METHOD OF EXTRACTING A PIGMENT FROM MICROALGAE

Abstract

The present invention relates to a method of extracting a pigment from microalgae.

Claims

1. A method of extracting a pigment from microalgae, comprising the following steps: a) providing microalgae in an aqueous culture medium, wherein said microalgae are enriched with a pigment, b) inducing said microalgae to enter a flagellated stage using a germination-inducing condition, and/or disrupting said microalgae resulting in a suspension comprising said disrupted microalgae and said pigment, and c) extracting said pigment from said flagellated microalgae and/or from said suspension using a liquid-liquid extraction system comprising a solvent, wherein the liquid-liquid extraction system is selected from a countercurrent chromatography system, a centrifugal partition chromatography system, and a membrane-assisted liquid-liquid extraction system.

2. The method according claim 1, wherein said method comprises the following steps: a) providing microalgae in an aqueous culture medium, wherein said microalgae are enriched with a pigment, b) inducing said microalgae to enter a flagellated stage using a germination-inducing condition, and c) extracting said pigment from said flagellated microalgae using a liquid-liquid extraction system comprising a solvent, wherein the liquid-liquid extraction system is selected from a countercurrent chromatography system, a centrifugal partition chromatography system, and a membrane-assisted liquid-liquid extraction system.

3. The method according to claim 1, wherein said method comprises the following steps: a) providing microalgae in an aqueous culture medium, wherein said microalgae are enriched with a pigment, b) disrupting said microalgae resulting in a suspension of said disrupted microalgae and said pigment, and c) extracting said pigment from said suspension using a liquid-liquid extraction system comprising a solvent, wherein the liquid-liquid extraction system is selected from a countercurrent chromatography system, a centrifugal partition chromatography system, and a membrane-assisted liquid-liquid extraction system.

4. The method according to claim 1, wherein said microalgae are initially in a cyst stage and are induced to enter a flagellated stage by means of a germination-inducing condition.

5. The method according to claim 1, wherein said germination-inducing condition is selected from a phototrophic condition, a mixotrophic condition, and a heterotrophic condition.

6. The method according to claim 1, wherein said microalgae are disrupted mechanically.

7. The method according to claim 1, wherein said step c) is followed by step d) obtaining said pigment by lyophilization, freezing, or vaporization of said solvent, or by dissolving said pigment in a nutritional oil, or another solvent.

8. The method according to claim 1, wherein said microalgae have become enriched with said pigment by nutrient depletion, excessive light exposure, high salinity, and/or overexpression resulting from genetic modification of said microalgae.

9. The method according to claim 1, wherein said microalgae are Chlorophyta.

10. The method according to claim 1, wherein said microalgae are selected from Haematococcus pluvialis, Chlorella zofingiensis, Neochloris wimmeri, and Chlamydomonas nivalis.

11. The method according to claim 1, wherein said liquid-liquid extraction system is a membrane-assisted liquid-liquid extraction system.

12. The method according to claim 1, wherein said liquid-liquid extraction system is a liquid-liquid chromatography system selected from a centrifugal partition chromatography system and a countercurrent chromatography system.

13. The method according to claim 1, wherein said solvent has a vapor pressure of at least 10 mbar at 25° C. and ambient pressure.

14. The method according to claim 1, wherein said solvent is selected from methyl-tert-butyl ether, ethyl acetate, butan-i-ol, dichloromethane, chloroform, diethyl ether, ethyl methyl ether, toluene, benzene, ketone, 1,1-dichloroethane, cyclohexane, isopropyl acetate, 2-methyltetrahydofuran, methyl ethyl ketone, methylcyclohexane, 2,2,4-trimethylpentane, xylene, pentan-i-ol, dodecane, decane, acetone, ethanol, propan-2-ol, propan-i-ol, methanol, tetrahydrofuran, tert-butanol, acetonitrile, dimethyl sulfoxide, acetic acid, ethylene glycol, n-alkanes, and oil such as nutritional oil, or a combination thereof.

15. The method according to claim 1, wherein said pigment is a keto-carotenoid.

16. The method according to claim 1, wherein the solvent is an organic solvent, a water based solution, a plant oil, or a deep eutectic solvent, or a combination thereof.

17. The method according to claim 1, wherein the microalgae are Haematococcus pluvialis.

18. The method according to claim 13, wherein the solvent has a vapor pressure of at least 64 mbar at 25° C. and ambient pressure.

19. The method according to claim 14, wherein the solvent is selected from ethyl acetate and methyl-tert-butyl ether, or a combination thereof.

20. The method according to claim 15, wherein said pigment is astaxanthin.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0084] The present invention is now further described by reference to the following figures.

[0085] FIG. 1 is a schematic representation of extracting a substance according to the present invention using a CPC system.

[0086] In the beginning of the astaxanthin-extraction, the CPC system is filled with a solvent or solvent mixture and the rotation is started. Subsequently, water is pumped into the CPC system to act as mobile phase, and a fraction of the solvent i.e. the stationary phase is replaced by said mobile phase within the CPC system, wherein said replacement depends on the flow rate of the pump and the rotation speed (FIG. 1A).

[0087] Fermentation broth is subsequently pumped into the CPC system. Mass transfer occurs within the CPC system, so that astaxanthin is extracted from microalgae in flagellated stage into the solvent (FIG. 1B).

[0088] Alternatively, in one embodiment, said fermentation broth can contain disrupted cells, so that astaxanthin is extracted from said fermentation broth which contains astaxanthin released by disrupted cells. Alternatively, in one embodiment, said fermentation broth can contain both intact cells in flagellated stage and disrupted cells, so that astaxanthin is extracted both from intact flagellated cells and the fermentation broth containing astaxanthin released by disrupted cells.

[0089] The fermentation broth is pumped into the CPC system, until the solvent is saturated with astaxanthin (FIG. 1C-E). The extracted cells exit the CPC system from the last chamber at the outlet (FIG. 1D,E).

[0090] Once the solvent is saturated with astaxanthin, water is pumped out of the last chamber to collect the saturated solvent from the first chambers (FIG. 1F). Astaxanthin-containing solvent is completely pushed out of the CPC system, and thus collected. The solvent can be evaporated to obtain astaxanthin (FIG. 1G).

[0091] The extraction process can be repeated any number of times.

[0092] FIG. 2 is a schematic representation of an exemplary membrane-assisted extraction system. The transfer of astaxanthin from the cell broth through the pores of the membrane to the solvent is presented. At time t.sub.start an exemplary solvent or solvent mixture is in contact with an astaxanthin containing fermentation broth. As the time progresses, astaxanthin penetrates through the pores and dissolves in the solvent respectively solvent mixture. After an infinite long time t.sub.∞, the astaxanthin distributes between the phases according to its partition coefficient.

[0093] In a typical operation, one fluid phase (wetting phase) fills the membrane pores due to capillary forces and the other fluid phase is non-wetting. In one embodiment, a hydrophobic membrane is used. An exemplary solvent (or solvent mixture) is the wetting phase, while the aqueous algae broth is the non-wetting phase. The solvent (or solvent mixture) fills the pores of the membrane. In one embodiment, to stabilize the liquid-liquid interface at the pore mouth, the non-wetting fluid is held at a slightly higher pressure, resulting in a transmembrane pressure of approximately 0.1 bar. In a preferred embodiment, to stabilize the liquid-liquid interface at the pore mouth, the non-wetting fluid is held at a slightly higher pressure, resulting in a transmembrane pressure within the range of 0.01-0.1 bar.

[0094] FIG. 3 shows astaxanthin extraction yield from germinating H. pluvialis cells using methyl-tert-butyl ether as solvent at 0 h, 8 h, 16 h, 24 h, 32 h, 40 h, 48 h, 56 h and 64 h after inducing the germination. The yield was calculated according to equation 2.

[0095] FIG. 4 shows microscope images of partially extracted H. pluvialis cells 48 h after the germination was induced. For the extraction 1 mL germinated algae broth was mixed with 5 mL methyl-tert-butyl ether.

[0096] FIG. 5 shows astaxanthin concentrations in the extract, 0 h (immediately after inducing the germination) and 24 h after inducing the germination obtained with different solvents (n-heptane, butan-1-ol, ethyl acetate, methyl-tert-butyl ether (MTBE) and dichloromethane).

[0097] FIG. 6 depicts shake flask experiments with the solvents butan-1-ol, heptane, methyl-tert-butyl ether, ethyl acetate and dichloromethane (from left to right) at t=0 h and 24 h after inducing the germination.

[0098] FIG. 7 shows the extracted amount of astaxanthin in the 1st collected fraction obtained using operating conditions A, the extracted amount of astaxanthin in the shake flask experiment and the yield Y.sub.extract for an injection volume of 0.5 mL and three different elution times 6.9 min, 18.9 min and 36.9 min.

[0099] FIG. 8 shows resulting yields Y.sub.extract of the three different injection volumes 2 mL (t.sub.elution=8.40 min), 5 mL (t.sub.elution=11.4 min), and 10 mL (t.sub.elution=16.4 min) of operating conditions C. The astaxanthin contents of all the collected fractions and of the injected sample (amount of astaxanthin in the injected fermentation broth) were quantified to calculate the yield (Y.sub.extract).

[0100] FIG. 9 shows the astaxanthin concentration of the injected algae broth of fraction 1, fraction 2, and the remaining fractions of the three different injection volumes 2 mL, 5 mL, and 10 mL of the operating conditions C. With increasing injection volume, an increase of the astaxanthin concentration in the collected fractions 1 and 2 can be seen. The original feed astaxanthin concentration of 65 mg L.sup.−1 was concentrated to 507 mg L.sup.−1 and 302 mg L.sup.−1 in fraction 1 and 2 with an injection volume of 10 mL.sup.−1.

[0101] FIG. 10 shows the principle of membrane-assisted liquid-liquid extraction.

[0102] A) Schematic representation of the cross-section of a parallel tube hollow fiber contactor allowing for a non-dispersive contact of two fluids via a microporous membrane. At each pore mouth of the membrane a fluid-fluid interface is formed. The fluid-fluid interface is stabilized by a slight overpressure on one side of the membrane. The membrane serves as a physical separation barrier between the feed and extracting phase (solvent or mixture of solvents). The concentration gradient of the solute in the two phases is the driving force for the mass transfer across fluid-fluid interface and extraction of a solute from one fluid phase (feed) in the other fluid phase (extracting phase).

[0103] B) Flow sheet of a pilot plant of a hollow fiber contactor used for the extraction of astaxanthin from algae broth. The algae broth in the right reservoir was pumped in the tubes of the hollow fibers, the solvent in the left reservoir was pumped at the shell side. Both streams flow co-currently and are recirculated.

[0104] FIG. 11 shows a further schematic representation of extracting a substance according to the present invention using a CPC/CCC system.

[0105] In the beginning of the astaxanthin-extraction, the CPC/CCC system is filled with a solvent or solvent mixture and the rotation is started. Subsequently, water is pumped into the CPC/CCC system to act as mobile phase, and a fraction of the solvent i.e. the stationary phase is replaced by said mobile phase within the CPC/CCC system, wherein said replacement depends on the flow rate of the pump and the rotation speed (FIG. 11A).

[0106] Fermentation broth is subsequently pumped into the CPC/CCC system (FIG. 11B). Mass transfer occurs within the CPC/CCC system, so that astaxanthin is extracted from microalgae in flagellated stage into the solvent (FIG. 11C,D). Alternatively, in one embodiment, said fermentation broth can contain disrupted cells, so that astaxanthin is extracted from said fermentation broth which contains astaxanthin released by disrupted cells. Alternatively, in one embodiment, said fermentation broth can contain both intact cells in flagellated stage and disrupted cells, so that astaxanthin is extracted both from intact flagellated cells and the fermentation broth containing astaxanthin released by disrupted cells.

[0107] The fermentation broth is pumped into the CPC/CCC system, until the solvent is saturated with astaxanthin (FIG. 11C-E). The extracted cells exit the CPC/CCC system from the end of the outlet (FIG. 11E).

[0108] After a predefined switching time, t.sub.elution, the stationary phase is pushed out of the column. This can be achieved by changing the flow direction of the mobile phase (water), switching from the descending mode to the ascending mode (FIG. 11F,G option 1) or by pumping solvent in the descending mode (FIG. 11F,G option 2). In both cases, astaxanthin-containing solvent is completely pushed out of the CPC/CCC system and collected. In FIG. 11F,G, the fractionated stationary phase is shown, numbered starting with the most concentrated fraction. The solvent can be evaporated to obtain astaxanthin.

[0109] The extraction process can be repeated any number of times.

[0110] FIG. 12 shows the astaxanthin concentrations in fraction 1, fraction 2, fraction 3 and the remaining fractions for an injection volume of 2 mL, 5 mL and 10 mL of a high-pressure homogenized biomass at 200 bar, the injected astaxanthin concentration and the calculated yield Y.sub.feed. The original feed astaxanthin concentration of 254 mg L.sup.−1 was concentrated to 2771 mg L.sup.−1 and 2376 mg L.sup.−1 in fraction 1 and 2 with an injection volume of 10 mL.

[0111] FIG. 13 shows the collected fraction 1, fraction 2, fraction 3 and the remaining fractions for an injection volume of 10 mL of a high-pressure homogenized biomass at 200 bar. The original feed astaxanthin concentration of 254 mg L.sup.−1 was concentrated to 2771 mg L.sup.−1 and 2376 mg L.sup.−1 in fraction 1 and 2 with an injection volume of 10 mL.

[0112] FIG. 14 shows the astaxanthin oleoresin concentration in the water rich phase and the solvent using a membrane-assisted liquid-liquid extraction. The astaxanthin oleoresin concentration decreases from 23.77 mg L.sup.−1 in the water to 5.05 mg L.sup.−1 after an extraction time of 1410 min, while a final concentration of 31.7 mg L.sup.−1 is reached in the solvent.

[0113] FIG. 15 shows the astaxanthin content of the algal broth (germinated) and in the solvent, using a membrane-assisted liquid-liquid extraction. The astaxanthin content decreases from 10.9 mg in the algal broth to 0.32 mg after an extraction time of 165 min, while the content in the solvent increased to 1.4 mg.

EXAMPLES

Example 1: Microalgae Culture

[0114] Haematococcus pluvialis (SAG number 192.80) was procured from the Culture Collection of Algae at the University of Göttingen, Germany (SAG). As culture medium, Bold Modified Basal Freshwater Nutrient Solution (BBM) was used. It was prepared by diluting 20 mL Bold Modified Basal Freshwater Nutrient Solution (50× concentrate) from Sigma-Aldrich (Taufkirchen, Germany), with 980 mL de-ionized water, obtaining the following composition (per liter): 11.42 mg H.sub.3BO.sub.3, 25.0 mg CaCl.sub.2.2H.sub.2O, 0.49 mg Co(NO.sub.3).sub.2.6H.sub.2O, 1.57 mg CuSO.sub.4.5H.sub.2O, 50.0 mg EDTA (free acid), 4.98 mg FeSO.sub.4.7H.sub.2O, 75 mg MgSO.sub.4.7H.sub.2O, 1.44 mg MnCl.sub.2.4H.sub.2O, 0.71 mg MoO.sub.3, 0.003 mg NiCl.sub.2.6H.sub.2O, 31.0 mg KOH, 0.003 mg KI, 175.0 mg KH.sub.2PO.sub.4, 75 mg K.sub.2HPO.sub.4, 25 mg NaCl, 250.0 mg NaNO.sub.3, 0.002 mg Na.sub.2SeO.sub.3, 0.001 mg SnCl.sub.4, 0.0022 mg VOSO.sub.4.3H.sub.2O, and 8.82 mg ZnSO.sub.4.7H.sub.2O. Additionally, 1.64 g of sodium acetate (Molecular biology grade, >99.0%) was added to the culture medium. The pH was adjusted to 6.8.

[0115] Parts of the H. pluvialis colonies were transferred from the agar-plate into a 250 mL Erlenmeyer flask and cultivated in 150 mL BBM+20 mM sodium acetate. The culture was cultivated at a shaking plate for 16 days at 24° C. until an optical density (OD) of 0.6 at 750 nm was reached. The light was continuously supplied by one cool-fluorescence lamp with a light intensity (photon flux density) of 50 μmol m.sup.−2 s.sup.−1. Subsequently, the broth was transferred in into a 2000 mL Erlenmeyer flask, filled up with fresh culture medium (working volume of 1600 mL) and incubated at the previous conditions for 14 days. This broth was used as an inoculum for the cultivation in a self-designed open pond with a total volume of 22 liter. The initial OD at 750 nm was adjusted to 0.1 and using a working volume of 8 liter. Water loss by evaporation was compensated once every 24 h by adding distilled water. The open-pond was illuminated continuously with two cool-fluorescence lamps with a light intensity (photon flux density) of 100 μmol m.sup.−2 s.sup.−1 at a constant room temperature of 24±1° C. for 14 days.

[0116] The induction of astaxanthin synthesis (enrichment of astaxanthin in the cells) was performed in the open pond at an OD of 0.8 at 750 nm by increasing the light intensity (photon flux density) to 250 μmol m.sup.−2 s.sup.−1 for 7 days.

Example 2: Induction of Germination

[0117] To induce germination of H. pluvialis cyst cells, 400 mL of the cyst culture broth was transferred into a 500 mL Erlenmeyer flask and placed 24 hours on the shaking plate (175 rpm) at a light intensity (photon flux density) of 50 μmol m.sup.−2 s.sup.−1 and a temperature of 24±1° C. Afterwards the broth was centrifuged at 5500 rpm for 2 min and the supernatant was discarded. The cyst biomass was suspended into fresh BBM+20 mM sodium acetate and 30 mL were transferred into a 50 mL Erlenmeyer flask with an OD of 4 at 750 nm. Culture conditions at the shaking plate were the same as described in example 1.

Example 3: Solvent suitability

[0118] Relevant physical properties of several solvents are reported (Table 1). Solvents were chosen regarding their ability to extract astaxanthin from the germinated algae cells, the maximal solubility in water, their hydrophobicity and enthalpy of vaporization.

TABLE-US-00001 TABLE 1 Physical properties of the tested solvents [6]. butan- methyl-tert- ethyl n-heptane 1-ol butyl ether acetate dichloromethane solubility in 0.00024 7.4 4.2 8.08 1.73 water/wt % (25° C.) (25° C.) (20° C.) (25° C.) (25° C.) solubility in 0.0024 80 44 87.9 17.6 water/g l.sup.−1 (25° C.) (25° C.) (20° C.) (25° C.) (25° C.) log P.sub.octanol/water/— 4.5 0.84 0.94 0.73 1.25 boiling point at 1 bar/° C. 98.4 117.73 55.0 77.11 40 enthalpy of vaporization Δ.sub.vapH, 36.57 52.35 29.82 35.60 28.82 (101.325 kPa, T = 25° C.)/ kJ mol.sup.−1

Example 4: Determination of the Biomass Concentration

[0119] The dry weight (DW) biomass concentrations were determined in quadruplicates. 1 mL culture aliquot was transferred into 2 mL micro-centrifuge tubes, centrifuged at 5500 rpm for 5 min and the supernatant was discarded. The biomass was washed with distilled water, which was discarded after centrifugation at 5500 rpm and the moist biomass was stored at −80° C. and freeze dried subsequently. The freeze-dried samples were weighed and the biomass concentration was calculated. Therefore each Eppendorf tube was weighed empty before and with biomass after freeze drying. The dry weight biomass (DW) of each tube was divided through 1 mL.

Example 5: Astaxanthin Quantification

[0120] For the determination of the astaxanthin content in the algae broth, 5 mg of the DW was weighed in with a balance of Satorius (Göttingen, Germany). For cell homogenization the biomass was processed with mortar and pestle. Extraction of the astaxanthin out of the broken cells was achieved by adding 10 mL of dichloromethane. The extraction was repeated three times, until the cell debris was left colorless. The astaxanthin-rich dichloromethane extract was evaporated with a rotary evaporator from Heidolph Instruments (Schwabach, Germany) and saponified for 3 h at room temperature in the dark using the method of Taucher et al. [7]. Therefor 2.25 mL of acetone, 0.25 mL of MeOH and 0.5 mL of 0.05 M NaOH in MeOH were added to the extracted astaxanthin. Afterwards 3 mL petroleum ether were added. The mixture was washed with 3 mL of a 10 wt % aqueous NaCl solution and centrifuged for 2 min at 5500 rpm and the lower phase was discarded. The washing step with the NaCl solution was repeated two more times. The organic phase was evaporated and the extracted astaxanthin was dissolved in 3 mL of solvent B (methanol, MTBE, water, 8:89:3, v/v), which was used in the HPLC method, and filtrated through a 0.22 μm disposable nylon syringe-filter from Berrytec (Grünwald, Germany).

[0121] The de-esterified astaxanthin samples were analyzed with a high-performance liquid chromatography unit (HPLC unit) (LC-20AB prominence Liquid chromatography, Shimadzu, Japan) consisting of an YMC Carotenoid column (C30, 3 m, 150×4.6 mm, YMC Co., Japan) and a diode-array detector (SPD-M20A prominence diode array detector, Shimadzu, Japan). As mobile phase, solvent A (methanol, MTBE, water, 81:15:4, v/v) and solvent B (methanol, MTBE, water, 8:89:3, v/v) with the following gradient were used: 2% solvent B for 11 min, a linear gradient from 2% solvent B to 40% solvent B for 7 min, 40% solvent B for 6.5 min followed by a linear gradient to 100% solvent B in 2.5 min, 100% solvent B for 3 min, a linear gradient to 2% solvent B in 3 min, held for 7 min. The flow rate was 1 mL min.sup.−, the injection volume was 10 μl and the column temperature was kept at 22° C. For the astaxanthin quantification a calibration curve was created with the chemical standard from Dr. Ehrenstorfer GmbH (Augsburg, Germany). The signal of the diode-array detector was recorded at 478 nm.

Example 6: Optimum Extraction Time for Germinated H. pluvialis Cells

[0122] For the determination of the ideal time for the extraction of astaxanthin from the germinated cells, with a time gap of 8 hours two cultures for the germination, culture 1 and culture 2, were prepared as described in the Example 2. For the determination of the astaxanthin yield, one shake flask extraction experiment was performed 0 h, 16 h, 24 h, 40 h, 48 h and 64 h (culture 1) and 8 h, 32 h and 56 h (culture 2) after the germination was initiated. At every of the mentioned time points 1 mL of the algae broth was transferred into a 15 mL falcon tube. 5 mL of methyl-tert-butyl ether were added and the binary mixture was shaken intensively for 30 min with the Multi Bio RS-24 from bioSan (Riga, Latvia) at a room temperature of 24±1° C. After that, to separate the phases, the sample was centrifuged for 2 min at 5500 rpm. 4 mL of the astaxanthin-rich methyl-tert-butyl ether phase were withdrawn and evaporated with a rotary vacuum evaporator from Heidolph Instruments (Schwabach, Germany). The astaxanthin content in the extracts was quantified using the HPLC method described in Example 5. The biomass concentration and astaxanthin content of the algae broth for all studied time intervals was quantified as described in Example 4 and Example 5, respectively. The mass of astaxanthin in the extract, in the algae broth and the yield are presented in FIG. 3.

[0123] The optimal time for the extraction of astaxanthin from the germinated cells was determined to be between 24 h and 32 h after the germination was induced (FIG. 3). The extraction yield, calculated according equation 3, was nearly constant between 24 h and 32 h reaching yields of 56 to 60% of the total astaxanthin available in the algal broth. After 40 h the yield decreased to 31% and further to 17%, 64 h after the germination was induced. The astaxanthin content in the algal broth remained constant within the investigated time range. The decrease of the yield after 32 h was caused by morphological changes of the germinated cells. An increasing number of germinated cells lost their flagella and built up a robust cell structure, with reduced permeability for methyl-tert-butyl ether (FIG. 4).

[0124] The time points t=0 h (extraction was performed immediately after adding new medium) and t=24 h after the induction of the germination were taken to determine the extraction efficiency of the solvents shown in Table 1. FIG. 5 shows the astaxanthin concentration in heptane, butan-1-ol, ethyl acetate, methyl-tert-butyl ether (MTBE), and dichloromethane of the extract phase, after mixing 5 mL of each solvent with 1 mL feed at the mentioned times. The extraction efficiency of each solvent is increasing between t=0 h and t=24 h, due to the increasing number of germinated algae cells. 24 h after inducing the germination, the extraction efficiencies of the tested solvents was in the order [methyl-tert-butyl ether]>[dichloromethane]>[ethyl acetate]>[butan-1-ol]>[heptane] (FIG. 5 and FIG. 6).

Example 7: Extraction with Countercurrent Chromatography (CCC) and Centrifugal Partition Chromatography (CPC) System

[0125] CCC experiments were carried out on a countercurrent chromatography column, model HPCCC-Mini Centrifuge (0.8 mm i.d.) with a β-value between 0.5 to 0.78 from Dynamic Extractions (Wales) and a column volume of 18.2 mL. Two isocratic Gilson 306 pumps (Gilson, USA), equipped with an 806 Manometric Module (Gilson, USA), were used for delivering the mobile and stationary phases.

[0126] CCC extraction experiments were conducted using methyl-tert-butyl ether as an extraction solvent. Therefore, methyl-tert-butyl ether was stirred for two hours with BBM+20 mM sodium acetate culture medium at a room temperature of 24±1° C. The equilibrated system was split into the upper solvent-rich phase and the lower culture medium-rich phase using a separatory funnel. The phases were degassed in an ultrasonic bath. The CCC unit was prepared by filling the column with the solvent-rich phase i.e. the stationary phase. Rotation was set at 1900 rpm and the culture medium, saturated with methyl-tert-butyl ether (the mobile phase) was pumped in the descending mode with a flow rate F of 1 mL min.sup.−1. After equilibrium conditions in the column were reached, the germinated biomass was injected to the column via an injection loop. After the corresponding elution time (shown in Table 2 for each operating condition), the stationary phase was pushed out of the column. That was done by switching from the descending mode to the ascending mode. The stationary phase was fractionated into 2 mL HPLC vials until no more stationary phase was coming out of the column. Defined parts of the collected fractions were pipetted into round bottom flasks, evaporated and further processed for the HPLC analysis as explained in Example 5.

[0127] The CPC experiment was performed in the CPC 250 PRO SPECIAL BIO VERSION column with twin cells, with a total column volume of 250 mL, from Gilson Purifications SAS (formerly Armen Instruments, France):

[0128] The CPC column has 12 disks, where each disk contains 20 engraved twin-cells; in total 240 cells. The column was connected to a pressure vessel (Apache Stainless Equipment Corporation, USA) with a total volume of 5 Liter for pumping the algae broth into the CPC. An overpressure of up to 7.3 bar was provided by the in-house compressed air line.

[0129] The CPC experiment was carried out, using ethyl acetate as the extraction solvent. Ethyl acetate was stirred for 2 hours with deionized water at 24±1° C. The separated phases were degassed with an ultrasonic bath. The CPC unit was filled with solvent-rich phase i.e. the stationary phase. After the rotational speed of the system had been set to 1350 rpm, the mobile phase (water saturated with ethyl acetate) was fed to the CPC unit at a pressure of 7.3 bar in the descending mode, what results in a flow rate of 30 mL min.sup.−1 at the set pressure. After that, 720 mL of the germinated algae broth with an OD of 4 at 750 nm was pumped into the CPC column with the same pressure. After 24 min (corresponds to 720 mL algae broth at this flow rate), the flow of algae into the CPC was stopped. The pressure vessel was filled with deionized water (saturated with ethyl acetate) and the stationary phase was pushed out of the column in the ascending mode. The stationary phase was fractionated into 15 mL falcon tubes until no more stationary phase was coming out of the column. Defined parts of the collected fractions were pipetted into round bottom flasks, evaporated and further processed for the HPLC analysis as explained in Example 5.

[0130] The elution time t.sub.elution is the time span between the start of the injection (i.e. pumping) of the biomass into the CCC (CPC) column and switching from descending to ascending mode to push out the stationary phase from the column. Looking at the injected biomass as a tracer, equation 1 gives the minimum time required for the extracted biomass (cells) to leave the column.

[00001]$\begin{array}{cc}{t}_{\mathrm{elution},\min }=\frac{V_{\mathrm{MP}}+V_{\mathrm{injection}}}{F}& \mathrm{equation}1\end{array}$

[0131] V.sub.MP is the volume of the mobile phase and V.sub.injection is the injected volume.

[0132] After the elution time, t.sub.elution, the stationary phase was pushed out the column in the ascending mode by pumping the culture medium (CCC), respectively water (CPC)-rich phase. In both cases the phase was saturated with the solvent used for the extraction, methyl-tert-butyl ether for CCC and ethyl acetate for CPC. The fractions of the astaxanthin dissolved in the solvent coming out from the column were collected, evaporated and the astaxanthin content determined according to the procedure described in Example 5.

[0133] The yield Y.sub.feed was calculated as quotient of the mass of astaxanthin in the collected fractions m.sub.astaxanthin,fraction to the astaxanthin mass in injected feed biomass m.sub.astaxanthin,feed, as shown in equation 2.

[00002]$\begin{array}{cc}{Y}_{\mathrm{feed}}=\frac{m_{\mathrm{astaxanthin},\mathrm{fraction}}}{m_{\mathrm{astaxanthin},\mathrm{feed}}}.Math.100& \mathrm{equation}2\end{array}$

[0134] Additionally, at every CCC (CPC) experiment, three shake flask experiments were performed, in order to determine the extractable amount of astaxanthin from the cells in the corresponding cell stage. Therefore 1 mL of the algae broth was mixed with 5 mL methyl-tert-butyl ether (CCC experiments) respectively ethyl acetate (CPC experiment) for 30 min. After centrifugation at 5500 rpm for 5 min, 4 mL of the solvent was taken and the astaxanthin content was determined according to the procedure described in Example 5. A yield Y.sub.extract, which takes the extractable amount of astaxanthin from the cells in the corresponding cell stage into account, was defined according to equation 3,

[00003]$\begin{array}{cc}{Y}_{\mathrm{extract}}=\frac{m_{\mathrm{astaxanthin},\mathrm{fraction}}}{N.Math.m_{\mathrm{astaxanthin},\mathrm{extract},\mathrm{shake}\mathrm{flask}}}.Math.100& \mathrm{equation}3\end{array}$

wherein m.sub.astaxanthin,extract,shake flask is the extracted amount of astaxanthin in the extract of the shake flask experiment. As the shake flask experiment was always performed with 1 mL algae broth, the factor N is needed to adjust the value to the feed injected into the CCC/CPC. For example, if 5 mL algal broth was injected into the CCC/CPE and the shake flask experiment was carried out with 1 mL algae broth, this results in a value of 5 for N.

Example 8: CCC and CPC Experiments

[0135] According to the results shown in the previous example such as example 6, methyl-tert-butyl ether was used as the extraction solvent in all CCC experiments and ethyl acetate in the CPC experiment. The germination was induced according to the conditions described in the previous examples. The following five operating conditions were examined in the CCC and one in the CPC column (Table 2). The obtained results are summarized in Table 2.

TABLE-US-00002 TABLE 2 Operating conditions for the experiments A, B, C, D, F in the CCC column and E in the CPC column. A B C D E c.sub.biomass injected/mg 4.95 2.95 2.95 4.97 16.8 6.72 8.65 mL.sup.−1 V.sub.inj/mL 0.5 0.5 2 2 5 10 2 5 720 m.sub.biomass injected/mg 2.5 1.48 5.9 10.0 24.9 49.8 33.6 6055 m.sub.astaxathin injected/mg 0.026 0.027 0.108 0.1 0.33 0.66 0.57 63.95 t.sub.elution/min 6.9 18.9 36.9 6.9 18.9 36.9 8.4 20.4 38.4 8.4 11.4 16.4 8.4 11.4 24 c.sub.astaxanthin, injected/ 52 54 65.5 260 104 91.4 mg.sub.astaxanthin L.sup.− V.sub.column/mL 18.2 18.2 18.2 18.2 18.2 250 c.sub.1st fraction/ 2.5 2.5 2.5 4.9 5.6 5.0 29.5 29.8 25.5 46.9 124.8 507.6 195.7 179.6 338.8 mg.sub.astaxanthin L.sup.−1 Y.sub.extract/% 61.3 76.1 58.9 47.7 47.7 48.6 48.4 72.0 53.7 113.8 129.1 113.1 44.2 44.9 21.9 Y.sub.feed/% 11.1 13.8 10.7 27.5 27.5 28.0 27.9 41.5 31.0 65.9 74.7 65.5 39.1 39.1 6.4 F c.sub.biomass injected/mg mL.sup.−1 8.91 17.82 26.56 V.sub.inj/mL 2 5 10 2 5 10 2 5 10 m.sub.biomass injected/mg 17.8 44.6 89.2 35.6 89.3 178.2 53.1 132.8 265.6 m.sub.astaxathin injected/mg 0.17 0.43 0.86 0.34 0.86 1.71 0.51 1.27 2.55 t.sub.elution/min 7.5 11 15.5 8 11 16 9.5 12 17.5 c.sub.astaxanthin, injected/mg.sub.astaxanthin 85.5 171 254.9 L.sup.−1 V.sub.column/mL 18.2 18.2 18.2 c.sub.1st fraction/mg.sub.astaxanthin L.sup.−1 58 225 460.2 238 607 1554 393 1337 2771 Y.sub.extract/% 50 52.4 48.8 94.8 87.3 109.9 99 111 150 Y.sub.feed/% 37.9 69.18 63.9 71.8 66.1 83.2 76 85 115

[0136] Operating Conditions A

[0137] In the operating conditions A, three different elution times were examined, namely 6.9 min, 18.9 min and 36.9 min. The injection volume (germinated algae broth) was 0.5 mL resulting in an injected amount of astaxanthin of 0.026 mg. Here, the influence of increasing elution times on the yield was investigated. Only the first and most concentrated fraction was analyzed for its astaxanthin content and used for the calculation of the yield. As presented in FIG. 7, an increase of t.sub.elution from 6.9 min to 36.9 min did not affect the yield Y.sub.extract of extracted astaxanthin. The calculated yields Y.sub.extract were in the range of 60 to 76%.

[0138] Operating Conditions B

[0139] To verify the results from operating condition A, in operating condition B three different elution times were examined for the injection volumes of 0.5 mL and 2 mL. For 0.5 mL the elution times were chosen similar to operating conditions A, were the column was emptied 6.9, 18.9 and 36.9 min after the injection of 0.5 mL germinated algae cells. For the injection volume of 2 mL of biomass, the elution times 8.4 min, 20.4 min and 38.4 min were examined. Only the first and most concentrated fraction was analyzed for its astaxanthin content and used for the calculation of the yield.

[0140] Similar to the results of operating conditions A, the increase of the elution time did not affect the yields Y.sub.extract and Y.sub.feed. For the injection of 0.5 mL, the values obtained for Y.sub.extract were around 48%, values for Y.sub.feed were around 28%. The injections of 2 mL germinated algae broth show a similar trend. For an elution time of 8.4 min and 38.4 min, Y.sub.extract is 48% and 54%. For an elution time of 20.4 min, Y.sub.extract was calculated to be 72%. For Y.sub.feed, similar trends can be seen, yields of 27%, 42% and 31% were obtained for the elution times of 8.4 min, 20.4 min and 36.4 min, respectively.

[0141] Operating Conditions C

[0142] In the operating conditions C, the influence of three different injection volumes, 2 mL, 5 mL and 10 mL on the yield and concentration of astaxanthin was examined. The astaxanthin content in the first two collected fractions was determined and the content of the low concentrated remaining fractions was determined after pouring these fractions together. For the calculation of the yields with equation (2) and (3), the mass of astaxanthin (m.sub.astaxanthin,fraction) in fraction 1, fraction 2 and in the remaining fractions were summed up. As shown in the previous operating conditions A and B, an elution time longer than calculated with equation (1) doesn't affect the extracted amount of astaxanthin respectively the achieved yields. Consequently, t.sub.elution were determined to be 8.4 min, 11.4 min and 16.4 min according to equation (1) for the injected volumes of 2 mL, 5 mL and 10 mL. The calculated yields Y.sub.extract were 113% for the elution times of 8.4 min and 16.4 min and 130% for 11.4 min and are presented in FIG. 8. The achieved yields Y.sub.feed were 65% for the elution times of 8.4 min and 16.4 min and 75% for an elution time of 11.4 min.

[0143] In FIG. 9 the concentrations of the injected algae broth and the collected fraction 1, fraction 2 and remaining fractions of the three injection volumes are shown. As it can be seen, the starting concentration of 65 mg.sub.astaxanthin L.sup.−1 in the injected algal broth is concentrated to 500 mg.sub.astaxanthin L.sup.−1 and 300 mg.sub.astaxanthin L.sup.−1 in fraction 1 and fraction 2 when 10 mL were injected.

[0144] Operating Conditions D

[0145] In operating conditions D, two different injection volumes of the germinated algae broth, 2 mL and 5 mL, were injected with two different biomass concentrations. For an injection volume of 2 mL, 16.8 mg mL.sup.−1 biomass and for an injection volume of 5 mL, 6.72 mg mL.sup.−1 biomass were injected. Thus, the injected amount of biomass respectively astaxanthin was the same in these experiments. The achieved Y.sub.extract was 44%, Y.sub.feed was 39% for the injected volumes. In the run, where 2 mL algae broth were injected, the concentrations reached in the first and second fraction were 195 mg.sub.astaxanthin L.sup.−1 and 48 mg.sub.astaxanthin L.sup.−1. Similar values were reached, when 5 mL were injected resulting in concentrations of 180 mg.sub.astaxanthin L.sup.−1 and 50 mg.sub.astaxanthin L.sup.−1 for fraction 1 and 2.

[0146] Operating Conditions E

[0147] In the CPC run, 720 mL germinated cell broth was injected into the CPC, corresponding to a biomass of 6055 mg and 63.95 mg astaxanthin, respectively. In the first collected fraction an astaxanthin content of 338 mg L.sup.−1 was measured. The yield Y.sub.extract was 21.9%.

[0148] Operating Conditions F

[0149] In the operating conditions F, three different biomass concentrations of mechanically disrupted cyst cells were injected into the CCC unit. For every biomass concentration, three different injection volumes, 2 mL, 5 mL and 10 mL were examined. The mechanical cell disruption was carried out with a high-pressure homogenizer (APV 1000, APV Systems, Denmark), in which the algae broth was pressed through a gap at a pressure difference of 200 bar. This pressure difference causes the cell wall to burst and the cytoplasm together with AXT is released into the medium. After injection of the biomass, the solvent was fractionated after the time t.sub.elution as shown in FIG. 11F,G (option 2), by pumping the solvent in the descending mode.

[0150] The astaxanthin content in the first three collected fractions was determined and the content of the low concentrated remaining fractions was determined after pouring these fractions together. For the calculation of the yields with equation (2) and (3), the mass of astaxanthin (m.sub.astaxanthin,fraction) in fraction 1, fraction 2, fraction 3 and in the remaining fractions were summed up. For an injection volume of 10 mL, the astaxanthin concentration in fraction 1 increased from 420 mg L.sup.−1 to 1554 mg L.sup.−1 and 2771 mg L.sup.−1 with an increase of the injected biomass from 89 mg to 178.2 mg and 265 mg. In FIG. 12, the astaxanthin concentration of the first three fractions, the remaining fractions and the injected astaxanthin concentration for an injection volume of 2 mL, 5 mL and 10 mL with a concentration of the injected biomass of 26.6 g L.sup.−1 are presented. In FIG. 13, the collected fractions of an injection volume of 10 mL and a biomass concentration of 26.6 g L.sup.−1 are presented.

Example 9: Membrane-Assisted Liquid-Liquid Extraction

[0151] A pilot plant with PTFE hollow fibers with a total surface area of 0.1619 m.sup.2 was used for membrane-assisted liquid-liquid extraction. The solvent ethyl acetate and the homogenized H. pluvialis cysts were placed in two separate reservoirs. The solvent ethyl acetate was pumped in to the shell side and homogenized H. pluvialis cysts were pumped in the lumen side (in the tubes) of the hollow fiber membrane module. The two streams, ethyl acetate and the homogenized H. pluvialis cysts broth were pumped concurrently and circulated at the respective site of the membrane (FIG. 10). The run was stopped after 3 h. Astaxanthin was extracted from the fermentation broth into the solvent. Before the run, a shake flask experiment was performed similar to the CCC/CPC experiments. The absorbance of the extracted pigments was measured with UV/Vis spectroscopy at 478 nm to be 23.5. Three hours after starting the run, an absorbance of 0.333 at 478 nm was measured for the solvent circulated in the shell side of the plant. Assuming an infinite long extraction time, the same yield as in a shake flask experiment i.e. around 90%, this means a one stage extraction, can be expected.

Example 10: Membrane-Assisted Liquid-Liquid Extraction

[0152] A plant with seven PTFE hollow fibers with a total surface area of 0.00838 m.sup.2 was used for the membrane-assisted liquid-liquid extractions. The solvent was pumped with the centrifugal pump Iwaki MD-15RV-220N (Iwaki, JPN), the fermentation broth was pumped with a two channel peristaltic pump Verderflex EZi C (Verder GmbH u. Co. KG, DE). Ethyl acetate was used as a solvent and saturated with water prior to the extraction with the membrane-assisted liquid-liquid unit. The fermentation broth was equilibrated with ethyl acetate by adding defined amounts of said solvent. The unit was started by pumping the fermentation broth at the shell side of the unit. Subsequently, the solvent rich phase was pumped at the lumen side (inside the fibers). An overpressure of approximately 40 mbar on the water side was set. Samples of the feed and the solvent were taken at regular intervals. Table 3 shows two operating conditions of the two experiments V1 and V2 performed with membrane-assisted liquid-liquid extractor.

TABLE-US-00003 TABLE 3 Operating conditions of the two experiments V.sub.1 and V.sub.2 performed with membrane-assisted liquid-liquid extractor. V.sub.water/algal broth/ V.sub.solvent/ F.sub.water/algal broth/ F.sub.solvent/ c.sub.astaxanthin, startwater/mg mL V.sub.water/algal broth mL V.sub.solvent ml min.sup.−1 ml min.sup.−1 L.sup.−1 V.sub.1 867 Shell 650 Lumen 265 13.3 23.7 V.sub.2 867 Shell 640 Lumen 260 13.1 10.9

[0153] V1

[0154] In V1, an astaxanthin oleoresin from NATECO.sub.2 (Hopfenveredlung St. Johann GmbH, DE) was dissolved in water equilibrated with ethyl acetate, reaching a starting concentration of 31.7 mg L.sup.−1 of the astaxanthin oleoresin. The flow rate of the water enriched with the astaxanthin oleoresin was set to 265 mL min.sup.−1. The solvent was pumped with a flow rate of 13.3 mL min.sup.−1. The pressure difference was set to 40 mbar. FIG. 14 shows the astaxanthin oleoresin concentration in the solvent and water rich phase from the beginning of the extraction until a concentration of 31.7 mg L.sup.−1 of astaxanthin oleoresin was reached in the solvent after 1410 min.

[0155] V2

[0156] In V2, an algal broth with germinated algal cells was used for the extraction of astaxanthin. The algal broth was pumped with a flow rate of 260 mL min.sup.−1. The solvent was pumped with a flow rate of 13.1 mL min.sup.−1. The pressure difference was adjusted to 30 mbar. FIG. 15 shows the astaxanthin content of the algal broth and the solvent vs. the extraction time. The astaxanthin content from the algal broth decreased from a starting value of 10.9 mg to 0.32 mg after 165 min. In the same time period, the astaxanthin content in the solvent increased to 1.4 mg. Deposit of algal biomass in the death areas of the shell was observed.

REFERENCES

[0157] [1] Koller M, Muhr A, and Braunegg G. Microalgae as versatile cellular factories for valued products. Algal Research, 2014; 6, 52-63. [0158] [2] Shah M M R, Liang Y, Cheng J J, Daroch M. Astaxanthin-Producing Green Microalga Haematococcus pluvialis: From Single Cell to High Value Commercial Products. Frontiers in Plant Science. 2016; 7. [0159] [3] R. Praveenkumar, K. Lee, J. Lee and Y.-K. Oh, Breaking dormancy: an energy-efficient means of recovering astaxanthin from microalgae, Green Chemistry, 2014; 17, 1226-1234. [0160] [4] Marchal L, Mojaat-Guemir M, Foucault A and Pruvost J. Centrifugal partition extraction of β-carotene from Dunaliella salina for efficient and biocompatible recovery of metabolites, Bioresource Technology, 2013; 134, 396-400. [0161] [5] Xiping Du, Congcong Dong, Kai Wang, Zedong Jiang, Yanhong Chen, Yuanfan Yang, Feng Chen, Hui Ni. Separation and purification of astaxanthin from Phaffia rhodozyma by preparative high-speed counter-current chromatography. Journal of Chromatography B. 2016; 1029-1030, 191-197. [0162] [6] David R. Lide e. CRC Handbook of Chemistry and Physics, Internet Version 2005. 2005. [0163] [7] Taucher J, Baer S, Schwerna P, Hofmann D, Hümmer M, Buchholz R, et al. Cell Disruption and Pressurized Liquid Extraction of Carotenoids from Microalgae. Journal of Thermodynamics & Catalysis. 2016; 7(1):7.

[0164] The features of the present invention disclosed in the specification, the claims, and/or in the accompanying figures may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof.