus, Jerusalem 91904, Israel.
obtained Protozoa,
of
113
METHODS
and growth media H. pluvialis 34/7 was from the Culture Collection of Algae and Windermere, U.K. The alga was cultivated in
Organism
* Corresponding
AND
114
HARKER
ET AL.
Bold’s Basal Medium (11) modified to pH 7.0 (NaN03 2.9 mM; MgS04. 7Hz0 0.3 mM; NaCl 0.43 mM; K2HP04 0.43 mM; KH2P04 1.29 mM; CaCl*. 2H20 0.17 mM; ZnS04.7Hz0 30.7 PM; MnC12.4H20 7.3 /-‘M; Moo3 4.9 /rM; CuS04.5H20 6.3 /IM; CoN03.6H20 1.7 /jM; H1B03 0.18mM; EDTA 0.17mM; KOH 0.55 mM; FeS04. 7Hz0 17.9 /lM; HzS04 10 /*M). The photobioreactor In the closed photobioreactor the alga was grown under semi-axenic conditions throughout the batch-run. Prior to inoculation of the algae in the photobioreactor, the reactor was sterilised by means of a 0.2% sodium hypochlorite solution or high-pressure steam. The photobioreactor (30 I medium volume) was of a cylindrical construction and made of glass (see Fig. 1 for a schematic representation and dimensions). The photobioreactor was equipped with an external illumination system composed of four fluorescent tubes, which supplied 50 ,nmol rnp2 ssl (PAR) to the surface of the culture vessel (Fig. 1). The photobioreactor was surrounded by a polythene insulating sheet in an effort to maintain a relatively constant temperature within the environment closely surrounding the reactor. No other means of temperature control within the vicinity of the photobioreactor were available. Mixing and aeration were achieved by bubbling ambient air through a sparger unit located in the base of the photobioreactor (Fig. 1). Air, sterilised using filters (0.2 I’m, Millipore), was passed via the air inlet tube (Fig. 1) into the sparger, from which the air penetrated into the culture medium through a series of capillary holes. The turbulence caused by air passing up through the culture vessel was enough to mix the medium sufficiently to keep the cells in constant suspension in the culture medium. The aeration supplied the cells with a continuous source of ambient levels of COz. The air was pumped through the sparger at a rate of 1.5-3.01 per min, depending upon the age of the culture so that aeration was increased during the formation of the nonmotile astaxanthin-rich aplanospores in order to keep them in suspension. A silicon-based antifoam (Basildon Chemicals Ltd.) was added to the culture at 0.2ml per liter of algal culture when required. When the alga had reached the stationary phase of growth NaCl was added to the culture medium. Equal amounts of NaCl were added at 30, 35 and 40d to achieve a final concentration of 40mM NaCl in the reactor on day 40. The application of NaCl in this manner (three separate stages over a period of ten days to yield a final concentration of 40 mM) was used as the results of earlier experiments demonstrated that this would induce the culture to produce higher yields of astaxanthin per unit volume of culture medium rather than if the salt was added in a single application by minimising high rates of cell death (12). When the astaxanthin levels of the cells in the reactor had reached the stationary phase of accumulation (as determined by HPLC analysis-see below), aeration of the culture vessel was stopped. The algal culture was allowed to settle on the base of the reactor and then harvested as a thick algal suspension by allowing the concentrated algal slurry to flow out of the reaction via the sample line (Fig. 1). The thick algal suspension was rapidly frozen at ~20°C and kept for subsequent analysis. Analytical methods Algal cell growth was determined either by cell number as determined using a Improved Neubauer haemocytometer, or by determining
J.FERMENT.BIOENG.,
Scale drawing of the pilot photobioreactor FIG. 1. Schematic representation of the 30 I air-lift photobioreactor constructed for the evaluation of growth and carotenoid production in the unicellular microalgae H. pluvialis. Numbers: @ air outlet; 2, air outlet filter; :T media, antifoam and inoculum inlet; 2 condenser; :.:I air inlet filters; #G one of four fluorescent tubes surrounding the bioreactor; 121 growth medium; C.$ air inlet; f.9 sparger; 10 sample line and bottle.
dry cell weight after centrifugation to separate the cells from the media followed by washing and drying at 100°C for 24 h. Pigments (chlorophyll and carotenoid) were extracted in 100% acetone using a tissue homogenizer (Mickle Engineering Co. Ltd., UK) and quantified by reversed-phase HPLC as described previously (13). A Spherisorb 0DS2 column (25.0~4.6cm) operating on a solvent gradient of O-100% (over 30 min) ethyl acetate in acetonitrile/water (9/l v/v) at 1.0 ml/min was used to separate carotenoids and chlorophylls. Nitrate levels were determined using selective ion probes (Orion) and appropriate standards. Phosphate assays were carried out using the heteropoly blue/ascorbic acid method (14). RESULTS AND DISCUSSION The carotenoid composition of the H. pluvialis cells in the reactor at different stages of the batch-run as shown in Table 1 indicate that the green vegetative cells contain those pigments characteristic of chloroplasts of the majority of green algae and higher plants. Such cells (examined during exponential growth of the culture) completely lack any ketocarotenoid (e.g. astaxanthin) and contain only the so-called ‘primary’ carotenoids (;3-carolutein and the xanthophyll cycle tene, neoxanthin, carotenoids-violaxanthin, antheraxanthin and zeaxan-
VOL. 82, 1996
TABLE
ASTAXANTHIN
1.
Carotenoid
SYNTHESIS
BY HAEMATOCOCCUS
composition (expressed as a percentage of total carotenoid) of three different different stages of the life cycle as determined by reversed-phase HPLC (present Percentage
Carotenoid Beta-Carotene Echinenone Canthaxanthin Adonirubin Astaxanthin di-ester Astaxanthin mono-ester Astaxanthin Lutein Violaxanthin Neoxanthin Total carotenoid ,% of dry wt.
Vegetative Cells CCAP 34/l
Aplanospores MUR 145 (15)
5 4 4
70 10 12 0.5-1.0
of H. pluvialis
during
of total carotenoid
Aplanospores CCAP 34/7
8
strains study)
115
1
Palmella stage NIVA CHL 9 (16) 1
3 7 76 1 7 2 1 -
34 46 1 6 -
2.1
0.7
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).
of which lutein was the most abundant carotenoid. In contrast, highly encysted cells (aplanospores) of W. pluvialis (harvested during the stationary phase) were primarily composed of astaxanthin (in esterified form) 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 cultures 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 in Fig. 2. During the early stages of the batch-run the number of cells (Fig. 2A) increased at a relatively rapid rate which corresponded to high rates of chlorophyll synthesis. However during this growth phase the level of total carotenoid increased only slightly (Figs. 2A, B) and represented - 1.O% of algal dry weight. The addition of NaCl (between day 30-40) to the culture had a significant effect on the growth characteristics of the culture and the rate of synthesis of carotenoids. NaCl was added to the reactor when the cells had reached the stationary phase of growth and had the effect of causing an increase in the cell mortality rate within the culture (Fig. 2A). This caused a significant reduction in the levels of chlorophyll but stimulated a rapid increase in carotenoid synthesis (Figs. 2A, B). Chlorophyll levels in the bioreactor only began to fall after the addition of NaCl on day 30 (the level on day 29 was the highest recorded and that on day 35-after NaCl addition-was lower than that measured on day 14). Previous studies on a lab-scale had shown that NaCl addition was an effective way of accelerating the process of encystment and concomitant synthesis of astaxanthin in H. pluvialis (and in a number of other fresh-water microalgae, e.g. Oocystis spp.), although its addition will result in some cell death (12). Those cells which survive the addition of 40mM NaCl are capable of increasing rates of astaxanthin synthesis per cell (3-4 x greater than the rates in the absence of NaCl; see Ref. 12). Levels in excess of 40 mM NaCl (the concentration used in this study) will further increase the rate of astaxanthin synthesis (on a per cell basis) but will also induce higher rates of cell death in the culture and therefore the levels of astaxanthin per unit volume of thin),
culture are substantially reduced compared to either the control (no NaCl) or the optimum value of 40 mM NaCl (12). The effect of NaCl has been shown to be one of the most important factors in governing astaxanthin syn-
_._
0
20
40
60
80
100
Day 50
B
E
40 30 20 10
a-
0
20
’ 40
60
n
80
loo-
Day
70 60 50 40 30 Day FIG. 2. Growth and carotenoid accumulation of H. pluvialis cells in the photobioreactor. Symbols: (A) 0 , cell number (S.E. C4.9?4; n=3); 0, dry cell weight (S.E.k3.3,‘; n=3); (B) 0, chlorophyll (S.E.f3.6%; n=3); 0, carotenoid (mg/l) (S.E.i4.7!4; n=3); (C) q , chlorophyll (S.E. *3.9x; n=3); 0, carotenoid (pg per cell) (S.E.+3.1%; n=3). Arrows indicate first and final applications of NaCl to the photobioreactor.
116
HARKER
J. FERMENT.BIOENG.,
ET AL.
thesis in Haematococcus and can nearly be as effective as exposure to high irradiances in production per algal cell (exposure to high irradiances can also induce very high levels of cell death; Ref. 12). Effectively, two different stages can be promoted in the culture by the addition of NaCl: in the first (before NaCl is added) the vegetative cells lack astaxanthin whilst in the second (after NaCl addition) growth ceases (indeed cell numbers actually decline), aplanospores are formed which continue to increase in size and astaxanthin content (non-motile adult palmella cells are only transiently observed after the initial addition of NaCl). Thus, the addition of NaCl simply serves to accelerate the natural transition to the second (stationary) stage of the culture and subsequent astaxanthin synthesis in Haematococcus (12). The change in the carotenoid composition of the alga during the full course of the batch-run is shown in Fig. 3. A reduction in the levels of ‘primary’ carotenoids (especially lutein which declined steadily throughout the experiment) after the first addition of NaCl (Fig. 3A) was accompanied by an increase in levels of total ‘secondary’ carotenoid (especially di-esters of astaxanthin). The increase in carotenoid content during the batch run is primarily due to the synthesis of astaxanthin (in esterified form; Figs. 3B, C) which correlated well with an increase in dry cell weight (Fig. 2A). During the batch-run the total carotenoid content increased by 8-10 fold when expressed on a per unit column or per cell basis (Figs. 2B, C, respectively). In comparison, total carotenoid per dry cell weight only increased from -1,Od to 2.7x, reflecting (at least in part) the synthesis of the sporopollenin-based cyst wall. Indeed, throughout the course of the batch-run the dry cell weight of the culture (Fig. 2A) increased at a constant rate, indicating that cells of H. pluvialis which survived the addition of NaCl continued to increase in size as they accumulated astaxanthin esters. Table 1 shows that mono-esters of astaxanthin were the dominant pigments in the previous study by Renstr$m et al. (14), who analysed adult palmella cells (in nitrate-depleted cultures) immediately prior to encystment. In the present study the data clearly shows that esterification of astaxanthin and of the astaxanthin monoesters continued during the process of secondary carotenoid accumulation with the ratio of di-esters; monoesters of astaxanthin increasing after NaCl treatment (Fig. 3C; see also Ref. 15). Only relatively small amounts of both canthaxanthin and echinenone were measured, even in highly encysted cells and ‘free’, or unesterified, astaxanthin accounted for <2% of total carotenoid in encysted cells. Not only does the carotenoid content and composition of aplanospores of Haematococcus change with the age of the culture, but there also appears to be considerable inter-strain variation in the relative amounts of the ‘minor’ keto-carotenoids (e.g. canthaxanthin) in encysted cells. The ratio of chlorophyll a : b during the exponential and stationary phases of the batch run was constant at a relatively low value of 1.5 : 1 (data not shown). The highest carotenoid content obtained in this reactor was 2.7% (cell dry weight) which is markedly lower than the levels regularly obtained in laboratory-scale cultures (>5.5% carotenoid on a dry wt. basis; data not shown). Such a reduction upon scale-up of algal cultivation is not altogether unexpected and similar trends have
0
20
40
60
80
100
60
80
100
60
80
100
Day
2
4o
3
30
8 B B 20 U
c
4
10
8 h: rn
0 0
20
40 Day
30 ,
0
20
40 Day
FIG. 3. Change in carotenoid content and composition of cells of H. pluvialis during the course of the batch-run. (A) Changes in the levels of lutein ( n ), $-carotene (O), neoxanthin (a) and the xanthophyll cycle carotenoids (violaxanthin + antheraxanthin + zeaxanthin: q ); (B) increase in total secondary carotenoid content of the culture; (C) increase in levels of astaxanthin mono-esters ( l ), astaxanthin di-esters (0) and canthaxanthin (0). Levels of ‘free’ (unesterified) astaxanthin accounted for <2% of total carotenoid. Arrows indicate first and final applications of NaCl to the photobioreactor.
been reported for other algal production systems, including ,3-carotene synthesis in Dunaliella spp. On scale-up the level of environmental control that can be exerted over the algal culture decreases and sub-optimal conditions may therefore result. In the present study, for example, irradiance levels could not be efficiently maintained at the high levels (approximately 1,500 /Imol m 2 s i) previously identified as leading to maximum rates of astaxanthin accumulation (17). The pattern of cell growth and carotenoid accumulation was as expected for an algal batch production system, thus it can be concluded that one or more components of the system was a limiting factor in cell growth/carotenoid production during the batch-run. Figure 4 indicates that the alga soon depleted the phosphate and particularly the nitrate present in the medium during the exponential growth phase which resulted in a
VOL. 82, 1996
5 4
a .c ._
.Z
“0 # ; L .Z z
ASTAXANTHIN
SYNTHESIS BY HAEMATOCOCCUS
117
a 100 80
60 40 20 0 0
10
20
30
40
50
60’
g 0
Day Effect of nitrate and phosphate concentrations on H. growth in the photobioreactor. (A) A, cell number (S.E.t4.9%; n=3); (B) q , nitrate; 0, phosphate. Arrows indicate first and final applications of NaCl to the photobioreactor.
20
40
60
80
100
FIG. 4.
pluviulis
reduction and finally cessation of the growth of the algae. The depletion of the nitrate content of the medium coincided with the onset of the stationary growth phase and the increased synthesis of carotenoids (which was accelerated by the addition of NaCl-see above). Nitrate levels were depleted by day 20 (down to <20x of the starting level-see medium composition above) by which time the number of cells had reached the stationary phase but the total biomass continued to increase (Figs. 2A and 4). The depletion of nitrogen does not specifically appear to be a causative agent in the reduction of total chlorophyll. The level of Fe also declined during the exponential phase falling from 1.79~ 10m5 M to 0.6 X 10e5 M (data not shown). During the exponential growth period the density of the culture greatly increased restricting the amount of light each cell received. The bioreactor constructed for the present study was of a typical air-life design with a relatively small ratio of surface area (available for illumination): reactor volume. This is an important implication considering the importance of light for algal growth and carotenoid production (1). Green, vegetative, cells of EL pluviafis require a relatively low light intensity [-40-50pmol rn-* SK’ (PAR)] to achieve high rates of growth, whilst very high light intensities [--l,SOO2,00O/*mol m-* s-l (PAR)] significantly improve the rate of astaxanthin synthesis in the aplanospores (17). Rather than considering the total light energy impinging on the culture surface, the most important factor is the quantity of energy available at the cellular level. These concepts of ‘light per cell’ and ‘light regime’ expressed by Richmond (18), describe the duration of each exposure of the average cell in the photic zone, below the compensation point, or in darkness. Soeder (19) was among the first to elaborate the idea of ‘area1 density’, thereby taking into account the mutual shading of cells. Thus, an efficient growth system for large-scale cultivation of algal biomass (requiring relatively low irradiances and producing high cell densities) will generally be unsuited to a process such as carotenogenesis which is optimised at high irradiances. To partly overcome the phenomenon of light limita-
FIG. 5. Temperature and pH of the H. pluvialis culture medium in the photobioreactor. (A) A, pH; (B) 0, maximum temperature; A, minimum temperature (“C). Arrows indicate first and final applications of NaCl to the photobioreactor. and to obtain the best light regime, high levels of mixing were required to reach a turbulent flow of the culture. In microalgal mass culture, productivity is affected by the mixing system and when light is considered as a limiting factor of growth, the effective self shading of a cell has a significant effect on the growth rateicarotenoid production. This means that an efficient turbulent flow (high liquid velocity) must be maintained within the culture, so each cell is able to receive enough light in order to improve the photosynthetic efficiency of the culture (20). The mixing system employed in the present study had to take into account the current phase in the life cycle of the alga, so that the culture of green vegetative cells in the exponential phase of growth required a lower liquid velocity because of their fragility and the relatively low cell concentration. The aplanospores are more robust than the green vegetative cells due to their thick cell wall and their culture in the stationary phase required a higher liquid velocity to prevent cell sinking, and to ensure the best light regime for carotenogenesis. Nevertheless the high cell densities of Haematococcus produced in the batch-run would affect the subsequent ability to optimise high irradiance levels for carotenoid synthesis in the same reactor vessel. An alternative light ‘induction’ system would be preferable, perhaps incorporating a reactor design that, unlike a conventional airlift reactor, maximises light interception and is combined with dilution of the culture prior to the stage of astaxanthin accumulation. The results also pose the question of when is the best time to harvest such a fermentation process? It has been suggested that it is commercially viable to harvest Haematococcus once the carotenoid content of the culture has reached -2.0% of the dry cell weight of the culture (2, 18). In the present study this would have meant harvesting the bioreactor earlier in the batch-run. During the batch-run the pH of the medium in the bioreactor did not deviate significantly from the optimal pH for cell growth (pH 7.0) (Fig. SA) and no attempt was therefore made to control this. Similarly the temperature of the culture medium was not subject to any tion
118
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J. FERMENT.BIOENG.,
ET AL.
degree of control as the temperature of the environment surrounding the bioreactor could not be strictly maintained due to practical considerations and, as a result, fluctuated in the region 14-27°C during the full course of the batch-run (Fig. 5B). However it is important to note that these temperatures are not lethal to this strain of Haematococcus and are within the range to allow for optimum rates of cell growth and carotenoid production (17). The bioreactor was not completely axenic throughout the batch-run but only minimal contamination by microorganisms i.e. protozoan predators, bacteria and fungi was observed by light-microscopic examination of the culture. It is thought that algal growth was not greatly affected as a result. In contrast, when the same strain of Haematococcus was cultivated in a 250 1 open-raceway pond high levels of cyanobacterial contamination and predation by protozoans, resulting in poor algal growth, were documented (data not shown). Conclusions This investigation allowed a preliminary evaluation of the potential of an air-lift photobioreactor for the mass cultivation of the freshwater microalga, H. pluvialis, for carotenoid production. The technical feasibility of the mass cultivation of H. pluvialis in a conventional air-lift bioreactor has been demonstrated with the addition of NaCl being used to accelerate the natural transition from an initial stage of green vegetative cells to a final stationary phase consisting of large red (astaxanthin-rich), immobile, aplanospores. However, this study has also demonstrated that such a growth system (producing a high algal biomass) is not well suited to carotenoid production due to the lightrequirement for this biosynthetic process. Thus, the relatively low light levels within the bioreactor meant that the rate of carotenoid synthesis by the alga was low, and that the length of time required by the alga to synthesize significant quantities of astaxanthin was relatively long. The addition of NaCl was effective in stimulating carotenogenesis but (on a small-scale) final astaxanthin yields were improved when combined with exposure to high irradiances (17). This indicates that a separate ‘carotenoid-induction’ system is preferentially required to ensure high rates of carotenoid synthesis. Currently, a 100f bioreactor of similar design is used as a draw/fill operation where the alga is allowed to accumulate biomass in the bioreactor and then drawn off and subjected to carotenoid inducing conditions in a different system (unpublished data). ACKNOWLEDGMENT This work was supported by a John Moores University Grant and by the European Agriculture and Fisheries Programme (AIR2 CT94-1283).
Research Research
REFERENCES 1. Johnson, E. A. and An, G. H.: Astaxanthin from microbial sources. Crit. Rev. Biotech., 11, 297-326 (1991). 2. Meyers, S. P.: Developments in world aquaculture, feed formulations, and role of carotenoids. Pure Appl. Chem., 66, 10691076 (1994). 3. Miki, W.: Biological functions and activities of animal carotenoids. Pure Appl. Chem., 63, 141-146 (1991). 4. Kobayashi, M., Kakizono, T., Nishio, S., and Nagai, S.: Effects of light intensity, light quality and illumination cycle on astaxanthin formation in the green alga Haematococcus pluvialis. J. Ferment. Bioeng., 74, 61-63 (1992). 5. Boussiba, S., Fan, L., and Vonsbak, A.: Enhancement and determination of astaxanthin accumulation in green alga Haetnatococcus pluvialis. Meth. Enzymol., 213, 386-391 (1992). 6. Elliot, A.M.: Morphology and life history of Huematococcus pluvialis. Arch. Protistenk., 82, 250-272 (1934). 7. Burczyk, J.: Cell wall carotenoids in green algae which form sporopollenins. Phytochemistry, 26, 121-128 (1987). 8. Lang, N. J.: Electron microscopic studies of extraplastidic astaxanthin in Haematococcus. J. Phycol., 4, 12-19 (1968). 9. Santos, F. M. and Mesquite, J. F.: Ultrastructural study of Haematococcus lactcstris (Girod.) Rostafinski (Volvocales) I. Some aspects of carotenogenesis. Cytologia, 49, 215-228 (1984). 10. Bubrick, P.: Production of astaxanthin from Haematococcus. Bioresource Technol., 38, 237-239 (1991). 11. Nichols, H. W. and Bold, H. C.: Trichsarcina polvmorpha gen. et sp. nov. J. Phycol., 1, 34-38 (1964). 12. Harker, M., Tsavalos, A. J., and Young, A. J.: Factors responsible for astaxanthin formation in Huematococcus pluviulis. Bioresource Technol., 55. 207-214 (1996). 13. Tsavalos, A. J., Harker, M., and Young, A. J.: Secondary carotenoid synthesis in microalgae, p. 47-51. In Murata, N. (ed.), Research in photosynthesis, vol. 3. Kluwer Academic Publishers, Dordrecht (1992). 14. Murphy, J. and Riley, J. P.: A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta, 27, 31-36 (1962). 15. Grung, M., D’Souza, M. L. D., Borowitzka, M., and LiaaenJensen, S.: Algal carotenoids 51. Secondary carotenoids 2. Huematococcuspluvialis aplanospores as a source of (3S, 3’S)-astaxanthin esters. J. Appt. Phycol., 4, 165-171 (1992). 16. Renstr#m, B., Borch, G., Skulberg, M., and Liaaen-Jensen, S.: Optical purity of (3S, 3’S’-astaxanthin from Haematococcus pluvialis. Phytochemistry, 20, 2561-2564 (1981). 17. Harker. M., Tsavalos, A. J., and Young, A. J.: Use of response surface methodology to optimise carotenogenesis in the microalga, Haematococcus pluvialis. J. Appl. Phycol., 7, 399-406 (1995). 18. Richmond, A.: Outdoor mass cultivation of microalgae, p. 285-330. In Richmond, A. (ed.), Handbook of microbial mass culture. CRC Press, Boca Raton, Florida (1986). 19. Soeder, C. J.: Massive cultivation of microalgae: results and prospects. Hydrobiol., 72, 197-209 (1980). 20. Gudin, C. and Chaumont, D.: Cell fragility: the key problem of microalgae mass production in closed photobioreactors. Bioresource Technol., 38, 145-151 (1991).
Autotrophic Growth and Carotenoid Production of Haematococcus pluvialis in a 30 Liter Air-Lift Photobioreactor MARK HARKER,§
ALEX J. TSAVALOS,
ANDREW
AND
J. YOUNG*
School of Biological and Earth Sciences, John Moores University, Byrom Street, Liverpool, Received
28 August
1995/Accepted
UK
21 May 1996
by the fresh-water green unicellular alga HaeDue to the different culture requirements 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 phosphate) 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 monoand especially di-esters), partly overcoming the need to increase irradiance levels. H. pluvialis exhibited relatively 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. Growth
and ketocarotenoid
(astaxanthin)
production
matococcus pluvialis has been evaluated in a 301 air-lift photobioreactor.
[Key words:
astaxanthin,
carotenoid,
Haematococcus] This is in part due to several disadvantageous characteristics of the alga when compared to other micro-algae (e.g. the halotolerant microalga, Dunaliella spp.), including: (i) a relatively slow growth rate; (ii) a preference for low growth temperatures; (iii) a requirement of light for cultivation, and (iv) susceptibility to contamination and predation that may require the use of closed-growth system (cf. open-pond cultivation of Dunaliella spp. and Spirulina spp.). Commercial cultivation of H. pluvialis for the production of astaxanthin has, to date, been limited to the growth of the alga in raceway ponds, containing 30,000 to one million liters of algal culture (10). The major problem encountered when operating this open pond system is the contamination of the ponds by bacteria, fungi and other faster growing algae, as well as protozoan predators which have been reported to eliminate 90% of the algal biomass within 72 h (Spencer, K. G., US patent no. 4871551, 1989). The work reported in this study was conducted to evaluate the use of a conventional, reliable autotrophic system for the production of carotenoids from H. pluvialis, which would minimise or eliminate the threat posed by potential predators and competitors. For this purpose a 30 I glass column, bubble airlift-photobioreactor (with conventional design features) was constructed (Fig. 1). A two-stage batch process was adopted for the production of astaxanthin from H. pluvialis designed to allow for biomass production followed by an astaxanthin accumulation stage. This involved manipulating the culture conditions by the addition of NaCl at the onset of the stationary phase of algal growth when levels of phosphate and, especially nitrogen had been depleted, in order to promote astaxanthin production in the alga.
The occurrence of astaxanthin [(3S, 3’S)-3,3’-dihydroxy-/3,/3-carotene-4,4’-dione] in the freshwater microalga Haematococcus pluvialis has been subject to increasing experimental investigation in recent years due to great commercial interest in the pigmenting qualities of this carotenoid. Astaxanthin is used as a source in pigmentation of fish in aquaculture (especially salmonids; 1 and 2) and is also recognized as having superior antioxidant activity when compared to other carotenoids (e.g. p-carotene) and also to rw-tocopherol (3). Haematococcus spp. accumulate astaxanthin when exposed to stress conditions, generally high irradiances (4), usually in combination with nutrient deprivation (e.g. 5). The accumulation of astaxanthin in H. pluvialis (containing carotenoids typical of higher plants and the majority of green algae) is associated with a morphological transformation from green vegetative cells to deep-red, astaxanthin-rich, immobile aplanospores (6). These aplanospores are surrounded by a thick cell wall composed of the bio-polymer sporopollenin (7). The astaxanthin is accumulated in lipid bodies which coalesce and eventually totally fill the cell (8, 9). In recent years there has been considerable interest in the synthesis of astaxanthin by Haematococcus, and knowledge about the influence of abiotic factors such as irradiance levels, nutrient depletion and the use of exogenous compounds such as salt and ferrous forms of iron in controlling carotenogenesis in this alga is now better understood. There is still, however, some debate as to the most suitable conditions (e.g. enrichment or depletion of nitrogen and phosphate in the culture medium) which will allow for both algal growth and astaxanthin production (4, 5). Few studies have, however, considered the practical aspects concerned with the scale-up of astaxanthin production in this freshwater microalga.
MATERIALS
author. 5 Present address: Dept. of Genetics, The Hebrew University Jerusalem, Givat Ram Campus, Jerusalem 91904, Israel.
obtained Protozoa,
of
113
METHODS
and growth media H. pluvialis 34/7 was from the Culture Collection of Algae and Windermere, U.K. The alga was cultivated in
Organism
* Corresponding
AND
114
HARKER
ET AL.
Bold’s Basal Medium (11) modified to pH 7.0 (NaN03 2.9 mM; MgS04. 7Hz0 0.3 mM; NaCl 0.43 mM; K2HP04 0.43 mM; KH2P04 1.29 mM; CaCl*. 2H20 0.17 mM; ZnS04.7Hz0 30.7 PM; MnC12.4H20 7.3 /-‘M; Moo3 4.9 /rM; CuS04.5H20 6.3 /IM; CoN03.6H20 1.7 /jM; H1B03 0.18mM; EDTA 0.17mM; KOH 0.55 mM; FeS04. 7Hz0 17.9 /lM; HzS04 10 /*M). The photobioreactor In the closed photobioreactor the alga was grown under semi-axenic conditions throughout the batch-run. Prior to inoculation of the algae in the photobioreactor, the reactor was sterilised by means of a 0.2% sodium hypochlorite solution or high-pressure steam. The photobioreactor (30 I medium volume) was of a cylindrical construction and made of glass (see Fig. 1 for a schematic representation and dimensions). The photobioreactor was equipped with an external illumination system composed of four fluorescent tubes, which supplied 50 ,nmol rnp2 ssl (PAR) to the surface of the culture vessel (Fig. 1). The photobioreactor was surrounded by a polythene insulating sheet in an effort to maintain a relatively constant temperature within the environment closely surrounding the reactor. No other means of temperature control within the vicinity of the photobioreactor were available. Mixing and aeration were achieved by bubbling ambient air through a sparger unit located in the base of the photobioreactor (Fig. 1). Air, sterilised using filters (0.2 I’m, Millipore), was passed via the air inlet tube (Fig. 1) into the sparger, from which the air penetrated into the culture medium through a series of capillary holes. The turbulence caused by air passing up through the culture vessel was enough to mix the medium sufficiently to keep the cells in constant suspension in the culture medium. The aeration supplied the cells with a continuous source of ambient levels of COz. The air was pumped through the sparger at a rate of 1.5-3.01 per min, depending upon the age of the culture so that aeration was increased during the formation of the nonmotile astaxanthin-rich aplanospores in order to keep them in suspension. A silicon-based antifoam (Basildon Chemicals Ltd.) was added to the culture at 0.2ml per liter of algal culture when required. When the alga had reached the stationary phase of growth NaCl was added to the culture medium. Equal amounts of NaCl were added at 30, 35 and 40d to achieve a final concentration of 40mM NaCl in the reactor on day 40. The application of NaCl in this manner (three separate stages over a period of ten days to yield a final concentration of 40 mM) was used as the results of earlier experiments demonstrated that this would induce the culture to produce higher yields of astaxanthin per unit volume of culture medium rather than if the salt was added in a single application by minimising high rates of cell death (12). When the astaxanthin levels of the cells in the reactor had reached the stationary phase of accumulation (as determined by HPLC analysis-see below), aeration of the culture vessel was stopped. The algal culture was allowed to settle on the base of the reactor and then harvested as a thick algal suspension by allowing the concentrated algal slurry to flow out of the reaction via the sample line (Fig. 1). The thick algal suspension was rapidly frozen at ~20°C and kept for subsequent analysis. Analytical methods Algal cell growth was determined either by cell number as determined using a Improved Neubauer haemocytometer, or by determining
J.FERMENT.BIOENG.,
Scale drawing of the pilot photobioreactor FIG. 1. Schematic representation of the 30 I air-lift photobioreactor constructed for the evaluation of growth and carotenoid production in the unicellular microalgae H. pluvialis. Numbers: @ air outlet; 2, air outlet filter; :T media, antifoam and inoculum inlet; 2 condenser; :.:I air inlet filters; #G one of four fluorescent tubes surrounding the bioreactor; 121 growth medium; C.$ air inlet; f.9 sparger; 10 sample line and bottle.
dry cell weight after centrifugation to separate the cells from the media followed by washing and drying at 100°C for 24 h. Pigments (chlorophyll and carotenoid) were extracted in 100% acetone using a tissue homogenizer (Mickle Engineering Co. Ltd., UK) and quantified by reversed-phase HPLC as described previously (13). A Spherisorb 0DS2 column (25.0~4.6cm) operating on a solvent gradient of O-100% (over 30 min) ethyl acetate in acetonitrile/water (9/l v/v) at 1.0 ml/min was used to separate carotenoids and chlorophylls. Nitrate levels were determined using selective ion probes (Orion) and appropriate standards. Phosphate assays were carried out using the heteropoly blue/ascorbic acid method (14). RESULTS AND DISCUSSION The carotenoid composition of the H. pluvialis cells in the reactor at different stages of the batch-run as shown in Table 1 indicate that the green vegetative cells contain those pigments characteristic of chloroplasts of the majority of green algae and higher plants. Such cells (examined during exponential growth of the culture) completely lack any ketocarotenoid (e.g. astaxanthin) and contain only the so-called ‘primary’ carotenoids (;3-carolutein and the xanthophyll cycle tene, neoxanthin, carotenoids-violaxanthin, antheraxanthin and zeaxan-
VOL. 82, 1996
TABLE
ASTAXANTHIN
1.
Carotenoid
SYNTHESIS
BY HAEMATOCOCCUS
composition (expressed as a percentage of total carotenoid) of three different different stages of the life cycle as determined by reversed-phase HPLC (present Percentage
Carotenoid Beta-Carotene Echinenone Canthaxanthin Adonirubin Astaxanthin di-ester Astaxanthin mono-ester Astaxanthin Lutein Violaxanthin Neoxanthin Total carotenoid ,% of dry wt.
Vegetative Cells CCAP 34/l
Aplanospores MUR 145 (15)
5 4 4
70 10 12 0.5-1.0
of H. pluvialis
during
of total carotenoid
Aplanospores CCAP 34/7
8
strains study)
115
1
Palmella stage NIVA CHL 9 (16) 1
3 7 76 1 7 2 1 -
34 46 1 6 -
2.1
0.7
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).
of which lutein was the most abundant carotenoid. In contrast, highly encysted cells (aplanospores) of W. pluvialis (harvested during the stationary phase) were primarily composed of astaxanthin (in esterified form) 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 cultures 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 in Fig. 2. During the early stages of the batch-run the number of cells (Fig. 2A) increased at a relatively rapid rate which corresponded to high rates of chlorophyll synthesis. However during this growth phase the level of total carotenoid increased only slightly (Figs. 2A, B) and represented - 1.O% of algal dry weight. The addition of NaCl (between day 30-40) to the culture had a significant effect on the growth characteristics of the culture and the rate of synthesis of carotenoids. NaCl was added to the reactor when the cells had reached the stationary phase of growth and had the effect of causing an increase in the cell mortality rate within the culture (Fig. 2A). This caused a significant reduction in the levels of chlorophyll but stimulated a rapid increase in carotenoid synthesis (Figs. 2A, B). Chlorophyll levels in the bioreactor only began to fall after the addition of NaCl on day 30 (the level on day 29 was the highest recorded and that on day 35-after NaCl addition-was lower than that measured on day 14). Previous studies on a lab-scale had shown that NaCl addition was an effective way of accelerating the process of encystment and concomitant synthesis of astaxanthin in H. pluvialis (and in a number of other fresh-water microalgae, e.g. Oocystis spp.), although its addition will result in some cell death (12). Those cells which survive the addition of 40mM NaCl are capable of increasing rates of astaxanthin synthesis per cell (3-4 x greater than the rates in the absence of NaCl; see Ref. 12). Levels in excess of 40 mM NaCl (the concentration used in this study) will further increase the rate of astaxanthin synthesis (on a per cell basis) but will also induce higher rates of cell death in the culture and therefore the levels of astaxanthin per unit volume of thin),
culture are substantially reduced compared to either the control (no NaCl) or the optimum value of 40 mM NaCl (12). The effect of NaCl has been shown to be one of the most important factors in governing astaxanthin syn-
_._
0
20
40
60
80
100
Day 50
B
E
40 30 20 10
a-
0
20
’ 40
60
n
80
loo-
Day
70 60 50 40 30 Day FIG. 2. Growth and carotenoid accumulation of H. pluvialis cells in the photobioreactor. Symbols: (A) 0 , cell number (S.E. C4.9?4; n=3); 0, dry cell weight (S.E.k3.3,‘; n=3); (B) 0, chlorophyll (S.E.f3.6%; n=3); 0, carotenoid (mg/l) (S.E.i4.7!4; n=3); (C) q , chlorophyll (S.E. *3.9x; n=3); 0, carotenoid (pg per cell) (S.E.+3.1%; n=3). Arrows indicate first and final applications of NaCl to the photobioreactor.
116
HARKER
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ET AL.
thesis in Haematococcus and can nearly be as effective as exposure to high irradiances in production per algal cell (exposure to high irradiances can also induce very high levels of cell death; Ref. 12). Effectively, two different stages can be promoted in the culture by the addition of NaCl: in the first (before NaCl is added) the vegetative cells lack astaxanthin whilst in the second (after NaCl addition) growth ceases (indeed cell numbers actually decline), aplanospores are formed which continue to increase in size and astaxanthin content (non-motile adult palmella cells are only transiently observed after the initial addition of NaCl). Thus, the addition of NaCl simply serves to accelerate the natural transition to the second (stationary) stage of the culture and subsequent astaxanthin synthesis in Haematococcus (12). The change in the carotenoid composition of the alga during the full course of the batch-run is shown in Fig. 3. A reduction in the levels of ‘primary’ carotenoids (especially lutein which declined steadily throughout the experiment) after the first addition of NaCl (Fig. 3A) was accompanied by an increase in levels of total ‘secondary’ carotenoid (especially di-esters of astaxanthin). The increase in carotenoid content during the batch run is primarily due to the synthesis of astaxanthin (in esterified form; Figs. 3B, C) which correlated well with an increase in dry cell weight (Fig. 2A). During the batch-run the total carotenoid content increased by 8-10 fold when expressed on a per unit column or per cell basis (Figs. 2B, C, respectively). In comparison, total carotenoid per dry cell weight only increased from -1,Od to 2.7x, reflecting (at least in part) the synthesis of the sporopollenin-based cyst wall. Indeed, throughout the course of the batch-run the dry cell weight of the culture (Fig. 2A) increased at a constant rate, indicating that cells of H. pluvialis which survived the addition of NaCl continued to increase in size as they accumulated astaxanthin esters. Table 1 shows that mono-esters of astaxanthin were the dominant pigments in the previous study by Renstr$m et al. (14), who analysed adult palmella cells (in nitrate-depleted cultures) immediately prior to encystment. In the present study the data clearly shows that esterification of astaxanthin and of the astaxanthin monoesters continued during the process of secondary carotenoid accumulation with the ratio of di-esters; monoesters of astaxanthin increasing after NaCl treatment (Fig. 3C; see also Ref. 15). Only relatively small amounts of both canthaxanthin and echinenone were measured, even in highly encysted cells and ‘free’, or unesterified, astaxanthin accounted for <2% of total carotenoid in encysted cells. Not only does the carotenoid content and composition of aplanospores of Haematococcus change with the age of the culture, but there also appears to be considerable inter-strain variation in the relative amounts of the ‘minor’ keto-carotenoids (e.g. canthaxanthin) in encysted cells. The ratio of chlorophyll a : b during the exponential and stationary phases of the batch run was constant at a relatively low value of 1.5 : 1 (data not shown). The highest carotenoid content obtained in this reactor was 2.7% (cell dry weight) which is markedly lower than the levels regularly obtained in laboratory-scale cultures (>5.5% carotenoid on a dry wt. basis; data not shown). Such a reduction upon scale-up of algal cultivation is not altogether unexpected and similar trends have
0
20
40
60
80
100
60
80
100
60
80
100
Day
2
4o
3
30
8 B B 20 U
c
4
10
8 h: rn
0 0
20
40 Day
30 ,
0
20
40 Day
FIG. 3. Change in carotenoid content and composition of cells of H. pluvialis during the course of the batch-run. (A) Changes in the levels of lutein ( n ), $-carotene (O), neoxanthin (a) and the xanthophyll cycle carotenoids (violaxanthin + antheraxanthin + zeaxanthin: q ); (B) increase in total secondary carotenoid content of the culture; (C) increase in levels of astaxanthin mono-esters ( l ), astaxanthin di-esters (0) and canthaxanthin (0). Levels of ‘free’ (unesterified) astaxanthin accounted for <2% of total carotenoid. Arrows indicate first and final applications of NaCl to the photobioreactor.
been reported for other algal production systems, including ,3-carotene synthesis in Dunaliella spp. On scale-up the level of environmental control that can be exerted over the algal culture decreases and sub-optimal conditions may therefore result. In the present study, for example, irradiance levels could not be efficiently maintained at the high levels (approximately 1,500 /Imol m 2 s i) previously identified as leading to maximum rates of astaxanthin accumulation (17). The pattern of cell growth and carotenoid accumulation was as expected for an algal batch production system, thus it can be concluded that one or more components of the system was a limiting factor in cell growth/carotenoid production during the batch-run. Figure 4 indicates that the alga soon depleted the phosphate and particularly the nitrate present in the medium during the exponential growth phase which resulted in a
VOL. 82, 1996
5 4
a .c ._
.Z
“0 # ; L .Z z
ASTAXANTHIN
SYNTHESIS BY HAEMATOCOCCUS
117
a 100 80
60 40 20 0 0
10
20
30
40
50
60’
g 0
Day Effect of nitrate and phosphate concentrations on H. growth in the photobioreactor. (A) A, cell number (S.E.t4.9%; n=3); (B) q , nitrate; 0, phosphate. Arrows indicate first and final applications of NaCl to the photobioreactor.
20
40
60
80
100
FIG. 4.
pluviulis
reduction and finally cessation of the growth of the algae. The depletion of the nitrate content of the medium coincided with the onset of the stationary growth phase and the increased synthesis of carotenoids (which was accelerated by the addition of NaCl-see above). Nitrate levels were depleted by day 20 (down to <20x of the starting level-see medium composition above) by which time the number of cells had reached the stationary phase but the total biomass continued to increase (Figs. 2A and 4). The depletion of nitrogen does not specifically appear to be a causative agent in the reduction of total chlorophyll. The level of Fe also declined during the exponential phase falling from 1.79~ 10m5 M to 0.6 X 10e5 M (data not shown). During the exponential growth period the density of the culture greatly increased restricting the amount of light each cell received. The bioreactor constructed for the present study was of a typical air-life design with a relatively small ratio of surface area (available for illumination): reactor volume. This is an important implication considering the importance of light for algal growth and carotenoid production (1). Green, vegetative, cells of EL pluviafis require a relatively low light intensity [-40-50pmol rn-* SK’ (PAR)] to achieve high rates of growth, whilst very high light intensities [--l,SOO2,00O/*mol m-* s-l (PAR)] significantly improve the rate of astaxanthin synthesis in the aplanospores (17). Rather than considering the total light energy impinging on the culture surface, the most important factor is the quantity of energy available at the cellular level. These concepts of ‘light per cell’ and ‘light regime’ expressed by Richmond (18), describe the duration of each exposure of the average cell in the photic zone, below the compensation point, or in darkness. Soeder (19) was among the first to elaborate the idea of ‘area1 density’, thereby taking into account the mutual shading of cells. Thus, an efficient growth system for large-scale cultivation of algal biomass (requiring relatively low irradiances and producing high cell densities) will generally be unsuited to a process such as carotenogenesis which is optimised at high irradiances. To partly overcome the phenomenon of light limita-
FIG. 5. Temperature and pH of the H. pluvialis culture medium in the photobioreactor. (A) A, pH; (B) 0, maximum temperature; A, minimum temperature (“C). Arrows indicate first and final applications of NaCl to the photobioreactor. and to obtain the best light regime, high levels of mixing were required to reach a turbulent flow of the culture. In microalgal mass culture, productivity is affected by the mixing system and when light is considered as a limiting factor of growth, the effective self shading of a cell has a significant effect on the growth rateicarotenoid production. This means that an efficient turbulent flow (high liquid velocity) must be maintained within the culture, so each cell is able to receive enough light in order to improve the photosynthetic efficiency of the culture (20). The mixing system employed in the present study had to take into account the current phase in the life cycle of the alga, so that the culture of green vegetative cells in the exponential phase of growth required a lower liquid velocity because of their fragility and the relatively low cell concentration. The aplanospores are more robust than the green vegetative cells due to their thick cell wall and their culture in the stationary phase required a higher liquid velocity to prevent cell sinking, and to ensure the best light regime for carotenogenesis. Nevertheless the high cell densities of Haematococcus produced in the batch-run would affect the subsequent ability to optimise high irradiance levels for carotenoid synthesis in the same reactor vessel. An alternative light ‘induction’ system would be preferable, perhaps incorporating a reactor design that, unlike a conventional airlift reactor, maximises light interception and is combined with dilution of the culture prior to the stage of astaxanthin accumulation. The results also pose the question of when is the best time to harvest such a fermentation process? It has been suggested that it is commercially viable to harvest Haematococcus once the carotenoid content of the culture has reached -2.0% of the dry cell weight of the culture (2, 18). In the present study this would have meant harvesting the bioreactor earlier in the batch-run. During the batch-run the pH of the medium in the bioreactor did not deviate significantly from the optimal pH for cell growth (pH 7.0) (Fig. SA) and no attempt was therefore made to control this. Similarly the temperature of the culture medium was not subject to any tion
118
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ET AL.
degree of control as the temperature of the environment surrounding the bioreactor could not be strictly maintained due to practical considerations and, as a result, fluctuated in the region 14-27°C during the full course of the batch-run (Fig. 5B). However it is important to note that these temperatures are not lethal to this strain of Haematococcus and are within the range to allow for optimum rates of cell growth and carotenoid production (17). The bioreactor was not completely axenic throughout the batch-run but only minimal contamination by microorganisms i.e. protozoan predators, bacteria and fungi was observed by light-microscopic examination of the culture. It is thought that algal growth was not greatly affected as a result. In contrast, when the same strain of Haematococcus was cultivated in a 250 1 open-raceway pond high levels of cyanobacterial contamination and predation by protozoans, resulting in poor algal growth, were documented (data not shown). Conclusions This investigation allowed a preliminary evaluation of the potential of an air-lift photobioreactor for the mass cultivation of the freshwater microalga, H. pluvialis, for carotenoid production. The technical feasibility of the mass cultivation of H. pluvialis in a conventional air-lift bioreactor has been demonstrated with the addition of NaCl being used to accelerate the natural transition from an initial stage of green vegetative cells to a final stationary phase consisting of large red (astaxanthin-rich), immobile, aplanospores. However, this study has also demonstrated that such a growth system (producing a high algal biomass) is not well suited to carotenoid production due to the lightrequirement for this biosynthetic process. Thus, the relatively low light levels within the bioreactor meant that the rate of carotenoid synthesis by the alga was low, and that the length of time required by the alga to synthesize significant quantities of astaxanthin was relatively long. The addition of NaCl was effective in stimulating carotenogenesis but (on a small-scale) final astaxanthin yields were improved when combined with exposure to high irradiances (17). This indicates that a separate ‘carotenoid-induction’ system is preferentially required to ensure high rates of carotenoid synthesis. Currently, a 100f bioreactor of similar design is used as a draw/fill operation where the alga is allowed to accumulate biomass in the bioreactor and then drawn off and subjected to carotenoid inducing conditions in a different system (unpublished data). ACKNOWLEDGMENT This work was supported by a John Moores University Grant and by the European Agriculture and Fisheries Programme (AIR2 CT94-1283).
Research Research
REFERENCES 1. Johnson, E. A. and An, G. H.: Astaxanthin from microbial sources. Crit. Rev. Biotech., 11, 297-326 (1991). 2. Meyers, S. P.: Developments in world aquaculture, feed formulations, and role of carotenoids. Pure Appl. Chem., 66, 10691076 (1994). 3. Miki, W.: Biological functions and activities of animal carotenoids. Pure Appl. Chem., 63, 141-146 (1991). 4. Kobayashi, M., Kakizono, T., Nishio, S., and Nagai, S.: Effects of light intensity, light quality and illumination cycle on astaxanthin formation in the green alga Haematococcus pluvialis. J. Ferment. Bioeng., 74, 61-63 (1992). 5. Boussiba, S., Fan, L., and Vonsbak, A.: Enhancement and determination of astaxanthin accumulation in green alga Haetnatococcus pluvialis. Meth. Enzymol., 213, 386-391 (1992). 6. Elliot, A.M.: Morphology and life history of Huematococcus pluvialis. Arch. Protistenk., 82, 250-272 (1934). 7. Burczyk, J.: Cell wall carotenoids in green algae which form sporopollenins. Phytochemistry, 26, 121-128 (1987). 8. Lang, N. J.: Electron microscopic studies of extraplastidic astaxanthin in Haematococcus. J. Phycol., 4, 12-19 (1968). 9. Santos, F. M. and Mesquite, J. F.: Ultrastructural study of Haematococcus lactcstris (Girod.) Rostafinski (Volvocales) I. Some aspects of carotenogenesis. Cytologia, 49, 215-228 (1984). 10. Bubrick, P.: Production of astaxanthin from Haematococcus. Bioresource Technol., 38, 237-239 (1991). 11. Nichols, H. W. and Bold, H. C.: Trichsarcina polvmorpha gen. et sp. nov. J. Phycol., 1, 34-38 (1964). 12. Harker, M., Tsavalos, A. J., and Young, A. J.: Factors responsible for astaxanthin formation in Huematococcus pluviulis. Bioresource Technol., 55. 207-214 (1996). 13. Tsavalos, A. J., Harker, M., and Young, A. J.: Secondary carotenoid synthesis in microalgae, p. 47-51. In Murata, N. (ed.), Research in photosynthesis, vol. 3. Kluwer Academic Publishers, Dordrecht (1992). 14. Murphy, J. and Riley, J. P.: A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta, 27, 31-36 (1962). 15. Grung, M., D’Souza, M. L. D., Borowitzka, M., and LiaaenJensen, S.: Algal carotenoids 51. Secondary carotenoids 2. Huematococcuspluvialis aplanospores as a source of (3S, 3’S)-astaxanthin esters. J. Appt. Phycol., 4, 165-171 (1992). 16. Renstr#m, B., Borch, G., Skulberg, M., and Liaaen-Jensen, S.: Optical purity of (3S, 3’S’-astaxanthin from Haematococcus pluvialis. Phytochemistry, 20, 2561-2564 (1981). 17. Harker. M., Tsavalos, A. J., and Young, A. J.: Use of response surface methodology to optimise carotenogenesis in the microalga, Haematococcus pluvialis. J. Appl. Phycol., 7, 399-406 (1995). 18. Richmond, A.: Outdoor mass cultivation of microalgae, p. 285-330. In Richmond, A. (ed.), Handbook of microbial mass culture. CRC Press, Boca Raton, Florida (1986). 19. Soeder, C. J.: Massive cultivation of microalgae: results and prospects. Hydrobiol., 72, 197-209 (1980). 20. Gudin, C. and Chaumont, D.: Cell fragility: the key problem of microalgae mass production in closed photobioreactors. Bioresource Technol., 38, 145-151 (1991).