Yuji Morimura

Some data on the growth physiology of Chlorella studied by the technique of synchronous culture

Abstract 1. 1. Using the technique of synchronous culture, biochemical events occurring in the life cycle of Chlorella were investigated with reference to the change in contents of major elements (N, P, K, Mg, and S), crude protein, lipides, carbohydrates, and ash, and also with special reference to the effect of deprival of each major element from the medium. 2. 2. The life cycle of algal cells was divided in seven successive stages as follows: ( a ) “nascent dark cells” ( D n ) which are the young cells newly produced from mother cells; ( b ) “active dark cells” ( D a ), the most chlorophyll-rich and photosynthetically active cells which are derived from nascent dark cells when illuminated; ( c ) cells of transient stage between “dark” and “light” cells ( D ~ L ); ( d ) immature light cells ( L 1 ) which are large in size but not yet ripe enough to perform cell division when incubated in the dark; ( e ) half-mature light cells ( L 2 ) which can only partially divide when kept in the dark; ( f ) mature light cells ( L 3 ) which can completely divide when incubated in the dark; and ( g ) fully ripened light cells ( L 4 ) which are at the stage immediately prior to the process of cell division. 3. 3. The major nutrient elements were assimilated by algal cells in more or less different manners during the course of normal life cycle. In terms of percentage of dry weight, nitrogen content was the least variable, showing only a slight decrease at the stage of D ~ L ; the phosphorus content decreased at stages D a and D ~ L , and increased at stages L 1 and L 2 ; the sulfur content decreased considerably as the cells changed from D - to L -stage, and increased markedly as L 3 transformed into L 4 . 4. 4. The percentage of crude protein remained almost constant throughout the life cycle, while lipide and carbohydrate contents varied more or less irregularly. 5. 5. The effect of the deficiency of each major element was investigated using normally grown D a -cells as the starting material of synchronous culture. It was revealed that the growth of D a -cells was sooner or later retarded in the absence of major elements. The retardation of growth occurred most strongly in the N-free and P-free media, less markedly in the K-free and S-free media, and most insignificantly in the Mg-free medium. Except in the case of S-deficiency, the cells grown in the absence of each major element could perform cell division, giving rise, however, to more or less unhealthy daughter cells. The average number of daughter cells produced from one mother cell was: 2.4 in N-free medium, 3.5 in P-free medium, 5.1 in K-free medium, and 6.4 in Mg-free medium, compared with 6.0–6.5 in the case of normal culture. The daughter cells formed in N-free and Mg-free media were profoundly etiolated, while those produced in P-free and K-free media looked normal in color. They were either unable to grow further (as in the case of N-deficiency) or could only slightly grow, and were all incapable of performing further cell division. 6. 6. A peculiar phenomenon was observed when the synchronous culture was run in S-free medium. In this case the starting D a -cells grew apparently normally until the earlier stage of light cells, but unlike the cells grown in the absence of other major elements, they were entirely incapable of performing cell division. This fact, together with the observation that the assimilation of sulfur occurred mainly at the stage of light cells, indicates that sulfur plays some essential role in the process of cell division. The capacity of cell division, which had been lost in the absence of sulfur, could be restored by subsequent supply of sulfate and nitrate and by illuminating the cells with the provision of CO 2 -enriched air.

The role of sulfur in the cell division of chlorella

Summary1.The role of sulfur in the process of cell division of Chlorella was studied using the technique of synchronous culture. When the “dark cells” (smaller and strongly photosynthesizing cells), which had been grown in a normal nutrient medium, were further cultured in an S-deficient medium under photosynthesizing conditions, the cells grew up to some extent, showing about two-fold increase of DNA-content followed by the division of nucleus into two. At this stage, however, the cells fell into a stalemate, being unable to complete cellular division.2.When such cells were transferred to a medium containing potassium sulfate only, some synthesis of DNA and further division of nuclei occurred. Under non-photosynthesizing conditions, the process of cellular division ensued, giving rise to a formation of small daughter cells. Under photosynthesizing conditions, on the other hand, the cells increased appreciably in size, without, however, being able to perform cellular division. The process of cellular division thus halted could be resumed when the cells were further supplied with nitrate and sulfate under photosynthesizing condition.3.Based on these observations, it was concluded that sulfur plays, in cooperation with some nitrogenous substance(s), an essential role in the process of DNA-formation and nuclear division as well as in the process of cellular division.

Effects of various antimetabolites upon the life cycle of Chlorella.

Since the technique of synchronous culture of Chlorella was developed in our laboratory in 1953 (TAMIYA et al.), extensive studies have been performed to investigate the effects of various environmental factors upon the life cycle of algal cells. The experimental organism used throughout in our studies was Chlorella ellipsoidea, and the environmental factors or conditions, whose effects had been investigated were: temperature, light intensity (Mo~IMURA 1959), deprival of each essential mineral nutrient (ItAs~ et al. 1957), and irraditation with ultraviolet light (SAsA 1961). During the course of these studies the technique of synchronization of algal culture has been greatly improved (MoRI~U~A 1959; TAMIYA et al. 1961), and it has now become possible to synchronize the algal growth not only at the cellular level but also at the level of nuclear development and division (TAMIYA et al. 1961). The general conclusions we have arrived at by these investigations were that the normal course of the life cycle of ChloreUa eUipsoidea proceeds in a manner as schematically illustrated in Fig. 1. The cycle starts from a young and small cell, referred to in the figure as the Dn-cell (nascent D-cell) which derives from the fully matured mother cell denoted by L 4. When grown under light of saturating intensity (with provision of CO s and mineral nutrients) the Dn-cell grows and, after passing through successively the stages denoted by Da (active Dcell), D-L (transition between Dand Lcells), L 1 (unripened L-cell), and L 2 (half-ripened L-cell), it attains the stage of La-eell (ripened L-cell). The L3-cell further ripens, without requiring the light, into L4-eell which in turn, also independently of light, divides into four ])n-cells. By ~he Feulgen staining it was revealed 1 that the cells at the stages of Dn,

[6] Synchronous culture of Chlorella

Publisher Summary This chapter discusses the two methods of synchronous culture, which have been most widely in use for Chlorella. In the first method, the cultures are started from a homogeneous population of small young cells—called Ds cells—which are separated from less homogeneous populations by differential centrifugation. When the cells reach a mature stag—called L3 cells—after which they do not require light for further development to complete cellular division, the light is turned off. When all the cells have completed division, the light is turned on to start the second cell cycle. In the other method, called “the method of programmed light-dark regimes,” a random culture is subjected, without being fractionated beforehand into a homogeneous population, to a programmed light and dark alternation, in which the lengths of the light and dark periods are adequately chosen according to the algal species and culture conditions. The synchrony of growth and cell division is attained on repeating the light and dark regimes several times. The technical procedures and some precautions to be taken in running the synchronous cultures by the former method are described.