PHOSPHORUS EXCHANGE IN MARINEPHYTOPLANKTON

BY THEODORE R. RICE

FISHERY BULLETIN 80

UNITED STATES DEPARTMENT OF THE INTERIOR, Douglas McKay, Secretary

FISH AND WILDLIFE SERVICE, John L. Farley, Director

ABSTRACTPhosphorus exchange in Nitzschia· clostai'iu.In, isoiated and grown in pure culture,

was demunstrated by using rudioactive phosphorus find was ~h()\\"n to "IlI'Y withchanges in the phosphorus concentration of the medium amI with the ph~'sioiogical

conditions of t,he cells. A modification of MiCluel's nutrient solutions was madeto prevent the formation of precil)itates when autoclavecl, since it was necessarythat'the radioactive phusphorus he eithel' in solution or within the ('elll>, Completerecovery of cells fur radioactive l\SSll~' was accomplished b~' using n harium-sulfatefilter pad in a detachable tiltel'iug apparatus. The gl'entest exchange occurredwhen cells gl'own in high concentrntions (If phosphorus were tilter-washed. Alsoexchange between ('ells and medium wus determined while the cells were photo­synthesizing, and when they wei'€' kept in the dark. A redistribution of intra­cellular phosphorus between the inorganic and organir fractions occul'l"ed whencells wel'e placed in the light in medium .containing only a tmce of phosphorus..Little phosphorus was converted into ~he organic state by cells kept in the dal'k.

UNITED STATES DEPARTMENT OF THE INTERIOR, Douglas McKay, Secretary

FISH AND WILDLIFE SERVICE, John L. Farley, Director

PHOSPHORUS EXCHANGE IN MARINEPHYTOPLANKTON

BY THEODORE R. RICE

FISHERY BULLETIN 80

From Fishery Bulletin of the Fish and Wildlife Service

VOLUME S4

UNITED STATES GOVERNMENT PRINTING OFFICE • WASHINGTON: 1953

For sale by the Superintendent of Documents, U. S. Government Printing Office, Washington 25, D. C.Price 15 cents

CONTENTSMaterials and methods _

Culture mediunL _Cultlll'e procedurc _Phosphorus measurements _Determination of population size _Preparat.ion of filters _Fractionation of cells _

Experiments _Phosphorus absorpt.ion " ~ __ - - _Conditions affecting phosphorus exchange by cells when filter-washed " _

Exchange by 1I0mlcficient cells _Exchange by phosphate-deficient cells _Effect of exposure time on exchange _

Redistribut.ion of intracellular phosphol'us _Exchange by cells suspelllied in culture mediunL _

Exchange in t.he light_~ _Exchange in the dark _

Exchange determined with radioactive phosphorus ill the mediullL _Exchange determined with radioactive phosphorus in the cells _

Discus~oll _Summary _Literature citecL _

II

Page77777878787979797981818283838484858586868989

PHOSPHORUS EXCHANGE IN MARINE PHYTOPLANKTON

By Theodore R. Rice, Fishery Research Biologist

The labeling of plankt.onic algae with radio­active phosphorus was investigat.ed as one phaseof a study of the food of filter-feeding inverte­brates. The phytoplankton first had to be grownunder conditions that. would result in the cellsabsorbing and ret.aining large quant.it.ies of act.ivephosphorils before it could be used as labeledfood. This required t.hat the processes of phos­phorus exchange in phytoplankton be analyzed.

The relative abundunee of phosphorus has beenknown to be a factor limiting the growt,h ofphytoplankton in the sea since the time of Brandt(1899, 1902). Studics in the laboratory withpure cultures, as well as cOJTclat,ions resultingfrom work in the ficld, have proved phosphorusto be of critieal importanee. Its abundance innatural bodies of watcr previous to rapid inereasesin population sizes and its seasonal depletion withan inerease of phytopla.nkton have been shownmany times.

A decrease in the phosphorus concentrat.ion ofthe medium following an increa.se in the phyto­plankton population has been relatively easy todemonstrat.e in the laboratory. Detection ofphosphorus exchange was not possible untiltracer techniques for active phosphorus weredeveloped. By using It culture medium con­taining 11 mixture of radioactive llnd nonradio­active phosphorus, a measurement, of phosphorusexchange is possible. The normal movement ofphosphorus can be traced by following the move­ment of radioactive phosphorus, since it is gen­erally accepted that eells cannot distinguishbetween the two isotopes of phosphorus andthat isotopic effeets can be disregarded. Further­more, the addition of small amounts of radio­active phosphorus, in these studies, has notchanged the physiology of the eells throughradiation, nor the rate of uptake of phosphorusby them.

Using radioactive phosphorus, Gest and Kamen

NOTE.--This study was part or a project earl'ied on under a cooperative.\greement between thc United States Fish and Wildlife Service aud theUnited States Atomic Energy Commission.

(1948) and Goldberg, Walker, and Whisenand(1951) have demonstrated that phosphorus isexchanged by planktonie algae. In both of theseinvestigat.ions, exehange was tested for only shortintervals of timc by eit.her centrifuging or filter­washing the eells containing radioaetive phos­phorus. The appearance of radioactive phos­phorus in t.he washings demonstrat.ed. exehange.Although filter-washing also has been used inthis investigation to determine fac.tors influencingexehange, emphasis has been placed on measuringexchange over longer periods of time while theceUs were maint,ained under various environ­mental condit.ions.

MATERIALS AND METHODS

CULTURE MEDIUM

The addition of nut.rient.s t.o natural sea wut,eris still apparently the best method o~ preparingculture medium for murine planktonic algae.One disadvunt.a.ge of this method is that precipi­t.ates often form when the enriched sea water isautoelaved. It was important that no preeipi­tates form in the medium used in these experi­ments, since the radioactive phosphorus must becontained either in the eeUs or in the medium.Sea water enriched with Miquel's solut.ions, usmodified by Ketchum and Redfield (1938), wasused since Nitzschia closterium grows well in thismedium. It was found that their solutions Aand B caused precipitates to form when added tosea water and autoelaved. ,By adding iron asFe(NH4) (804)2 instead of as FeCl3 in solution B,and by removing the phosphate compound fromsolution B and adding it as solution C, the for­mation of precipitates was avoided. The sameconcentration of phosphorus, 56 JLgAP/L addedas KH2P04 instead of as Na2HP04 '12H20, wasused to prepare solution C. The import.antchange is that. solution C was not added until afterthe sea water containing solution A and themodified solution B hud been autoclaved undallowed to cool. If solution C was added to the

77

78 FISHERY BULLETIN OF THE !"ISH AND WILDLIFE SERVICE

autocll1ved sea wat.er before it had cooled com­plet.ely, precipit.at.es still formed .

.The three solut.ions used in the preparation ofthis culture medium were-Solution A__ KN03 20.2 grams.

Dist.illed wateL ___ To make 100 cubiccent.imeters ofsolution.

Solution B_ _ MgSO,_ __________ 4 grams.CaCb____________ 4 grams.Fe(NH4) (SO,h____ 1 gram.HCl (conc.)_______ 2 cc.Distilled water____ To make 100 cc. of

solution.Solution C__ KH2PO,__________ 1.53 grams.

Distilled water____ To make 100 cc. ofsolution.

Particulate matter was first removed from thesea water by filtering it through cotton. Then,0.55 cc. of solution A and 0.50 cc. of solution Bwere added to each liter of sea water. Thismedium was autoclaved and, after cooling com­pletely, 0.50 cc. of solution C, which had beenautoclaved separately, was added aseptica.lly.

CULTURE PROCEDURE

Nitzschia closterium. was isolated into pureculture and used in these experiments. It wasmaintained in pure culture on agar containing 2percent glucose find 0.5 percent peptone in addi­tion to Miquel's inorganic nutrients. Tests forpurity were not mnde on cultures during or afterthe experiments since sterile techniques andinorganic media were used and since most experi­ments were completed within a short period oftime.

The cultures were grown in a refrigeratedcabinet (fig. 1) at a temperature of 20° ± 2° C.

. The cabinet was equipped with adjustable woodenshelves to each of which six 40-watt daylightfluorescent lights, spaced 2 inches apart, wereattached. Approximately 2 inches above thelights were glass shelves upon which the cultureswere placed.

PHOSPHORUS MEASUREMENTS

The radioactive phosphorus used in theseexperiments came from Oak Ridge NationalLaboratory. 1 Activity was determined by meansof a conventional dip-type Geiger-Muller countingtube connected with a scaler-of-64 circuit. The

I Su~plied on authorization from the Isotopes Division, United StatesAtomle Energy Commission.

FIGURE I.-Culture cabinet showing illumination ofcultures.

geometry was constant, and corrections for decayand coincidence loss were made. The radio­active phosphorus was standardized by comparisonwith a simulated standard of known microcuriestrength. Inorganic-phosphorus concentrationswere determined by the At..kins-Dcniges molybdatemethod as modified by Wattenberg (1937). Salt­error corrections were made and algal cells wereremoved by filtering before making determina­tions. The intensity of the blue color was meas­ured with a Klett-Summerson photoelectric color­imeter which had been standardized with solutionsof known phosphorus content. Klett filter No. 66and a 4.5 x 2.5 centimeter cell were used. Sincethe intensity of the blue color changes with time,determinations were made approximately 10minutes after the addition of reagents. The con­centration of phosphorus is expressed as micro­gram atoms of phosphate phosphorus per liter(",gAP/L) as recommended by the InternationalAssociation of Physical Oceanography (Sverdrup,et a1. 1942, table 34).

DETERMINATION OF POPULATION SIZE

The number of cells per liter was determinedby removing approximately 5 cc. of mediumafter vigorously shaking the culture to distributethe cells evenly throughout the medium. Theaverage of two cell counts, made with an improvedNeubauer haemocytometer, was taken as thepopulation size. A careful check has shown that

PHOSPHORUS EXCHANGE IN PHYTOPLANKTON 79

FIGURE 2.-St,ainless-steel filter.

FRACTIONATION OF CELLS

The cells were centrifuged from the culturemedium and extracted in the cold with 10 ceo of

this method is very aceurate for determining uni­algal populat,ion sizes, the aeeuracy agreeingfavorably with that found by other investigators(Pearsall and Loose 1937, Pratt 1940, andWinokur 1948).

PREPARATION OF FILTERS

A stainless-steel filter (fig. 2), which could betaken apnrt for the insertion of specially eutWhatman No. 4:2 filter paper, was used with avacuum pump. The barium-sulfate preeipitatewas formed by heating 15 cc. of one-fifth normalH 2S04 and adding 0.6 ceo of normal BaCI2• Theprecipitate was filtered onto the filter paper andrinsed with distilled water before filt.rations weremade.

~FILTERPAPER

eold 10-percent trichloraeetic acid (TCA) forabout 1 hour. After cent,rifuging, extraction wasrepeated with a new volume of TCA. The TCA­insoluble residue was dissolved in HNOa anddiluted with dist.illed water for counting. TheTCA-soluble frnct.ion was neutralized to phenol­phthalein with NaOH and the inorganie phos­phorus fraction was precipitated with lO-pereentCaC12 in saturated CaOH2 • The preeipitate,after centrifuging, was dissolved in a small amountof dilute HCl, made to volume with distilled water,and the ra.dioactive disintegrations were eounted.The supernatent was also made to volume withdistilled water and countl')d. Thus the radio­active-phosphorus content of the inorganic phos­phorus, the TCA-insoluble phosphorus (eon­sidered as protein-bound phosphorus), and theTCA-soluble organic phosphorus (ester phos­phorus) were measured. The protein-boundphosphorus and the ester phosphorus were alwayscombined and reported as the organic fraction.

EXPERIMENTSPHOSPHORUS ABSORPTION

Phosphorus in culture medium containingdividing cells ean be reduced below a coneentra­tion detectable with the colorimetric method ofannlysis if no other factor limits eell division.Consequently, it has been assumed that phos­phorus completely disappears from the medium.By using radioactive phosphorus, the reduction ofphosphorus in the culture medil~m can also bedetermined, provided no exchange of phosphorusoccurs between the cells and the medium. Toaccomplish this requires only that a knownamount of radioact,ive phosphorus be added tomedium containing nonradioactive phosphorusprior to the addition of cells. Since the cellscannot distinguish between these two isotopes,the same percentage of each should be absorbedin any period of time. Thus, measurements ofthe radioact,ive phosphorus absorbed can beconverted to represent inorganic phosphorusabsorbed, since the initial ratio of the two isknown.

To compare the absorption curves of phosphorusas determined with radioactivity measurementsand with colorimetric analysis, culture mediumwas prepared with enough radioactive phosphorusto give 11,000 counts per minute per 25 cc. ofmedium and an inorganic phosphorus concentl'a-

80 FISHERY BULLETIN OF THE FISH AND WILDLIFE SERVICE

tion of 44 J£gAP/L. Nitzschia cells wero added tothis medium in sufficient quantity to give n, popu­lation of 18.5 X 107 cells pOl' liter and the cult,urewas placed in the cultum cabinet in the light.Counts for the radioactive phosphorus contentof cells and colorimetric analysis for the inorganic­phosphorus content of the medium were madeperiodically. This colorimetric analysis also in­cluded the radioactive phosphorus in the medium,but. there was such a small number of radioactiveatoms that little, if n.ny, change was caused bytheir presence or absence.

In figure 3, the amounts of radioactive andnonradioactive, or inorganic, phosphorus ab­sorbed by the cells are shown. Since both radio­active and inorganic phosphorus are plotted onthe vertical scale so that a certain percentage ofone is equal to the same percentage of the other,the determinations for radioactive phosphorus

also represent microgram-atoms of phosphol"Us.The cells continued to absorb phosphorus fromthe medium until it. was reduced below a concen­tration detectable with the colorimetric method.However, radioactive determinations for phos­phorus made at tho end of this experiment showedthat a trace of phosphorus still remained in themedium. This may be due to radioactive deter­minations being many times more sensitive thancolorimetric determinations. It is believed thatcontamination can be ruled out. The authorsuggests that this trace of radioactive phosphorusin the medium is due to exchange from the cells.

From the two curves in figure 3 it can be seenthat at first the absorption of radioactive phos­phorus by the cells was greater than absorptiondetermined by the decrease of inorganic phos­phorus in -the medium. This differencE:' must bethe result of exchange or of errors in measurement

II 44

.10 40

en!oJ 9 36

::)

~ • It:::) 0 0z 8 x:

32 0..::::E en

0 0..... :E:7 • 28 0..en0z enoct 6 24 ::::Een 0J

~~

05

octx: 20 I~

::::Ez 4 oct

16 It:

(I) • (!)

0~

:3 0 It:Z • 12 0::)

0 ::::E0 2 0 B

4

0

TIME IN HOURS

FIGURE 3.-Phosphorus absorption by Nitzschia cells as determined by both colorimetric and radioactivity measure­ments. Circles show amounts of inorganic phosphorus measured colorimetrically; dots show amounts of inorganicphosphorus from radioactivity observations.

PHOSPHORUS EXCHANGE IN PHYTOPLANKTON 81

or of a combinat,ion of t,he two. When exchangeoccurs, the specific activity, or ratio of radio­active to total phosphorus atoms, of the mediumis changed. In this experiment only nonradio­active phosphorus could initially be exchangedfrom the cells since the cells had previously grownin medium containing only nonradioactive phos­phorus. That the observed differences in thetwo curves could have been caused by exchangeseems improbable, when the accura.cy of thecolorinietric method is considered. With thismethod small changes cannot accurately bedetermined. This is especially so when thcmedium contains large amounts of phosphorusrequiring dilution to obtain satisfactory colorintensities for measurement.

Even if all the difference "between the twoabsorption curves could be attributed to ex­change, which it cannot, the total amount ofphosphorus exchanged would still be very small.From this experiment it can be concluded that,since the metabolic need for phosphorus by divid­ing cells is so great and the errors of the colori­metrie method for measuring phosphorus are solarge, any movement of phosphorus due to ex­change cannot be detected with a reasonabledegree of accuraey.

CONDITIONS AFFECTING PHOSPHORUS EX­CHANGE BY CELLS WHEN FILTER-WASHED

Another way to detect exchange is to incorpo­rate radioactive phospllOrus into the cells beforeplaeing them in a medium which is continuallyrenewed and which contains only no"nradioactivephosphorus. If an exchange of phosphorus occurs,the radioactive phosphorus leaving the cells isremoved before it can be reabsorbed by the cells.The appearance of radioactive phosphorus in themedium would indicate that phosphorus was beingexchanged between eells and medium. Sincethe washing JP.edium in these experiments con­tained the same concentration of nonradioactivephosphorus as the medium in which the cells hadbeen growing, no differentiation could have beenmade between phosphorus originally in thewashing medium or in the cells without the use ofradioactive phosphorus.

In experiments conducted by Gest and Kamen(1948) on exchange by fresh-water algae, washingswere made by centrifuging the cells containingradioactive phosphorus from the medium, deeant-

ing the medium, resuspending the cells in newmedium, and repeating "the process several times.Tl1is method was first used as a means of washingin these investigations, but it was later found thatfilter-washing was more suitable. Goldberg, et al.(1951) found that 50 percent of the radioactivephosphorus in the diatom Aste./'ionella japonica islost when the organism is washed while suspendedon a filter. However, it will be shown that severalfactors may influenee the. amount of phosphorusexchanged from phytoplankton when filter­washed.

In t,he following experiments, Nitzschia cells intwo different physiological states, nondeficient andphosphate-deficient. were used to follow phos­phorus exchange. The nondefieient cells weregrown in a north window in culture mediumcontaining phosphorus, while the phosphate­defieient cells were grown for several days in theculture cabinet in medium containing no addedphosphorus, following methods developed byKetchum (1939b). The deficient cells continueto divide in the absence of phosphorus in the

.medium until the phosphorus in eaeh cell isgreatly reduced and a eondition is reached wherefurt.her division without phosphorus is impossible.Exchange by nondeficient cells

The required number of cells were centrifugedfrom stock culture medium eontaining phosphorus.These cells were then resuspended in new culturemedium and divided into two equal cultures. 1'0one culture enough radioactive and nonact,ive

. phosphorus was added to give a concentration of45.5 p.gAP/L, and enough to give 1,137.5 p.gAP/Lwas added to the remaining culture. To eachculture, radioactive phosphorus was added insufficient amounts to give a specific activity of0.1624 p.c/p.gAP. Both cultures were placed inthe culture cabinet in a constant source of lightfor 20 hours. During this time the cells took upphosphorus and became radioactive. At the endof 20 hours the cells from 50 cc. of each of thesecultures were filtered by means of a BaSO, filter,and a radioactive count was made on them todetermine the total radioactive phosphorus ab­sorbed. To measure the loss of phosphorus, cellsfrom another 50-ce. portion were then filteredand washed five times with 50-cc. por~ions ofculture medium containing the same concentrationof phosphorus as the medium in which these cells

30.6

92.7

2.672

111.670

Adh'ity losthy cells Crom

50 cc. 01 Percentagemedium in excbangedcounts per'

minute

8.730

185.100

Activity incells Crom50 cc, oC

medium incounts per

minute

i'Uediulll

Low concentratiun (45.5,.gA PILLHil(h concentration (1.137.5,.gAP/Lj . . .

Exchange by phosphate-deficient cells

Ketchum (1939 b) has shown that phosphat,e­deficient cells cont.ain only a small fraction of t.he .phosphorus found in nondeficient cells, aud thatmore phosphorus is absorbed by deficient cellswhen again plnced in medium containing phos­phorus. Since the physiology of phosphat.e­deficient cells must be different, from that. ofnondeficiellt cells, the amount of phosphorus ex­changed by this t.ype of cell when filter-washedwas t.esteel. The effects of low and high phos­phorus concent.rations on t.he amount of phospho­rus exchanged was also determined.

The phosphate-defieient cells used in the exper­iment were prepared by suspending cells inmedium t.o which no phosphorus had been added.After plaeing the culture in the cabinet in thelight for 3 dll,yS, the population had inc-reased from45 X 107 to 82 ;< 107 cells per liter. Since t.heculture medium cont.ained only a t.race of phos­phorus, t.he increase in the number of cells ~ro?­

ably had reduced t.he amount of phosphorus wlthmeacil cell to a point. where furt.her division wasimpossible. From this eulture t.he cells were cen­trifuged, resuspended in medium cont.aining radio­active phosphorus, and then filter-washed as de­scribed for the nondeficicnt. cells.

Phosphate-deficient c.eUs grown in the higherconcentration of phosphorus absorbed more phos­phorus than tho~e grown in the lower concentra­tion (table 2). Those grown in the higher con­centration also lost a larger percentage of phos­phorus when filter-washed. Thus, the phosphorusconcentration of the medium in which the cellsare grown controls the amount of phosphorus

TABLE I.-Exchange by non-deficient cells grown- in low undhl:gh phosphorus concentrations for 20 h01If.~ before .filter­washing

[Inithl rell count was 46 X lO' cells per liter. SrCCifiC acth·it~· was 0.1624, ,.c/,.gAP in botb media

--------------------_.---

FISHERY BULLETIN OF THE FISH AND WILDLIFE SERVICE

concent.ration of phosphorus in the medium inwhich cells are grown cont.rols the amount ofphosphorus in the cells available for exchange.

82

had been growing hut. to which no radioac.t.ivephosphorus had been added.

The 45.5 and 1,137.5 /lgAP/L in t.hese experi­ment.s will be referred to as low and high phos­phorus concentrations. It is rcalized that t·he45.5 /lgAP/L is not a low concentration whencompared with eoncentrntions occurring in n~ture.

However, t.he quantity of phosphorus avaIlableper cell i~ more important than t.he concentrution

.per unit volume of medium. Since some popula­tions of cells used are often many times greaterthan those ordinarily occurring in nature, theconditions in these cultures with t.h£' low con­cent.rations may after all be very similar to naturalconditions.

In all filter-washing experiments, the activityin the washings plus the act.ivity still remainingin the cells after washing always amounted to atleast 90 percent of the tot.al activity containedin the same quantity of cells before washing.Thus never more than 10 percent. of the radio-,active phosphorus was unaccounted for event.hough six dift'erent activit.y readings were maoein the washing procedure.

Although the medium with the high phosphorusconcent.ra.tion contained 25 times more phosphorusthan that of the low concentration, it also con­tained 25 times more radioactive phosphorus,specific act.ivity being t.he same in both cultures.Like amounts of radioactive phosphorus absorbedor exchanged in 'either culture would thereforerepresent the same amount of phosphorus.

Loss of radion-ctive phosphorus to the mediumshowed t.hat nondeficient cells grown in bot.h thelow and high phosphorus concentrations ex­changed phosphorus with the .medium whenfilter-washed (table 1". The cells grown in thehigh concentration not only absorbed more phos­phorus from the medium in which they were grownbut also exchanged more phosphorus with themedium when filter-washed. Ketchum (1939 a)has previously shown that Nitzschia. cells absorbmore phosphorus when grown in medium con­taining high phosphorus concentrations. Sincethe amount of phosphorus entering the cells isproportional to t.he concentration in the medium,it ne.cessarily follows that. any phosphorus enteringthe cells in 'excess of that which the cells can con­vert into the organic st.at.e, will remain in the in­organic state. This may explain the observationthat, when other conditions are similar, the

PHOSPHORGS EXCHANGE IN PHYTOPLANKTON 83

-----------1-------------

TABI,E 2.-Exchange by phosphate-deficient cells grown inlow and high phosphorus concentrations for :eo hOllrsbefore jilter-washing

[Initial cell count was 45 X 10' cells per liter. Speciftc activity was 0.3538p.e/p.gA P in both medial

entering and exchanged by the phosphate-defieientcells, the same as for the nondeficient cells.

Comparison wit,h the nondeficient cells of thepreceding experiment, (table 1) shows that phos­phate-deficient cells grown in low concentrationsof phosphorus absorbed more phosphorus thannondeficient cells grown in a similar concent,rationof phosphorus. Since the initial population sizeswere approximately the same in this and theprevious experiment, it can be assumed that themetabolic rate of the phosphate-deficient cells washigher than that of the nondeficient cells. Also,less .phosphorus was exchanged by deficient eellswhen filter-washed even though they had absorbedmore phosphorus. This was probnbly due to moreof the phosphorus taken into the cells during thisperiod of time being converted into the organic~tate.

Effect of exposure time on exchange

The smaller amounts of phosphorus exchangedby phosphate-deficient cells as eompared with t.hatexchanged b~T nondeficient cells in the previousexperiment was due ,to an altered physiologicalcondition. This difference in phosphorus ex­change in the two different, types of cells wasexpressed as the percent of the total radioactivephosphorus contained in the cells. Even thoughexchange between cells in the same physiologicalcondition and the medium remains constant itwould appear to decrease with time if expressedas a percent of the total radioactive phosphorusin the cells. This would be due to the amountof labeled phosphorus in the nonexchangeable frac­tion increasing during a longer growing period.This experiment only demonstrates the effect oftime upon the amount of phosphorus exchangedwhen computed in the above manner and empha­sizes the importance of considering the conditionsof the experiment when exchange is expressed asa percent of total radioactive phosphorus.

Activity ill Acti"ity lostcells from by cells from

Medium 50 cc. of 50 cc, of Percentagemediulll in mctliunl in exchangedcounts per count.~ per

minute minute

Low concentration (45.5p.gAP/LJ_ 43,980 986 2.2High concentration 0,137.5

17.3p.gAP/L) .... _______________ . ____ 465.700 80,716I

TABLE a.-Exchange by nondejicient cells grown in lowand high phosphorus concentratiolls for 7 days prior tofilter-washing

(Exposure to radioactive phos\,horus was also lor the 7 days. Initial cellcount was 10 X 10' t..Us per ht.rr. Specific r.ctivity WaS 0.21123 p.c!p.gAP inboth merlia)

REDISTRIBUTION OF INTRACELLULARPHOSPHORUS

Phosphorus exchange is difficult to detect in aculture with a growing population; however, inthe filter-washing experiments, exchange wasshown. It was further found that the quantity of

The cells were taken from a culture similar tothat used in the previous experiment with non­deficient cells. The cells were suspended in twocultures, the one with a low and t,he other with ahigh phosphorus concent,ration, and grown for 7days in the culture cabinet in the light. The initialpopulation of cells in these cultures was 10 X 107

cells per liter. a much smaller number of cells t,hanwas used in the previous experiments. Thissmaller initial population was used in order thatthe cells could continue to divide and convertinorganic phosphorus into organic-phosphoruscompounds. After 7 days in the culture cabinet,the cells were fiIt.er-washed as in the two previousexperiments.

The cells grown in the high concentration ofphosphorus absorbed more phosphorus t,han thosegrown in the low concentration, as in t.he previousexperiments (table 3). Similarly, the cells grownin t.he high concentration of phosphorus exchangedmore phosphorus with the medium when filter­washed. Since the cells used in this experimentcame from the same type of culture as those cellsused in the first of these filter-washing experi­ments, a comparison of the results is possible. Aswould be expected, more radioaetive phosphoruswas absorbed by the cells during the 7-day periodthan by the cells grown for only 20 hours. Sineemore radioactive phosphorus entered the cellsduring this longer period, more was converted intoorganic compounds. Consequently, when filter­washed, a smaller percent of radioactive phos­phorus was lost by the cells.

5.1

25.4

2,094

101,604

Acti"ity lostby eells from

50 cc. of Percentagemedium exchangedin counts

per minute

40,850

399,375

Activity incells from50 cc. ofmediumin counts

per minute

Medium

Low conccn tration (45.5 p.gA1'/1.) .High concentmtion (1,1:17.5p.gAP/L) .. . _

84 FISHERY BULLETIN OF THE FISH AND WILDLIFE SERVICE

phosphorus exchanged depended on the amount ofphosphorus in the inorganic fraction in the cells.Whether or not the small amount of phosphorusremaining in the medium at the end of the uptakeexperiment was due to exchange is not known. Forthis trace of phosphorus to be the result of ex­change would require that some phosphorus alwaysremain in an inorganic sta.te in the cells.

To prepare cells with the inorganic fractionreduced to a minimum necessitates growing themin the light in medium containing no phosphorus.This was the metllOd used to prepare phosphate­deficient cells in the previous experiment. Underthese conditions the cells reduce the intracellularphosphorus to a point where further cell divisionis impossible. Theoretically, under these comli­tions the inorganic fraction ·has been reduced to assmall an amount as possible.

The rearrangement of intracellular phosphoruswas traced in Nitzschia cells by following themovement of radioactive phosphorus. To mediumcontaining 14.7 X 107 cells per liter were added8.75 microcuries of radioactive phosphorus. Thesame amount of radioactive phosphorus wasadded again the following day. On the second daythe cells were centrifuged from this active mediumand resuspended in medium with less than" 0.5p.gAPjL of nonactive phosphorus. This mediumwith cells containing radioactive phosphorus wasdivided into two cultures. One culture was keptin the light while the other was placed in the dark.Ketchum (1939b) has shown that nondeficientcells similar to these will not absorb phosphorusfrom the medium in the dark, but it was not shownwhether changes occur in the distribution ofintracellular phosphorus.

The distribution of intracellular radioactivephosphorus between the organic and inorganicfractions was determined at the time the cellswere suspended in the new medium and again onthe sixth, tenth, and fourteenth days. The cellswere fractionated with trichloracetic acid, asdescribed in Materials and Methods, and radio­activity counts were made on each fraction. Theresults of this experiment are shown in table 4.The cells kept in the light continued to combineinorganic phosphorus into the organic' fractionfor most of the 14 days. At the end of this time,14.7 percent of the radioactive phosphorus stillremained in the inorganic state. Under theconditions of this experiment., cell division had

stopped by the sixth day. It can be seen intable 4 that little inorganic phosphorus wasconverted into the organic fraction after celldivision stopped. The ceasing of cell divisioncould not have been due to the lack of some othernutrient since the medium contained sufficientamounts of all nutrients except phosphorus.The occurrence of phosphorus in the inorganicfraction at the end of 14 days may indicate thatsome inorganic phosphorus is always present inNitzschia cells and that division is stopped notby a complete disappearance of inorganic phos­phorus but rather by the reduction of the inorganicphosphorus below a threshold amount. Fromthese findings it appears that the trace of phos­phorus remaining in the medium in the phos­phorus-uptake experiment can be attributed toexchange between the cells and medium.

TABLE 4.-DistribuUon of intracellular radioactive phos­phorus in Nitzschia cells si/spended in mediwn containingonly a trace of nonradioacUve phosphorus

[The cells had previously grown for 2 days In medium with phosphorus ofhigh speciftc activity]

-Culture placed in light Culture placed in dark

Length of time in newmedium Inorganic Organic Inorganic Organic

fraction fraction fraction fraction

Percent Percent Perct7lt Percenlodays .. _______ .. ________ 163.0 137.0 163.0 137.06 days. ________ . _________ 19.1 80.9 59.2 40.810 days ________ . ______ •__ 20.7 79.3 66.0 34.014 days __________________ 14.7 85.3 53.4 46.6

1 Determined before dividing cells into two cultures.

There was not as much change in the distribu­tion of phosphorus between the inorganic andorganic fractions in t.he cells kept in the dark asin those grown in the light, yet there was aslight decrease in the inorganic fraction except onthe tenth day (table 4). This one determinationwas undoubtedly in error. From the data forcells grown in the dark, it can .now be surmisedthat a slight metabolic activity is going on whichrequires the redistribution of intracellularphosphorus.

EXCHANGE BY CELLS SUSPENDED IN CULTURE. MEDIUM

Exchanae in the liaht

The difficulty of detecting phosphorus exchangebetween cells and medium when phosphorus isbeing absorbed rapidly by the cells has been shownpreviously. However, if large amounts of radio­active phosphorus can be incorporated into the

PHOSPHORUS EXCHANGE IN PHYTOPLANKTON 85

TABLE 5.-Loss of radl:oactille phosphoru.s to mediu'ln byphotosynthesizing and dividing cells

[The cells of 25 ce. of medium contained 11.728 counts per minute)

Phosphorus exchange did occur, since radio­active phosphorus from the cells appeared in themedium. The -increase in radioactivity in themedium was rapid at first and then decreasedwith time. Thus, it has been shown that cells,

cells in a short enough period of time to preventmuch from being combined into organic compounds,there is greater possibility of detecting exchangeby measuring the loss of radioactive phosphorusfrom the cells. It must also be considered that,since each cell takes part in the exchange process,the total amount of radioactive phosphorusexchanged from the eells will depend upon thenumber of cells used. If a large number ofdividing cells are used, a high concentration ofphosphorus in the medium is needed. Further,if the concentration of phosphorus in the mediumis not sufficient, the radioactive phosphorusexchanged from the cells to the medium will bereabsorbed in too large an amount to permit adetection of exchange.

A culture of Nitzschia with 22.7 X 107 cells perliter in medium containing 2 microcuries of radio­active phosphorus per 100 cc. and a phosphorusconcentration of less than 0.5 p.gA/L was prepared.It was found that cells could absorb this amountof phosphorus within 20 hours and yet retainmost of the radioactive phosphorus in the inor­ganic state. The cells were then centrifuged fromthis medium and resuspended in new mediumcontaining 2,000 p.gAP/L of nonradioactive phos­phorus. It. has been found" that very little reduc­tion in the division rate of Nitzschia occurred inthis coneentration. This new culture was placedin the culture cabinet in the light. Aliquotsamples of 25 cc. each were removed periodically,and the cells were filtered from the medium sothat radioactive measurement could be made onthe culture medium. The sampling was con­tinued over a period of 14 days (table 5).

169 50.0 hours _202 80.0 hours. _212 150.0 hours _202 198.0 hours. _234 246.0 hours. _231 342.0 hours. . _

while absorbing and converting phosphorus intoorganic compounds, do exchange phosphorus withthe medium.. The radioactive phosphorus in themedium continued to increase until 6.97" percentof the total radioactive phosphorus contained inthe ceBs had been exchanged to the medium at theend of 14 days.

Exchange in the dark

Ketchum (1939 b) has demonstrated thatphosphate-deficient eells pla.ced in the da.rk willcontinue to absorb phosphate only for about 10hours. He also showed that the amount of phos­phate absorbed in the dark by phosphorus­deficient cells depends on the length of time theeells have been grown in the light in me diumcontaining no phosphate. From these findings itcan be eoncluded that any uptake of radioactivephosphorus by cells can be at,tributed to exchangeif radioactive phosphorus is added to the mediumafter the phosphate deficiency of the cells hasbeen replaced.

Excha;nge determined with radioactive phos­phorus in the medi.um.-A culture of 45 X 107 cellsper liter was prepared in medium containing46 p.gAP/L. The cells used to prepare this culturecame from a medium containing phosphorus.However, to be sure that the eells had no phos­phate debt, they were placed in the dark fi)r 16hours in medium eontaining phosphorus. Thecells were then centrifuged from this medium andresuspended in identical medium to which 0.45mic.roeuries of radioactive phosphorus per 100 cc.had been added. The culture was again plaeedin the dark and only removed periodically forsampling. The cells were filtered from aliquotsamples of medium, and counts were made forradioactivity in the cells. Inorganic-phosphorusdeterminations were also made on the mediumfrom each sample. Exchange does occur, asshown by radioactive phosphorus appearing inthe cells (table 6). Although the uptake ofradioactive phosphorus is small, it, is believed tobe represe~tative of the amount exehanged, sineecorreetions ,for contamination were made. Thiswas accomplished by filtering the medium con­taining the same quantity of radioactive phos­phorus and determining the activity retained onthe filter, in the BaSO. precipitate, and on thefilter paper. The counts appearing in table 6were then obtained by subtracting the counts

219368493601621678

Activity in 25ce. of mediumin counts per

minuteLength of time

Activity in 25ce. of mediumin counts per

minuteLength of time

0.5 hour__ . .1.0 hour • __4.0 hours • _7.0 hours __ . _

14.0 hours .•25.0 hours _

86 FISHERY BULLETIN OF THE FISH AND WILDLIFE SERVICE

[The medium contained 9,887 counts PCI" minute]

[The cells from 25 ec. of medium contained 22,570 counts per minute]

--------------------

TABLE 7.-LoS8 of rad·ioaetiut! phosphorus to medil"n by cellskept in thp. dark

DISCUSSIONSome investigators have found it necessa.ry to

wash the eells before using them for chemicalanalysis. According to Gest and Kamen (1948),the washing liquid is usually distilled water,0.85 percent saline, or less frequently, freshculture medium. Washing with a saline solutionis less drastic than with distilled water. Evenso, many organisms have been found to losephosphorus when washed with saline solutions.A young culture of Bacteri'um coli, after two wash­ings with a saline solution, showed a rapid releaseof inorganic phosphorus in the absence of glucose(Macfarlane 1939). Also an appreciable quantityof the original phosphorus of Trypanosoma eq-uip­erdum is lost, when the organisms are washedwith saline solut,ion (Moraczewski and Kelsev1948). It must be pointed out, however, that thistype of experiment, does not demonstrate exchange.For phosphorus exchange to oceur, there must be amovement of equal numbers of phosphorus atomsboth into and out of the cells. This naturallyprecludes interpreting the above observations asbeing exehange, since neither distilled water norsaline solutions ordinarily contain phosphorus.

Fresh-water algae grown in high-phosphatemedia have been shown to store an appreciablequantity of soluble phosphate which is readilylost when the cells are washed with medium(Gest and Kamen 1948). Aiso marine speciesof algae exchange phosphate to a nutrient mediumwhen filter-washed, as has been shown by Gold­berg, et 81. (1951) and in the experiments reportedin this paper. Even with the use of radioactivephosphorus which makes possible an altering of·the isotopic distribution of phosphorus in eitherthe medium or the c.ells, exchange is still difficultto demonstrate, espee-ially in rapidly dividingunicellular forms. This is because the amountof phosphorus absorbed by the cells is many timesgreater than that leaving the cells throughexchange.

Goldberg, et al. (1951) stated that as muc.h as50 percent of the phosphorus is removed from themarine diatom Asterionella when it is washedwith sea water; but the factors c.ontrolling thisremoval of radioactive phosphorus from the cellswere not discussed. In the filter-washing ex­periments conducted in this investigation,Nitzschia cells were always washed with a culturemedium containing the same concentration of

4646464646

519615746735

154178224148245

C~I~~i;:~~ ir5 Inorganiccc. 01 medium phosp~orU!lin counts pcr . 01 medIUm III

minute pgAP/L

Length of time

87 12.0 hours • _155 24.5 hours _175 :!R.O hours _210 34.0 hours _325

Activity in25 cc. 01 me­

(Hum incounts pc·r

minute

Length 01 ti me

Length of time

0.5 hour _1.2 hours _2.0 hours _3.5 hours _5.5 hours •__

0.5 hour • • •• _2.5 hours • _5.0 bours __ • __ • . . _22.5 hours .• _26.0 hours . _

TABLE 5.-Uptake of radioactive phosphorus by cells frommedium. when kept in the dark

due to contamination from the observed cell­activity count. Of the total radioactive phos­phorus in the medium, about 2.5 percent wasexchanged with the cell~. There was no detect­able change in the inorganic-phosphorus concen­tration of the medium.

~~~.v~v~~_diumin

counts pel"minute

------1----- -.----------

Exchange determined with radioac#lle phosphol'U8in the cells.-Sinee a tendency for the cells toclump in the previous experiment possibly causedone determination to be low, this experiment wasdevised with the active phosphorus inside thecells so that determination could be made on themedium. To test for the loss of radioactivephosphorus from the cells through exchange, aculture of 40 X 107 cells per liter was prepaledwith 1 microcurie of radioac.tive phosphorus per100 cc. of medium and less than 0.5ILgAPjL.After 16 hours the eulture was cent,rifuged, andthe cells containing radioactive phosphorus weresuspended in new medium containing 56 ILgAPjLof nonradioactive phosphorus. This culture wasplaeed in the dark and samples were removedperiodically. There was a loss by exchange ofradioactive phosphorus from t,he cells to themedium (table 7). Of the total radioactivephosphorus in the eells, 3.26 percent was exchangedwith the medium.

PHOSPHORUS EXCHANGE IN PHYTOPLANKTON 87

nonradioact,ive phosphorus as that of the mediumin which the cells had previously grown. Theamount of phosphorus exchanged wit.h the mediumwhen filter-washed in this manner was found tovary with the physiological condition of the ceUs.Phosphate-deficient cells grown for the sameperiod of time in medium containing radioactivephosphorus not only absorbed more phosphorusthan nondeficient cells, but also exchanged asmaller percentage of that absorbed when filter­washed. Regardless of the condition of the cells,the amount exchanged varied with the phosphorusconcentration of the medium in which the cellswere grown. Those grown in a high phosphorusconcentration not only absorbed more phosphorusbut also exchanged more phosphorus with thewashing medium.

The percent of radioactive phosphorus ex­changed by the eells also depends on the lengthof time the eells have been grown in radioactivephosphorus. In the filter-washing experiments,nondeficient cells grown for 7 days in the presenceof radioaetive phosphorus exchanged a smallerpercent of radioaetive phosphorus than the sametype of cell grown in a simila.r medium for 20 hours.This was the result of more radioactive phosphorusbeing converted into organic or nonexchangeablecompounds in cells grown for the longer period oftime. Thus, a smaller percent of the radioaetivephosphorus in the cells was exchanged with themedium even though the tota.l phosphorus ex­changed in both instances possibly was the same.

In cultures of rapidly dividing unicellularforms, the organie matter inereases at a fast rate,quickly removing both radioactive and nonradio­active phosphorus from the medium and thuscomplicating the measurement of exchange. It.is less diflicul t to deteet exchange by measuringthe movement of radioactive phosphorus from thecells to a medium continually renewed and con­taining only nonradioactive phosphorus, providedthe specific activity of the exchangeable phos­phorus of the cell is high. These were the con­ditions in the filter-washing experiments. Since

. under' most conditions some radioactive phos­phorus remains in the inorganie fraetion of thecell and the addition of large amounts of phos­phorus to the medium causes an increase in theamount of phosphorus exchanged by the cell,exchange of radioactive phosphorus from the cellcan also be detected hy increasing the phosphorus

concentration of the medium instead of continuallyrenewing it. By following this procedure, phos.,phorus exchange was measured between dividingcells maintained in the light and the culture me­(Hum. While exchange is easily demonstrated byfilter-washing, the experiments conducted in thelight and in the da.rk may be more representativeof normal exchange.

In the exchange experiment in the dark thecells were in equilibrium with phosphorus in themedium before the radioaetive phosphorus wasadded. The actual amount of phosphorus addedwith the radioact.ive phosphorus in terms ofp.gAP/L was insignificant. Since the cells hadremained in the culture eabinet long enOl.:gh forany phosphate deficiency to have been repaidand, t.herefore, any absorption of phosphorusfrom the medium to have stopped, the appearanceof radioactive phosphorus in the cells indicatedthat phosphorus exchange had occurred betweencells and medium. In some instances it is onlypossible to measure exchange over short periodsof time; however, it should not be concluded that.exchange ceases when it is no longer possible todetect it, but rather that both the radioactiveand nonradioact.ive phosphorus of the mediumand the cells have rea.ched equilibrium. Thus,neither a further, absorption. of phosphorus fromthe medium nor exchange would alter the specificactivity, wit,hout which exchange cannot, bedemonstrated.

In the experiments conducted in the light andin t,he dark the amount of phosphorus exchangedwas very small. It may be questioned whethert,here was not an exchange between the phosphorusof the medium and that adsorbed on the surfaceof the cells instead of the phosphorus from insidethe cells. That some of the phosphorus exchangedin these experiments came from inside the cells issupported by several observations. In relation tothe total amounts of phosphorus adsorbed, theamounts exchanged in the filter-washing experi­ments were too large to have been exchanged onlywith the phosphorus adsorbed on the cell surface.Also it was possible to measure exchange for aslong as 24 hours in some experiments and for alonger period of time in the experiment tested for14 days. It is possible that exchange betweenintracellular phosphorus and the medium is com­plicated by biological or other processes of t,hecell, resulting in a longer time being required for

88 FISHERY BULLETIN OF THE FISH AND WILDLI"FE SERVICE

an equilibrium to be reached than if the phospho­rus were exchanged only from the surface.

The amount of phosphorus on t,he surfaee of thecell may also be directly related to the manner inwhich phosphorus enters the cell. Two ways inwhich phosphorus may enter a eell are diseussedcy Kamen and Spiegelman (1948). One methodis believed to be diffusion through the eell mem­brane of phosphorus as inorganic orthophosphateto combine with the intracellular orthophosphate.Inorganic orthophosphate is assumed to be thesource of phosphorus for the various organic phos­phates in the cell. The other method is the entryof phosphorus into the cell through esterifieationat the cellular interfaee. Intracellular inorganicorthophosphate would then arise primarily fromthe breakdown of organic phosphate. From theirexperimental data with yeast these investigatorsconcluded that the primary meehanism of theentrance of phosphate is by esterification.

Phosphorus probably enters algae by one or acombination of these two methods. The mecha­nism of entry is of importance in phosphorus­exchange studies only if it requires that phosphorusremain on the surfaee of the eells for any periodof time. If the entry of phosphorus into the cellis by diffusion instead of by esterification, it mightbe very difficult for much pho~phorus to be ad­sorbed on the eell surfaee, sinee there would be atendency for any phosphorus eoming in eontactwith the cell surfaee to be taken into the cell.The process of esterifieation, on the other hand,would probably require that phosphorus remainon the surface of the cell for a longer time. Sincea population of unicellular algae presents a largesurface area, exehange can take place between anyphosphorus adsorbed .on the surface of the cellsand the medium. As far as I can determine therehas not been sufficient consideration of the partadsorption ·plays in phosphorus exchange in micro­organisms. It is impracticable to distinguish thephosphorus exe-hanged from the cell surface fromthat exehanged from inside the cells. Thus, ex­change demonstrated in these studies should beconsidered to include phosphorus both from thecell surface and from inside the cell.

The amount of phosphorus in phytoplankton. varies with the concentration of phosphorus in the

medium and some investigators have consideredthat the minimum phosphorus which can exist inthe cell is "bound" phosphate, presumably organic

phosphorus. Several observations were made inthis investigation which possibly show that all ofthe minimum phosphate in the cell does not neces­sarily exist as an organic fraction. It was observedin the phosphorus-absorption experiment that atrace of phosphorus remained in the medium.For this phosphorus to remain in the mediumwould require that some phosphorus, which couldbe exchanged with the medium, still remain in thecells. Since a sufficient amount of all othernutrients were present to allow the completeutilization of all the phosphorus, it appears thatsome phosphorus was still present in the inorganicfraetion of the cells. It is well known that cellsplaced in culture medium containing no phos­phorus will continue to divide until they beeomephosphorus deficient. These cells then eould con­tain only a minimum amount of phosphorus. Cellseontaining radioactive phosphorus and placed inmedium containing only a trace of phosphoruscontinued to divide and reduce the inorganic­phosphorus fraction for several days. However,after all division had stopped some radioactivephosphorus remained in the inorganic fraction.Thus it appears that some of the minimum phos­phorus of the eell may remain in the inorganicfraction.

Hutchinson and Bowen (1950) added radioactivephosphorus at the surface of Linsley Pond andstudied its movement over a 4-week period insummer. It appeared that practically all the radio­active phosphorus entered the phytoplanktonimmediately and that the rate of regeneration wasrapid. This continual release of phosphorus fromdecomposing organisms, which could be mistakenfor exchange, is one of several factors in operationtending to complicate exchange studies undernatural conditions. Of course, phosphorus lost inthis manner could only remain in the water andsubsequently be mistaken for exchange when theconcentration of phosphorus in the water wasalready greater than the demands of the organ­isms. If a lack of phosphorus limited cell division,any phosphorus returned to the water througheither regeneration or exe-hange would be absorbedimmediately and any possibility of its detee-tionwould be eliminated. However, with culturesgrown in the laboratory it is possible to reduce

. decomposition of cells to an insignificant pointand thus eliminate the effect of regeneration. Also,it is possible to e-ontrol the phosphorus concentra-

PHOSPHORUS EXCHANGE IN PHYTOPLANKTON 89

Hon in the medium so that any radioactive phos­phorus exchanged from cells to medium Jan bemeasured before being reabsorbed by the cells.Thus it can be concluded that while phosphorusabsorption and regeneration can be followed innature, as shown by Hutchinson and Bowen, themeasurement of exchange is limited to t.he labora­tory where conditions can be more closely con­trolled.

SUMMARY

1. Phosphorus exchange between culture mediumand marine plankt.onic algae was demon­st.rated.

2. The amount of phosphorus exchanged byNitzschia cells when filter-washed variedwit.h the concentration of phosphorus in themedium in which the cells were grown andwith t.he physiological condition of the cells.

3. The percent of radioactive phosphorus ex­changed from cells depends upon the lengthof time the cells are grown in the presence ofradioactive phosphorus. It was found thatcells grown over longer periods of time con­verted more radioactive phosphorus intoorganic or nonexchangeable compounds.

4. A redistribution of intracellular phosphorusbetween the inorganic and organic fractionsoccurred when cells were placed in the lightin medium containing only a trace of phos­phorus. Cells kept in the dark convertedvery little intracellular inorganic phosphorusinto the organic state.

5. Exchange between photosynthesizing and divid­ing cells and culture medium was demon­strated by placing cells containing radioactivephosphorus in nonactive medium and meas­uring the radioactive phosphorus exchanged.from the cells to the medium.

6. Exchange between cells and medium was shownwhile the cells were not photosynthesizingand dividing.

LITERATURE CITED

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suntersuch. Abt. Kiel, vol. 4, pp. 213-230.

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GEST, HOWARD, and MARTIN D. KAMEN.1948. Studies on the phosphorus metabolism of green

algae and purple bacteria in relation to photosynthesis.Jour. BioI. Chem., vol. 176, pp. 299-318.

GOLDBERG, EDWARD D., THEODORE J. WALKER, andALICE WHISENAND.

1951. Phosphate utilization by diatoms. BioI. Bull..vol. 101, No.3, pp. 274-284.

HUTCHINSON, G. EVELYN, and VAUGHAN T. BOWEN.1950. Limnological studies in Connecticut. IX. A

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unicellular organisms. Cold Spring Harbor Symposiaon Quantitative Biology, vol. 13, pp. 151-163.

KETCHUM, BOSTWICK H.1939 a. The absorption of phosphate and nitrate by'

illuminated cultures of Nitzschia closterium. Amer.Jour. Bot., vol. 26, pp. 399-407.

1939 b. The development and restoration of deficienciesin the phosphorus and nitrogen composition ofunicellular plants. Jour. Cell. and Camp. Physiol.,vol. 13, pp. 373-381.

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of marine diatoms by culture. BioI. Bull., ·vol.75, pp. 165-169.

MACFARLANE, MARJORIE G.1939. The phosphorylation of carbohydrate in living

cells. Biochem. Jour., vol. 33, pt. I, pp. 565-578.MORACZEWSKI, S. A., and F. E. KELSEY.

1948. Distribution and rate of metabolism in phos­phorus compounds of Trypanosoma equiperdulli.Jour. Infectious Diseases, vol. 82, pp. 45-51.

PEARSALL, W. H., and L. LOOSE.1937. The growth of Chlorella vulgaris in pure culture.

Proc. Roy. Soc. London, ser. B, vol. 121, pp. 451­501.

PRATT, R.1940. Influence of the size of the inoculum on the

growth of Chlorella vulgaris in freshly preparedculture medium. Amer. Jour. Bot., vol. 27, pp.52-56.

SVERDRUP, H. V., M. W. JOHNSON, and R. H. FLEMING.1942. The Oceans. Prentice-Hall, Inc., New York,

1087 pp.\VATTENBERG, H.

1937. Bestimmung von Phosphat, Silikat, Nitrat andAmmoniak im Seewasser. Rapp. Cons. Explor.Mer, vol. 103, pp. 1-26.

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U. S. GOVERNMENT PRINTING OFFICE, 1953 0-243945