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REMOTE SENSING, 1996, VOL. 17, No.8, 1609-1615

Reflectance spectrophotometry of cyanobacteria within soil crustsa diagnostic tool A. KARNIELI The Remote Sensing Laboratory, J. Blaustein Institute for Desert Research, Ben Gurion University, Sede-Boker Campus 84990, Israel

and V. SARAFIS Center for Biostructural and Biomolecular Research, University of Western Sydney, Hawkesbury, Richmond 2753, NSW, Australia (Received 15 March 1995; in final form 1 December 1995)

Abstract. Identification of cyanobacterial soil crusts is important for mapping apparently barren soils. The letter presents a diagnostic tool for the identification of cyanophyte within soil crusts by means of in vivo reflectance spectrophotometry measurements. Spectral reflectance spectra of the crust samples were measured under several conditions-dry, after wetting, after their phycobilin pigments had been removed, and after the crust had been immersed in paraffin oil. Differences in the reflectance were enhanced by calculating ratio spectra. It is shown in this letter that relative higher reflectivity of the crust in the blue region is caused by the spectral characteristics of the phycobilins. This work is a first step towards mapping biogenic crusts by using airborne or spaceborne sensors which have the capability to detect in the blue band.

1. Introduction Apparently barren desert soils may contain significant microphytic components. An important group of organisms comprising soil crusts in such habitats are cyanobacteria (West 1990). These organisms contribute to soil stability (Danin 1991), soil build up (Shield and Drouet 1962), soil fertility (Zobeck and Fryrear 1986), and to the soil water regime (Verrecchia et al. 1995). The identification of cyanobacteria within microphyte crusts by indirect methods poses a problem. Brock (1973) asserts the difficulty an ecologist would have in identifying a cyanophyte. According to him, the blue green colour is hardly diagnostic. Although Rippka (1988) mentions the phycobilins as major determinants of cyanobacterial colour she also drew attention to some of the difficulties in identifying cyanophytes in nature by their colour alone. In the marine environment some efforts have been recently made to identify a single species of cyanobacteria using satellite sensor data (Borstad et al. 1992, Sathyendranath et al. 1994, Subramaniam and Carpenter 1994). Karnieli and Tsoar (1995) showed that the spectral reflectance of the cyanobacterial crust is lower than that of the active sand dune in the green-, red- and near-infrared spectral bands (500-600, 600-700 and 700-1100 nm, respectively), but somewhat higher in the blue (400-500nm). This phenomenon has been observed in many other spectral measurements of biogenic crusts where cyanobacteria are present, either as free algae or in conjunction with lichens (Karnieli et al. 1995, 0143-1161/96 $12.00 © 1996 Taylor & Francis Ltd


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Stroosnijder and Hoogmoed 1995). In all these cases the reference substrates (either soil or rock without crust) and the crust spectra curves cross each other at about 500 nm. We hypothesized that the relative higher reflectivity of the crust in the blue region is probably caused by the spectral characteristics of cyanophytes. Here we investigate the potential of in vivo reflectance spectrophotometry as a diagnostic tool. The purpose of the experiments was to demonstrate the uniqueness of cyanobacterial crusts. These contain, besides chlorophyll a and assorted carotenoids and xanthophylls, phycobilins. Phycobilins are generally not detectable in higher plants. They are protein pigments containing a linear tetrapyrrol as the chromophore. Removal of the phycobilins (Siegelman and Kycia 1973) was carried out in order to assess their contribution to the total reflectance spectra. Note that this method leaves other pigments in situ. The area under investigation is located in the northern Negev desert along the Israel-Egypt border and will be referred to as the 'Halamish' site. The geographic coordinates are 34°23' E/30057' N, the elevation is 200m above MSL, and the mean annual rainfall is 100 mm. This area was selected because it is characterized by a large spread of cyanobacteria (= blue-green algae) crust as described by Karnieli and Tsoar (1995). The crust consists mostly of Microcoleus »aginatus accompanied with Scytonema, Schizothrix, Calothrix, Chroococcidiopsis, Nostoc, and Phormidium (Danin et al. 1989, Danin 1991). 2. Materials and methods Microphytic crusts from the Halamish site were removed by a spatulate trowel, in order to maintain intactness of the crust. Measurement by reflectance spectrophotometry was carried out using the Li-Cor LI-1800 field-portable spectrometer. In the current research the spectrometer was applied as a laboratory instrument (for more details see Karnieli et al. 1995). It was equipped with a 15° sensing head and using a Tungsten Iodide (Osram) 1000 W lamp as a laboratory light source. Reference calibration was done using a cosine detector under the same conditions. Four samples of each treatment and reference were recorded. Spectra were observed at 1 nm intervals from 400 to 1100 nm and were averaged over four readings with a 90° rotation between readings. Spectra were obtained from active sand dunes from the same region as the crusts. All spectra were read both before and after wetting by distilled water. Wetted samples were recorded immediately after wetting as well as after about 12h which included a period of sunlight illumination. The presence of random noise in the all spectra was minimized using two average filters sequentially, consisting of moving windows of 5 and 3 points successively (Mather 1987, p.245). A reference crust lacking phycobilins was obtained according to a modification of the methods described by Siegelman and Kycia (1973). The method involves taking a crust and inundating it in a 0·05 M potassium phosphate buffer at pH 6·8. The sample was then frozen and thawed twice in the buffer. After thawing, the sample was washed for 15 min in the buffer and then treated in 2 mg ml- I of egg white lysozyme (L6876 Sigma) in the same buffer for 8 h. A phosphate buffer rinse was given for 15 min and the sample was then placed in the fresh lysozyme solution as above for about 2 h. The sample was then frozen and thawed once more and rinsed in distilled water. Reflectance spectra were obtained from the reference crust prior to wetting, after wetting, after the first two freeze thaw cycles and the last freeze thaw cycle to determine the loss of phycobilins. Bowers and Hanks (1965) has already shown that the presence of moisture decreases spectral reflectance as a


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function of wetting. Knipling (1970) also showed that the substantial reduction of refraction index differences in an air filled scattering structure by infiltration of water also decreases reflectance. In order to reduce the scattering in the dry crust samples as much as possible, mineral paraffin oil was used as an immersion medium. 3.

Results Inspection of the surface of the crust by a binocular dissecting microscope showed the presence of cyanobacteria on the surface of the crust. Microscopic examination of the crusts by means a compound microscope confirmed the present of cyanophytes mainly Microcoleus vaginatus with some Nostoc punctiforme being present as well. Figure 1 presents two reflectance spectra, the wetted crust (the reference crust), the phycobilin extracted crust, and the ratio between them. After removing the phycobilins a decrease in the reflectance is noted in the blue region (at about 430 nm). This indicates successful leaching of phycobilin pigments (but not of the chlorophyll pigments and other accessory pigments) and confirms the contribution of the phycobilins to the (blue) colour of the crust. A comparison of the dry and wetted crust is shown in figure 2. The ratio spectrum shows an enhanced reflection in the blue, mainly between 400-500nm, while the absorbance in the red (at about 600 nm) increased. A close view of the natural surface in the Halamish site after rain, demonstrates the difference in colour between dry and wet crusts (figure 3). Danin (1991) observed the same cyanobacterial habitat and noted that after wetting the crust, some phototactic cyanobacteria move out of their sheaths and above the thin layer of soil covering them. A similar phototaxis is known in cyanobacterial mats in other habitats (Gabai 1985, Richardson and Castenholz 1987). This process suggests that upon wetting cyanobacteria from within the crust are exposed to in increased incident radiation. We interpret the reflectance spectrum of the immediately wetted crust as indicative of the improved penetration of light into and out of the crust due to a reduction of scatter. This is because of the closer matching of refractive indices of water, algae and sand. Figure 4 shows the spectra of a crust immediately after wetting (to), 12 h 22.00

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A. Karnieli and V. Sarafis 40.00

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Figure 3. Close view of dry (upper right side) and wet (lower left side) cyanobacterial crusts in Halamish site, western Negev, Israel. afterwards (tl), and their ratio spectrum. Increased reflectance in the blue region accompanied by an increase the absorbance in the red region is noted. Where the dry crust is compared with one immersed in paraffin oil the even closer matching of the refractive index of the oil to that of the sand and dry algae shows an even greater improvement of penetration of light into and out of the crust. Figure 5 demonstrates this effect. Here the crust is compared dry against a crust immersed in paraffin oil of a refractive index more closely matching that of sand and dry algae in the crust.

4. Discussion III vivo spectrophotometry of cyanophytes as parts of microphytic crusts has rarely been carried out. The presence of cyanobacteria may be inferred from the spectral signatures of phycobilin pigments present in these organisms. Other organisms


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Remote Sensing Letters 22.00 20.00

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Figure 5. Spectra of dry cyanobacterial crust, oily crust, and the ratio between them. possessing such pigments are the eucaryotic Cryptophyceae and Rhodophyceae (Bold and Wynne 1985). These algae have not however been reported from microphytic crusts and were not observed in the samples investigated here. Spectroscopy of intact cyanophytes in transmission was studied by a number of authors (Hattori and Fujita 1959 a.b, Haxo 1960, Jones and Myers 1965). Absorption peaks due to phycoerythrin at -580nm and at -630nm due to phycocyanin were discernible. A preponderance of phycocyanin is the main contributor to the bluish hue noted by us for the crusts described above. We thus interpret (in our data) the blue reflectance maximum at - 430 nm as support for the use of reflectance spectrophotometry in vivo for the diagnosis of cyanophyte crusts. The peak broadening noticed by Duysens (1956) and by Fukshansky (1981) due to the distribution error introduced by spatial non-uniformity of absorbers (known as the sieve effect) was not sufficient to change any of the conclusions in this study. The spectral change in the blue reflectance upon phycobilin extraction in figure 1 serves to confirm our


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A. Karnieli and V. Sarafis

inference as to the characteristic reflectance of phycobilins since the chlorophyll absorbance at ~ 685 nm remains virtually unchanged. The spectral data presented here demonstrate the applicability of ratio reflectance spectrophotometry as may be used in imaging spectrometry to selectively reveal the presence of the cyanobacteria microphyte crusts. An extension of the methodology to active reflectance spectrophotofluorimetry may improve the selective detection of cyanobacteria crusts. The use of DCMU as a poison of the electron transport chain in cyanobacteria (Black et al. 1963, Duane et al. 1965, Hoare et al. 1967) should enhance the sensitivity further as shown by Lichtenthaler and Rinderle (1988). Andersson et al. (1994) used imaging spectrofluorimetry of higher plant leaves to study the effects of DCMU as a photosynthetic poison as a further development of photosynthetic poison studies and demonstrated the expected enhancement of fluorescence at 685 nm. In conclusion, it is asserted here that cyanobacterial soil crusts can be identified from reflectance data and may serve for ground truth verification of remote sensed data. The results of this study can be used for biogenic crust mapping by using airborne or spaceborne sensors and can be applied by the numbering of current and future satellite sensors such as Landsat TM, the VEGETATION instrument, MOMS, SeaWiFS, MODIS and others. All have the capability to detect reflectance data in the blue region.

Acknowledgment This research was supported by the Israel Science Foundation administrated by the Israel Academy of Science and Humanities. We would like to thank Dr. Inka Dor of the Hebrew University, Jerusalem for determining the cyanobacteria.

References ANDERSSON, M., EDNER, H., JOHANSSON, J .. RAGNARSON, P., SVANBERG, S., and WALLINDER, E., 1994, Remote monitoring of vegetation by spectral measurements and multi-colour fluorescence imaging. Physical Measurements and Signatures in Remote Sensing. Proceedings of the Sixth International Symposium held in Val d'lsere. France, on 17-21 January, 1994 (Paris: CNES), pp. 835-842. ' BLACK, C C, FEWSON, C A., and GIBBS, M., 1963, Photochemical reduction of triphosphopyridine nucleotide by cell-free extracts of blue-green algae. Nature, 198, 88. BOLD, H. C, and WYNNE, J. W., 1985, Introduction to Algae: Structure and reproduction (Englewood Cliffs, NJ: Prentice-Hall, Inc.), 706 p. BORSTAD, G. A., GOWER, J. F. R., CARPENTER, E. J., and REUTER, J. G., 1992, Development of algorithms for remote sensing of marine Trichodesmium blooms. In Marine Pelagic Cyanobacteria: Trichodesmium and other Diazotrophs, edited by E. J. Carpenter, D. G. Capone and J. G. Reuter (Dordrecht: Kluwer Academic Publishers), pp. 193-210, BOWERS, S. A., and HANKS, R. J., 1965, Reflectance of radiant energy from soils. Soil Science, 100, 130-138. BROCK, T. D., 1973, Evaluation and ecological aspects of the cyanophytes. In The Biology of Blue-Green Algae, edited by N. G. Carr and B. A. Whitton (Oxford: Blackwell Scientific Publications), pp.487-500. DANtN, A., 1991, Plant adaptation in desert dunes. Journal of Arid Environment, 21, 193-212. DANIN, A., BAR-OR, Y., DOR, I., and YISRAELI, T., 1989, The role of cyanobacteria in stabilization of sand dunes in southern Israel. Ecologia Mediterranea, 15, 55-64. DUANE, W. C, HOHL, SR. M. C, and KROGMANN, D. W., 1965, Photophosphorylation activity in cell-free preparations of a blue-green alga. Biochimica Biophysica Acta, 109, 108-116. DUYSENS, L. N.' ,M., 1956, The flattening of the absorption spectrum of suspensions as compered to that of solutions, Biochimica Biophysica Acta, 19, 1-12,


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FUKSHANSKY, L., 1981, Optical properties of plants. In Plants and the Day Light Spectrum, edited by H. Smith (London: Academic Press), pp. 21-40. GABAI, V. L., 1985, A one-instant mechanism of phototaxis in the cyanobacterium Phormidil/m unicinatll/n. Federation of European Microbiological Societies Microbiology Letters, 30, 125-129. HATTORI, A., and FUJITA, Y., 1959 a, Formation of phycobilin pigments in blue-green alga Tolypothrix tenuis as induced by illumination with colored lights. Journal of Biochemistry, Tokyo, 46, 521-524. HATTORI, A., and FUJITA, Y., 1959 b, Crystalline phycobilin chromoprotein obtained from a blue-green alga. Journal of Biochemistry. Tokyo, 46, 633-644. HAXo, F. T., 1960, The wavelength dependence of photosynthesis and the role of accessory pigments. In Comparative Biochemistry of Photoreactioe Systems, edited by M. B. Allen (New York: Academic Press), pp. 339-359. HOARE, D. S., HOARE, S. L., and MOORE, R. B., 1967, The photoassimilation of organic compounds by autotrophic blue-green algae. Journal of General Microbiology, 49, 351-370. JONES, L. W., and MYERS, J., 1965, Pigment variations in Allacystics nidulans induced by light of selected wavelengths. Journal of Phycology, I, 7-14. KARNIELI, A., SHACHAK, M., TSOAR, H., ZAADY, E., KAUFMAN, Y., and PORTER, W., 1995, The effect of microphytes on the spectral reflectance of vegetation in semi-arid regions. Remote Sensing of Environmellt, (in press). KARNIELI, A., and TSOAR, H., 1995, Spectral reflectance of biogenic crust developed on deseret dune sand along the Israel-Egypt border. Illternariollal Journal of Remote Sensing, 16, 369-374. KNIPLING, E. B., 1970, Physical and physiological basis for the reflectance of visible and nearinfrared radiation from vegetation. Remote Sensing of Environment, I, 155-159. LICHTENTHALER, H. K., and RINDERLE, U., 1988, The role of chlorophyl fluorescence in the detection of stress conditions in plants. CRC Critical Reviews ill Analytical Chemistry, 19, S29-S85. MATHER, P. M., 1987, Computer Processing of Remotely Sensed Images (Chichester: John Wiley & Sons). RICHARDSON, L. L., and CASTENHOLZ, R. W., 1987, Diel vertical movements of the cyanobacterium Oscillatoria terebriformis in a sulfide-rich hot spring microbial mat. Applied and Enoironmental Microbiology, 53, 2142-2150. RIPPKA, R., 1988, Recognition and identification of Cyanobacteria. In Methods of Enzymology. Vol. 167. Cyanobacteria, edited by L. Packer and A. N. Glazer (San Diego: Academic Press, Inc.), pp. 28-93. SHIELDS, L. M., and DROUET, F" 1962, Distributions of terrestrial algae within the Nevada test site. American Journal of Botany, 48, 547-554. SIEGELMAN, H. W., and KYCIA, J. H., 1973, Algal biliproteins. In Handbook of Phycological Methods Physiological Methods and Biochemical Methods, edited by J. A. Hellebust and J. S. Craigie (Cambridge: Cambridge University Press), pp. 71-79. STROOSNIJDER, L., and HOOGMOED, W., 1995. Physical measurements on degraded watersheds under silvo-pastoral land use. Proceedings of the lnternational Symposium on Remote Sensing and GIS as Tools for Monitoring Soils in the Enoironment, held in Ouagadougou, Burkina Faso, SUBRAMANIAM, A., and CARPENTER, E. J., 1994, An empirically derived protocol for detection of blooms of the marine cyanobacterium Trichodesmium using CZCS imagery. International Journal of Remote Sensing, IS, 1559-1569. VERRECCHIA, E., YAIR, A., KIDRON, G. J., and VERRECCHIA, K., 1995, Physical properties of the psammophile cryptogamic crust and their consequences to water regime of sandy soils, north-western Negev Desert, Israel. Journal of Arid Environmellts, 29, 427-437. WEST, N. E., 1990, Structure and function of microphytic soil crusts in wildland ecosystems of arid to semi-arid regions. Advances in Ecological Research, 20, 179-223. ZOBECK, T. M., and FRYREAR, D. W., 1986, Chemical and physical characteristics of windblown sediments II. Chemical characteristics and total soil and nutrient discharge. Transactions of American Society of Agriculture Engineering, 29, 1037-1041.