Planta (Berl.) 75, 161--163 (1967)
The Effect of Peptone on the Growth of Heterotrophic Cultures of Chlorella vulgaris (Emerson Strain)* D. J. GRIrrITnS Received February 21, 1967
Summary. Peptone is more effective than nitrate as a nitrogen source for heterotrophic cultures of Chlorella vulgaris (Emerson strain). It allows the production of an increased amount of algal material and supports an enhanced rate of cell division. Peptone-supplied heterotrophic cultures have a significantly higher content of protein and of soluble nitrogenous substances. The relation between these observations and the growth behaviour of this strain is referred to. The E m e r s o n strain of Chlorella vulgaris has been well documented for its inability to perform active cell division when cultured in the dark on a medium containing glucose (FINKLE et al., 1950; GRIFFITHS, 1961; KAI~LANDEIr and K~Avss, 1962; KILLAM and MYERS, 1954). I n an a t t e m p t to overcome the "dark-block" KILLAM and MYwns (1954) added various substances to the medium. Of the substances tried, only casein-hydrolysate (acid-hydrolysed, vitamin-free) was found b y t h e m to have a significant effect, producing a marked increase (up to ten fold) of the mean cell volume. As part of an investigation of certain aspects of the nitrogen metabolism of this strain (Cambridge collection No. 211/11 n), peptone (BDH, bacteriological) was tested for its ability to sustain growth and cell division under heterotrophic conditions. The peptone was supplied at a concentration of 0.5 per cent. in an otherwise nitrogen-free inorganic medium supplemented b y 0.05 M glucose, and the cultures aerated (10 ccs/min) in the dark for seven days at 22 o C (G~IF~THS, 1965). B y initiating experiments with a relatively high initial population of " s t a r v e d " cells (i.e. cells kept in the dark on an inorganic medium before inoculation) it is possible to eliminate cell division almost entirely from nitrate-supplied (0.025 M potassium nitrate) heterotrophic cultures, but still allow cell growth to continue. Under these conditions, peptone is a far more effective nitrogen source t h a n nitrate (Table 1). I t allows the production of a greatly increased a m o u n t of algal material as measured b y d r y weight (dried at 100 ~ C to constant weight) or total * The experimental work described in this communication was carried out whilst the author was on the staff of the Botany Department, University College of North Wales, Bangor.
162
D.J. GmFFIT~S:
Table 1. A comparison o/ the growth o/ nitrate- and peptone-supplied heterotrophic cultures
Mean population density (106 X cells/ml) Algal volume (Iza x 106/ml) Algal d.ry weight (rag x 10-3/ml)
l~itrate cultures
Peptone cultures
0.266 219 (823)* 169 (6.4)**
3.136 1,522 (485)* 811 (2.6)**
* Mean cell volume (>a). ** Mean cell dry weight (mg • 10-7).
volume (calculated from the population density and mean cell diameter, measured microscopically). More significantly, however, peptone also supports an enhanced rate of cell division, reflected as an increase in population density and a decrease in mean cell volume and dry weight. Table 2. Nitrogen content o/autotrophic and heterotrophic cultures Nitrogen content expressed as a percentage on a dry weight basis
Autotrophic cultures tteterotrophic cultures: (a) nitrate-supplied (b) peptone-supplied
Soluble nitrogen
Insoluble nitrogen
1.2
7.4
0.2 0.9
1.3 2.6
The nitrogen content of the cells was estimated as ammonia, after micro-Kjeldahl digestion, by a colorimetrie method using Nessler reagent (BDH). Table 2 shows the nitrogen content of nitrate- and peptonesupplied heterotrophic cultures compared with t h a t of corresponding actively growing autotrophie cultures growing in the light on an inorganic nitrate medium. Assuming t h a t the insoluble nitrogen is derived largely from cell protein, it is clear t h a t whilst in nitrate-supplied heterotrophic cultures, protein synthesis has lagged well behind the synthesis of the non-protein component, in the peptone-supplied heterotrophic cultures the lag is appreciably less. The difference between the two types of heterotrophic cultures is even more marked when the soluble nitrogen (extracted with 0.1 per cent. trichloracetie acid at 80~ for three separate periods of 15 rain) is considered, the value in peptone cultures falling only slightly below t h a t in the autotrophic cultures. Estimation of nitrate/nitrite nitrogen as ammonia following reduction by Devarda's alloy showed t h a t there is an insignificant accumulation of these forms
The Effect of Peptone on the Growth of Chlorella
163
of nitrogen (less t h a n 0.02 per cent. of the dry weight) under all three culture conditions. The Emerson strain has been reported as having a m a x i m u m growth rate in the light of 2.6 doublings per day (KARLANDER and KlCAUSS, 1966) and values of this order have been obtained b y the author over short periods. Under the present conditions, however, the average daily rate of growth in autotrophie cultures over the seven d a y growth period is m u c h lower. The values for population density, total algal volume, d r y weight and nitrogen all lie between 0.6 and 0.7. I n nitrate-supplied heterotrophic cultures, the d r y weight is the only component to increase at this rate (0.62) whilst the corresponding value in peptone-supplied heterotrophic cultures is 0.86. Total nitrogen and population density have a very low rate of doubling (0.26 and 0.08 respectively) in nitratesupplied heterotrophie cultures. W i t h peptone, the corresponding values are 0.48 and 0.29. Clearly, active cell division and nitrogen assimilation in this strain require some specific kind of nitrogenous or other materials which are limiting during heterotrophie growth on a nitrate medium but which, under the conditions specified here, are at least partly available from peptone. A more detailed investigation of the nature of the peptone effect is in progress. References
FI~:LE, B. J., O. APt'L]~)IA~, and F. K. ~LEISCtKER: Growth of Chlorella vulgaris in the dark. Science l l l , 309 (1950). GRI~FITHS, D. J. : Light-induced cell division in Chlorella vulgaris, BEIJEEINCK (Emerson strain). Ann. Bot., N. S. 25, 85 (1961). - - The accumulation of carbohydrate in Chlorella vulgaris under heterotrophie conditions. Ann. Bot., N. S. 29, 347 (1965). KARLANDEE, E.P., and R. W. KR~uss: Effect of monochromatic light on the growth of Chlorella vulgaris. Proc. Ann. Meetings, Plant Physiol. 37, 1962). -- - Responses of heterotrophic cultures of Chlorella vulgaris BEYE~I~CK tO darkness and light. I. Pigment and pH changes. Plant Physiol. 41, 1 (1966). K~LA~I, A., and J. ~[YERS: A special effect of light on the growth of Chlorella vulgaris. Carnegie Inst. Washington Ybk. 54, 167 (1954). Dr. D. J. GI~IFFITttS School of Biological Sciences La Trobe University Melbourne, Australia
]2a
Planta (BerI.), Bd. 75
The Effect of Peptone on the Growth of Heterotrophic Cultures of Chlorella vulgaris (Emerson Strain)* D. J. GRIrrITnS Received February 21, 1967
Summary. Peptone is more effective than nitrate as a nitrogen source for heterotrophic cultures of Chlorella vulgaris (Emerson strain). It allows the production of an increased amount of algal material and supports an enhanced rate of cell division. Peptone-supplied heterotrophic cultures have a significantly higher content of protein and of soluble nitrogenous substances. The relation between these observations and the growth behaviour of this strain is referred to. The E m e r s o n strain of Chlorella vulgaris has been well documented for its inability to perform active cell division when cultured in the dark on a medium containing glucose (FINKLE et al., 1950; GRIFFITHS, 1961; KAI~LANDEIr and K~Avss, 1962; KILLAM and MYERS, 1954). I n an a t t e m p t to overcome the "dark-block" KILLAM and MYwns (1954) added various substances to the medium. Of the substances tried, only casein-hydrolysate (acid-hydrolysed, vitamin-free) was found b y t h e m to have a significant effect, producing a marked increase (up to ten fold) of the mean cell volume. As part of an investigation of certain aspects of the nitrogen metabolism of this strain (Cambridge collection No. 211/11 n), peptone (BDH, bacteriological) was tested for its ability to sustain growth and cell division under heterotrophic conditions. The peptone was supplied at a concentration of 0.5 per cent. in an otherwise nitrogen-free inorganic medium supplemented b y 0.05 M glucose, and the cultures aerated (10 ccs/min) in the dark for seven days at 22 o C (G~IF~THS, 1965). B y initiating experiments with a relatively high initial population of " s t a r v e d " cells (i.e. cells kept in the dark on an inorganic medium before inoculation) it is possible to eliminate cell division almost entirely from nitrate-supplied (0.025 M potassium nitrate) heterotrophic cultures, but still allow cell growth to continue. Under these conditions, peptone is a far more effective nitrogen source t h a n nitrate (Table 1). I t allows the production of a greatly increased a m o u n t of algal material as measured b y d r y weight (dried at 100 ~ C to constant weight) or total * The experimental work described in this communication was carried out whilst the author was on the staff of the Botany Department, University College of North Wales, Bangor.
162
D.J. GmFFIT~S:
Table 1. A comparison o/ the growth o/ nitrate- and peptone-supplied heterotrophic cultures
Mean population density (106 X cells/ml) Algal volume (Iza x 106/ml) Algal d.ry weight (rag x 10-3/ml)
l~itrate cultures
Peptone cultures
0.266 219 (823)* 169 (6.4)**
3.136 1,522 (485)* 811 (2.6)**
* Mean cell volume (>a). ** Mean cell dry weight (mg • 10-7).
volume (calculated from the population density and mean cell diameter, measured microscopically). More significantly, however, peptone also supports an enhanced rate of cell division, reflected as an increase in population density and a decrease in mean cell volume and dry weight. Table 2. Nitrogen content o/autotrophic and heterotrophic cultures Nitrogen content expressed as a percentage on a dry weight basis
Autotrophic cultures tteterotrophic cultures: (a) nitrate-supplied (b) peptone-supplied
Soluble nitrogen
Insoluble nitrogen
1.2
7.4
0.2 0.9
1.3 2.6
The nitrogen content of the cells was estimated as ammonia, after micro-Kjeldahl digestion, by a colorimetrie method using Nessler reagent (BDH). Table 2 shows the nitrogen content of nitrate- and peptonesupplied heterotrophic cultures compared with t h a t of corresponding actively growing autotrophie cultures growing in the light on an inorganic nitrate medium. Assuming t h a t the insoluble nitrogen is derived largely from cell protein, it is clear t h a t whilst in nitrate-supplied heterotrophic cultures, protein synthesis has lagged well behind the synthesis of the non-protein component, in the peptone-supplied heterotrophic cultures the lag is appreciably less. The difference between the two types of heterotrophic cultures is even more marked when the soluble nitrogen (extracted with 0.1 per cent. trichloracetie acid at 80~ for three separate periods of 15 rain) is considered, the value in peptone cultures falling only slightly below t h a t in the autotrophic cultures. Estimation of nitrate/nitrite nitrogen as ammonia following reduction by Devarda's alloy showed t h a t there is an insignificant accumulation of these forms
The Effect of Peptone on the Growth of Chlorella
163
of nitrogen (less t h a n 0.02 per cent. of the dry weight) under all three culture conditions. The Emerson strain has been reported as having a m a x i m u m growth rate in the light of 2.6 doublings per day (KARLANDER and KlCAUSS, 1966) and values of this order have been obtained b y the author over short periods. Under the present conditions, however, the average daily rate of growth in autotrophie cultures over the seven d a y growth period is m u c h lower. The values for population density, total algal volume, d r y weight and nitrogen all lie between 0.6 and 0.7. I n nitrate-supplied heterotrophic cultures, the d r y weight is the only component to increase at this rate (0.62) whilst the corresponding value in peptone-supplied heterotrophic cultures is 0.86. Total nitrogen and population density have a very low rate of doubling (0.26 and 0.08 respectively) in nitratesupplied heterotrophie cultures. W i t h peptone, the corresponding values are 0.48 and 0.29. Clearly, active cell division and nitrogen assimilation in this strain require some specific kind of nitrogenous or other materials which are limiting during heterotrophie growth on a nitrate medium but which, under the conditions specified here, are at least partly available from peptone. A more detailed investigation of the nature of the peptone effect is in progress. References
FI~:LE, B. J., O. APt'L]~)IA~, and F. K. ~LEISCtKER: Growth of Chlorella vulgaris in the dark. Science l l l , 309 (1950). GRI~FITHS, D. J. : Light-induced cell division in Chlorella vulgaris, BEIJEEINCK (Emerson strain). Ann. Bot., N. S. 25, 85 (1961). - - The accumulation of carbohydrate in Chlorella vulgaris under heterotrophie conditions. Ann. Bot., N. S. 29, 347 (1965). KARLANDEE, E.P., and R. W. KR~uss: Effect of monochromatic light on the growth of Chlorella vulgaris. Proc. Ann. Meetings, Plant Physiol. 37, 1962). -- - Responses of heterotrophic cultures of Chlorella vulgaris BEYE~I~CK tO darkness and light. I. Pigment and pH changes. Plant Physiol. 41, 1 (1966). K~LA~I, A., and J. ~[YERS: A special effect of light on the growth of Chlorella vulgaris. Carnegie Inst. Washington Ybk. 54, 167 (1954). Dr. D. J. GI~IFFITttS School of Biological Sciences La Trobe University Melbourne, Australia
]2a
Planta (BerI.), Bd. 75
