APPLIED MICROBIOLOGY, JUly, 1965Copyright © 1965 American Society for Microbiology

Vol. 13, No. 4Printed in U.S.A.

Gas Exchange of AlgaeI. Effects of Time, Light Intensity, and Spectral-Energy Distribution on the

Photosynthetic Quotient of Chlorella pyrenoidosa

ELIZABETH C. B. AMMANN AND VICTORIA H. LYNCH

Research Laboratories, Lockheed Missiles & Space Company, Palo Alto, California

Received for publication 18 February 1965

ABSTRACTAMMANN, ELIZABETH C. B. (Lockheed Missiles & Space Co., Palo Alto, Calif.), AND

VICTORIA H. LYNCH. Gas exchange of algae. I. Effects of time, light intensity, andspectral-energy distribution on the photosynthetic quotient of Chlorella pyrenoidosa.Appl. Microbiol. 13:546-551. 1965.-Continuously growing cultures of Chlorella pyreno-

idosa Starr 252, operating at constant density and under constant environmental con-

ditions, produced uniform photosynthetic quotient (PQ = C02/02) and 02 values dur-ing 6 months of observations. The PQ for the entire study was 0.90 + 0.024. The PQremained constant over a threefold light-intensity change and a threefold change in 02

production (0.90 + 0.019). At low light intensities, when the rate of respiration ap-

proached the rate of photosynthesis, the PQ became extremely variable. Six lamps ofwidely different spectral-energy distribution produced no significant change in thePQ (0.90 i 0.025). Oxygen production was directly related to the number of quantaavailable, irrespective of spectral-energy distribution. Such dependability in producinguniform PQ and 02 values warrants a consideration of algae to maintain a constant gas

environment for submarine or spaceship use.

Unicellular algae have been considered as ameans of providing for the long-term oxygenrequirements of man in a closed environment. Abasic problem in the design of a successful photo-synthetic gas exchanger is to maintain oxygen andcarbon dioxide concentrations within a physio-logically desirable range; neither gas can be al-lowed to accumulate or be depleted beyond levelstolerable to man or to the alga. A thorough studyof the algal photosynthetic quotient (PQ =C02/02) may facilitate the maintenance of a con-trolled gas environment. Emphasis should beplaced on determining whether the ratio of C02to 02 iS constant over long periods, and whethercontrolled changes in the ratio can be achievedand maintained. The ideal end result would be asystem in which the PQ could be made just tocompensate a man's respiratory quotient (RQ =C02/02) during all metabolic fluctuations.

MATERIALS AND METHODS

Culture organism and media. Chlorella pyreno-idosa 252, obtained from the Starr culture collec-tion (Starr, 1960), was used for all experiments.The alga was grown on a basal salt medium (Am-mann and Lynch, 1964) with 1 g of nitrogen perliter in the form of urea. The inorganic salts weresterilized by autoclaving, and the urea and ironcomplex were filtered and added. Contaminationwas checked by plating 0.2 ml of algal culture

medium on Antibiotic Medium 3 (Difco), in-cubating the plates at 37 C, and noting growth24 and 48 hr later. Incubation was continued atroom temperature for 1 week. Pure algae cultureswere maintained during all experiments.

Culture apparatus. The algal growth unit con-sisted of a fluorescent lamp (A) surrounded by acirculating water layer 5 mm thick and an algallayer 7 mm thick (Fig. 1). The lamp was 47 cm longand was formed by concentric 80-mm, 70-mm, and60-mm Pyrex tubing. The volume of the algallayer was 700 ml. Aluminum foil was used to coverthe outer tube to increase the irradiation and toisolate the algae from room light. Temperature ofthe water-cooling layer was controlled to ±-1 C bya Forma unit (B).The culture was aerated with compressed air

enriched with 2% C02, which was admittedthrough two sintered-glass filters (C) of mediumporosity (10 to 15 ,I) located at the bottom of thegrowth tube. The gas left the culture unit througha condenser (D) and a Drierite drying column,and passed into a flowmeter and 02 and C02analyzers. The culture was kept sterile by passagethrough cotton at both the entrance (E) and exit(F). A series of differential pressure valves main-tained a constant flow through the unit. Flow ratesup to 700 ml/min could be achieved.The culture was maintained at a constant opti-

cal density. The difference in radiation hittingphotocells (G and H; CL 604, Clairex Inc., NewYork, N.Y.) produced a current which activated a

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solenoid valve (I) at the optical density desired,allowing nutrient medium (J) to dilute the culture.The overflow left via a U tube. The amount ofgrowth under constant density conditions was

determined by clamping the unit below the gradu-ated cylinder (K) and determining the volume ofculture collected for a measured time. Waste cul-ture was collected in a carboy (L).The entire growth unit was assembled asep-

tically. The culture medium and large receivingbottle were autoclaved, and the other componentswere sterilized in place with an ethylene oxide-'Freon mixture (11% ethylene oxide, 54% Freon-11,and 35% Freon-12). All sampling was done througha rubber diaphragm stopper port (M) with sterilesyringes.Measurement of experimental parameters. Essen-

tial parameters for calculating 02 production, CO2utilization, and the PQ were measured on a digitalmultimeter model 881 (Electro Instruments, Inc.,San Diego, Calif.) and recorded on a Dura Mach10 typewriter. Oxygen was analyzed with a Beck-man F3 paramagnetic analyzer with an 18 to 23%range. Carbon dioxide was determined with a

Beckman 15A infrared analyzer by use of a 0 to 3%scale. A Metrohm combination electrode (N, Fig.

1) linked with a Beckman Zeromatic pH meter wasused to measure pH (i0.1 units). A thermistor-type thermometer (0, Fig. 1) recorded the tem-perature of the culture solution to 4-0.1 C. Thedifferential current produced by photocells (G andH) was used to measure cell density. Flow rate wasdetermined by use of a Statham 197 pressure trans-ducer and a fixed-resistance Whatman no. 1 filterpaper in the air stream (±0.05 ml/min).

Gas calculations. Oxygen production and CO2utilization of the system, in milliliters per minute,was determined by the difference between inputand effluent gases expressed as a fraction andmultiplied by the gas-flow rate in milliliters perminute. The PQ, as used here, equals the ratio ofthe CO2 consumed to the 02 produced. To obtainthe A02 and ACO2 of the gas stream, the followingmeasurements and corrections were made. (i) 02and CO2 concentrations of the exit gas, havingpassed through the algal column, were determinedby calculating the average value of 10 measure-ments taken over a 10-min period. (ii) A correctionfactor for instrument drift was obtained by meas-uring the percentage of 02 and CO2 of a calibrationgas approximately equal to the exit gas. The dif-ference between the true and observed values wasadded to, or subtracted from, the exit-gas valuesobtained in (i). (iii) Entrance gas, before passingthrough the column, was measured and correctedin the same manner, with a second calibration gasapproximating the entrance gas. (iv) All valueswere corrected for barometric pressure fluctua-tions. (v) The difference between the correctedentrance and exit gases equaled A02 and ACO2.Standard deviations of the instruments were: A02,0.03%; ACO2, 0.01%; and PQ, 0.013. These valueswere obtained by substituting a tank gas for thealgae exit gas and measuring all parameters asusual.The entrance and exit calibration-gas values

were determined with a Scholander gas analyzer.Replicate values determined over a period of 1year and compared with the original values usedthroughout the experiments agreed to 0.05%. Thus,the 02, C02, and PQ values are directly compara-ble to one another, although the absolute valuesmay show some deviation when compared withother workers' data.

Accurate photosynthetic quotient values areobtained only under conditions of constant tem-perature and pH when this method of calculatinggas changes is used. The solubility of 02 and CO2is affected by temperature, and the solubility ofCO2 is influenced by pH; measured gas changeswould reflect both physical and biological changeswith temperature and pH fluctuations.

Energy-spectrum determination. A BeckmanDK2A recording spectrophotometer was set up tomake spectroradiometric measurements (Trujillo,1962), with a General Electric F40 warm whitefluorescent lamp as the comparison lamp and en-ergy values from a General Electric data sheet.Bausch & Lomb neutral density filters of 0.3 (Y2%transmission), 0.6 (94% transmission), 0.9 (h%

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transmission), and 1.2 (116% transmission) wereused when lamp energies saturated the photocell.Energy spectra were compared from 400 to 700 nmand expressed in quanta per second per 10 nm percentimeter.

RESULTS

Gas measurements under constant environmentalconditions. The 02 production, CO2 consumption,and PQ of C. pyrenoidosa growing under constantenvironmental conditions were determined andcompared for 6 months. The environmental con-ditions were as follows: irradiation, General Elec-tric F40 cool white fluorescent lamp operating at120 v; gas, 2.0 to 2.2% CO2 in air; flow rate, 95to 110 ml/min; pH, 5.53 to 6.29; temperature,23.2 to 25.9 C; cell count, 43,250 to 53,000 cellsper mm3. A series of two to six determinations ofall experimental parameters was made per day.Five cultures were used during the 6-month inter-val, because the temperature control malfunc-tioned twice, and the culture became contami-nated twice. The longest time that any unit wasrun was 3 months; this culture was inoculated on30 September 1964. Results for it are shown from17 November 1964, on (Fig. 2). (Note added inproof: The latter culture has now been runningfor 8 months with no change in 02 or PQ values.The algal photosynthetic gas exchanger, oper-

ating under constant environmental conditions,remained dependable during the 6-month period(Fig. 2). A gradual decline in 02 production from1.36 ± 0.052 to 1.17 + 0.037 ml/min was seenfrom mid-August to November. The decline was

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due to a corresponding energy decrease, 176 X101' to 123 X 1015 quanta (400 to 700 nm) persecond per centimeter, in the single lamp usedduring this period, rather than to any change inalgal metabolism. In November, the introductionof a lamp of higher energy, 137 X 1015 quanta(400 to 700 nm) per second per centimeter, in-creased the 02 production, showing that it wasdirectly dependent upon the available lightenergy (Fig. 3). Some of the 02 production stand-ard deviation was due to instrument error. Thestandard deviation due to instrumentation was±0.030. This value was obtained by substitutinga tank gas for the algae exit gas and measuringall parameters as usual. The difference betweenthe algal and instrument standard deviationsindicated that the algal culture had a very small02-production fluctuation, which was < 0.03ml/min.Changes in PQ were also small under the same

environmental conditions, ranging from 0.97 to0.85 during the 6-month interval, with an averageof 0.90 i 0.024. The standard deviation due toinstrumentation alone was ±0.013. The differenceof +0.011 represented the PQ fluctuation of thebiological system.

Gas exchange and light intensity. C. pyrenoidosawas grown under the environmental conditionslisted in the preceding section. For 2- to 8-hrintervals, the lamp intensity was changed, andmeasurements were made. Two lamps were usedto obtain a 12-fold difference in light energy ofthe same relative spectral output (Fig. 4). ASylvania F48 warm white lamp with very high

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ENERGYFIG. 3. Oxygen production as a function of total

lamp energy. Energy is expressed in quanta (400 to700 nm) X 1015 per second per centimeter of lamplength. Six-month cool white lamp study (A); light-intensity study with warm white lamp (0); lampsof different spectral-energy distribution (0). (1)Cool white lamp, (2) gro-lux, (8) red, (4) red +filter, (5) gold, (6) green. (See Fig. 6.)

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output gave energy levels of 293 X 101' to 130X 1015 quanta (400 to 700 nm) per second percentimeter with the use of a Variac. A SylvaniaF40 warm white lamp produced 130 X 1015 to24 X 101' quanta (400 to 700 nm) per second percentimeter. A screen was placed around the latterlamp to obtain the lowest energy.A threefold decrease in light intensity from

293 X 1015 to 90 X 1015 quanta (400 to 700 nm)per second per centimeter did not change the PQof C. pyrenoidosa (Fig. 5), even though the 02production decreased approximately threefoldduring the same interval (Fig. 3). At each energylevel, the 02 production appeared to stabilizeimmediately. The average PQ was 0.90 i 0.019,which matched the quotient calculated for the6-month constant environmental study. Below90 X 101' quanta (400 to 700 nm) per second percentimeter, the observed PQ did not remain con-stant. A decrease to an average of 0.83 was seenduring the 8-hr interval when the energy was at45 X 1015 quanta (400 to 700 nm) per second percentimeter. This change was not due to tempera-ture or pH effects on the soluibility of gases in theliquid, as they remained constant. A further de-crease to 24 X 1015 quanta (400 to 700 nm) persecond per centimeter caused an even greaterdecrease in PQ to an average value of 0.71, whichfluctuated markedly during the 8-hr measurementinterval. At this low light intensity, the amountof 02 evolved (0.21 ml/min) approximated theamount of 02 consumed (0.18 ml/min) when thealgae were carrying out respiration in the absenceof light.Gas exchange and spectral-energy distribution of

light. General Electric and Sylvania fluorescent

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lamps of different spectral output (Fig. 6) weresubstituted for the standard General Electric F40cool white lamp for 8-hr periods in the continu-ous-culture apparatus. The optical density meterand solenoid valve were disconnected from the

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system during this time. The average algal gen-eration time in this system was 34 hr. Oxygen,C02, and PQ measurements were made. Gas ex-change with the cool white lamp was measuredbefore and after the experimental lamp runs toobtain control values.No change in the PQ was observed when C.

pyrenoidosa was exposed to light of widely differ-ent spectral-energy distribution (Fig. 7). Thedaily average of quotients for each lamp (0.88,0.87, 0.87, 0.92, 0.90) matched the average valueobtained with cool white light (0.90, 0.88, 0.92).The average value of 0.90 + 0.025 was identicalto the quotient obtained during the 6-month con-stant environmental study.Oxygen production was related to the total

number of available light quanta irrespective ofits spectral-energy distribution (Fig. 3). Most ofthe light was absorbed in one pass through thedense culture of algae used. Measurements madeby use of the DK2A spectrophotometer with anintegrating sphere showed nearly complete ab-sorption from 690 to 650 nm and from 500 to 400nm. An 85% absorption was seen at 500 nm, theminimal wavelength absorption range of chloro-phyll. The aluminum foil reflected this energyback into the culture.

DISCUSSION

A satisfactory photosynthetic gas exchangermust be a stable and reliable system which per-forms in a predictable manner. When combinedwith man in a small enclosed area, it must main-tain a physiologically desirable gas environmentfor both organisms for long periods of time. Thepresent study indicates that such dependabilitycan be achieved. Individual gas exchangers, con-taining cultures of C. pyrenoidosa maintainedunder constant environmental conditions, haveproduced uniform 02 and PQ values for 3-monthperiods. No manifestation of the large PQ- and02-production fluctuations observed in syn-chronous growth cultures is seen (Nihei, 1954;Sorokin, 1957); the cells continually exist in allphases of development. Varying the light condi-tions does not change the reproducibility of the02 production and PQ values; neither a threefolddecrease in light intensity nor changes in its spec-tral-energy distribution altered the PQ, and theincrease or decrease in light energy changed the02 production of the system in a predictablemanner. Such dependability in producing 02 andmaintaining a uniform PQ warrants the use ofalgae in maintaining a closed gas environment.

Artificial illumination is subject to deteriora-tion, which affects the productivity and reliabilityof the gas-exchange system. During 4 months of

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continuous operation, one of the cool white fluo-rescent lamps was observed to decrease 30% inquanta emitted, with a concomitant decrease in02 production. Lamp replacements will impose asevere weight penalty on a photosynthetic gasexchanger. For space applications, serious con-sideration should be given to the use of naturalsunlight.The capability of achieving a controlled bal-

ance between man's 02 requirements and RQ, andthe 02 production and PQ of the plant system,may be the key to success of a photosyntheticgas exchanger. During the present 6-month study,C. pyrenoidosa produced a PQ of 0.90 ± 0.024,with urea as a nitrogen source. This value wasremarkably constant under changing light inten-sity and spectral-energy distribution. The averagePQ value from gas measurements in the ElectricBoat Program was 0.87 (Benoit, 1964), whereasa calculation of the measurements of Hannon andPatouillet (1963) for 02 and CO2 gas exchangegave 0.95 + 0.020. These values were obtainedwith the thermophilic Sorokin strain of C. pyre-noidosa 7-11-05 grown on urea. The above valuesdo not appear to miatch man's RQ. Resting RQvalues of man are usually cited between 0.80 and0.82 (Benedict, 1918; Rossier, Buhlmann, andWeisinger, 1960; Consolazio, Johnson, andPecora, 1963). At the onset of exercise, Issekutzand Rodahl (1961) observed an initial RQ greaterthan one, which then declined to a value of ap-proximately 0.75 during steady moderate activ-ity. Data on man's average RQ during moderatework activity, as anticipated in a space environ-ment for 24-hr periods, are required before precisedesigns can be made for a practical photosyn-thetic gas exchanger. Additional investigations ofalgal PQ, with emphasis on determining whethercontrolled changes in the ratio can be achievedand maintained, also seem desirable.The very low light intensities at which changes

in the PQ were observed have no practical valuefor a photosynthetic gas-exchange system. Theamount of 02 produced is necessarily small, andthe PQ fluctuates markedly. In all probability,the dense culture would not maintain equilib-rium conditions for gas exchange and wouldslowly stabilize at a much lower cell density.-

Current research on light reactions in photo-synthesis suggests that more than one pigmentsystem may be involved as a primary photocat-alyst and be responsible for different chemicalreactions (French, 1961). No difference in PQ wasobserved by use of C. pyrenoidosa and fluorescentlamp systems with bandwidths of 100 nm orgreater. The two pigments of C. pyrenoidosa,chlorophyll a and b, have closely overlapping ab-

sorption spectra; it is impossible, with the wide-band illumination used, to alter greatly the rela-tive absorptions of the individual pigments. Thetwo photosynthetically active pigments of theblue-green alga, Anacystis nidulans, and the redalga, Porphyridium cruentum, have absorptionspectra with less overlap. Perhaps the PQ of thesealgae can be changed with wide-band illumi-tion.

ACKNOWLEDGMENTWe thank William Page for the design and fabri-

cation of the electronic systems used to measurethe experimental parameters, John Durichek forthe mechanical assembly and maintenance of theculture apparatus, and Robert Galvin and DonaldPeeler for the Scholander gas analyses.

LITERATURE CITEDAMMANN, E. C. B., AND V. H. LYNCH. 1964. Purinemetabolism by unicellular algae. II. Adenine,hypoxanthine, and xanthine degradation byChlorella pyrenoidosa. Biochim. Biophys. Acta87:370-379.

BENEDICT, F. G. 1918. A portable respiration ap-paratus for clinical use. Boston Med. Surg. J.178:667-678.

BENOIT, R. J. 1964. MIass culture of microalgae forphotosynthetic gas exchange, p. 413-425. In D.F. Jackson [ed.], Algae and man. Plenum Press,Inc., New York.

CONSOLAZIO, C. F., R. E. JOHNSON, AND S. J.PECORA. 1963. Physiological measurements ofmetabolic functions in man, p. 53. McGraw-HillBook Co., Inc., New York.

FRENCH, C. S. 1961. Light, pigments and photo-synthesis, p. 447-471. In W. D. McElroy and B.Glass [ed.], Light and life. Johns HopkinsPress, Baltimore.

HANNAN, P. J., AND C. PATOUILLET. 1963. Gasexchange with mass cultures of algae. II. Reli-ability of a photosynthetic gas exchanger.Appl. Microbiol. 11:450-452.

ISSEKUTZ, B., JR., AND K. RODAHL. 1961. Respira-tory quotient during exercise. J. Appl. Physiol.16:606-610.

NIHEI, T. 1954. Changes of photosynthetic activ-ity of Chlorella cells during the course of theirnormal life cycle. Arch. Mikrobiol. 21:155-164.

RoSSIER, P. H., A. A. BUHLMANN, AND K.WIESINGER. 1960. Normal physiology of respira-tion, tissue respiration, p. 113. In P. C. Luch-singer and K. M. Moser [ed.], Respirationphysiologic principles and their clinical applica-tions. C. V. Mosby Co., St. Louis.

SOROKIN, C. 1957. Changes in photosynthetic ac-tivity in the course of cell development inChlorella. Physiol. Plantarum 10:659-666.

STARR, R. C. 1960. The culture collection of algaeat Indiana University. Am. J. Botany 47:67-86.

TRUJILLO, E. F. 1962. Spectroreflectometry, p.37-38. In Model DK-A ratio recording spectro-photometers. Beckman Instruments, Inc. Ful-lerton, Calif.

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