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The measurement and significanceof ATP pools in filamentous blue-green algaeP.J. Bottomley a & W.D.P. Stewart aa Department of Biological Sciences , University of Dundee ,Dundee, DD1 4HN, Scotland, U.K.Published online: 17 Feb 2007.

To cite this article: P.J. Bottomley & W.D.P. Stewart (1976) The measurement andsignificance of ATP pools in filamentous blue-green algae, British Phycological Journal, 11:1,69-82, DOI: 10.1080/00071617600650111

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Br. phycol. J. 11:69-82 1 March 1976

THE MEASUREMENT AND SIGNIFICANCE OF ATP POOLS IN FILAMENTOUS BLUE-GREEN ALGAE

BY P. J. BOTTOMLEY and W. D. P. STEWART Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, Scotland, U.K.

A modified luciferin-luciferase assay has been developed for measuring ATP pools in fila- mentous blue-green algae. The assay, which should be applicable to studies on algae in general, is simple, reliable, inexpensive, sensitive at the pmole level and can be used in any laboratory with a suitable liquid scintillation counter. Studies using the two blue-green algae, Anabaena cylindrica and Anabaenopsis circularis (strain 6720) show that in steady state cultures, good correlations exist between the size of the extractable ATP pool and algal biomass measured on the basis of cellular dry weight, chlorophyll, total nitrogen and total carbon. This correlation holds at measured ATP levels up to 2400 pmoles of ATP extracted or up to 400 pmoles per ml of culture. At higher densities of algae the relationship is non-linear. The size of the extractable ATP pool of A. cylindrica and A. circularis is usually in the range of 150-200 nmoles ATP mg chl a -1 which represents approximately 0-20-0.25 ~ of the total cellular carbon.

There is evidence of a rapid turnover of ATP molecules in blue-green algae in the light and dark and that the algae show a remarkable capacity to maintain their ATP pool. However certain changes in environmental or metabolic conditions can result in rapid changes in the ATP pool which are not manifested by corresponding rapid changes in algal biomass. The findings are discussed in relation to the use of ATP analysis in physiological and ecological studies on blue-green algae.

Adenosine triphosphate (ATP) is an essential constituent of all living organ- isms, and its free intracellular level has often been measured using the firefly luciferin-luciferase assay first developed by Strehler & Trotter (1952). Within recent years with the introduction of more readily available and sensitive equip- ment for measuring light scintillations, the technique has become increasingly popular compared with other methods of measuring rates of ATP synthesis, pools and utilization (see Simonis & Urbach, 1973). In addition to its use in the study of physiological mechanisms, there has also been the suggestion that it may offer a highly sensitive assay for measuring the biomass of living organisms in natural ecosystems (Holm-Hansen & Booth, 1966; Daumas & Fiala, 1969; Holm-Hansen, 1969, 1970; Brezonik, Brown & Fox, 1975).

However, the method has not been used extensively with blue-green algae, which are the dominant forms of phytoplankton in many mesotrophic and eutro- phic fresh waters (see Fogg, Stewart, Fay & Walsby, 1973), neither has there been a detailed description of the method as applied to blue-green algae. The technique has, however, been used in laboratory studies on the unicellular blue-green alga, Anacystis nidulans by Bornefeld, Domanski & Simonis (1972), Bornefeld & Simonis (1974) and by Bedell & Govindjee (1974). In this paper we provide details of a simple, reliable and economical modification of the luciferin-luci- ferase assay for use with blue-green algae which can be used in any laboratory with a suitable liquid scintillation counter. Data, obtained with the two nitrogen- fixing filamentous blue-green algae, Anabaena cylindrica and Anabaenopsis circularis 6720, are also presented on ATP pool sizes and on the ways in which ATP pools correlate with algal biomass under steady state and fluctuating environmental conditions.

69

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70 P. J. BOTTOMLEY AND W. D. P. STEWART

MATERIALS A N D M E T H O D S

ALGAE Two filamentous heterocystous blue-green algae were used as experimental material:

Anabaena cylindrica Lemm. (CU 1403/2a) obtained from the Culture Centre of Algae and Protozoa, Cambridge and Anabaenopsis circularis (strain 6720) kindly supplied by Professor R. Y. Stanier, Institut Pasteur, Paris.

GROWTH CONDITIONS The algae were grown axenically in continuous culture, unless otherwise stated, in medium

BG-11 of Stanier, Kunisawa, Mandel & Cohen-Bazire (1971) at 27°C and at a light intensity of 200tzW cm -2. The cultures were stirred magnetically and bubbled with humidified air, unless otherwise stated, at a rate of 1-2 1 min -1. The mean generation time of the alga was maintained at 24 h and the cell density was 2-3 ~.g chlorophyll a ml-L

MEASUREMENT OF CHLOROPHYLL a 5 ml aliquots of culture were filtered through Whatman 2-5 cm glass fibre circles, washed

with distilled water, and placed in Universal bottles with 5 ml of 100 % methanol and extracted for 12 h at 4°C in the dark. Absorbance of the solution was measured at 663 nm with a Unicam SP600 spectrophotometer and chlorophyll a was measured by the method of Mackinney (1941).

CARBON AND NITROGEN ANALYSIS 50 ml aliquots of culture were filtered onto preweighed 2.5 cm glass fibre circles, washed with

distilled water and dried at 80°C to constant weight. Per cent carbon and nitrogen were then estimated using a Hewlett-Packard model 185B CHN analyser.

THE LUCIFERIN-LUCIFERASE ASSAY The analysis of ATP depends on the fact that the enzyme luciferase in the presence of luci-

ferin, ATP, and Mg ++ catalyses the formation of adenyl-luciferin, and that adenyl-luciferin is oxidised in the presence of oxygen to produce adenyl-oxyluciferin and light thus:

Mg ++ (1) ATP + Luciferin ~- Adenyl-luciferin + pyrophosphate

Luciferase (2) Adenyl-luciferin + 42Oz -+ adenyl-oxyluciferin + hv. The amount of light emitted is proportional to the concentration of ATP. It is therefore

necessary to quantitatively extract the ATP from the algae, prepare ATP standards and enzyme- substrate, and then to measure luminescence using a suitable photometer.

EXTRACTION OF ATP FROM THE ALGAE Extraction was carried out using the method of Cole, Wimpenny & Hughes (1967) as

modified by Welsch & Smith (1969). 6 ml aliquots of algal suspension were injected rapidly into 1.5 ml aliquots of ice-cold 3 M perchloric acid (HC104). The samples were mixed vigorously and allowed to extract for 10 rain. The pH of the mixture was adjusted to pH 7.4 by the addition of 3"5 ml of the following mixture: 1 M KOH, 22.5 ml; 1 M KC1, 17.5 ml; 2 M Tris, 60 ml. The samples were then centrifuged at 30,000 × g for 30 min at 4°C to remove cell debris plus pre- cipitated chlorate salts and the supernatant was stored at 4°C. All samples were assayed for ATP on the day of extraction since, in our hands, prolonged storage of algal ATP even at -- 10°C caused erratic results.

ATP STANDARDS An accurately known stock solution of approximately 1 mM ATP (ATP Na2.3H20) was made up in 25 mM Hepes MgSO4 buffer, pH 7.4 (St. John, 1970). Calibration curves were constructed for each experiment by diluting 50 ~tl of the standard ATP solution into 50 ml of Hepes buffer and mixing thoroughly. Aliquots of this were then used for preparing the calibration curve.

PREPARATION OF ENZYME-SUBSTRATE 50 mg of firefly-lantern extract (Sigma FLE-250) were suspended in 5 ml of deionised dis-

tilled water and rotated at 78 r.p.m, for 24 h at 4°C. The suspension was centrifuged at 30,000 × g for 15 min, the pellet discarded and the supernatant, which contained 1-5-2 mg protein m1-1 was stored at 4°C for 1-2 h until required. The latter procedure tended to decrease the background fluorescence of the preparation. Fresh enzyme substrate was reconstituted for each experiment and used immediately.

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ATP pools in blue-green algae 71

THE MEASUREMENT OF ATP ATP luminescence was measured using a Tracerlab Corumatic 200 liquid scintillation counter

(International Chemical and Nuclear Corporation) which was operated manually with the gain setting (which regulates the sensitivity of the counter to pulses of a particular energy) at 6, with a threshold value of 0.025, and with the coincidence control switched out so that luminesence could be measured. For the assay of each sample, 0.9 ml Hepes, pH 7.4 and 100 ~tl luciferin- luciferase were added to the inner chamber of the vial shown in Fig. 1 using clean 100 t~l glass syringes. This modified scintillation vial was designed to minimise the quantities of reagents required for each assay. Prior to assaying each test sample, the vial was placed in the counting chamber and a blank value of counts obtained. An aliquot of the test sample or of the ATP standard was then injected into the vial, mixed once, and returned to the counting chamber. Counting was carried out over a 10 sec period (see Results).

;eol

orion viol

lube

FIG. 1. The modified scintillation vial used to measure ATP. Note the inner glass tube where the reaction is carried out.

REAGENTS Firefly lantern extract (FLE-250) and Hepes buffer were obtained from Sigma Ltd, London.

Adenosine triphosphate (ATP Na2.3H20) was obtained from Boehringer Corporation (London). All other reagents were obtained from British Drug Houses, Poole, and were the finest grade commercially available. Gases were obtained pre-mixed from Air Products Ltd.

RESULTS

ASSESSMENT OF THE METHOD

Sensitivity The general usefulness of the luciferin-luciferase assay depends largely on the

sensitivity of the instrument used to detect the light pulses. In studies using the Tracerlab Corumatic 200 liquid scintillation counter, as sensitivity increases, so also does the background count and thus it is essential that the instrument is operated at a gain setting which allows maximum sensitivity with minimum relative background. It was found that a gain setting of 6 allowed the accurate measurement of ATP levels as low as 10 pmoles with background counts amounting to less than 5% of the counts obtained with the test material.

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72 P. J. BOTTOMLEY AND W. D. P. STEWART

THE TIME COURSE OF LUMINESCENCE Figure 2 presents data on the time course of luminescence from a standard

amount of ATP. It is seen that there is a rapid decrease in luminescence for the first 20 sec after mixing the reactants and that this is followed by a much slower decrease over the subsequent 20-180 sec period. That is, maximum sensitivity and proportionality (i.e. counts measured perpmole of ATP) are obtained during the early stages of the luciferin-luciferase reaction. With the scintillation counter employed it takes 8 sec from the time of mixing the reactants in the vial to the commencement of counting in the instrument. Measurements were therefore routinely carried out over the 8-18 sec period after mixing.

x

o

I00

80

60

4O

0

\

\ \ \

\ \

m~--1 • • • m ~ n ~ m _ _ m ~ n

' 0 'o ' ' . . . . 2 4 6 80 I00 120 140 160 180 Time (sec)

FIG. 2. The time course of decay in luminescence observed on injecting (at 0 time) 20 ~1 containing 27 pmoles of ATP into a reaction vial containing 0"9 ml of buffer and 100 ~tl of enzyme-substrate mixture. In this and in subsequent figures, each counting period was 10 sec.

CALIBRATION CURVES Figure 3 presents data on a typical calibration curve for ATP. It is seen that

there is a linear relationship between luminescence and added ATP up to 35 pmoles ATP. At higher ATP values the response changes and the previous linea- rity is lost. These results indicate that measurements should be carried out within the 1-30 pmole range of the curve. Figure 4 shows the results obtained on using four different batches of luciferin-luciferase. Although there is some variability in the response obtained from batch to batch, a linear relationship between luminescence and ATP is always obtained at ATP concentrations up to 30 pmoles per sample. However, because of the variability from preparation to preparation of enzyme-substrate, standard curves were routinely prepared for each batch of enzyme-substrate.

EXTRACTION OF ATP Various methods have been used to extract ATP from micro-organisms in-

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A T P pools in blue-green algae 73

120

100

n

/ 6o

8

4O

0 - - i i i i I I

5 I0 15 20 25 50 55 40 ATP (pmoles)

FIG. 3. A cal ibrat ion curve showing scintillations measured on adding different a m o u n t s o f ATP. In this a n d in subsequen t Figs. and Tables, all values given are the m e a n s o f triplicate de terminat ions .

80~ •

o 6O x

u 40

2O

; I t I i I 0 4 8 12 i6 20 24 28

ATP (pmoles)

FIG. 4. Cal ibra t ion curves obta ined us ing different batches of commercia l ly supplied enzyme-subs t ra te mixture .

cluding direct heating at 100°C (Strehler, 1953), boiling in Tris buffer (Holm- Hansen & Booth, 1966), chloroform plus heat treatment (Dhople & Hanks, 1973), boiling in ethanol (St. John, 1970), the use of trichloroacetic acid (Okkeh, Tillberg & Kylin 1975) and perchloric acid (Cole et al., 1967). In our studies,

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74 P. J. BOTTOMLEY AND W. D. P. STEWART

~ 200 a c

~.. 160

b--

120

J / /

[ ]

I i i I l

6 12 18 24 30

Time (rain)

FIG. 5. Time course of extraction of ATP with perchloric acid (3 M). Each sample of 6 ml of algal suspension was injected, within 5 sec of sampling, into ice-cold perchloric acid, and extracted for the varying times shown in the Fig. The material was then neutralised to pH 7.4 and the supernatants were assayed for ATP.

extraction with ice-cold 3M perchloric acid was used routinely. As Fig. 5 shows extraction is complete within 5 min. This procedure does not result in any de- composition of ATP, as evidenced by the fact that known quantities of ATP are recovered quantitatively after being subjected to the extraction procedure.

IONIC QUENCHING Bornefeld & Simonis (1974) reported that the growth medium of Kratz &

Myers (1955) caused some quenching of luminescence in their studies using the unicellular Anacystis nidulans. We found that there was no difference in the counts obtained when ATP was analysed in medium BG-11 (Stanier et al., 1971), Allen and Arnon's medium (Allen & Arnon, 1955) or in Hepes -MgSO4 buffer.

ATP POOLS OF THE ALGAE

The ATP pool and its turnover time

With the heterotrophic bacterium Klebsiella aerogenes, Harrison & Maitra (1969) found that the ATP pool size may change if the time taken to transfer the test samples from the reaction vessel to the terminating solution exceeds the turnover time of the pool. Figure 6 presents data which show that this aid not occur in the case of blue-green algae when a glass pipette, which allowed the light to penetrate, was used for sampling. Indeed steady state ATP pools were obtained even if the transfer time varied between 5 and 30 sees. Similar results (not shown) were obtained on sampling in the dark. These findings provide evidence that the ATP extracted during an average sampling time of 5 sec is a true estimate of the ATP pool in these blue-green algae.

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ATP pools in blue-green algae

\ 24o

200

~ 160

0 fO 2 30

Time (sec)

FIG. 6. The measured ATP pools of Anabaena cylindrica obtained after different times were taken to transfer the algal samples from the continuous culture vessel to the extraction vials.

75

Figure 7 shows the results which are obtained when algal biomass is measured on the basis of the following parameters: extractable ATP [Fig. 7 (a)], chloro- phyll a [Fig. 7 (b)], dry weight [Fig. 7 (c)], total N content [Fig. 7 (d)] and total carbon [Fig. 7 (e)]. It is seen that with all the parameters measured there is a similar response to increase in concentration of algae. Similar findings were obtained with Anabaenopsis 6720 (Fig. 8). Such data provide evidence that extractable ATP is a valid measure of algal biomass in material taken from steady state cultures. Calculations from such data show that extractable ATP repre- sents 0-13% of the total dry weight, and that the ATP content is in the range of 150-200 nmoles mg chl a -1. Similarly, since cellular carbon and nitrogen repre- sent approximately 50% and 8% respectively of the dry weight in both algae, ATP represents 0.20-0.25 % and 1-1-1.5 % of the cellular carbon and nitrogen respectively.

In the above experiments, using steady state cultures, ATP pool and algal biomass showed, as expected, a constant relationship. Figure 9 shows the effect of transferring metabolically active algae growing under standard aerobic conditions at a light intensity of 200~zW cm -z to a lower light intensity (20EzW cm -2) and subsequently into the dark. It is seen that in these short-term experiments with each change in environmental conditions there is a transient drop in the ATP pool but that within minutes of each change the pool returns to its original level.

Table 1 on the other hand, shows the results which are obtained when A. cylindrica is incubated in air in the dark for a prolonged period. It is seen that the ATP pool falls off with increase in the time of incubation in the dark and that the time during which the pool remains high is dependent on the environmental conditions under which the alga is incubated during the previous period of incu- bation in the light. Furthermore when the ATP pool decreases to very low levels, the chlorophyll concentration decreases only slightly. Figure 10 shows a second

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76 P . J . BOTTOMLEY AND W. D. P. STEWART

TABLE 1. The effect of pretreatment in the light on the ATP pool of A. cylindrica subsequently incubated in the dark

Pretreatment Air + 5% CO2 Air Time (h) ATP pool % of initial ATP pool % of initial

pool pool

0 125 100 127 100 1 130 104 125 98 2 137 110 130 102 4 145 116 75 59 6 135 108 45 35 8 125 100 30 24

10 110 88 26 21 12 50 40 25 20

Aliquots of algae (200 ml) were taken from continuous culture and gassed in batch culture in the light (200 FW/cm -2) with air + 5 % CO2, or in air, at a rate of 1 1 min -1 for 24 h. The algae were subsequently placed in the dark for 12 h. Samples were taken at intervals for ATP and chlorophyll a analyses. The chlorophyll a concentration decreased by 8 % over the 12 h incu- bation period in the dark in both series.

"~ (0)

~ 2O

- × I0 ~

'~ ' ' ib)

(c

~ 12 = / = '_o ./ / = ~ / ' ~ , ,

' (d ) '11

-=T° 6 " / ~ 1 i i i

8 ~m/m o 4 / m /

Volume of algae (ml)

FIG. 7. Biomass of Anabaena cylindrica measured on the basis of: a, extracted ATP; b, chlorophyll a; c, dry weight; d, total nitrogen, and e, total carbon.

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ATP pools in blue-green algae 77

20

o

g 4

~ 8

~ 4 c:;

/ i i i i i

(d)

c T --~ o_

~ i I i i

(el

Volume of algae (ml)

FIG. 8. Biomass of Anabaenapsis circularis measured on the basis of: a, extracted ATP, b, chlorophyll a; c, dry weight; d, total nitrogen, and e, total carbon.

example of the drop in ATP level which can occur without a corresponding decrease in chlorophyll content. Here the alga was placed under dark anaerobic conditions, and the ATP pool fell within seconds to negligible levels and then recovered partially over a period of 2 h when substrate phosphorylation only was operating. Figure 11 shows another example of conditions under which the ATP pool may drop and remain low without a corresponding drop occurring in the chlorophyll a concentrations. In this experiment the alga was subjected to phosphorus starvation over a period of 96 h. It is seen that with time the extrac- table ATP pool declines to negligible levels whereas the chlorophyll concentra- tion actually increases over the first 72 h of the experiment and thereafter decreases only slightly. Thus, under varying environmental conditions large fluctuations in the ATP/chlorophyll a ratio can occur as the ATP pool changes rapidly without similar rapid changes occurring in the chlorophyll content of the alga.

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78 P. J. BOTTOMLEY AND W. D. P. STEWART

I 2

, oo o °

\ _c 0 120 /

o _~ / P

o []

80

o -

I J I I I

2 4 6 8 IO 12

Time (rain)

Fl6.9. Transient fluctuations in the ATP pool of Anabaena cylindrica in response to rapid changes in environmental conditions. The alga was grown initially in air at a light inten- sity of 200 ~W cm-Z; at arrow 1, the light intensity was reduced to 20 v.W cm 2; at arrow 2, the alga was placed in the dark.

i

E

o_

2oo 8O

60

40

20

, , ,~ , ,

20 4 6 80 I00 120

Time (rain)

FIG. 10. The change in the ATP pool observed on transferring dark aerobically incu- bated Anabaena cylindrica to dark anaerobic conditions (bubbling with N2/CO.% 99.96:0 04, v/v) at 0 time.

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ATP pools in blue-green algae 79

i -/

v

g~

ID ~ m l

/

200

4O

0

O

i 24 48 72 96

Time (h'~

FIG. 11. The effect of phosphorus starvation on the ATP pool and chlorophyll a con- centration of Anabaena cylindrica. The alga was transferred to phosphorus-free medium at 0 time.

DISCUSSION

In this paper information is presented on how the luciferin-luciferase assay may be applied to the measurement of ATP pools, transitions and turnover in fresh-water blue-green algae. The method is highly sensitive, measuring ATP concentrations in the pmole range and because of this it is not necessary, for accurate assay, to concentrate by filtration or other means, algal suspensions with chlorophyll a concentrations of 1 #g m1-1 or more. This facilitates the speed of assay and has the advantage that although the apparent turnover rate of ATP molecules in blue-green algae is lower than in bacteria (approximately 20 turn- overs min -1 in Anabaena cylindrica and Anabaenopsis circularis growing photo- autotrophically in continuous culture, compared with approximately 300 turn- overs min -1 in E. coli, Holms, Hamilton & Robertson, 1972) possible changes in the ATP pool which may arise during filtration and concentration of the algae are avoided. In addition, the use of modified scintillation vials (Fig. 1) has resulted in considerable savings in reagents, particularly luciferin and luciferase, with the quantities of enzyme-substrate used in each assay in this study costing

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80 P. J. BOTTOMLEY AND W. D. P. STEWART

only 10-20% of that of equivalent material recommended for use with some commercially available ATP kits.

The reproducibility of the technique is excellent with maximum variation from sample to sample being less than 10% of the mean value, and with 100% extraction being obtained with perchloric acid. This contrasts with the findings of Dhople & Hanks (1973) who encountered problems in the use of perchloric acid with certain bacteria.

The size of the extracted ATP pool in Anabaena cylindrica is 160 4- 35 nmoles ATP mg chl a -1 or 2.4 ± 0.5/~moles ATP g d wt -1. The corresponding values for Anabaenopsis circularis are 180 4- 30 nmoles ATP mg chl a -1 and 2.7 4- 0.45 #moles ATP g d wt -~. These pool sizes are rather similar to those reported for other photosynthetic prokaryotes, for example Anacystis nidulans (3 /~moles g d wt -1, Biggins, 1969; 200 nmoles mg chl a -1, Bornefeld & Simonis, 1974) and Rhodospirillum rubrum (4/zmoles g d wt -~, Welsch & Smith, 1969).

In steady state culture the ATP pool remains steady and shows strong positive correlations with the total nitrogen, total organic carbon, chlorophyll content, and dry weight of the algae (Figs 7 and 8). Thus, in such circumstances, it is an excellent measure of algal biomass. Under fluctuating conditions, on the other hand, changes in the ATP pool do occur. Often, these are transient changes as the algae switch from one phosphorylating mechanism to another as environ- mental conditions change (see Fig. 9). Similar changes have been noted pre- viously by Holm-Hansen (1970) in studies on Euglena. He emphasised that since the ATP pools tend to re-establish themselves quickly they may provide a rela- tively reliable measure of biomass, particularly in situations where alternative methods are either poor or inapplicable (see e.g. Holm-Hansen & Booth, 1966). Such transient changes are discussed more fully elsewhere (Bottomley & Stewart, 1976).

Although the ATP pool often returns to its normal level within seconds, various results obtained with Anabaena and Anabaenopsis show that this is not always the case and that very large variations in ATP/biomass values sometimes occur. First, in short-term experiments, if the alga is transferred from aerobic to anaerobic conditions in the dark (conditions which may be encountered in eutrophic waters with algal blooms) the ATP pool falls within seconds and recovers only very slowly, although biomass measured on a chlorophyll basis does not change (Fig. 10). Second, in longer term experiments, when the algae are transferred from light aerobic conditions (usual day-time conditions in natural ecosystems) to dark aerobic conditions (usual night-time conditions) the ATP pool shows a transient drop and recovers as photophosphorylation ceases and oxidative phosphorylation supplies the ATP to the pool. However with time, as Table 1 shows, the ATP pool again falls off in the dark as the substrates for oxidative phosphorylation are used up, and during this time biomass, estimated as chlorophyll a concentration, changes only slightly. Furthermore, the period of time during which the ATP pool remains high prior to falling depends, among other things, on how much carbon reserves are available as a result of photo- synthesis during the preceeding light period. Third, under conditions of nutrient- deficiency the ATP/chlorophyll ratio declines (Fig. 11) as noted by other workers, for example Hoim-Hansen (1970) and Brezonik et al. (1975). In our experiments we have obtained ATP/chlorophyll ratios ranging from 0.005q3.12 depending on

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ATP pools in blue-green algae 81

the envi ronmenta l conditions. These compare with values of 0.04-0.20 obtained by Holm-Hansen (1970) with a variety of algae.

Thus, al though the extracted ATP pool in blue-green algae is steady under constant condit ions and often under fluctuating environmenta l conditions, there are various circumstances when the A T P pool, relative to the chlorophyll con- tent, fluctuates greatly. Such latter occasions are sufficiently c o m m o n to suggest that A T P measurements as an assay of biomass be used with caution, as in fact Ho lm-Hansen (1970) recommended. In particular, its general applicabili ty and usefulness in fresh-water ecosystems requires further investigation, especially since the relationship between biomass and A T P pools in natural ecosystems has yet to be established over prolonged periods and under the varying environ- mental condit ions so characteristic of most freshwater habitats.

ACKNOWLEDGEMENTS

This work was made possible through research support to W.D.P.S. from the Natural Environmental Research Council, the Science Research Council and the Royal Society.

REFERENCES

ALLEN, M. B. ~¢. ARNON, D. I., 1955. Studies on nitrogen-fixing blue-green algae. I. Growth and nitrogen fixation by Anabaena cylindrica Lemm. PI. Physiol., Lancaster 30: 366-372.

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