Production of intracellular phytochemicals in Chlorella under heterotrophic conditions

A heterotrophic synchronous culture (HSC) of Chlorella regularis S-50, a strain with a high growth rate, and one for its mutant were established by alternately culturing for 6 h on medium containing glucose and for 3 h on medium without glucose. The changes in cellular components and respiratory activity during the course of cell cycling in the HSC were investigated. The synchronized daughter cells (small cells) produced by the HSC contained 2-3 times as much intracellular phytochemicals (carotenoids, chlorophyll, tocopherols, and others typical of green plants) as the glucose-metabolizing cells or the non-synchronous cell mass. To attain the HSC at high cell density, the effects of glucose and oxygen on growth rate, synchronous growth, and production of intracellular phytochemicals were revealed. During the increase in cell mass, when a glucose concentration of 0.5-10 g l(-1) in the culture fluid was maintained by glucose feeding, and the (Qo2)(max) in cells was kept constant by supplying oxygen, cell mass increased synchronously with a specific growth rate, mu, of over 0.2 h(-1). In the HSC, when the late glucose-metabolizing cells were aerated by supplying oxygen to maintain over a half of the (Qo2)(max) under glucose-deficient conditions, intracellular phytochemicals increased rapidly in parallel with cell division. On the basis of these results, the HSC system at high cell density was attained by a glucose-limited fed-batch culture that involves three controlled conditions; glucose concentration, glucose feeding time, and oxygen supply. The system maintained synchronous growth for more than three generations until the cell density reached about 90 g l(-1) with a mu of 0.2 h(-1). In the HSC system, synchronized daughter cells were obtained at a cell productivity of 84 g l(-1) (30 h)(-1). The cell yield for glucose was 0.45. A weight of 100 g of dry cells contained 710 mg carotenoids, 350 mg lutein, 50 mg alpha-carotene, 60 mg beta-carotene, 3.3 g chlorophylls, 23 mg tocopherols, and others, with 63 g of protein rich in essential amino acids. Industrial-scale HSC systems made it possible to steadily produce Chlorella, containing 10-50 times as much phytochemicals as green and yellow vegetables, regardless of the weather.