THE CULTURE OF DUNALIELLA TERTIOLECTA BUTCHER-A EURYHALINE ORGANISM'

Abstract Dt~zaliel la lerliolccta Butcher was found to be a euryhaline organism which

grew a t salinities ranging from 3.75 to 120°/,,. All the coliservative elements of sea water, with the exception of chlorine, were found necessary for growth of the alga. The niinirnum req~~irement for sodiuln mas much greater than that for any other element, and it was not possible to substitute other monovalent cations for the minimurn requirement. Also, the alga c o ~ ~ l d tolerate high concentrations of sodi~lnl chloride. The potassiu~il and sulphur concentrations of the medium could be reduced to very low levels. Dl~nalielln. COLIICI also tolerate high concentra- tions of these two elements. The addition of lithium to the lnedi~lm inhibited the growth of the alga. High concentrations of sodium could partially eliminate the inhibition clue to lithi~lm. The ~ninimurn concelitratiolis of calcium and mag- nesium necessary for growth approached the concentrations fo~llid in fresh waters. Calci~lrn and magnesiuni were inhibitory a t high concentrations, but the inhibition a t high concentrations co~lld be prevented if a AIIg/Ca ratio of 4 was maintained over a wide range of concentrations in the medi~lrn.

Introduction

The genus Di~nalzella il~cludes both fresh and salt water species (22), a situatio~l which is not uncommon (9). This genus is of worlcl-wide distribution and is generally present in brine lakes (4, 15, 29). Diinaliella has been observed to bloo~n under conclitions of high salt concenti-ation (20), and some strains are able to grow in inedia saturated with sodium chloride (14, 29).

Seine species of Dz~naliella, inclucling D. tert~olccla, occur in sea water (cf. ref. 8). As a medium, sea n7ater is a complex solution of salts, and final analysis may reveal the presence of all elements of the periodic table (21). The conservative elements3 in sea water are present i l l large concentration and occur a t a constant ratio regardless of the salinity of the water. Most of these elements are know^^ to be essential constit~~ents in plant nutrition. Kalle (21) considers sea water as an ideal medium for growth of marine plants, but very little is Irnown of the relationship between the concentration of conservative elements and growth of microorgailisms (10, 38). The few studies which have been made (1, 10, 17, 33, 34) have indicated considerable liberty can be taken with the composition of the medium. A number of fresh-water algae have been found to grow well and tolerate conditions of high salt con- centration (13, 36). I t has been suggested that, as a group, green algae seem to be most adaptable to a wide variety of conditions (32).

Previous work with Dunalzella tertiolecta has shown that the alga could be c~iltured equally well in an artificial medium ancl in an enriched sea water

IManuscript received February 24, 1960. Contributioli No. 1097 from the Woods I-Iole Oceanographic Institution, Woods Hole, - .

Massachusetts, U.S.A. ZPul~lic I-14th Service Research Fellow of the National Institutes of Health. Present

address: Division of Aoolied Biolocrv, National Research Council. Ottawa. Canada. aConservative clern&~'ts as used this paper refer to C1-, SO.,', Na+, I\;Ig++, Ca++, and K+.

I n sea water the ratio of these ions remains virtually unchanged regardless of the salinity, or through the activity of plants and animals.

Can. J. Microbiol. Vol. 6 (1960)

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368 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 6 . 1960

medium (24). I t was the purpose of this s tudy to investigate the concentration limits of the various conservative elements of sea water which are required for growth or are tolerated b y D. tertiolecta, ant1 to determine if the alga had any requirements which would clearly distinguish i t as a marine organism.

Materials and Methods

An axenic culture of Dz~naliella tertiolccta Butcher" was used thro~ighout this investigation. T h e alga had been originally isolated from sea water b u t was cultivated on an artiiicial medium for approximately a year prior to the experimental worl;. T h e basic artificial medium, \vhich contained no organic growth factors, was the same as used in previous work (24). The compositioil of this medium (clesignated rl meclium) and of two modifications which were also used (designated B meclium and C medium) are presented in Table 1. In I3 meclium only the soclium chloride concentration was I-eclucecl; in C medium the concentrations of sodium chloride, magnesium sulphate, magne- sium chloride, and calcium chloricle were reduced as coinpared with A medium.

TABLE I

Composition of thc basic artificial media*

A medium

NaCl 410 mil l FeC13 1 .5 /JIM- MeS0.t 24 mill IHd30~ 185.0 uilf M ~ C I ? ' 2 o Ill IM CaCI? 10 I U M ICNO, 1 mill IC2H POI 100 /J i l l NaaSiOa 100 u M

NaCl 15 mil l Other salts as in A medium

C rnediulu

NaCl 50.0 luill MgCl? 2.75 m M MgS0.j 2.0 lniM CaCI? 1.25 ~niM

Other salts as in mcdiurn

*Clllorides standardized by titration.

The alga was cultivated in 125-ml Erlenmeyer flnslts containing 50 ml of ~uedium. T h e flasks were plugged with cotton and were s11al;en by hand several tiines daily. Illumination was provided by cool-white 40-w fluorescent lamps with a n intensity a t the level of the culture shelf of approximately 3500 meter-candles, as measured by a General Electric type DW-68 light meter. The temperature was mai~ltained a t 18' C.

Cells were harvested from either two or three flaslts for each treatment \vithin a n experiment. T h e cells were ltilled and stained with - Iz - acetic

'Previously this strain was referred to as D. ez~chlora WHOI-1 (24, 25, 26), but recently was identified corrcctly by Dr. R. W. Butcher as D. tertiolecta. This type was first isolated from Oslo Fjord (8).

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McLACALAN: CULTURE O F DUNALIELLA TERTIOLECTA 369

acid. The rate of growth was determined perioclically by malcing four to eight replicate cell cou~lts per treatment using a Levy hemocytometer counting chamber. The most rapid rate of growth in each treatment is reportecl. The cultures \\:ere harvested in the log phase of growth; the c o ~ ~ c e ~ ~ t r a t i o ~ ~ of cells a t this time was usually between 100 X 10"and 200 X 10.' cells per milliliter. The maximum cell concentrations obtainecl under the experimental condi- tions were approximately 5 X 1 0 6 cells per milliliter. The rate of growth is expressed as the number of divisio~ls per clay using the equation: 10% ( N 1 / N o ) / t where No is the concentration of cells a t inoc~~lation and Nt the concentration of cells a t time t (27).

Results 1. Salini ty

The concentl-ations of sodium chloride, magnesium chloride, magnesium sulphate, and ca l c i~~m chloride \\rel-e varied from 1 / 1 6 X to 4 X the concentra- tion of A medium. Conce~ltrations greater than 4 X that of A mccli~~m prc- cipitated and mere therefore not usccl. The rates of growth from 1 / 2 X to 2 X were approximately the same (Fig. 1) . The rates of growth a t concentratiolls of 1 / S X a11d 4 X were consiclerablp reduced, but these treatments ultimately achieved the same final cell concentration as the treat~ncnts a t concentratiolls from 1 / 4 X to 2 X . Yo growth occurred a t 1 / 1 6 X concentration. Growth occurrecl over a range of salinities from 3.75 to 120700. These results suggest that the rate ol growth may have been limited by high or low total salt con- centration of thc medium.

CONCENTRATION O F B A S I C SALTS

FIG. 1. The rate of growth of the alga a t var io~~s salinities. X represents the concentra- tion of basic salts (salinity) in A medium.

2. Sodilmz Chloride Preliminary results showed that the concentration of sodium chloride in

A medium could be varied from 25 to 1000 m M without affecting the rate of growth or the final cell concentration.

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370 CANADIAN JOURNAL O F MICROBIOLOGY. VOL. 6. 1960

In A medium containing more than 1000 m M sodiu~n chloride, the rate of growth was reducecl, ancl growth was completely suppressed a t 2000 mil4 (Fig. 2). However, in all treatments in which growth occurred, the same final cell concentration was obtained. The rate of growth in the 1800 m M sodium chloride treatment was approximately the same as the rate of growth of the 4 X treatment of the previous experiment (Fig. 1). The total salt concentra- tion of the 1800 i n M s o d i ~ ~ m chloride and 4 X treatments was approximately the same (ionic concentration ca. 3800 mM), which indicates the osmotic pressure bet~veen 1000 and 1800 i n M sodium chloride affected the rates of growth. The sharp breal; in the response curve between 1800 and 2000 mil4 sodium chloride may suggest tha t .sodium chloride is intrinsically toxic at higher concentrations.

The treatments containing 0 to 25 m M sodium chloride were iiloculated froin a stock culture grown on B medium, so very little soclium cl~loricle was cal-ried over with the inoculum. The rates of growth decreased from 25 to 10 m d l sodium cliloride, ancl more rapidly from 10 to 0 m M sodium chloride (Fig. 2). These results suggest tha t the clecrease in the total salt concentr a t ' ion between 25 and 10 mild sodiuin chloride affected the rate of growth. The rate of gro~vth between 0 and 10 m M was also affected by the decreased salt concentration of these treatments. However, the total salt concentration in treatineilts from 0 to 10 m M soclium chloride exceeded the total salt concen- tration of the 1 / 8 X treatment of the previous experiment. As none of the treatments from 0 to 8 11111d sodium chloride obtaiiled a maximuni cell con- centration, these results suggest that sodium chloride per se limited growth.

::: y A I 0 ' " " ' 4 8 I2 16 20 24 V I I I , \o

1000 1200 1400 1600 I800 2000

S O D I U M CHLORIDE - rnM

FIG. 2. The rate of growth of the alga in various concentrations of sodium chloride. The concentration of the other salts was maintained a t the concentration of A medium.

The rate of growth in C medium was unaffected between 80 and 100 mil4 sodium chloride (Fig. 3). A decrease in the rate of growth occurred in all treatments contai~l i~lg less than SO nil1J sodium chloride, but the maximum cell conceiitrations were obtained in all treatments in which growth occurred.

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McLACHLAN: CULTURE O F DUN.\LIELLA TERTIOLECT.1 371

In the lowest sodium chloride conceiitration in which growth occurred (30 mM), the total salt concentration was coiisiderably less than that of the 0 m M sodium chloride treatment of the previous experiment. Therefore, growth in this experiment was suppressed by a low total salt concentration before sodiuin chloride became deficient.

0 1

0 20 4 0 60 a o l o o 5 0 1 0 0 150 2 0 0 250 300 350

S O D I U M C H L O R I D E - rnM P O T A S S I U M C H L O R I D E - mM

FIG. 3. The rate of growth of the alga in various concentratio~ls of sodiun~ chloride. The concentration of the other salts was maintained a t the concentration of C medium.

FIG. 4. The rate of growth of the alga in various concentrations of potassium chloride in A, B, and C media.

3. Potassiz~m Chloride The addition of 50 m1VI potassium chloride to I3 and C media increased the

rate of growth over that of the controls containing 1.2 inM potassium (as potassium nitrate and potassium phosphate). Concentrations exceeding 50 mill potassium chloride depressed the rate of growth (Fig. 4). The rate of growth in A medium was unaffected until the potassium chloride concentratioi~ exceeded 150 mM. These results suggest a slight antagonism between potas- sium and sodium between 50 and 150 mild potassium as B and C media contained considerably less sodi~lin than A mediu~n. The inhibitio~i in all cultures containing more than 150 inM potassium chloride was probably clue to potassium toxicity per se.

Stock cultures in A medium were grown without the addition ol potassium; nitrate and phosphate were added as sodium salts. The stock cultures were allowed to grow for 12 days and then inoculated into media containing micro- molar concentrations of potassium chloride. The results (Fig. 5) show that 40 to 50 p M potassium were necessary to permit a normal rate of growth. Essentially the same final cell concentration was obtained in treatments containing 30 p M or more potassium. Cell division could be directly related to the concentration of potassium in the medium (Table 11). Fi-om these data it was possible to calculate that the alga required approximately 6.OX pg-atoms of potassium per cell. These results also show that sodiuin could not be substituted for potassiuin in growth of the alga.

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372 CANADIAN JOURNllL O F MICROBIOLOGY. VOL. 6 . 1960

TABLE I1

Thc crfect of various concentrations of potassi~lrn on the maximum cell concentration

pg-atonis potassiunl p M potassi~1111 added No. cells/ml X 10' Per cent of control per cell X

No growth 168

4. Sorlium Clzloi.ide am1 Potass i l~m Chlorirlc Prcliminarq results inclicatecl that it was not possible to substitute potas-

sium chloride foi- sodium chloride. T ~ v o series of cultures were established \\-it11 the coi1centr;ltion of sodiuin chloricle varied in each series from 0 to 25 mil4; magnesium ant1 calcium salts urei-e added a t the concentration of B medium. In one series potassium chloride \\-as added a t the concentration of B mecliuin (1.2 mM), luncl in the other series a t r~ concentration oi 50 111111.

P O T A S S I U M CHLORIDE - ,uM SOD1 U M CHLORIDE - mR/

FIG. 5. The rate of gro~vth of thc alga in various micromolar concentrations of potassium chloride in A medium.

FIG. 6. The rate of gro\vth of the alga in various concentrations of sodiuni chloride in B niedi~1111 to \vIiich either 50 mllfor 1.2 n~ilfpotassi~lni chloride was added.

XI1 cultures uere inoc~~latetl froin B medium stoclc cultures to reduce the amount of socli~~m chloride cru-I-ied over ~vitln the inoculum. KO increase in the rate of grout11 \\-as effected b). the addition of 50 in144 potassium chloride in cultures containing 10 m-11 or less sodium chloride (Fig. 6), even though the total salt conccnti-ation of the incclium \\-as sufficicllt to permit a more rapid rate of g ~ o n th. The rate of gi-o~vtln was, therefore, limited by the sodium chloride concentration. 1;ifty milliino1a1- potassium chloricle stim~llatccl the rate of g~-o\vtll a t sodium chloi-icle conccntl-ations of 15 and 20 mild. The rate of gi-011 th in the 20 S a c 1 - 50 I<CI treatment 11 as greater than that of thc other

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McLACIIL.AS: CULTURE O F DUNXLIELLA TERTIOLECTA 373

treatments, and was the most I-apicl rate recorded cluring this stucly. The rates of growth in the 15 NaC1 - 50 I<C1 and 25 NaCl - 50 I(C1 treatments werc comparable with the highest rates obtained in other experiments in this stucly.

5. Li th ium Chloride All concelltratiolls of litl~ium cl~loricle aclclecl to -4, B, ancl C media inhibited

the rate of growth (Fig. 7). A ~naximuin cell concentration was obtaincd only in A mcdiu~n containing 50 mild lithium chloride. These results suggested an antagonism betwecn sodium and lithiunl as A medium contains consider- ably more sodiu~n than B and C meclia. Cells of treatinents in which rnaxirnuln ccll concentrations werc not obtainecl became greatlj. enlarged, and after a short pcriod of gro\vth, cl~lorotic.

In treatments containing 50 mdil lithium chloritlc ancl various concentl- a t ' ions of socliurn chloride, the rate of gro\vth nras depenclent up011 the sodium chloride concentration (Fig. 8). IHo\vever, concentrations of socliu~n chloricle greater than 400 lnild did vcry little to enhance the rate of growth. Maximum cell concentrations \\-ere obtai~lecl only in treatments containing 200 m M or more soclium chloride. As in the previous experiment, cclls in the lower sodium chloride concentrations e~llargecl and so011 turncd cl~lorotic.

L I T H I U M C H L O R I D E - m M S O D I U M C H L O R I D E - mM

FIG. 7. The rate of growth of the alga in various concentrations of lithium chloride in A, 13, and C ~nedia.

FIG. 8. The rate of growth of the alga in various concentrations of sodium chloride in I3 medi~lm with all treatments containing 50 milllithium chloride.

6. S~llfihclr Cultures containil~g micromolar concentrations of sodium sulphate were

i~loculatecl from a stocl; culturc \\rhich contained 0.25 in111 sodium sulphate. 111 all treatments, magnesium was acldccl as magnesium chloricle a t the con- centration of X medium. No grou th occurred in the treatment in \\~hich sulphur had been omittccl. A rnininlunl concentration of appl-oximately 150 plld was necessal-JJ to effect a normal rate of growth (Fig. 9). All treatments colltaining 50 p11d or more sodium sulphate reachecl the same final cell concentration.

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374 CAE.4DIAN JOURNAL OF &IICROBIOLOGY. VOL. 6 . 1960

'The filial cell concentration of the 25 pill treatmelit was orlly 26% of tlie final cell co~lcentration of tlie 50 yAd treatment. This ma). suggest that sulphi~r is not directly involved in cell clivision. Hase ef al. (16) fou~icl a sulphur cle- ficiency unique in causing a complete cessation of cell clivisio~i.

I t was possible to add sodium sulphate LIP to a concentration of 300 m111 i l l A medium witliout arfecting the rate of gro~vth or the final cell concentration. Cotice~iti-ations esceecling this resulted in a precipitation of calcium sulphate. I t was :ilso possible to replace sodiuin chloride in A, B, and C media ivith sodium sulphate without affecting gro~vth. Soine stuclies (20) have inclicated that it is not possible to replace socliiln~ chloricle ~vi th other sodium salts.

S U L P H U R ( A S S U L P H A T E ) - p M

FIG. 9. The rate of of the alga in various ~i~icromolar concentr;ltions of sodium sulphate in A med i~~rn .

7. Calcium Chloride Calcium chloride was added a t various conce~itrations to A , B, aiid C meclia.

The carry-over of calciiiin with the inoculum was about 200 p d l in treatments based on A ancl B media, and about 25 p,ld i l l treatments based on C medium. The I-ate of growth was inhibited a t 50 inn{ i l l A medium, a t 40 mllf in C medium, a i d a t 30 mAd in B medium (Fig. 10). These results suggest that the inhibitory effects of excess calcium \\-ere antagonizecl b), sodium since one of the important differences between 13 medium and C aiid A media is that the socliiim chloride concentratioi~ is more t h a i ~ three to allllost 30 times higher in the latter two than in the former. All treatmeiits containing added calcium reached the same final cell coiicentration. The low rate of growth i l l A medium with low calcium concentration may also indicate an antagonism between calcium and sodium chloride. I n B and C media, the reduced rate of growth a t the lower calcium concentrations undoubteclly reflects the low total salt concentration of these treatinents.

8. Calcium CIzloride and Sodizim Chloride Treatments were prepared with 60 111M calcium chloride and concentrations

of sodium chloricle from 25 to SO0 inn{. At concentrations of 25 and 50 mM sodium chloride, the reduced rate of growth was similar to that of the previous

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>IcL.-\CIILAN: CULTUKIS OF DUSAL1ELL.A TERTIOLECTA q - -

313

experiment, i.c. in the B and (' ~nerlia (Fig. 10). 'I'hc rate of growth fro111 100 to SO0 mrl4 sodium chloride \\;as the same and comparable with the 60 n ~ l i c a l c i ~ ~ m ch101-ide (A medium) of thc previous experiment. Therefore, inhibition due to excess calcium can be somewhnt mitigated by increasing the s o d i ~ ~ m concentratio~i, but the inhibition of the rate of gro\vth could not be cntircly overcome by an ir~creasecl socli~lm conce~itration.

C A L C I U M C H L O R I D E - m M M A G N E S I U M C H L O R I D E - r n M

FIG. 10. The rate of growth of thc ~ l g a in various corlcentrations of calcium chloride in A, R , and C incdia.

FIG. 11. The rate of groivth of the alga in varioos co~lcentratio~ls of m a g n e s i ~ ~ ~ n chloride in A, B, and C media.

9. ilgaagncs.iiim Clzloride A'lagnesium chloride inhibited the rate of growth a t co~icentratio~is esceeding

120 m M i l l A, B, ancl C media (Fig. 11). The degree of inhibition of the rate of gro~vth may be related to the h/lg/Ca ratio as C ~nedium contains considc~-ably less calciu~n than A ancl B media. All treatments based on A meclium and all treatments based 011 B ~nedium, except thc one containing no added mag- nesium, reacher1 the same final cell concentration. A maximum cell concentra- tion mas not obtained in treatments contai~i i~lg 240 ant1 280 I ~ M magnesium chloride in C medium. This again suggests inhibitioll clue to cscess mag- nesium wrhich was less antagonized b\- calcium.

10. C a l c i ~ ~ m and -4Jagnesil~nz Treatments nlcre establisher1 in which the l/Ig/Ca ratio was held constant

a t 4.0, b ~ ~ t the concentration of YIg/Ca was varied from 0.8/0.2 to 240/60 mrT1. There was 110 difference in the rate of growth over this rangc of magnesium and calcium concentrations, ,lnd all cultures reached thc same final cell con cent ratio^^. Thel-eforc, inhibitory effects of excess magnesinm ancl c a 1 clum '

coulcl be overcolnc by maintaining a favorable YIg/Ca ratio.

Discussion

The results of this investigation have show11 that Dlinaliella tert~olecta is clefi~litely an euryhaline organism. Other s t ~ ~ d i e s with this genus (14, 22) and with other orgallisms (cf. I-efs. 6, 7, 35) have demonstrated tolerances to a

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376 CANADIilN JOURNAL O F MICROBIOLOGY. VOL. 6 . 1960

wide range of salinities. b,Iost organisms studied, though, do not grow well a t salinities much greater than normal sen water (35700). Some iilvcstigators (1, 13) were able to grow their organisms a t concentrations varying from those present in fresh mrnters to full strength sea water. Hornever, D,~llzaliella t c ~ - tiolecta could not tolerate ail osmotic pressure comparable with fresh \\raters.

Dzlnaliclln teitiolccta has a conspicuous requirement for soclium. Sodium could be proviclcd either as sodium cl~loricle or soclii~in sulphate. Presumably other sodiuin salts coulcl also suffice as long as the anion was not toxic. The requirement for socliu~n nras specific, ancl none of the other rnonovalcilt cations examined were able to replace the minimum sodiiim I-cquirement.

Osterhout (28) reportecl a socliu~n I-equireme~lt in ~naci-oscopic marine algae, and several sti~clies have confirmed a reqi~ire~nent in microscopic marine algae (10, 17, 15, 19, 31). 111 some of these stuclies it was shown tha t some organic compouncls mere able to substitiitc 11a1-tially, but not completely, for sodium. 12ungi (38) and bacteria (20) have a soclii~iu requirement. ~11cl Stiles (37) has i~ldicatecl a requirement for sodiu~n may be a ge~lcral phenom- enon among marine plants. 011 the other hand, other stuclies have show11 that not all marine algae have an obvious sodium requirement. Allen (1) and Georgc (13) nrerc ostensibly able to grow several marine species in soclium- free media, a~icl ;\/layer (23) has fou~lcl a marine Cl~~lorelln \vhich grows bcttcr when sodium is oinittcd from the inediu~n.

In blue-green algae, a group virti~ally unk~low~l from the pla~llcton of the sea ( S ) , soine fresh-water species have an absolute sodiuin requii-ement (2 , 3). Other studies ~vi th fl-esh-water algae have sholvn a stimulation from the addition of sodium \vl~ilc others have show~l that sodium can psi-tiall\, rcplacc potassium (37).

Potassium was fou~ld ~lecessal-y for thc growth of Dl~naliclla, but only in cluantitics which presumablj. fi~lfillcd the metabolic rcquireine~lts of the alga. There mas no indication that soclium coulcl I-eplace potassium as a metabolite. 'I'he quantity of potassium 1lecess;u-y for normal growth was far less than the concentration founcl in sea water. Pintnel- a~lcl Provasoli (31) a~icl Vishniac (35) also dcmonst~-atecl that the pot:lssii~m I-equiremc~lt for several other marine orgallisms could bc greatly recluced from that of sea water. At the other extreme, D. teitiolecta was able to tolerate a potassium co~lcentration many times that of sea water.

Osterhout (25) clemonstl-atccl that lithium was 11ot able to replace sodium. 111 the present study, it was also s l ~ o ~ v n that lithium could not replace sodium in the growth of Duwnlbclla. Litllii~nl was f o u ~ ~ d to bc toxic a t all co~lcenti-ations usecl. H beneficial action fro111 the aclclitio~l of lithium has been observed in the growth of some plants, but more generally the effect is depressing (37). Frerking (12) has claimccl that l i t h i ~ ~ m is toxic to plants requiri~lg calciu~u, but not to algae and fu~lgi supposedly free from calcium.

If chloride is Ilecessary for gro~vth of D~~n.al.iella, it is in much less clcmn~ld tha11 sodium. I t was not possible to I-eplacc a11 chloricle with su l~~ha te s clue to difficulties with precipitation. I t Ilas not been clcmo~lstrated that ch1o1-ide is

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McLACHLAN: CULTURE OF DUNALIELLA TERTIOLECT.2 377

necessary for the gromtll of marine algae, but it has bee11 founcl essential in the development of sonle land plants (37). E>-ster (11) clitl fincl that the gro\vth of a fresh-water alga under ,ultotrophic conclitions was stimulated by the addition of chloride.

I t was possible to obtain a normal cell concentration of D~~naliella a t a calciunl concentratioi~ co~llparable ulitll that of fresh \vaters. Droop (10) and IIutner (17) have also reported calciun~ I-equirements of the same order of magnitude. Contrari~vise, Allen (1) louncl little flexibility nrith regard to ca l c i~~m, and reduced gro\vtl~ resulted \vheil the co~~centration iell below that of normal sea water.

Growth of D~~mzliella occul-recl over a wicle range of magnesi~~m coi~ceiitra- tions varying in concentration fronl that characteristic of fresh waters to a concentration g rea t l~ exceecling that of normal sea water. Si~nilar results were also obtained by Droop (10) and Hutner (17), but one of the organisms studied by Droop had a inagnesium optiinum higher than that of Korth Atlantic nTatei-s. Pintiler and Provasoli (31) found their organisms were inhibitecl a t a magnesium concentration greater than tha t of normal sea water.

Vishniac (38) and Vollen~veider (39) have reported that magnesi~~m and calci~im could I-eplace each other to a certain extent. Several experiments were carried out to cletermine i f either ele~llent c o ~ ~ l d substitute for the other in the g r o ~ ~ - t h ol D. tertiolecta. All experiments were negative and indicated substitutioil did not occur. The alga could tolerate, ho~vever, wide variations in the hIg/Ca ratio as loilg as one or the other was not liinitii~g. This also seeins to be true ol other organisllls (32). Toxic effects of high collcei~tratiol~s ol calci~im or rnagi~esi~~in could be eliminatecl by maintaining a proper JlIg/Ca ratio. Pintiler ancl Provasoli (30) also inclicated that toxic effects of high magnesiuiu concentratioi~s in tlleii- medium could be overcome by incre,lsing the ca l c i~~m co~~centratioii.

IVith the possible eaceptioil of chloride, all major elements in sea water were founcl necessary for the groxvth of Dz~~zaliella tertiolecta. Apal-t from sodi- um, the concentrations necessary for the development of this alga were of the same 01-cler of magnitude as tllose liecessary for the growth of fresh-water algae. Tlle osmotic and s o d i ~ ~ m req~~ireiilents are the principal features which clisting~~isl~ this alga as a mai-ine organism. The socli~~m requii-einent is not merely one of osmoreg~~lation as othei- salts cannot substitute for sodiu~n. Other salts can substitute for sodium as os~noregulators as long as a miiiimum concentration of s o d i ~ ~ n l (ca. 10 mdJ) is maintained in the mecli~~i-rl. I t can be concl~~cled that, when culturing this 01-ganism, the first cleficiency to be encountered will be sodium. Droop (10) has reached a sinlilar conclusion wit11 other organisms.

The alga also sho~x~ecl considerable tolerailce to increasecl concentrations of other elements in sea water. 111 concentratillg sea water, the osmotic pressure of the inedi~~in limited growth before a single eleinent became toxic. By increasing the concentration of each element singly, magnesium was foui~d to be no st toxic. Geiierally the dominzult ion in alkali lalies is magi~esium which

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3 78 CANADIAN JOURPI'AL O F MICROBIOLOGY. VOL. 6 , 1960

may explain the absence of this species fro111 these habitats, particularly if the calcium concentration is low. The alga was more tolerant tolvards high concentrations of potassiunl than towards the other elements.

I t was not possible to increase significantly the rate of growth over that of enriched sea water media (24) under the various conditions used in this study. This would tend to support the conclusion of I<alle (21) that sea water is an ideal medium for plant growth.

Acknowledgments

Grateful appreciation is exteilcled to Dr. Paul R. Gorhaill and Dr. Bosturiclc H. I<etchuin for reading the manuscript.

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