Journal of Applied Phycology 9: 445450, 1997.

c 1997 Kluwer Academic Publishers. Printed in Belgium.

445

Heterotrophic production of lutein by selected Chlorella strains

Xian-Ming Shi, Feng Chen , Jian-Ping Yuan & Hui Chen
Department of Botany, the University of Hong Kong, Pokfulam Road, Hong Kong ( Author for correspondence; phone: 852-28591945; fax: 852-28583477; email: sfchen@hkusua.hku.hk)
Received 7 May 1997; revised 12 September 1997; accepted 14 September 1997

Key words: Chlorella protothecoides, heterotrophic cultivation, biomass, lutein

Abstract Seven Chlorella strains representing three species obtained from culture collections and research laboratories were screened for their potential of heterotrophic production of lutein on two different media (Basal and Kuhl) containing glucose. While both media supported good growth and lutein formation of the seven strains in darkness, higher biomass concentrations and lutein content were achieved on Basal medium. Chlorella protothecoides CS-41 was chosen from the seven strains for further investigation due to its higher productivities of both biomass and lutein. The maximal biomass concentration and lutein content of C. protothecoides cultivated heterotrophically with 9 g L 1 glucose in a 3.7-L fermentor were respectively 4.6 g dry cells L 1 and 4.60 mg lutein g 1 dry cells on Basal medium, and 4.0 g dry cells L 1 and 4.36 mg lutein g 1 dry cells on Kuhl medium. The heterotrophic cultivation process was scaled up successfully to 30 L using a fermentor, in which the Basal medium containing 36 g L 1 glucose was used; the maximal biomass concentration of 16.4 g dry cells L 1 , specic growth rate of 0.92 d 1 , lutein content of 4.85 mg lutein g 1 dry cells, growth yield of 0.47 g dry cells g 1 glucose and lutein yield of 1.93 mg lutein g 1 glucose were respectively achieved. Introduction Microalgae have long been recognised as an efcient biological system to harvest solar energy for producing biomass and a great variety of metabolites (Myers & Clarck, 1944; Borowitzka, 1994). More recently, signicant attention has been drawn to the use of microalgae as a source of feedstuff for animals due to the accumulation of high-value nutrients in the cells (Chaumont, 1993), such as astaxanthin (Kobayashi et al., 1991) and xanthophylls (Hilaly et al., 1994). Lutein is not only one of the most prominent carotenoids in human serum and foods (Bieri et al., 1985; Khachik et al., 1986), but also the representative of  , -carotenoids (Craft & Soares, 1992). Lutein has been used for the pigmentation of animal tissues and products, as well as for the coloration of foods, drugs and cosmetics (Klaui, 1982). In the USA, two lutein-containing products, Aztec Marigold and Tagetes have recently been commercialized (Rayner, 1993). Microalgae are one of the major sources of naturally occurring lutein (Goodwin, 1980). To date, most industrial microalgal processes have been based on the open pond technology (Borowitzka, 1994). The inability to maintain pure culture is a major unsolved problem in large-scale open systems (Richmond & Vonshak, 1991). There is also little scope for controlling the culture conditions so as to optimize cell and product yields (Borowitzka, 1994). For this reason, an achievable cell concentration is usually less than 1 g dry cells L 1 , which means that the downstream processing costs are high (Borowitzka, 1994). Although the development of closed photobioreactors, especially a tubular photobioreactor, has been able to overcome some of the problems encountered in pond systems (Richmond, 1987; Richmond & Vonshak, 1991; Lee & Low, 1992; Borowitzka, 1994), other problems associated with the operation of photobioreactors remain, including reduction in light penetration (Chaumont, 1993), the difculty in controlling

446 oxygen concentrations (Weissman et al., 1988), and overheating in summer (Torzillo et al., 1986). An alternative to overcome or minimize the problems associated with the photoautotrophic strategies is to develop heterotrophic culture technology which takes advantage of the extra capacity of the existing fermentation industry. The theoretical possibility has been demonstrated for certain microalgae including Chlorella to grow under heterotrophic conditions (Theriault, 1965; Endo et al., 1977; Chen & Johns, 1991; Lalibert & de la No e, 1993; Barclay et al., 1994; Running e u et al., 1994; Day & Tsavalos, 1996). The advantages and disadvantages of heterotrophic growth of microalgae have recently been reviewed (Chen, 1996). Only limited information is available about the heterotrophic production of lutein by microalgae. In this work, two most widely used media, Basal and Kuhl for Chlorella were employed (Sorokin & Krauss, 1958; Kuhl, 1962; Theriault, 1965; Chen & Johns, 1991; Gouveia et al., 1996; Tam & Wong, 1996). The Basal medium contains Co, which is absent in the Kuhl medium. A survey of literature indicates that Co may not be essential to most Chlorella species (Oh-hama & Miyachi, 1988; Chen & Johns, 1991; Javanmardian & Palsson, 1991; Lee & Palsson, 1995). A high cell dry weight concentration of 30 g L 1 was achieved for Chlorella vulgaris in N-8 medium without Co (Javanmardian & Palsson, 1991). The aim of this study was to screen Chlorella strains for heterotrophic production of lutein, and to develop a high rate production system.
Table 1. Chlorella strains and their sources. Name and Catalogue No. Chlorella pyrenoidosa 15-2070 Chlorella pyrenoidosa 15-2071 Chlorella pyrenoidosa HKU-003 Chlorella vulgaris HKU-004 Chlorella vulgaris 15-2075 Chlorella protothecoides CS-41 Chlorella vulgaris CS-42 Strain code 1 2 3 4 5 6 7 Sourcesa Carolina Carolina HKU HKU Carolina CSIRO CSIRO

a Carolina = Carolina Biological Supply Co., Burlington, USA. HKU = Department of Botany, the University of Hong Kong. CSIRO = CSIRO Marine Laboratory, Hobart, Australia.
Table 2. Composition of Basal and Kuhl media. Ingredients glucose KNO3 KH2 PO4 NaH2 PO4 H2 O Na2 HPO4 2H2 O MgSO4 7H2 O EDTA H3 BO3 CaCl2 2H2 O FeSO4 7H2 O ZnSO4 7H2 O MnCl2 4H2 O MnSO4 H2 O MoO3 (NH4 )6 Mo7 O24 4H2 O CuSO4 5H2 O Co(NO3 )2 6H2 O pH Basal (mg L 9000 1250 1250
1)

Kuhl (mg L 9000 1011.1 621 89 246.5 9.3 0.061 14.7 6.95 0.287 0.169

1)

1000 500 114.2 111 49.8 88.2 14.2 7.1 15.7 4.9 6.1

0.01235 0.00249 6.1

Materials and methods The strains used and their sources are summarized in Table 1. The compositions of the two media Basal (Sorokin & Krauss, 1958) and Kuhl (Kuhl, 1962) used for the cultivation in both asks and 3.7-L laboratory fermentors are listed in Table 2. For 30-L fermentor cultivation, 36 g L 1 glucose and 7 g L 1 KNO3 were used in the medium. Sterilized medium (121  C, 15 min) was inoculated with 5% exponentially growing inocula. Heterotrophic cultivation of the 7 Chlorella strains was initially carried out in a 250-mL Erlenmeyer ask containing 100 mL medium at 26  1  C under continuous shaking (180 rpm) in the dark. Heterotrophic cultivation of the selected strain, C. protothecoides (CS-41), was carried out using a 3.7-L fermentor (Bioengineering

AG, Wald, Switzerland) and the scale-up experiment was performed using a 30-L fermentor of the same type (Bioengineering AG). The cultivation conditions in the fermentors were computer controlled as follows: pH, 6.1  0.1; temperature, 26  C; agitation, 480 rpm; D.O., 50% saturation. The dry cell weight concentration was determined according to Chen et al. (1996). A DX 500 HPLC system (Dionex Co. Sunnyvale, California, USA) was used to determine glucose in the culture uids. The system comprised an ED 40 electrochemical detector, a GP 40 gradient pump and an LC 20 chromatography enclosure, equipped with a 4  250 mm CarboPacTM PA1 column (Dionex) and an LC30 stainless steel automated Rheodyne injection

447 valve with a 25 L xed loop. PeakNet software and a DX LANTM computer interface card (Dionex Co.) were used for the system. The mobile phase consisted of 200 mM NaOH-distilled water (8:92, v/v). The ow rate was set at 2.0 mL min 1 . The column was kept at room temperature (2225  C). The samples were ltered through a 0.22 m lter (Millipore) before injection. Standard glucose (Sigma Chemical Co.) was used for the identication and quantication. Nitrate concentration was determined by a Beckman HPLC system, equipped with a 125 Solvent Module pump and 166 UV-VIS detector (System Gold, Beckman Instruments, Inc.). The samples were injected through a Rheodyne 7725i valve with a 20 L loop after they were ltered through a 0.45 m lter, and then separated at room temperature (2225  C) by a 250  4.6 mm Vydac 302 IC column (Beckman), packed with a low capacity anion exchange support carrying a quaternary ammonium phase bonded to spherical silica. The eluent solution (mobile phase) consisted of 0.5 g L 1 phthalic acid and 0.5 g L 1 sodium tetraborate aqueous solutions. The ow rate was set at 2.0 mL min 1 . Chromatographic peaks were identied by comparing retention times against the standard. The anion was detected by indirect ultraviolet absorption detection at 290 nm. All chemicals were purchased from Sigma Chemical Co. The amount of nitrate was expressed in the form of KNO3 . Lutein concentration was determined according to the method described by Shi and Chen (1997). Specic growth rate,  was determined according to Chen and Johns (1996).

Figure 1. Maximum biomass production by seven Chlorella strains in shake asks under heterotrophic conditions.

Figure 2. Maximum lutein content in dry cells of seven Chlorella strains in shake asks under heterotrophic conditions.

Results Seven Chlorella strains were tested for their ability to produce lutein heterotrophically on both Basal and Kuhl media containing glucose in ask culture. Both media supported good growth (Figure 1) and lutein formation (Figure 2) in all seven strains in darkness. Strain 5 (C. vulgaris 15-2075) and strain 6 (C. protothecoides CS-41) produced the highest biomass (4.7 g dry cells L 1 ) on Basal medium, while only strain 6 gave the highest biomass (4.4 g dry cells L 1 ) on Kuhl medium. The biomass concentrations of strain 2 (C. pyrenoidosa 15-2071) and 3 (C. pyrenoidosa HKU-003) were the lowest on both media (Figure 1). Strain 6 had the highest lutein content, 4.48 and 4.33 mg g 1 dry cells on Basal and Kuhl media, respectively, whereas, strain 2 had the lowest content of lutein (1.741.82 mg

g 1 ) on both media. Similar results were also obtained from mixotrophic cultivation of the seven Chlorella strains (data not shown). For the seven strains tested, Basal medium was superior to Kuhl medium in terms of biomass concentration and lutein content, although differences were very small (Figures 1, 2). Since C. protothecoides CS-41 (strain 6) had the highest biomass concentration and lutein content, this strain was chosen for further investigation using a 3.7-L fermentor under controlled culture conditions. As shown in Figure 3A, the maximum biomass concentration (4.6 g dry cells L 1 ) on Basal medium in the 3.7-L fermentor was obtained at the third day, and the maximum lutein content (4.60 mg g 1 dry cells) and yield (15.95 mg L 1 ) were achieved at the 8th and 6th day, respectively. The cell growth yield and lutein yield on glucose were 0.48 g g 1 and 1.73 mg g 1 , respectively (Table 3). Figure 3B shows that the max-

448

Figure 4. Heterotrophic growth of C. protothecoides on Basal medium and its consumption of C- and N-sources in a 30-L fermentor.

Figure 3. Heterotrophic production of biomass and lutein by C. protothecoides on the two different media in a 3.7-L fermentor. A: Basal. B: Kuhl.

imum biomass concentration (4.0 g dry cells L 1 ) on Kuhl medium was achieved on the 5th day, 2 days later than on Basal medium. Maxima of both lutein content (4.36 mg g 1 dry cells) and lutein yield (14.82 mg L 1 ) were obtained at the 8th day (Figure 3B). The consumption of carbon and nitrogen sources by C. protothecoides responded well to the algal growth. It was found that NO3 in both culture broths was completely consumed by the third day, while glucose was also exhausted, respectively, at the third day in Basal culture broth and at the fourth day in Kuhl culture broth (data not shown). Like the ask cultures, the concentrations of both biomass and lutein on Kuhl were lower than those on Basal in fermentor cultures. These results further conrmed that Basal medium was more suitable for C. protothecoides CS-41. The Basal medium was thus selected for the scale-up experiment. Figure 4 shows the algal growth and its corresponding consumption of C- and N-sources in a 30-L fermentor. The highest biomass concentration (16.4 g L 1 ) was obtained at 136 h, when nitrate was total-

Figure 5. Heterotrophic production of lutein by C. protothecoides on Basal medium in a 30-L fermentor.

ly consumed though 1.1 g L 1 glucose in the medium remained, which indicated that nitrate was the limiting factor. As shown in Figure 5, the highest lutein content (4.85 mg g 1 dry cells) and yield (66.31 mg L 1 ) appeared at a much later stage (262 h and 190 h, respectively) than that for the maximum biomass concentration (Figure 4). These results were in agreement with those obtained from the 3.7-L fermentor (Figures 3). The specic growth rate of C. protothecoides CS-41 in the 30-L fermentor was 0.92 d 1 and the cell growth yield and the lutein yield on glucose were 0.47 g g 1 and 1.93 mg g 1 , respectively (Table 3). The former was comparable to that observed in the 3.7-L fermentor, while the latter was higher (Table 3).

449
Table 3. Heterotrophic growth and lutein production of C. protothecoides in fermentorsa . Fermentor size(L) Medium Maximal biomass Dry wt. Time (g L 1 ) appeared 3.7 3.7 30 Basal Kuhl Basal 4.6 4.0 16.4 3d 5d 136 h Maximal lutein in dry cells Content Time (mg g 1 ) appeared 4.6 4.4 4.9 8d 8d 262 h Maximal lutein in uids Yield (mg L 16.0 14.8 66.3
1)


(d
1)

Yg (g g

Yltn (mg g

Time appeared 6d 8d 190 h ND ND 0.92

glucose)

glucose)

0.48 0.42 0.47

1.73 1.62 1.93

a ND = not determined.  = specic growth rate. Yg = cell growth yield on glucose. Yltn = lutein yield on glucose.

Discussion In this study, both Basal and Kuhl media supported good growth of C. protothecoides CS-41, but higher concentrations of biomass and lutein were obtained on Basal medium. The Basal medium contained a much higher concentration of EDTA (500 mg L 1 compared to 9.3 mg L 1 ) and higher concentrations of minerals than the latter (Table 2). EDTA is the most commonly used chelating agent in media, which plays an important role in stablizing the sufcient supply of trace metal elements, and in the prevention of inhibitory effects of some metals (Oh-hama & Miyachi, 1988). Although Chlorella is popular in the eld of microalgal biotechnology, there have been no reports of either signicant breakthroughs in its large scale production or new application and potential uses in the last decade (Vonshak, 1990). The development of microalgal biotechnology depends in particular on the identication of more high-value products from algal cells and the improvement of cultivation systems (Chaumont, 1993). It was found in this study that C. protothecoides CS-41 could produce comparatively high concentrations of lutein heterotrophically, which has not been reported to our knowledge. Samuel and Bose (1993) investigated the effect of SANDOZ 9789 on growth and pigment (including lutein) content of C. protothecoides CS-41, but the alga was cultivated under photoautotrophic conditions. Wu et al. (1993) compared liposoluble compounds in photoautotrophic and heterotrophic cultures of C. protothecoides CS-41, but no individual carotenoids were separated. C. pyrenoidosa was investigated for the production of xanthophylls (containing lutein) by Theriault (1965). Our results demonstrate that C. protothecoides CS-41 was a better alga than C. pyrenoidosa. This was reected by the

higher growth yield (0.47 g g 1 compared to 0.36 g g 1 ) and higher lutein content (4.85 mg lutein g 1 dry cells compared to 4.67 mg xanthophylls g 1 dry cells) achieved in the present study. Research in process optimization and strain improvement is continuing in our laboratory to further enhance the lutein productivity in heterotrophic cultures.

Acknowledgements This research was supported by the Research Grant Council of Hong Kong and the University of Hong Kong Committee on Research and Conference Grants. The authors wish to thank Dr L. Ramsden for his assistance in the use of the HPLC system for the determination of glucose concentrations.

References
Barclay WR, Meager KM, Abril JR (1994) Heterotrophic production of long chain omega-3 fatty acids utilizing algae and algae-like microorganisms. J. appl. Phycol. 6: 123129. Bieri JG, Brown ED, Smith JC (1985) Determination of individual carotenoids in human plasma by high performance liquid chromatography. J. liq. Chromatogr. 8: 473484. Borowitzka MA (1994) Large-scale algal culture systems: the next generation. Australasian Biotechnol. 4: 212215. Chaumont D (1993) Biotechnology of algal biomass production: a review of systems for outdoor mass culture. J. appl. Phycol. 5: 593604. Chen F (1996) High cell density culture of microalgae in heterotrophic growth. Trends Biotechnol. 14: 421426. Chen F, Johns MR (1991) Effect of C/N ratio and aeration on the fatty acid composition of heterotrophic Chlorella sorokiniana. J. appl. Phycol. 3: 203209. Chen F, Johns MR (1996) Relationship between substrate inhibition and maintenance energy of Chlamydomonas reinhardtii in heterotrophic culture. J. appl. Phycol. 8: 1519.

450
Chen F, Zhang YM, Guo SY (1996) Growth and phycocyanin formation of Spirulina platensis in photoheterotrophic culture. Biotechnol. Lett. 18: 603608. Craft NE, Soares JH (1992) Relative solubility, stability, and absorptivity of lutein and  -carotene in organic solvents. J. agric. Food Chem. 40: 431434. Day JG, Tsavalos AJ (1996) An investigation of the heterotrophic culture of the green alga Tetraselmis. J. appl. Phycol. 8: 7377. Endo H, Hosoya H, Koibuchi T (1977) Growth yield of Chlorella regularis in dark-heterotrophic continuous culture using acetate. J. Ferment. Technol. 55: 369379. Goodwin TW (1980) The Biochemistry of the Carotenoids, 2nd edn. Vol. I, Plants. Chapman and Hall, London. Gouveia L, Veloso V, Reis A, Fernandes H, Novais J, Empis J (1996) Evolution of pigment composition in Chlorella vulgaris. Biores. Technol. 57: 157163. Hilaly AK, Karim MN, Guyre D (1994) Optimization of an industrial microalgal fermentation. Biotechnol. Bioeng. 43: 314320. Javanmardian M, Palsson BO (1991) High-density photoautotrophic algal cultures: design, construction, and operation of a novel photobioreactor system. Biotechnol. Bioeng. 38: 11821189. Khachik F, Beecher GR, Whittaker NF (1986) Separation, identication, and quantication of the major carotenoid and chlorophyll constituents in extracts of several green vegetables by liquid chromatography. J. agric. Food Chem. 34: 603616. Klaui H (1982) Industrial and commercial uses of carotenoids. In Britton G, Goodwin TW (eds), Carotenoid Chemistry and Biochemistry. Pergamon Press, Oxford: 309328. Kobayashi M, Kakizono T, Nagai S (1991) Astaxathin production by a green alga, Haematococcus pluvialis accompanied with morphological changes in acetate media. J. Ferment. Bioeng 71: 335 339. Kuhl A (1962) Zur Physiologie der Speicherung kondersierter anorganischer Phosphate in Chlorella. Vortr. Botan. hrsg. Deut. Botan. Ges. (N.F.) 1: 157166. Lalibert G, de la No e J (1993) Auto-, hetero-, and mixotrophic e u growth of Chlamydomonas humicola (Chlorophyceae) on acetate. J. Phycol. 29: 612620. Lee CG, Palsson BO (1995) Continuous medium perfusion leads to long-term cell viability and oxygen production in high-density photobioreactors. Biotechnol. Letts 17: 11491154. Lee YK, Low CS (1992) Productivity of outdoor algal cultures in enclosed tubular photobioreactor. Biotechnol. Bioeng. 40: 1119 1122. Myers L, Clarck LB (1944) Culture conditions and the development of the photosynthetic mechanism. II. An apparatus for the continuous culture of Chlorella. J. gen. Physiol. 28: 103112. Oh-hama T, Miyachi S (1988) Chlorella. In Borowitzka MA, Borowitzka LJ (eds), Microalgal Biotechnology. Cambridge University Press. Cambridge: 326. Rayner PB (1993) Food & drink colors from natural sources. Food Marketing & Technol. International 7: 910. Richmond A (1987) The challenge confronting industrial microalgal culture: high photosynthetic efciency in large-scale reactors. Hydrobiologia 151/152: 117121. Richmond A, Vonshak A (1991) Preface of the special issue of Biores. Technol. 38: 8384. Running JA, Huss RJ, Olson PT (1994) Heterotrophic production of ascorbic acid by microalgae. J. appl. Phycol. 6: 99104. Samuel K, Bose S (1993) Effects of SANDOZ 9789 on growth and pigment content of Chlorella protothecoides grown under two different light intensities. J. envir. Biol. 14: 231241. Shi XM, Chen F (1997) Stability of lutein under various storage conditions. Nahrung/Food 41: 3841. Sorokin C, Krauss RW (1958) The effect of light intensity on the growth rates of green algae. Plant Physiol. 33: 109113. Tam NFY, Wong YS (1996) Effect of ammonia concentrations on growth of Chlorella vulgaris and nitrogen removal from media. Biores. Technol. 57: 4550. Theriault RJ (1965) Heterotrophic growth and production of xanthophylls by Chlorella pyrenoidosa. Appl. Microbiol. 13: 402416. Torzillo G, Pushparaj B, Bocci F, Balloni W, Materassi R, Florenzano G (1986) Production of Spirulina biomass in closed photobioreactors. Biomass 11: 61-74. Vonshak A (1990) Recent advances in microalgal biotechnology. Biotech. Adv. 8: 709727. Weissman JC, Goebel RP, Benemann JR (1988) Photobioreactor design: mixing, carbon utilization and oxygen accumulation. Biotechnol. Bioengng 31: 336344. Wu QY, Sheng GY, Fu JM (1993) Comparative study on liposoluble compounds in autotrophic and heterotrophic Chlorella protothecoides. Acta Bot. Sinica 35: 849858.