Changes in Chlorophyll a/b Ratio and Products of '4C02 Fixationby Algae Grown in Blue or Red Light'
J. L. Hess and N. E. TolbertDepartment of Biochemistry, Michigan State University, East Lansing, Michigan 48823
Received May 1, 1967.
Sutmmary. Chlamydomonas and Chlorella were grown for 10 davs in white light.955 Uw/cm2 blue light (400-500 m,u) or 685 Juw/cm2 red light (above 600 mb). Ratesof growth in blue or red light were initially slow, but increased over a period of daysuntil normal growth rates were reestablished. During this adaptation period in bluelight, total chlorophyll per volume of algae increased 20 % while the chlorophyll a/bratio decreased. In red light no change was observed in the total amount of chlorophyllor in t'he chlorophyll a/tb ratio. After adaptation to growth in blue light and uponexposure to "CO, with either blue or white light for 3 to 10 minutes, 30 to 36 % ofthe total soluble fixed 1"C accumulated in glycolate-14C which was the major product.However, with 1 minute experiments, it was shown that phosphate esters of the photo-synthetic carbon cycle were labeled before the glycolate. Glycolate accumulation byalgae 'grown in blue light occurred even at low light intensity. After growth of theal,gae in red light, 1"C accumulated in malate, aspartate, glutamate and alanine, wherea;glycolate contained less than 3 % of the soluble 14C fraction.
Several groups of insvestigators have reported aneffect of blue light upon the rate of photosvnthesisand upon the distribution of 14C among the productsof 14CO9 fixation. Initially, Warburg et al. (24,25)reported a stimulation of photosynthesis by Chlorellain red light upon addition of blue light. In a recentconfirmation of this phenomenon with Acetabularia,Tenborgh (19) provided reasons why t-his blue lightpotentiation is different from the Emerson enhance-ment effect associated with the 2 pigment systemsfor electron transport. When photosynthesis was re-stricted to blue light only, Roux et al. (17), Tyszkie-vicz (22) and Voskresenskaya and Grishina (23)all fotund an increased proportion of amino acids,particularly glycine and serine, and generallv lessstarch synthesis. More recently Zak (28), usingChlorella, and Andreeva and Korzheva (1), usingsunflower leaves, have made similar observations.Cayle and Emerson (4) in 1957 using Chlorella re-ported that glycine was labeled in the C-2 carbonafter 5 minutes of 14CO2 fixation in blue light butuniformly labeled from experiments in white light.Thus, the general trend of these investigations hasbeen that blue light affected glycine and serine bio-synthesis and perhaps other amino acids and protein.However, Krotkov's group (6, 7, 21) also usingChliorella as well as tobacco leaves, found that blue
1 Supported in part by NSF grant GB 4154 and pub-lished as journal article No. 4064 of the Michigan Agri-cultural Experiment Station. This investigation was re-ported at the Fourth International Photobiology Con-gress, Oxford, England, 1964.
light, although stimulatory, promoted 14C labeling ofaspartate and glutamate. In some of the above ex-periments, light intensity may have been a limitingfactor. Horvath and Szasz (10) have reported thatamino acids were major products of photosynthesisat low light intensity and that sugars were formed athigh light intensity.
Since blue light appeared to effect glycine andserine, we reasoned that glycolate formation wouldalso be affected, for in the higher plant glycolate isa precursor for glycine and serine (16). When thework was initiated in 1963, we could confirm withChlorella the stimulation of glycine and serine forma-tion in blue light, but there was no pronounced effectupon glycolate. Afterwards it was discovered thatalgae, unlike most higher plants form serine fromP-glycerate (9). A CO pathway may be functioningin algae (26), however, most of the free glycolate isnot metabolized to serine but excreted, since the algaelack a normal glycolate oxidase (8. 9). However,the algae, after growth for several days in the bluelight, altered their photosynthetic or metabolic processin such a way that glycolate became the single majorproduct of short periods of 14CO, fixation. Conse-quently, this report is concerned with algae grownfor several days in blue light in contrast to all previouswork with algae grown in white light and onlv ex-posed to blue light during the test period.
In this investigation, we also examined the algaefor any gross spectrophotometric changes duringg.rowth in blue light. Fuj ita and Hattori (5) re-ported that changes in chlorophyll a and b concentra-tions in Tolypothrix responded to light quality rather
tlianlight intensity. Jones andMAIN-asimiiar conclulion from work withintensitv apparently affects only thCOInv'rS1io01.
Materials and Met
.i/go(I. Cultures wvere obtainedC'ollection of Algae" at Indiana Lingtol, Indiana. Chrorclla pyrenioidson) ( No. 39)5) xas cultured inmediumll (14 ) x ith Hoagland%Chlamvdomionas r-eibhardtii Dangeatwas grown on the high phosphateby Orth et al. (15). Cells vereot mlediuimi in 2.8 L 'Lowx form'afitted w-ith air inlets and agitatedslhaker b)v abotut 60 excursions pertUringof algae inxwhite light, thesa controlled environnment chamber ataimied a temperature in the culturConti11uo,LS light at the surface o1200 ft-c froimi WVestinghouse cool.fluores-cent bulbs (F96T12/CW/51Hwere gassed with 0.2 % CO., v/v iralso cultured in light passed by a bsvsteni wxhich transmitted light as sThe red filter was Fire Red" No. ISta-e anid Lighting Company. Chiclight Nvas obtained from a bank ofriescent light's General Electric Ilight wvas obtained from a bankfluoresceint lights ( General ElectSylvania F15>T8-B) and wras filtercelluloid filter aind 5 CI of a Cutaining 30 g I1 (27). The transnblue and red commlercial filters watrophotonmetrically by us. The cccelluloid and CuSO, solution passesioIn whichl was predominately bet500 imi/t anid contained no light al
1001
1I0
50-
_II
300 400 500
Wavelength, mjp
Fic;. 1. Absorption spectra offor photos nthesis experiments. TI-transmitted onlv those wvaveleng( .-- ). The blue celluloid filt
-ers( 11 ) reached of the culturing and photosynthesis experiments inA nacvstis. Light blue or red light N-ere performed in dark rooms.e rate of pigmnent 'rhese cultures wveregrowvn under continuous light,
aerated with 0.2 % CO. in air, anidmaintained at20 to 210 by a water bath.
:hods Culturesgrow blue light received
955 ,t\v/cnl2 asmeasured by a recording spectro-fromii the Culture radiometer, model SPR fromInstrumentation Special-
,niversitv.Bloom- ties Company, Inc. This intensity of blue light also
'OSaIChick;(Enmer- measuredl400 ft-c bya Weston IlluminationNlMeter,anl inorganic salt model 756with a quartz filter. Values in the tablesmiicronutrients. are expressed in ft-c to reflect the actual mea.sure-
rd(-) (No. 90) ments because the spectroradiometer was not yetmedium described availab!e when the research was done. Cultuirescultured in 1.5 L grown in continuous red light received 685 uv/cm'
' ernbach flasks between610 to 00m,u or 765 Mxw/cm2 between 610
on a reciprocating to 720mru. As measured by our Weston Illuminationminiute. For cul- MIeter this intensity- was equal to 200 ft-c. Thebiaker was kept irn cultures weredliluted every second dav with freshLt 15° xvhichmain- nutrient to an absorbance of 0.2 at 680my as measurede imieditutmi of 200. in a 1 cm cuvette with a Beckman DU spectropho-f the culture wxas tometer. Aliquotswvere removed at various timesxvhlite. super-high, a-fter innoculation to determine the rate of growtth-0). The cultures as expressedin increase absorbance at 680nml.
l air. Algaexvere '4CO2 Plhotosynthesis Experimnents. Cells wveretlue or a red filter remoxed in the dark from thegrowth media by cen-
hoxvn in figuire 1. trifugation, and a 1 % (v/v) suspension of cells was
[10 gelatin (Grand prepared in 1 m-t phosphate with a final pH of 6.5.ago.Illinois), and A 15 ml suspension, in lollipop fitted with large
15 iattred fluo- bore stopcock for rapid removal of aliquots, was
1F15T12-1R). Blue maintained at 20° in a Xx'ater bath. After 5 minutespreillumination in a designated light, a NaH14CO3
:ric F15T12-B or (2-5,umole) solution was added and samples were
ed throug-h a blue removed at 1, 3, and 10 imiinutes. Aliquots were
ISO, solution con- dunmped directly into warm methanol and furthernission of both the heate(l. The 14C products in methanol-water extracts.sdetermined spec- were separatedl by chromatography and radioautog-
mbination of blue raphy (3).d a band of einis- Extraction and Determiniation of Chlorophyll.xveen 400 mytand Ten ml of 0.5 algal suspension centrifuged
)ove 570 mM,. All in a clinical centrifuge at maximum speed for 3minutes. The supernatant fraction was discarded andthe tube inverted on an absorbant surface to eliminate
/Red Gelatin excess Xvater. After several minutes, the cells wereresuspended in 3 ml of absolute methanol and placed
/ in a stoppered centrifuge tube in the dark for atleast 2 hours to insure complete extraction of thepigments. After centrifugation. the absorbance ofthe green supernatant fluid was measured and clhloro-phyll a and b concentration calculated (12).
Measuremient of in vizo Absorption Spectra. Theneutral-density, filter technique of Shibata et al.
zl_<Sj-<- (18) xvas employed in which a filter paper saturated600 700 with mineral oil was placed betxveen the light source
and the cuvette. We found that a double thicknessfilter systems used of waxed paper (Schleicher and Schuell Co. No.
h red gelatin filter B-2). placed next to the cuvette on the face towardrths above 600 m,u the ligtht source, most effectively elucidated the fineinsmitted only those structure of the in vivo chlorophyll spectrum. The
tted nio red or far ratio of chlorophyll a/b absorption was evaluated fromspectra measured oIn the Carv 15 spectrophotomiieter
'HESS AND TOLBERT-PHOTOSYNTHESIS BY ALGAE IN BLUE LIGHT
while using a slit idth of approximately 0.7 mm.Under these conditions a 1 % algal suspension gaveapproximatelyt 0.5 absorbancy units at 750 my.
Results
Growth of Algae. Cultures, when removed fromwhite light and put in either red or blue light (asdescribed in the Methods section) grew more slowlyfor 4 or 5 days before attaining a relatively constant,rapid grow-th rate which approximated that of thecultures in w-hite light of similar intensities. Thus,Chlamydornonas and Chlorella in blue light grew
slowlv at first and then more rapidly, as measuredby the culture's absorbance at 680 m,u (fig 2). Allmeasurement-s were made on non-synchronized or
random cultures which initially had similar cell popula-tions as judged from approximately similar absorbancevalues. Chlauiydomonas in red light also grew more
slowly at first, but after several days in the red light,they too grew rapidly. We think that the slow re-
covery of the growth rate in blue or red light was an
adaptation rather than a mutation, since all the resultspresented in this paper were similarly reproduceablewhen starting oxer again with stock cultures kept inwhite light.
Rate of 14COo, Fixation. With Chlorella or
Chlamydomionas fully adapted after at least 10 days
growth in either white, blue, or red light, experimentson the rate of 14CO fixation were performed withincreasing time and intensity of red, blue or whitelight. The available intensity of the blue light wasthe limiting factor, but representative data in figure 3indicated that 400 ft-c was approaching light satura-tion. In the experiments nearly linear 14C fixationrates over the 10 minute period were observed, andthus CO2 availability did not become limiting. Thefixation rates with Chlorella grown in blue light wereunusual since relatively high levels of fixationoccurred in 10 minute periods at low blue light in-tensities.
Distributtion of 14C Amtong Soluble Products ofPhotosynthesis. For the purpose of presentation, the]4C distribution among the products of CO2 fixationhas been divided into 3 groups of compounds: A) phos-phate esters representing components of the photo-synthetic carbon cycle, B) malate, aspartate, gluta-mate, and alanine which are the compounds associatedwith the citric acid cycle that accumulate "-C, andC) glycolate and glycine plus serine which are asso-
ciated with the glycolate pathway (16). The percentdistribution of 14C among each product of "CO2fixation by paper chromatography is on file alongwith results from other variations of light intensityand quality (8).
Algae grown in either white, blue or red light
=L 10th day~~~~~~~~~~~~~~~~~~~0hE dthdyy0.6|
~~~~~~~~~~5th day
0
.0
0.4-0
0
Time (hours)FIG. 2. Rate of growth of Chlorella pyrciioidosa and Chlaniydomonas reinhardtii after designate days in blue
light. Vertical litnes designate the variation of rate of growth from repeated experiments after 10 days culturein blue light.
FIG. 3. Rate of total 14C fixation by algae in increasing intensities of blue light. In part A, C/ilo cll//a pyrenoi-dosa were grown in white light and in part B in blue light for 10 days before the experimiient. In part C,Chlamydomonas reinihardtii were adapted for 10 days in blue light. Ordiniate values are total 14C in the solublefraction from 15 ml of a 1 % algal suspension.
and allowed to fix 14C02 in the same tyrpe of lightincorporated initially the highest percentage of 14Cinto the phosphate esters of the photosynthetic carboncycle. Typical data for Chlamydomonas are sum-
marized in figure 4. As indicated by the rapid re-
duction in the percent of the total 14C fixed whichaccumulated in these esters, the pool sizes in the blueadapted algae appeared smaller than in the algae
grown in red light.Chlamydomonas grown in blue light rapidly accu-
mulated 31 % of the newly fixed 14C into glycolateduring photosynthesis with 350 ft-c of blue light. Ifthe algae were grown in red light, they accumulatedonly a trace of 14C labeled glycolate (fig 4). Similarresults were obtained with Chlorella except that thetotal percentage of fixed 14C in glycolate was onlyabout half that found with Chlamydomonas.
For Chlamydomonas the percent '4C in glycine andserine was not greatly altered by their growth ineither blue or red light. Chiorella, however, showeda 1 to 2 fold increase in the percent of the total 14Cin serine immediately after culture in blue light was
initiated. This result was similar to earlier experi-ments cited in the introduction with Chlorella. Simul-taneously with increase serine-14C, we observed thatthe percent of 14C accumulating in P-glycerate de-creased about half.
For both algae the percent of the total 14C incor-porated into malate, aspartate, glutamate. and alaninewas somewhat greater with aligae grown in red lightthan white light and much greater than with algaegrown in blue light (fig 4). Thus, grovth in bluelight promoted 14C accumulation during the initialminutes of 14C0. fixation into glvcolate while growthin red light resulted in 14C accunmlation in malate,aspartate, glutamate, and alanine.
If blue light were used for 14CO2 fixation experi-ments with algae grown in white light or during thefirst to third day after initiation of their growth inblue light, the large significant changes in the per-centage of 14C incorporated into glycolate x ere notobserved. Since accumulation of a large percentageof the 14C in glycolate in blue light did not occur withalgae growrn in white light, glycolate accumulationwas probably not related to possible alterations inassimilatory power produced by the blue liglht.
Algae grown for 10 days in blue or red lightproduced about the same products regardless ofwhether 14C02 fixation was measured in white, blueor red light. Thus, the same accumulatioil of 14C-glvcolate bv algae grown in blue light occurred whenthe 10 minute '4C0 fixation period waS run in eitherblue or white light. These facts support the hypo-thesis that the cultures growin in blue light actually
HESS AND TOLBERT-PHOTOSYNTHESIS BY ALGAE IN BLUE LIGHT
experienced a period of slow cellular adaptation whichwas not rapidly reversed by white light.
The data in figure 5 emphasizes the effectivenessthat growth of Chlamilydomonas in blue light has onglycolate production. It is established that the amountof glycolate production by algae increases at higherlight intensities (2, 13, 20,26). In the present ex-periments glycolate production at 1 10 ft-c by Chlamnv-donmonas grown in either red or white light was lessthan 3 % after 10 minutes (see fig 5B for algaegrown in red light), but increasing the light intensityto 1200 ft-c resulted in considerable formation ofglvcolate-14C. However, if the Chlamnydomionas weregrown in blue light for 10 days, then they formed in110 ft-c of blue (fig 5A) or white light (data notshown) nearly the same amount of glycolate-14C whichthe algae grown in white or red light could only pro-duce at 1200 ft-c. For Chla;nndomonas grown in bluelight '4C in glycolate amounted to 15 to 36 % of thetotal 14C fixed between 3 and 10 minutes, and con-sequently glycolate-'4C was the major soluble productof CO,, fixation. This amount was 3 to 4 times more14C than in any other single product, yet as seen fromfigure 4, during the first minute of 14CO2 fixation,there was more 14C in P-glycerate or sugar phos-
o 60 \ Phosphate Esters -0
40-
.0
%_ ~~~~lue\420-0/0
phates than in glycolate. Thus, algae grown in bluelight accumulated large amounts of glycolate-14C evenat low light intensities and high light intensity furtherincreased the amount of glycolate which was formed.It is also apparent in figure 5, that glycine and serineaccumulation was not affected by growing theChla-mi,domuonas in blue light. This is consistent withthe fact that serine and glycine synthesis is independentof glycolate formation in algae (9), in contrast toplants in which serine and glycine are formed fromglycolate ( 16).
Chlorophyll Coniteiit. Using a Carv 15 recordingspectrophotometer, absorption spectra were measuredon cell suspensions as described in the 'Methods sec-tion. The spectra in figure 6 have equal absorbancevalues at 550 my, but, for clarity of presentation, thecurves have been separated in the figure. The changeof the 680 mrp/7655 mu absorbance ratio, which repre-sent in vivo maxima for chlorophylls a and b re-spectively, indicated a significant decrease in thechlorophyll a/b ratio as the period of culture in bluelight increased. No significant changes in these re-gions of the spectrum were observed for algae grownin red light. Other portions of the spectrum didreflect absorption changes caused by growing the cells
* Glycolatea Glycine +
Malate, Aspartate,Glutomate + Alanine
Red _.
- -
I 3 10 1 3Time (min)
I0 I 3
FIG. 4. Percent distribution of 14C among soluble products formed by Chlaiamydomlonias after adaptation toblue or red light for 10 days: (0- )) Cells grown in blue light anid 14CO. photosynthesis was performed in 350ft-c blue light; ( 0----0) Cells grown in red light and 14CO, photosynthesis was performed in 200 ft-c of redlight ( >600 mu).
FIG. 5. Total 14C in glycolate or glycinehardtii grown in (A) Blue light or (B)(* -U) in 1200 ft-c white light.
in red light, but these were not consistent and toocomplicated for a careful evaluation by this technique.
The above in vivo measurements were verified byresults from spectral measurements of chlorophyll inextracts from the algae. The total chlorophyll con-tent on the basis of the cell volume increased about20 % during the first 6 days of culture in blue light,and a significant decrease appeared in the chlorophylla/b ratio (fig 7). Although the ratio of chlorophylla/b varied from 1 to 2.8 for different starting cul-tures which had been grown in white light, a con-sistent trend was a decrease in the chlorophyll a/bratio during culture of the algae in blue light. Similarresults were obtained for Chlorella. The data fromChlamydomonas extracts, however, were more con-sistent than with Chlorella, perhaps, because the
10 I 3 10Time (minutes)
plus serine formed by 0.5 % (v/v) suspension of Chlaiiiydomonas reini-red light; ( * 0 ) after 14CO. fixation in 350 ft-c blue light oi
pigments from Chlorella were extracted less qulantita-tively.
Discussion
Three major changes were observed when Chlanty-domonas or Chlorella were grown in 955 ILw/Cm2 ofcontinuous blue light (425-540 mu) or in 765 uw/cm2red light (above 600 mu). A) Growth rate slowedfor 3 to 5 days, but returned after 5 to 10 days torates equal to those with similar amounts of whitelight. B) After 5 to 10 days adaptation to bluelight the algae incorporated about 30 % of the total'4C fixed in 10 minutes into glycolate at low (110ft-c) blue or white lilght intensities and even more at1200 ft-c light. This phenomena did not occur until
HESS AND TOLBERT-PHOTOSYNTHESIS BY ALGAE IN BLUE LIGHT
Y1-
s
A,m,uFIG. 6. In vivo spectra and chlorophyll a/b ratio
for Chlamydomonas reinhardtii grown for designatednumber of days in blue light. T(he spectra were mea-
sured on approximately 1 % algal suspensions and re-
corded with a Cary 15 Spectrophotometer. All absor-bance values at 550 mA were approximately equal. Theabsorbance ratio a/b equals the absorbance ratio 680mnu/655 mg which was indicative of the chlorophyll a/chlorophyll b ratio.
= 3.0-
cg \ Chlomydomonas0
3%2.0 0
06
0
DaysFIG. 7. Changes in the chlorophyll content of
Chlartydomiionias reinhardtii as determined by the amount
of chlorophyll extracted with methanol. The changein the chlorophyll a/b ratio is plotted as a function ofthe increasing nuimber of days of growth in blue light.
after the initial period of adaptation. After growthin blue light, 14CO2, fixation in white light producedthe same "lC distribution among products as in bluelight. Algae grown in red light incorporated more
1"C into malate, aspartate, glutamate and alanine andonly trace amounts into glycolate. C) During growthin blue light, chlorophyll content increased 20 % whilethe chlorophyll a/b ratio decreased. The results were
reproduceable when starting again with fresh cultureswhich had been grown in white light. The resultssuggest a slow environmental adaptation over several
generations to the specific light quality. The datainvite speculation that both CO, fixation and thepigments for electron transport in photosynthesis areso intimately interdependent that complementarychanges in both systems compensate for environmentalalterations. These changes could maximize thecapacitv of an organism to utilize light and CO,.The accumulation of such large amounts of 14C in
glycolate by algae grown in blue light does not indi-cate that glycolate was being formed by a separatepathway of CO, fixation. In all experiments P-glyc-erate and sugar phosphates contained the most 14Cduring the first minute of 14CO2 fixation, whilesamples taken at 3 minutes contained much moreglycolate-14C than P-glycerate-"-C or any sugar phos-phate (fig 4). The results suggest that glycolate-14Cwas accumulating as an end product of photosynthesis,and probably it was being excreted (9, 20).
Our experiments do not prove that the increasedchlorophvll b content with respect to chlorophyll a islinked to the altered 14CO2 fixation. Both changesoccurred upon growing algae in blue light, but theyneed not be associated. The decrease chlorophyll a/bratio during growth in blue light might be expectedfrom the data of Fujita and Hattori (5). The Soretband absorption for chlorophyll b is approximately5so % greater than the absorption for chlorophyll abetween 400 to 500 m/u. Plants grown in shade alsoseem to have more chlorophyll b.
Unlike glycolate-14C formation, the rate of label-ing of glycine and serine did not increase whenChlamydomonas were adapted oil blue light. WhenChlorella were placed in blue lighlt 14C-label in serineincreased immediately and before similar changesoccurred for glycolate-_4C. With Chlorella, increasedserine-14C was accompanied by decrease 'IC inP-glycerate. These results appear consistent with theabsence of a typical glycolate oxidase in algae andwith the formation of serine from P-glycerate ratherthan from glycolate as occurs in higher plants (16).Movement of 14C from P-glycerate to serine mayoccur due to limited availability in low blue light ofNADPH and ATP which is needed to convertP-glycerate to triose phosphate. Although not in-vestigated, one might predict with higher plants. whichrapidly metabolize glycolate to serine and then tosucrose, that growth in blue light would also increasethe pool size of glycine and serine and sugars. Thusglycine and serine accumulation by higher plants inblue light could arise from both P-glycerate andglycolate.
Cavle and Emerson (4) ran "-CO, fixation ex-periments with Chlorella pyrenoidosa in the presenceof 8 microeinstein/cm2/min of blue or red light.Their algae had been grown in white light and onlythe subsequent 5 minute experiment was done inmonochromatic light. As we also observed, theyfound no significant difference in total amount of"CO, fixation or distribution of 1"C between aminoacids and phosphate esters. However, they observedthat the specific activity of the alanine, glycine and
serine XXhlicil WaCs prod(uTced in ; minutes. 0o blue lightxvas greater than that fromii red liglht. Further, thedistribution of 14C in gIlvcine, but not in alanine, wasaltered b)v the blue light. ft would appear thatmetabolic clhanges in blue lighlt begin immediately.The large clhange in 1 4C distribution which leads toan accumila6tiot of glvcolate b1 algae in blue light,as. observed 1b us, seemis to orcctr only after e-everaldav-s of grovth in blue liglt.
In a series of palp)ers frolmi Krotkov's laboratory.it hals been reporte(I that the addition of blue lightto red light stimulated 1 4C(O, inicorporationi intoaspartate. malate anti glitamllate duir-inig 30 iminluteexperimilenlts and (lecreased 14(7 in glycine and glvcolate6, 7, 21). Although1 these resUlts appear the opposite
fromii ours, the 2 tyl)es of experimenits are not colm-parab'e witlh respect to grovth of the algae or typeof im0onoclrromatic light emliloved. Krotkov's groul)use(d algae gro\Xn in white light with 5 % CO., rathel-than in the blue or redl light vith 0.2 % CO. Fur-tlher, they used a (lark plretreatment p)eriod whichvarie(l fromii 1 .5 to 20 hours. l171eir blue light onlysupplemente(l red lighlt and( even when their 1luefilter alonie wXas used, it tralusmittecltimuclh light be-twveen 500 to 600 muw, some at 680 M/1, an(d all liglitabout 710 minu (21 ). Our- u1se of a1 (uSO4 Solution,as emiiphasizdcd by \Withrov ( 27), comipletely elimi-nates red light witl blue p)igment filters.
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