JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 19, No. 5, 453-457. 1995 Promotive Effect of SAminolevulinic Acid on the Growth and Photosynthesis of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Spirulina platensis zyxwvutsrqponmlkjihgfedcbaZYXWVU KEN SASAKI,‘* FACLJNDO J. MARQUEZ,* NAOMICHI NISH10,2 AND SHIRO NAGAI* Hiroshima-Denki Institute of Technology, Nakano, Aki-ku, Hiroshima 739-03’ and Department of Fermentation Technology, Faculty of Engineering, Kagamiyama, Higashi-Hiroshima 739,* Japan Received 31 October 1994/Accepted 19 January 1995 Photosynthetic activity in terms of 02 evolution and the growth of Spirulina platensis was stimulated by adding 5aminolevulinic acid (ALA, 500 mg/n to photoautotrophically growing cells. After ALA was added to the medium, intracellular accumulations of phycocyanin and chlorophyll were stimulated simultaneously, followed by enhancement of the photosynthetic activities of photosystems I and II, and lastly, growth was promoted. ALA did not directly activate the photosynthetic electron transport system. However, during a 3-h incubation of intact cells with ALA, photosynthetic activity was enhanced. [Key words: 5-aminolevulinic acid, Spirulina platensis, phycocyanin, chlorophyll] SAminolevulinic acid (ALA) is an essential precursor for the biosynthesis of tetrapyrrols such as heme, chlo- rophyll and vitamin BL2. Recently, ALA has attracted at- tention as a biodegradable herbicide which is not harm- ful to humans or animals (1). On the other hand, Tanaka et al. (Proc.19th Annu. Meet. Plant Growth Regulator Sot. of America, San Francisco, p. 237-241, 1992) re- ported that a suitable concentration of ALA sprinkled on the leaves of plants such as rice, barley, potato and garlic, had promotive effects on the rates of growth and photosynthesis. Also, crop yields were enhanced by the application of ALA at the leaf-stage in the life cycle of these plants. However, the mechanism of these promo- tive effects resulting from ALA application has yet to be elucidated. It has also been found that in algae such as Chlorella protothecoides (2) and Scenedesmus obliquus (3), addition of ALA to the medium stimulates chloro- phyll accumulation. The cyanobacterium Spirulina platensis accumulates phycocyanin and chlorophyll intracellularly (4). Phycoc- yanin has an open tetrapyrrol chromophore (phycobilin) and plays an important role in photosynthesis as a light- harvesting pigment, mainly in photosystem II (PS II) in the phycobilizome, in cells of cyanobacteria and red algae (4). ALA seems to be an important precursor for phycocyanin biosynthesis. In addition, Cornet et al. (5) found a close correlation between the intracellular accu- mulation of phycocyanin and the growth rate of S. platensis. Here, the effects of ALA addition on growth, photo- pigment accumulation and photosynthesis are investi- gated in the photoautotrophic growth of S. platensis in order to elucidate the overall mechanism of growth pro- motion by ALA for this cyanobacterium. MATERIALS AND METHODS Organisms and culture medium Axenic and clonal strains of S. platensis NIES-39, NIES-46 (National Insti- tute for Environmental Studies, Environmental Agency, Tsukuba, Japan) and IAM- (Institute of Applied Microbiology, University of Tokyo) were used. The me- * Corresponding author. dium used throughout was modified SOT medium (6) in which the B6 solution consisting of Ni, Cr, V, Wo and Ti was reduced to l/IO concentration. Cultivations Stock cultures were carried out as de- scribed by Ogawa and Terui (6). Preculture was per- formed in a lOO-ml conical flask (50ml of medium, 10 to 20% inoculum) illuminated under tungsten bulbs (200 W, Toshiba Leff lamp, Toshiba Co. Ltd., Tokyo) at 4 klx (7.73 x 1O-3 kJ/cm*.h) for 7d at 30°C. The flasks were shaken manually once a day to maintain a homogeneous condition. Cultivation was carried out in a 1.2-1 Rhoux bottle (500ml of medium) for 6 d at 30°C at a light intensity of 4 klx on the surface of the bottle while the culture was mixed with a magnetic stirrer (ca. 200 rpm). The ini- tial cell concentration was 0.03 g dry cells/l. The initial pH was adjusted to 9.2. ALA was added aseptically into the medium at the beginning of the cultivation within the range O-500mg//. ALA stock solution (20 g/l) was filtrated through a cellu- lose nitrate filter (0.45 pm, Toyo Roshi, Tokyo). Photosynthetic activity The specific oxygen evolu- tion rate (Qo2P, mg 0*/g cell. min) was determined as the whole photosynthetic activity by measuring the in- crease of the dissolved oxygen (DO, mg/l) concentration due to oxygen evolution by S. platensis (7). The cells were harvested by centrifugation (10,000 X g, 20 min) and washed twice with 10 ml of 20 mM HEPES-NaOH buffer (pH 8.0). Cells were resuspended in fresh modified SOT medium with a density of 0.05 g/l (ODS60=0.072) and placed in a water-jacketed cylindrical glass (3 cm in di- ameter and 11.5 cm in height) followed by pure NZ bub- bling for 5 min to remove the oxygen. The cell suspen- sions were incubated at 30°C under strong illumination (20 klx, 200 W, incandescent bulb) and O2 evolution was measured by an oxygen electrode (Type 5G, Able Co. Ltd., Tokyo) for 30min. Under this very low cell concen- tration and strong light illumination, the light energy seems to be sufficient for a short period of photosyn- thesis. The activities of photosystems I and II (PS I and II) were determined according to the method reported by Schor et al. (8). Cells were harvested and washed twice with 10 ml of 0.01 M Tris-HCl buffer (pH 7.2) and resus- 453