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Chapter 11

Production, Extraction, and Quantification of Astaxanthin by Xanthophyllomyces dendrorhous or Haematococcus pluvialis: Standardized Techniques
Alma Rosa Domínguez-Bocanegra
Abstract
For many years, benefits and disadvantages of pigments production either by microalgae or yeasts have been under analysis. In this contribution we shall deal with Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) and Haematococcus pluvialis, which are known as major prominent microorganisms able to synthesize astaxanthin pigment. Then, the usual trend is to look for optimal conditions to conduct astaxanthin synthesis. From one side, pigment production by H. pluvialis is promoted under cellular stress conditions like nutrient deprivation, exposition to high light intensity, aeration. On the other side, X. dendrorhous is able to show significant increase in astaxanthin synthesis when grown in natural carbon sources like coconut milk, grape juice. The main aim of this chapter is to describe optimal environmental conditions for astaxanthin production by X. dendrorhous or H. pluvialis. Key words: Haematococcus pluvialis, Phaffia rhodozyma, Xanthophyllomyces dendrorhous, Astaxanthin synthesis, Coconut milk

1. Introduction
Astaxanthin is an abundant carotenoid pigment that can be synthesized by few microorganisms, among which are Brevibacterium, Mycobacterium lacticola (1), Agrobacterium auratim (2), Haematococcus pluvialis (3), and the yeast Xanthophyllomyces dendrorhous (4). Several marine species get the color by ingestion of suitable microorganisms (5–7). Due to its antioxidant properties, there is a growing economic interest in aquaculture, chemical, pharmaceutical, and alimentary industries (8, 9). The renewed interest in natural sources of the pigment is twofold: from one side synthetic versions of astaxanthin are rather expensive ($2,500– 3,000/kg) and on the other may induce human cancer (10).

José-Luis Barredo (ed.), Microbial Carotenoids From Fungi: Methods and Protocols, Methods in Molecular Biology, vol. 898, DOI 10.1007/978-1-61779-918-1_11, © Springer Science+Business Media New York 2012

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Moreover. YM: 10 g/L of dextrose (glucose). Domínguez-Bocanegra Microalgal astaxanthin purified from H. Let’s mention that carotenoid biosynthesis is regulated by singlet oxygen and peroxide radicals (7).1. H. sugar cane juice (17). pluvialis are detailed here. and 3 g/L of malt extract. attention is paid to culture conditions like aeration. . YPG: 20 g/L of dextrose (glucose). etc. alfalfa residual juice (19).172 A. 29). a high C/N ratio may enhance astaxanthin accumulation according to Kakizono et al. Culture Media and Reagents 1. IL. 2. astaxanthin production by H. light intensity (continuous or photoperiod). pluvialis NIES-144 (National Institute for Environmental Studies (NIES). pluvialis. 3 g/L of yeast extract. X. Viability of large scale production for commercial purposes seems to be a realistic issue (12–14). systematic shaking of the culture. Among the recommended media for cellular growth and pigment production. and light (13. pluvialis is enhanced by oxidative stress. 2. pluvialis presents extremely complex relationships between nutrients. 2. pluvialis is indeed a powerful antioxidant agent in vitro under both hydrophobic and hydrophilic conditions (11). pluvialis may induce changes in primary metabolism (28. dendrorhous NRRL-10921 (ARS culture collection. 3 g/L of yeast extract. Peoria. But also. namely X. 14). cell yield. Finally. Complementary descriptions can be found (23. Materials Materials and methods for both X. 1). as a secondary metabolite. and to produce carotenoids such as astaxanthin (15). 2. and 10 g/L of bacto-peptide. we can mention the following: cane molasses (16). H. and coconut milk (23). standardization of the extraction. corn wet-milling by-products (18). high astaxanthin accumulation in H. (31). 2). quantification. claiming that could remove free radicals (30). the main issue is the selection of culture media. Microorganisms 1. When dealing with X. hydrolyzed peat (21. pluvialis. USA) (Fig. cell type. and astaxanthin formation (24–27). dendrorhous and H. Japan) (Fig. grape juice (20). For instance. University of Tokyo. The aim of this chapter is to propose a systematic methodology to set experimental parameters which play a key role in astaxanthin synthesis by two prominent microorganisms. 22). growth. X. 5 g/L of bacto-peptide. United States Department of Agriculture USDA.R. dendrorhous. dendrorhous and H.2. 2. and production techniques for astaxanthin is proposed here. In the case of H. dendrorhous is able to ferment glucose and other sugars. 32).

8 × 10−3 g/L H3BO4 (see Note 2). X. Fig. 0.075 g/L MgSO4·7H2O. Coconut milk with this composition: 16 g/L of total carbohydrates.025 g/L NaCl. 0. 0.4 g/L K2HPO4. 0.001 g/L EDTA.0 g/L NaNO3. 0.6 × 10−5 g/L CuSO4·5H2O. and 2. 2. . 4.49 g/L of potassium.25 g/L of sodium. BBM: 1.6 × 10−3 g/L MnCl2·4H2O.7 × 10−3 g/L H3BO4 (see Note 2). BG-11: 1. 4. 0.006 g/L citric acid. and 1 g/L of calcium (see Note 1). 1. and Quantification of Astaxanthin… 173 Fig.8 × 10−5 g/L CuSO4·5H2O. 0. 1. 0. 3.075 g/L K2HPO4.036 g/L CaCl2·2H2O. 4. 0. H.2 g/L Na2CO3.005 g/L FeCl3·6H2O.075 g/L MgSO4·7H2O. 1. 0. 3. 2. and 5.1 × 10−5 g/L NaMoO4.5 g/L NaNO3. dendrorhous view under microscope Zeiss model Axios II Plus (augmentation ×40). pluvialis view under microscope Zeiss model Axios II Plus (augmentation ×40).2 × 10−4 g/L ZnSO4·7H2O.2 × 10−5 g/L NaMoO4.175 g/L KH2PO4.4 × 10−4 g/L ZnSO4·7H2O.11 Production. 0. 0. 2.1 g/L of magnesium.025 g/L CaCl2·2H2O. 2. 0. 0. Extraction. 0. 5.8 × 10−3 g/L MnCl2·4H2O.

7. High-performance liquid chromatography (HPLC). BAR: 0. and 1. Incubator. 6. Microscope. 4. The recommended inoculum appearance is shown in Fig. 3. Analytic balance. Methods 3. 2.0 g/L CH3COONa (see Note 2). 9. for 48 h. 0. 8. 0. 3. inoculate 1 mL of H. 0. 7. 2. .025 g/L NaCl. 11. Vortex.075 g/L MgSO4·7H2O. 10. MA. Inoculum Preparation of H.7 × 10−4 g/L CuSO4·5H2O. 0. Incubate the flask in an orbital shaker at controlled temperature of 22°C and stirring speed of 150 rpm.2.175 g/L KH2PO4.174 A. place aseptically the cell suspension in a 250-mL baffled Erlenmeyer flask containing 50 mL of YM medium.3 × 10−4 g/L MnCl2·4H2O.3 × 10−4 g/L CoCl3. Incubate at 22°C and 150 rpm for 48 h (see Note 3).3. USA). Dinitrosalicylic (DNS) acid reagent (34). 2. 4. dendrorhous 1. NaCl 20% w/v. 7. Orbital shaker. Centrifuge. 9. Switzerland). 7. Methanol: water: hexane (95:4:1 v/v/v). 10. Within test tubes.1. 12. 2. 4. Glass pearls (425–600 μm diameter). 5.075 g/L K2HPO4. Solvent A: 100:70:70 of hexane: acetone: ethyl alcohol. Convey the obtained cellular growth in a second 250-mL baffled Erlenmeyer flask keeping a relation of 10% volume of seed culture.025 g/L CaCl2·2H2O. Inoculum Preparation of X.25 g/L NaNO3.4 × 10−3 g/L ZnSO4·7H2O. 3. 0. 3. 8. Equipment 1. Billerica.5 × 10−4 g/L NaMoO4. 11. 3. Astaxanthin standard (Hoffmann-La Roche. 3. 2.025 g/L EDTA. pluvialis type strain in a total volume of 10 mL of BG-11. Domínguez-Bocanegra 6. Basel. Spectrophotometer. 0. Maintain the cultures with continuous light (177 μmol photon/m2s) for 120 h at room temperature (28°C). pluvialis 1. Neubauer haemocytometer.0025 g/L FeCl3·6H2O. From a storage tube. 0.R. 0. Artificial lighting equipment.45-μm Filter (Millipore.

4. dendrorhous standardized inoculum and astaxanthin synthesis. 5. H. X.500 × g. Incubate at 28°C. 2. 6. Extraction. 4. 3. . Convey the cultures to 250-mL Erlenmeyer flasks with 100 mL of BG-11 medium. Keep the same conditions of continuous lighting (177 μmol photon/m2s) and temperature (28°C) for 120 h. Fig. and continuous illumination (177 mol photons/m2s) for 120 h (see Note 4). taking care of stirring by hand once a day for 10 s. 3.45-μm pre-weight filter. and Quantification of Astaxanthin… 175 Fig. room temperature (28°C). wash it twice with 2 mL of distilled water and filter it through a 0.3. pluvialis standardized inoculum. The recommended inoculum appearance is shown in Fig. dendrorhous 1. Centrifuge samples of 2 mL from the cultured medium for 5 min at 3. 3.5 vvm. Inoculate again with the seed culture at 10% v/v in an Erlenmeyer flask of 250 mL with 100 mL of BG-11.11 Production. 4. Dry Weight Analysis of X. Suspend again the cell pellet. aeration of 1.

Then. 1 and 2. Dry Weight Analysis of H. Domínguez-Bocanegra 3. dendrorhous. Then.6.000 × g in a clinical centrifuge. 3. Perform this analysis every 24 h. dendrorhous and H. 2. 3. pluvialis) in a Neubauer haemocytometer. 2. Heat the cell package at 80% in a suspension. after which 8 mL of distillated water is added. pluvialis 1. Measure optical density (OD) in a spectrophotometer: OD650 for H. as long as the experiment requires. 90:10 (v/v) methanol:water. 4.8. Quantify and register the dry weight on a chosen analytic balance. pluvialis To determine chlorophyll by the APHA technique (33) proceed as follows: 1. Cellular Number of X. 3. Chlorophyll Content Determination H.000 × g for 5 min. dendrorhous The DNS method (34) is used to determine residual reducing sugars in the culture media. 2. Suspend again the cell pellet. Filter the cellular package through a 0. Incubate a 1 mL sample and centrifuged at 3. 4. leaving them to dry for 24 h at 60°C. Perform previous operation two times more. 3. pluvialis With a Pasteur pipette. supply 1 mL of DNS reagent to the supernatant. 3. Quantify and register the dry weight on a chosen analytic balance. as follows: 1. Sugar Content Evaluation X. inoculate a sample of the culture (either X.176 A. as long as the growth kinetics and pigment production processes are carried out. Determine OD665 in a spectrophotometer. pluvialis and OD470 for X. place them immediately in an ice bath for rapid cooling.4. Optical Density of X. dendrorhous and H. Cover the tubes and heat them up to boiling point for 5 min. typically 120 h for X. 3. dendrorhous and 168 h for H. pluvialis. Centrifuge samples of 5 mL from the culture medium for 5 min at 3. 3.000 × g for 5 min.5. dendrorhous or H. 1. pluvialis 3. wash it with 3 mL of distilled water and centrifuge again under the same conditions. Take a sample of 5 mL culture. 3.7. perform the cellular counting every 24 h with a microscope. for 3 min in the dark and centrifuge once more at 3. .R.45-μm pre-weight filter. Microorganisms’ cellular shapes are shown in Figs.500 × g for 5 min. Incubate triplicates of 5 mL samples from three different Erlenmeyer flasks and centrifuge the samples at 3. Dry the filters containing the biomass at 60°C for 24 h. 2.

pluvialis Here pigments are determined using a modification of the Britton procedure (37). to separate the petroleum ether containing the colorant. Different kinetic states of X.500 × g. Now. 4. wash the cell pellets twice with 1 mL of distilled water and 1 mL of acetone. Pigment Extraction from H. 7. 5. OD575 measurement. In order to extract the colorant from its organic phase. 1 mL of acetone. Add 1 mL of glass pearls (425–600 μm diameter) together with 1 mL of dimethylsufoxide (DMSO) at 60°C. 2. Centrifuge at 3. quantify astaxanthin with a commercial standard using the HPLC method (36). 1 mL of petroleum ether. for 5 min.10.11 Production. subsequently. for 5 min. 3. Add. Check the presence of carotenoids by OD474 using an extinction coefficient of 1% = 2. Inoculate triplicates with 5 mL samples from three different Erlenmeyer flasks and centrifuge the samples at 900 × g for 5 min. and Quantification of Astaxanthin… 177 4. .9. 8. Then. Shake the samples with a vortex for 5 min.500 × g triplicate samples of 1 mL aliquots of the saturated culture from three different Erlenmeyer flasks. Pigment Extraction from X. dendrorhous 1. 3. until solvent evaporates. Centrifuge the samples during 5 min at 3. To calibrate the spectrophotometer use a standard reference curve. 6.5 mL/min. 5. break the cells by vigorous shaking using a vortex.100 (35). and 1 mL of NaCl at 20% w/v. The recommended culture appearance is shown in Fig. The eluting solvent is methanol: water: hexane (95: 4: 1 v/v/v) with a flow rate set at 0. 3. dendrorhous in astaxanthin production within coconut milk. Extraction. 5. Fig. 5. 1.

178 A. 6. Growth Kinetics of H.2 and Note 4).5 for 5 days. Wash the cell pellet twice with 5 mL of distilled water and add the same volume of glass pearls together with 5 mL of solvent A (100:70:70 of hexane: acetone: ethyl alcohol). dendrorhous The optimal conditions for cellular production of X. 6. dendrorhous are as follows: 1. 3.R. 3. Place samples of 5 mL from standardized inoculum in 250-mL baffled Erlenmeyer flasks containing 50 mL of coconut milk. pluvialis are as follows: 1. or YM medium (see Subheading 3. . From cellular growing in medium BBM to pigment cyst formation in BAR medium. YPG. Parallel experiments must be run by triplicate. 2. Monitor the presence of carotenoids by OD474 using an extinction coefficient of 1% = 2. Place samples of 70 mL from standardized inoculum in 1. and pH = 4.600 for astaxanthin (35). Growth Kinetics of X. 5.12. Break the cells by vigorous shaking using a vortex in order to extract the colorant from the organic phase. Ensure a homogeneous mixing using a shaker at 150 rpm. H. pluvialis kinetics. Domínguez-Bocanegra Fig.1 and Note 3). The recommended culture appearance is shown in Fig. 4. 2.100 for the total carotenoids and 1% = 1. pluvialis The optimal conditions for cellular production of H.11. quantify astaxanthin using a commercial standard by the HPLC method (36). 3. 22°C. Finally.000mL Erlenmeyer flasks containing 700 mL of BBM or BG-11 media (see Subheading 3.

suitable for astaxanthin production in different media (YM. 2. 4. Usually. Raw residual coconut milk was obtained from a local candy industry located in Mexico City. The key difference is the use of coconut milk as a carbon source without any additional nutrient (see Note 5). The transfer of H.11 Production. Figure 5 shows the desired culture pigmentation. Extraction. and Quantification of Astaxanthin… 179 2.12. dendrorhous The pigment production goes together with the growth of the yeast. Pigment Production of X. BG-11 (39). 2. previously fitted with 700 mL of BAR medium. It was sterilized by filtration and no nutrient was added.14. Pigment Production of H.000-mL Erlenmeyer flask. FAB is as BBM medium but without element trace. or coconut milk). aeration. 1. and light intensity. continuous illumination or 12 h light/12 h dark cycle (177 μmol photon/m2s). to inoculate 20% v/v of the seed culture optimally grown according to Subheading 3. Maintain the following culture conditions: Non-aeration. 3. continuous illumination at 345 μmol photon/m2s. This step is mandatory. Figure 6 shows the desired culture pigmentation. pluvialis 4. Normally. maximal cell growth is reached between 7 and 8 days. constant room temperature (28°C). dendrorhous in a second 250-mL baffled Erlenmeyer flask at 10% volume for 48 h is critical to ensure recommended exponential phase growth conditions. The conditions are then exactly the same as described in Subheading 3.5% CO2. The transfer of X. and manual agitation every 24 h. the maximal pigment figures are obtained between 168 and 192 h. Figure 3 shows the standardized inoculum.11. It is worth noticing that conditions for cellular growth are completely different for pigment production. 3. this second treatment is mandatory to get high pigment production (see Note 6). pluvialis seed in a second 250-mL baffled Erlenmeyer flask at 10% volume for 120 h is critical to ensure . Take a 1. and BAR (40). 3. optimal pigment production is reached after 120 h. daily manual agitation. YPG. Ensure regulated temperature at 28°C. In this sense. The media reported here are widely used and may be found as follows: BBM (38). aeration of 1. Usually. in terms of the media.13. Notes 1.

the absence of aeration. Torres-Muñoz for the fruitful discussions along the preparation of the manuscript. This step is mandatory as far as further microorganism growth is very sensitive initial conditions of inoculation. grape juice. the formation of aplanospores with high contents of astaxanthin (42–48). i. Schroeder WA. Jorge A.R. Such factors corrupt the cell cycle and therefore limit its growth and photosynthetic process. J Appl Bacteriol 70:181–191 2. such stress growing conditions enhance the process of cyst formation. pp 119–178 . Figure 4 shows the standardized inoculum. promote both the cellular growth and the synthesis of astaxanthin in different X. and the use of sodium acetate as a source of inorganic carbon in the BAR medium. Indeed. dendrorhous yeasts. Schroeder WA (1995) Microbial carotenoids. Lotan T.e. Gu WL. or coconut milk. J Ind Microbiol 14:502–507 7. Andrews AG. J Ind Microbiol Biotechnol 19:114–117 6. I would also like to thank Dr. vol 53. Miki W (1995) Composition and presumed biosynthetic pathway of carotenoids in the astaxanthin producing bacterium Agrobacterium aurantiacum. Hirschberg J (1995) Cloning and expression in Escherichia coli of the gene encoding β-C-4-oxygenates. Berlin. Acknowledgments The author expresses her gratitude to TESE for the experimental facilities and to CINVESTAV for giving me a non-salary work permit along the development of this research work. Neils HJ. Optimal cellular growth versus pigment production in H. that converts β-carotene to the ketocarotenoid canthaxanthin in Haematococcus pluvialis. References 1. Johnson EA (1995) Carotenoids protect Phaffia rhodozyma against singlet oxygen damage. An GH. The very rich nutrient composition of coconut milk avoids the need of additional nutritional compounds added to the media. FEMS Microbiol Lett 28:139–144 3. Domínguez-Bocanegra recommended exponential phase growth conditions. Phaff HJ.. Johnson EA. dendrorhous: Natural carbon sources present in cane sugar molasses. In: Fiechter A (ed) Advances biochemical engineering and biotechnology. pluvialis: Optimal factors in astaxanthin production are exposure to a high light intensity (345 μmol photon/m2s). Starr MP (1976) Carotenoids of Phaf fi a rhodozyma red pigmented fermenting yeast. Phytochemistry 15:10003–10007 5. Optimal cellular growth versus pigment production in X. Leenheer AP (1991) Microbial sources of carotenoid pigments uses in foods and feeds. Yokayama T. Further improvements of astaxanthin production may be obtained with the use of mutated version of the yeast (41). FEBS Lett 364:125–128 4. Springer. 5. 6.180 A. Johnson EA (1997) Ethanol increases carotenoid productin in Phaffia rhodozyma.

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