Photoacoustic spectroscopy of Anacystis nidulans

ARCHIVESOFBIOCHEMISTRY AND BIOPHYSICS Vol. 222, No. 2, April

15, pp. 411-415, 1983

Photoacoustic II. Characterization

Spectroscopy

of Anacystis

of Pigment Holochroms

nidulans

and Thermal Deactivation

ROBERT CARPENTIER, BERNARD LARUE,

AND

Spectrum

ROGER M. LEBLANC

Centre de Reckmche en Photobiqphysique, Univemiti du Qu&c Trois-Rivi&es, Trois-Rivi&res, Q&bec G9A 5H?‘, Canada

d

Received July 8, 1982, and in revised form December 20, 1982

The photoacoustic spectrum of Anacystis nidulans recorded at room temperature is qualitatively similar to low-temperature absorption or fluorescence excitation spectra. The bands of pigment holochroms are well resolved compared to room-temperature absorption spectra. The thermal deactivation spectrum obtained by extrapolating acoustic data for an infinitely thin sample indicates that the photosynthetic efficiency decreases from phycocyanin to chlorophyll a and carotenoids.

which deals more specifically with A. nidulans itself, reports the photoacoustic properties of thin algal layers. Our main conclusions are (i) pigment forms of A. nidulans which are detected only at low temperature by conventional spectroscopic techniques can be identified at room temperature by photoacoustic spectroscopy; (ii) the action spectrum for the thermal deactivation of algal pigments is obtained by extrapolating the spectrum of an infinitely thin sample; and (iii) phycocyanin acts as the major light-harvesting pigment in A.

Photoacoustic spectroscopy measures the heat production of an absorbing sample irradiated with a frequency-modulated light beam. When the thermal deactivation of excited molecules competes with other light-driven processes, such as fluorescence and chemical energy storage, this technique provides an indirect estimate of the yield of these processes. Indeed, acoustic data are currently used in the determination of fluorescence quantum yields (l-4) and a theoretical approach has been developed for the kinetic analysis of multistep photochemical processes (5). However, quantitative comparisons with absorption spectra are often complicated by the optical absorption saturation which occurs with optically thick material and may lead to featureless photoacoustic spectra (6, 7). In the accompanying paper, we have reported measurements performed on layers of the cyanobacteria Anacystis nidulans having a controlled thickness. It was concluded on a general level that the relationship between sample thickness and photoacoustic signal intensity can be predicted from the optical and thermal properties of the sample. The present study,

nidulans. MATERIALS

AND

METHODS

Growth and filter deposition of algae, recording of spectra, and photoacoustic data treatment are described in the accompanying article. All the photoacoustic spectra are recorded at 100 Hz, using a spectral bandwidth of +2.5 nm. The spectrum of an infinitely thin sample is calculated according to the following extrapolation procedure. Carbon black normalized spectra taken from a series of samples having a thickness comprised between 1 and 6 pm are first equalized with each other at 490 nm. Next, the spectrum is extrapolated to zero thickness for wavelengths comprised between 400 and 700 nm and selected at 5-nm intervals. 411

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Copyright0 1933by Academic Press,Inc. All rightsof reproduction in anyformreserved.

412

CARPENTIER, RESULTS

AND

LARUE,

DISCUSSION

Although the pigment content of algae, in particular the phycocyanin-to-chlorophyll ratio, varies slightly from one culture to another, all the photoacoustic spectra recorded from the same batch of algae exhibit striking similarities (Fig. 1). Some of the minor spectral details which are not always obvious in thin samples usually become more apparent in data obtained from thick samples (>lO pm). As shown in the preceding article, this result is expected for a material which is optically thick, but not opaque. Given the previous considerations, the data from Table I constitute a composite of several experiments and summarize the typical features of most spectra. Photoacoustic spectra of A. nidulans reveal a variety of details which are not normally detected in absorption spectra recorded at room temperature (see, for instance, Fig. 2 and Refs. (8, 9)). The possibility that instrumental artifacts could produce such details is ruled out by their absence from photoacoustic spectra of other types of materials having broad absorption bands in the spectral regions investigated (the authors, unpublished observations). Moreover, our identification of

0 450

550 WAVELENGTH

s50 , nm

FIG. 1. Carbon black normalized photoacoustic spectra of A. nidulans layers. L, sample thickness in pm.

AND

LEBLANC

pigment holochroms is usually corroborated, without large wavelength shifts, by low-temperature measurements of absorption or fluorescence excitation spectra (Table I). As already reported for 7YK absorption spectra (8), the c-phycocyanin band is resolved here into two peaks at 625 and 635 nm. This doublet likely reflects the particular environment of phycocyanin in intact algae, since, upon cooling extracted phycocyanin from 295 to 7’7”K, one observes only a single fluorescence excitation peak which shifts from 625 to 620 nm (8). We also suspect the presence of several shoulders between 550 and 610 nm, but a single one, in the range 580-590 nm, is prominent in most spectra. The chlorophyll peak at 678 nm is accompanied by a shoulder at 670 nm which was already reported not only in low-temperature measurements (8, 91, but also in absorption spectra recorded at room temperature with a special equipment (10). Finally, at least four carotenoid peaks or shoulders are detected between 455 and 500 nm, while 7’7°K absorption spectra resolve only two (8,9). This unexpectedly high sensitivity of photoacoustic spectroscopy, which occurs in spite of the spectral bandwidth of 22.5 nm used here, has already been noticed by Ortner and Rosencwaig (11). The effect of optical absorption saturation is quite evident in the series of spectra shown in Fig. 1. In fact, when normalized with each other at a common wavelength, no two spectra from samples differing in thickness can be exactly superposed. Those from the 0.5- and l-pm samples constitute an exception. This indicates, in agreement with the known diameter of A. nidulms cells (12), that a nominal thickness comprised between 1 and 2 pm corresponds to a monolayer of algae. Below this value, the signal intensity should be strictly proportional to the fraction of the filter surface actually covered with algae and no variation in the shape of the spectrum would then be expected. As shown in the accompanying article, optical absorption saturation distorts the photoacoustic spectrum and prohibits, in principle, quantitative comparisons with absorption spectra. This problem can be

PHOTOACOUSTIC

SPECTROSCOPY TABLE

PHOTOACOUSTIC

AND ABSORPTION

Absorption Pigment

680 (P)

Carotenoids

460-465 (S)

Phycocyanin

nidulans,

413

II

I

PEAKS IN SPECTRA OF Anacgstis

nidulans

spectrum’

298-305°K

Chlorophyll a (red region)

OF Anacystis

Photoacoustic spectrum (298°K)

WK 670 (L-3) 679 (P) 686 (S) 705 (S) 465-470 (S)

490-505 (S) 580 (S) 625 (P) 635 (S; weak)

Allophycocyanin

502 (P) 580 (S) 625 (P) 634 (PI 650 (S)

670 (S)* 678 (P) 690 (S; weak)* 455 (P/S) 480 (P) 490 (P) 500 (P) 550-610 (Several 625 (P)” 635 (P)* 650 (S; weak)

shoulders)

Note. S = shoulder, P = peak. Values in nm. a Data from Cho and Govindjee (8) and from Fig. 2. *Best seen with sample thickness > 10 pm.

partly alleviated by diluting the sample with nonabsorbing material (6, 7) or, alternately, by calculating the spectrum from the phase of the signal instead of its amplitude (13,14).l Using an approach somewhat similar to the dilution method, we have extrapolated the spectrum at an infinitely thin sample, a procedure which is expected to cancel out the distortion due to optical absorption saturation (see Appendix). Since the absolute intensity of such a spectrum would be zero, one must use for this purpose a set of spectra which have all been equalized with each other at a common wavelength. To minimize the errors involved in the extrapolation procedure, a prior study of the saturation effect has been performed over the spectral range investigated (accompanying article). We have selected 490 nm as the common reference wavelength since the magnitude of the saturation effect is then in-

termediate to the extremes found at the maximum and minimum of absorption, respectively. Also, for the reasons specified above, the spectrum from the 0.5-pm sample was omitted from the calculation. The e,xtrapolated spectrum in Fig. 2 is nearly identical to the one obtained from

’ One could also increase the frequency of the modulated light beam, such that heat diffusion rather than light absorption becomes the limiting factor with respect to signal intensity. However, this method is quite impractical here since, with increasing frequency, the signal intensity decreases much faster than the thermal diffusion length.

FIG. 2. Action spectrum for thermal deactivation in A. niduhns. Absorption spectrum from a liquid suspension of algae (. . e), carbon black normalized photoacoustic spectrum from a l-pm sample (-), or for an infinitely thin sample (0). The acoustic spectra are equalized with the absorption spectrum at 630 nm.

1.5-

- o.“%icTrx7 WAVELENGTH,

“m

414

CARPENTIER,

LARUE,

a l-pm sample, except that the latter gives a weaker signal at 440 nm. This wavelength corresponds to the maximum of absorption in the visible range, which results in a very early saturation of the photoacoustic signal intensity. On the other hand, the extrapolation procedure is very tedious and introduces “noise” which blurs the fine details of the spectrum. In practice, the l-pm spectrum remains a very satisfactory approximation above 460 nm. Provided that a prior normalization against a carbon black sample is performed, the extrapolated signal intensity defines an action spectrum for the thermal deactivation of incident photons. Consequently, the action/absorption ratio is a relative measurement of the thermal quantum yield of absorbed photons. Attributing an arbitrary value of 1 to phycocyanin at 630 nm, one calculates from Fig. 2 ratios of 1.4 and 1.25 at 490 and 680 nm, respectively. We can assume that the fluorescence decay of excited pigment molecules is negligible at room temperature in A. nidulans (8). The above figures are thus proportional to the fraction of absorbed energy which is unavailable for the photosynthetic process. Our results indicate that the photosynthetic efficiency of the algal pigments decreases according to phycocyanin > chlorophyll

a > carotenoids.

The high photosynthetic efficiency of phycocyanin, as deduced from measurements of quantum yields for oxygen evolution and chlorophyll fluorescence excitation, has already been known for some time (for a review, see Ref. (15)). This fits with the concept that the maximum photosynthetic yield is reached when the absorbed energy is distributed equally between photosystems I and II reaction centers (16). As shown by Wang et al. (17) who have measured actions I and II independently from each other, phycocyanin acts as an antenna for both photoreactions, while chlorophyll is associated almost exclusively with reaction I. However, the thermal yield must be expressed here in

AND

LEBLANC

arbitrary units since the relative magnitude of competing photochemical processes cannot be estimated from the present data alone. As previously suggested (5, 18), this information could be obtained through the use of photosynthetic inhibitors by comparing the spectra from active and inactive algae. Such an approach, which requires that the experimental fluctuations of the signal intensity remain smaller than the difference to be measured, is feasible only with thick algal layers. We have recently collected data under these conditions and our results will soon be reported. APPENDIX

The extrapolation procedure is justified by the following treatment. According to the accompanying article, which also defines the symbols used below, the photoacoustic signal intensity, Q, is given by

Q=

Acu(r - 1) [l - e@+@]. k&a2 - 4)

[l]

If L is small when compared to l/(a! + us), one can use the approximation e++“s)L N 1 - (a + u,)L.

PI

The term AL/k,a, will be constant for a given sample thickness and chopping frequency and it can be seen from Eqs. [l] and [2] that Q then becomes proportional to a(r - l)(cu + a,) [31 cl”-4 7 which is equal to (1 - i)a cl ~ - 1 [a + (1+ i)aJ [ 2as ’ a2 - (1 + i)“az

1

r43

The algebraic development of the latter expression gives a[(r2(1 - i) - (1 + i)Zaa . (a2 - 2iaf)2as

r51

PHOTOACOUSTIC

SPECTROSCOPY

Dividing both the numerator and denominator by (1 - i) yields (1 + i) a(1 - i) cY2- (1 _ i) 2a:

1

(a2 - 2ia32a,

,

WI

then

(a” - 2iu32a,

m

(1 - i) T-iy

PI

and finally

If the thermal deactivation yield is constant at all wavelengths, it is thus seen that the extrapolated signal is simply proportional to (Y,the optical absorption coefficient, irrespective of the value of a,, the thermal diffusion coefficient. Consequently, the extrapolation procedure may be applied to any type of sample which is not entirely opaque. ACKNOWLEDGMENTS We thank Claire Rousseau for her technical assistance and Alain Tessier for his critical reading of the manuscript. R.C. was supported by a postgraduate scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC). The authors are grateful for financial support from the NSERC and the “Minis&e de 1’Education du Quebec.”

OF Anacystis

niddans,

II

415

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