HYDROGEN ION BUFFERING OF CULTURE MEDIA FOR ALGAE FROM MODERATELY ACIDIC, OLIGOTROPHIC WATERS

88 RUSSF.LL L. CHAPMAN AND MARGARET C. HENK

Graham, L. E. Se McBride, G. E. 1978. Mitosis and cytokinesisin sessile sporangium of Trentepohlia aurea (Chlorophyceae),J. Phycoi 14:132-7.

Marchant, H.J.& Pickett-Heaps.J. D. 1973. Mitosis and cyto-kinesis in Cottochaete scutata, j . Phytoi. 9:461-71.

Maltox.K. R. & Stewart. K. D. 1984. Classification of the greenalge: a concept based on comparative cytology. In Irvine, D.Y..O.St}o\\x\.\i.}A.\^^.\ThtSjstrmaticsof Green Algae. Univ.Cambridge Press, Cambridge, pp. 29-72.

O'Kelly, C.J.& Floyd, G. L. 1984. Correlations among patternsof sporangial structure and development, life histories, andultrastructural features in green algae, with special referenceto ordinal circumscriptions and phytogenies in ihe Ulvophy-ceae. tn Irvine, D. E. G. &John, D. M. [Eds.] TheSysltmaticsof Green Atgae. Univ. Cambridge Press. Cambridge, pp. 121 -56.

Picltett-HeapsJ. D. 1967. Ulinutructure and differeniiaiion inCAara sp. 11. Mitosii. AusL J. Biol. Sci. 20;883-94.

1975. Green Algae. Sinauer Aisoc., Sunderland. Massa-chusetts, 606 pp.

Pickeit-Heaps,J.D.«f Marchant. H.J. 1972. The phylogeny ofihe green algae: A new proposal. Cytobios 6:255-64.

Roberu. K. R. I9S4. The flagellar apparatus in Batophora andTrtntepohtia and its phylogenetic significance. In Irvine. D.E. G. Se John, D. M. JEds.] The Systematics of Green Algae.Univ. Cambridge Pres*. Cambridge. London, pp. 331-41.

Roberts, K. R., Stewart. K. D.. Se Mattox. K. R. 1982. Structureof the anisogametes of ihe green siphon Pseudobryopsis sp.(Chlorophyta)./ Phycoi 18:498-508.

Sluiman. H.J. 1983. The flagellar apparatus ofthe zoospore ofihe Blamentous green alga Coleochaete pulvinata: absolute con-figuration and phylogenetic significance. Protoplasma 115:160-75.

Sluiman, H. J., Roberts, K. R.. Stewart, K. D. Sc Matiox. K. R.1983. Comparative cytology and taxonomy ofthe Ulvophy-ceae. IV. Mitosis and cytokinesis in Utothrix (Chlorophyta).Ada Bot. Seeri 32:257-69.

Stewart, K. D. & Mattox, K. R. 1975. Comparative cytology,evolution and classification of the green algae with someconsideration ofthe origin of other organisms with chloro-phylls A & B. Bot. Rev, 41:104-35.

Stewart, K. D., Mattox, K. R., Se Flovd, G. L. 1973. Mitosis,cytokinesis, the distribution of plasmodesmata. and othercytological characteristics in the Ulotrichales, Ulvales. andChaetophorales: phylogenetic and taxonomic consider-ations./ Phycoi 9:128-41.

Thompson. R. H. 1959. The life cycles of Cephaleuros and Sto-matochroon. Proc. int. Bot. Congr. 9:397,

Turner. F. R. 1968. An ullrastruciural study or plant spermato-genesis; Spermaiogenesis in Is'itellaj- Cell Biol. 37:370-93,

J. Phycoi 21,

HYDROGEN ION BUFFERING OF CULTURE MEDIA FOR ALGAE FROMMODERATELY ACIDIC, OLIGOTROPHIC WATERS'

John D. Wehr,^ Lewis M. Brown and Ingrid E. VanderelstDepartment of Plant Sciences. University of Western Ontario. London. Ontario N6A 5B7, Canada

ABSTRACT

Five hydrogen ion buffers were compared for their use-fulness in regulating pH in a model oligotrophic, mod-erately acidic (phi 6.0) algal growth medium. These were3,3-dimethylglutaric acid (DMGA), tricarbaHylic add(TCA), trans-aconitic acid (tAA), N-2-hydroxyethylpiper-azine-W-2-ethanesulfonic acid (HEPES) and 2'(N-mor-pholino)ethanesulfonic acid (MES). All buffers (2.5 mM)except HEPES limited the reduction of pH in a NH^*-based medium during growth of Chrysochromulinabreviturrita Nich. to less than 0.12 uniLs, compared withmore than 2 units in an unbuffered medium. Long termgrowth ofC. breviturrita in these media was significantlyinhibited (P < 0.05) by TCA and tAA. MES was able tocontrol pH with the minimum amount of NaOH (1.0 mM)added to the medium to adjust to pH 6.0. Four of fivebacterial isolates were capable of utilizing tAA as a soleorganic-C source, and no isolate could metabolize HEPESor MES. No significant differences (? > 0.05) were foundin the maximum growth rates of six algal species (fromfive classes) in a medium with or without MES buffer,although significantly greater cell yields of Ochromonasdanica Prings. were obtained in the buffered medium.

' AccebUd: 14 Scrvrmber 1983.* Addreu for reprint re<iueus.

MES (pK, = 6.15) was considered to be the most usefulbuffer in the pH range 5.0-6.5, due to ils biological in-ertness, buffering capacity, the minimal requirement forexcess base to adjustpH and its minimal metal complexingability.

Key index words: algae: culture medium; 3,3-dimethyl-glutaric acid; HEPES; 2-{N-morpholino)ethanesulfonicacid; MES; pH; pH buffer; softwater

Until recently most freshwater algal media did notinclude a buffer to control hydrogen ion concentra-tions. Many of these relied on the presence of suf-ficient HCOa" and/or PO4*" to minimize shifts inpH during growth. However, biologically mediatedalkalinity and pH changes nonetheless occur withnitrogen uptake. Growth in NO," or NH,* mediaresults in the generation of strong base or acid, re-spectively (Brewer and Goldman 1976), CO, uptakemay also cause a reduction in pH. This is a particularproblem in more dilute media which attempt to ap-proximate the chemistry of oligotrophic waters(Wehretal. 1985).

In a compilation of methods for preparing fresh-water growth media (Nichols 1973), only one recipe(Woods Hole Freshwater) included a pH buffer. Thisbuffer, TRIS (tris[hydroxymethyllaminomethane)has been used widely but has poor buffering capacity

HYDROGEN ION BUFFERING OF MEDIA 89

below pH 7.8 (Perrin and Dempsey 1974). Otherbuffers which have been employed in this pH rangeinclude triethanolamine and glycylglycine, althoughthe former suffers both from an apparent toxicityto certain algal species and cation interactions (Pro-vasoli and Pintner 1960), while the latter can bemetabolized by bacteria (McLachlan 1973). For me-dia which are alkaline or near neutral pH, the zwit-lerionic buffer HEPES (N-2-hydroxyethylpipera-2ine-N'-2-ethanesulfonic acid; pKa 7.55: Good et al.1966) has been shown to be superior to TRIS andother buffers in buffering capacity near pH 7.0 andits lack of inhibitory physiological effects (Smith andFoy 1974). These characteristics and its negligiblemetal complexing ability have resulted in HEPESnow being utilized in a variety of algal media (e.g.Bekheet and Syrett 1977, Healey 1977. Francke andten Cate 1980, Whitton and Shehata 1982).

In algal culture studies of species from less alkalineenvironments, particularly in the pH range 5.0-6.5,the choice of a buffer is less clear. In early studiesof "oligotrophic species" in this pH range H* levelscould not be regulated adequately (Moss 1973). Theamino acid histidine (pKa, = 5.97) has been pro-posed as a useful pH buffer in this range (Hutner1972). but it has been found to chelate metals almostas strongly as EDTA (Provasoli and Pintner 1960)and it can serve as the sole carbon source for somebacteria (Stanier et al. 1970). A few other pH buffersappear to be potentially useful. These are DMGA(3.3-dimethylglutaric acid; pKa, = 6.31), TCA (tri-carballylic acid; pKa, = 6.22), tAA (trans-aconiticacid; pKa, = 5.89) (Mallette 1967) and MES (2-(N-niorpholino)cthanesulfonicacid; pKa, = 6.15) (Good^t al 1966). The first three ofthese are notable inthat they do not contain N, P or S, but are not"vitterions. HEPES may be compared with this groupbecause of its proven biological inertness, althoughIts buffering capacity at pH <6.5 is limited.

In the present study we investigate the suitabilityof several buffers in terms of H+ buffering ability,microbial degradability (for xenic and axenic cul-tures), stimulatory and/or inhibitory effects on algalSrowth and usefulness in a model softwater system.

MATERIALS AND METHODS

Mosi of ihe experiments were carried out using an axenic cul-^^*^^f Chrysochromulina brei'iturrita Nich. which was isolated from

an oligotrophic, moderately acidic lake in south-central Ontariojoinder Lake: 78*56* W, 45"04* N). Details of isolation are given^ Wchr et al. (1985). Growth of several other specie* was also«ied. The choice of species followed a preliminary screening of

Cie and other cultures for comparable growth at pH 6.0 and• The original sources of these isolates were as follows: Uni-

" of Texas Culture Collection (UTEX nos.): Cryptomonas.yw/uj/m Prings. (358). Mougeotia sp, (LB 758). OchromonasPringi. (LB 1298). Synura petersenii Korsch. (LB 239): Car-

a Biological Supply: Pendinium cf. cmctum (Mull.) Ehr.; Uni-y ^ Western Ontario Culture Colleclion; Euglena grarilisJ " ' isolate from soil/hay enrichment culture by V. Zva-

: U.W.O. no. 40). Chrysochromulina. Cryplomonas and Ochro-J cultures were axenic, the other four contained bacteria.

HEPESA MES• DMGAO TCAV tAA

(NoOHl (mM)

FIG. 1. pHtitration curves of 2.5 mM HEPES. MES, DMGA.TCA and tAA buffers versus NaOH. Solid symbols indicate pKavalues (average of duplicate litraiions).

Growth of bacteria was tested using the pH buffers as the soleorganic-C source. The medium «2 of Stanier ei al. (1970: p. 79)was employed, using either glucose or ihe buffers, supplied at0.3'X (standard medium concem ration) and 0.05^ (approximateconcentration used in algal cultures). The basal medium to w hichorganic carbon was added, contained 1 g/L NH^Cl, I g/LK,HPO«, 200 mg/L MgSO«.7H,O. 10 mg/L FeSO,.2H,O, 0.02mg/L CuSO,, 0.02 mg/L CoCl,.6H,0 and 0.01 mg/L ZnCl,.For comparability with algal tests, the pH was adjusted to 6.0.The bacteria used (University of Western Ontario Culture Col-lection) were Escherichia coh. .\Ucrococcus luleus. Pseudomonas fiuo-rescens, Serratia marcescens and an unknown gram negative rodbacterium which was isolated from the lake water from which C.breviturrita was isolated.

The culture medium previously used for C. brmturnta, Mus-koka #42, which was designed lo approximate Canadian ShieldWaters influenced by acidic precipitation, has been described indetail previously (Wehr et al. 1985). In the presenl study onlyhalf Ihe normal N and P (Muskoka «112 medium) was supplied(50 fiM (NH4)H,PO0. The pH of the medium was 6.0, ihe con-ductivity was ca. 70 pS/cm and ihe toial alkalinity was ca. 12 *iEqHCO,-/L. For the other algal species lhis medium was supple-mented wilh 50 ^M CaNO, and 20% soil extract (prepared fromnatural loam: 100 g/L, twice autoclaved. then 0.45 fim membranefiltered). The addition of soil extract contributed an average of15 nEx\ HCO,"/L alkalinity to the medium, which was negligiblein combination with MES buffers.

Algal cultures were grown as described previously (Wehr et al.1985) at 20* C. with an irradiance of 60 ME m-' s"' on a 12:12h LD photoperi<xl. The cultures were grown in 125 mL FJirlen-meyer flasks containing 50 mL of media. All treatments were runin triplicaie. Cell counts of unicellular algae were carried oulusing a Biophysics systems laser-based cell counter which wascalibrated against hemacytometer counu. Mougeotia growth wasmeasured using hand counts in a Sedgwick-Rafter cell after ho-mogenizing in a blender. Bacterial cultures were grown in 5 mLof liquid media in tesi tubes, Eschenchia coli, M, luteus and theunknown bacterium were grown at 37" C: P. Jfuorescem and S.marceicrns were grown at 25* C. Cell densities were estimated byturbidity using a spectrophotometer at 420 nM. The measure-ments were corrected for background turbidity using carbon de-pleted cultures.

90 JOHN D. WEHR F.T AL.

T A B U 1. Gnrwth a/Chr>-sochromutina breviturrita in Muskoka til 12 medium. pH 6.0. supplied wilh different H' buffen (2,5 mM). Buffersluted in descending order oJ pKa value nearest 6,0 (italic). Growth data ugniftcantly different from unbuffered controls 0' < 0.05) indicated byasteruk (*); n ' 3. SL ± SD.

BufTcr trraimmi"

UnbufferedHEPES"DMGA'TCA'MES*lAA'

1

—

3.03.723.67

<4.03.04

2

7.556.314.85&/54.25

3

6.22

5.89

(itiv./iby)

0.72 ± 0.120.73 ± 0 . 1 40.64 ± 0,040.70 ± 0.040,77 ± 0.070.62 ± 0.08

Virld(IO«(dl»/ml.)

3.04 + 0.073.01 ± 0,042.83 + 0.173.53 ±0 .11*2.94 ± 0.072,85 ± 0.20

• For explanation of abbreviations see Introduction."pKa dau from Good et al. (1966).< pKa data from Mallette (1967).

The pH buffers HEPES. MF^. DMGA. rAA and TCA wereobtained from Sigma Chemical Company, and solutions of the^were prepared directly from the purified acids as supplied. Equi-molar buffering capacities (2.5 mM) were measured using titra-tions against 1.0 M NaOH in duplicate. Changes in pH weremeasured using a "Ross" (Orion model 810310) semi-micro pHelectrode.

RESULTS

The relative buffering capacities ofthe five buff-ers (each 2.5 mM) tested (Fig. 1) indicate the ex-pected differences in pH optima and also the amountsof NaOH required for each to set the pH to theendpoint of 6.0. All were titrated from the initialpH of the buffer in solution to pH lO.O. Both TCAand tAA (forabbreviations see Introduction) provedto be efficient buffers from pH 3.0 to over 6.0, henceboth required more than 5 mM NaOH to reach thedesired endpoint. HEPES required only 180 MMNaOH, but the buffering capacity at pH 6.0 wasslight. The pKa of MES (6.15) is near the desiredrange and required approximately 1.0 mM NaOHto reach pH 6.0.

Growth of C. breifiturrita in the Muskoka #112medium (pH 6.0) was compared using these fivebuffers versus the unbuffered medium (Table I).There were no significant differences in maximumdivision rates or yield of the alga, with the exceptionof one significant (P < 0.05) increase with the TCA-buffered treatment. The pH of the unbuffered me-dium (supplied with NH.,* as the sole N-source)dropped an average of nearly 2 pH units (Fig. 2).HEPES had only a moderate buffering capacity, whileall others limited the shift to less than 0.12 units.Although the slopes of the titration curves (Fig. I)would indicate greater buffering by TCA and tAA,in this regime they were insignificantly different fromDMCA and MES.

Long term growth of C. breviturrita in the pres-ence of pH buffers was examined by subculturingthis alga in the treatments shown in Table I fourconsecutive times (Fig. 3). No significant changes incell yield over time were detected in the unbufferedor the HEPES and MES-buffered treatments. Amarked decline was observed in the TCA treatment.

in which there was initially a stimulation, ln the tAAtreatment a strong decline in yield was also found.

Biodegradability of these buffers by bacteria wascompared by replacing glucose with each buffer asthe sole organic-C source (Table 2). The gram pos-itive bacterium, Micrococcus luteus, was capable ofmetabolizing three of the five buffers: DMGA, TCAand tAA, to different extents. Three of the otherspecies (all gram negative) could grow on tAA as acarbon substitute, but Escherichia coli grew only onglucose. Most metabolizable buffers supported somegrowth at the lower concentration tested (0.05%),which is comparable to that frequently employed(2-3 mM) in algal growth media. Neither of thebuffers (HEPES, MES) of Good et al. (1966) sup-ported bacterial growth.

As MES appeared to be the most practical bufferat pH 6.0, growth of six algal species, representingfive classes, were compared in buffered media (Table3). All ofthese species were screened for the abilityto grow equally well at pH 6.0 and 7.0. None of thespecies grew less well in the buffered medium. How-ever, cell yields oi Ochromonas danica in the unbuff-ered medium were half {P < 0.05) that measuredin the buffered medium. This was the only regimein which the pH declined during growth. Net pHchanges in all unbuffered media were significantlygreater (in absolute value) than in the buffered me-dia.

Buffer concentration had a significant effect finthe magnitude of the net pH change during growthof C breviturrita (Fig. 4a). MES concentrations £ 1.0mM were sufficient to limit pH reduction to less than0.2 units. Maximum division rates and cell yieldswere unaffected over the concentration range 0-25mM MES (Fig. 4b, c). Growth was inhibited at great-er concentrations. No evidence was observed for adose-response growth stimulation in C. breviturritadue to the buffer.

DISCUSSION

A major concern in many physiological studies ofalgae in culture is maintaining the pH at a desiredlevel. Although the most widely used means has been

HYDROGEN ION BUFFF.RINC OF MEDIA 91

X

DMGA

5 -

4 ^

TCA MES tAA

10 20 10 20 10 20

t i m e ( d a y s )iRes in pH in Muskolta « U 2 with difTerent buffer treatments {2.5 mM or no buffer) over time during growth of CSD, n - 3). SD bar* less ihan symbol siw not shown.

through a COj-HCO,- system, this is of limited val-^ ' " "^°^^ oligotrophic, moderately acidic media

attempt to model freshwaters in the earlyg of acidification (Wchr et al. 1985). In nature,

such softwater lakes in the pH range 5.0-6.5 fre-quently contain <100 MF.q HCO5 / L (Dillon et at.^^78, Schindler et al. 1980).

The principal criteria for a pH buffer in cultureedia include: (1) a pKa within the physiologically

or ecologically relevant range; (2) high solubility inwater and minimal solubility with other (esp. non-polar) solvents; (3) an inability to cross biologicalmembranes (hence low biological reactivity); (4) weakor negligible complexing with metals and (5) chem-ical stability (Good et al. 1966. Perrin and Dempsey1974). In addition, a buffer in more dilute media(e.g. Muskoka #112: conductivity = 70 ^S/cm)should ideally provide adequate buffering without

tnicteria. Escherichia coli, Micrococcu* luteus. Pseudomonas fluorescens. Serratia marcescent and anbuffers as the sole carbon source. Grtm-th expressed as a percentage of growth in glucose controit. measured i-wi

ce at 420 nm and corrected for background turhiHity using carbon depleted cultures (for explanation of buffer abbreviations see Introduction),

-—• p"*"""*"E, coli

W. tuteus

P. fiuorfscens

"• marcesffiu

"nktiown

0.05030,050.3

0.30,0$b.30.050.3

(-130 nm)

0.0710.08G0.0400.0410.4880.3550.7090.7770.S080.224

UF.PJ-S

0000

0000

fl0

A

tlMC.A

000

47

0600d

14

bMwtMfwr ( 1 control)

TCA

0

28106

000Q

a0

MES

0000

0

00000

lAA

00

ioo138MO

18810&

0

92 JOHN D. WEHR ET AL.

Ml

atu

o

0-3 -

0-2 -

unbuffered HEPES DMGA

0.3 -

0.2 -

0.1 -

TCA MES t A A

U 1

n u m b e r of

1

su b c u t t u r e s

Ftc. 9. Influence of long-term growth in media supplied with different buffers (2.5 mM) on final cell yield of C. brrviturrita viarepeated subcultures (x ± SO, n " 3). Each culture wan sequentially transferred Four times into an identical medium every seven days.Nutrient deficiencies may have developed in media with TCA and tAA due to altered chemical activities or solubilities of some nutrients.

excess amounts of neutralizing salts (e.g. NaOH) toachieve the desired pH.

The present results suggest that ofthe five bufferstested, tAA is least suitable in several regards. Itsbiodegradability (Table 2) renders it unsuitable fornon-axenic cultures. TCA and DMGA may also beunsuitable in this regard. Both tAA and TCA are

plant products. Long term inhibitory effects of tAAand TCA on C. breviturrita (Fig. 3) are clearly un-favourable. Previous studies (e.g. Provasoli and Pint-ner 1960, Smith and Foy 1974) which have exam-ined the use of pH buffers in algal growth mediatypically use replicate treatments, but have not con-sidered potential longer term effects over several

TABLE 3. Comparison of pH change and growth of six algal speaei at initial pH 6,0 in a buffered (2.5 mM MES) and an unbuffered medium.Data lignifitantty different (P < 0.05) from unbuffered controls indicated by asterisk fn — ?, 8 ± SD; ND • no data avaitabte).

Spnrtn

Cryptomonas ovata v. palustris

Eugtma gracilis

Mougeotia sp.

Ochromonas danica

Peridinium cf, dnclum

Synura pet*rsmu

MFJ buffer

—

—

-

—

0.130.66O.II0.570.020.48

-0.51-t.6O

0.050.610.191.15

pH

±±±±±±±±±±±±

0.01*0.100.02*0.020.02*0.04O.IO»0.070.02*0.15O.Q2»0.16

MJK. diviikin rair

0.77 ± 0.040.74 ± 0.020.64 ± 0.020.65 ± 0.02

NDND

0.65 ± 0.060.61 ± 0.010.41 ± 0.050,46 ± 0.060.58 ± 0.070.59 ± 0.02

Ylrld(IO*

299363275271

3241

3311652322

259274

c:rl

±±±

±±±±±±±±

l .mL- ' )

25528210820*1032518

HYDROGEN ION BUFFERING OF MEDIA

1.5 - O

1" 0.5 H

a —.

c o

>' C

o

o .Z

E

1.0 -1

0.5 -

0.3 n

0.2 •

10

[MES] (mM)

100

'^o. 4. Influence of MF^ buffer concentration in the growth•*iedium on (a) net pH change over time, (b) maximum divisionrates and (c) final cell yield in the growth of C. bmttumta (.5 ±*u, n - 3), SD ijars !<•«, than symbol size not shown.

subcultures. Trace metal or other nutrient deficien-* 'cs may develop upon repeated subcultures as aresult of interactions with some of ihese buffers (e.g.chelation or other effects). Such nutrient deficien-^'es may develop only after several subcultures, asstored reser%*es are exhausted. We have demonstrat-^asuch responses previously for N-deficiency (Wehr^J^al. 1985) and Sc-deficiency (Wehr and Brown1985). The two zwitterions. H F.PES and M ES. lackedthese negative biological characteristics. However,r»EPES h less suitable in ihe present context due tons more alkaline pKa (7.55) and weaker bufferingcapacity below pH 7.0. 3,3-Dimethylgluiaric acid"lay be useful, particularly where a broad pH range

3.5-6.0) is of interest and greater concentra-of Na- (or K*) are not a concern. It has beento buffer low pH (<5.0), metal enriched mediaerson 1983, Wehr and Whitlon 1983). How-it is metabolized by some bacteria, so it is un-bl in xenic cultures. The lack of a dose-re-

sponse stimulation to C breviturrita by MES (Fig. 4)is of particular importance. A significantly loweryield (approx. 50*^) of O. danicn in an unbufferedmedium when compared with the MES-buffered me-dium (Table 3). is likely due to a large negative pHshift (-1.6 units), which may have reached levelswhich were too acid for this species. The presentdata (Figs. 1-4; Tables 2, 3) show that MES satisfiesthe criteria for a buffer of dilute media in the pHrange 5.0-6.5 mosi fully. There have been uses ofMES for buffering microbial media (e.g. Epstein andLockwood 1984) and it has proven useful in con-trolling pH during photosynthesis experiments (Ra-ven and Beardall 1981).

One of the advantages of Cood*s zwitterionic buff-ers is that several ofthese form relatively weak com-plexes with metal ions (Good et al. 1966). This isperhaps a key reason for the use of HEPES in manyalgal culture media. Other buffers in ihis group,such as bicine and TES. may form considerablystronger metal complexes than does HEPES (Goodetal. 1966, Nakonand Krishnamoorthy 1983). Themetal binding constants for MES with Mg'*, Ca**.Mn'*and Cu** were found by Good and coworkers(1966) to be low or negligible. .\ further study, usingspecific ion techniques, has found that stability con-stants for MES with Cu**. Zn**, Cd«* and Pb»* wereless than those measured for HEPES (R. Schenck,D. Huizenga and P. G. C. Campbell, Universite duQuebec, pers. comm.). Another study, using the zin-con method (Collier 1979), has shown that bothHEPES and MES bind Zn** weakly, considerably lessihan several other widely used buffers.

As a pH buffer MES has several advantages foralgal growth media in the pH range 5.0-6.5. Typicalof N-substituted amitioelhanesulfonic acids, ii haslow biological activity, low temperature and con-centration effects on buffering capacity and weakmetal complexing abilities (Good et al. 1966. Perrinand Dempsey 1974, Vega and Bates 1976). MES.when used in a medium designed to approximatemoderately acidic, sofiwater lakes (Muskoka #112:Wehr et al. 1985), proved to control pH adequatelywith several algal species, had no apparent stimu-latory or inhibitory effects on growih and was foundsuitable for both axenic and non-axenic cultures.

Financial support provided by the Natural Science* and Engi-neering Research Council ni Canada, the Ontario Ministrj of theEnvironment and the Academic Development Fund of the Uni-versity of Western Ontario is gratefully acknowledged. Technicalassistance provided by D. J. Row was most appreciated. Com-ments and data provided by Dr*. P. G. C. Campbell. R. Schenckand D. Huiienga (University du Quebec) on buffer complexingwere most appreciated.

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Brewer. P. C. Sc Goldman. J. C. 1976. Alkalinity changes gen-erated by phytoplankion growth. Limnot, Oceanogr. 21:108-

94 JOHN D, WEHR ET AL.

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Dillon, P. j . . Jeffriei. D. S.. Snyder. W.. Reid. R.. Van. N. D..Evans, D., Moss. J. Se Scheider. W. A. 1978. Acidic precip-itation in south-central Ontario: recent observations. / Fish.Res. Board Can. 35:809-15.

Epstein. L. 8c Lockwood. J. L. 1984. Suppression of conidialgermination of Helminthasponum vitoriae in soil and modelfungifttatic systems. Phytopathology 74:90-4.

Francke, J. A. & ten Cate. H.J. 1980. Ecotypic differentiationin response to nutritional factors in the algal genus Stigeo-climicum Kuiz. (Chlorophyceae). Br. Phycoi f. 15:343-55.

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Healey. F. P. 1977. Ammonium and urea uptake by some fresh-i*-ater algae. Can.f, Bot. 55:61-9.

Hutner. S. H. 1972- Inorganic nutrition. Annu. Rer: Microbiol.26:313-46.

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Nakon. R. & Krishnamoorthy, C. R. 1983. Free-metal ion de-pletion by "Good's" buffers. Science [Wash. D.C) 221:749-50.

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Phycot, 22,94-102(1986)

GROWTH RATE DEPENDENT OPTIMUM RATIOS IN SELENASTRUM MINUTUM(CHLOROPHYTA): IMPLICATIONS FOR COMPETITION, COEXISTENCE

AND STABILITY IN PHYTOPLANKTON COMMUNITIES'*

David H. TurpinDepartment of Biology. Queen's Universiiy. Kingston. Ontario. Canada K7L 3N6

ABSTRACTSteady-State growth equations predict that the optimum

C:P ratio (RJ o/Selenastrum minutum (Naeq.) Collinsshould change by a factor of 20 over the growth range ofthis organism. Chemostat cultures were established at fixedinfiaw C:P ratios chosen such that a transition from ca rbonto phosphorus limitation should occur solely as a result ofincreasing the steady-state growth rate. Measurements ofresidual dissolved inorganic carbon (DIC), cellular C:P,the kinetics of photosynthesis with respect to [DlCj and

• Accepud: 15 Soi'ember 1985* Dedicated lo Dr. N. J. Antia for his contributions to the field

of algal physiology and ecology on the occasion of his retirement.

the response of culture biomass lo DIC or KjHPO^ ad'ditions were obtained. These results show that optimumratios are growth rate dependent and that this dependencycan be predicted based on steady-stale algal growth equa-tions.

A theoretical analysis was undertaken evaluating therange of growth rate dependent changes in the optimumratio which could be expected for different nutrient pairs.Further analysis showed that, under certain conditions,thegrowth rate dependence of the optimum ratio may alterthe breadth of zones of stable coexistence between speciesand allow for either the formation or complete eliminationof such zones.

Key index words: carbon limitation; coexistence; competi'