The relation between the rate of photosynthesis and the pigment content was studied in seedlings of Avena sativa var. Victory. Etiolated seedlings were illuminated during different periods. Next, the rate of photosynthesis as well as the pigment concentrations were determined in the primary leaves.


The saturation rate of photosynthesis proved to increase with increasing pigment concentrations until the ratio chlorophyl b / chlorophyll a approached its “normal” value. Although the amounts of both pigments still keep on increasing, the saturation rate of photosynthesis remains the same.


In agreement with other authors we could observe that the formation of chlorophyll a starts immediately upon illumination, while b is formed only after a lapse of time.


Xanthophylls are present in etiolated seedlings in relatively large amounts; their formation is not influenced by light.


Other carotenoids are also present in non-illuminated plants, though in small amounts. Light, however, accelerates their development.


As far as it was studied the quotient chlorophyll a / carotenes remains constant except for very low pigment concentrations

Spruit-van der Burg, A.

Utrecht University Repository

VOL. 5 (195o) BIOCmMICA ET BIOPHYSICA ACTA I75  EMISSION SPECTRA OF LUMINOUS BACTERIA  by A. SPRUIT-VAN DER BURG Biophysical Research Group under the direction o/A. J. KLUYVEI~, Delft and o/ J. M. W. MILATZ, Utrecht (Netherlamls) INTRODUCTION In 1936 and 1937, EYMXRS AND VAN SCHOUVCENBURG 1, 2 published their work on the emission spectra of Photobacterium phosphoreum and two other species of luminous bacteria as well as observations on the spectra of some chemoluminescent reactions. In the course of our own investigations we became aware of the fact that a considerable error may be caused by the absorption of emitted light by the emitting material itself. It is well known that many if not all organisms contain compounds exhibiting selective absorption in the visible region. As such may be mentioned riboflavin and the cyto- chromes. The error due to absorption is composed of two factors. One of these must be attrib- uted to absorption of the light by the emitting bacterium itself, the other to repeated absorption by surrounding cells. As regards the error caused by the absorbing compounds within the emitting cell, a rough calculation indicates that this is negligible if one may assume that the coloured compounds are distributed homogeneously over the cell. The second of these two causes of error can be made negligible by using very dilute suspen- sions as will be shown below. This method has the disadvantage that the light intensity to be measured is small, so that long exposures axe needed. Still this is the only way of avoiding the application of corrections to the measured spectrum. Such corrections cannot be made accurately, as we are dealing here with a combination of absorption and scattering. As we will show, EYMERS AND VAN SCHOUWENBURG did not pay sufficient attention to these sources of error which have rather seriously affected their results. EXPERIMENTAL METHODS The organisms used are kept in the culture collection of the Biophysical Research Group under the following names: Photobactevium phosphoveum, Ph. splendidum, Ph. fischeri. The bacteria were cultivated in liquid media of the following composition: Ph. phosphoveum in: peptone 2~to, sodium nitrate x~/o, sodium chloride 2~/o, Ph. fischeri in: peptone x%, glycerol 1%, sodium chloride 3% to which small pieces of limestones were added. Both species were cultivated in aeration flasks. Ph. splendidum was grown in flasks with shallow layers of a medium containing: yeast autolysate x : xo, sodium nitrate I ~/o, sodium chloride 2~/o . After about twenty hours when maximum luminescence had developed the bacteria were separated by centrifuging, washed with 3~o sodium chloride solution, and suspended in o.x25 M phosphate buffer containing in addition: in the case of PA. pAosphoveum and Ph. splendidum glucose 3~o, sodium nitrate i~/o, sodium chloride 2~/o, in the case of PA. #sc/wr/glucose 3~o, sodium chloride 3~o- The suspensions were placed before the slit of the spectrograph in a LXYBOLD gla~ box with a Re]erences p. x 7& z76 A. SPRUIT-VAN DER BURG VOL. 5 (I950) depth of ~ or I cm, the back wall of which had been painted black to avoid an increase in light path by reflection. During the exposure air was bubbled through the bacterial suspension. The emission spectra were obtained by a photographic method. Two spectrographs were used, viz., a Fuzss glass spectrograph f/9 in which another lens had been placed behind the camera objec- tive in order to bring the opening to f/4.5 appr. The other was a VAN HEZL  spectrograph f]2 with a prism filled with ethyl cinnamate. With this instrument special precaution had to be taken to keep the temperature of the prism constant during the long exposures, Pk. phosphoreum and Ph. splen- didum were photographed with both instruments, Ph. [ischevi with the f/2 instrument only. The photographic material was Agia Isopan SS, pre-illuminated with red.light to give a density of 6% after development. By this treatment threshold difficulties are overcome, thus increasing the sensitivity for small fight intensities. In order to obtain density marks, spectra were photographed for comparison on the same plates, from a calibrated band lamp with the aid of a step slit. The actual emission spectra were also photo- graphed through a step slit. In this way on each plate several spectra of the same bacterial suspension were obtaincd. The conversion of transmission into intensity of the spectra was carried out according to the method described more in detail  by EYMERS AND VAN SCHOUW]tNBURG 1 (see also referenceS). The spectra reproduced in the following section are averages of several measurements ,  INFLUENCE OF DENSITY OF SUSPENSIONS ON THE EMISSION SPECTRA This is illustrated in Fig. i. Curve B was obtained with a density of 8 F1]ml wet bac- teria of Pk. phosphoreum per ml liquid and with a vessel with black back wall, curve C with a density of 4 °/A]ml and a vessel with white back wall. The difference in the two spectra is caused by the increased absorption i  the suspension with the highest density. This absorption is especially marked in the blue region. Above ~ = 560 m/t the two curves are in fact identical. Curve A was taken at a density of 2/A/ml. The difference with curve B is only slight, and amounts to zo- i5% for wave-lengths below 460 m/t. As in the case of curve B the density is four times that of curve A, the absorption present in curve B must be more than four times that of curve A. We conclude that the /nt A °-A,B I 400 420 440 460 480 500 520 540 560 580 600 620 tnt~ Fig. z. Inf luence of the densi ty  upon the emission spect rum of Ph. phosphoreum intensity drop of xo-x5% corresponds to more than three times the absorption still present in curve A. It follows that this absorption must be less than 3-5%. As the Re/erences p. r78. VOL. $ (z95o) EMISSION SPECTRA OF LUMINOUS BACTERIA z77 accuracy of the measurement is about 3%, the curve A may be considered to give the most accurate representation f the emission spectrum of Ph. l~hosphoreum obtainable with the method employed 4. In the same way we found that spectra of Ph. splendidum at densities 6 and I8/A/ml and a depth of o. 5 cm were identical within the experimental error, and of Ph. fischeri at densities 5 and I5 ffl/ml. Consequently the curves given in Fig. 2 apply to these densities. Curve D of Fig. z is the spectrum published previously by EYMERS AND VAN SCHOUWENBURC (l.c.). They used a vessel which was painted completely white with the exception of the wall facing the spectrograph. From the data given in their paper it may be concluded that they have used a density of approximately 2o pl/ml. Their curve fits in between our curves B and C, as can be expected. The secondary maximum in curve D at approximately 54 ° mff is probably due to an irregularity in the photo-  graphic material. RESULTS AND DISCUSSION The final results of our measurements are given in Fig. 2. The wave-lengths of maxi- mum emission are approximately: Pk. t~hosplwreum 472 raft, Ph. sl~lendidum 489 mff and Ph. fisckeri 496 raft. For comparison the spectrum of the fungus Armillaria mellea, with maximum at 526 raft, published previously 4 and discussed in detail elsewhere 5 is also included. There is no doubt that the spectra of the luminescent organisms investi- gated are different. Apparently the conclusion of E~ERS AND VAN SCHOUW~NBURC" that the spectra of Pk. phosphoreum and Ph. splendidum are the same is not correct. Finally we wish to make some remarks regarding the attempted analysis of emission /nt 400 / '~ . . .  /~'~.~ ------ Ph. phosphoreum / / / \  \ " . /  .,, ph ,~e.~,~u~. I / . . ."" " :  X.''.. ., .......... I / ':/\".. " . . . . .  " " " ' "  "... \\ • - \ • ... \ %,. °~ • ... ",~. ". .  ~ .  42o 44o 46o ~ 50o 5zo 540 56o 58o 6o0 6,'o 6~ n,p Fig. z. Emission spectra of luminous organisms spectra by EYMERS AND VAN SCHOUWENBURG 1' I. These authors believe to have given evidence that emission spectra of chemo- and bioluminescent reactions as well as References p. I78. i78 A. SPRUIT-VAN DER BURG VOL. 5 (I950) fluorescence spectra, plotted versus frequency, could be analysed into a discreet number of symmetrical curves, each representing a broadened, so-called fundamental  frequency. Considering the fact that emission spectra of large molecules are superpositions of vibra- tional and rotational bands, the position of which in the spectrum is determined by the magnitude of the corresponding electronic transition, there is little reason to expect the same frequencies to occur in the spectra of different sources. The spectra of bio- luminescent reactions published in this paper give no support to this hypothesis of EYMERS AND VAN SCHOUWENBURG. It  is to be regretted that this idea still receives attent ion in the l iterature 6,7 Owing to various circumstances this work, the experimental part of which was carried out during I943, could not be published unt i l  now. SUMMARY Emission spectra have been determined of three species of luminous bacteria. It is shown that in this determination special attention has to be paid to the absorption of the emitted light by the bacterial suspensions themselves. R]~SUM]~ L'auteur addtermind les spectres d'dmission de trois esl~ces de bactdries lumineuses. I1 a montrd q'il est n6cessaire de tenir compte de l'absorption de la lumi~re dmise par les suspensions bactdriennes. ZUSAMMENFASSUNG Der Verfasser hat die Emissionsspektren von drei Arten yon Leuchtbakterien bestimmt. Es erwies sich hierbei als notwendig, die Absorption des emittierten Lichtes durch die Bakteriensuspen- sionen selbst besonders zu berUcksichtigen. REFERENCES 1 j. G. EYMERS AND K. L. VAN SCHOUWENBURG0 Enzymologia , I (I936) IO 7. * j. G. EYMERS AND K. L. VAN SC~[OUWENBURO, ibid., 3 {I937) 235. 8 L. S. ORNSTEIN, J. W. MOLL, AND H. C. BURGER, Obiektive Spektralphotometrie, Samml. Vieweg, Berlin I932. 4 A. VAn DER BURG, Thesis, Utrecht I943. s E. C. WASmNK AND A. SPRUIT-VAN DER BURG, in preparation. 6 E. N. HARVEY, Living Light, Philadelphia 194 I. 7 F. H. JOHNSON, Advances in Enzymology, 7 (x947) 2x5. Received November zst, I94 9 

Emission spectra of luminous bacteria

The relation between the rate of photosynthesis and the pigment content was studied in seedlings of Avena sativa var. Victory. Etiolated seedlings were illuminated during different periods. Next, the rate of photosynthesis as well as the pigment concentrations were determined in the primary leaves.




The saturation rate of photosynthesis proved to increase with increasing pigment concentrations until the ratio chlorophyl b / chlorophyll a approached its “normal” value. Although the amounts of both pigments still keep on increasing, the saturation rate of photosynthesis remains the same.




In agreement with other authors we could observe that the formation of chlorophyll a starts immediately upon illumination, while b is formed only after a lapse of time.




Xanthophylls are present in etiolated seedlings in relatively large amounts; their formation is not influenced by light.




Other carotenoids are also present in non-illuminated plants, though in small amounts. Light, however, accelerates their development.




As far as it was studied the quotient chlorophyll a / carotenes remains constant except for very low pigment concentrations

Emission spectra of luminous bacteria

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