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Nitrogen and phosphorus metabolism during synchronous growth of Chlorella pyrenoidosa
Experimental
209
Cell Research 23, 209-217 (1961)
NITROGEN AND PHOSPHORUS METABOLISM DURING SYNCHRONOUS GROWTH OF CHLORELLA PYRENOIDOSA1 R. R. SCHMIDT Department
of Biochemistry and Nutrition, Virginia t Blacksburg, Va., U.S.A.
Polytechnic
Institute,
Received May 4, 1960
METHODS have 17-19,
been devised in this and other 221 to obtain cultures of algae that would
laboratories simultaneously
[l-5, 8, 11, 12, begin growth
and progress through all stages of cellular growth and division almost synchronously. In essence, the synchronized culture magnifies the metabolic changes that accompany the growth of a single cell enough to be measured easily by macro- or semi-micro quantitative biochemical techniques. The present investigation was made to obtain additional information con(Van cerning the synchronous growth of algae using Chlorella pyrenoidosa Niel strain, code No. Z, 2.2.1). Total cellular nitrogen, residual or crude protein nitrogen, nitrate and phosphate uptake, cellular dry weight and cell number
were
measured.
MATERIALS
AND
METHODS
Synchronization procedure.-Synchronization was initiated by maintaining cultures at high cell densities (1 g fresh weight per 100 ml of culture) under low light intensity (500 f.c.). Cells from these cultures developed more or less synchronously when placed under conditions for maximal growth (25”C, light intensity of 1500 f.c., aeration with 5 per cent CO, in air, initial cell density of 5 x lo6 cells per ml for culture tubes 2” in diameter). When the majority of these cells exhibited signs of cellular division, the cultures were placed in the dark. Cells which were in stages of nuclear or cellular division completed division and released daughter cells. However, these daughter cells, perhaps due to lack of reserve material, remained at a maintenance state after their release. Cells which had completed less than one-third of their development likewise remained at a maintenance state while cells beyond this point of development were often able to continue growth to various stages of nuclear or cellular division. Thus, a net gain in synchrony was accomplished by alternate periods of 18 hours of light and 12 hours of darkness. After four cycles of intermittent 1 This paper constitutes part of the thesis submitted to the faculty of the Graduate School, University of Maryland, August 1957, in partial fulfillment of the requirements for the M.S. degree. 14 -
61173260
Experimental
Cell Research 23
210
R. R. Schmidt
illumination the degree of synchrony was evaluated during one synchronous growth cycle by the method of Spencer [20]. The cultures were found to have a maximum degree of synchrony characterized by a standard deviation of cellular division times of 1.00 hour with a coefficient of variation of 5.20 per cent and a “range coefficient” of 0.792. To further improve synchrony, the daughter cell suspension resulting from the fourth dark period was subjected to fractional centrifugation. In two centrifugation steps in Goetz centrifuge tubes (1160 g for 1; min, and 4 min respectively), daughter cells of homogeneous density were separated from older cells of higher densities and from cellular debris and dead cells of lower density. The resulting daughter cell suspension was transferred to a culture vessel made of Plexiglass ($” thickness) having the following inside dimensions: 4%” width, :” thickness, and 146” height with the bottom 2” tapering to a point. Owing to the greatly increased surface to volume ratio, a high initial cell density (15-20 x 106 cells per ml of culture) was possible. It was assumed that light intensity or nutrient concentration never became a limiting factor to growth during synchronous development because steady-state cultures of the same initial cell concentration exhibited logarithmic or exponential growth for periods in excess of one synchronous growth cycle. Conditions for steady-state growth.-Cells were cultured under conditions for maximal growth for a minimum of seven days. Periodic dilutions were made with fresh culture medium to prevent light intensity or nutrient concentration from limiting the growth of the cells. Such cultures reflected logarithmic or exponential growth typical of “random” or steady-state cultures. Media.-The nutrient medium for synchronized cells contained 1.25 g KNO,, 0.5 g MgSO, * 7 H,O, and 1.25 g KH,PO, per liter. The final solution contained four p.p.m. Fe as EDTA (ethylenediaminetetraacetic acid) * NaFe, and one ml of a micronutrient stock solution containing: 1.81 g MnCl, * 4 H,O, 0.222 g ZnSO, .7 H,O, 0.079 g CuSO, * 5 H,O, 2.86 g H,BO,, 0.176 g (NH,),Mo,O,, * 4 H,O, 0.023 g NH,VO, (meta), 0.049 g Co(NO,), * 6 H,O, and 1.39 g CaCl, per liter. After autoclaving, the pH of the culture medium was adjusted with sterile KOH to pH 6.8. In nitrogen balance experiments, the phosphate buffer was increased to 2.5 g KH,PO, per liter. In phosphate deficiency experiments the buffer was “Tris” buffer ((tris (hydroxymethyl) aminomethane)) at 1.25 g per liter. Micro-Kjeldahl procedure.-A modification of Johnson’s procedure [7] was used for the quantitative estimation of organic N. A sample containing 10 to 60 pg of organic nitrogen was pipetted into an 18 x 150 mm Pyrex test-tube, one ml of 5 N H,SO, containing 0.20 g CuSO, and 0.129 g selenious acid per liter was added, and the sample was evaporated to constant volume at 100°C. Large glass marbles were placed on top of the test tubes; then, the tubes were placed in an oven or sand bath at 300°C with approximately one-third of each tube exposed to the room to act as an air condenser. The digestion period was 12 hours. To the tube, after digestion, was added in order: 5 ml of water, 3 ml of 3.3 N NaOH, and 2 ml of color reagent containing 7 g K,HgI, (“Purified” Reagent Grade, Fisher Scientific Co.), and 1.75 g of gum ghatti per liter. The reaction mixture was mixed and allowed to stand five tube at 490 rnp in a B and L minutes before reading in a 13 x 100 mm calorimeter Spectronic 20 spectrophotometer. The variation between quadruplicate determinations was not greater than one to three micrograms of nitrogen within a 10 to 60 microgram range. Experimental
Cell Research
23
Metabolism during synchronous growth
211
nitrogen.-Cells were separated from the culture medium by centriresuspended in an equivalent volume of distilled water, and analyzed by the micro-Kjeldahl method. Soluble reduced nitrogen.-Several methods were used to extract the cells after separation from the culture medium by centrifugation: (1) three 10 minute extractions with 10 per cent trichloroacetic acid at 25”C, (2) three 10 minute extractions with 10 per cent trichloroacetic acid at 0°C (3) one 10 minute extraction with one per cent acetic acid followed by three 10 minute extractions with water at X0X, (4) four 10 minute extractions with distilled water at 25°C and (5) four 10 minute extractions with aqueous EDTA . Na. In each case extracts were analyzed by the modified microKjeldahl procedure. Cellular dry weight.-The cells were separated from the culture medium on tared Millipore filters 22 mm in diameter with a pore size of 0.45~. The cells together with the filter were oven-dried at 80°C for 12 hours, cooled to room temperature in a desiccator over P,O,, and weighed. Phosphate analysis.-Phosphate uptake was measured by a modification [14] of Kitson and Mellon’s [9] vanadate method. Nitrate analysis.-Nitrate remaining in the culture medium, a measure of nitrate uptake, was measured by a modified phenoldisulfonic acid method in which the reagent was added to not more than one ml of the culture medium or standard nitrate solution. Reagents and procedures were otherwise the same as those of Johnson and Ulrich [6]. All nitrate analyses reported in this paper were made with the above method. The method of Swann and Adams [al] proved to be excellent for culture medium containing high concentrations of nitrate. Cellular growth.-In certain experiments, the growth of synchronously developing cells was followed with periodic readings of optical density at 550 rnp in a B and L Spectronic 20 spectrophotometer. Nuclear stage.-The beginning of nuclear division during synchronous growth was determined by the staining procedure of Schaechter and DeLamater 1161. Polyphosphate granules.-The cells were filtered from the culture medium on Millipore filters, then smeared on coverslips, placed in Carnoy’s fixative or Farmer’s fixative for five minutes, and hydrated through seven steps (100, 95, 90, 70, 50, 30, 0 per cent ethanol). The cells were stained for one to five minutes in a 0.10 per cent aqueous solution of toluidine blue, then dehydrated back through the ethanol series to xylene and mounted in Permount. Cell number.-Cell counts were made in a Levy-Hausser hemacytometer. Total
organic
fugation, modified
RESULTS
AND
DISCUSSION
The difference between synchronous growth and the typical logarithmic or exponential growth of a steady-state culture is emphasized in Fig. 1. Although all of the cells divided during a synchronous growth cycle, there vvas on the average less than an eight-fold increase in cell number because some of the cells divided into less than eight daughter cells. A similar observation was matie by Ivvamura et 01. [5] for Chlorelln ellipsoidea. It will probExperimental
Cell Research 23
212
R. R. Schmidt
ably be impossible to obtain a quantitative and reproducible picture of the detailed and subtle biochemical and physical events of nuclear and cellular divison using Chforella until a system can be devised where all the cells in a culture divide into the same number of daughters.
I.61 0
:
3
:
6
:
9HO”PAZ
:
:
15
:
18
:
21
4
24
Fig. 1. Fig. l.-Growth
of Chlorelfa pyrenoidosa.
Fig. 2. a-0-0,
steady state; A---A,
synchronous.
Fig. 2.-Total reduced cellular nitrogen during synchronous growth (C. pyrenoidosa). Exper. No. 1; A-A-A, Exper. No. 2; O-0-0, Exper. No. 3.
O-0-0,
The cells in synchronized cultures of Chlorella pyrenoidosa (Van Niel strain) began nuclear division between the 14th and 15th hours. Daughter cell release was initiated at approximately the 18th hour and usually had completed by the 21st hour. Total cellular reduced nitrogen increased logarithmically or exponentially during synchronous growth (Fig. 2). The residual nitrogen remaining after cells were extracted with 10 per cent trichloroacetic acid at 0°C was used as a rough approximation of the crude protein fraction of the cells. This nitrogen fraction represented approximately 79-80 per cent of the total cellular nitrogen. The exponential character of residual cellular nitrogen strongly indicated that no pool of soluble nitrogen intermediates accumulated in the cells prior to nuclear division (Fig. 3). Other extraction procedures also failed to reveal any accumulation of soluble reduced nitrogen immediately prior to nuclear division (Table I). The possibility still remains, however, that composition of the soluble nitrogen pool changes during cellular development. Daughter cells coming out of a dark period usually contained less soluble nitrogen than daughter cells which were formed and released in the light. Undoubtedly, the soluble nitrogen pool is utilized for cellular maintenance during dark periods. Trichloroacetic acid extraction at 25°C probably resulted Experimental
Cell &search
23
h4efabolism during synchronous growfh
213
in some hydrolysis of residual cellular nitrogen because continued extraction at this temperature always yielded more soluble nitrogen. At 0°C with trichloroacetic acid only little nitrogen was extractable after 3-4 repeated extractions. In an effort to extract water soluble intermediates which were TABLE I. Extructable
cellular nitrogen during synchronous (C. pyrenoidosa).
Extractable
nitrogen
Time hours
10 % TCA, 25”
10 % TCA, 00
0 3 6 9 12 15 18 21
11.9 20.6 32.6 28.2 30.5 28.6 27.9 34.0
8.2 15.0 22.0 22.0 20.0 21.0 21.0 -
as per cent of total 1 % HAc, distilled water, 80”
10.4 12.1 14.2 15.6 16.5 18.0 20.6 -
cellular
Distilled water, 25”
13.3 4.9 1.7 2.5 3.8 2.1 0.0 -
growth
nitrogen Aqueous EDTA.Ka, 25”
10.0 2.4 6.9 5.0 7.5 6.3 0.0 -
acid insoluble, the cells were extracted with distilled water. To facilitate the water extraction, the sodium salt of ethylenediaminetetraacetic acid was added to chelate the divalent cations which are known to decrease the permeability of cell membranes. As presented in Table I more nitrogen was extractable with aqueous EDTA . Na than with distilled water. Very recent evidence indicates that double distilled water or deionized water can extract as much as 22 per cent of the total cellular nitrogen near the end of cellular development. Extraction of cellular nitrogen with deionized water can be prevented by the addition of Ca2+ to the system. The equivalence between the disappearance of nitrate nitrogen from the culture medium and the increase in total reduced cellular nitrogen is illustrated in Fig. 4. Since the modified micro-Kjeldahl method used to analyze for total cellular nitrogen only measured reduced cellular nitrogen and not oxides of nitrogen, the equivalence of the two curves indicated that nitrate was quickly reduced as it was absorbed and did not accumulate as nitrate. Difficulty was encountered in obtaining a nitrogen balance until the reagent grade potassium nitrate and ammonium sulfate salts were recrystallized three times in deionized water and dried in ~ncuo before use as nitrogen standards. Experimental
Cell Research 23
214
R. R. Schmidt
As a further measure of synchronous growth cellular dry weight was analyzed. The increase in cellular dry weight was not a logarithmic or exponential function as was the increase in total cellular nitrogen (Fig. 5). Near the end of a synchronous growth cycle, an odor similar to that of freshly mown grass was evolved from the culture. When the air stream coming 2.4
2.4 t
1
I.40L5-
6
12 --77 HOURS
I8
21
Fig. 4.
Fig. 3. Fig. 3.-Residual cellular nitrogen after cells were extracted acid at 0” during synchronous growth (C. pyrenoidosa).
9
with
10 per cent tricholoracetic
Fig. 4.-Total reduced cellular nitrogen and nitrate nitrogen uptake during synchronous growth (C. p~w~oidosa). To each value for nitrate nitrogen uptake was added the initial concentration of total reduced cellular nitrogen. o-O-0, cellular nitrogen; A-A-A, nitrate nitrogen uptake.
from the culture was bubbled through 1 N sulfuric acid, approximately 0.03 to 0.05 per cent of the total nitrogen of the culture was trapped. White needlelike crystals were precipitated from the acid solution by ether. The crystals are being collected for future identification. Measurement of phosphate uptake indicated that the time and amount of phosphate absorbed by the cells during synchronous growth was related to the nitrogen source (Fig. 6). When ammonium was the sole nitrogen source, the cells not only absorbed a larger quantity of phosphate than cells cultured on nitrate nitrogen but they absorbed it earlier in cellular development. The rate of phosphate absorption decreased during nuclear and cellular division on both nitrogen sources. Periodicity in phosphate metabolism has also been observed in synchronously dividing cultures of the protozoan, Tetrahymena. Hamburger and Zeuthen [23] observed that the rate of absorption of 32P was highest before and lowest immediately after synchronous division. Plesner [13] observed that the cellular adenosine triphosphate concentration reached a maximum before and a minimum after cellular division. Experimenlal
Cell Research 23
215
Metabolism during synchronous growth The phate, or two During 20 or
cells were stained with toluidine blue, a stain specific for polyphosduring synchronous growth. Daughter cells were found to contain one staining granules tentatively identified as containing polyphosphate.. periods of high phosphate uptake numbers of granules increased to more per cell. It seemed likelyithat the one or two polyphosphate
Fig. 6.
Fig. 5. Fig. 5.-Cellular No. 1; A---A--A, Fig. 6.-Effect nitrogen; Q-0,
dry weight during Exper. No. 2.
synchronous
growth
(C. pyrenoidosa).
Q-O-0,
of nitrogen source on phosphate nitrate nitrogen.
uptake
(C. pyrenoidosa).
A-A,
0+
BtT
15
I8
Exper. ammonium
21
HOURS
Fig. 7.-Effect of phosphate starvation medium; A-f&-A, no phosphate.
on synchronous
growth (C. pyrenoidosa).
O-0,
complete
granules in daughter cells could serve as a phosphagen for a certain amount of cellular growth. To test this hypothesis, synchronized daughter cells were placed on phosphate deficient culture medium with nitrate as the nitrogen source. Synchronous growth of the phosphate deficient culture was compared to that of a culture on complete medium (Fig. 7). Growth in the two cultures was almost parallel up to the stage of nuclear division when the growth of Experimenial
Cell Research 23
R. R. Schmidt the phosphate-deficient culture practically ceased with only a few cells actually completing nuclear or cellular division. Nihei [ 11 ] observed that Chlorella ellipsoidea accumulated phosphorus, chemically identified as polyphosphate, by a photosynthetic phosphorylation process occurring during the latter stages of cellular development. This photosynthetic phosphorylation process probably corresponds to the period of high phosphate uptake and metachromatic granule accumulation prior to nuclear division in Chlorella pyrenoidosa. Sal1 et al. [15] found, by cytochemical and microchemical procedures, that polyphosphate accumulations were highest immediately prior to and lowest after cell division of synchroMudd et al. [lo] demonstrated that nously dividing cells of Corynebcrcterium. polyphosphate could be used for nucleic acid synthesis and cellular growth for Mycobncterium. These present observations indicate that polyphosphate probably also plays an important role in the biochemistry of cell growth and division of Chlorella
pyrenoidosa.
SUMMARY
Cultures of the alga Chlorella pyrenoidosa have been synchronized and examined for shifts in metabolism during cellular development. Total cellular nitrogen, crude protein nitrogen, and nitrate uptake increased exponentially, while dry weight did not increase exponentially during synchronous growth. The air stream coming from the culture was found to contain an unidentified organic nitrogen compound. Cytochemical and physiological evidence indicated that polyphosphate accumulated in the cells and was important for cellular growth and division. The author wishes to acknowledge his indebtedness to Dr. Robert Krauss, Department of Botany of the University of Maryland, in whose laboratory the study was initiated and to Drs. R. W. Engel and K. W. King for encouraging the continuance of the research in the Department of Biochemistry and Nutrition of the Virginia Polythechnic Institute. Gratitude is also extended to Drs. W. E. C. Moore and M. S. Read for their review and criticisms of the manuscript. REFERENCES 1. 2. 3. 4.
HASE, E., MORIMURA, Y., MIHARA, S. and TAMIYA, I-I., Arch. Mikrobiol. 32, 87 (1958). HASE, E., MORIMURA, Y. and TAMIYA, H., Arch. Biochem. nnd Biophys. 69, 149 (1957). HASE, E., OTSUKA, H., MIHARA, S. and TAMIYA, H., Biochim. et Biophys. Acta 35, 180 (1959). IWAMURA, T., J. Rio&m. Japan 42, 575 (1955).
Experimenlal
Cell Research 23
Metabolism during synchronous growth
217
5. IWAMURA, T., HASE, E., MORIMURA, Y. and TAMIYA, H., A. I. Virtanen Homage Volume. Biochemistry of Nitrogen, p. 89. Helsinki, Finland, i955. 6. JOHNSON, C. M. and ULRICH, A., Anal. Chem. 22, 1526 (1950). 7. JOHNSON, M. J., J. Biol. Chem. 137, 575 (1941). 8. KANAZAWA, T., J. Gen. Appl. Microbial. 4, 102 (1958). 9. KITSON, R. E. and MELLON, M. G., Ind. Eng. Chem. Analyt. Ed. 16, 379 (1944). IO. MUDD, S., YOSHIDA, A. and KOIKE, M., J. Bacteriot. 75, 224 (1958). 11. NIHEI, T., J. Biochem. Japnn 42, 245 (1955). 12. NIHEI, T., SASA, T., MIYACHI, S., SUZUKI, K. and T.%MIY.~, H., Arch. Mikrobiol. 21, 155 (1954). 13. PLESSER, P. E., Exptl. Cell Research 29, 462 (1958). 14. RICHARD, L. A., ed., U.S. Dept. Agr., Agriculture Handbook 60, 160 (1956). 15. SALL, T., MUDD, S. and TAKAGI, A., J. Bacteriot. 76, 640 (1958). 16. SCHAECHTER, M. and DELAMATER, E. D., Am. J. Botany 42, 417 (1955). 17. SCHMIDT, R. R., M.S. Thesis in Botany, Univ. of Mary&d, College Park, Md., 1957. 18. SOROKIN, C., PhgsioZ. Pkmtarum 10, 659 (1957). 19. SOROKIN, C. and MYERS, J., J. Gen. Physiol. 40, 579 (1956). 20. SPENCER, H. T., %LS. Thesis in Biochemistry, Virginia Polytechnic Institute, Blacksburg, Va., 1960. 21. SWANN, M. H. and ADAIMS, M. L., Anal. Chem. 28, 1630 (1956). 22. TAMIYA, H., IWAMURA, T., SHIBATA, K., HASE, E: and Gu,H& T., Biochim. et Biophys. Aetu 12, 23 (1953). 23. ZEUTHEN, E.., Advances in Biot. and Med. Phys. 6, 37 (1958).
Experimental
Cell Research 23
209
Cell Research 23, 209-217 (1961)
NITROGEN AND PHOSPHORUS METABOLISM DURING SYNCHRONOUS GROWTH OF CHLORELLA PYRENOIDOSA1 R. R. SCHMIDT Department
of Biochemistry and Nutrition, Virginia t Blacksburg, Va., U.S.A.
Polytechnic
Institute,
Received May 4, 1960
METHODS have 17-19,
been devised in this and other 221 to obtain cultures of algae that would
laboratories simultaneously
[l-5, 8, 11, 12, begin growth
and progress through all stages of cellular growth and division almost synchronously. In essence, the synchronized culture magnifies the metabolic changes that accompany the growth of a single cell enough to be measured easily by macro- or semi-micro quantitative biochemical techniques. The present investigation was made to obtain additional information con(Van cerning the synchronous growth of algae using Chlorella pyrenoidosa Niel strain, code No. Z, 2.2.1). Total cellular nitrogen, residual or crude protein nitrogen, nitrate and phosphate uptake, cellular dry weight and cell number
were
measured.
MATERIALS
AND
METHODS
Synchronization procedure.-Synchronization was initiated by maintaining cultures at high cell densities (1 g fresh weight per 100 ml of culture) under low light intensity (500 f.c.). Cells from these cultures developed more or less synchronously when placed under conditions for maximal growth (25”C, light intensity of 1500 f.c., aeration with 5 per cent CO, in air, initial cell density of 5 x lo6 cells per ml for culture tubes 2” in diameter). When the majority of these cells exhibited signs of cellular division, the cultures were placed in the dark. Cells which were in stages of nuclear or cellular division completed division and released daughter cells. However, these daughter cells, perhaps due to lack of reserve material, remained at a maintenance state after their release. Cells which had completed less than one-third of their development likewise remained at a maintenance state while cells beyond this point of development were often able to continue growth to various stages of nuclear or cellular division. Thus, a net gain in synchrony was accomplished by alternate periods of 18 hours of light and 12 hours of darkness. After four cycles of intermittent 1 This paper constitutes part of the thesis submitted to the faculty of the Graduate School, University of Maryland, August 1957, in partial fulfillment of the requirements for the M.S. degree. 14 -
61173260
Experimental
Cell Research 23
210
R. R. Schmidt
illumination the degree of synchrony was evaluated during one synchronous growth cycle by the method of Spencer [20]. The cultures were found to have a maximum degree of synchrony characterized by a standard deviation of cellular division times of 1.00 hour with a coefficient of variation of 5.20 per cent and a “range coefficient” of 0.792. To further improve synchrony, the daughter cell suspension resulting from the fourth dark period was subjected to fractional centrifugation. In two centrifugation steps in Goetz centrifuge tubes (1160 g for 1; min, and 4 min respectively), daughter cells of homogeneous density were separated from older cells of higher densities and from cellular debris and dead cells of lower density. The resulting daughter cell suspension was transferred to a culture vessel made of Plexiglass ($” thickness) having the following inside dimensions: 4%” width, :” thickness, and 146” height with the bottom 2” tapering to a point. Owing to the greatly increased surface to volume ratio, a high initial cell density (15-20 x 106 cells per ml of culture) was possible. It was assumed that light intensity or nutrient concentration never became a limiting factor to growth during synchronous development because steady-state cultures of the same initial cell concentration exhibited logarithmic or exponential growth for periods in excess of one synchronous growth cycle. Conditions for steady-state growth.-Cells were cultured under conditions for maximal growth for a minimum of seven days. Periodic dilutions were made with fresh culture medium to prevent light intensity or nutrient concentration from limiting the growth of the cells. Such cultures reflected logarithmic or exponential growth typical of “random” or steady-state cultures. Media.-The nutrient medium for synchronized cells contained 1.25 g KNO,, 0.5 g MgSO, * 7 H,O, and 1.25 g KH,PO, per liter. The final solution contained four p.p.m. Fe as EDTA (ethylenediaminetetraacetic acid) * NaFe, and one ml of a micronutrient stock solution containing: 1.81 g MnCl, * 4 H,O, 0.222 g ZnSO, .7 H,O, 0.079 g CuSO, * 5 H,O, 2.86 g H,BO,, 0.176 g (NH,),Mo,O,, * 4 H,O, 0.023 g NH,VO, (meta), 0.049 g Co(NO,), * 6 H,O, and 1.39 g CaCl, per liter. After autoclaving, the pH of the culture medium was adjusted with sterile KOH to pH 6.8. In nitrogen balance experiments, the phosphate buffer was increased to 2.5 g KH,PO, per liter. In phosphate deficiency experiments the buffer was “Tris” buffer ((tris (hydroxymethyl) aminomethane)) at 1.25 g per liter. Micro-Kjeldahl procedure.-A modification of Johnson’s procedure [7] was used for the quantitative estimation of organic N. A sample containing 10 to 60 pg of organic nitrogen was pipetted into an 18 x 150 mm Pyrex test-tube, one ml of 5 N H,SO, containing 0.20 g CuSO, and 0.129 g selenious acid per liter was added, and the sample was evaporated to constant volume at 100°C. Large glass marbles were placed on top of the test tubes; then, the tubes were placed in an oven or sand bath at 300°C with approximately one-third of each tube exposed to the room to act as an air condenser. The digestion period was 12 hours. To the tube, after digestion, was added in order: 5 ml of water, 3 ml of 3.3 N NaOH, and 2 ml of color reagent containing 7 g K,HgI, (“Purified” Reagent Grade, Fisher Scientific Co.), and 1.75 g of gum ghatti per liter. The reaction mixture was mixed and allowed to stand five tube at 490 rnp in a B and L minutes before reading in a 13 x 100 mm calorimeter Spectronic 20 spectrophotometer. The variation between quadruplicate determinations was not greater than one to three micrograms of nitrogen within a 10 to 60 microgram range. Experimental
Cell Research
23
Metabolism during synchronous growth
211
nitrogen.-Cells were separated from the culture medium by centriresuspended in an equivalent volume of distilled water, and analyzed by the micro-Kjeldahl method. Soluble reduced nitrogen.-Several methods were used to extract the cells after separation from the culture medium by centrifugation: (1) three 10 minute extractions with 10 per cent trichloroacetic acid at 25”C, (2) three 10 minute extractions with 10 per cent trichloroacetic acid at 0°C (3) one 10 minute extraction with one per cent acetic acid followed by three 10 minute extractions with water at X0X, (4) four 10 minute extractions with distilled water at 25°C and (5) four 10 minute extractions with aqueous EDTA . Na. In each case extracts were analyzed by the modified microKjeldahl procedure. Cellular dry weight.-The cells were separated from the culture medium on tared Millipore filters 22 mm in diameter with a pore size of 0.45~. The cells together with the filter were oven-dried at 80°C for 12 hours, cooled to room temperature in a desiccator over P,O,, and weighed. Phosphate analysis.-Phosphate uptake was measured by a modification [14] of Kitson and Mellon’s [9] vanadate method. Nitrate analysis.-Nitrate remaining in the culture medium, a measure of nitrate uptake, was measured by a modified phenoldisulfonic acid method in which the reagent was added to not more than one ml of the culture medium or standard nitrate solution. Reagents and procedures were otherwise the same as those of Johnson and Ulrich [6]. All nitrate analyses reported in this paper were made with the above method. The method of Swann and Adams [al] proved to be excellent for culture medium containing high concentrations of nitrate. Cellular growth.-In certain experiments, the growth of synchronously developing cells was followed with periodic readings of optical density at 550 rnp in a B and L Spectronic 20 spectrophotometer. Nuclear stage.-The beginning of nuclear division during synchronous growth was determined by the staining procedure of Schaechter and DeLamater 1161. Polyphosphate granules.-The cells were filtered from the culture medium on Millipore filters, then smeared on coverslips, placed in Carnoy’s fixative or Farmer’s fixative for five minutes, and hydrated through seven steps (100, 95, 90, 70, 50, 30, 0 per cent ethanol). The cells were stained for one to five minutes in a 0.10 per cent aqueous solution of toluidine blue, then dehydrated back through the ethanol series to xylene and mounted in Permount. Cell number.-Cell counts were made in a Levy-Hausser hemacytometer. Total
organic
fugation, modified
RESULTS
AND
DISCUSSION
The difference between synchronous growth and the typical logarithmic or exponential growth of a steady-state culture is emphasized in Fig. 1. Although all of the cells divided during a synchronous growth cycle, there vvas on the average less than an eight-fold increase in cell number because some of the cells divided into less than eight daughter cells. A similar observation was matie by Ivvamura et 01. [5] for Chlorelln ellipsoidea. It will probExperimental
Cell Research 23
212
R. R. Schmidt
ably be impossible to obtain a quantitative and reproducible picture of the detailed and subtle biochemical and physical events of nuclear and cellular divison using Chforella until a system can be devised where all the cells in a culture divide into the same number of daughters.
I.61 0
:
3
:
6
:
9HO”PAZ
:
:
15
:
18
:
21
4
24
Fig. 1. Fig. l.-Growth
of Chlorelfa pyrenoidosa.
Fig. 2. a-0-0,
steady state; A---A,
synchronous.
Fig. 2.-Total reduced cellular nitrogen during synchronous growth (C. pyrenoidosa). Exper. No. 1; A-A-A, Exper. No. 2; O-0-0, Exper. No. 3.
O-0-0,
The cells in synchronized cultures of Chlorella pyrenoidosa (Van Niel strain) began nuclear division between the 14th and 15th hours. Daughter cell release was initiated at approximately the 18th hour and usually had completed by the 21st hour. Total cellular reduced nitrogen increased logarithmically or exponentially during synchronous growth (Fig. 2). The residual nitrogen remaining after cells were extracted with 10 per cent trichloroacetic acid at 0°C was used as a rough approximation of the crude protein fraction of the cells. This nitrogen fraction represented approximately 79-80 per cent of the total cellular nitrogen. The exponential character of residual cellular nitrogen strongly indicated that no pool of soluble nitrogen intermediates accumulated in the cells prior to nuclear division (Fig. 3). Other extraction procedures also failed to reveal any accumulation of soluble reduced nitrogen immediately prior to nuclear division (Table I). The possibility still remains, however, that composition of the soluble nitrogen pool changes during cellular development. Daughter cells coming out of a dark period usually contained less soluble nitrogen than daughter cells which were formed and released in the light. Undoubtedly, the soluble nitrogen pool is utilized for cellular maintenance during dark periods. Trichloroacetic acid extraction at 25°C probably resulted Experimental
Cell &search
23
h4efabolism during synchronous growfh
213
in some hydrolysis of residual cellular nitrogen because continued extraction at this temperature always yielded more soluble nitrogen. At 0°C with trichloroacetic acid only little nitrogen was extractable after 3-4 repeated extractions. In an effort to extract water soluble intermediates which were TABLE I. Extructable
cellular nitrogen during synchronous (C. pyrenoidosa).
Extractable
nitrogen
Time hours
10 % TCA, 25”
10 % TCA, 00
0 3 6 9 12 15 18 21
11.9 20.6 32.6 28.2 30.5 28.6 27.9 34.0
8.2 15.0 22.0 22.0 20.0 21.0 21.0 -
as per cent of total 1 % HAc, distilled water, 80”
10.4 12.1 14.2 15.6 16.5 18.0 20.6 -
cellular
Distilled water, 25”
13.3 4.9 1.7 2.5 3.8 2.1 0.0 -
growth
nitrogen Aqueous EDTA.Ka, 25”
10.0 2.4 6.9 5.0 7.5 6.3 0.0 -
acid insoluble, the cells were extracted with distilled water. To facilitate the water extraction, the sodium salt of ethylenediaminetetraacetic acid was added to chelate the divalent cations which are known to decrease the permeability of cell membranes. As presented in Table I more nitrogen was extractable with aqueous EDTA . Na than with distilled water. Very recent evidence indicates that double distilled water or deionized water can extract as much as 22 per cent of the total cellular nitrogen near the end of cellular development. Extraction of cellular nitrogen with deionized water can be prevented by the addition of Ca2+ to the system. The equivalence between the disappearance of nitrate nitrogen from the culture medium and the increase in total reduced cellular nitrogen is illustrated in Fig. 4. Since the modified micro-Kjeldahl method used to analyze for total cellular nitrogen only measured reduced cellular nitrogen and not oxides of nitrogen, the equivalence of the two curves indicated that nitrate was quickly reduced as it was absorbed and did not accumulate as nitrate. Difficulty was encountered in obtaining a nitrogen balance until the reagent grade potassium nitrate and ammonium sulfate salts were recrystallized three times in deionized water and dried in ~ncuo before use as nitrogen standards. Experimental
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214
R. R. Schmidt
As a further measure of synchronous growth cellular dry weight was analyzed. The increase in cellular dry weight was not a logarithmic or exponential function as was the increase in total cellular nitrogen (Fig. 5). Near the end of a synchronous growth cycle, an odor similar to that of freshly mown grass was evolved from the culture. When the air stream coming 2.4
2.4 t
1
I.40L5-
6
12 --77 HOURS
I8
21
Fig. 4.
Fig. 3. Fig. 3.-Residual cellular nitrogen after cells were extracted acid at 0” during synchronous growth (C. pyrenoidosa).
9
with
10 per cent tricholoracetic
Fig. 4.-Total reduced cellular nitrogen and nitrate nitrogen uptake during synchronous growth (C. p~w~oidosa). To each value for nitrate nitrogen uptake was added the initial concentration of total reduced cellular nitrogen. o-O-0, cellular nitrogen; A-A-A, nitrate nitrogen uptake.
from the culture was bubbled through 1 N sulfuric acid, approximately 0.03 to 0.05 per cent of the total nitrogen of the culture was trapped. White needlelike crystals were precipitated from the acid solution by ether. The crystals are being collected for future identification. Measurement of phosphate uptake indicated that the time and amount of phosphate absorbed by the cells during synchronous growth was related to the nitrogen source (Fig. 6). When ammonium was the sole nitrogen source, the cells not only absorbed a larger quantity of phosphate than cells cultured on nitrate nitrogen but they absorbed it earlier in cellular development. The rate of phosphate absorption decreased during nuclear and cellular division on both nitrogen sources. Periodicity in phosphate metabolism has also been observed in synchronously dividing cultures of the protozoan, Tetrahymena. Hamburger and Zeuthen [23] observed that the rate of absorption of 32P was highest before and lowest immediately after synchronous division. Plesner [13] observed that the cellular adenosine triphosphate concentration reached a maximum before and a minimum after cellular division. Experimenlal
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215
Metabolism during synchronous growth The phate, or two During 20 or
cells were stained with toluidine blue, a stain specific for polyphosduring synchronous growth. Daughter cells were found to contain one staining granules tentatively identified as containing polyphosphate.. periods of high phosphate uptake numbers of granules increased to more per cell. It seemed likelyithat the one or two polyphosphate
Fig. 6.
Fig. 5. Fig. 5.-Cellular No. 1; A---A--A, Fig. 6.-Effect nitrogen; Q-0,
dry weight during Exper. No. 2.
synchronous
growth
(C. pyrenoidosa).
Q-O-0,
of nitrogen source on phosphate nitrate nitrogen.
uptake
(C. pyrenoidosa).
A-A,
0+
BtT
15
I8
Exper. ammonium
21
HOURS
Fig. 7.-Effect of phosphate starvation medium; A-f&-A, no phosphate.
on synchronous
growth (C. pyrenoidosa).
O-0,
complete
granules in daughter cells could serve as a phosphagen for a certain amount of cellular growth. To test this hypothesis, synchronized daughter cells were placed on phosphate deficient culture medium with nitrate as the nitrogen source. Synchronous growth of the phosphate deficient culture was compared to that of a culture on complete medium (Fig. 7). Growth in the two cultures was almost parallel up to the stage of nuclear division when the growth of Experimenial
Cell Research 23
R. R. Schmidt the phosphate-deficient culture practically ceased with only a few cells actually completing nuclear or cellular division. Nihei [ 11 ] observed that Chlorella ellipsoidea accumulated phosphorus, chemically identified as polyphosphate, by a photosynthetic phosphorylation process occurring during the latter stages of cellular development. This photosynthetic phosphorylation process probably corresponds to the period of high phosphate uptake and metachromatic granule accumulation prior to nuclear division in Chlorella pyrenoidosa. Sal1 et al. [15] found, by cytochemical and microchemical procedures, that polyphosphate accumulations were highest immediately prior to and lowest after cell division of synchroMudd et al. [lo] demonstrated that nously dividing cells of Corynebcrcterium. polyphosphate could be used for nucleic acid synthesis and cellular growth for Mycobncterium. These present observations indicate that polyphosphate probably also plays an important role in the biochemistry of cell growth and division of Chlorella
pyrenoidosa.
SUMMARY
Cultures of the alga Chlorella pyrenoidosa have been synchronized and examined for shifts in metabolism during cellular development. Total cellular nitrogen, crude protein nitrogen, and nitrate uptake increased exponentially, while dry weight did not increase exponentially during synchronous growth. The air stream coming from the culture was found to contain an unidentified organic nitrogen compound. Cytochemical and physiological evidence indicated that polyphosphate accumulated in the cells and was important for cellular growth and division. The author wishes to acknowledge his indebtedness to Dr. Robert Krauss, Department of Botany of the University of Maryland, in whose laboratory the study was initiated and to Drs. R. W. Engel and K. W. King for encouraging the continuance of the research in the Department of Biochemistry and Nutrition of the Virginia Polythechnic Institute. Gratitude is also extended to Drs. W. E. C. Moore and M. S. Read for their review and criticisms of the manuscript. REFERENCES 1. 2. 3. 4.
HASE, E., MORIMURA, Y., MIHARA, S. and TAMIYA, I-I., Arch. Mikrobiol. 32, 87 (1958). HASE, E., MORIMURA, Y. and TAMIYA, H., Arch. Biochem. nnd Biophys. 69, 149 (1957). HASE, E., OTSUKA, H., MIHARA, S. and TAMIYA, H., Biochim. et Biophys. Acta 35, 180 (1959). IWAMURA, T., J. Rio&m. Japan 42, 575 (1955).
Experimenlal
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Metabolism during synchronous growth
217
5. IWAMURA, T., HASE, E., MORIMURA, Y. and TAMIYA, H., A. I. Virtanen Homage Volume. Biochemistry of Nitrogen, p. 89. Helsinki, Finland, i955. 6. JOHNSON, C. M. and ULRICH, A., Anal. Chem. 22, 1526 (1950). 7. JOHNSON, M. J., J. Biol. Chem. 137, 575 (1941). 8. KANAZAWA, T., J. Gen. Appl. Microbial. 4, 102 (1958). 9. KITSON, R. E. and MELLON, M. G., Ind. Eng. Chem. Analyt. Ed. 16, 379 (1944). IO. MUDD, S., YOSHIDA, A. and KOIKE, M., J. Bacteriot. 75, 224 (1958). 11. NIHEI, T., J. Biochem. Japnn 42, 245 (1955). 12. NIHEI, T., SASA, T., MIYACHI, S., SUZUKI, K. and T.%MIY.~, H., Arch. Mikrobiol. 21, 155 (1954). 13. PLESSER, P. E., Exptl. Cell Research 29, 462 (1958). 14. RICHARD, L. A., ed., U.S. Dept. Agr., Agriculture Handbook 60, 160 (1956). 15. SALL, T., MUDD, S. and TAKAGI, A., J. Bacteriot. 76, 640 (1958). 16. SCHAECHTER, M. and DELAMATER, E. D., Am. J. Botany 42, 417 (1955). 17. SCHMIDT, R. R., M.S. Thesis in Botany, Univ. of Mary&d, College Park, Md., 1957. 18. SOROKIN, C., PhgsioZ. Pkmtarum 10, 659 (1957). 19. SOROKIN, C. and MYERS, J., J. Gen. Physiol. 40, 579 (1956). 20. SPENCER, H. T., %LS. Thesis in Biochemistry, Virginia Polytechnic Institute, Blacksburg, Va., 1960. 21. SWANN, M. H. and ADAIMS, M. L., Anal. Chem. 28, 1630 (1956). 22. TAMIYA, H., IWAMURA, T., SHIBATA, K., HASE, E: and Gu,H& T., Biochim. et Biophys. Aetu 12, 23 (1953). 23. ZEUTHEN, E.., Advances in Biot. and Med. Phys. 6, 37 (1958).
Experimental
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