Chlorophyll production from Spirulina platensis: cultivation with urea addition by fed-batch process Carlota de Oliveira Rangel-Yagui, Eliane Dalva Godoy Danesi, Jo~ ao Carlos Monteiro de Carvalho * , Sunao Sato Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of S~ ao Paulo, Av. Prof. Lineu Prestes, 580, B-16, 05508-900, S~ ao Paulo-SP, Brazil Received 1 February 2002; received in revised form 24 July 2003; accepted 7 September 2003 Abstract The cyanobacterium Spirulina platensis is an attractive alternative source of the pigment chlorophyll, which is used as a natural color in food, cosmetic, and pharmaceutical products. In this work, the influence of the light intensity and urea supplementation as a nitrogen source using fed-batch cultivation for S. platensis growth and chlorophyll content was examined. Cultivations were carried out in 5 l open tanks, at 30 ± 1 °C. Response surface methodology was utilized for analysis of the results, and models were obtained for biomass productivity, nitrogen-cell conversion factor and chlorophyll productivity. The best cellular growth was observed with 500 mg/l of urea at a light intensity of 5600 lx, whereas the highest concentration of chlorophyll in the biomass was observed with 500 mg/l of urea at a light intensity of 1400 lx. Overall, the best chlorophyll productivity was observed with 500 mg/l of urea at a light intensity of 3500 lx, providing the optimal balance between the cellular growth and the biomass chlorophyll content. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Spirulina platensis; Biomass production; Chlorophyll; Fed-batch process; Urea; Light intensity 1. Introduction Chlorophyll is a naturally occurring pigment present in photosynthetic plants, including algae, and in some photosynthetic bacteria, known as cyanobacteria. The greater part of industrially prepared chlorophyll-deriv- atives is destined for the increasing demand for natural colorants for food and beverages. Some of the industrial production is also destined for the cosmetic and toiletry market, and to the pharmaceutical market (Hendry, 1996). Currently, most of the commercially produced chlorophyll is obtained from vegetable sources (Gross, 1991). Nevertheless, there is a growing interest in the biotechnology field for obtaining non-vegetable sources of colors. The use of fermentation processes possess a number of advantages when compared to vegetable sources, including the possibility of continuous cultiva- tion, and the rapid multiplication of microorganisms (Taylor, 1984). Spirulina platensis is a cyanobacterium that has been largely studied due to its commercial importance as a source of protein, vitamins, essential amino acids, and fatty acids (Ciferri and Tiboni, 1985; Zhang et al., 1999). More recently, special attention has been given to S. platensis as a potential source of pharmaceuticals, and other high value products such as chlorophyll (Boro- witzka, 1995; Chen and Zhang, 1996). The utilization of chlorophyll from S. platensis is an attractive alternative that should be considered due to its high content of this pigment, and ease of cultivation. The cyanobacterium S. platensis possesses a high tolerance to alkaline pH, for ease of cultivation; a large size for its cell aggregates for ease of harvest; and an easily digestible cell wall (Jensen and Knutsen, 1993). It has been shown that the composition of the cultiva- tion medium, cellular age, and light intensity are the main factors influencing chlorophyll content in S. platensis biomass. Cultivations carried out under poor illumination conditions present higher biomass chlorophyll content than cultivations carried out under high illumination conditions, suggesting an inverse proportional relation- ship between light intensity and chlorophyll content * Corresponding author. Fax: +55-11-38156386. E-mail address: jcmdcarv@usp.br (J.C.M. de Carvalho). 0960-8524/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2003.09.002 Bioresource Technology 92 (2004) 133–141