Phosphate uptake by blue green algae in vitro and in a lake during an algal bloom:Useful application of a force-flow relationship

Gernot Falkner, Peter Strasser' & Dietmar Graffius l

Osterreichische Akademie der Wissenschaften, Institut ftr Limnologie, Abteilung Mondsee, A-5310 Gais-berg 116, AustriaI Botanisches Institut, Lehrkanzel II, Universitiit Salzburg, Lasserstrasse 39, A-5020 Salzburg, Austria

Keywords: algal bloom, blue green algae, force-flow relationship, phosphate uptake, threshold value

Abstract

A bioenergetic model, developed by M. Thellier, was applied to the 3 2 P-phosphate uptake data obtainedwith blue green algae and allowed the determination of a threshold concentration for the absorption ofphosphate.

Data obtained during an algal bloom of Oscillatoria rubescens showed that algal growth ceased when thephosphate concentration in the lake dropped below this threshold value. The resulting starvation led to anincrease in the permeability of the membrane for phosphate.

Introduction

It is generally acknowledged that the use of 3 2 p_

phosphate for the evaluation of phosphorus-bio-mass-formation by algae in the field raises severalproblems. First of all, phosphate incorporation isnot strictly coupled to the primary production.Under extreme conditions, at least for limited peri-ods, phosphate uptake without growth ('phosphateover plus phenomenon') as well as growth withoutphosphate uptake, with increasing phosphate defi-ciency, is possible. (For review, see Harold, 1966).The uptake rate itself is a function of the phosphatecontent of the algal cell, which itself can vary con-siderably depending on the'phosphorus deficiency'of the algae (Fuhs, 1969).

For these reasons attempts have been made tocorrelate phosphate uptake with growth by apply-ing a modified Michaelis-Menten-equation whichtakes into consideration the phosphorus content ofthe algae (Fuhs, 1969; Rhee, 1973; Droop, 1975).However, deviations from the Michaelis-Mententype of curve have been reported for very low andvery high phosphate concentrations. These devia-tions are explained by the existence of more than

one uptake system operating simultaneously(Brown et al., 1978). Furthermore, from growthkinetics of Selenastrum Brown & Button (1979)suggested the existence of a shutdown of transportsystems at low growth rates.

A further complication arises from the possibleefflux of non-labelled phosphorus during the 32p-phosphate uptake which prevents the measure-ments of net incorporation rates (Nalewajko &Lean, 1978). Added to this is the difficulty to evalu-ate in a simple way the orthophosphate concentra-tion in the field.

In order to describe phosphate uptake in thefield, we have taken another approach that does notinvolve the use of the Michaelis-Menten-equation.Instead we have attempted to correlate the concen-tration dependence of the phosphate inflow withthe absorbing force, a parameter which is definedlater. In these investigations the main attention isfocused on the conditions which for energetic rea-sons still allow a phosphorus incorporation andthose that make it impossible.

Hydrobiologia 108, 265 271 (1984).© Dr W. Junk Publishers, The Hague. Printed in The Netherlands.

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Materials and methods Field experiments

Laboratory experiments

Anacystis nidulans Richt. Dr. strain 1402-1 (Al-gal Culture Collection G6ttingen) and Nostoc mus-corum Allens strain 1013, kindly provided by Prof.Dr E. Kusel-Fetzmann, were cultured in Kratz andMyer's medium (1955) at 38 0C modified by theaddition of 0.106 g/ I Na 2CO 3 and 0.84 g/ 1 NaHCO 3(Essl, 1969). The phosphate concentration in thegrowth medium was 400 iM. The algal culture wasilluminated with fluorescent lamps (Osram 40W/22-1 weil3 de luxe, cool) at an intensity of 250/IEm-2 s I and gassed with air containing 5% (v/ v)CO 2.

For measurement of phosphate uptake the algaewere washed free of phosphate with a HEPES-NaOH buffer (50 mM, pH 7.6) containing thegrowth medium without phosphate. The uptakestudies were performed by a filtering centrifugationtechnique (Werdan & Heldt, 1972) using 0.4 mlpolyethylene tubes (W. Sarstedt, Niimbrecht-Rom-melsdorf). The tube contained 20 l of 10% (w/v)trichloroacetic acid at the bottom, an overlayer of70 l silicon oil (AR 75, Wacker Chemie Mtinchen)and a top layer of 0.3 ml of the algal suspension:The reaction was started by the addition of 32p_phosphate (Amersham, specific activity: 2- 1013 Bqmol- ') at concentration levels as indicated in thelegends and terminated by centrifugation through asilicon oil layer into (10% (w/v)) trichloroaceticacid. The amount of radioactivity in the pellet andin the supernatant was determined with a BeckmanLS 230 scintillation counter.

The decrease of the external phosphate concen-tration in algal cultures during growth was followedwith the aid of 32 P-phosphate in parallel cultures inwhich algae grew under exactly the same condi-tions. For phosphate determination the algal mate-rial was separated from the growth medium bycentrifugation of an aliquot of the culture in aBeckman microfuge. From the radioactivity in thesupernatant the orthophosphate concentration wascalculated after extraction with isobutanol-benzene(see below).

The silicon oil filtration centrifugation methodcould not be applied to algae cultivated under con-ditions where the uptake rates were very high. Herewe used the standard method for field measure-ments.

Phosphate uptake by phytoplankton (mainlyOscillatoria rubescens) was determined in the fol-lowing way: Different amounts of 3 2 P-phosphatewere added to 60 ml aliquots of lake water to give afinal additional concentration and a radioactivityof 3.7 105 Bq/ 60 ml of phosphate ranging from 0.0to 2.0 ,uM. The lake water contained between 5 and18 g chlorophyll a/1. Appropriate correction wasmade for the phosphate content in lake water. Afterincubation for 5 minutes 20 ml aliquots were takenand filtered through glass fiber filters (Schleicherund SchUll, GF 92) to terminate the uptake process.The filters carrying the algae were washed with amedium containing a 20 j/M phosphate concentra-tion and carefully dried by suction. Filter absorp-tion of 3 2 P-phosphate was determined for each con-centration by using the filters with algae of 20 mlaliquots taken immediately after phosphate addi-tion (i.e. at zero time). The separation of the incor-porated phosphate into the metabolized and thenon-metabolized fraction was performed by extrac-tion of an ammonium molybdate orthophosphatecomplex from the resuspended pellet with isobuta-nol-benzene as described by Nielsen & Lehninger(1955). The total amount of phosphorus com-pounds in the lake was determined after hydrolysiswith sulfuric acid by an ammonium molybdatemethod according to Strickland & Parsons (1972).The dissolved inorganic phosphate in the lake wasmeasured enzymatically following a procedure giv-en by Pettersson (1979). Chlorophyll was evaluatedaccording to the method of McKinney (1941).

The phosphate uptake measurements were per-formed in the field in daylight in 100 ml bottles,immersed in lake water and in the laboratory at250 ,uEm 2 s .

Results

1. Rationale of the experimental approach to ana-lyze the uptake behaviour of blue green algae in thefield

In our analysis of phosphate uptake under steadystate conditions we have adopted a steady statemodel, developed in previous studies on the phos-phate incorporation by Anacystis nidulans (Falk-ner et al., 1980). According to this model phosphate

267

is taken up in the light in an energy dependentmanner but not by a process involving an activetransport. Instead it is incorporated via facilitateddiffusion into the cell, where it is converted byphotophosphorylation into the metabolized frac-tion (mainly polyphosphates) according to the fol-lowing scheme:

out in ADP (P - P)n

Pi Pi \ P PATP (P )n+"-F

cellmembrane

light ADP

For the type of reaction sequences outlined aboveThellier (1973) has proposed and applied a linearforce-flow relationship for the description of theabsorption process, leading to the equation:

RT (P) 0v = L ED = L 2,3- log B

nF (Pi)i

Thus the driving force for phosphate flow (v) throughthe membrane is the diffusion potential

RT (Pi).ED = -In B, which is controlled by the ex-

nF (Pi)i

ternal concentration of phosphate in the medium(Pi)o, and the internal concentration (Pi)i resultingfrom photophosphorylation in the cytoplasm. Pa-rameter B includes the membrane potential and thecoupling with further metabolic processes. L is aphenomenological coefficient which reflects thepermeability of the membrane, and the constantsR, T, n, and F have the usual meaning.

In order to use this equation one may assumewith Thellier that (Pi)i remains approximatelyconstant when (Pi)o is changed since the higheruptake rate caused by an increase in the phosphateconcentration in the medium is compensated for bya correspondingly more rapid metabolism. A plotof the velocity of the phosphate absorption versusthe logarithm of the phosphate concentration givesa straight line with a slope reflecting the permea-bility coefficient L and an intercept that gives thevalue for the fraction (Pi) i/B (Thellier, 1970).Therefore we have chosen to present the data in theform of a plot of v versus the logarithm of theexternal phosphate concentration and we shallrefer to this as a 'Thellier plot'.

As is clear from this analysis the term log B/(Pi)ipermits an evaluation of the'absorbing force' of thealgal cell under various ecological conditions. Thevalue of (Pi)i/ B represents a threshold concentra-tion which must be exceeded for an uptake to occur.Thus the smaller the value for (Pi)i/ B, the higherwill be the velocity of uptake for a given phosphateconcentration. As long as the phosphate concen-tration is above this value, the net uptake will bepossible for energetic reasons. When (Pi)o equals(Pi)i/ B, the velocity of the uptake becomes zero.Since it is not possible to determine the value for(P)i and B separately, the ratio (Pi)i/ B (= r- 1 inThellier's designation) corresponding to the thresh-old concentration shall be referred to henceforth as'A'.

The bioenergetic relation described in this sec-tion expresses the net uptake rates in terms of an'absorbing force'. Its application to an analysis ofthe concentration dependence of 32 P-phosphate up-take is thus only possible if net uptake rates can bemeasured, that is, if the efflux of non radioactivephosphate during the incorporation of 32 P-phos-phate can be neglected.

In an earlier report (Falkner et al., 1974) weshowed for external concentrations in the micro-molar range that in the presence of significantphosphate uptake, phosphate efflux was belowexperimentally detectable levels. Also, under theusual growth conditions in batch cultures with highphosphate concentrations, the influx appeared tobe far in excess of efflux. In the latter case it wasalso possible with the aid of 32 P-phophate to meas-ure the net phosphate incorporation: Accordingly itwas shown that the influx of tracer occurs at thesame rate as the formation of phosphorus biomass(Falkner, unpubl.). Since similar uptake and re-lease characteristics were established by the bluegreen alga Nostoc muscorum we may conclude thatunder the experimental conditions employed thenet uptake of phosphate in the light can be readilyfollowed with 3 2 P-phosphate as has been shown tobe the case also with diatoms (Perry, 1976).

Assuming that the measurement of net incorpo-ration in the micromolar range is possible using atracer the validity of the derived bioenergetic modelmay be tested directly. From the model we canpredict that phosphate incorporation will proceedup to the point where the external phosphate con-centration reduces to the threshold value. We can

reach this threshold value in two ways. First by adirect calculation from the uptake kinetics and se-cond from a determination of the limiting phos-phate concentration in the external medium atwhich phosphate incorporation ceases.

Figure 1 shows the kinetic analysis, in the form ofa Thellier plot, of the phosphate uptake by algaefrom a culture in which all available phosphate hadbeen incorporated and thus no further decrease ofthe external phosphate concentration could be ob-served. The uptake kinetics yielded a thresholdvalue of 20 nM phosphate. From direct measure-ment of the phosphate concentration remaining inthe growth medium after the end of growth a sim-ilar value of 19 ± 2 nM was obtained. This closecorrespondence in the values for the threshold con-centration indicates the applicability of the bio-energetic model.

Figure 2 shows the effect of increasing phosphatedeficiency on the threshold value and the permea-bility of the cell membrane which is proportional tothe slope of the straight line as determined from aThellier plot. In this experiment the algae werewashed by centrifugation and resuspension in agrowth medium without phosphate and then fur-ther cultivated in the phosphate free medium underthe usual growth conditions. It can be seen that the

phosphateuptake 50-

1 4.0-

3.0

iE

10

/ oZZ

log [P]

-1.5 -10 -05 0 PM

Fig. 1. Thellier plot of the concentration dependence of phos-phate influx, performed with algae immediately after phosphateuptake had ceased. 500 ul of the algal suspension (0.1 pg chloro-phyll/ ml) were injected into bottles containing the growth medi-um and different 32 P-phosphate concentrations between 0.1 and1.0 pM. The reaction was terminated by filtration through aglass fiber filter. Temperature: 38 ° C. Illumination. Intercept onthe x-axis: -1.71 = log 0.0195 pM.

, 60-E

E

/40

a 20-

/==rj- -~ ~,w~:~:~~~~~~~~~~~~~~~~~~~~~~~

phosphateuptake

/

log [Pi]

-20 -10 0 PM

Fig. 2. Effect of prolonged cultivation of Anacstis nidulans(0 30 min., 0 60 min., 22 hours) on a phosphate free mediumon the influx pattern as analyzed by a Thellier plot. Chlorophyllcontent: 0.3 g chlorophyll/ ml at the beginning and 0.6 ygchlorophyll/ml at the end of the experiment. 38 o C. Illumina-tion.

A-value decreases first during the subsequent oc-curring process of phosphate depletion with essen-tially no change in the permeability of the mem-brane. This indicates a continuously increasingfaculty for absorption presumably in connectionwith the mobilization of endogenous polyphos-phate resources. Later the slope increases in re-sponse to phosphate depletion, indicating the in-creasing activity of the phosphate carrier in themembrane. The increase in the A-value seen afterprolonged incubation can be attributed to the onsetof deterioration of the algae.

2. Changes in the absorption of phosphate duringthe course of an algal bloom

The absorption capacity of an algal populationseems to be a variable parameter that shows astrong dependence of the phosphate nutrition stateof the alga. It was therefore of considerable interestto establish the extent to which the absorption ca-pacity changed during an algal bloom and whethera relationship could be established between thisabsorbing capacity and the altering phosphate con-centration. For this investigation we chose an eu-

268

- ~ L

269

trophic lake (the Obertrumer See near Salzburg,Austria) in which the spring circulation causes anannual algal bloom of Oscillatoria rubescens inApril and May. During the development of thisbloom the phosphate concentration sinks below thelimits of detection (<10 nM) while the content ofNO3 and NH- remains above 2 jumol/ 1 (A. Jagsch,pers. commun.).

All measurements described here were perform-ed with an algal biomass consisting of more than95% of Oscillatoria rubescens.

The first experiment shows a time dependence ofthe steady state influx of phosphate with a lakesample of Oscillatoria rubescens (Fig. 3) in thepresence of two different concentrations of the ion.The 3 2 P-phosphate taken up was separated in themetabolized and non-metabolized fraction imme-diately after the experiment. The fact that practical-ly all the incorporated tracer is found in the metab-olized fraction, matches with the reaction schemeproposed above. The uptake rate remained con-stant throughout the day in line with the data ob-tained using diatoms by Perry (1976).

Figure 4 shows a series of Thellier plots from dataaccumulated in the year 1981, which demonstratethat in the field, too, the concentration dependenceof the uptake rate obeys to a logarithmic function.It is striking (Table I) that the algae could grow aslong as they were able to compensate for the de-creasing phosphate concentration in the lake by aneven greater reduction of the A-value. The end ofthe algal bloom was then characterized by a drop inthe lake phosphate concentration below the A-value (Table I) which occurred between the 15th of

phosphate

50 uptake

. 40 V5 V

E 30 V

o 20

10 0

0 10 20 30 min

Fig. 3. Time dependence of phosphate influx into Oscillatoriarubescens at 0.1 (circles) and 10.0 (triangles) MiM external phos-phate concentration in lake samples. Open symbols: totalamount of incorporated phosphate, closed symbols: metabol-ized phosphate.

4.0

30

.'X20E

* E

/ 10

phosphateuptake

,__. ltog [Pi ]

-20 -1.0 0 10 iM

Fig. 4. Thellier plots of phosphate influx for lake samples ofOscillatoria rubescens taken in 1981. · 9th of April; O 15th ofApril; A 26th of May; 2nd of July.

April and the 7th of May 1981. Concomitantly,there was no increase in the mean chlorophyll valueper liter lake water (18 Mg/ 1) after the 15th of April,as determined in a series of vertically taken samples,indicating that the algae had actually stopped grow-ing. The following period of phosphate starvationthen leads to a dramatic increase of the value for

RT2,3 L as is observed in cultures which were pro-

nF

longed incubated in a phosphate free medium (Fig.2). Obviously phosphate starvation in the lake hada similar effect on the absorption capacity as aphosphate depletion caused experimentally in thelaboratory by washing and subsequent cultivationin a phosphate free medium.

Discussion

In order to describe the steady state phosphateuptake we have assumed - according to non-equili-brium thermodynamics - that for the small valuesof the net fluxes a linear relationship exists betweenthe net uptake flux and the absorbing force. Byplotting the uptake rate versus the phosphate con-centration a logarithmic curve is obtained whichhas a shape similar to the Michaelis-Menten hyper-bola. This logarithmic curve is characterized by two

Pv t - -

270

Table 1. Change of the bioenergetic properties of Oscillatoria rubescens during an algal bloom.

Date Lake phosphate A-value RT Mean chlorophyll2.3 -Lconcentration (nM) (nM) nF value (jg/l)

4.4.81 740 182 43 129.4.81 250 140 63 17

15.4.81 30 12 65 187.5.81 <10 8 25 16

26.5.81 <10 56 653 112.7.81 <10 70 2915 6

parameters which have an intuitive biological mean-ing: A phenomenological coefficient, which reflectsthe permeability of the membrane and a thresholdvalue, below which no net uptake occurs. It is to beexpected that this value is influenced by variousecological conditions. At the end of the algal bloomthe threshold value for Oscillatoria rubescens wasof the same order of magnitude as that obtained inpractice for Selenastrum capricornutum in culture(Brown & Button, 1979).

It must be stressed, however, that this kineticapproach yields useful results only under condi-tions for which the net uptake is measured in thepresence of different amounts of phosphate. In con-trast to our results, Nalewajko & Lean (1978) ob-served a considerable efflux accompanying the up-take process. The kinetics of phosphate uptake canbe explained using the bioenergetic mcdel in thefollowing way: Due to the constant internal phos-phate concentration, the efflux is always held at aminimal predetermined level. This level is preciselydefined by the threshold'A'-value, at which concen-tration the influx is equal to the efflux. At externalphosphate concentrations in the micromolar rangethis results in an influx rate several orders of magni-tude greater than the efflux rate. Therefore Nale-wajko & Lean (1978) could only observe efflux atextremely low phosphate concentrations (some-times <0.01 uM). For high concentrations theseauthors (Nalewajko & Lean, 1978) also detected anuptake of 3 2 P-phosphate that was directly propor-tional to the net rate of 3 'P-phosphate removalfrom the medium. Thus net uptake can only bemeasured, either in the laboratory or in the field,when algal growth is limited by phosphate and if theexternal phosphate concentration is several foldhigher than the A-value. If these conditions arefulfilled, the values for A and 2.3 RTL/ nF can beobtained from the described kinetic analysis. Once

these values are available phosphorous biomassformation at low phosphate concentrations forwhich a measurement of net uptake rates by traceris impossible, can be readily estimated by extrapo-lation of the logarithmic function. For example,with the values for the 9th of April 1981 in Table 1an incorporation rate of phosphate of 0.95 #mol/mg chlorophyll per hour can be calculated. Sincethe total phosphorous content of Oscillatoria ru-bescens at this time of the bloom was 180,umol permg chlorophyll the algae would need nine days todouble this phosphorous biomass, assuming 14hours photophosphorylation per day. Consideringthe low growth rate of Oscillatoria rubescens(Staub, 1961) this value appears to be quite realisticunder the given conditions in the lake. With the aidof the tracer we have obviously been able to meas-ure the net phosphate uptake in the lake. Theuncertainties in the estimation of biomass forma-tion by 32 P-phosphate addition in this study weretherefore rather due to the well known difficultiesof the determination of orthophosphate in the lakethan to a possible isotope exchange. However, al-though the enzymatic phosphate determinationsuffers from inaccuracy subject to the organicphosphorus content in the lake the values obtainedcould adequately indicate whether or not the or-thophosphate concentration is higher or lower thanthe A-value and thus whether or not algal growth,expressed as phosphorus biomass formation pertime unit, occurs.

Summary

A bioenergetic model, proposed by M. Thellier,which connects uptake rates with an 'absorbingforce', was applied to phosphate uptake data gainedwith blue green algal cultures in the laboratory and

271

- during an algal bloom - with lake samples con-

taining Oscillatoria rubescens. According to this

model is the uptake velocity v = k log--. Thus byA

plotting v against the phosphate concentration Pi alogarithmic curve is obtained which has a shapesimilar to the traditional Michaelis-Menten hyper-bola. The constant k reflects the permeability of themembrane and the term A is a threshold value forphosphate uptake, which must be exceeded to allowphosphate incorporation.

In order to test the validity of this model forphosphate incorporation, experiments were per-formed with algal cultures, in which the algae wereallowed to incorporate all available phosphate.Here the threshold value, calculated by the bio-energetic model actually corresponded to the re-maining phosphate concentration in the medium.The subsequent phosphate starvation caused anincrease of the permeability of the membrane.

During an algal bloom in the Obertrumer See asimilar picture emerged. The bloom lasted as longas the phosphate concentration was above thethreshold value of the algal material under the re-spective ecological condition. Algal growth ceasedwhen the phosphate concentration dropped belowthe threshold value. During the following weeks thepermeability of the membrane increased dramati-cally due to an activation or additional induction ofthe phosphate carrier.

Acknowledgements

The authors are greatly indebted to Prof. Dr M.Thellier, Rouen, and Dr J. V. Small, Salzburg, forthoroughly reading and improving the manuscript;to Prof. Dr O. Kiermayer, Salzurg, for his encour-agement; to Prof. Dr E. Kusel-Fetzmann, Wien, forkindly providing Nostoc muscorum, and to theFonds zur Frderung der wissenschaftlichen For-schung in Osterreich for financial support.

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Received 6 February 1983; accepted 5 May 1983.