Studies on Chlorella vulgaris. X.

Influence of the Age of the Culture on the Accumulation of Chlorellin Author(s): Robertson Pratt, John F. Oneto, Jane Pratt Source: American Journal of Botany, Vol. 32, No. 7 (Jul., 1945), pp. 405-408 Published by: Botanical Society of America Stable URL: http://www.jstor.org/stable/2437358 Accessed: 06/08/2010 05:02
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STUDIES

ON CHLORELLA VULGARIS. X. INFLUENCE OF THE AGE OF THE CULTURE ON THE ACCUMULATION OF CHLORELLIN 1 Robertson Pratt, John F. Oneto, and Jane Pratt

CHLORELLIN, AN antibiotic substance or complex of substances active against a number of Grampositive and Gram-negative bacteria has been extracted fromcultures of Chlorella in inorganic nutrientsolutions (Pratt et al., 1944) and some of its biological properties have been described (Pratt, 1942, 1943). To date, however, the yields of chlorellin have been relativelysmall. The present study was undertaken, therefore, to ascertain the course of accumulation of this antibioticagent in growingcultures of Chlorella vulgaris with a view to determiningthe age at which the maximumquantity of the active compound may be obtained fromthe external solution. MATERIALS AND METHODS.-The organismused in these studies was Chlorella vulgaris. The cells were cultured as described previously (Pratt, 1943) in 500 ml. Florence flasks, each of which contained 300 ml. of nutrientsolution. The initial density of population in each culture was 100 cells/cu.mm. At suitable intervals during a period of nearly two months, duplicate cultures were prepared for testing as follows: 1. Cells removedby filtration of culture through No. 5 Whatman paper. 2. Volume of clear filtratereduced to about onetenth by distillation under reduced pressure (temperature did not exceed 35?C.). 3. Residue fromNo. 2 evaporated to drynessover CaCl2 in evacuated desiccator. 4. Residual salt cake extractedwith 10 ml. CHC13 which had been redistilled recentlyfromK2CO3. 5. Suspension from No. 4 filteredthroughNo. 5 Whatman paper. 6. Filtrate from No. 5 evaporated to dryness in vacuo withoutheating. 7. Ten ml. one per cent potassium phosphate buffer(pH 7) added to residue.

dotted sigmoid curve, representing the idealized growthcurve for Chlorella (Pratt, 1940), is drawn according to the equation x
log A-X = K (t-t1) (1)

The solutions that were obtained were tested for antibacterial activity by the cylinder plate method (Abraham et al., 1941) using Staphylococcus aureus NRRL strain No. 313 (same as F.D.A. strain No. 209) as the test organism and an incubation period of 18 hours at 370C. Extracts prepared fromblank cultures which were not inoculated showed no antibiotic activity. The diameter of the cylinders employed in these tests was 8 mm. Therefore,a preparation lacking antibiotic activity (as measured by this test) is indicated by a value of 8 mm. RESULTS.-The principal results from the first group of experiments are shown in figure 1. The solid curve in the lower portion of the figureshows that the increase in cell numberfollowed the normal course that has been described previously. The
1 Received for publication May 10, 1945. 405

where X represents the density of population at time,t; A representsthe maximumdensityof population that is attained; t1 representsthe time when X - A/2; and K is a constant.Numerical values for the terms in the equation are from earlier work (loc. cit.). The solid curve in the upper portion of figure 1 represents the diameters of the zones of inhibitionthat extracts fromthe cultures of different ages caused on plates of Staph. aureus. As was anticipated from earlier work, it was foundthat solutionsfromculturesthathave attained their full growth-i.e., two weeks of age or older under the conditionsof these experiments-are relativelyrich in chlorellin.The entirelyunexpected result was the discovery that almost as much chlorellin can be extracted fromvery young cultures-i.e., cultures about 2 days old. Between the second and sixth days the chlorellin contentof the cultures decreased rapidly and then increased abruptly fron the sixth day until about the twelfthor fourteenth day after which time it remained at approximately the same relativelyhigh level. It is noteworthy that the period of rapid decrease in chlorellinconcentration coincided with the phase of growth during which the rate of growthwas continuouslyincreasing and that the lowest concentrationcoincided approximatelywith the time of most rapid increase in density of population. This relation is emphasized by comparison of the curves for growth and for diameterof zones of inhibition withthe dottedcurve in the centersection of figure1. The timeaxis is the same for all curves. The dotted curve in the center section is drawn according to the equation dx = k x (AX)(2) dt and representsthe differential formof equation (1). To verifythe observation that the concentration of chlorellinin the externalsolutiondecreases during periods of rapid increase in population,the following experimentwas performed.Cells were removed by centrifugalization from several thirty-one-day old culturesof the same series,and the cell-freesolutions were pooled.2 To this 1500 ml. pool of pale vellow solution20 Gm. Norite "A" were added and the mixture was stirred for 20 minutes. Then the carbon was removed by filtration.A small portion of the 2 After the normal maximum of population density has been attained(ca. 95,000-100,000 cells/cu. mm.underthe conditions of theseexperiments) the population remains staticforseveralweeks. relatively

406 25 -

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) Diameter of zone of inhibiFig. 1. ( Up per portion. ages tion caused by extractsfromculturesof different no on platesof Staph. aureus. A value of 8 mm.indicates of thestandsincethatis thediameter zone of inhibition, of ard cylinder. (Centerportion.)Increasein population of time-ata culturesas a function Chiorella vulgyaris Pratt (1940). (Lower portion.) Population in Chiofrom of different ages. Dottedcurvereprellasvulgaris cultures Pratt curve-data from normal growth resents theoretical timeat which Noritetreat(1940). The arrowrepresents mentwas used. See text for explanation.

clear, colorless filtratethat was obtained was used to prepare a suspension of the cells that had been removed fromthe original cultures,another portion was set aside for extractionand the remainderwas dispensed in the culture vessels (300 ml./fiask). A volume of the cell suspension was added to sufficient each flaskto give a population of 2.79 X 1010 cells per flaskwhich was equivalent to the mean populaThe tion in the culturesat the timeof the treatment. time at which cultures were subjected to this treatment is indicated by an arrow (fig. 1). of this procedureon subsequentdensity The effect of population in the cultures and on the antibiotic activity of extracts prepared from them is shown by the dashed curves. First, as has been described previously for cultures treated in this manner ( Pratt, 1944) there was a renewed activity of growth,the course of which tended to duplicate thlat of the firstgrowth period. Concomitant with this

there was a slight rise in the pH value of the solutions. The curves in the upper portion of figure 1 show that the Norite treatmentremoved an appreciable portion, although not all, of the antibiotic observamaterial fromthe solutions. The significant tion is the fact that again a decrease in antibiotic activitv accompanied the firstpart of the growth cycle and that as the rate of increase in population declined there was an increase in the antibiotic activityof the extracts prepared fromthe different solutions. In another set of experimentstwo series of cultureswere inoculated. Series A was inoculated in the regular way and served as a control. Series B was inoculated with cells which had been soaked for one hour in two changes of distilled water immediately priorto theirinoculationintothe culture.Thus, cells of this series should have been virtually devoid of chlorellin.The results of the experimentsare shown in figure2. It is noteworthy that in the cultures of series B the initial rise in chlorellin concentration of the external solutionduringthe first36 hours was lacking and that the curve representingantibiotic activity of the extracts from the solutions of this series remained at a relativelylow level throughout the firsthalf of the growthperiod, the time during which the rate of increase in density of population was continuously accelerating. During the latter half of the growth cycle when the rate of increase in density of population was declining,the concentration of chlorellin in the external solution increased. DISCUSSION.-The facts observed in these experiments are that when cells froma four-dayold culture of Chlorella vulgaris are placed directly in fresh nutrientsolutions there is a rapid increase in the chlorellincontentof the external solutionduring the firstthirty-six to forty-eight hours followed by a sharp decline during the next two to four days. The period of this decline is concomitantwith the period during which the rate of increase in density of population is continuouslyaccelerating and the time at which the lowest concentrationof chlorellin is present in the external solution seems to coincide approximately with the peak of the differential growth curve. Following this stage, and coincident rates of with the period of continuouslydiminishing increase in density of population is a period during which the chlorellin concentrationin the external solution increases. When, however, the inoculum consists of cells that have been soaked in distilled water to remove the chlorellin, the initial increase and subsequent diminutionin chlorellin content of the external solution are eliminated. Only the final increase in concentrationthat accompanies the declining rate of growthin the later stages occurs. It is possible to make several interpretationsof these facts that are consistentwith the present data and with previously published reports. It has been suggested in previous work that chlorellin is toxic for cells of Chlorella as well as for certain other

July, 1945]

PRATT,

ONETO,

AND PRATT-CHLORELLIN

407
-,

3 Exceedingly low concentrations may be stimulating for Chlorella. (See Pratt, 1942.)

organisms.3 Therefore,it may be hypothesizedfrom ~~25 thepresent experiments thatif thisis true, actively growing and dividing eells are capable of destroy/ ZONES OF 20 INHIBITION ing,inaetivating, or detoxifying chlorellin, butthat static (non-dividing) cells possessthisabilityonly ifat all. Therefore, slightly, whenonlya smallfractionof thecells in a culture is dividing, production _ _ 0 t/ of chlorellin exceedsthedetoxifying or inactivating capacityof the cells and a surplusis available to diffuse from the cells and to accumulate in the ex10' XODWH 0 /GROWTH ternalsolution. This condition prevailsin the culI/ S E R IESA z tures during thefirst inoculation. twodaysfollowing 0 75 Evidencealreadyin theliterature showsthatchloI ~~~~~~~F: rellindiffuses readilythrough cell membranes and is probablydistributed betweenthe cells and exI ~~~z ternalsolution according to the concentration gradient(Pratt, 1942, 1944). I~~~~ when a During the next phase of development verylarge fraction of the cells is dividing actively 10~~~~~~~~ and the actual numbers of cells in thecolonyis inSERIES A creasingveryrapidly, the detoxifying or inactivatx ~~~~~~~~~B ing capacityof the cultureis large and chlorellin disappearsfrom theexternal medium. Later,when the rate of increasein density of population is de25 clining, an ever increasing proportion of the cells o 8 1 N chlobecomes staticand divides seldom. At thistime rellin is againproduced in excessand so accumulates oncemorein thecells and thendiffuses intotheexternalsolution. If, on the otherhand,later studiesshouldshow thatchlorellin is an essential forcells of metabolite DAYS in the Chlorellavulgaris, theobservations recorded present work couldbe accounted forwith equal facilFig. 2. (Upper port!ion.) Diameter of zone of inhibiitv. The data in thatcase couldbe takento indicate tion caused by extracts from cultures of differentages ornon-dividing thatonlystatic cellsproduce chlorel- on plates of Staph. auxreus.A value of 8 mm. indicates lin in excessoftheir needs.Therefore, it no zone of inhibition,since that is the diameter of the physiologic would be only whencells were dividing slowlyor standard cylinder. (Lower portion.) Population in CXhlowhena relatively smallproportion ofthecellsin the rella vul1garis cultures of different ages. See text for exculturewas dividingthat production of chlorellin planationof two series. would exceed current demandsand that a surplus wouldbe availableto diffuse from thecellsandtoac- Ofdividing cells per thousand cells present is a conin theexternal cumulate This wouldapply stantlydecreasing functionof the age of the culture solution. to the two days immediately inoculation. (Pratt, 1940). Hence, as timeprogresses,the age of following Duringthesecond period ofdevelopment theproduc- the "average" cell must continuously increase, at tionof chlorellin well fall short of current re- least until sometimeafter the maximumpopulation might if the phenomenonrequirements andall orsomeofthepreviously accumu- has beenattained. Tberefore, lated chlorellin mightbe metabolized by the very ported by Maximov and Mozhaeva should be found rapidly increasing number ofnewcells,thuscausing to apply to the unicellular cryptogamsas well as to its disappearance from theculture solution. During the cells of phanerogams, it might assume maj'or thefinal chlorellin would importancein the finalelucidationof the mechanisms stagesofthegrowth curve, accumulate again becauseof theincreasing propor- that are responsible for the changes in the concentionof static, inactive cells in theculture. tration of chlorellin that occur in aging cultures of relatively Another factor thatshouldnotbe neglected is the Chlorella. SUMMARY thatchangesin the permeability possibility of the cellsoccurwith increasing age ofthecultures. MaxiCultures of Chlorella vulgaris were grown in conmovand Mozhaeva (1944) reported thattheaging tinuous light in an inorganic nutrientsolution enprocessin cells of oats and broadbeans is charac- riched by a mixtureof 5 per cent C02 in air. At interized first byreduction and laterby an increase in tervals over a period of about two montbsthe cells oftheprotoplasm. permeability In colonies of Chlo- were removed fromi different and extracts cultures., rella vulqaris, morethanthree daysold,thenumber prepared fromthe cell-freesolutions were tested by
__
_

the cylinder plate metbod for antibiotic activitv against Staph. aureus.

408

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hours immeto forty-eight During the thirty-six diately following inoculation of the cultures chlorellin accumulates in the external solution. This is followed by a sharp decrease in chlorellin concentrationduringthe next two to fourdays. From about the fifthor sixth day to about the fourteenthday, there is a continuousrise of chlorellinconcentration which then remains relatively constant for at least six weeks. Other experimeiits showed that chlorellin accuLITERATURE

mulates in the culturesolutionwhengrowthin population is relativelyslow. During the periods of most rapid increase in density of population chlorellin disappears from the external solution. Possible interpretationsof the data are discussed.
UNIVERSITY COLLEGE THE SAN CITED OF CALIFORNIA, OF PHARMACY, MEDICAL CENTER, 22, CALIFORNIA FRANCISCO

ABRAHAm, E. P., ET AL.

1941. Further observations on penicillin. Lancet 141:177-188. MAXIMOV,N. A., AND L. V. MOZHAEVA. 1944. Age variations in the colloid-chemical properties of protoplasm of vegetable cells. -II. Variations in permeability and viscosity in the leaf cells of broadbeans and oats. Compt. Rend. (Dok.) Acad. Sci. U.R.S.S. n.s. 42:277-280. PRATT, R. 1940. Influence of the size of the inoculum on the growth of Chlorella vulgaris in freshlyprepared culture medium. Amer. Jour. Bot. 27: 52-56. -. 1942. Studies on Chlorella vulgaris. V. Some

properties of the growth inhibitor formed by Chlorella cells. Amer. Jour. Bot. 29:142-148. . 1943. Studies on Chlorella vulgaris. VI. Retardation of photosynthesisby a growth-inhibiting substance from Chlorella vulgaris. Amer. Jour. Bot. 30: 32-33. . 1944. Studies on Chlorella vulgaris. IX. Influence on growthof Chlorella of continuous removal of chlorellin from the culture solution. Amer. Jour. Bot. 31:418-421. 1944. Chlorellin, an antibacterial sub, ET AL. stance from Chlorella. Science 99:351-352.

A VIRUS TUMOR DISEASE L. M. Black

THE DISCOVERY of a new plant virus,Aureogenus magnivena Black, and data on its specifictransmission by the agallian leafhoppers Agallia constricta Van Duzee, Agallia quadripunctata (Provancher) and Agalliopsis novella (Say) were reported in earlier papers (Black, 1943, 1944). It is the purpose of this publication to describe the symptoms produced by this virus in a numberof plants. To do this, experimentselucidating the host range of the virus will be brieflydescribed first. Then will follow a descriptionof the various symptoms,including tumorsformedby the virus on roots of many of the suscepts and tumors formedon the stems, petioles and leaves of certain others. In an earlier paper (Black, 1944) the appellations "clover big-vein virus" and "clover big-vein" were suggested for the pathogen and the disease it causes in Trifolium incarnatum L. It now seems that the terms "wound-tumor virus" and "wound-tumor disease" may be more appropriate and distinctiveand they are proposed here as alternativenames preferable to those originally suggested. The reasons for choosing these names will be discussed in greater detail later. HOST RANGE.-The host range studies reported in this paper constituteonly a preliminarysurvey of the possible suscepts of the virus. The species tested were selected on the basis of theiravailability and their adaptation to greenhouse culture. Many were ornamentals, some were vegetables and some were common weeds. The weed seeds were kindly 1 Receivedforpublication April9, 1945.

OF PLANTS'

provided by Dr. S. G. Younkin; the seeds of cultivated plants were purchased fromvarious seed companies. Leafhoppers of the species Agallia constricta, fed on diseased crimson clover for two to three weeks and then kept on healthy clover for an additional week, were used to inoculate the plants. Three experiments,each of which included most of the species, were conducted. In each experimentfive insects were placed on each of two seedlings of a species and allowed to feed on the plants fora week. The insects were then removed and the plants observed in a greenhouse during a period of not less than six weeks. Some were observed for more than a year. Two uninoculated plants were retained as controls. If one or more of the inoculated plants developed symptomstypical of the disease the species was judged to be susceptible. The symptomsof wound-tumordisease were so distinctive that this procedure was considered sound, especially since any doubtfulcases were omittedfromthe list of suscepts presented in table 1. The 43 suscepts listed in the table belong to 20 families. The authoritiesfor the names of the cultivated plants are those assigned by Bailey (1925) while the authoritiesfor the wild species are those given by Gray (1908). Of the species listed,Brachycome iberidifolia, Chrysanthemumleucanthemum var. pinnatifidum, Melilotus alba, Trifolium incarnatum, Rumex acetosa, Linaria maroccana, and Schizanthus wisetonensis appeared to be especially easy to infectby means of insects. It is evident that the virus has a wide host range. As the three tests