AU606458B2 - Method and device for producing carotenoids and particularly astaxanthin by culture of microalgae - Google Patents

Description

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cs'~ VO i--32l8 ~RANISATIONNiviN,'ALE DELA PROPRIETE INTELLECTUELLE Bureau internatijonal DEMANDE INTERNATIONALE PUBLIEE EN VERTU DU TRAITE DE COOPERATION EN MATIERE DE BREVETS (PCT) (51) Classification internationale des brevets; 4 Numno de publication internationale: WVO 89/ 01977 C12P 23/00, C12M 1/00 Al(43) Date de publication iiiternationale: 9 mars 1989 (09.03.89) (21) Num~ro de Ia demnande internationale: PCT/FR88/00431 (74) Mandataire: BREVATOME; 25, rue de Ponthieu, F- 75008 Paris (22) Date de dip6t international: 31 aocit 1988 (31.08.88) (31) Etats disignis: AT (brevet europ~en), AU, BE (brevet (31) Numnio de la demnande prioritaire: 87/12250 europ~en), CR (brevet europ~en), DE (brevet europ~en), FR (brevet europ~en), GB (brevet europ~en), (32) Date de priorit6: 3 septembre 1987 (03.09.87) IT (brev;et europ~en), JP, LU (brevet europ~en), NL 1 (brevet europ~en), NO, SE (brevet europ~en), US.

Pays de priorit6: FR, Publi6e (71) Diposant (pour tous les Etals dgsign~s sauf US): COM- A4tec rapport de recherche internationale.

MISSARIAT A L'ENERGIE ATOMIQUE [FR/FR]; 3 1-33, rue de Ia F~d~ration, F-75015 Paris (FR1).

(72) Inventeurs; et Inventeurs/Diposants (US seulernent) GUDIN, Claude [FR/FR]; Le Parc 4, Traverse Ste-Anne, F- 13 100 Aixen-Provence (FIR). JUNGAS, Colette [FR/FR]; Lotis- M..JP. 1_~AY 1989 sement du Pont-de-I'Arc, B~timent B 5, F-13090 en-Provence VAILLANT, Jean-Franqois [FR/ UTRLA FR]: 11, rue Delaunoy, BAtiment F-77000 Nelun is corrct for 3I MAR 1989 amendinents mnade under F Al. WT OF printin;), (54)Title: METHOD AND DEVICE FOR PRODUCING CAROTENOIDS AND PARTICULARLY ASTAXAN- THIN BY CULTUREv OF MICROALGAE (54) Titre: PROCEDE DE PRODUCTION DE CAROTENOIDES ET NOTAMMENT D'ASTAXANTHINE PAR CULTURE DIE MICROALGUES ET DISPOSITIF POUR LA MISE EN CEUVRE DU PROCEDE (57) Abstract hr Thle method comprises the cultivation of the microalgae Haernatococcus pluvials v Ip 4 in a photoreactor which is conitinuously Isupplied (18, 12, 11, 6) with C0 2 and with 12 J a Inutrient substrate (14, 11, 6) containing ni- 4 3 Itrates, the ratio C/N in the culture medium i being such that 2 <C/N 120 at the end of -2 the growth of the microalgae, the C concen- tration in the culture medium being from I- -4 0.22 to 12 mg/I and the N concentration in a 4 the medium at the end of the growth bein 8 6 close to 0 and at the beginning of the growth being from 40 to 120 mg/I. (57) Abrig6 -2 Ce proc~cd6 consiste 6 cultiver Ia microalgue Haemnatococcus pluvialis dans un photor~acteur aliment& en continu 18. 12, 11, 6) en CO, et en substrat nourricier (14, It, 6) renfermant des nitrates, le rapport C/N dans le milieu de culture tant tel que 2<C/N7< 120 en fin de croissance de Ia microalgu-. Ia concentration en C dans le milieu de culture allant de 0.22 6 12 mg/I et Ia concentration en N dans le milieu en fin de croissance 6tant voisine de 0 et en de~but de croissance a]lant de 40 d 120 mg/I.

de la Propriet6 industt .fi

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Signed i Pierre CHAUMUEjE 7; D eclaran t's N am e E B. RICE CO PATENT ATTORNEYS This form is suitable for any typo of Patent Application. No legalisation required.

j i VERIFIED TRANSLATION OF 2 522Z/9 1 PROCESS FOR THE PRODUCTION OF CAROTENOIDS AND IN PARTICULAR ASTAXANTHIN BY THE CULTURE OF MICROALGAE AND APPARATUS FOR PERFORMING THE PROCESS

DESCRIPTION

The present invention relates to a process for the production of carotenoids and more particularly astaxanthin by the culture of microalgae, as well as to an apparatus for performing the process. It advantageously applies to the agro-alimentary field and more specifically in pisciculture for the production of food for fish and crustaceans, as well as in pharmacology. More particularly, astaxanthin permits the pink pigmentation of the flesh of shrimp, trout, salmon, etc.

The colouring of different classes of microalgae is due to three main categories of pigments leading to photochemical reactions of the photosynthesis, namely chlorophylls, phycobilins and carotenoids. The latter are liposoluble, isoprenic and result from the bio-.

synthesis of mevalonic acid. These pigments or carotenoids ati of two types i.e. carotenes and xanthophylls.

Each species of microalgae contains 5 to carotenoids, whereof 1 or 2 preponderate.

At present, about sixty different carotenoids are known in microalgae and the most common are B-carotene, fucoxanthin, zeaxanthin, dinoxanthin, violaxanthin, antheraxanthin, myxoxantophyll, lutein, perydinine, neoxanthin, canthaxanthin and astaxanthin.

B-carotene more particularly represents 10% by weight of the microalgae Dunaliella bardwill and Dunaliella salina. Lutein can be produced by Chlore.lla pyrenoidosa at a rate of 1 g/L of culture. Diadino- L*iL4 xanthin in particular represents 2.4% by weight of B 9492.3 LC 2 Vacularia virescens.

With regards to astaxanthin, i.e. the pigment to which the invention more particularly applies and which is of formula C4 H520 with a molecular weight of 59 6 g and the developed formula:

O

CH3 Ch, CH3 CH3 CH3

OH

CH

3

C

H 3

CH

3

CH

3 OCH3

O

microalgae containing this pigment are Chlamydomonas nivalis, Euglena heliorobuescens and sanguinea, Balticola droebakensis, and Haematococcus pluvialis also known under the name Haematococcus la custris.

Astaxanthin represents up to 1% by weight of the three first microalgae and up to 5% by weight of the fourth.

For further information concerning the microalgae and the synthesis of pigments by these microalgae reference can be made to the article by C. Gudin and C. Thepenier "Bioconversion of Solar Energy into Organic Chemicals by Microalgae", published in Advances in Biotechnological Processes 6, pp.73-110, 1986.

Moreover, certain yeasts contain certain of the aforementioned pigments. In particular, Pfaffia rhodozyma contains up to 0.1% of its dry material S weight as astaxanthin.

The aforementioned pigments can be extracted from microalgae containing them or animals which have eaten these microalgae, or obtained by chemical synthesis.

In the particular case of astaxanthin, it is also L| possible to produce the latter by the treatment of S4shrimp waste.

B 9492.3

LC

3 The different presently known processes for the production of carotenoids, no matter whether by chemical synthesis or the treatment of microalgae containing carotenoids, are generally long and costly, whilst their efficiency is limited.

The present invention particularly relates to a process and to an apparatus for the production of carotenoids making it possible to obviate these disadvantages. It more particularly makes it possible to produce astaxanthin in simple manner and with a high yield.

Rather than extract the carotenoids naturally present in microalgae, the inventors have sought to cultivate these microalgae and optimize the parameters acting on the said culture with a view to increasing the production of carotenoids by the corresponding microalgae.

In particular, the Inventors have found that the production of carotenoids by Haematococcus pluvialis in the presence of at least one nitrogen-containing compound and at least one carbon-containing compound can be significantly increased if the concentration ratio C/N in the culture medium, at the end of the microalgae growth phase, is at least equrl to 2 and is at the most equal to 120.

The C/N ratio can be modified either by modifying the carbon quantity present in the culture medium, or by modifying the nitrogen quantity present in the culture medium, or by simultaneously acting on the carbon quantity and the nitrogen quantity.

The quantity of carotenoids produced by Haematococcus pluvialis increases with the Haematococcus quantity present in the culture medium. In addition, the growth phase of the microalgae must be optimized.

LI 35 To this end, the C/N ratio at the start of culture SB 9492.3 LC N iA is advantageously chosen between 0.00018 and 0.3, the dissolved nitrogen concentration ranging between and 120 mg/L and preferably 80 and 120 mg/L, whilst the dissolved carbon concentration ranges between 0.22 and 12 mg/L and preferably 4.4 and 12 mg/L. For these C/N values, the production of carotenoids is equal to that naturally found in Haematococcus and is therefore low.

The Inventors have found that a relatively large dissolved nitrogen quantity was necessary for the production of the biomass. However, a nitrogen deficiency in the culture medium for a given biomass quantity was favourable to the production of carotenoids.

Moreover, the nitrogen concentration in the culture medium, at the end of the culture cycle, is advantageously chosen close to 0. (The lower the nitrogen concentration, the higher the quantity of carotenoids produced, for a same carbon quantity in the culture medium).

In practice, the nitrogen concentration at the end of culture is equal to 0.1 mg/[ the carbon concentration during the carotenoid production phase being maintained at between 0.22 and 12 mg/L in the culture medium.

Advantageously, the carbon-containing compound are introduced into the culture medium in the form of pure carbon dioxide gas or mixed with air. However, it is also possible to introduce them in the form of carbonates and in particular calcium, magnesium and similar carbonates.

In the same way, the nitrogen-containing compounds are advantageously introduced into the culture medium in the form of at least one compound chosen from among the ammonium salts, nitrates and urea. Among the ammonium salts reference is made to NH4CL, whilst as nitrates reference is uade to sodium, potassium and similar nitrates.

4) B 9492.3 LC 5 The pH of the culture medium is maintained at a neutral pH between 6.5 and 7.5 and is e.g. equal to 7.

The carbon dioxide dissolved in the aqueous medium and designated CO d is responsible for the carbon supply to the microalgae and is subject to the following chemical equilibria I: CO2d H CO HCO CO 2 2 2 3 03

I)

3E which are dependent on the pH between 4 and 12.

The addition of CO 2 to the culture medium would normally lead to an acidification thereof. However, in view of the fact that the microalgae very rapidly absorbed the CO 2 (the kinetics of the photosynthesis reaction is at the most equal to 1 second), the chemical equilibria I tend to move from right to left, which corresponds to an alkalinization of the medium.

In other words, if the demand of the microalgae for CO is higher than the CO 2 supply to the medium, the pH of the latter is displaced towards values higher than In order to bring the pH to between 6.5 and 7.5, it is possible to increase the CO 2 supply and obtain a pH stabilization if the demand and supply of CO d are equal, 2 or it is possible to add to the culture medium a strong acid such as HCL, HNO or H PO The normality of the added acid varies from 2 to 4.

Apart from the pH, the addition of nitric acid modifies the nitrogen quantity present in the culture medium, which can be a problem for the continuous control of the nitrogen supply to the microalgae.

The acidification of the culture medium is preferably carried out with orthophosphoric acid which, as a result of these three dissociation constants, has an excellent buffer capacity. Moreover, the PO ion in highly appreciated by the microalgae and constitutes r, :i :1: ii ;4 6 i ti

-I.

B 9492.3 LC

.L~

6 one of the ions necessary for their growth.

Apart from the dependence of the CO 2 photosynthesis demand, the pH of the culture medium is dependent to a certain extent on the nitrogen supply, as is apparent from the two following biological equations II, III given in the case of astaxanthin.

(II) 106CO 2 138H20 16NO 13802 160H (III) 106C02 106H20 16NH 1060 16H 2 microalgae biomass microalgae biomass The above displacement of the equilibria I ia also dependent on the light intensity of the incident light necessary for the growth of the microalgae. Light can be natural or artificial and its intensity ranges between 30 and 400 W/m 2 per day.

Another factor acting on the above equilibria I is the cellular concentration of the culture medium, which can be measured by the number of cells per volume unit (taking a sample which is placed in a microscope), by the dry matter weight per volume unit (corresponding to taking a sample, filtering the sample, drying at 100°C and then weighing the material obtained), or by the optical density of the culture medium.

The cellular concentration of a culture medium estimated by measuring the optical density OD follows Beer's law, whose principle will now be given. The light function LnI/Io absorbed by a culture sample of thickness e (expressed in cm) of dry weight PS (expressed in g/L) for a length is: OD LnI/Io e(X)xPSxe in which es() represents the molecular extinction coefficient at wavelength X of the culture medium studied, Io represents the light intensity of the instant light, I represents the light intensity on leaving the SB 9492.3 LC 7 sample and Ln represents the natural logarithm.

This is followed by the plotting of the absorbtion spectrum E(x) of the studied culture and measurements are carried out at the wavelength corresponding to the absorbtion maximum, i.e. OD and at the wavelength max corresponding to the absorbtion minimum, i.e. OD On assuming E(x) to be constant for a given culture, the optical density OD makes it possible to follow the evolution of the total biomass for the number of microalgae cells per volume unit expressed in dry weight.

Advantageously, carbon dioxide gas is introduced into the culture medium mixed with air. The air quantity used preferably represents 5 to 10 volumes of air per volume of culture and per hour.

In order to aid the growth of microalgae and therefore the production of carotenoids, the culture medium is advantageously kept at a temperature of 20 to 0 C and preferably at 20 0

C.

According to the invention, the carbon quantity and nitrogen quantity present in the culture medium are continuously checked.

Thus, the CO 2 quantity dissolved in the culture medium is constantly measured either with the aid of a dissolved CO probe, or by measuring the gaseous CO on I 2 2 entering and leaving the culture reactor, said measurement being performed by infrared spectrometry, or by determining the CO 2 quantity actually consumed by the microalgae by measuring the 02 quantity emitted by the latter. Thus, whenever a CO 2 molecule is photo- J synthetically fixed by a microalga, a 02 molecule is emitted. Following the dissolved oxygen concentration of the culture medium with a dissolved 02 electrode (Ingold) makes it possible to carry out the CO 2 check.

These different means can be used alone or in redundancy, in order to perfectly control the carbon supply to the microalgae, everything obviously being j j B 9492.3 LC il L. i P-8 *i| I8 brought to the cellular concentration unit. The latter can be stabilized by continuous application of the i culture principle using a constant dilution level corresponding to a residents time in the reactor between two to 20 days. The dilution level is linked with the growth rate of the microalgae and corresponds to the nutrient medial quantity introduced every hour.

The different possible checks of the carboncontaining supply of the microalgae are obviously only valid when the pH is maintained between 6.5 and i The inventive process is advantageously applied to the production of astaxanthin responsible for the pink colouring of certain crustaceans and fish.

The astaxanthin quantity produced is in particular determined by spectrophotometry, i.e. by measuring the optical density of astaxanthin at 478 nm present in the culture medium. The relationship between the OD and the astaxanthin concentration is given by Beer's law.

Although the invention is applied with particular advantage to astaxanthin production, it also applies to other carotenoids. In particular, a culture according to the invention of Haematococcus pluvialis permits the production of B-carotene, adoirubin, lutein, violaxanthin and neoxanthin, apart from astaxanthin, which can in particular be used in the agricultural industry and in pharmacology.

The invention also relates to an apparatus for performing the inventive process and which is characterized in that it comprises a photobioreactor able to contain the culture medium, means for supplying the culture medium with the carbon-containing compound, means for checking the carbon quantity present in the culture medium, means for supplying the culture medium with the nitrogencontaining compound and means for checking the nitrogen quantity present in the culture medium.

I' 4 B 9492.3 LC I I 3 _l l 9 Other features and advantages of the invention can be gathered from the following illustrative and nonlimitative descripA'., with reference to the attached drawings, wherein show: Fig. 1 Figs. 2 and 3 Diagrammatically an apparatus for performing the inventive process.

Curves giving the optical density variations of the astaxanthin and the culture medium over time for different values of the C/N ratio respectively at 478 and 760 nm.

A Haematococcus pluvialis strain, which is a microalga of the microchlorophyta type is cultured in a reactor 2 having a culture photobioreactor 4 provided in its lower part with a pipe 6 for supplying the nutrient substrate necessary for the growth of the microalgae present in the photobioreactor 4. Pipe 6 is equipped with a pump 8 for the circulation of the nutrient substrate 11.

At its top, the photobioreactor 4 is provided with a pipe 10 for the discharge of the depleted nutrient substrate, the latter issuing at the top of a carbonator 12 or CO reactor. This carbonator is formed by a glass or plastic aerating column. The base of carbonator 12 is connected to supply pipe 6.

The arrows in Fig. 1 indicate the flow direction of the different substances necessary for the growth of the microalgae contained in the photobioreactor 4.

For further details concerning the construction of the carbonator and the photobioreactor, reference can be made to the aforementioned article by C. Gudin and C. Thepenier.

At its top, carbonator 12 is supplied via a supply pipe 14 with the nutrient substrate 11 constituted by B 9492.3

LC

Bristol medium, whose composition is given in the article in Amer j. Botany de Starr RC, 1964, no.51, pp.1013- 1 04 4 and entitled "Culture Collection of Indiana". In addition, said solution contains the complex of iron and ethylenediaminetetracetic acid (EDTA). A pump 16 mounted on the supply pipe 14 injects the Bristol medium into carbonator 12.

A pipe 18 issuing at the base of the carbonator 12 makes it possible to supply the latter with a mixture of air and CO 2 This mixture represents 5 to 10 times the colume of the culture per hour and contains 0.1 to by volume of CO 2 Pipe 18 is equipped with a flowmeter making it possible to regulate the CO 2 feed flow.

The base of the carbonator 12 is equipped with a diffuser 22 or aerator, made from fritted glass and j permitting the diffusion of the gaseous mixture into the i Bristol medium 11.

The CO 2 quantity dissolving in the nutrient medium 11 is dependent on the shape and dimensions of the carbonator 12 and in particular its surface to volume ratio, as well as the shape of the diffuser 22 and in particular its porosity and surface.

The stage of dissolving CO 2 in substrate 11 (corresponding to the passage from the gaseous phase to the liquid phase) is the longest in the entire process and corresponds to the hydration of the CO ion, which is 2 approximately 26.6 seconds at 25°C. The other stages -15 in the process take approximately 10 seconds, excepting the CO consumption dissolved by the microalgae i 30 contained in photoreactor 4, which is at the most equal to 1 second.

Reactor 2 is placed under natural or artificial light and a detector 24 located in the vicinity of the i photobioreactor 4 and of the Eppley pyranometer type, makes it possible to measure the light intensity to which SALI the microalgae contained in photobioreactor 4 are exposed.

B 9492.3 LC L li_ I 11 Detector 24 is connected to a computer 30, such as that sold by Hewlett Packard under the reference HP85 and to which it supplies the said measurement.

The photobioreactor 4 is also located in a thermostatically controlled bath 26 in which circulates the water which can be heated or cooled at random. The water supply 27 issuing into the base of the enclosure 26 's equipped with an electrovalve 28, whose contr,:l is ensured by computer 30, as a function of the temperature of the water measured with the aid of a probe 32 immersed in the enclosure 26 and supplying the measurement signal to the computer The circulating water cooling and heacing system, which is known to the Expert, is not shown in Fig. 1 and is positioned outside the reactor 2.

As the pH of the solution tends to become alkaline due to the CO 2 consumption by the microalgae, an orthophosphoric acid supply pipe 34 issuing at the top of the carbonator 12 must be provided. Pipe 34 is equipped with an e'lectrovalve 36, whose opening and closing are rendered dependent, via computer 30, on the measurement of the pH of the culture medium performed by a probe 38 immersed in the photobioreactor 1. Supply pipe 18 is provided with a branch 40 making it possible to sample the mixture injected into carbonator 12, to which is fitted an infrared analyzer 412 for measuring the gaseous 'i CO quantity present at the intake of carbonator 12, said analyzer being connected to computer In the same way, an outlet 144 for the gases introduced into the carbonator 12 is located on the top of the latter. Outlet pipe t44 is equipped with an infrared analyzer 46, which is also connected to computer 30 and is used for measuring the CO 2 quantity not dissolved in the culture medium. By comparison of the signals supplied by analyzers 12 and 46, it is B 9492.3 LC M I ~fk 0 12 possible to know the CO quantities dissolved in the culture medium and which can consequently be photosynthesized by the microalgae contained in the photobioreactor 11.

The nitrogen check of the culture medium is carried out with the aid of an electrovalve 48 mounted on the nutrient substrate supply pipe IlI and whose opening and closing are controlled by computer 30. The opening and closing of valve 48 fix the flowrate of the fresh nutrient substrate, i.e. the dilution level of the culture medium for a continuous production of carotenoids.

The nitrogen quantity present in the culture medium is regularly measured (twice daily) by taking a culture medium sample, which is introduced into a known nitrogen measuring chain, such as the TECHNICON analyzer.

The extraction of the microalgae enriched with carotenoids and in particular astaxanthin takes place in known manner by centrifuging, ultrafiltration or, as described in the article by B. Renstrom et al published in Phytochemistry, vol. 20, no.11, pp.2561-25 6 4, entitled "Optical Purity of Astaxanthin from Haematococcus pluvialis".

The microalgae are received in a container J connected to the carbonator medium, via a supply pipe 52.

EXAMPLE

a) Growth of Haematococcus pluvialis Using at the start of culture a nutrient substrate constituted by Bristol medium of the following composition: NaNo 0.25 g/L CaCL 2 2H20 0.025 g/l MgSO 4 7120 0.075 g/L 30 K21-1 PO 0.075 g/l S:H H 0 1.75 g/L aCL 0.025 g/l B 91192.3 LC

A

13 a pH of 7, a temperature of 200C, continuous artificial 2 lighting of 30 W/m per day, 5 volumes of air per culture volume and per hour containing 5% of CO 2 roughly 20 hours are necessary for reaching the stationary Haematococcus pluvialis growth phase.

The exponential growth of the microalgae corresponda to the following Myers biochemical equation IV: (IV) 6.1' CO 2 3.65 1120 NI 3 6. 110.3 22 6.82 0 2 in which the microalgae biomass is green.

The C/N ratio in the above culture medium at the start of the cycle is equal to 0.3.

The cellular concentration is determined by spectrophotometry, i.e. as a function of the optical density of the culture, at 760 nm corresponding to the maximum 15 absorbtion of Haematococcus pluvialis.

b) Astaxanthin Production At the end of 20 days, the microalgae are pigmented red as a result of a carotenoid identified as astaxanthin.

the astaxanthin concentration is measured by the optical density of the culture at 478 nm, which corresponds to the astax:anthin abscrzbtion maximum.

The maximum pigmentation of the microalgae and therefore the concentration of astaxanthin and other carotenoids synthesized by Haematococcus pluvialis is in the stationary growth phase of the microalgae corresponding to a nitrogen deficiency in the culture medium and the more the culture medium is concentrated with microalgae, the higher the concentration of astaxanthin and other carotenoids.

Through increasing the initial nitrogen concentration of the culture medium, .t is possible to significantly increase the production of astaxanthin by microalgae, as can be clearly gathered from Figs. 2 and 3.

Fig. 2 gives the variations of the optical density B 91192.3 1, .E i 14 at 478 nm expressed in arbitrary units and therefore the astaxanthin concentration variations as a function of time, expressed in days. The operating conditions are those given in parag.-aph a) with a varying NaNO concentration in the Bristol medium, the CO percentage in the 5 air volumes being equal to 0.1.

Curve A corresponds to an initial culture medium containing 0.25 g/L of NaNO 3 curve B to an initial quantity of 0.5 g/L of NaNO and curve C to an initial concentration of 0.75 g/L of NaNO 3 At the start of the culture cycle, curve A corresponds to a C/N ratio of 0.0055, curve B to a C/N ratio of 0.00275 and curve C to a C/N ratio of 0.0018. At the end of 24 days, i.e. at the end of culture, the optical density of the culture medium has only doubled for curve A, but has tripled for curve B and quadrupled for curve C, which respectively corresponds to a doubling, tripling and quadrupling of the astaxanthin concentration. At the end of the cycle, the C/N ratio for the three curves is approximately 2.2.

The fact of doubling or tripling the initial nitrate dose and therefore the nitrogen dose of the culture medium leads to an important increase in the astaxanthin production by increasing the growth or development of microalgae.

Hereinafter is given the calculation making it possible to find the C/N ratio in the culture medium, x g/L of NaNO corresponding to 14'x/85 g/b of nitrogen 3 30 and y% of gaseous CO in the air CO 2 mixture corresponds S'2 2 to O0'y/5 mg/b of dissolved CO (measured with infrared 2 analyzers and therefore 12*y/5 mg/L.

It is also possible to increase the astaxanthin production by -aematococcus pluvialis by keeping constant the nitrogen quantities dissolved in the culture medium and by increasing the carbon quantity dissolved B 9492.3 LC c

C

i Dunaliella salina. Lutein can be produced by Chlorella pyrenoidosa at a rate of 1 g/b of culture. Diadinoxanthin in particular represents 2.4% by weight of B 9492.3 ~I I I 15 in said medium. This can be clearly gathered from Fig. 3, which gives variations of the optical density at 478 nm, designated OD478, i.e. those of the 478 astaxanthin concentration and the variations of the optical density at 760 nm, designated OD 7 6 0 i.e. those the microalgae concentration, as a function of time.

The culture conditions are the same as those given in a with a variable CO quantity injected into the 2 culture.

Curves E, I and G correspond to an injection into the culture medium of 5 volumes of air per volume of culture and per hour, enriched with 1% CO 2 and curves F, J and H to air enriched with 2% of CO 2 Curves E and F give the optical density at 478 nm and respectively correspond to a C/N ratio at the start of the culture cycle of 0.055 and 0.11 and at the end of the culture cycle of 2.2. On the basis of these curves, it is apparent that the optical density and therefore the astaxanthin concentration increases with the C/N ratio, i.e. when C increases.

The curves of Figs. 2 and 3 were made in a discontinuous culture or batch system by adjustment of the initial cellular concentration to approximately at an optical density value of 478 nm. This culture was stopped after 24 hours following the total consumption of the nitrogen and therefore the nitrates dissolved in the culture mediur.. This is clearly apparent from curves G and H of Fig. 3, which relate to the disappearance of the nitrogen in the culture medium and are measured by the reduction of the OD of the medium to 220 nm.

Curves I and J of Fig. 3 give the optical density at 760 nm respectively for a C/N ratio of 0.055 and 0.11 at the start of culture. Through increasing the 35 C quantity for a fixed value of N leads to an increase -i \k B 9492.3 LC vvwaxicu vy uiieiIicaL syn nesis.

In the particular case of astaxanthin, it is also possible to produce the latter by the treatment of shrimp waste.

B 9492.3 LC L- Ii I' CI i i-i l_ i 1 16 in the cellular concentration and therefore in the astaxanthin concentration.

Figs. 2 and 3 were plotted with a doublebeam spectrophotometer with a linear response between 0 and 3 marketed under the name UVICON. The calibration of the measuring and reference cells was carried out at 478 nm using the Bristol medium given in a, said medium then being used as the reference solution for determining the concentration of microalgae and the astaxanthin concentration.

The cells used had a thickness e of 1 cm. The molecular extinction coefficient of Haematococcus pluvialis at 760 nm is approximately 3, but that of astaxanthin is not definitely known. Thus, the Haematococcus pluvialis PS concentration is governed by the equation PS OD760 x 3.3. Moreover, for OD60 0.8 given on curve J of Fig. 3, PS equals 2.6 g/L.

For the continuous production of astaxanthin, the dissolved carbon concentration is chosen as constant 20 throughout the production period. This concentration is equal to 12 mg/L 5% of CO 2 mixed with 5 volumes of air per culture volume) and it is introduced continuously into the culture medium.

For the dissolved nitrogen quantity, a discontinuous supply takes place following each introduction period of the Bristol medium into the culture medium, the dissolved nitrogen concentration in the culture being 120 mg/i 0.75 g/L of NaNO 3 and before each introduction period of the Bristol medium, the dissolved nitrogen concentration in the culture is equal to 0.1 mg/L.

The Bristol medium replenishment period (or the dilution rate of the culture medium) is 20 days.

The above description has shown the key role of the C/N ratio in the culture medium on the production of astaxanthin from Haematococcus pluvialis. Up to now, no publication relating to this strain of microalgae mentions the role played by this ratio on the pigmentation of the Smicroalgae.

/91 B 9492.3 LC

Claims (21)

1. Process for the production of carotenoids consisting of culturing the microalga Haematococcus pluvialis in the presence of at least one nitrogen- containing compound and at least one carbon-containing compound, the C/N ratio of the carbon and nitrogen concentrations in the culture medium at the end of the microalgae growth phase being such that 26C/N120.

2. Process according to claim 1, characterized in that the nitrogen concentration at the end of the growth phase is close to 0.

3. Process according to claims 1 or 2, characterized in that the carbon-containing compounds are introduced into the culture medium in the form of carbon dioxide gas (CO

4. Process according to any one of the claims 1 to 3, characterized in that the nitrogen-containing compounds are introduced into the culture medium in the form of at least one compound chosen from among ammonium salts, nitrates and urea.

5. Process according to any one of the claims 1 to 4, characterized in that the nitrogen-containing compounds are in the form of sodium nitrate.

6. Process according to any one of the claims 1 to characterized in that the nitrogen concentration at the start of growth is in the range between 40 and 120 mg/L.

7. Process according to any one of the claims 1 to 6, characterized in that the carbon concentration at the start and end of growth is in the range between 0.22 and 12 mg/L.

8. Process according to any one of the claims 1 to L Li 7, characterized in that the culture medium is maintained D 91192.3 LC 0F N r U 18 at a pH between 6.5 and

9. Process according to any one of the claims 1 to 8, characterized in that the culture medium is maintained at a temperature between 20 and 250C.

10. Process according to any one of the claims 1 to 9, characterized in that the culture medium is kept at a temperature of

11. Process according to any one oi the claims 1 to 10, characterized in that the culture medium is maintained at a neutral pH with orthophosphoric acid (H 3PO4)

12. Process according to any one of the claims 1 to 11, characterized in that the culture medium is aerated with 5 to 20 volumes of air per culture volume and per hour.

13. Process according to any one of the claims 1 to 12, characterized in that the microalgae culture takes place in the presence of light whereof the light intensity is between 30 and 400 W/m4 per day.

14. Process according to any one of the claims 2 to 13, characterized in that the carbon dioxide gas quantity consumed by the microalga is determined by measuring the oxygen quantity emitted by the microalga, reduced to a cellular concentration unit.

15. Process according to cl. im 14, characterized in that the cellular concentration is determined by measuring the optical density of the culture medium at 760 nm.

16. Process for the production of astaxanthin using the process according to any one of the claims 1 to 15, 1 characterized in that the astaxanthin quantity produced is determined by measuring the optical density of the B 91492.3 LC 19 culture medium at 478 nm.

17. Apparatus for performing the process according to any one of the claims 1 to 16, characterized in that it comprises a photobiorector able to contain the culture medium, means (18, 12, 6) for supplying the culture medium with the carbon-containing compound, means (20, 18, 42, 46, 30) for checking the carbon quantity present in the culture medium, means (14, 11) for supplying the culture medium with the nitrogen-containing compound and means (48, 30) for checking the nitrogen quantity present in the culture medium.

18. Apparatus according to claim 17, characterized in *o :o that means (30, 36, 38) for checking the pH of the culture medium are provided. S

19. Apparatus according to claims 17 or 18, characterized in that the means (28, 30, 32) for checking the temperature of the culture medium are provided.

Apparatus according to any one of the claims 17 to 19, characterized in that means (24, 30) for checking the light intensity received by the culture medium are provided.

21. Process for the production of carotenoids substantially as hereinbefore described with reference to the example. oDATED this 19 day of October 1990 COMMISSARIAT A L'ENERGIE ATOMIQUE Patent Attorneys for the Applicant: F.B. RICE CO. i V IC- These different means can be used alone or in redundancy, in order to perfectly control the carbon supply to the microalgae, everything obviously being B 9492.3 20 DESCRIPTIVE ABSTRACT Process for the production of carotenoids and particularly astaxanthin by the culture of microalgae and apparatus for performing the process. This process consists of culturing the microalga Haematococcus pluvialis in a photobioreactor (4) continuously supplied (18, 12, 11, 6) with CO 2 and nutrient substrate (14, 11, 6) containing nitrates, the C/N ratio in the culture medium being 2.C/N.120 at the end of growth of the microalga, the C concentration in the culture medium ranging between 0.22 and 12 mg/L and the N concentration in the medium at the end of'the growth being close to 0 and between 40 and 120 mg/L at the start of growth. (Fig. 1) -r i 'I i t i i 11 B 9492.3 LC 44 636 L, i