Journal of Biotechnology 115 (2005) 81���90 Production of Dunaliella salina biomass rich in 9-cis- -carotene and lutein in a closed tubular photobioreactor Mercedes Garc��a-Gonzaleza, �� �� Jose �� Morenoa, J. Carlos Manzanob, F. Javier Florencioa, Miguel G. Guerreroa,��� a Instituto de Bioqu�� ��mica Vegetal y Fotos�� ��ntesis, Consejo Superior de Investigaciones Cient�� ��ficas-Universidad de Sevilla, Centro de Investigaciones Cient�� ��ficas Isla de la Cartuja, Avda. Am�� erico Vespucio, s/n, 41092 Sevilla, Spain b Consejer�� ��a de Agricultura y Pesca. Junta de Andaluc�� ��a, C/Juan de Lara Nieto s/n, 41013 Sevilla, Spain Received 8 March 2004 received in revised form 20 July 2004 accepted 30 July 2004 Abstract Performance of Dunaliella salina cultures outdoors in a closed tubular photobioreactor has been assessed. Optimization of conditions involved verification of the effect of several determining factors on the yield of both biomass and carotenoids. Maximal biomass productivity (over 2 g (dry weight) m���2 d���1 or 80 g m���3 d���1) was achieved at 38 cm s���1, flow rate 2 �� 109 cells l���1, initial population density 25 ���C, temperature semi-continuous regime, keeping a cell density interval between 2 �� 109 and over 4 �� 109 cells l���1. Coverage of the tubular loop with a sunshade screen to avoid light-induced damage of cells was essential to maintain growth performance. The cellular -carotene level increased significantly during the light period, as also did that of lutein. The rise in the -carotene level could be accounted by the 9-cis-isomer, with all-trans- -carotene remaining steady during the light period. By sunset, the ratio between 9-cis- and all-trans-isomers of -carotene amounted to 1.5, with over 60% of total -carotene corresponding to the 9-cis-isomer. Removal of sunshade enhanced carotenoid accumulation by cells to reach up to 10% of dry biomass. Cultivation of Dunaliella in closed tubular photobioreactor, thus represents a suitable approach for the production of a high-quality microalgal biomass enriched in the valuable 9-cis-isomer of -carotene and lutein. �� 2004 Elsevier B.V. All rights reserved. Keywords: Biotechnology -Carotene isomers Lutein Closed tubular photobioreactor Dunaliella salina Microalgae Outdoor culture ��� Corresponding author. Tel.: +34 954489506 fax: +34 954460065. E-mail address: mgguerrero@us.es (M.G. Guerrero). 1. Introduction The halophilic green biflagellate microalga Dunaliella salina has since long been recognized as an efficient biological source of -carotene, a pigment of increasing demand and a wide variety 0168-1656/$ ��� see front matter �� 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2004.07.010

82 M. Garc�� ��a-Gonz�� alez et al. / Journal of Biotechnology 115 (2005) 81���90 of market applications: as food colouring agent as pro-vitamin A (retinol) in food and animal feed as an additive to cosmetics, multivitamin preparations, and in the last decade as a health food product under the antioxidant claim (Edge et al., 1997 Johnson and Schroeder, 1995). Many epidemiological and oncological studies suggest that humans fed on a diet high in carotenoid-rich vegetables and fruits, which maintain higher than average levels of serum carotenoids, have a lower incidence of several types of cancer and degenerative diseases (Ben-Amotz, 1999). Under given stress conditions more than 10% of algal dry weight can be -carotene, which has been shown to be composed of mostly two stereoisomers: all-trans and 9-cis (Ben-Amotz et al., 1982). The pig- ment quality depends on the predominance of 9-cis- isomer, more efficient in protecting against oxidative damage (Jimenez and Pick, 1993). The extent of - carotene accumulation and the 9-cis to all-trans ratio has been claimed to be a direct function of the inte- gral amount of light to which the algae are exposed during a division cycle (Ben-Amotz and Avron, 1983 Ben-Amotz et al., 1988). Open ponds, without or scarce process control, rep- resent the conventional method used in commercial production plants for Dunaliella (Borowitzka, 1992). However, these systems seem to have reached their technical limits (Chaumont, 1993). High CO2 con- sumption with low efficiency, impractical control of some environmental factors, contamination problems, and requirement for high amounts of salt, water, land and solar irradiance have probably limited the expan- sion of mass cultures of this microalga (Ogbonna and Tanaka, 2000). As a matter of fact, only in Israel, China, USA and Australia are nowadays plants commercially producing -carotene from Dunaliella (Ben-Amotz, 1999). The use of closed tubular photobioreactors for microalgae culture represents a very interesting alter- native to outdoor open ponds, since they offer high values of both photosynthetic efficiency (PE) and pro- ductivity (Torzillo et al., 1986). These systems allow operation under continuous culture protocols, dimin- ishing the operational inputs (CO2, water, salt, nutri- ents) and providing steady and controlled conditions (Tredici and Zitelli, 1997). This paper reports on the performance of Dunaliella cultures in an outdoor closed tubular photobioreactor, in an attempt to identify the most appropriate condi- tions for producing high-quality biomass, enriched in specific carotenoids, and to evaluate the efficiency of this alternative culture system. 2. Materials and methods 2.1. Organism and culture medium D. salina UTEX 2538, from the Texas University Culture Collection, Texas (USA), was grown on the medium described by Shaish et al. (1992). The inocu- lum for the photobioreactor was grown aseptically in- doors, in 20 l glass containers, bubbling through the medium air (about 50 l (l culture)���1 h���1) supplemented with 1% (v/v) CO2, at 25 ���C under continuous illumina- tion (fluorescent lamps, 200 mol photon m���2 s���1). In outdoor cultures, f2 medium, described by Guillar and Ryther (1962) and modified to contain 2 mM NaHCO3, 5 mM NaNO3 and 2 M NaCl, was used. 2.2. Outdoor culture system and operation mode The culture system employed has been previously described (Del Campo et al., 2001), and consisted es- sentially in a 55 l closed polymethyl metacrylate tubu- lar photobioreactor with an airlift system to recirculate the cell culture and an horizontal loop, consisting of tubes (90 m long, 2.4 cm i.d. and 2.2 m2 surface), which acts as solar receiver, submerged in a thermostatic pond of water. The airlift was made up of a degasser (in which the pH and temperature probes were inserted) and two tubes 3 m high (the riser and the downcomer). Com- pressed air was supplied into the riser tube to move the cell suspension through the tubes and provide turbu- lence to the culture. The culture pH (7.5 �� 0.5) was controlled by the addition of CO2 gas and temperature was maintained at about 25 ���C, except when indicated. Aliquots of the culture were manually sampled once a day, early in the morning, to assay for biomass and -carotene. The system was operated in semi-continuous regime, sup- porting a pre-established cell density by removal of part of the cell suspension and replacement with fresh medium. The increase in biomass and -carotene be- tween two consecutive dilutions was taken as measure- ment of productivity.