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Studies on growth and Phycobilin pigments of the Cyanobacterium Westiellopsis iyengarii.

Introduction

Cyanobacteria are the oxygenic photosynthetic prokaryotes that occur worldwide in a range of terrestrial, fresh water and marine environment [1]. Cyanobacteria are morphologically diverse and they consist of unicellular, colonial, pseudofilamentous, pseudoparenchymatous uniseriate, filamentous forms unbranched, undifferentiated, differentiated with specialized cells called heterocyst and akinetes and heterotrichous branched forms [2]. Cyanobacteria, like higher plants and eukaryotic algae, perform photosynthesis with the help of two photosystems known as photosystem II and photosystem I. In cyanobacteria the major light harvesting antenna consists of a supra-molecular protein-chromophore complex known as phycobilisome, which is anchored onto the cyanobacterial thylakoid membrane from the cytoplasm. For an optimal performance of photosynthesis, not only the photosystems (PS II and PS I) have to be coordinated, but the accessory light harvesting complex too has to be coordinated with the photo systems.

Generally cyanobacteria have the pigments chlorophyll a, carotenes and phycobillins. The absorption of light energy by cyanobacteria is based upon the occurrence of one or two forms of chlorophylls together with carotenes and phycobilins. Chlorophylls are the pivotal photosynthetic pigments. Chlorophyll a and chlorophyll b are the major chlorophylls present in the thylakoids of cyanobacteria. Carotenoid found in cyanobacteria includes variety of xanthophylls and [beta] carotene. These pigments are located in thylakoids alongside with chlorophylls.

Phycobilisome is composed of chromophoric and non-chromophoric proteins arranged in a definite order to maximize energy trapping and eventually channeling it to PSII reaction center. The chromophoric proteins are those attached with linear tetrapyrroles and are called phycobiliproteins, while non-chromophoric proteins are called as colorless linker polypeptides. The three major phycobiliproteins commonly found in cyanobacterial phycobilisomes are; Phycocyanin (PC), Allophycocyanin (APC) and Phycoerythrin (PE). Cyanobacteria that do not produce chlorophyll b typically possess water-soluble phycobilin pigments. Early studies on cyanobacterial pigments have shown that, light qualities alter the relative levels of phycobiliproteins. In the natural environment, quantity of the photsynthetically active radiation (PAR) changes from time to time. Thus, it becomes necessary to study the effects that the light intensity can bring about, changes on the cyanobacterial light harvesting accessory pigments.

Phycobiliproteins produced by Cyanobacteria are used in the food, drug, and cosmetic industries as natural coloring agents. They are used in clinical diagnostics as fluorescent reagents. Phycofluors primarily Phycoerytherin and Allophycocyanin are used in fluorescence-activated cell sorting, flow cytometric analysis and histochemistry. Screening of additional cyanobacteria for the presence of biliproteins with desirable characteristics likely will result in additional discoveries of this nature [1,3].

Cyanobacteria now find wide use in agriculture and hold great promise in the field of bioremediation. They are used as producers of single cell proteins, biofertilizers, animal feed and biocontrol agents. Cyanobacteria, especially those capable of diazotrophic growth offer distinct advantages as potential biodegradation organisms, since their survival is not dependent on the presence of high concentration of organic compounds. Bioaccumulation of metals by cyanobacteria is used for removal of metal ions from polluted water. Cyanobacteria have some medicinal effects such as anticancer, antibacterial and antifungal activity. Cyanobacteria are one of the most promising groups of microorganisms finding new bioactive natural products. This present study was aimed to find the differences in protein and pigment of Westiellopsis iyengarii which was first isolated by Madras University botany laboratory, under various cultural and nutritional conditions.

Materials and Methods

Cyanobacterial strain

Westiellopsis iyengarii was obtained from the Center for Advanced study, University of Madras, Chennai.

Growth conditions

Westiellopsis iyengarii is a heterocystous blue-green alga. Its stock clonal axenic culture has been maintained on agar slants (1% v/v) at 28 [+ or -] 2[degrees]C, illuminated with 1500 lux of incandescent light, culture was grown and maintained in B[G.sup.11] medium [4] has been used as the basal medium in all experiments.

The pH should be 7.1 after sterilization

Culture conditions

Culture was grown and maintained at 25 [+ or -] [degrees]C under fluorescent illumination of 15500 luxs. Only liquid cultures were used throughout the investigation. Clumping of cells was reduced by gentle shaking of the flasks manually once daily. The experiments were grown under different light intensities (Low, Medium and High ie 10, 20 and 40 [micro][Em.sup.-2][S.sup.-1] respectively) and different light qualities (Blue, Green and Red). The exponential phase 18th day cultures were harvested and used for the all the experiments.

Extraction of protein from culture

Culture grown under different experimental conditions was centrifuged and the pellet was washed with Tris-HCl buffer (50 mM pH 7.5). Then the pellet was resuspended in 5 mL of the same buffer and was kept in deep freezer. Cells were thawed and sonicated in Labsonic 2000 sonicator until the cells were completely. This sonicated cell were centrifuged for 15 minutes and the clear supernatant was carefully removed and used for the protein estimation

Protein determination

Protein concentration in samples was determined by the method of Bradford [5] using bovine serum albumin as a standard

Estimation of Chlorophyll a

Chlorophyll a was estimated by the method of Mackiney [6]. Harvested cells from each treatment were suspended in 1 mL of distilled water. Then 4 mL of methanol was added and the tubes were incubated in a water bath at 60[degrees]C for 30 min. The tubes were then centrifuged for 10 min and the supernatant was used to read the absorbance at 663 nm.

Estimation of the phycobillin pigments

Phycobiliproteins were extracted by subjecting the cells to mild sonication in phosphate buffer and repeated freezing and thawing; the amounts of Phycocyanin (PC), Allophycocyanin (AC) and Phycoerythrin (PE), absorption maxima of the supernatant were determined spectrometrically and a quantitative estimation was done according to Bennett and Bogorad [7].

Results

Westiellopsis iyengarii, a Filaments with true branching; made of prostrate and erect systems, prostrate system multiseriate, erect system uniseriate; branches short, arising on both the sides: individual sheath around filaments absent; heterocysts intercalary, quadrate or cylindrical, was investigated for its protein and pigment contents at a rangeof different light intensities (low, medium and high) and qualities (Blue, Green and Red). Under both conditions the influence of nitrogen was also studied.

Growth characteristics of Westiellopsis iyengarii at different light intensities and light qualities

Growth characteristics of Westiellopsis iyengarii grown in BG11 medium with and without nitrogen under different light intensities (10, 20 and 40 [micro][Em.sup.-2] [S.sup.-1]). The organism showed very good growth in medium light intensities and poor growth at very low and high light intensities. Maximum chlorophyll a content (0.9 mg/mL) was attained by culture grown under medium light intensity (40 [micro][Em.sup.-2][S.sup.-1]) in nitrogen amended medium (Fig-1). Chlorophyll a obtained in the highest light intensity (40 [micro][Em.sup.-2][S.sup.-1]) was maximum the cultures were grown in nitrogen amended medium (0.12 mg/mL). The chlorophyll a content of cultures in low light intensity was less as 0.06 mg/mL in both medium with and without nitrogen amended medium. W. iyengarii studied under different qualities of light (Blue, Green and Red). Culture in blue light showed maximum amount of chlorophyll a than the other light qualities. The chlorophyll content of W. iyengarii in blue light was 0.013 mg/mL (with nitrogen) and 0.12 mg/mL (without nitrogen). But under the same light intensity the absence of nitrogen source Chl a amount was 0.83 mg/mL. Chl a synthesis under green filter condition in nitrogen amended medium cells showed 0.07 mg/mL and in the absence of nitrogen it was 0.5 mg/mL red filter maximum Chl a of .08 mg/mL was obtained in the presence of nitrogen (Fig-2).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Protein content of W. iyengarii at dfferent light intensities

Protein content of W. iyengarii was estimated from 18th day culture. Maximum amount of protein was obtained from the cultures were grown under medium light intensity. Moreover, the cultures grown on nitrogen supplemented medium was found to have more protein when compared with cultures grown under medium light intensities was 820 [micro]g/mL (with nitrogen) and 480 [micro]g/mL (without nitrogen) (Fig-3). This is merely twice the amount as estimated in cultures grown under high light intensities. When compared with cultures grown under medium and high light intensities. The protein content of cultures grown under low light intensity was very less. This indicates that the medium light intensity and nitrogen supplemented medium greatly increases the protein content of the test isolate.

[FIGURE 3 OMITTED]

Protein content of W. iyengarii at different light qualities

W. iyengarii was studied under different qualities of light (blue, green and red). The culture had maximum level of protein (440 [micro]g/mL) in blue light with nitrogen supplemented medium (Fig-4). Secondly the higher protein content was obtained in red light 400 [micro]g/mL (with nitrogen) and 290 [micro]g/mL (without nitrogen). While comparing the three light qualities green filter grown cultures showed very less protein content

[FIGURE 4 OMITTED]

Phycobillin pigments at different light intensities and qualities.

At medium light intensity all three pigments was high especially in the nitrogen supplemented medium (PC 0.005 mg/mL, APC 0.0089 mg/mL and PE 0.0039 mg/mL). in the high light intensity also Phycobillin pigment synthesis were high in the presence of nitrogen PC 0.004 mg/mL, APC 0.003 mg/mL and PE 0.002mg/mL. while comparing the three light intensities pigment was less in low light intensity (Fig-5).

A Phycocyanin content of W. iyengarii was highest in red light condition (0.005 mg/mL) and the Phycocyanin pigment was totally absent in green light both in the presence and absence of nitrogen medium. In blue light the cultures grown in medium with nitrogen shoed higher Phycocyanin content 0.004 mg/mL than in the medium without nitrogen. Allophycocyanin level was maximum at red light 0.005 mg/mL than in blue light 0.003 mg/mL (Fig-6). Phycoerythrin content was highest in blue light (0.002mg/mL) than in red light condition (0.0006 mg/mL) in blue light and red light. Phycoerythrin content was high in cultures grown in the medium with nitrogen. No phycoerythrin content was observed in green light condition. All the three Phycobillin pigments were absent in green light condition

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

Discussion

The influence of light intensity on Phycobilisome content has been reported in several organisms [8-10],. Changes in light intensities caused dramatic changes in the synthesis of Chl-a and protein. In W. iyengarii Chl-a and protein content was high in medium light condition 20 [micro][m.sup.-2][s.sup.-1]. This reflects the adaptation of the organism to that light intensity. Similar results were reported by [10,11]. The results suggest that a diverse, Physiological adaptation by the organism leads to increased protein synthesis. Photochemical activity of PS II of medium light adapted W. iyengarii cells increased upon illumination of the cells in Phycocyanin and Allophycocyanin absorption region compared to that of low and high light adapted cells. Organisms grown in the presence of nitrate source showed enhancement in the growth compared to nitrate absent medium. Which clearly indicate the essential of nitrate source for the growth of the organism.

Better growth rate of W. iyengarii in red light than blue and green light was probably due to the higher photosynthetic efficiency and quantum yield in the red light grown cells. Chl-a concentration was high in blue filter grown cells as reported earlier [12]. Significant production was observed in cultures grown under blue light [13]. Found among the Synechococcus strains growth was greatest under red and white light but significantly less under green light condition. Enhancement of PE production by W. iyengarii was observed in blue light than red light probably due to an acclimation phenomenon at low irradiance.

Less PE synthesis resulted in decreased growth under green light but blue light stimulated the accumulation of pigments like Chl-a, PE and PC [12]. PC and APC were obviously high in red light grown cells than blue light. Blue light enhance PE was reported by Hauschild et al [13] in marine Synechococcus sp. Higher production of protein in blue light compared to Red light indicated the well known enhancement of protein production by blue light in the presence of nitrated and its assimilation is similar to that observed in shade condition in green micro algae [14]. However the chlorophyll a and protein content was high in blue light but the phycobilipigment was high under red light reflecting the necessity of high wavelength for the Phycobilin pigment synthesis especially PC and APC. Because PE was as high in blue light than red light shows the need of low light for its synthesis. The W. iyengarii showed growth in green filter but the Phycobilin pigment content was totally nil which reflects the organism prefers light of 20 [micro]E [m.sup.-2][s.sup.-1], low wage length suppressed the color of the Phycobilin pigment. These results suggest that light quality, and quantity; through its effect on growth rate, may be an important factor controlling the distribution and abundance of the various pigments type of W. iyengarii.

References

[1] Patterson, G. M. L., 1995, Biotechnological Applications of Cyanobacteria, Journal of Scientific and Industrial Research, 55, pp. 669-684.

[2] Desikachary, T. V, 1959, Cyanophyta. Indian council of Agricultural Research, New Delhi, India, pp. 686.

[3] Graham, Linda, E, and Wilcox, L. W., 2000, Algae. Prentice Hall: Upper Saddle River, NJ, pp. 97-131.

[4] Rippka, R., Deruellss, J.R., Waterbury, M., Herdman, and Stanier, R. Y., 1979, Generic assignments, strain histories and properties of pure cultures of cyanobacteria, J. Gen. Microbiol, 111, 1-61.

[5] Bradford, M.M., 1976, A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem, 72, 248-54.

[6] MacKinney, G., 1941, Absorption of light by chlorophyll solution, J. Biol. Chem, 140, 315-322.

[7] Bennett, A., and Bogorad, L., 1973, Complementary chromatic adaptation in a filamentous blue-green alga, J Cell Biol, 58, 419-435.

[8] Yamanaka, G., Glazet, A.N., 1980, Dynamic aspects of phycobillisoma structure phycobillisome turn over during nitrogen starvation in Synechococcus , Arch. Microbiol, 124, 39-47.

[9] Granier, F., Dabacq, J. P., and Thomas, J. T., 1994, Evidence for the transient association of new proteins with the Spirulina maxima phycobilisome in relation to light intensity, Plant Physiol, 160, 747-754.

[10] Nomsawai, P., Tandeau, de Marsac, N., Thomas, J.C., Tanticharoen, M., and Cheevadhanarak, S., 1999, Light regulation of phycobilisome structure and gene expression in Spirulina platensis C1 (Arthrospira sp. PCC 9438), Plant Cell Physiol, 40, 1194-1202.

[11] Parameswaran, P., 1993, Light responses and physiological characterization of cyanobacteria (blue-green algae) from rice fields, Ph.D., thesis, University of Madras.

[12] Figueroa, F. L., Jimenez, C., Vergara, J.J., Robles, M.D., and Niell, F. X., 1995, Growth, pigment synthesis and nitrogen assimilation in the red alga Porphyra sp. (Bangiales, Rhodophyta) under blue and red light, Science, 59, 9-20.

[13] Hauschild, C. A., McMurter, H. J. G., and Pick, F. R., 1991. Effect of spectral quality on growth and pigmentation of picocyanobacteria, J. Phycol, 27, 698-702.

[14] Senge, M., Senger, H., 1991, Adaptation of photosynthetic apparatus of Chlorella and Ankistrodesmas to blue and red light, Bot. Acta, 104, 39-143.

Periyasamy Ashokkumar (1) * and Narayanaswamy Anand (2)

(1) * Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 600 025, India E. Mail: ashokkumarps@yahoo.co.in

(2) Vice-Chancellor, VELS University, VelanNagar, PV Vaithiyalingam Road, Pallavaram, Chennai 600 117, India
NaN[O.sup.3] 1.5 g
[K.sup.2]HP[O.sup.4] 0.04 g
MgS[O.sup.4] x 7[H.sub.2]O 0.075 g
Ca[Cl.sub.2] x 2[H.sub.2]O 0.036 g
Citric acid 0.006 g
Ferric ammonium citrate 0.006 g
EDTA (disodium salt) 0.001 g
NaC[O.sub.3] 0.02 g
Trace metal mix 1.0 mL
Agar (if needed) 10.0 g
Distilled water 1.0 L

Trace metal mix:

[H.sub.3]B[O.sub.3] 2.86 g
Mn[Cl.sub.2] x 4[H.sub.2]O 1.81 g
ZnS[O.sub.4] x 7[H.sub.2]O 0.222 g
NaMo[O.sub.4] x 2[H.sub.2]O 0.39 g
CuS[O.sub.4] x 5[H.sub.2]O 0.079 g
Co(N[O.sub.3])2 x 6[H.sub.2]O 49.4 mg
Distilled water 1.0 L
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Author:Ashokkumar, Periyasamy; Anand, Narayanaswamy
Publication:International Journal of Biotechnology & Biochemistry
Date:May 1, 2010
Words:2752
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