Environmental Conditions and the Pattern of Metabolism in Algae

G. E. Fogg

Department of Botany Westfield College London, England

A single strain of an algal species may show remarkable varia­tion in the intensity and pattern of its metabolic activitie'l according to the conditions to which it is exposed. This is sometimes obvious to the unaided eye-as, for example, when a green, actively growing culture of Botryococcus braunii is compared with a nitrogen-deficient one in which the cells have accumulated carotenoids and lipoids­and impressive evidence of it is given by many sets of analytical data for Chlorella and other unicellular algae. Nevertheless this variability is often overlooked, especially in biochemical studies on algae, and we are far from a complete understanding of how it occurs.

Much is known of the effects of conditions such as light inten­sity, temperature, and hydrogen ion concentration on the rates of individual metabolic processes and on their final expression in terms of growth in cell numbers; yet we are largely ignorant of the differential effects that these conditions may have on different processes. Sorokin (1960) discussed the effect on the over-all growth rate of Chlorella of the different effects of light and temperature on accumulation of cell material and on cell division but did not consider effects on metabolic patterns. Spoehr and Milner (1949), in an important paper, showed that the chemical composition of Chlorella, expressed in terms of carbohydrate, fat, and protein, varies according to the light intensity and temperature at which cultures are grown; yet, as they pointed out, the time factor must be taken into account in evaluating such results. If cultures are made in a limited volume of medium and the cell material is

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D. F. Jackson (ed.), Algae and Man© Plenum Press 1964

78 Fogg

harvested after a relatively long period of growth it is difficult to distinguish between primary and secondary effects when assessing the effect of a given factor on the balance of metabolic processes. For example, temperature undoubtedly has direct effects; in cul­tures that have grown for some time, however, these effects may be masked by those produced by the different effective light inten­sities and nutrient deficiencies resulting from the different cell populations that have developed at the different temperatures. The method of continuous culture enables the direct effects of environ­mental conditions on metabolic pattern to be studied but, although Myers (1946) used this method to determine the effects of light intensity on various cellular characteristics of Chlorella, this tech­nique has not been used, as far as I am aware, for any detailed studies of this sort.

The extent of the variation which may occur is perhaps best illustrated by some results of Fogg and Than-Tun (1960) for the nitrogen-fixing blue-green alga Anabaena cylindrica, which, although not obtained with continuous cultures, were obtained with cultures grown for a sufficiently short period (48 hr) for secondary effects arising from different rates of growth to be at a minimum. Cultures were grown at temperatures of 15, 20, 30 and 35C and light inten­sities of 2,000, 5,500, and 10,000 lux, in a purely mineral medium with elementary nitrogen as the only nitrogen source, and the changes in cell carbon and nitrogen determined. The assimilation of carbon showed the expected relationships to light intensity and temperature, with saturation at 5,500 lux, temperature limitation at 20C and below, and no inhibition at either the highest light in­tensity or the highest temperature. The assimilation of nitrogen showed a high temperature coefficient (QIO= 5 for the range 20-30C) but was strongly inhibited at 35C; it showed much the same rela­tionship to light intensity as did carbon assimilation-in agreement with the idea that nitrogen fixation in blue-green algae is closely dependent on photochemical reactions. Thus the ratio of carbon assimilated to nitrogen assimilated varied little with respect to light intensity but greatly with respect to temperature, being 40 or more at both 15 and 35C as compared with 10 at 30C, the optimum tem­perature for nitrogen assimilation. As a result the nitrogen content, expressed as a percentage of total dry weight, of cells which in all cases were actively growing varied from about 2.3 at 15C to 4.5 at 30C. These results are for a nitrogen-fixing alga; a parallel study

Environmental Conditions and the Pattern of Metabolism in Algae 79

with the organism growing on combined nitrogen was not carried out, but it is thought that the observed changes in rate of nitrogen assimilation result from effects on the later stages rather than on the fixation process. They may thus perhaps be taken as indicating that, in general, changes in temperature may produce marked alterations in the balance of the major anabolic processes in algae.

Changes in the supply or consumption of metabolites may have considerable effects on metabolic pattern, quite apart from those on the over-all rate of metabolism. The processes of intermediary metabolism are effected by reversible reactions and although we arbitrarily distinguish sequences of reactions such as glycolysis, the tricarboxylic acid cycle, and the carbon dioxide fixation cycle, these are so intermeshed through common intermediates as to form a single flexible system through which material can flow along various paths, largely according to supply and demand. The con­cepts of "overflow" and "shunt" metabolism which Foster (1949) put forward to explain organic acid production and other metabolic features of fungi are equally applicable to algae.

In an algal population growing exponentially, synthesis of pro­teins and other protoplasmic constituents predominates and directly utilizes intermediates of the photosynthetic carbon cycle. There is little accumulation of cell-wall materials or of reserve products, nitrogenous or nonnitrogenous. Transfer of such cells to a medium lacking a nitrogen source, as is commonly done for experiments on photosynthesis, does not have any immediate effect on the quantum efficiency or rate of photosynthesis but results in a drastic diver­sion of the intermediates produced to pathways other than those of protein synthesis. The enzyme system leading to carbohydrate synthesis accepts the major part of this overflow, as is shown both by the value of the photosynthetic quotient under such conditions and by direct analysis (for references see Fogg, 1959). More de­tailed evidence exists regarding the change in the pathway of carbon occurring when a nitrogen supply is restored to such cells. Fogg (1956) showed that the supply of ammonium nitrate to photo­synthesizing nitrogen-deficient cells of the diatom Navicula pellicu­losa brought about a dramatic change in the distribution among various cell fractions of radiocarbon supplied as bicarbonate, 87% of the carbon fixed then entering the fraction soluble in 80°;'; ethanol .but insoluble in benzene as compared with 24% in cells with no sup­plied nitrogen source. Holm-Hansen et al. (1959) carried out similar

80 Fogg

experiments with Chlorella, identifying individual intermediates by autoradiography. They found, for example, that in one experiment the supply of ammonium chloride increased the pro­portion of radiocarbon fixed in amino acids from 9.9 to 57% of the total while the radiocarbon incorporated in sugar phosphates correspondingly fell from 64 to 7%. That these switches should occur seems obvious now, yet until comparatively recently it was tacitly assumed that the pathway of carbon was the same in growing cells as in cells in a medium devoid of a nitrogen source. From this assumption arose the idea, still widely held, that carbohydrate is always the immediate product of photosynthesis.

A less obvious effect of the same sort is perhaps involved in the excretion of glycolic acid from photosynthesizing cells. Using radiocarbon as a tracer, Tolbert and Zill (1957) found that during short-term photosynthesis experiments Chlorella pyrenoidosa liberates glycolic acid as an extracellular product, its concentration in actively growing cultures reaching 3 to 8 mg/liter. Pritchard, Griffin, and Whittingham (1962), who have made a detailed study of the production of glycolate by Chlo rella , conclude that it is derived from the carbon dioxide acceptor in photosynthesis, ribu­lose diphosphate, and that it accumulates only when photosynthesis is carbon dioxide limited. One would expect that glycolate would escape from the photosynthesizing cells until equilibrium was estab­lished between the intracellular and extracellular concentrations and it seems possible that under some circumstances glycolate might be the sole product of photosynthesis. Warburg and Krippahl (1960) have indeed reported a stoichiometric equivalence between glycolate excreted and carbon dioxide taken up by Chlorella such that one mole of glycolate is produced for every two moles of carbon dioxide assimilated. This would be expected to happen when cells begin photosynthesis in fresh medium. Only when steady dif­fusion gradients of glycolate have been established can the assimi­latory power of the photochemical reaction become available for the synthesis of materials for the production of new protoplasm. Nalewajko, Chowdhuri, and Fogg (1963) have found that con­centrations of glycolate of the order of 1 mg/liter do, in fact, abolish the lag phase in the growth of a planktonic strain of Chlorella pyrenoidosa under light-limited conditions. This addition has no appreciable effect on the relative growth rate, whereas additions of substances such as glucose, acetic acid, and pyruvic acid under

Environmental Conditions and the Pattern of Metabolism in Algae 81

similar conditions increase the relative growth rate but have no effect on the lag. Production of glycolate rather than carbohydrate may result in discrepant results in determinations of the quantum efficiency of photosynthesis. It may also have its ecological impli­cations; phytoplankton may not be able to begin growth until steady diffusion gradients of glycolate have been established around the cells. This might partly explain the abruptness with which growth begins in spring in temperate waters and also its dependence on decrease in turbulence.

The availability of particular metabolites in the environment may thus have considerable immediate effects on metabolic pattern but nevertheless this pattern will be determined primarily by the relative activities of the various enzyme systems in the organism. The availability of a particular metabolite may, however, have longer term effects by producing alterations in the balance of these enzyme systems. This is most clearly illustrated by the effects of nitrogen deficiency. As we have seen, carbohydrate is the major product of metabolism immediately following the withdrawal of the nitrogen supply but it is clear that fat synthesis comes to predomi­nate in most algae if the deficiency continues for several days.

The changes occurring during prolonged incubation in a medium with no nitrogen source, but otherwise complete, have been the subject of studies with Chlorella spp. (e.g., Bongers, 1956), Navicula pelliculosa (Fogg, 1956), and Monodus subterraneus (Fogg, 1959). The total amounts of protein and nucleic acids in such cultures remain constant or increase slightly at the expense of the soluble nitrogenous fraction of the cells and other constituents such as chlorophyll. The rate of photosynthesis declines and after a few days remains more or less constant at a value about 5% of that shown initially by the cell suspension. As a result of this continued photosynthesis the total dry weight of cell material increases and, cell division being limited, there is an increase in dry weight per cell. The proportion of fat synthesized shows no immediate altera­tion, evidently being limited by enzyme activity rather than by avail­ability of intermediates. After about three days of nitrogen starva­tion the fat content on a percentage dry weight basis begins to increase. This change coincides with a climacteric in respiration and is evidently the result of a partial breakdown in organization of the cells. In Navicula the amount of fat expressed on a cell volume basis shows no increase and radiocarbon studies show a fall in the

82 Fogg

proportion of photosynthetically fixed carbon entering the fat frac­tion at this stage. It seems that the observed increase in fats on a dry weight basis is mainly the result of hydrolysis of other materials and their consequent loss by respiration or diffusion from the cells, the fats themselves remaining unchanged. In Chlorella and Monodus, however, there is a definite increase in the total amount of fat in cultures at this stage. The explanation of this is perhaps that a differential inactivation of enzymes has occurred with the result that the fat-synthesizing system is better able to compete for the available intermediates. Even in Navicula there appears to be some such change since nitrogen-starved cells of this diatom returned to a medium containing nitrate were found to continue to synthesize the same high proportion offat for 24 hr although the rate of photo­synthesis was nearly trebled. Only after two days in the nitrate medium, by which time, we may suppose, synthesis of fresh enzyme had altered the original balance, did it fall to that characteristic of cells grown with an adequate nitrogen supply.

Metabolic changes in the later stages of cultures in which growth is limited by the nitrogen supply are not necessarily of the same nature as those which occur when exponentially growing cells are transferred to a nitrogen-deficient medium. Among oJher things, the presence of metabolic products in the medium in the former situation and their absence in the latter may lead to important differences. Both direct analysis (e.g., Spoehr and Milner, 1949; Collyer and Fogg, 1955; Bongers, 1956) and tracer studies (Fogg, 1956) show that fat accumulation is characteristic of unicellular species belonging to the Chlorophyceae, Xanthophyceae, and Bacillariophyceae under such conditions. There does not seem to be any preliminary phase in which carbohydrate synthesis predomi­nates, for this falls off concurrently with protein synthesis. The accumulation of fat is not directly determined by the concentration of available nitrogen (Fogg, 1959) and, again, seems to depend largely on the enzymic balance of the cells. The possibilities of alteration in enzymic balance appear to be rather different in ex­ponentially growing cells transferred to a nitrogen-deficient medium from those in cells in a culture in which the nitrogen supply has been exhausted in the course of growth. As Tamiya and his colla­borators have shown, the pattern of metabolism of Chlorella under­goes marked changes during the cycle of cell development and division. An exponentially growing population consists largely of

Environmental Conditions and the Pattern of Metabolism in Algae 83

the cell type distinguished by Tamiya et ai. (1953) as D-cells, the transformation of which into L-cells is not dependent on a nitrogen source. The L-cells produced in the nitrogen-deficient medium can undergo division but the second generation of D-cells is unable to develop normally and the metabolic pattern becomes fixed at that characteristic of this developmental stage, with carbohydrate syn­thesis predominating (Nihei et aI., 1954). Cells in a culture in which the nitrogen supply becomes exhausted as a result of growth, on the other hand, become arrested in the L-stage, perhaps because cell division is more sensitive than other processes to the by­products of metabolism which accumulate.

Nihei et ai. (1954) found that whereas the photosynthetic quotient (02 / -C02) of D-cells is about unity, that of L-cells may be as much as 3.3, indicating that the products of metabolism at this stage are more reduced than they are at other stages. If it is correct that fat accumulation in nitrogen-deficient cultures is the result of the halting of the developmental cycle at a point at which fat synthesis predominates, then other factors stopping the cycle at tht: same point should have a similar effect. It is not clear whether this is so. Phosphorus deficiency brings cell development to a stand­still apparently at this stage but may not always induce fat accumu­lation (Spoehr and Milner, 1949).

Otsuka (1961), however, has found that sulphur-deficient cells of Chiarella, which become arrested at a stage when some cell ex­tension has occurred and the nucleus has divided once, accumulate abnormally large amounts of fat. Also, von Denffer (1948) observed that fat accumulation in Nitzschia palea was the result of the accu­mulation of a staling product which blocked mitosis and was not necessarily confined to conditions of nitrogen deficiency. Probably, in addition to the effect of halting cyclic changes in enzymic balance at a particular point, other factors are involved in fat accumulation. Synthesis and degradation of enzymes in cultures of limited volume take place under the progressively modifying influence of falling nutrient concentrations and rising concentration of staling products. As in populations transferred abruptly to con­ditions of nitrogen deficiency, the activity of the fat-synthesizing system may be less susceptible to such adverse conditions than is that of other competing systems. Nitrogen deficiency evidently does not induce fat accumulation in Myxophyceae and Rhodo­phyceae (Collyer and Fogg, 1955), but it is not known what the

84 Fogg

basis of this difference from the other algal groups is. Finally there are the changes in metabolic pattern resulting

from adaptive enzyme formation. The classic instance of the ap­pearance of a new pattern of metabolism in an alga following its adaptation to a new environment-the hydrogenase activity of hydrogen-adapted Scenedesmus (Gaffron, 1940)-is probably a matter of activation of an already existing enzyme rather than of the synthesis of new enzyme molecules, since similar adaptation in Chlamydomonas moewusii has an adaptation period ofless than 10 min (Frenkel and Rieger, 1951). A few instances of adaptation by algae to carbohydrate substrates have been reported (for references see Fogg, 1953), and Anabaena cylindrica evidently needs to be adapted to nitrate before it can be utilized (Fogg and Wolfe, 1954), but in no case does any very thorough investigation of the adapta­tion appear to have been carried out. The indications are that algae are as well able to produce adaptive enzymes as other microorgan­isms; investigations of this question should be rewarding.

LITERATURE CITED

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Collyer, D. M., and Fogg, G. E. 1955. Studies on fat accumulation by algae. J. Exp. Bot. 6:256-275.

Denffer, D. von. 1948. Uber einen Wachstumshemmstoff in alternden Diatomeen­kulturen. BioI. Zentr. 67:7-13.

Fogg, G. E. 1953. The metabolism of algae. Methuen and Co. Ltd., London. Fogg, G. E. 1956. Photosynthesis and formation of fats in a diatom. Ann. Bot. 20:

265-285. Fogg, G. E. 1959. Nitrogen nutrition and metabolic patterns in algae. Symp. Soc.

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(Myxophyceae). Symp. Soc. Gen. Microbial. 4:99-125. Foster, J. W. 1949. Chemical activities of fungi. Academic Press, Inc., New York. Frenkel, A. W., and Rieger, C. 1951. Photoreduction in algae. Nature 167:1030. Gaffron, H. 1940. Carbon dioxide reduction with molecular hydrogen in green algae.

Am. J. Bot. 27:273-283. Holm-Hansen, 0., et al. 1959. Effects of mineral salts on short-term incorporation

of carbon dioxide in ChIarella. J. Exp. Bot. \0:\09-124. Myers, J. 1946. Culture conditions and the development of the photosynthetic

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Environmental Conditions and the Pattern of Metabolism in Algae 85

Nihei, T., et al. 1954. Change of photosynthetic activity of Chlorella cells during the course of their normal life cycle. Arch. Mikrobiol. 21 :155-164.

Otsuka, H. 1961. Changes oflipid and carbohydrate contents in Chlorella cells during the sulfur starvation, as studied by the technique of synchronous culture. J. Gen. Appl. Microbiol. 7:72-77.

Pritchard, G. G., Griffin, W. J., and Whittingham, C. P. 1962. The effect of carbon dioxide concentration, light intensity and isonicotinyl hydrazide on the photosyn­thetic production of glycolic acid by Chlorella. J. Exp. Bot. 13: 176-184.

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Spoehr, H. A., and Milner, H. W. 1949. The chemical composition of Chlorella; effect of environmental conditions. Plant Physiol. 24: 120-149.

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Tolbert, N. E., and Zill, L. P. 1957. Excretion of glycolic acid by Chlorella during photosynthesis, pp. 228-231. In H. Gaffron (ed.). Research in Photosynthesis. Interscience Publishers, Inc., New York.

Warburg, 0., and Krippahl, G. 1960. Glykolsaurebildung in Chlorella. Z. Natur­jorschung. 5b:197-199.