photo acclimation of chlorella vulgarus - lee, 1996

8/3/2019 Photo Acclimation of Chlorella Vulgarus - Lee, 1996

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P h o t o a c c l i m a t i o n o f C h l o r e l l a v u l g a r i s t o R e d Lig h t f ro mLi g h t - Em i t t i n g D i o d e s Le a d s t o A u t o sp o r e R e l e a se Fo l l o w i n g Ea c hC e l lu l a r D i v i si o n

C ho ul - G y un Le e a nd Be r nha r d . P a l s s o n*

Depart ment of Chemical E ngineering, Un iversity of Michigan, Ann Arbor, Michigan 48109-2136

The detailed light requirement for photosynthesis and photoautotrophic cell growthcan be a ssessed u sing solid sta te t echn ology. Advan ced light-emitting diodes (LEDs),constru cted with double-power double-heterostructure (DDH) gallium aluminumarsenide (GaAlAs) chips, were examined for their ability to support mass culture ofthe eucaryotic alga Chlorella vulgaris. LEDs with peak emittan ce of 680 nm (withhalf-power band width of 20 nm) were used as a sole light source for the cultivationof C. vulgaris. Fluorescent light (FL) served as a contr ol. The final cell mass a ndspecific cellular growth rat e u nder LEDs were compara ble to th ose obtained u nderfull-spectr um light (FL). The na rr ow-spectr um m onochromatic red light was foun dto reduce the average cell volume from 60 m 3 t o 3 0 m 3, a n d t o m a k e t h e s i z edistribution an d t he per cell DNA distribution na rrower, but did not affect the totalbioma ss production. By switching light sour ces, th e two distin ct cell populat ion sta tes(obtained u nder red LEDs a nd FL, respectively) were found to be interchangeable.Two par am etric flow cytometric analyses showed tha t t he cells grown un der red LE D

light had a more un iform DNA content at all cell sizes, as compar ed to cells grownund er FL. These results sh ow tha t th e critical cell size for releasing au tospores un derred LED is smaller tha n th at under F L. The number of aut ospores in one mother cellwhen grown u nder LED light appeared t o be two, so that the m other cells break upafter only one roun d of DNA replication. Alth ough the solid state LE D light sour cealtered the cell cycle behavior ofC. vulgaris, it can be used as an effective light sourcefor au totrophic growth . Use of LEDs th erefore promises to advan ce th e cur rent sta teof algal ph otobioreactors due to t heir efficiency, sma llness, reliability, long lifetime,and desirable light characteristics.

I n t r o d u c t i o n

The long history of photosynthetic cultures has re-sulted in detailed chara cterization of algal metabolic andgrowth responses to light intensity (Falkowski a ndLaRoche, 1991). However, much less is known about t heeffect of spectral quality on these processes. Full-spectrum light (FL) is normally used for algal growthstudies, about half of which is photosynthetically useful(400-700 nm). The action spectra of green a lgae (Em-e r s on a n d L ew is , 1 94 3) s h ow p r e fe r r e d a b s or p t i onran ges: blue (420-450 nm) and red (660-700 nm). Ithas been known for some time that Chlorella can growunder red light (Wassink and van der Scheer, 1950).Subsequent studies used either broad red light (forexample, all the wavelengths above 600 nm) by usingcutoff filters (Cayle an d E merson, 1957; Haus child et al.,

1962; Hess and Tolbert, 1967; Tregunna et al., 1962;Wassink a nd van der Scheer, 1950; Wilhelm et a l., 1985)or monochr omatic red light by inter ference filter s but forshort periods ranging from 30 s to 1 day (Bader et al.,1992; Grotjohann and Kowallik, 1989; Senge and Senger,1990; Terborgh, 1966). The latt er appr oach was u sed formeasur ing photosynth esis-related a ctivities. Extensivework on the effect of spectral quality on photosyntheticmetabolism has been performed by Kowallik and hiscolleagues (Kowallik, 1962, 1965; Kowallik and Grotjo-han n, 1988; Kowallik et al., 1990). They found th at blue

light delayed th e cell division burst after a dark periodand produced fewer and larger aut ospores (Kowallik,

1963; Pirs on and Kowallik, 1960, 1964). These st udiesused monochromatic red light as a means t o study theprocess of photosynthesis but not as an alternative lightsource for algal mass cultur es.

A reliable, highly efficient, an d defined light source isdesired for a lgal growth stu dies and for mass cultivat ion.If the light source used has a narrow spectral output thatoverlaps th e photosynth etic absorption spectrum, theemission of light at photosynthetically less efficientfrequencies (and thus unnecessary heat generation)would be eliminated and thus the overall light conversionimproved. One light source tha t meets t hese criteria islight-emitt ing diodes (LEDs) (Schreiber, 1983). LEDs ar elight and small, they have a very long life expectancy,an d t hey h ave a highly efficient conversion of electr icity

to light so tha t heat generation is minimized. LEDs havea half-power bandwidth (HPBW) of 20-30 nm which canbe mat ched with the photosynthetic action spectra .

In spite of all these advantages, the low light intensityof conventional LEDs makes their use in algal culturesystems un at tr active. Recent ly, however, double-powerdouble-heterostructure (DDH) gallium aluminum ars-enide (GaAlAs) chips which can em it light a t m uch higherintensity and efficiency have been developed (Cook et al.,1988). A dense ar ra y of th is DDH GaAlAs LEDs can emitphoton fluxes as high as 900 mol/(m2s) or 14 m W/cm2

(Barta et al., 1990), which is sufficient to supportphotosynthesis in man y plant s (Walker, 1989).

In t he present study, red GaAlAs LEDs (HPBW of 20nm) were examined for their ability to support growth

* Address correspondence to Bernh ard . Palsson at Depart-ment of Bioengineering, University of CaliforniasSan Diego, LaJolla, CA 92093-0412.

249Biotechnol. Prog. 1996, 12, 249256

8756-7938/96/3012-0249$12.00/0 1996 American Chemical Society and American Institute of Chemical Engineers


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of the green alga Chlorella vulgaris. Ph ysiological cha ngesduring t he long-term photoacclimation to na rrow ra ngered light will be described.

M a t e r i a l s a n d M e t h o d s

Cell Line an d Culture Medium. C. vulgaris (UTEX398, recently r enamed as C. kessleri (Kessler a nd H uss,1992)) was obta ined from Th e Cult ur e Collection of Algaeat UTEX (Austin, TX) on proteose agar. The culturemedium used was N-8 (Vonshak, 1986).

L E D s , L i g h t Me a s u r e m e n t , a n d P o w e r S u p p l i e s .Red DDH GaAlAs LEDs were obtained from QuantumDevices Inc. (Bar neveld, WI). These LEDs ha ve narr owspectral outputs that peak a t wavelengths of approxi-mat ely 630, 660, 680, 700, and 730 nm. Blue LEDs (470nm peak wavelength) were obtained from DigiKey (ThiefRiver Fa lls, MN). The red LEDs were powered by simpledc power supplies (GP-233, GoldStar Pr ecision, Cer ritos,CA) at a const ant voltage between 1.6 and 2.1 V. Highervolta ge (2.6-3.5 V) was applied for blue LEDs. Fluores-cent lights (22 W Cool White Circline, General ElectricCo.) were used as a full-spectrum control.

The light intensity of the LED units was measuredusing a silicon ph otocell (Model 0560.0500, Testoterm

GmbH & Co., Germa ny). By monitoring the output ofthe photocell located at the bottom of the culture flasks,the int ensity of each LED u nit in t he same experimentalset could be matched approximately to that of the redlight portion from a full-spectr um fluorescent light (FL).

Heat generated by th e LEDs was found to be negligiblein the normal operating range.

C e l l a n d D N A A n a l y s i s . The samples for measure-ment were prepared by diluting the cell cultur e with th eappropriat e am ount of isotonic diluent (Fisher S cientific,Pittsbur gh, PA). The cell concentr ation was counted bya microprocessor-controlled electronic particle counter,Coulter Counter model ZM with a Channelizer modelC256 (both from Coulter Electronics, Inc., Hialea h, F L),a s w el l a s b y u s in g a h e m a cy t om e t er . T h e C ou l t er

Counter counts the number of particles an d measuresthe size of the part icles at the same t ime by detectingthe changes in electrical resistance induced by a sus-pended particle (or a cell) in a conductive liquid (isotonicdiluent) while the particle is traversing a small aperture.T h e n u m b e r of t h e p u ls es i n r e si st a n ce (h e n ce t h enumber of valleys in th e current profile) is the numberof particles in t he am ount of liquid tha t passed th roughth e orifice. The ar ea of each pulse is proport iona l to th esize of t he particle (after calibration with standardspherical particles with a uniform diameter and volume)since the detection is continuous a nd t hus in an integralmann er. The C256 Channelizer a cquired pulse-heightsignals from th e Coulter Counter and displayed the cellsize distribution as a h istogram after filtering t he noise

out from the signal via a predetermined gate. It alsocalculated th e cell volume using t he par amet ers obtainedfrom calibration. All the da ta, including th e histogramand other parameters, could be further analyzed byCoulter AccuComp Softwar e after t ra nsferring dat a fromth e Coulter Ch ann elizer to an IBM-compat ible PC. Thisprogram could show all the stat istical par ameters (suchas mean, median, skewness, and kurtosis from bothvolume and diameter statistics) as well as differentialand cumulative displays for the histograms.

All growth rates were calculated on the basis of thetotal biomass concentration ()cell concentration aver-age cell volume) inst ead of on th at of simple cell concen-tra tion, since th e average cell size was highly variable.It was m ore meaningful t o compare the actual growth

performance with the specific volumetric growth rat erather than the specific cellular growth rate.

The DNA content of th e cells was deter mined by usin ga flow cytometer (Coulter EPICS 751, Coulter Coopera-tion, Hialeah, F L) at 488 nm after fixing with meth anolfollowed by stain ing with propidium iodide (PI). To fixth e cells, roughly 106 cells were stored at 4 C until theywere analyzed (at least 2 days) after being mixed with 1mL of ice-cold meth an ol. Sta ining was perform ed the dayprevious to operat ing the flow cytometer. The detailed

staining pr ocedure is described elsewhere (J avanma rdianand Palsson, 1992). The stained samples were keptovernight at 4 C. The following day, they were centr i-fuged and resuspended in isotonic solution (Hematall,Fisher Diagnostics, Pitt sburgh, P A) to remove excess P I,then immediately analyzed. Cells in t heir stationaryphase were used as an external standard for a singleD N A e qu i va l en t s in ce a n i n t er n a l s t a n d a r d s u c h a schicken red blood cell overlapped with sample DNAhistograms.

C u l t u re C o n d i t i o n s . Three LEDs were used forilluminat ion of a single 125 mL Er lenmyer flask. EachLED un it of thr ee LEDs consisted of either t hree of ak in d or t w o L E Ds of o n e w a ve le n gt h a n d a t h i r d o f another. The 125 mL flasks used for cultivation were

doubly wrapped with heavy duty aluminum foil in orderto block th e ambient light. Each LED un it, mount ed ontop of an E rlenmyer flask, provided a light intensity of40 W/cm 2 (or 3 30 l x) a t t h e b ot t om of t h e fl a sk s .However, a similar unit of the brightest blue LEDsavailable could only emit the maximum light intensityof 1.2 W/cm2 (or 7 lx) at th e bott om of the flask. Theilluminat ion of each 22 W F L could give 4000 lx a t thebottom of the flask (about 40 W/cm 2 of the red lightportion). All of the experiments were performed batch-wise with 50 mL of N-8 medium per flask a t a consta nttemperat ure of 25 C and a n initial pH of 6.2. A magneticmultistirrer (Bellco, Vineland, NJ) was used to keep thecells in suspension. The seed cultures were preparedeither in 125 mL flasks with a 50 mL working volume

using 680 nm LED units or in a 3 L glass fermentor(BioFlo II, New Brunswick Scientific, Edison, NJ) witha 2.5 L working volume, usin g four of 32 W circline F Ls(General Electric Co., Cleveland, OH). The inoculumdensity ranged from 1 10 5 t o 4 10 5 cell/mL.

R e s u l t s

Five different red GaAlAs LEDs were tested (630, 660,680, 700, and 730 nm ) for their ability t o support algalgrowth (Lee, 1994). Those LEDs having t heir peakwavelengths in the range 660-700 nm could support t hegrowth ofC. vulgaris, without any blue light su pplement.The 680 nm LEDs provided the best support for growth.Neither 630 nor 730 nm LE Ds could support growth. The

average specific volumetric growth rates over the first24 h after inoculation were approximately 0.042, 0.047,0.030, and 0.063 h-1 (or doubling times of 23.8, 21.3, 27.8,and 15.9 h, respectively) for 660 nm, 680 nm, 700 nm,and FL, respectively. This result, that C. vulgaris cangrow un der highly efficient r ed light as well as under afull spectrum, holds pr omise for the future of sma ll-to-intermediate size, and perhaps large-scale, cultures ofmicroalgae.

L E D s A l t e r t h e C e l l S i z e D i s t r i b u t i o n s . The ex-periments described above showed that growth underLEDs leads to smaller average cell size and a decreasedaverage DNA cont ent per cell with a sha rper dist ribution(Lee, 1994). The avera ge cell size under t he na rr ow redspectra of LEDs was always much smaller than that

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observed under a FL. The average cell size under LEDswas consistently in t he na rrow ran ge of 25-35 m 3/cell,

while tha t un der FL was in th e wider ran ge (50-

120 m3

/cell), with a mean value of approximately 60 m 3/cell.

To further examine this phenomena, several experi-ments were performed using inocula with different initialavera ge cell sizes. Average cell sizes of 33, 54, and 75m 3 /cell were obtained from flask culture with LEDs, aBioFlo fermentor culture with FL, and a flask culturewith FL, respectively. All of these inocula were inlogarithmic growth phase when inoculated into flasksilluminat ed with t hree 680 nm LEDs each. In all cases,the average cell size converged within 2 days t o thetypical cell size (25-35 m 3 /cell) obtained using LEDs(Figure 1). Increasing the light intensity furt her led toa still smaller cell size (Figure 1 at 160 h).

T h e C e l l S i z e I s N o t a S i m p l e F u n c t i o n o f L i g h t

I n t e n s i t y . An experiment was designed to determine iflight intensity was the cause of the reduction in averagecell volum e. Three flasks, each with a F L, were supple-ment ed with different inten sities of red light from LEDs.Two additional flasks using either only FL or only redlight served as controls. The cell growth under FLsmainta ined the same a verage cell size regardless of theintensity of the supplemental red light (Figure 2A).However, upon removal of the FLs (arrow in Figure 2A)from t he t wo of thr ee flasks using both light s ourcessthusthe cells in these flasks were exposed to the LED lightalonesthe average cell volume quickly decreased to thetypical average cell size obtained under red LED lightalone. Alth ough the a verage cell size was redu ced by one-half, the total biomass r emained about th e same (Figure

2C) due to a doubling in th e cell concentr at ion. Note theearly burst of cell concentration under LED only andimmediate bursts in th e two flasks after t he F Ls wereremoved at the end of the culture (Figure 2B).

A confirming result was obtained when a cultur e tha thad been adapted to a light source (either LED or FL)was switched to the other light source (Figure 3). A seedcultu re grown under LEDs was divided into two cultur es.One of the cultu re was switched to FL (4) while the otherwas maintained under LEDs (O). Similarly, another seedcultu re prepar ed under F L was divided into two cultures;one mainta ined under FL (]) and the other under LE Ds(0). Fi gu r e 3 s h ow s t h a t t h e s h i ft i n c el l s iz e w a sreversible; the sma ller cells cultur ed under LEDs becamelarger when th ey were cultured under FLs an d vice versa.

From these observations, t he cell size was found notto be a simple function of light intensity. If it were afunction of the intensity only, the stabilized cell sizes inthe experiment shown in Figure 2A should have beendistributed over the range (from 30 to 60 m 3 /cell). Thecell concent ra tion of C. vulgaris culture grown under

F i g u re 1 . Time courses of cell concentrations (open symbols)an d their avera ge cell sizes (closed ma rker s) of the th ree inocula,whose original a verage cell sizes at inoculat ion were different :32.53 m 3 /cell (O), 54.19 m 3 /cell (0), and 73.82 m 3 /cell (4).The intensity of LEDs was increased from 30 to 40 W/cm 2 a t160 h (indicated by the arrow).

F i g u re 2 . Changes in the average cell size over time (A), thecell concentrat ion (B), and the total biomass (C). Lightingschemes: red LEDs only at h igh intensity (O); a FL per flaskplus high in ten sity (powered at 2.0 V) red (b), medium-intensityred (1.8 V) (0), and low intensity (1.6 V) red (9), respectively;and F L only (4). The inoculum was prepared un der a FL. Thearrow indicates the point when FLs were removed from twoflasks (b a n d 9, see text for details).

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FL was doubled when the cells were exposed to narrow-

ran ge red light by reducing their sizes by one-half (b a nd9 in Figure 2 and 0 in F igure 3).

E a r l y A u t o s p o r e R e l e a s e W a s t h e C a u s e o f R e -d u c e d C e l l S i z e . To understand the shift in averagecell size better, an alternating illumination scheme withLEDs and FLs was designed. Lighting scheme A (O) wasto illuminate t he culture first with FL, then with LEDs,and finally back to under F L. The reverse scheme toscheme A was scheme B (]). The profiles of th e averagecell sizes (closed symbols) and the total biomass (opensymbols) are shown in Figure 4; 4 a n d 0 were grownunder LEDs and FL, respectively, at all times.

Consistent with th e results pr esented above, dram atica n d q u ick d ecr e a se s i n t h e a v er a g e ce ll s iz es w er eobserved shortly after the exposure to 680 nm LEDs

(refer O a t 4 5 h a n d ] at 190 h in Figure 4, as well asFigures 1 and 2A). This shift suggests an altered patt ernof breakup of mother cells t o autospores. The presenceof r ed light seemed to facilitate mother cell breakupirrespective of number of unreleased autospores presentin the mother cell at the time of exposure to red light.However, in scheme B, th e avera ge cell volume in creasedgradually when LE Ds were r eplaced by FL (see ] after45 h and O after 190 h in F igure 4), indicating m ultiplerounds of DNA and nu cleus r eplicat ion before in creasingthe nu mber of aut ospores per mother cell. Since the t otalbiomass did not actively grow as the cell concentrationincreased (due to substrate limitation), the changes inaverage cell sizes after 1 week were not a s dra mat ic asthe ear lier dat a. However, by compar ing the results from

the t wo schemes, it was evident th at, in the pr esence ofnarrow-spectrum red light (or absence of another color),the cells went through only one round of r eplicationbefore mother cell breakup, t hus accounting for lowavera ge cell sizes compared to those under FL. In otherwords, t he critical cell size to release au tospores seemedto be smaller un der red light. A much more detailedstudy is necessary to elucidate the changes in metabolismand gene expression during photoacclimation to mono-chromatic r ed light.

D N A C on t e n ts Ch a n g e u n d e r R e d Li g h t. Tofurt her verify the number of autospores r eleased dur ingburst, t he per cell DNA content under the two differentlight sources was determined. The DNA histogramsobtained under FL showed wider distributions as well

as larger conten ts th an t hose under LE Ds (Figure 5). Thes m a ll er ce ll s gr ow in g u n d er L E Ds h a d a n a r r ow erdistribution with a small average per cell DNA content.

Thus, t he larger a verage cell size would be expected t olead to h igher per cell DNA content .

The t wo-para meter ana lysis of size a nd DNA contentcon fi r m ed t h i s e xp ect a t i on . I n Fi gu r e 6 , t h e x-axisrepresents the amount of the DNA (LINPI; amount of PI) per cell and the y-axis represents the forward anglelight scattering (FALS), whose value is pr oportional t oth e part icle size. Significan t difference in size and DNAcontent was found between the cells grown under LEDsand t hose grown un der FLs (Figure 6). For easy com-parison, two reference grid lines were drawn at the samelocation in all graphs and each region was numbered 1thr ough 16. The significant ly larger populations abovethe horizonta l grids in F igures 6A,D indicate the larger

a v er a g e ce ll s i ze u n d e r FL s . O n t h e ot h e r h a n d , t h eportions on the right side of th e vertical grid linesrepresent a larger amount of DNA per cell. Under fullspectra, a number of large cells with high DNA contentswere present (regions 2 a nd 14 in F igure 6). However,for cells grown un der LED light, there seemed to be anupper limit on cell size, since there were few cells overthe r eference horizonta l grid. As can be seen in Figure6B, t he cells with the higher amount of DNA showedalmost constant FALS (along the reference horizontalgrid between regions 6 and 8). Fr om these observat ionsand the given fact that the cells analyzed in Figure 6Bwere tak en in th e logarith mic phase (see Figure 4 at 42.8h), one can conclude t hat only a small portion of C.vulgaris seems t o undergo mult iple rounds of replicat ion

under r ed light, and the cells appear t o break up aftereach round of DNA replication.

L a c k o f B l u e L i g h t D i d N o t C a u s e C e l l C y c l eAlterations. To determine whether C. vulgaris growthunder r ed LEDs with blue light supplement is similar totha t grown under a F L, the brightest blue LEDs availablew er e u s ed t o p r ov id e b l u e l ig h t t o t h e cu l t u r e. T h er e su l t s s h ow ed t h a t b lu e l ig h t d id n ot i n cr e a se t h eaverage cell volume nor the per cell DNA content.Furth ermore, far less efficient blue LEDs failed tosupport the algal cultur e without red light (Lee, 1994).Per cell chlorophyll content however wa s increased withblue light supplement. The increase was more easilydistinguishable when the cells were actively growing(data not shown).

F i g u re 3 . Reversibility of the a verage cell size (solid mar kers)and the corresponding biomass profiles (open markers) whenthe light source is changed. The light sources were changed asfollows: LED to LED (O), FL to LED (0), LED to F L (4), andFL to FL (]). The first light source represent s th e source fromwhich those cells were previously adapted, and the graph shownhere is from the data with the second light source after switchingthe light source.

F i g u r e 4 . Reversible shift of the average cell size (solidmarkers) and the corresponding biomass (open markers). LED(4 ); FL (0); scheme A (O): start ed with F L, to LED after 45 h,back to FL a fter 190 h; scheme B (]): LED for the first 2 da ys,FL for following 6 days, back to LED after 8 days. The timingat which the light sources were chan ged is indicat ed by arrows.



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D i s c u s s i o n

Th e C. vulgaris cell cycle consists of sequential andsynchronized rounds of DNA replicat ion r esultin g in cellbursts t hat give 2n aut ospores (Donna n et al., 1985). Thenumber of replications prior to a burst can be as high as

four (n ) 4) resulting in the release of 2, 4, 8, or 16

au tospores. The present s tu dy is focused on th e influence

of t he spectra l composition of light on the cell cycle

behavior ofC. vulgaris. Two light sources were used; full-

spectr um fluorescent light (FL) and na rr ow-spectr um red

F i g u re 5 . Comparisons of DNA histograms between the cells under red light and those under fluorescent light. Refer to text andlegend to Figure 4 for the illumination scheme. The x-axis is the channel number (indicating fluorescence intensity) from the flowcytometer analysis, which is proportional to the amount of DNA per cell, and the y-axis is the percent of the cell population in thatchannel, resulting in t he a rea of any histogram being constant ()100). The numbers under the time represent the peak channels.



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light obtained from double-power double-heterostructure(DDH) gallium aluminum arsenide (GaAlAs) light-emit-ting diodes (LEDs) with peak emittance in the range630-730 nm.

C. vulgaris could grow on red light only with peakemittance in the range 660-7 0 0 n m f o r m o r e t h a n 1month. The best results were obtained at 680 nm. Thisresult was expected given the overlap with the photo-synthetic action spectrum which has a peak at 680 nm(Emerson an d Lewis, 1943). The main r esults from t hiss t u d y a r e t h a t , w h e n C. vulgaris is grown under redLEDs as the sole light source, its cell cycle is shortenedso that typically only one (n ) 1) round of replicationtak es place prior to mother cell burst . Consequent ly, th eaverage cell size is smaller but the cell mass growth rateis unaltered.

Cells were found to grow differently under LED lightthan under F L light. Although th e final biomass con-centrations were similar using either light source, the

initial volumetric growth r ate u nder r ed light was about60% of that under a white light. The average cell sizewas significantly smaller when the cells were grown withLEDs. Fur ther, their size distribution was narr ower. Ahigher intensity of red light resulted in smaller averagecell volume. It was r eported t hat blue light delayed thecell division (Munzner and Voigt, 1992; P irson andKowallik, 1960, 1964) and produced fewer and largerau tospores (Kowallik, 1963; Pirson an d Kowallik, 1964).The results reported here suggest that red light stimu-lates cell breakup and t hus t he cells under LEDs r eleaseabout twice as many autospores when switched to FLthan the cells maintained under full-spectrum light.

When the DNA content of th e cells was examinedsimultaneously with size using t wo-para meter flow cy-tometric analysis, it was found that cells grown underFL at tained higher average per cell DNA content. Thetotal DNA and RNA contents per culture volume has

F i g u re 6 . Bivariant dot plots showing the DNA conten t (PI fluorescence) and s ize (FALS). Cells grown u nder the light schemes Aand B described in the text an d legend to Figure 4: (A and D) cells grown un der a FL (at 42.8 h from scheme A and 92.5 h fromscheme B, respectively); (B and C) cells grown un der a red light (at 42.8 h from scheme B an d 92.5 h from scheme A, respectively).



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been reported earlier (Pirson an d Kowallik, 1964). How-ever, there were no data reported on the DNA contentper cell, or per cell DNA distr ibutions. Two-para met ricflow cytometry analysis showed that the cells with highavera ge DNA cont ent were th e larger cells. Conversely,cell populations grown under LED light contained rela-tively few cells th at att ained h igh average DNA cont ents.Their DNA distribution was much narrower comparedto that of cells grown under the FL.

Taken t ogether, these results show that the r elease of

aut ospores is dependent on the spectra l composition ofthe light. Cells grown under r ed LED light are smallerand have more u niform cell size an d DNA distribution.Thus the cells appear to break up after each division andfor m t w o a u t o sp or e s w h en g r ow n u n d er r e d L E Ds ,whereas a significantly larger portion of cells undergomultiple replications prior to releasing autospores whengrown under full-spectrum light. The r eason for thisdifference is unknown, but this phenomenon was foundto be highly reproducible and cell size can be controlleddirectly by switching between LED a nd FL. It h as beenreported t ha t wh ite light dela yed cell division (compa redt o r e d or fa r -r e d l igh t ) of a u n i ce ll u la r g r ee n a l gaChlamydomonas reinhardtii, though the extent of thedelay was less than that with blue light (Munzner and

Voigt, 1992). Red light is known to stimu late glycolat esynthesis (Becker et al., 1968; Lord et al., 1970) orpreserve stored carbohydrates (Kowallik, 1987; Szasz andBarsi, 1971). However, further studies are needed toresolve the reason for the smaller critical cell size fordivision under red light.

O n t h e o t h e r h a n d , t h e f i n d i n g t h a t t h e b l u e l i g h tsupplement did not improve growth performance wasunexpected. It is generally believed that long-termphotoaut otr ophic culture r equires both red an d blue lightfor algae (Clauss, 1970; Terborgh, 1966) as well as forother higher plants (Bula et al., 1991; Hoenecke et al.,1992; Schmidt, 1984; Voskr esenska ya et a l., 1968). Ther e su l t t h a t Chlorella cou l d gr ow on r e d L E Ds w a sencouraging since no highly efficient blue LEDs are

curr ently available (Barta et a l., 1990). However, bluelight did stimulate chlorophyll differentiation, as previ-ously reported (Hess an d Tolbert , 1967; Schm idt, 1984).Detailed r eviews on t he effect of blue light on photosyn-thetic carbon metabolism can be found elsewhere (Kow-allik, 1982; Senger 1987; Voskresenska ya, 1979).

From a practical sta ndpoint, a n LED light source hasseveral importan t a dvant ages for ph otobioreactor designan d opera tion. Their power convers ion efficiency is high,resulting in better use of electricity and less heat dis-sipation. LEDs are small and thus offer significantflexibility in the design of culture appara tus for photo-synthet ic organisms. For basic scientific studies, one canuse a well-defined light source easily without the use ofsophisticated light filters and cooling systems. Thisa b il it y s h ou l d r e s u lt i n s t u d ie s t h a t h e lp u s b et t e runderstand and define the interactions between thewavelength of incoming light and metabolic and growthchar acteristics. Fur ther , solid state t echnology is reliableand offers interesting possibilities with respect to t em-poral a nd spectral contr ol of the light source. Given a llthese advanta ges, new generations of photobioreactors(Lee and Palsson, 1994, 1995) should accelerate thepractical use of high-density algal cultures.

C o n c l u s i o n

C. vulgaris was able to grow under red light emittedby LEDs, over the range 660-700 nm, without an y bluelight su pplements. The growth kinetics based on total

biomass concentration of the cells grown with LEDs weresimilar t o those with F L. However, the avera ge cell sizesunder LEDs were smaller and consistently ranged be-tween 25 and 35 m 3/cell as compa red to cont rols un derfull-spectrum lights, which were 2-3-fold larger. Thecells photoacclimated to the light from LEDs had lessD N A p er ce ll a n d a p p ea r e d t o g o t h r ou g h on l y o n edivision before breaking u p and releasing aut ospores.Despite these differences under red light, LEDs couldsuccessfully replace the conventional lights for long-termalgal mass cultures with unique benefits.

A c k n o w l e d g m e n t

This project was funded in part by NASA Grant No.G-NAGW-2608 and DOE Grant No. DE-FG22-93PC93212.The authors wish to thank Mr. Mehran Shahabi for hishelp with the flow cytometry.

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Accepted November 20, 1995.X

BP950084T

X Abstract published in Advance ACS Abstracts, February 15,1996.


photo acclimation of chlorella vulgarus - lee, 1996

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