THE COMPARATIVE PHYSIOLOGY AND BIOCHEMISTRY OF THE BLUE-GREEN ALGAE G. E. FOGG Department of Botany, University College, London, England

CONTENTS Introduction................................................................................... Cell Physiology ..................................... The Structure of Myxophycean Protoplasm................................................. Resistance to Extremes of Temperature ..................................... Drought Resistance...... ....................................................... Effects of Hydrogen Ion Concentration ..................................... Movement.................................................................................. Nutrition....................................................................................... Mineral Nutrition .....................................

Phototrophy................................................................................ Chemolithotrophy ..................................... Chemo-organotrophy ..................................... Growth-Factor Requirements............................................................... Chemical Composition.......................................................................... General Considerations .....................................

Pigments................................................................................... Carbohydrates.............................................................................. Fats and Lipoids........................................................................... Nitrogenous Substances..................................................................... Discussion...................................................................................... References.....................................................................................

INTRODUCTION The blue-green algae, otherwise known as the Myxophyceae or Cyanophyceae, are in many ways exceptional forms of life. This is most obvious in their cell structure, which differs in many important respects from that of other organisms. Thus, while it is now accepted that blue-green algae deoxypentose nucleic acids and while there is no general agreement on the organization of the region of the protoplast in which these are present, it is clear that they have neither nuclear membranes nor bodies strictly comparable with the chromosomes of other organisms (1-5b). The photosynthetic pigments of bluegreen algae are contained in the peripheral region of the protoplast, or chromatoplasm, which, although it has a lamellar structure resembling that of a chloroplast, differs from the plastids of other algae and of higher plants in having no sharp boundary with the rest of the protoplasm (5a, b). In these features there is some resemblance to the bacteria but blue-green algae differ from these and all other organisms with the ex-

148 148 148 149 151 151 151 152 152 153 154 155 155 156 156 156 158 158 159 160 162

ception of the Rhodophyceae in completely lack. ing flagella (6). The occurrence in certain species of the enigmatical structures known as heterocysts appears to be a cytological peculiarity unique to the Myxophyceae (7). There is much to suggest that the physiology and biochemistry of blue-green algae are as distinctive as their cell structure, but these aspects have received little critical consideration although a moderate amount of relevant information is now available. In this review some of the physiological and biochemical features of blue-green algae will be discussed with the particular object of establishing the extents of the correspondences and differences between them and other organisms. The classification used is that of Fritsch (1), to which authority the reader is also referred for a general account of blue-green algae. CELL PHYSIOLOGY The Structure of Myophycean Protoplasm Detailed accounts of the protoplasm of bluegreen algae being already available (1, 2), it is

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only necessary here to draw attention to its more outstanding features. It is normally in the state of a gel, possessing a high degree of viscosity and showing no streaming, in which respects it contrasts with the protoplasm of most other organisms. Brownian movement of included particles cannot be observed in the healthy protoplast and prolonged action of strong centrifugal forces on an Osclatoria has been found to cause no displacement of the cell contents. Vacuoles are not conspicuous in healthy cells but the protoplasts of many species contain curious structures, socalled pseudovacuoles or gas-vacuoles, which from the available evidence appear to be gasfilled spaces stabilized by the presence of a lipoid membrane (8). Similar gas-vacuoles have not been recorded as present in any other kind of organism with the possible exception of bacteria (one bacterial species in which they have been found, Thiothfrix, is probably a colorless member of te Mxophcea;seshop plasmolysis 54).Thecell of of ofblue-greenhalgaed how the plantsbecause ellm brane usually fails to separate from the protoplast and because large sap-vacuoles are not present. The water relations are further complicated by marked swelling on imbibition of some material, probably myxophycean starch, present mn the protoplasm (2). Among various records of the effects of chemical reagents on myxophycean protoplasm, an observation of Drawert's (2) that a molar solution of urea causes it to form a tougher gel seems to be of particular significance. It is a matter of simple experiment to verify this, preferably using a more concentrated solution of urea, and to show that, by contrast, the protoplasts of other algae are more easily broken up after such treatment. Since concentrated solutions of urea are known to break hydrogen and other secondary bonds between peptide chains this finding suggests that in the protoplasm of blue-green algae the protein chains are linked to a considerable extent by main valence bonds rather than by secondary bonds such as appear to predominate in other kinds of protoplasm. This idea is borne out by the observation that treatment with a one per cent solution of thioglycollate, which disrupts the main valence bond So--S-, renders the protoplasts of blue-green algae more fluid and causes the formation of large vacuoles. It thus appears that in blue-green algae the

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protoplast normally has a rather rigid basic structure of protein molecules linked by main valency bonds, which does not permit streaming and which immobilizes inclusions. Such a structure might account on the one hand for the lack of conspicuous vacuoles, since it would reduce the tendency of these to coalesce, and on the other hand for the formation of gas-vacuoles, since it might reduce the diffusion of gas produced in metabolism sufficiently for the partial pressure to rise locally to the level required for bubbleformation. The absence of relatively large cell organelles, such as nucleus and chromatoplasts, demarcated by distinct membranes is also an indication that the pattern of diffusion within blue-green algae is different from that in other organisms. Resistance to Extremes of Temperature

Blue-green algae are well known for their to withstand extremes of temperature. ability Thus, together with green algae and diatoms, they

are usually conspicuous in collections of algae from polar regions. The abundance of Phormidium and Lyngbya spp. at latitudes of 71 and 78S has been described as "astonishing" (9). Such abundance is evidently achieved by slow growth in the absence of competing or predatory organisms combined with resistance to the unfavorable conditions. The absence of blue-green algae from the flora of snow fields (1, 10) is perhaps to be ascribed to the paucity of nutrients in such a habitat rather than to the low temperatures. On the other hand blue-green algae are the of life in hot springs. Our of these thermal forms has been knowledge g recently by Gesner (10). Investigators have not always appreciated the steepness of the temperature gradients in and around hot springs so that reported temperatures may be quite different from those at which algae are actually growing but, nevertheless, it is certain that many species of blue-green algae live and grow at

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tolerated by the other main group of hot prting algae, the diatoms. In terms of number of species about 35 C is the optimum for development of blue-green algae but there is trustworthy evidence of two species, a Phormidium and an Oscillatoria, persisting at a temperature as high as 85 C while Synechococcus spp. are frequently found

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at temperatures over 60 C. Bacteria are the only other forms of life which can tolerate these physiologically high temperatures. Few laboratory studies of the effects of temperature on the growth of blue-green algae have been made. Bumining and Herdtle (11) obtained growth of thermal species in culture at the following temperatures (all in Centigrade) FischereUa sp., 55; Mastigocladus laminosus, 55; Nostoc muscorum, 55; Oscillaoria anguina, 0. acuminata, 0. formosa and 0. boryana, 50; 0. geminati, 45; Phormidium luridum, 45; P. ambiguum, 48. These temperatures are not necessarily the highest which can be tolerated by these species, since resistance is evidently dependent on factors such as light intensity, carbon dioxide supply (11) and the reaction of the medium. Thus the temperature limit for Mastigocladus laminosus has been found to lie at 40 C at pH 5.5 and 7.3 but to be 54 C at pH 6.5 (Prat, quoted in 10). Determinations of the relative growth constants (k, expressed in logio units per day) of blue-green algae at different temperatures under conditions of light- and carbon dioxide-saturation of growth have been made by Kratz and Myers (12). Anabaena variailis was found to have a maximum value for k, 1.20, at about 35 C, Gerloff's strain of Nostoc muscorum, a maximum value for k, 0.86, at about 32.5 C, while Anabaena cylindrica and Allison's strain of N. muscorum both grew more rapidly at 32.5 C than at 25 C. All four of these algae failed to grow at 41 C but these are non-thermal forms isolated from temperate soils or freshwater; it should be noted that the temperatures which they are able to tolerate are much higher than those for algae of other groups obtained from similar environments. Thus, for most species of Chlorella, 25 C is the optimum for growth and temperatures above 30 C are lethal. A thermal species, Anacystis nidulans, was found by Kratz and Myers (12) to survive at 45 C and to have a maximum of k = 3.55, or a two-hour generation time, at 41 C. This is the highest growth rate yet recorded for an alga. The means whereby blue-green algae are enabled to survive extreme temperatures have been insufficiently studied. Neither in the amino acid composition of their bulked proteins nor in their enzymes do blue-green algae differ radically from other forms of life, and there is no reason to suppose that polar or thermal species are sharply differentiated in such respects from other

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members of the class. In an investigation by paper electrophoresis of enzymes extracted from Oscillooria princeps, Fredrick and Mancini (13) found that a low temperature strain contained a slow moving protein fraction not present in the normal strain but the significance of this observation remains to be determined. Resistance to high temperatures has been supposed to depend on special temperature relations in protoplasmic coagulation. Biinning and Herdtle (11), who found that both the absolute rate and the temperature coefficient of heat-killing of Oscillroa gemirnat, a hot spring alga, were approximately the same at given temperatures as for normal plants, considered that they had disproved this hypothesis. However, as Gessner (10) has pointed out, more extremely thermophilic species than 0. geminata must certainly have protoplasm which coagulates less readily at high temperatures than that of normal plants. Buinning and Herdtle determined the rates of photosynthesis and respiration of 0. geminata and found the temperature coefficients to be of similar magnitudes for corresponding temperature ranges so that assimilation was maintained in excess of respiration with rising temperature. These authors asserted that in this thermophilic alga respiration rate does not increase so rapidly with rising temperature as it does in plants restricted to living at normal temperatures. However, comparison of their values for temperature coefficients for 0. geminata with those for ordinary plants assembled by James (14, table 4) shows this conclusion not to be in accordance with the facts. The supposed sluggishness of the metabolism of thermophilic algae, which in view of the performance of Anacystis nidulans reported by Kratz and Myers (12) must be regarded as a myth, was attributed by Bunning and Herdtle to a protoplasmic structure hindering diffusion so that this process becomes rate-limiting at physiologically high temperatures. Restriction of free diffusion may be of some importance, but it seems likely that ability to withstand high temperatures is mainly due to a high proportion of heteropolar cohesive bonds or main valency bonds, as opposed to heat sensitive homopolar cohesive bonds, linking the structural peptide chains and so hindering denaturation. This idea is in accord with evidence discussed in the preceding section, but a similar hypothesis to explain heat resistance in higher plants is rejected by some authorities

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Drought Resistance The ability of many blue-green algae to survive prolonged desiccation cannot be doubted. Some species remain viable in sun-baked tropical soils and resume active growth in the rainy season or when samples brought into the laboratory are moistened. Successful cultures of many species may be obtained from herbarium specimens, spores of Nostoc commune, for example, having been germinated after 87 years in a dry condition in a herbarium (16). Ability to tolerate high concentrations of slts in the external medium is probably another manifestation of this property. Blue-green algae are often found growing in brine or on salt-marshes (1) and most species are able to tolerate media of up to eight atmospheres osmotic pressure (2). Spores are particularly resistant, but the vegetative cells of many species which do not produce spores are also able to withstand desiccation or high osmotic pressures. It appears likely that in blue-green algae, as o resistances esistances tto desiccation other i in organisms, other deslccation, cold and heat are due to the same underlying yg cause (15), but no physiological investigations of resistance to desiccation in blue-green algae appear to have been made. Effects of Hydrogen Ion Concentration Blue-green algae are most abundant in soils (17), freshwaters (18), hot springs and estuarine conditions (19) when the reaction is neutral or alkaline. A few species habitually grow in waters of around pH 5.0, e.g., Schizothriz spp. in Sphagnum bogs, but an alga containing phycocyanin and reported as growing in strongly acid solutions (20) is probably not a member of the Myxophyceae (see p 157). When final yield is used as a measure of growth, the pH optima for various species have been determined as: Allison's strain of Nostoc muscorum, 7-8.5 (21); Gloeothece linearis (Coccochloris peniocystis), 10 (22); Microcystis aeruginosa, 10 (23). Measurements of the effect of varying pH on relative growth rates, which are of more value in showing direct effects on growth, have been made for three species by Kratz and Myers (12). Their results may be summarized as follows, the pH values given being those of the medium at the end of the growth period: Anabaena variabilis, optimum pH 6.9-9; Anacystis nidulans, optimum pH 7.1-8.7; Gerloff's strain of N. muscorum, optimum pH 6.9-7.7. Alkaline

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conditions favor other activities of blue-green algae besides growth. Thus Burkholder (24) found the movement of Oscillatoria formosa to be inhibited outside the range pH 6.4-9.5, and Kratz and Myers (25) reported that the rate of photosynthesis of Anaboena variabilis in bicarbonate buffer decreased markedly below pH 7.0 whereas that of Chiorella pyrenoidosa under similar conditions remained constant from pH 6.0 to 7.5. The respiration of blue-green algae is not reduced, however, in slightly acid media (25). Experimental work thus confirms the impression given by ecological observations that a preference for alkaline conditions is a general characteristic of the group. In this respect the Myxophyceae differ from most other classes of freshwater algae, the species of which are distributed over wider pH ranges. Movement A s f e A exibited all blueby nearly lifein their some stage or other greenstriking algae atfeature hitr isis gingmv et.Teecasmo movement. The mechanismhas history gliding and our knowledge of it of this is obscure, advanced but little since it was reviewed by Burkholder (24) and Fritsch (1). A theory that the movement depends on rhythmic longitudinal waves, produced by changes in the shapes of the cells, traversing the algal filament has held first place for some time, largely because of the support given to it by measurements obtained by Ullrich (26) using an ingenious stereoscopic technique for the examination of kinematograph records of moving filaments. Nevertheless, in careful investigations by the same method, Schulz (27) failed completely to confirm Ulirich's findings. Schulz, who observed a correlation between the translatory movements of filaments and of carmine particles on their surfaces, is, together with some other recent workers, inclined to support the idea, first put forward by Correns in 1897, that movement is produced by secretion of mucilage. Electron micrographs of the cell membranes show structures which may be associated with such excretion (27, 28). Some species, Anabaena cylindrica, for example, show vigorous movements yet do not always secrete demonstrable amounts of mucilage. There is, however, ample evidence for liberation of soluble organic substances into the external medium by bluegreen algae (see p 159) and the adsorption of these at interfaces might well result in movement

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of the algal fiaments as suggested by Burkholder (24). This hypothesis, which is not necessarily incompatible with that invoking the secretion of mucilage, has not yet received direct experimental support. However it is produced, this type of movement is very characteristic of Myxophyceae and considerable importance has been attached to it by Pringsheim (6) as a means of distinguishing members of this class from the true bacteria. Apart from Myxophyceae and forms related to them, gliding movements, not necessarily effected by similar mechanisms, are shown also by members of the Rhodophyceae, Conjugalee, Bacillariophyceae, Chrysophyceae and Euglenineae. N

URITION

Mineral Nutrition From work such as that of Maertens (29) and Gerloff, Fitzgerald and Skoog (22, 23) it appears that the mineral requirements of blue-green algae are generally similar to those of other plants, and the attention of investigators has been largely focussed on one obviously exceptional feature of their nutrition; namely, the capacity of some species to assimilate or "fix" elementary nitrogen. Since nitrogen fixation by blue-green algae has been discussed in two recent reviews (30, 31) it need not be considered in detail here. Two points should, however, be mentioned. One is that, although some blue-green algae are unable to fix nitrogen, the property is of frequent occurrence in the class, being possessed by species belonging to the Rivulariaceae, Scytonmteae and Stigonemataceae, as well as by most members of the Nostocaceae. The other point is that, in its physiology and biochemistry, nitrogen fixation by blue-green algae does not appear to differ in any important respects from that effected by Azotobacter or by the Rhizobium-leguminous plant system. Recently, interest has developed in the trace element requirements of blue-green algae. Molybdenum was found by Bortels (32) to be essential for the growth on elementary nitrogen of seven species and the essentiality of this element for nitrogen fixation by Anabaena cylindrica has been confirmed by other workers (33, 34). For A. cylindrica molybdenum is not replaceable by vanadium (34). Wolfe (33) found molybdenum to be essential also for the growth of A. cylindrica on nitrate, saturation being reached

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at about 0.1 ppm of molybdenum in the medium whereas more than twice this concentration was required to give best growth on elementary nitrogen. Growth of A. cylindrica is possible in the absence of these relatively high concentrations of molybdenum when ammonia is supplied, but it remains undecided whether or not growth on this nitrogen source is possible in the complete absence of the trace element. Further work will no doubt show that in blue-green algae, as in other organisms, molybdenum is an essential component of nitrate reductase and possibly of other enzymes concerned in inorganic nitrogen metabolism, but evidence obtained by Wolfe (35) is consistent with the hypothesis that the predominating effect of molybdenum on the similation of elementary nitrogen and nitrate by A. cylindrica may be an indirect one exerted through inhibition of phosphatase activity (30). The necessity of sodium for the growth of A. cylindrica (36) and Nostoc muscorum (12) has been demonstrated in carefully controlled experiments. A. cylindrica requires a sodium concentration of at least 5 ppm for best growth and cannot utilize potassium, lithium, rubidium or caesium in place of sodium, nor sodium in the place of potassium (36). Since sodium has been found necessary for good growth of several other blue-green algae (20, 37), it appears that a requirement for sodium is a general characteristic of the class. In this respect these organisms may be exceptional, for there is no convincing evidence that omission of sodium from the nutrient medium in the presence of adequate potassium has adverse effects on the growth of other plants. The part played by sodium in the metabolism of blue-green algae is as yet unknown but it has been observed that sodium-deficient A. cylindrica contains less phycocyanin than that with adequate sodium, the chlorophyll content being unaffected (36). Also, it has been found necessary to supply cells of various species with sodium salts if high rates of photosynthesis are to be maintained (20, 25) although omission of this element seems to have a negligible effect on the respiration of these species (25). It has been stated that the growth of some blue-green algae is not reduced when calcium salts are omitted from the medium (22, 38), but it must be borne in mind that calcium is a common impurity in laboratory chemicals and for other species this element appears to be indis-

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pensable (29, 37). Carefully controlled experiments show calcium to be necessary for the growth of A. cylindrica on both free and combined nitrogen, the optimum concentration of calcium being at least 20 ppm and the element not being replaceable by strontium (34). Boron has been found essential for maximum growth of Nostoc muscorum, cultures deficient in this element becoming chlorotic (39). Experiments with N. muscorum, Calothric parietina, Coccochlorw peniocystt8 (Gloeothece linearis) and Diplocysti8 (Microcystdi) aeruginosa, have shown cobalt to be an essential element for these algae. Maximum growth of N. muscorum is still achieved if inorganic cobalt is replaced by much smaller amounts of the element in the form of cobalamin (40). From this it would seem that the essential metabolic role of cobalt is as a component of this growth factor.

Phototrophy Most members of the Myxophyceae are pigmented and many species have been grown in the light in purely mineral media so that there can be little doubt that phototrophy is their principal mode of carbon assimilation. A few detailed studies form a basis for comparison of the mechanism of photosynthesis with that in other plants. The photosynthetic apparatus of blue-green algae differs most conspicuously from that of nearly all other kinds of plants in having bilichromoproteins (such as phycocyanin) as well as characteristic carotenoids as accessory pigments. The chemistry and occurrence of these pigments will be discussed in a later section. Here it is only necessary to summarize present knowledge of their role in photosynthesis, a matter which has been considered in detail in several recent reviews (41, 42, 43). That light energy absorbed by the bilichromoprotein, phycocyanin, is utilized in photosynthesis was indicated by the work of Engelmann, published in 1883 and 1884, with 8Ociatoria and was confirmed by that of Emerson and Lewis (37) with Chroococcus. Arnold and Oppenheimer (44) have shown that the observed quantum efficiencies of photosynthesis by Chroococcus at different wave lengths can be accounted for in terms of transfer of energy from phycocyanin to chlorophyll by a process of resonance or "internal conversion" occurring when the pigment molecules concerned are

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separated by distances which are small compared with the wave length of the light. The results obtained with Chroococcus are thus reconcilable with the hypothesis that chlorophyll a is the principal photosynthetic pigment, in the sense that it is the one participating in the photochemical reaction. Haxo and his collaborators (45, 46) have reported action spectra for species of Osciatoriaceae and Anabaena, showing maximum photosynthetic efficiency at the absorption peak of the bilichromoproteins and indicating poor utilization of light absorbed directly by chlorophyll a. Nevertheless, the finding that in red algae, in which an analogous situation exists, the rates of photosynthesis at light saturation are the same whether the light is absorbed by the phycoerythrin or by the chlorophyll (43) and the results of fluorescence studies with Oscillatoria (47) suggests that chlorophyll a is always the pigment directly concerned in the photochemical reaction. Inactive absorption by chlorophyll a must then be attributed to the submicroscopic topography of the chromatophore or to a portion of the pigment not being combined in the appropriate manner for direct participation (41, 42, 43). Light absorbed by phycocyanin has also been found to be available for the Hill reaction carried out by cell homogenates of Synechococcus cedrorum (48). Light absorbed by the carotenoids of blue-green algae appears to be largely unavailable for photosynthesis (37, 45, 46). Both the proportions of the various pigments and the capacity to utilize the light absorbed by individual pigments may vary in an alga according to the intensity and wave length of the light with which it is illuminated (42). It has not been established unequivocally that such changes make for maximum efficiency in utilization of available light in the photosynthesis of blue-green algae, but that this is so seems likely from the facts presented in the preceding paragraph and from results obtained with red algae (42). For Anacystis nidulans it has been found that, as would be expected, the rate of light-limited photosynthesis is approximately a linear function of chlorophyll and phycocyanin contents whereas the rate of light-saturated photosynthesis shows no simple relation to pigment content (49). The evolution of oxygen by illuminated Oscillatoria was reported by Engelmann in 1883, and subsequent studies have confirmed the implication of this result that water is generally

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the hydrogen donor in the photosynthesis of blue-green algae. Nevertheless, there are indications that other substances may sometimes serve as auxiliary hydrogen donors. Many different species of blue-green algae are to be found in environments in which free hydrogen sulfide is present, and they often accompany photosynthetic bacteria (19). An Oscillatoria which grew in laboratory culture in the presence of 1 X 1M hydrogen sulfide was found by Nakamura (50) to evolve no oxygen but, instead, to accumulate sulfur within its cells when photosynthetically assimilating carbon dioxide under these conditions. Nakamura's results suggest that this Oscillatoria is able to use water or hydrogen sulfide in photosynthesis with equal facility, thus differing from the photosynthetic bacteria which always require an auxiliary hydrogen donor for reduction of the oxidizing moiety liberated by the photolysis of water. Another species, Synechococcus elongatus, resembles the green alga, Scenedesmus, in possessing a hydrogenase which becomes active when the alga is incubated under anaerobic conditions and which enables it to use elementary hydrogen in the reduction of carbon dioxide (51). What is evidently the same species is also reported as using sulfide as auxiliary hydrogen donor in photosynthesis, although if hydrogen is present as well this is used preferentially (52). The ability to use substances such as hydrogen and hydrogen sulfide in photosynthesis is not, however, invariably present in blue-green algae, even after anaerobic adaptation. Species of Oscillatoria, Nostoc and Cylindrospermum, for example, are evidently unable to use hydrogen in this way (53). The path by which carbon dioxide is fixed in photosynthesis is essentially the same in bluegreen algae as in other plants. Allison et al. (54) found the distribution of tracer among alcoholsoluble products in Nostoc muscorum after photosynthesis for 30 minutes in the presence of C14-labeled carbonate to be similar to that described for other organisms under comparable circumstances. Norris et al. (55) determined the distribution of C14, supplied as bicarbonate, after 5 minutes of photosynthesis in Phormidium sp., in species of Nostoc, including N. muscorum, and in Synechococcus cedrorum, and found it to be qualitatively similar to that in other kinds of plants except that an unidentified compound

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became labeled to a considerable extent in N. muscorum and Synechococcus but scarcely at all in the large number of other plants examined. Evidently, in blue-green algae as in other plants, phosphoglyceric acid is the first compound in which carbon fixed in photosynthesis appears but some variation may exist in the subsequent pathway. The photosynthetic assimilation of acetate by N. muscorum has been studied by Allison et al. (54), using radiocarbon as a tracer. Acetate was assimilated more rapidly by illuminated cells in the presence of carbon dioxide than it was in the dark or in the absence of carbon dioxide. The products of its assimilation were lipides, glutamate and carboxylic acids, the carbon of the carboxyl and methyl groups being incorporated into these to equal extents. Radiocarbon supplied as acetate was not incorporated into phosphoglyceric acid or phosphorylated intermediates of glycolysis during photosynthesis, a finding which suggests that the carbon of acetate is not oxidized to carbon dioxide prior to assimilation. In this respect the behavior of N. muscorum appears similar to that of other algae (56). Formate or urea supplied to N. muscorum in the light have been found to be converted to carbon dioxide by oxidation and hydrolysis respectively, this carbon dioxide being subsequently assimilated by normal photosynthesis (57). By the use of isotopically labeled molecular oxygen an apparent photo-inhibition of respiration has been demonstrated in Anabaena sp. This effect, which is not found in other algae such as Chlorella, appears to be the result of the close juxtaposition of the sites of oxygen consumption and production in blue-green algae (58).

Chemolithotrophy A critical consideration of the morphological and cytological characteristics of species of Beggiatoa, Thiothrix and Achromatium, which are usually assumed to be bacteria, led Pringsheim (6) to conclude that they are, in fact, colorless members of the Myxophyceae. This idea has been endorsed recently by other commentators on the taxonomy of bacteria (59). Two of these genera, Beggiatoa and Thiothrix, undoubtedly include chemolithotrophic (chemosynthetic) forms. In the dark these organisms develop in the absence of organic matter, deriving their energy from the oxidation of hydrogen

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sulfide to sulfur and of sulfur to sulfuric acid (6). Beyond this little is known of their biochemistry. Achromaium, a unicellular analogue of Beggiatoa, deposits sulfur within its cells (6) and is probably autotrophic but does not appear to have been studied critically by physiologists.

Chemo-organotrophy Certain blue-green algae appear to be obligate phototrophs and incapable of growth in the dark on any sort of organic substrate. Anabaena cylindrica (30), A. variabilis, Anacystis nidulans and Gerloff's strain of Nostoc muscorum (12) are of this type. Nevertheless, other species are frequently found in situations in which illumination is poor but organic matter abundant, and it thus appears that these may be chemo-organotrophic, i.e., capable of growth in the dark if supplied with suitable organic substrates. Studies with pure cultures confirm this impression. Harder (60) found that Nostoc punctiforme, an endophyte, can grow in the dark if supplied with any of a variety of organic compounds including glucose, galactose, sucrose, maltose, starch, inulin and citric acid. A strain of N. muscorum isolated by Allison is capable of growth in the dark on glucose or sucrose but at a much lower rate than under similar conditions in the light (21, 12). Allen (20) found various other species grew slowly in the dark if provided with suitable organic substrates. Such algae may be described as facultative chemo-organotrophs. Chemo-organotrophy is obligatory in other members of the class. A tendency towards this condition was evident in an endophyte, described as Nodoc puntiforme but perhaps more correctly identified as Anabaena cycadeae, which Winter (61) found to be scarcely capable of autotrophic growth although it was pigmented. There are, however, many colorless organisms which seem to be related to the pigmented Myxophyceae and in which the only mode of nutrition is chemo-organotrophy. Various authors have assigned organisms described as colorless to the Chroocales, Chamaesiphonaie and Nostocales, but it is not always clear whether these are completely devoid of photosynthetic pigments when in a healthy condition; moreover, nutritional studies with pure cultures have been carried out with only a few of them. Spirulina albida, however, is certainly free from chlorophyll and saprophytic (6). The Vitreoscillaceae, an

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assemblage of colorless Owilatoria-like organisms, several of which have been isolated in pure culture from habitats rich in decaying organic matter, are probably apochlorotic Myxophyceae (6, 62). These are undoubtedly chemo-organotrophic, but no detailed studies appear to have been made of their nutrition. Beggiatoa has been reported as being able to grow chemo-organotrophically as well as chemolithotrophically (6). There appears to be no reliable record of a member of the Myxophyceae absorbing particulate organic matter. It seems necessary to state this in view of the publication of a paper, the translated title of which is "The occurrence of carnivorous blue-green algae" (63). This paper, however, presents only some not completely convincing evidence that Ocalltoria 8plendida may produce extracellular products toxic to bacteria and subsequently benefit by absorption of nutritive substances released by the decomposition of these organisms.

u-Factor irents The blue-green algae studied in pure culture up to the present have been, almost without exception, unexacting species growing -readily in simple inorganic media. However, blue-green algae are of frequent occurrence in environments rich in organic matter and in freshwater lakes a distinct correlation exists between their abundance and the concentration of dissolved organic substances (18). Gloeotrichia echinulaa in impure culture has been found to require a thermolabile growth factor present in garden soil (64). However, such observations must not be taken as definite evidence that the species concerned have requirements for exogenous supplies of organic metabolites since some of them have been grown in pure culture in purely inorganic media (23) and there is evidence that organic substances may have indirect effects on the growth of algae as a result of complex formation with inorganic ions (65). Although interpretation of observations must be made with caution, it would be surprising were there no blue-green algae having growth factor requirements; definite evidence of the existence of one such species has recently been published by Provasoli and Pintner (66). These authors have found the marine alga, Phormidium persicinum, to have a purine requirement, satisfied by guanine, and vitamin requirements, satisfied by a mixture of thiamine, cobalamin,

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thymine and folinic acid, of which only thiamine, however, has so far been established as essential. CHE8MICAL COMPOSITION

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be the only green pigment in a number of samples of natural growths of blue-green algae (68, 69) and in Chroococcus (37) and Anacystis nidulans (49) grown in pure culture. Chlorophyll b has been reported as present in relatively small quantities in various Myxophyceae from hot springs (70) but the evidence for this is not quantitative and does not show that the samples contained no other types of algae. Carotenoids. Records of the occurrence of carotenoid pigments in Myxophyceae have been listed by Karrer and Jucker (71). a-Carotene appears always to be present, as it is in other kinds of algae (71, 72). a-Carotene has been reported as present in small amounts in

General Considerations In this section attention will be directed to the chemical composition of blue-green algae rather than to their metabolism. Available information concerned with the nitrogen metabolism (30) and acid metabolism (56) of these organisms gives little reason for supposing that these processes differ in any fundamental way from those in other forms of life. It is well established that the algal classes are each characterized by the general occurrence in their members of particular products, for example, specific accessory photosynthetic pigments. The following paragraphs will be concerned mainly with such qualitative biochemical characteristics of the Myxophyceae, but the possible existence of quantitative characteristics, in the sense that there may be a general tendency in the class towards the accumulation of the various types of metabolic products in particular proportions, should not be overlooked. The synthetic potential of an alga is directed along different paths at different stages in its development but, when this variation is allowed for, a distinct tendency for the compositions, in terms of proportions of major fractions such as carbohydrate, fat and protein, of species belonging to the same class to be similar may be seen in the few sets of analytical data that are so far available. Further work is needed to establish this point, but it seems that the blue-green algae may be characterized by a high cell nitrogen content and be different from most other kinds of algae, except the Rhodophyceae, in not showing an association of fat accumulation with low cell nitrogen contents (67). A high cell nitrogen content might be a consequence of the special type of protoplasmic structure possessed by the Myxophyceae.

were collected in the field, they may have been impure, and it remains to be shown conclusively that this pigment is characteristic of the class. A further pigment, flavacin, which is possibly a hydrocarbon, has also been obtained from A. flos-aquae (75). Myxoxanthin is a keto-carotenoid of formula C4oH4O, which has been found in Rivularia spp. (72), O8cillatoria rubescens (72,76) andOscillatoria sp. (77). It is perhaps the same pigment as that which Kylin (74) found in Calothrix scopulorum and described as calorhodin. A critical study by Goodwin and Taha (77, 78) makes it seem probable that myxoxanthin is identical with aphanin, a pigment obtained from Aphanizomenon by Tischer (73, 75), and with echinenone, a pigment isolated from the gonads of a sea-urchin and of limpets (78). From our present knowledge it appears that myxoxanthin is highly characteristic of blue-green algae, occurring otherwise only in the animal kingdom. Its preservation in lake deposits may afford a means of estimating the growth of blue-green algae in the past (79). A suggested structure for myxoxanthin (71) has been questioned by Goodwin and Taha (78) who consider that it is 4-keto-

coorofthebue-gren of the blue-green algae has always The color been one of the principal criteria used in distinguishing them from other algae. Now that more detailed information is available about the chemistry and distribution of algal pigments the assumption that certain pigments are characteristic of the class must be examined critically. Chlorophylls. Chlorophyll a has been found to

1I am grateful to Dr. B. C. L. Weedon for drawing my attention to this.

algaehasalwa

Aphanizomenon flos-aquae (73) and Calothrix

scopulorum (74), but, as the materials examined

,B-carotene. However, on reduction myxoxanthin yields a product identical with cryptoxanthin, which should, therefore, be 4-hydroxy-fl-carotene (77), but from results obtained by Wallcave and Zechmeister (80) this does not appear to be correcti. The structure of myxoxanthin thus remains uncertain.

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BLUE-GREEN ALGAE

Myxoxanthophyll [probably identical with the myxorhodin of Kylin (74)], which has the formula C*EH60, appears also to be characteristic of blue-green algae (71, 72). Aphanizophyll, isolated by Tischer (75) from Aphanimn, is related to, or is possibly identical with, this pigment. A tentative structural formula for myxoxanthophyll has been proposed (71) but remains unconfirmed. Other carotenoids reported from blue-green algae seem to be of more limited distribution within the class. Two that are perhaps peculiar to the Myxophyceae are aphanicin, isolated by Tischer from Aphanzomeno and regarded by him as a "dicarotenoid" of formula Ca0Hum0s, and oscillaxanthin, an acidic carotenoid of unknown constitution reported as occurring in small amounts in Oscillatoria rubewens (76). Besides these, lutein (81) and zeaxanthin (76), both of which occur in green algae and higher plants, have been reported as present in 0. rubescens. Bilichromoproteins. Blue-green algae are especially characterized by the presence in them of "phycobilins", more correctly known as bilichromoproteins (82), which are water-soluble pigments having a metal-free linear tetrapyrrolic chromophoric group attached to a protein of the globulin type. Such pigments seem otherwise to be regularly present only in the Rhodophyceae, but a few not-too-well substantiated reports of their occurrence in species belonging to other classes have been made. The existence of the "acid alga" studied by Allen (20), which has the morphology of a Chlorella but which lacks chlorophyll b and contains a bilichromoprotein, is, however, disturbing to those who believe the accessory photosynthetic pigments to be of taxonomic value. From its cell structure it is evident that this organism is not a member of the Myxophyceae and, furthermore, it does not contain appreciable amounts of diaminopimelic acid, which appears to be characteristic of this clas (83). Nor does it seem to have affinities with the Rhodophyceae. The Cryptophyceae, however, includes many blue-green and red forms, the nature of the pigments concerned being as yet unknown. Should it be shown that this class shares with the Myxophyceae and Rhodophyceae the distinction of producing bilichromoproteins, the difficulty presented by the "acid alga" might disappear since it might then be classed as a coccoid representative of the CrJptophyceae.

157

Rabinowitch (84) has reviewed the older literature on the bilichromoproteins. The two principal kinds, the red phycoerythrins and the blue phycocyanins, differ in the structure of their chromophores, the phycobilins 8senu stricto (85). The proteins linked to these chromophores have scarcely been studied. An amino acid analysis of the phycocyanin protein of 08cillatoria sp. has shown it to be similar in composition to the bulk proteins of other organisms except that it contains no appreciable amount of arginine (86). In this last respect the protein is not a typical globulin. Examination of a substantial number of species has shown that bilichromoproteins are invariably present in the Myxophyceae (69, 82). Phycocyanin is more characteristic of the class but phycoerythrin is often present in addition, and at least one species, Phormidium ectocarpii, seems to contain a phycoerythrin only (69, 82). The bilichromoproteins of the Myxophyceae differ distinctly from the corresponding pigments of the Rhodophyceae in the numbers and positions of their absorption peaks and are designated as c-phycocyanin (Xma- 620 mp) and c-phycoerythrin (X. - 557 to 560 mu). Chromatographic methods (82) facilitate the isolation and characterization of these pigments. By this means other bilichromoproteins besides the widely distributed pigments with the absorption maxima given above have been found in some species. "Allophycocyanin" Q(ma - 650 mju), once considered as arising from c-phycocyanin by denaturation, appears to be present in living material of Lyngbya lagerheimii as well as in various red algae. Phormidium ectocarpii was found to contain as its only detectable bilichromoprotein an apparently new phycoerythrin with absorption maxima at 542.5 and 565 mu. A rather similar pigment was obtained from P. fragile together with the conventional c-phycoerythrin and c-phycocyanin. The distribution of these various pigments among the species of Myxophyceae does not correspond with any taxonomic divisions. Other pigments. Other pigments besides those concerned in photosynthesis occur in Myxophyceae but have been little studied. Two pigments of undetermined chemical constitution have been obtained in crystalline form from the dark brown cell membranes of Calothrix 8copulorum and given the names of fuscochlorin and fuscorhodin (69, 74). Comparative studies with

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158

species of Gloeocapsa and Scytonema have shown that different species may have identical sheath pigments, their different colors depending on the reaction of the environment (87).

Carbohydrates Neither Payen (88) nor Kylin (74) could find more than traces of free reducing sugars in bluegreen algae. That such substances are actually present has been shown by paper chromatography of extracts of algae which had previously carried out photosynthesis in the presence of C1402. In this way, glucose, ribulose and other monosaccharides, both free and as their phosphate derivatives, have been detected in Nostoc muscorum and other species (55) through the incorporation of C14 into them. Sucrose was also detected in these species, but the most abundant non-reducing sugar in blue-green algae is evidently trehalose (74, 88), 1-[a-D-glucopyranoside]-a-Dgluco-pyranoside, which is otherwise to be found in fungi and freshwater Rhodophyceae but not in higher green plants. A polysaccharide, the presence of which causes the chromatoplasm to stain reddish-brown with iodine, occurs generally in blue-green algae (1) although Payen (88) was unable to detect it in any of the three species of which she made detailed analyses. This polysaccharide, which has been considered to be glycogen but which is perhaps better designated as myxophycean starch, evidently occurs in the cells in the form of submicroscopic granules (74). The main polysaccharide reserve of an Oscillatoria sp., a substance giving a reddish-brown color with iodine and readily soluble in cold water, has been shown to be a polyglucosan of the amylopectin type having an average chain length of 23-26 glucose units per one non-reducing end group (89). 0. princeps has been found to contain an enzyme synthesizing from glucose-i-phosphate, a glycogen-like polysaccharide containing from 14 to 16 glucose residue (90) which is evidently related to myxophycean starch. Fredrick and Mancini (13) tentatively attributed the production of a variant of this polysaccharide in a low temperature strain of 0. princeps to a decrease in concentration of branching enzyme relative to that of phosphorylating enzyme in the low temperature strain. Fritsch (1) and Drawert (2) have summarized the older work on cell membranes. Besides the

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"inner investment", which appears to be a coarse plasma membrane but of which analyses have not yet been published, there is generally a sheath, sometimes well defined but often diffluent. A substance reacting as cellulose when tested with iodine and sulfuric acid is occasionally present in this (74) but its principal components are mucilaginous polysaccharides. Metzner (28) has provided a valuable table showing the effects of various reagents on the sheaths of six different blue-green algae. Although the results of microchemical tests must be interpreted with caution, such work seems definitely to indicate pectins as the preponderating cell wall substances of blue-green algae. Payen (88) found that the gums obtained by aqueous extraction of Rivularia bulata, Calothriz pulvinata, and Nostoc commune yield on hydrolysis principally uronic acids and various sugars such as arabinose, glucose, or galactose. Kylin (74) reports a mucilage obtained from a marine species, Calothrix scopulorum, to yield galactose, glucose and a pentose on hydrolysis and to be esterified with sulfuric acid like certain of the polysaccharides of Rhodophyceae and Phaeophyceae. A mucilaginous polysaccharide obtained from Nostoc sp. has been found by Hough et al. (89) to be compounded of some 30 per cent of hexuronic acidsand perhaps 10 per cent of rhamnose and 25 per cent of D-xylose, the remainder being largely galactose with smaller amounts of glucose and an unknown sugar. A polysaccharide containing glucose, xylose, galactose, rhamnose, arabinose and glucuronic acid in the proportions 5:4:1:1:1:4 has been isolated from Anabaena cylindrica (91). Several electron microscope studies of the cell membranes of blue-green algae have now been made (e.g., 5a, 5b, 27, 28); these suggest that these complex polysaccharides form an amorphous filling in a framework provided by the interwoven fibrils of the cellulose-like components.

Fatsand Lipxdds The fatty acid content of blue-green algae has been found to be from 2 to 12 per cent of the total dry weight (67, 92) and in Gloeotrichia echinulata unsaturated acids have been found to form about 60 per cent of the total fatty acids (92). No more detailed analyses of the fats of Myxophyceae appear to have been reported. Among other substances soluble in fat solvents

BLUE-GREEN ALGAE

1956]

blue-green algae contain alcohols and hydrocarbons (92). It has been stated that sterols are totally lacking in these algae (72), but Goodwin and Taha (77) mentioned steroids as present in Oscilatoria sp., without, however, giving details of identification.

Nitrogenous Substances Amino acids. The amino acids of blue-green algae have been considered by Fogg and Wolfe (30). Since their review, Magee and Burris (93) have published analyses of the nitrogenous fraction of Nostoc muscorum. These latest results confirm the impression given by those of previous workers that there is no major difference between Myxophyceae and other classes of plants in respect to the amino acid composition of their bulked proteins. A minor nitrogenous constituent of blue-green algae, a-e-diaminopimelic acid, is of considerable interest since Work and Dewey (83) in a survey of 118 microorganisms representing most of the major groups found it to be present otherwise only in bacteria. This amino acid was present to the extent of 0.1 to 0.8 per cent of the dry weight in Anabaena cylindrica, OsciUatoria sp. (since reidentified as Microcoleu vaginatus Gom.) and Mastigocladus laminosus but was absent in amounts greater than 0.02 per cent from eight species of algae representing various other groups. The idea that a-e-diaminopimelic acid is found exclusively in bacteria and blue-green algae has to be modified in view of the identification of the amino acid in ChloreUa ellipsoidea by Fujiwara and Akabori (94). This identification has been confirmed, but it must be pointed out that the substance is present in this green alga in concentrations of the order of Mo of those in bacteria and blue-green algae. It is the meso- (DL) isomer of a-e-diaminopimelic acid which is found in A. cylindrica (Dr. E. Work, personal communication). In this alga it is not found in appreciable amounts as the free amino acid, but the nature of the form in which it is combined has not yet been determined. The amino acid may be important as a means of forming primary valence linkages additional to those formed by cystine between peptide chains (83) and thus contribute to the peculiar structure of the myxophycean protoplast. Polypeptide. The liberation in substantial amounts of soluble extracellular products in

159

healthy cultures of blue-green algae has been recorded many times; the phenomenon has been investigated in detail for Anabaena cylindrica (95). In this species, and probably in others, too, the principal substances concerned are polypeptides, free amino acids being generally present only in minute amounts (93, 95). The liberation of extracellular polypeptide is not due to autolysis and appears to be an invariable concomitant of growth, although the relative amount varies with cultural conditions (95). The relatively lower production of extracellular nitrogenous substances recently reported for Nostoc muscorum (93) and A. cylindrica (34) may be attributed to better supply of trace elements than was achieved in the experiments of earlier workers. Qualitative analyses showing a preponderance of hydroxyamino acids in the polypeptides -of A. cylindrica (95) have been confirmed by quantitative analyses. After hydrolysis, which was accompanied by considerable humin formation, the percentages of the total nitrogen of an impure preparation found in the various amino acids were: serine, 10.8; threonine, 6.4; glycine, 1.45; glutamic acid, 1.35; leucine and isoleucine, 1.3; aspartic acid, 1.1; alanine, 0.8; lysine 0.6; valine, 0.6; arginine, 0.4; with traces of cystine, proline and phenylalanine (Dr. L. Fowden, personal communication). No perceptible proportion of a-ediaminopimelic acid is present (Dr. E. Work, personal communication). The cells of A. cylindrica (35) contain appreciable concentrations of soluble peptide nitrogen, but it is not known whether any of the substances concerned are the same as those which appear in the medium. Because of their capacity to form complexes with various ions, extracellular polypeptides have important effects on the growth of algae in culture and probably also in natural environments, e.g., by solubilizing phosphates or rendering toxic ions harmless (65). Liberation of extracellular polypeptides is not peculiar to the Myxophyceae. There are numerous instances of its occurrence in the bacteria. It has been shown to occur in algae belonging to classes other than the Myxophyceae (65), and a detailed examination of the physiological relations in the excretion of nitrogenous products by various fungi has shown them to be essentially similar to those described for A. cylindrica (96). Proteim. Apparently, no investigations have been made of individual myxophycean proteins

G. E. FOGG

160

apart from those on the bilichromoproteins. Cyanophycin, a characteristic reserve product appearing as conspicuous granules in the cells of blue-green algae, is generally thought to be proteinaceous (1, 3) although there is little definite evidence for this. It does, however, contain a large proportion of arginine (7). Purine and pyrimidine bases. In Nostoc muscorum purine and pyrimidine bases account for 5.1 to 5.5 per cent of the total nitrogen, the bases detected being adenine, cytosine, guanine, thymine, uracil and xanthine. An unknown compound, detected by its absorption at 260 my&, presumably belongs to this class of compounds also (93). Nucleic acids. Deoxypentose- and pentosenucleic acids extracted from Nostoc muscorum are stated to be qualitatively similar, as far as the constituent bases are concerned, to those of other organisms (97). Cobalamin. Robbins et al. reported the synthesis by blue-green algae of factors having vitamin Bn activity [quoted in (98) and confirmed by Brown et al. (99)]. From 65 to 70 per cent of the vitamin B12 activity of Anabaena cylindrica is due to cyanocobalamin, the rest being due to pseudo-vitamin B12 and traces of factors A and B. The total concentration of such substances, which may correspond to over 1 ,ug of vitamin B12 per g dry weight in blue-green algae, is considerably greater than that found in other classes of algae or in higher plants and is of the order of that found in bacteria (98, 99).

DISCUSS8ION Although our knowledge of the metabolism of blue-green algae is still fragmentary, the information that is available gives little reason for doubting the correctness of the assumption that their life processes are based on chemical mechanisms essentially similar to those which have been found in other organisms. It seems worth while emphasizing this point since if fundamentally different kinds of biochemical machinery exist among the organisms on this planet they would be expected to occur in a group which is so exceptional in other respects. Nevertheless, many differences in physiological and biochemical detail exist to distinguish the Myxophyceae from other groups of organisms. Among substances which are especially characteristic of the class are bilichromoproteins,

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myxoxanthin, myxoxanthophyll, trehalose and diaminopimelic acid, and among enzyme systems there are those for nitrogen fixation and for utilization of auxiliary hydrogen donors, such as hydrogen in photosynthesis. On the other hand, blue-green algae are equally well characterized by the absence of certain substances, e.g., of chlorophylls other than a and, possibly, of sterols. Individually such characteristics are not exclusive to the Myxophyceae; thus bilichromoproteins are also found in the Rhodophyceae, trehalose in fungi, myxoxanthin in mollusks, and nitrogen fixation in certain bacteria. Thus, as in taxonomy based on morphological features, the class is best defined biochemically by a particular combination of characteristics rather than by the presence or absence of one particular substance. Another characteristic of blue-green algae, their peculiar protoplasm, seems remarkable as representing a different type of organization rather than for containing structural substances not present in other types of organism. This protoplasm is clearly different, in its rigid gellike structure and resistance to high temperatures, from that of other algae, higher plants and animals, but has features, such as resistance to high temperatures, in common with bacterial protoplasm. The requirements of blue-green algae for neutral or alkaline conditions-another feature also shown by bacteria-may possibly have some connection with this protoplasmic structure. No indication is evident that the characteristic manner of movement of bluegreen algae is directly related to their special protoplasmic structure, superficially similar movements being shown by several classes of organisms with more normal cytology, but not by bacteria. What part blue-green algae have played in the evolution of life is perhaps the most intriguing of the many questions that may be asked concerning these organisms. It is generally supposed that they represent one of the most ancient forms of life upon earth but no incontrovertible evidence exists for this. The fossil record is equivocal (1, p 859), and although the world-wide distribution of most of the species is some indication of antiquity, this indication is weakened by the demonstration that viable cells of a bluegreen alga can be of regular occurrence in the air (100). However, the comparative simplicity of their cell structure suggests for blue-green algae

1956]

BLUE-GREEN ALGAE

a position low in the evolutionary scale, and the assumption that this is their correct place opens up an attractive field for speculation. Present thinking favors the Haldane-Oparin hypothesis that life began under reducing conditions with the formation in the primitive hydrosphere of metastable organic molecules (see, for example 101, 102). It does not seem impossible that interaction among these molecules should have given rise to many biochemical systems, including self-reproducing ones, prior to the appearance of discrete organisms, nor that organisms, when they appeared, should have exchanged such systems between themselves and the environment just as present day bacteria are known to do (102). The fundamental uniformity in biochemistry which is shown by all living organisms, blue-green algae not excepted, no doubt dates from such a phase of evolution. Following the appearance of differentiated protoplasm, organisms resembling present day blue-green algae may have been among the first to evolve. Presumably the conditions prevailing would be less stable than they are now, and selection would be in favor of a type of protoplasmic structure, such as that of the blue-green algae, having greatest resistance to extreme chemical and physical conditions. Several other features of these algae seem to be related to conditions which probably prevailed at this stage in the earth's history. Modern blue-green algae attain their greatest development under warm, humid, subaerial conditions or in freshwater, and, although they can tolerate high salt concentrations, they are not usually abundant in marine habitats. This suggests that the major evolution of the class occurred before seawater had a composition resembling that which it now has. There is some evidence that reducing conditions persisted almost to the Cambrian (101); the fact that most blue-green algae are tolerant of reducing conditions (19) and the ability, already discussed, of some species to utilize reduced inorganic compounds in their metabolism suggests that the class appeared before the atmosphere became oxidizing. The high proportion of nitrogen-fixing species in the Myxophyceae supports the idea that the class underwent its greatest evolutionary development at a time when the onset of oxidizing conditions was resulting in the depletion of combined nitrogen reserves through denitrifi-

161

cation (31). The suggestion of Tilden (103) that the pigmentation of blue-green algae originated as an adaptation to the color and low intensity of the light available at the surface of the primitive earth is supported by recent work. Good reasons have been put forward for supposing that methane would give rise to substances which would impart a yellowish or red color to the primitive atmosphere (101). Since phycocyanin has maximum absorption for such wave lengths, and the evidence that light energy absorbed by it is available for photosynthesis is now substantial, it thus appears that blue-green algae would be particularly efficient in photosynthesis under primitive earth conditions. The reproduction of blue-green algae appears to be entirely asexual. It may be that their cell organization is inherently unsuited for a full sexual process. Although no processes similar to the transformation, transduction and recombination phenomena by which hereditary characters are acquired by bacteria have been described for blue-green algae as yet, it may be expected that these will be found in due course. Some such strictly limited capacity for recombination of hereditary characteristics is no doubt responsible for the low level of morphological organization that has been attained within the class. On the other hand, the biochemical variation shown both by the class as a whole and by individual species is considerable. Some species are able to synthesize all the metabolites they require from carbon dioxide, water, elementary nitrogen and mineral salts but are nevertheless able to utilize any of a wide variety of organic substrates if these are available. As in other types of microorganisms biochemical evolution from this self-sufficient primitive type appears to have taken place by loss of synthetic mechanisms. On the one hand, apochlorosis has given rise to obligately chemotrophic Myxophyceae, whereas on the other, some species have become obligately phototrophic. Loss of enzymes responsible for the synthesis of specific metabolites has undoubtedly led to the appearance of far more species requiring organic growth factors than have yet been discovered. The Myxophyceae appear to have existed apart from the main line of evolution from a very early stage so that relationships with other groups have become obscured. Although they have many biochemical and cytological features

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in common with the bacteria and with the Rhodophyceae, nevertheless important differences separate the class from these others. Thus flagella are present in the bacteria but absent from the Myxophyceae, and the Rhodophyceae have a much more highly organized cell structure thn than either Thell two uh of these two othergroups. other groups. The existence of such differences makes it clear that the relationship between these groups can only be through some extremely remote common ancestor.

mofrthee

structene

REFERENCES 1. F. E. 1945 The structure and reproduction of the algae. Vol. 2. Cambridge University Press, London, England. 2. DRA elRT, H. 1949 Zellmorphologische und sellphysiologische Studien an Cyanophyceen. I. Mitteilung. Planta, 37, 161

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209. 3 ZASTROW, E. M. VON 1953 tyber die Organisation der Cyanophyceenselle. Arch. Mikrobiol., 19, 17AW205. 4. CAssEL, W. A., AND HieTsoNsON, W. G. 1954 Nuclear studies on the smaller Myxophyceae. Exptl. Cell Research, 6,

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of Nostoc commune from a herbarium specimen eighty-seven years old. Bull. Torrey Botan. Club., 68, 664-6. 17. LUND, J. W. G. 1947 Observations on soil algae. II. New Phytologist, 46, 35-0. 18. PEARSALL, W. H. 1932 Phytoplankton in the Englishlakes. II. J. Ecol., 20, 241-262. 19. BAAs BECKING, L. G. M., AND WOOD, E. J. F. 1955 Biological processes in the estuarine environment. Proc. Acad. Sci. Amsterdam, B, 58, 160-181. 20. ALLEN, M. B. 1952 The cultivation of Myxophyceae. Arch. Mikrobiol., 17, 34-53. 21. ALLISON, F. E., HOOVER, S. R., AND MoRuRS. H. J. 1937 Physiological studies with the nitrogen-fixing alga, Nostoc muscorum. Botan. Gaz., 98, 433-463. 22. GERLOFF, G. C., FITZGERALD, G. P., AND SKOOG, F. 1950 The mineral nutrition of Coccochloris peniocystis. Am. J. Botany,

134-150. 5a. NIKLOWTZ, W., AND DilEws, G. 1956 37t, 835-840. Beitrfge zur Cytologie der Blaualgen. I 2. GERLOFF, G C., FITZGERALD, G. P., AND SKooG, F. 1952 The mineral nutrition of Arch. Mikrobiol., 24, 134 146. Microcystis aerupinosa. Am. J. Botany, Sib. DuEws, G., AND NIELOWITS, W. 1956 A, 26-32. Beitrfige sur Cytologie der Blaualgen. II. 24. BURHOLDER, P. R. 1934 Movementinthe Arch. Mikrobiol., 24, 147-162. 6. PRNGSIIEIM, E. G. 1949 The relationship Cyanophyceae. Quart. Rev. Biol., 9, 438-459. between bacteria and Myxophyceae. Bac25. K3U5TZ,W. A., AND Mrns, 3. 1955 Phototeriol. Revs., 13, 47-98. synthesis and respiration of three blue7. FOGO, G. E. 1951 The cytology of heterogreen algae. Plant Physiol., 30, 27tb280. cysts. Ann. Botany, N.S. 15, 23-35. reen H. an1930 tYber die Bewegungen 8. FOGG, G. E. 1941 The gas-vacuoles of the 26. der Beggiatoaceen und Oscillatoriaceen. Myxophyceae (Cyanophyceae). Biol. II. Mitteilung. Planta, 9, 144s194. Revs. Cambridge Phil. Soc., 16, 205-217. 27. SCHLZ, G. 9. FRITSCH, F. E. 1912 Freshwater algae. G. 1955 1955 Bewegungsstudien sowie 2 Lz, Beweg National Antarct. Exped., Nat. Hist., 6. tenuswe MembranunterGSSNE, F. 955 ydrootanie. Bnd Ielektronenmikroskopische 1955 10.10. F. Band I. Hydrobotanik. GESSNERg suchungen an Cyanophyceen. Arch. Energiehaushalt. Deutscher Verlag: der Mikrobiol., 21, 335-370. Wissenschaften, Berlin, E. Germany. 28. METZNER, I. 1955 Zur Chemie und sum 11. BtYNNING, E., AND HERDTLE, H. 1946 submikroskopischen Aufbau der Zellwande, Physiologische Untersuchungen an thermoScheiden und Gallerten von Cyanophyceen. philen Blaualgen. Z. Naturforsch., 1, Arch. Mikrobiol., 22, 45-77. . 93-99. 12. KRATZ, W. A., AND MYERS, J. 1955 Nutri- 29. MAERTENS, H. 1914 Das Wachstum von tion and growth of several blue-green Blaale in minerai1heN3 hrl6s algae. Am. J. Botany, 42, 282-287. Beitr. Biol. Pflans., 12, 439 496. 13. FREDRICK, J. F., AND MANCINI, A. F. 1955 30. Fog, G. E., AND WOLFE, M. 1954 The nitrogen metabolism of the blue-green Paper electrophoresis patterns of enzymes involved in polyglucoside synthesis in algae (Myxophyceae). In Autotrophic Oscillatoria princeps and its low temperamicroorganisms, pp. 99-125. Edited by

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