Journal not defined 9: 359–366, 1997. 359 c 1997 Kluwer Academic Publishers. Printed in Belgium. Sequential heterotrophic/autotrophic cultivation – An efficient method of producing Chlorella biomass for health food and animal feed James C. Ogbonna , Hiroyuki Masui & Hideo Tanaka Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan ( Author for correspondence; phone: +81-298-53-6646; fax: +81-298-53-4605; e-mail: jogbonna@sakura.cc.tsukuba.ac.jp) Received 15 July 1997; revised and accepted 13 September 1997 Key words: algae biomass, heterotrophic culture, autotrophic culture, carbon dioxide fixation, cellular components Abstract Sequential heterotrophic/autotrophic cultivation method was investigated for production of high concentration of Chlorella biomass with high cellular protein and chlorophyll contents. By using autotrophic growth medium, which contains glucose as organic carbon source, for heterotrophic culture, the protein and chlorophyll contents of the cells could be increased by simply illuminating the culture broth and aerating with CO 2 -enriched air at the end of the heterotrophic culture. A system was then constructed for continuous sequential heterotrophic/autotrophic production of algal biomass. The system was composed of the conventional mini-jar fermentor for the heterotrophic phase and a tubular photobioreactor for the autotrophic phase. The exhaust gas from the heterotrophic phase was used for aeration of the autotrophic phase in order to reduce the CO 2 emission into the atmosphere. With this system, it was possible to produce high Chlorella biomass concentration (14 g L 1 ) containing 60.1% protein and 3.6% chlorophyll continuously for more than 640 h. During the steady state, about 27% of the CO 2 produced in the heterotrophic phase was re-utilized in the autotrophic phase. When the tubular photobioreactor was replaced with a 3.5-L internally illuminated photobioreactor, the productivity increased from 2 g L 1 d 1 to 4 g L 1 d 1 . However, the chlorophyll content of the cells was lower due to the lower light supply coefficient of the photobioreactor. Introduction The potential of microalgae as a food staple in the human diet has been investigated for many years in dif- ferent countries (Vincent, 1969; Schwarz et al., 1995). Although research on the production of microalgae as protein supplement in foods is declining, the num- ber of malnourished children are still on increase in many developing countries while in many developed countries such as Japan, algal biomass such as Chlorel- la and Spirulina are produced commercially, primari- ly for consumption as health food. The high produc- tion cost currently prevents wide usage of microalgae as animal feed but numerous nutritional experiments clearly demonstrate the high value of some species of microalgae as protein supplement for fish, cattle, hogs, and chickens (Soeder, 1986). There is thus a need for development of efficient systems for algae biomass production. In commercial production of algal biomass, high cell density culture are desirable in order to reduce the cost for down-stream processing. Terry & Raymond (1985) made a detailed discussion on the historical background and various systems used for autotrophic production of microalgae. They classified them into ponds, channeled and shallow circulating systems and discussed the merits and demerits of each system. Because of variation in the climatic condition, growth characteristics of the microorganism used as well as methods of determining and reporting productivities, it is extremely difficult to compare the productivities of the various systems. However, the highest productivity reported was less than 30 g m 2 d 1 . This corresponds to a maximum productivity of 0.3 g L 1 d 1 assuming a uniform reactor depth of 10 cm. This is still very