Photooxidative Damage to the Cyanobacterium Spirulina platensis ... W/m 2) suggested that photodamage to the Spirulina cells was maximum at or beyond the.
CURRENTMICROBIOLOGYVol. 31 (1995), pp. 44-48
Current Microbiology An International Journal 9 Springer-Verlag New York Inc. 1995
Photooxidative Damage to the Cyanobacterium Spirulina platensis Mediated by Singlet Oxygen D.P. Singh, 1 Neeraj Singh, 2 Kavita Verma 2 1Department of Microbiology,Avadh University,Faizabad, 224 001 (U.P.) India 2Department of Microbiology,Barkatullah University,Bhopal 462 026 (M.P.) India
A b s t r a c t . Experiments on the specific growth rate, bleaching of pigments, O2 evolution, lipid
peroxidation, and loss of sulfhydryl ( - S H ) content in response to the varying light intensities (2-28 W / m 2) suggested that photodamage to the Spirulina cells was maximum at or beyond the photosynthesis saturating light intensity (12 W/m2). However, photobleaching of the chlorophyll a was relativeIy higher than [3 carotenoid. The results on the N,N-dimethyl-p-nitrosoaniline (RNO) bleaching in the presence of oxygen radical quenchers exhibited maximum effect of sodium azide and indicated about the generation of singlet oxygen. The chlorophyll a-sensitized production of singlet oxygen by a type II reaction cannot be ruled out because of maximum oxidative damage to the cells at or beyond the photosynthesis saturating light intensity, i.e., 12 W / m 2, when the availability of triplet chlorophyll is maximum.
In tropical countries, the main problem associated with mass cultivativation of Spirulina in an open system is its increased susceptibility to high light irradiance and buoyancy of the cells [1, 4]. Inactivation of photosynthesis under the above high irradiance is known to occur owing to the photodestruction of the photosynthetic apparatus by a variety of photosynthetically generated active oxygen species [2, 6, 20]. It has been suggested that the photoinhibition of PS II or degradation of D2 protein requires the presence of oxygen [10] or photosynthetically reduced oxygen [12]. Generation of superoxide ion is maximum in the presence of high oxygen and low CO2 concentrations [20]. Production of singlet oxygen ('02) is dependent on the availability of triplet sensitizers such as chromophores [15]. Nevertheless, production of both types of oxygen species in the photosynthetic organisms is related to the high light intensity. But the singlet oxygen differs from the superoxide anion with respect to its inability to react with the electron-rich molecules [15]. Application of radical quenchers has proved to be the only way to differentiate the type of oxygen species involved in the oxidative damage. Correspondence to: D.P. Singh
The present investigation on the photodynamic damage to the Spirulina platensis is an attempt to investigate the mode and type of oxygen species invoIved in the oxidative damage to the cells. Materials and Methods
Organism and growth conditions. T h e auxenic culture of Spirulina platensis was grown in Zarrouk's m e d i u m [26]. The cultures were grown in a culture room at a temperature of 24 _+ I~ and were illuminated by cool, white fluorescent tubes with approximate light intensity of 8 W / m 2 on the surface of culture vessels. Otherwise a
range of light intensities (2-28 W/m2) was used for the experimental purpose as and when required. Measurement of light intensity was done with a lux meter (Taiwan) capable of reading from 0 to 50,000 lux and was calibrated in terms of W/m2 for the measurement of light intensity. Measurement of pigments. The cells of Spirulinaincubated under varying light intensities (2-28 W/m2) for 24 h were taken for the measurement of absorption spectra (400-750 nm). The homogenized cell suspension in the phosphate buffer (pH 7.0, 20 mM)was scanned in DU-64, UV-visiblespectrophotometer(Beckman,Switzerland). The quantity of chlorophyll a and 13-carotene was calculated by employingthe formulagivenby Astier et al. [3]. Measurement of oxygen evolution and Hill activity. 02 evolution was measured in clark-type oxygen electrode (Hansatech, UK) fitted with a circulatingwater jacket. The exponentialphase cellsof Spirulinawere harvested and suspended in the sodium phosphate buffer (20 raM, pH 7.0) containing 10 mM of MgC12and 2 mM of
D.P. Singh et al.: Oxygen Damage to
45
S. platensis
nm. The radical quenchers like sodium azide, sodium formate, and histidine (10 mM each) were added to the mixture 10 min prior to the addition of RNO.
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Fig. 1. Effect of varying light intensities (2-28 W/m 2) on the specific growth rate (Ix/h) of the Spirulinaplatensis cells. NaHCO3. The varying light intensities (2-28 W/m 2) were provided by a halogen lamp (1000 watt). A zero oxygen level on the recorder was set by using the solid sodium dithionite. The concentration of oxygen in the air or water was noted down from the given chart. Rate of 02 evolution was calculated in terms of ixmol 02 evolved/nag protein/min. Hill activity in the permeabilized cells of Spimlina was measured by using 25 IxM of DCPIP (2,6 dichlorophenol,1indophenol) [23]. Diphenylcarbazide (DPC; 50 IXM)was used as an electron donor. Rate of Hill activity was expressed in terms of nmol DCPIP reduced/rag protein/min. Preparation of permeaplasts. Permeaplasts of the cells of Spirulina platensis were prepared by using 0.5% (wt/vol) lysozyme. Procedure for the preparation of the permeaplasts was the same as described by Wards and Myers [25] except that the EDTA was omitted from the reaction mixture. There was no measurable release of the phycocyanin pigment from the cells during preparation of the permeaplasts. Measurement of lipid peroxidation. Cells of Spirulina were suspended in the growth medium and were incubated under varying light intensities (2-28 W/m2). Samples were taken out at regular time interval for the measurement of lipid peroxidation. The rate of peroxidation was determined in terms of ~mol malonyldialdehyde (MDA) formed/mg protein/rain by using the method described by Heath and Packer [11]. The absorbance of MDA and thiobarbituric acid (TBA) adduct was read at 532 nm. However, absorbance of the same solution read at 600 nm was deducted from the absorbance recorded at 532 nm in order to correct for the nonspecific turbidity. An extinction coefficient of 155 mmol/em was used to calculate the amount of MDA present in the solution. Estimation of total sulfhydryl content. Total concentration of the sulfhydryl ( - S H ) content in the Spirulina cells was estimated by employing the Ellman's reagent [7]. Absorbance of the reaction mixture read at 410 nm was compared with the standard curve prepared by using the cystein (Sigma, USA). Amount of - S H was calculated in terms of nmol-SH/mg protein. Measurement of RNO bleaching. Rate of RNO (N,N-dimethyl-pnitrosoaniline) bleaching in the permeaplasts of Spirulina cells was measured [14]. The reaction was started by the addition of RNO (300 ixg/ml), and change in the absorbance was monitored at 440
Measurement of chlorophyll a fluorescence induction. Cells of Spirulina were grown at varying light irradiance (2-30 W/m 2) and were harvested in their exponential phase of growth (48 h old). The cells were suspended in Hepes-NaOH buffer (25 raM, pH 7.5) at a chlorophyll a concentration of 5 I~g/ml. The above cell suspension was used to measure the rise and decay kinetics of chlorophyll a fluorescence using a photofluorimeter-151 (Systronics, India) attached to a sensitive recorder (Lloyd, UK); the two broad band (20 raM) Corning filters of 435 nm and 680 nm were used for excitation and emission wavelength, respectively. The filter for the emission wavelengths was applied at a 90~ angle with respect to the excitation source. A shut-off filter was applied to regulate the incident light source (50 watt), which was operated manually. The variable fluorescence (Fv) was calculated by subtracting the value of Fo (original) from Fmax (maximal). Fo was the rise in fluorescence in the absence of DCMU, while Fmaxwas the rise in fluorescence in DCMU-supplemented condition. Measurement of growth rate. Growth of the Spirulina cells was monitored in terms of change in the protein concentration, and the specific growth rate was calculated [16]. Protein concentration was measured by the method of Lowry et al. [18] with the lysozyme as standard. Chemicals. All the chemicals at their analytical grade were obtained from Sigma Chemical Co. (USA), BDH and SRL (India).
Results Effect of light intensity on the growth and pigment content. The results (Fig. 1) showed an intensitydependent increase in the growth rate (0.03 w/h) up to 8 W/m 2 of light intensity. Further increase in the light intensity (from 8 to 28 W/m 2) resulted in a declining pattern in the specific growth rate (0.2 tx/h at 28 W/m2). The results indicated high light-induced photooxidative killing of the Spirulina cells beyond 8 W/m 2 of light intensity. Chlorophyll a and [3 carotenoid contents in the Spirulina cells also showed an intensity-dependent increase up to 8 W/m 2 of light intensity (Fig. 2). Thereafter the pigment level registered a gradual decline with increase in the intensity (from 8 to 28 W/m2). An enhanced sensitivity of the chlorophyll a compared with the [3 carotenoid content was probably responsible for the relatively higher loss of chlorophyll a. Effect of light intensity on the 02 evolution and RNO bleaching. Rate of O2 evolution was measured in the Spirulina cells as a function of different light intensities (Fig. 3). The rate of O2 evolution in the Spirulina cells increased with increase in the light intensity and was maximum (0.3 ~mol O2/mg protein/rain) at 12 W/m 2 of the light intensity. A further increase in the light intensity from 12 to 28 W/m 2 did not alter the rate of 02 evolution.
46
CURRENT MICROBIOLOGYVol. 31 (1995)
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Fig. 2. Effect of varying light intensities (2-28 W/m 2) on the photosynthetic pigments chlorophyll a ( ( 3 - - 0 ) and 13 carotenoid ( 0 - - 0 ) in terms of ~g/ml of Spirulina platensis cells. The cells grown at 8 W / m 2 for 3 days were transferred to varying light intensities for 24 h before the measurement of the pigment level.
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Fig. 4. Effect of varying light intensities (2-28 W/m 2) on the rate of lipid peroxidation (~mol MDA/mg protein/min) ( 9169 and on the sulfhydryl group content (nmol - S H group/mg protein/rain) ( 0 - - 0 ) of the Spirulina platensis cells. The cells of Spirulina were grown at 8 W / m z for 3 days and then transferred to varying light intensities for the measurement of the reaction. The rest of the experimental conditions were the same as described in Materials and Methods.
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