Hiroshi Tamiya

Correlation between photosynthesis and light-independent metabolism in the growth of Chlorella.

Abstract 1. 1. Observations on the culture of Chlorella have revealed that the algal cells assume two distinct forms in the course of their growth. One form, which we called “dark cells”, is smaller in size, richer in chlorophyll content, and stronger in photosynthetic activity than the other, which we referred to as “light cells”. When illuminated, dark cells grow and, with a substantial increase in mass, turn into light cells; the latter, when ripened, bear autospores in themselves (on the average 6–7 per cell) and eventually burst setting free the autospores which then become individual dark cells. The transformation of light cells into dark cells involves no increase of cell mass and occurs only under aerobic conditions, irrespective of whether the cells are in the light or in the dark. The dark cells freshly born from light cells are somewhat smaller in size and contain less chlorophyll than the “active” dark cells, into which the former turn rapidly under the influence of light. 2. 2. The processes of transformation between these types of cells were investigated separately under various experimental conditions, and it was concluded that (i) the main event occurring in the transformation of dark cells into light cells is photosynthesis, although it is accompanied by some other metabolic processes, which are to be distinguished from the photosynthetic process in the ordinary sense; and that (ii) the transformation of light cells into dark cells involves a light-dependent and accompanied also by some photosynthetic processes. 3. 3. The steady state of growth, as it is affected by light intensity and temperature, was investigated in detail, and its rate was compared with the photosynthetic rate using a common unit of measurement, i.e., in terms of weight of organic matter synthesized per unit time and per unit weight of cells. It was found that under weak light the rate of growth is exclusively determined by the photosynthetic process, whereas under strong light the light-independent metabolic processes become more or less significant in determining the rate of over-all growth. It was observed that the relative abundance of dark and light cells in the cultures varies considerably according to the culture condition, and indeed that the proportion of dark cells becomes larger in weaker light and at higher temperatures. 4. 4. Based on the experimental evidence mentioned above, the following formulae were set forth to describe symbolically the course of events occurring in the growth of algae. , where D and L represent dark and light cells, respectively, kp the rate constant of photosynthesis, by which dark cells are changed into light cells, kD the rate constant of increase in cell number in the dark process, and n the number of dark cells arising from one light cell. Steady state kinetics based on this scheme lead to the formulae representing the over-all growth rate and the relative abundance of dark and light cells in the culture as functions of (i) light intensity, (ii) the rate of dark process, and (iii) the light-saturated and light-limited rates of photosynthesis. Correspondence between the theory and the observations was found to be, by and large, satisfactory. 5. 5. It was revealed by this analytical study that the dark process involved in the algal growth has a remarkably large temperature coefficient, especially in the range of lower temperatures.

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.

Role of sulfur in the cell division of Chlorella, studied by the technique of synchronous culture

1. 1. Chlorella was grown synchronously under controlled supply of S- and N-sources so as to make the processes of nuclear and cellular division proceed stepwise, and by using 35S as a tracer the fates of S-compounds in various fractions of cell material (extracts with 70% ethanol, with 10% trichloroacetic acid, and the residue) were followed. 2. 2. In the cells grown in the normal medium, the S-content (in %) of the ethanol- and TCA-extracts decreased in the growing stage and increased markedly at the ripening stage. 3. 3. The ethanol extract contained an unknown S-compound and a non-S-containing peptide-like substance, the latter substance appearing only at certain stages of cell ripening. When chromatographed on paper the TCA extract gave only 1 radioactive (but ninhydrin-negative) spot, which, on hydrolysis with HCl, turned ninhydrin-positive owing to the liberation of several amino acids including cyst(e)ine. 4. 4. In the cells, whose nuclear and cellular division were controlled by limited supply of S- and N-sources, the quantity of the S-containing substance in the TCA-extract increased markedly before nuclear division, and the unknown non-S-containing substance in the ethanol extract appeared only before and concomitantly with, the occurrence of cellular division.

Sulfur-containing peptide-nucleotide complex isolated from Chlorella and yeast cells

Abstract 1. 1. From the cells of a green alga (Chlorella) and yeasts (Saccharomyces formosensis and S. cerevisiae), a new sulfur-containing peptide-nucleotide complex was obtained by extracting the cells with cold trichloroacetic acid (10%). The complex was revealed to contain cyst(e)ine as the major sulfur component in the peptide moiety, and adenine and uracil as the major bases in the nucleotide moiety. 2. 2. In the field of zone electrophoresis, the complex as a whole moved, unlike free nucleotides and polynucleotides, toward the cathode at pH 3.6 in the case of Chlorella and at pH 3.6–5.0 in the case of yeast. At pH 5.0, the complex obtained from Chlorella split into a number of components, the majority of which moved toward the anode. The unsplit complex obtained at pH 3.6 gave, on hydrolysis followed by paper chromatography, several unidentified components, besides adenine, uracil, and a number of amino acids. Some of these unidentified components gave a red color with ninhydrin reagent, and they were assumed to be the factors making the complex assume a positive charge as a whole. 3. 3. The complex obtained at pH 3.6 from Chlorella was also split into a number of subunits when it was subjected to an anion-exchange chromatography (Dowex 1). The complex has, thus, a property of being readily disintegrated by changes of pH or other conditions into its components of different degrees of complexity.

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,