PHYSIOEOGIA PEANTARUM 91: 729-734. 1994Pnltlfd In tX'Wimrk - ati r(};tll.\ reyen\'d

Effect of acetate on growth and ammonium uptake in themicroalga Scenedesmus obUquus

C Combres, G. Laliberte, J, Sevrin Reyssac atid J. de la Noiie

Combres, C , Laliberte, G., Sevrin Reyssae, J. and de la Noue, J. 1994. Effect of acetateon growth and ammonium uptake in the microalga Scenedestnus obliqtats. - Physiol.Flam. 91: 729-734.

The effect of acetate on growth and rate of ammonium uptake in Scetu'desmus obliquus(UTEX 78) was mvestigated under light-limiting conditions. Addition of acetate toautotropbic cells witb a grovttb constant of 0.71 day ' resulted in an increase in tbegrowtb rate (mixotropby. k = k3 day"'), and in tbe presertee of acetate, growthoccurred in the dark (heteiotropby, k = 0.44 day"'). Tbe rate of ammonium uptake inautotrophy (17.8 amol celL' min"') was similar to tbat in beterotrophy (17.4amol cell''min"') but was 3.7 times lower tban that in mixotrophy 165.9 amol cell"' min"'). Ingenera!, mixotrophie cells showed optimum ammonium uptake al the acetate concen-iration at wbich they were grown. In autotrophy. uptake of ammonium leveled off atabout 125 pA/ wbile no saturation was observed in mixotrophie cells. .An inerease in therate of uptake of ammonium was observed in autotrophic cells within I b after tbeaddition of acetate. Tbe activity of isocitrate lyase (EC 4.1.3.1). a key enzyme for tberegulation of the glyoxyiate cycle responsible for aeetate catabolism, showed a 3.9-foldincrease in activity after 24 h in tbe dark in tbe presence of aeetate. Tbe level ofisocitrate lvase activity in cells grown for 24 h in tbe dark in the presenee of 0-20 mMacetate also increased as a function of acetate concentration.

Kev words - Acetate, ammonium uptake, autotrophy, beterotrophy. isocitrate lyase,mixotropbv. Scenedc.'rnuL\ abliquits.

C. Combres, G. Lciiiberte. J. Sevrin Rev.s.sac and J. de la Noiie {corresponding author}.GREREBA, Utm: Uiral. Ste-Foy, Quebec. Canada. GIK 7F4.

Introduction

The genus Scenedesmus (Chlorococcales, Chlorophy-ceae) ts ubiquitous in various freshwater environments. Itplays a role in lake primary produetion and is often foundin eutrophic waters (Vincent 1980a,b). Its ability to grow-in eutrophic media makes il desirable lor use in high-rateoxidation ponds for the tertiary treatment of wastewaterlo remove nitrogen and phosphorus, atid for the concom-itant prodttctton of algal hiomass (Nair et al. 1981, Fin-gerhtJl et al, 1990), Scenedesmus is often the dominantspecies atnong microalgae present in high-rate oxidationponds such as those used for the treatment of pig wasteswhich are rich in organic and inorganic nitrogen and

phosphorus (Groeneweg et al. 1980). One of ihc reasonsfor the ability of this genus to grow in such environmentscould he its capacity to utilize organic compounds hoth inthe light (mixotrophy) and in ihe dark (heterotrophy)(Droop 1974 , Soeder and Hegewald 1988). Abelioviehand Weisman (1978) postulated that at least 15% of S.obliquus cellular carbon was derived from heterotrophicassimilation of glucose in high-rate oxidation ponds. Re-cently, Fingerhul et al, (1990) showed that at subopttmallight intensities S. fakcitus, isolated from oxidation pondsused for the treatment ol pig wastes, grows much better inthe presenee than in the absence of acetate, even though itcannot grow on acetate in the dark.

From these data, it is reasonable to assume that the

Reeeived 15 February. 1994: revised 25 April, 1994

Phy\rot Plant. 91. IW4 729

presence of organic compounds, in some environments,will favor the growth of many Scenedesmtis species. Thisenhancetnent of growth could he dtie to the additionalsupply of organic skeletons provided by the substrateand/or to the generation of energy by substrate oxidation.In addition, because the assimilation of nitrogen intoamino acids requires both energy and carbon skeletons,cells are ultitnalely dependent on photosynthesis for theassimilation of nitrogen (Turpin 1991). This is especiallyevident in cells grown in the presence of an excess ofnitrogen, where nitrogen assimilation stops in the darkbecause of a lack of carbon skeleton production (Schleeel al. 1985). However, if the nitrogen supply becomeslimiting, the levels of endogenous carbohydtate reservesincrease and eould subsequently be used as a source ofcarbon skeletons for nitrogen assimilation in the dark(Turpin 1991), The requirement of light for nitrogen assi-milation in nitrogen-replete cells eould be alleviated byan external souree of metabolizable organic carbon suchas glucose and acetate, which would permit nitrogena.ssimdation and growth in the dark (Schlee et al. 1985).For exatnple, Chlamydomonas reinhurdtii assimilatesammonium in the dark and in the light in the presence of3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU), uponaddition of acetate (Thacker and Syrett 1972).

Because our research group is interested in the uti-lization of microalgae lor wastewaster treatment from pigfarms, with a parallel biomass production (de la Noiie etal, 1992), and becau.se acetate is one of the most impor-tant low molecular weight compounds found in liquidmanure (Fingerhut et al. 1990), we decided to investigatethe effect of acetate on the growth of S. ohliqtms and onits rate of ammontum uptake. Since the utilization ofacetate in microalgae proceeds via the glyoxyiate cycleand because isocitrate lyase is largely responsible for thecontrol of this cycle (Syrett et al, 1963), we also eval-uated isocitrate lyase activity in S. obliquus in the pres-ence or absence of acetate, both in the light and in thedark.

Materials and method,s

Algal eulture

Scenedesmus nbliquus (UTEX 78) was axenieally grown,without aeration, in autoclaved Woods Hole MBL me-dium (Nichols 1973) supplemented with 5 m/W Tris-HClbufler (Tris-HCl, Sigma), pH 7.2. The cultures were sha-ken by hand once a day. The temperature was 22°C undera 14/10 h light/dark cycle and an illumination of 50 [jmolm-- s"' (Fhiltps F40D fluorescent, Scarborough, ON,Canada), Unless otherwise stated, 10 mM soditim acetatewas added to cells grown in heterotrophy and mixotro-phy, Funga! and bacterial contaminations of the cultureswere routinely screened hy incubating a culture aliquot inWoods Hole MBL medium supplemented with iO mMglucose. 10 mM acetate, 1 g I"' yeast extract (Difco,Detroit, MI, USA) and 1 g 1"' of nutrient broth (Difco).

Measurement of cell growth

For estimation of cell grow th, a culture aliquot was addedto 50 ml of medium and cell growth was followed bymeasuring the absorbance at 680 nm (LKB Ultrospec,Pharmacia, Cambridge, UK), Absorbances were takendaily in mixotrophy and every other day in heterotrophy.Growth constants, k (day"'), were estimated by least-squares fit of a straight line to the data, logarithmicallytransformed as described by Guillard (1973), On someoecasions, cells were counted with an Improved doubleNeubauer hemacvtometer.

Measurement of ammonium in the medium

All the experiments described below were performedwith cells in the exponential phase of growth. Cells wereharvested by centrifugation (4 000 g. 10 min, 4°C; RC5C,Sorvall Du Pont Canada, Inc., Mississauga, ON, Canada),washed twice in nitrogen-free Woods Hole MBL mediumand suspended in 20 to 30 ml of medium in a lOO-mlbeaker. Cells were exposed to a light intensity of 50 pmolm"- s"' (Sylvania GTE, 90 W 130 V, Scarborough, ON,Canada) and were under constant agitation with an orbitalshaker at 100 rpm. Every 15 min for a 3-h period, a 1-mlculture aliquot was harvested and transferred into a1,5-ml Ependorf centrifuge tube conlaining 500 p.1 ofw ater. After centrifugation (1 min, 13 000 g: HeraeusInstntment Biofuge 13, Hanau, Germany), 1 ml of thesupernatant was taken out and kept for determination ofammonium. Ammonium was measured by Nesslerizationas described in standard methods (APHA 1989). To a1 -ml sample, I ml of water was added followed by 40 |4lof Roche! le salt (50 g of potassium sodium tartrate tetra-hydrate dissolved in 100 ml of water) and 200 |jl ofNessier Reagent (Hach Company, Ames, IA, USA), withagitation after the addition of each reagent. After 5 min atroom temperature, the absorbance was read at 435 mn.The linear portion of a plot of ammonium concentrationleft in the medium as a function of time was used lor thecalculation of rates of ammonium uptake per cell. Todetermine the rate of amtnonium uptake on a ehlorophyilbasis, chlorophyll was extracted in 909^ melhanol at 65°Cand Its concentrasion was determined according to Porra(1990),

Isocitrate lyase activity

Isocitrate lyase activity (EC 4.1.3.1) was measured byfollowing the formation of glyoxyiate with its reactionwith 2,4-diuitrophenyl-hydrazine (Goulding and Merrett1970) using cells permeabilized with toltjene as describedby Lalibene and Hellebust (1989).

Results

S. obliquus can grow on acetale in the light or iti the dark(Fig. 1). The growth rate was dependent on acetate con-

730 Ptiysiot Ptanl 91. l'»4

o

2 0 2 S

Aeetate (mM)Fig. 1. Growtb rate (day"') of Scenedesmus obliquus as a func-tion of aeetate concentration imM) in tbe medium. Tbe ligbtintensity was 50 pmol n r ' s"' under a pbotoperiod of 14/15 hligbt/dark. Data points repre.sent tbe results of two separateexperiments in tbe ligbt (D, A) or in the dark ( • , A) witb themean (O, • ) ,

centration but the effect of acetate on cell growtb wasmore pronouneed with acetate concentrations up to about5 mM. Under the experimental conditions employed,rates of growth in mixotrophy were equal to the sum ofrates in auto- and helerotrophy. The growth rate was 0.71day ' in autotrophy (Fig. 1).

The presenee of acetate not only had an effect on cellgrowth, but il also influenced rates of ammonium uptakeexpressed on a cellular or a chlorophyll basis (Tab. 1). Inthe presence of 10 mM acetate the rate of ammoniumuptake was 3.7 times higher in mixotrophy (65.9 amolcell"' min"') than in autotrophy (17.8 amol cell"' min"')and helerotrophy (17.4 amol cell"' min"'). The same con-clusions were reached when rales of uptake were ex-pressed on a chlorophyll basis (Tab. I). Aiiquots takenevery hour over a 5-h period in auto-, hetero- and mix-otrophy showed that from an initial value of about 7.0 the

Acetate (mM)Fig. 2. Rate of ammonium uptake as a function of acetateeoncentration in the medium in cells grown in mixotropby on 10m.W aeetate. Data points represent tbe results of two separateexperiments ( C A) with tbe tnean (O).

pH never rose above 7.5 (data not shown). We thusconcluded that the disappearance of ammonium was due10 eellular uptake and that stripping of ammonium wasnegligible.

Interestingly, when mixotrophie cells grown on 10 mMacetate were transferred into media containing acetateconcentrations from 0 to 20 mM, the rale of ammoniumuptake was optimal at about 10 mM acetate (Fig, 2),Another picture emerged when we looked at rates ofammoniutn uptake in cells grown on different acetateconcentrations and transferred into their respective ace-tate medium during measurement of ammonium uptake.In that ease, rate of uptake was proportional to theamount of acetate present in the medtuni and did not leveloff up to 30 mM acetate (Fig. 3). It went from 23 amolcell"' min"' in aulotrophy up to nearly 100 amol cell"'min"' in the presence of 30 mM acetate.

The presenee of acetate also had an influence on thetransport kinetics of ammonium by 5. tihliquus. In au-

Tab. 1. Rate ol ammonium uptake in .S. obliquus grown on mineral mediutn (autotropbyl or on 10 mM aeetate in tbe lisiht(mixotropby) or in the dark (heterotropby). Tbe ammonium coneentration was 600 \iM and tlie cell density was about 35 x 10" eel!sml"'. Means ±SD (n = 3).

Acetate Ligbt intensityl m"- s"')

Rate of ammonium uptake(amol eet!'' min"')

Rate of ammonium uptake(anio! Ipg cblj ' min"')

500

500

17.8 ± 3.70

65.9 ± 3.917.4 ± 29

28.4 ± 7.40

94.4 ± 10.232.8 ± 5.5

Phvsiol. Piiim 91. 1 731

100 700

CB ,_^

cTo —

E li

Acetate (mM)Fig. 3. Rate of ammonium uptake as a function of acetateconcentration in the medium in cells grown in mixotrophy atdifferent acetate eoncentrations and suspended in tbe same aee-tate eoncentration used for growth. Data points represent theresults of two separate experiments (Q. A) witb tbe mean (O).

totrophy, cells showed a maximum rate of ammoniumuptake at about 125 (iM ammonium in the mediuttn and aslight inhibition above that (Fig. 4). Kinetic constantscalculated from a Lineweaver-Burk plot of the initialportion of the curve gave a K, of 30 iiM and a V,,,,,, of 26

120

100

c —

1::a o

SOO 1000 1500 2000 2500

Ammonium ()iM)

Fig. 4. Rate ot amnionium uptake as a function of ammoniumconcentration in autotiophie ( • . • ) or mixotropbic (O, CJ)cells. Aliquots of tbe medium were taken every 15 min, over a3-b period, and tbe hnear portion of the curve was used tocalculate rates of disappearance of anmioniuni from the me-dium. Data points represent tbe results of two separate experi-ments.

Time (h)Fig. 5. Time-dependent disappearance of ammonium from tbemedium witb (O) or witbout (D) 10 mM acetate added at thebeginning of the experiment to auUitropbic celks at a populationdensity of I5x 10'' cells ml '. Means ±SD (n = 4).

amol cell ' min '. In contrast, the K̂ and the V,,,,,, of cellsgrown in mixotrophy on 10 mM acetate were 121 \xM and86 amol cell"' min"', respectively (data nol shown). Inaddition, in the presence of acetate and contrary to whatis observed with cells grown in autotrophy, the rale ofammonium assimilation was not inhibited up to 2 000 nMammonium (Fig, 4),

All the preceding experiments in mixotrophy wereperformed with cells previously grown on acetate for atleast 3 days. We thus decided to look at the effect ofacetate on ammonium uptake in autotrophic cells notadapted to acetate. Acetate was added to autotrophic cellsat time zero and the disappearance of ammonium wasfollowed as a function of time. From Fig. 5, it is clear thatafter about 1 h the rate of ammonium uptake was signif-ieantly higher than in the eontrol. While the rate in au-totrophy waseonstantat2l atrtol cell'' min ' , it increased

Tab. 2. Isoeitrate lyase activity in S. oblicjuus cells grown inautotropby and transferred in the dark in tbe presence of 10 mMacetate. Means ±SD (n = 3). .At t, lOO'/r = .̂ 7± 14 amol giyoxy-late fonned cell"' mm"'.

Time Isocitrate lyase activity(% of t,,)

34

24

100 ± 0149 ± 24209 ± 12283 ± 60330 ± 273S9 ± 72388 + 21

732 Physiol Pbin. 91. iy94

Tab. 3. Isocitrate lyase aetivity in cells nrown in the dark during24 b in the presenee of different acetate concentrations. Means±.si> (n = 3). 100'/} = 7 1 + 2 0 amol of glyoxyiate formed cell"^min"'.

Acetate(mM)

isocitrate lyase aetivity(9; of heterotrophic cells without acetate)

(J13343

1020

lot)125 ± 30141 ± 2175 ± 61237 ± 62236 ± 52250 ± 79272 ± 44

to 64 amol cell ' min ' after about 1 h of exposure loacetate (Fig, 5) and stayed constant for at least 5 h.

When isocitrate lyase activity was measured in au-lotrophic ceils transferred to 10 mM acetate and the darkat time zero, it increa,sed 3,9-fold in a 24-h period, goingfrom an initial activity of about 37 amol cell"' min"' up to144 amol eell"' min~' (Tab. 2). In addition, when iso-citrate lyase activity was measured in heterotrophic cellskept in the dark for 24 h in the presenee of differentacetate concentrations from 0 to 20 mM, a significantincrease in activtty was observed as a function of acetateconcentration to a level of about 178 amol cell"' min"(Tab, 3).

Discussion

The presence of acetate enhanced the growth of 5. obli-qims, both in the light and in the dark. Under the experi-mental conditions used, the curve of the rate of hetero-trophic growth as a function of acetate concentration ranalmost parallel lo that of mixotrophic growth. This was incontrast to what has been observed in S. falcatus whichcan utilize acetate in the light but is unable to grow inheterotrophy (Fingerhut et al, 1990). The increase ingrowth rate of mixotrophie cells of 5. obliquus was prob-ably the consequence of the use of suboptimal light in-tensities, which is typical of those a few centimetersbelow the surface water of waste stabilization ponds.Generally, the addition of organic compounds such asacetate or glucose has no effect on algal growth rateunder light-saturating conditions (Fingerhut et al. 1990).

The response of tnicroalgae to the addition of a sourceof organic compound is dependent upon Iheir past expo-sure to nitrogen. In general, nitrogen-replete cells show arequirement for photosynthetically fixed CO2 in order totake up and assimilate nitrogen due to their low level ofendogenous carbohydrate reserves (Thacker and Syrett1972, Syrett 1981, Di Martino et al, 1991, Ttirpin 1991).This requirement for newly fixed carbon can be partiallyalleviated by an external source of carbon such as acetateor glucose. Our results corroborate these hypotheses andshow that nitrogen-sufficient cells of S. ohtiquus take upatnmonium only in the presence of light and/or acetate, in

addition, the rate of uptake of atnmonium in mixotrophy(65,9 amol cell"' min ') was higher than the sutn of therates reached in heterotrophy and autotrophy (17.4 -)- i7.S= 35.2 amol ceii ' min"'). The.se results resemble thoseobtained for Chktmvdtnmmas reinhardtii grown on ace-tate where the sum of the rates in heterotrophy andautotrophy were only 85% of those of tnixotrophic cul-tures (Tbacker and Syretl 1972).

The different rates of ammonium uptake by 5. obliquttsobserved as a function of acetate concentration indicatedthat amtifionium uptake tnight be regulated relative to thecarbon metabolism of the cell as proposed by Flynn(1991). Cells mixotrophically grown on 10 mM acetateshowed a quasi constant rale of ammonium uptake. Incontrast, cells exposed to different acetate concentrationsshowed a rate of ammonium assimilation proportional iothe amount of aeetate present in the medium.

Our results indicate that the rate of autotrophic ammo-nium uptake, at 50 pmol tn"" s"', was limited by thesupply of carbon skeletons so that a higher coneentrationof ammonium in the tnediutn did nol lead to a higher rateof assimilation. Upon the addition of acetate, the limita-tion in carbon skeletons was relieved and the rate ofammonium uptake increased up to at least 2 mM (Fig, 4),From the Henderson-Hasselbaeh equation, at pH 7.2abont 99.1 \9i of the ion ammonium is prtuonated (NHt)while 99.64'7f of the ion acetate is unprotonated(CHiCOO). Thus our data do not exclude the possibilityof a free diffusion of atnmonium acetate itiside the cellfollowing a favorable concentration gradient. That mightexplain why rates of ammonium assimilation did notlevel off as a function of ammonium concentration inmixotrophic cells since acetale vv-as always in excess ofammonium.

Many microalgae tililize acetate as a source of carbonboth in the light and in the dark (Droop 1974). In all algalstudies, acetate metabolistn proeeeds via the glyoxyiatecycle, a bypass to the decarboxylation reactions of theCalvin cycle. This anaplerotie sequence requires, in addi-tion to several enzymes of the Krebs cycle, the enzymesmalate syntha.se and isoeitrate lyase, the latter beinglargely responsible for the eontrol of the cycle (Syrett etal, 1963), Wbile its activity is absent in most microalgaegrown in autotrophy (Haigb and Beevers 1964, Martinez-Rivas and Vega 1993), the addition of acetate in the lightor in the dark results in increased levels of isocitrate lyasetnRNA and protein (Sehmidt and Zelsche 1990), In con-trast lo this general picture, isoeilrale lyase activity wasdetected in autotrophic cells of 5. obliqutis and this mightexplain the rapid increase in the rate of ammonium up-take upon addition of acetate to autotrophic cells. Inaddition, the presence of acetate resulted in a 3,9-foidincrease in isocitrate lyase activity over a 24-h period, aphenomenon also observed in many microalgae (Marli-nez-Rivas and Vega 1993), A higb correlation betweenScetu'destnus populations and isocitrate lyase activity inthe hypolimnion of a hypertrophic pond has been in-terpreted as an indication of the heterotrophic metabolism

Physiol. Planl. •.)!. !'»4 733

of Scenedesmtis (Vincent 1980b), Similarly, our datashow that isocitrale lyase activity is in part modulated bythe concentration of acetate present in the trtedium. Itwould be of interest to compare isocitrate lyase activity inScencdc.Krtttt.s cells grown in oxidation ponds to that ofautotrophic cells in order to evaluate the beterotrophicpotential of this alga in the field.

In summary, our data show thai acetate has importanieffects on both growth and ammoniutn uptake in S. obti-quiis. The presetice of this compound in pig waste and inother organically rich wastexvater might be part of theexplanation of the rapid proliferation of Scenedestnus in.such light-limited environments.

Aikmnvleilgftu'ins — This work wa.s supported by a FCAR-Action Spontanee and .NSERC-stategie grants to J. de la N., aN,SERC post-doctoraie scbolarsbip to G,L. and by a France-Quebec exebange projiram to J.S.R. and .1. de la N.

References

Abeliovieh, A, & Weisman, D. 1978. Role of beterotropbicnutrition in growth of tbe alga Scenedesmus ohliqutis inbigh-rate oxidation ponds. - Appl. Environ. Microbiol. 35:32-37.

APHA 1989. Standard metbods for tbe examination of waterand wastewater. - American Public Healtb .Association.Washineton, DC, pp. 117-120. ISBN 0-87553-161-x.

de la Noue,"]., Laliberte, G, & Proulx. D. 1992. Algae and wastewater. - J. Appl. Phycol. 4: 247-254.

Di Mattino Rigano, V,, Vona. V.. Esposito. S., Di Martino, C. &Rigano, C, 1991. Carbon skeletons sources for ammoniumassimilation in N-sufficient and N-limited cells of Cvani-dium caldarium (Rbodopbyta). - J. Pbycol. 27: 220-223.

Droop, M, R. 1974. Heterotropby of eavbon. - In Algal Physiol-ogy and Biocbemistry (W. D. P. Stewart, ed,). pp. 5.'!()̂ ,S,'i9.University of California Press. Los ,Angeies. CA. ISBN

Fingerhut, \i., Groeneweg, ]. & Soeder. C . I 1990. Acetateutilization in Scenedesmus falcatus. an alga from higb-rateponds. - Algol. Stud. 60: 57-64.

Flynn, K.J. 1991. Algal carbon-nitrogen metabolism: a bio-chemical basis for modelling tbe interactions between nitrateand animoninm uptake. - ]. Plankton Res. 13: 373-387.

Goulding, K. H. & .Menett. M.}. 1970. Experiments on enzymeinduelion in Chlorella pvretwidom - J Biol. Educ 4"43-52.

Groeneweg, J., Klein, B,, Mobn, F. H.. RunkeJ, K. H. & Stejigei,E. 1980. First resnits of outdoor treatment of pig manurewitb algai-baeterial systems. - /" Algae Biomass: Produc-

tion and Use (G. Shelef and C.J. Soeder, eds), pp, 255-264.Elsevier/Nortb-Holland Biomedital Press, New York, NY.tSBN 0-444-80242-8.

Guillard, R. R. L. 1973. Division rates. - In Handbook of Pbyc-ologieal Metbods. Culture Metbods and Growtb Measure-mems (J.R. Stein, ed,), pp, 289-31). Camhridse UniversityPress, Cambridge. ISBN 0-521-20049-0.

Haigb, W, G. & Beevers, H. 1964. The occurrence and assay ofisocitrate lvase in algae. - Areb. Biochem. Biopbys. 107:147-151.

Laliberte, G. & Hellebust, J. 1989. Regulation of proline contentof Chloreila autotrophica in response to cbanges in salinity.- Can. 1. Bot. 67: 1959-1965.

Martine/.-Rivas, J. & Vega, ]. M. 1993. Effect of eulttire condi-tions on the isocitrate debydrogenase and isocitrate lyaseactivities in Chlamvdomanas reinhardtii. - Pbysiol. Plant.88: 599-603.

Nair, K. V., Kannan, V. & Sebastian, S. 1981. Nutrient removalfrom sewace effluents using Scenedesmus obiiqtnis - IndianJ. EnvironT Healtb 23: 53-59.

Nicbols, H. W. 1973. Growtb (iiedia - freshwater. - fii Hand-book of Pbyeological Metbods. Culture ,Metbods andGrowth Mea.surements (J.R. Slein, ed.). pp. 7-24. Cam-bridge University Press, Cambridge. ISBN 0-521-20049-0.

Porra, R.J. 1990. A simple method for extracting chloropbylSsfrom reealcitrant alga, Nannochtoris atonius, witbout forma-tion of spectroseopieally-differem magnesium-rbodochlorinderivatives, - Bioebitn. Biopbys. Aeta 1019: 137-141.

Scblee. 3.. Cho, B.H. & Komor. E. 1985. Regulation of nitrateuptake by glucose in Chlorella. - Plant S'ci. 39: 25-30.

Scbmidt. .A. & Zetsche, K. 1990. Regulation of tbe syntbesis ofisoeitrate lyase and tbe corresponding mRNA in the greenalea Chlorogimiiim. - Bot. Acta !D3: 48-53.

Soeder, C,J. & Hegewald, E. 1988. Scenedesmus. - In Micro-Algal Biotecbnology (M. H. Borowitzka and L. J. Boro-witzka, eds), pp. 59-84. Cambridge University Press, Cam-bridge. ISBN 0-52l-32.'^49-5.

Syrett. P. L 1981. Nitrogen metabolism of microalEtte - CanBull. Fish. Aquat. ,Sei. 210: 182-210.

- , Merrell, M. J. & Bocks, S, M. 1963. Enzymes of the glyox-yiate cycle in Chlorella vuigaris. - J. Exp. Bot. 41: 249-264.

Tbacker. A. & Syrett, P. J. 1972. The assimilation of nitrate andamttnonium by Chtamvdomonas reinhardi. - New Phvtol.71 :423^33 . '

Turpin, D. H. 1991. Effects of inorganic N availability on algalpbotosyntbesis and carbon metabolism. - J. Phycol. 27:14-20.

Vincent. W. F. 1980a. The physiological ecology oi' a Scenedes-mus population in the bypolimnion of a hypertropbic pond.I. Pbotoautotropby. - Br. Phycol. J. 15: 27-34.

- 198()b. Tbe pbysiological ecology of a Scenedesmus popula-tion in the hypolimnion of a hypertrophic pond. 11. Hetero-tropby. - Br. Phycol. J. 15: 3 5 ^

Edited by C. P. Vance

734 Phvsiol. Plam. 1)). 1994