Biomacromolecules 2002, 3, 783-786
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Thermal Characteristics of Spirulina platensis Cells under Nongrowing Conditions at Various Values of pH Medium J. Monaselidze,* Sh. Barbakadze, Sh. Kvirikashvili, G. Majagaladze, D. Khachidze, and L. Topchishvili Institute of Physics of the Georgian Academy of Sciences, 380077 Tbilisi, Georgia Received February 8, 2002; Revised Manuscript Received April 26, 2002
The total value of heat (-Q) evolved by green-blue microalgae Spirulina platensis cells in a dark and stationary regime in the range of pH values 8.0-11.6 was determined. It was established that (-Q) reaches its maximum value at 360 ( 40 J/g of dry biomass in the pH range 9.3-10.3 and then sharply dropped relative to these values and reached zero at pH 7.5 ( 0.2 and 11.8 ( 0.2. It is affirmed that an optimum regime for preservation of Spirulina platensis cell viability in a dark and stationary regime is pH range 9.3-10.3. It was also shown that the peak of heat evolution with maximum about 45 °C, reflecting mainly the respiration of cells (oxygen absorption rate),3 did not displace along the temperature scale at a change of pH from 9.3 to 10.4 and slightly displaced lower and higher of these values of pH. It is supposed that the thermostability of biomacromolecules and their complexes responsible for cell respiration does not depend on pH medium in pH range 9.3-10.3. Introduction Fine works concerning the growth,1,2 physiology and biochemistry3 of blue-green microalgae Spirulina platensis, and also a role of this unique alga in water purification from various pollutants have been published for the past decades.4 A detailed analysis of dependences of photosynthetic answer on radiation, photosynthetic activity and growth rate on temperature,5 and also the influence of neutral salt concentration on cell growth in laboratory and outdoor conditions is given in this work. But, as far as we know, there are no data concerning the influence of pH medium on so important a parameter of S. platensis cell vital functions as heat production. Heat evolved by a living system is quantitatively connected with the sum of its metabolic processes. Therefore heat power or heat production rate is a useful parameter of quantitative characteristic of “biological activity”, and such studies were carried out on blood cells and on cells of cultivated tissues with the help of isothermic calorimetry.6-8 We show the possibility of S. platensis cell total heat production registration at pH 9.85 provided photosynthesis contribution close to zero.9-11 It enables us the possibility to question the influence of pH medium on S. platensis cell heat production for deeper understanding of physicochemical processes connected with vital functions of S. platensis cells. Materials and Methods Cellular Culture. The Institute of Plant Physiology, Russian Academy of Sciences, kindly granted strain IPPA B256. S. platensis cultivation was carried out on a special * To whom correspondence may be addressed. E-mail: mon@ iph.hepi.edu.ge.
Figure 1. Dependence of growth rate of native S. platenis on lightening spectrum of DNAT lamp (intensivity 4 klx).
photo-bioreactor created in the Institute of Physics, Georgian Academy of Sciences.12 S. platensis had been mixed for 30 min hourly at 30-34° C, under an illumination of 4 klx (Figure 1). An eight-day culture of S. platensis was used in experiments. S. platensis was grown in Zarrouk’s nutrient medium13 at pH 9.3-11.6 and at pH 8.0 in mineral medium (mineral water “Borjomi”) with content of 80 mg/L CaCl2, 1300 mg/L NaCl, 2000 mg/L KCl, 40 mg/L MgCl2, 4200 mg/L KCO3, and 8 mg/L SO4. Electrophoresis of S. platensis cells in 10% polyacrilamid gel PAGE at pH 10.0 and 11.55 was carried out in a Leammly system (Figure 2). The mix of marker proteins with molecular weight from 25 000 to 67 000 was used. Preparations of a firm SIGMA were used for electrophoresis. Micrographs of S. platensis cells were obtained by fluorescence microscopy (Saint-Petersburg, Luman U-2). The excitation of wavelength is 436 nm. The cells were gathered by centrifugation in Zarrouk’s medium at 100g during 5 min; the sediment was diluted with Zarrouk’s medium to a concentration 1.5-2% of dry biomass and then it was put into a calorimetric vessel. The increase of pH was reached spontaneously and with small steps
10.1021/bm025521o CCC: $22.00 © 2002 American Chemical Society Published on Web 07/08/2002
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Figure 2. Polyacrylamide gel electrophoresis of Sp. platensis cells at pH 9.3 (a), 10.05 (b), and 11.55 (c).
∼0.3-0.5 to keep the S. platensis suspension solution (pH 10.2) at 28-30 °C during 24 h in transparent retorts at illumination of 4 klx without nutrient medium. Dry weight of biomass was determined at 105 °C and weight of ash at 450 °C. Calorimetric Investigations. The calorimetric measurements were carried out on a differential scanning microcalorimeter (DSC)11,14,15 with sensitivity 10-7 cal/s; the value of measuring vessel was 0.30 mL. In the regime close to the isothermic one, the sensitivity is 2 × 10-8 cal/s. The change of fixed temperature (35 °C) is not more than 0.15 °C during 12 h.
Figure 3. Spirulina platensis filaments under fluorescent microscope in Zarrouk medium, pH 11.6: a, own fluorescence; b, ocidin orange (OO) 10 mg/mL (magnification 960×).
Results and Discussion Figure 4 shows the normalized curves of heat evolution of S. platensis cells at various values of pH medium. The curves show that the process begins about 5 °C and finishes at 52 °C. The curve profiles, in all cases, are characterized with an intensive maximum about 45 °C and shoulders about 25 and 38 °C, the intensity of which depends on pH. Total heat evolved in the temperature range 5-52 °C at a heating rate 7.0 °C/h, which was calculated from the area between baseline and dependence curve dQ/dT ) f(T) turned out to be dependent on pH medium. It was found out that the temperature of the main maximum (Tmax) was also dependent on pH medium. These results are summarized in Figures 5 and 6. It should be noted that in these experiments, a heating rate of 7.0 °C/h was chosen because the total value of heat evolution was not changed at pH values from 8.0 up to 11.5 at this and lower heating rates. As seen from Figure 5 Q ) f(pH) dependence has a bell-like form with a plateau on the top covering pH range 9.3-10.4. In this range of pH, heat evolved in the temperature range 5-52 °C equals 360 ( 40 J/g. -Q sharply drops at increase or decrease of pH, and at values 11.6 and 7.8 it is equal to 108 ( 15 J/g of dry biomass, accordingly. Extrapolation of -Q ) f(pH) dependence curve to zero values of -Q gives the values of pH 7.4 and 11.8, accordingly. This means that S. platensis cells stop respiring, their vital functions in a stationary regime, nonaerobic conditions and in dark (studies are carried out in a closed opaque vessel) are ceased.
Figure 4. Heat evolution curves of S. platenis cells in Zarrouk’s medium, calculated per gram of dry biomass at various values of pH medium: 1, pH 10.0; 2, 10.6; 3, 8.0. Scanning rate was 0.166 °C/ min.
As mentioned above, the heat production of eucaryotic cells and microorganisms is usually determined by isothermic calorimetry;6-8 therefore we decided to carry out similar measurements but with the help of a differential scanning microcalorimeter which can decrease rate so much that it makes it possible to carry out measurements in the isothermic regime. For determination of heat production at fixed temperature, the cell suspension was first scanned at a rate of 50 °C/h up to 35 °C and then the calorimeter was switched over to the regime close to isothermic one. In this regime the fixed temperature (35.05 °C) has been changed for 8 h not more than 0.1 °C. After the switch of calorimeter over the regime close to isothermic one, the system equilibrium is reached in 4-5 min, which leads to minimum loss of heat evolved
Thermal Characteristics of Spirulina platensis Cells
Figure 5. Influence of pH on heat evolution by S. platenis cells in Zarrouk’s medium: b, scanning regime; O, isothermic regime.
Figure 6. Influence of pH on temperature of maximum (Tmax) of main peak of heat evolution S. platenis cells in Zarrouk’s medium.
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-dQ/dT ) f(T) on elementary Caussian constituents is given. Thus, it is shown that the optimum regime of S. platensis cell vital functions is pH range 9.3-10.3 on the basis of S. platensis cell heat production measurements by two different microcalorimetric approaches. According to previous work,1 a 90% decrease of oxygen absorption by S. platensis cells in the dark and in a stationary regime is observed just in the temperature range 46-50 °C. This effect is interpreted by authors as a result of protein denaturation responsible for cell respiration. An analogous conclusion came also from the authors of ref 16 on the basis of a sharp decrease of respiration, growth rate, and heat production of Saccharomyces cereVisiae in nonaerobic conditions in the temperature range 36-39 °C. Therefore, the independence of Tmax of pH from 9.3 to 10.3 and weak dependence on the lower and higher values (Figure 6) obviously says that the proteins responsible for cell respiration are weakly sensitive to pH. It was of interest to find out if the changes occurred in protein composition of cells and in morphology of trichomes and cells of S. platensis in the pH range 7.8-11.6. For this we conducted electrophoresis and microcalorimetric studies. Figure 2 shows that high values of pH (e.g., pH 11.55) do not cause significant changes in protein composition of S. platensis cells, and micrographs presented in Figure 3 illustrate the identity of morphology of trichomes and cells that coincides with data obtained earlier in work17,18 at pH 10.0. Thus we conclude that an optimum regime for preservation of S. platensis cells viability in Zarrouk’s medium in stationary regime and in dark is pH range 9.4-10.3; thermostability of biomacromolecules and their complexes responsible for cell respiration weakly depends on pH; the morphology of trichomes, cells, and protein composition of S. platensis cells does not change in pH range 8.0-11.55. Acknowledgment. The work is supported by ISTC Project G-342.
Figure 7. Heat evolution curves as time function of S. platenis cells in Zarrouk medium: 1, pH 9.3; 2, pH 10.6; 3, pH 11.55.
by S. platensis cells. Actually, as seen from Figure 5, the heat evolution curves practically coincide with the baseline in 10-15 min. It should be noted that in a given case the value measured is a rate of heat evolution dq/dt. The integration of heat evolution curve gives the total quantity of heat (Qt) evolved in time t Qt )
dt ∫0t dq dt
The values of Qt obtained at different pH values are given in Figure 5. As can be seen, the profiles of dependences -Q ) f(pH) and -Qint ) f(pH) coincide with each other. A small difference in Qt and Qint values is connected with the fact that 12-20% of heat evolved in the temperature range 5-35% at scanning of a S. platensis suspension depends on scanning rate (ref 11). This value may be calculated from the curves presented in Figure 4 (see also work []) in which the deconvolution of a S. platensis suspension dependence
References and Notes (1) Vonshak, A. Spirulina: Growth, Physiology and Biochemistry. In Spirulina platensis (Arthrospira): Physiology, Cell-Biology and Biotechnology; Vonshak, A., Ed.; Taylor and Francis Ltd.: Basingstoke, U.K., 1997. (2) Vonshak, A. Outdoor Mass production of Spirulina: The basic Concept. In Spirulina platensis (Arthrospira): Physiology, CellBiology and Biotechnology; Vonshak, A., Ed.; Taylor and Francis Ltd.: Basingstoke, U.K., 1997; pp 79-99. (3) Cohen, Z. The Chemicals of Spirulina. In Spirulina platensis (Arthrospira): Physiology, Cell-Biology and Biotechnology; Vonshak, A., Ed.; Taylor and Francis Ltd.: Basingstoke, U.K., 1997; pp 175204. (4) Quing Hu; Westerhoff; Vermaas, W. Appl. EnViron. Microbiol. J. 2000, 66, 133-139. (5) Monanty, P.; Svirastava, M.; Krishna, B. The photosynthetic Apparature of Spirulina: Electron Transport and Energy Transfer. In Spirulina platensis (Arthrospira): Physiology, Cell-Biology and Biotechnology; Vonshak, A., Ed.; Taylor and Francis Ltd.: Basingstoke, U.K., 1997; pp 17-42. (6) Jonansson, P.; Wadso, I. Towards more specific information from isothermal microcalorimetric measurements on living systems. Therm. Anal. Calorim. J. 1999, 57, 275-281. (7) Wadso, I. Thermal and Energetic studies of Cellular Biological System; James, A. M., Ed.; Wright: Bristol, 1987; pp 34-67.
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(8) Wadso, I. Progress and problems in microcalorimetric work on mammalian cell systems. Thermochim. Acta 1988, 137, 1-10. (9) Monaselidze, J. R.; Barbakadze Sh.; Majagaladze, G. L.; Topchishvili, L. Biophys. J. Ru., in press. (10) Topchishvili, L.; Majagaladze, G.; Tananashvili, D.; Monaselidze, J. Abstracts, 5th Symposium/Workshops on Pharmacy and Thermal Analysis, Basel, Switserland, September 19-21, 2000. (11) Topchishvili L.; Barbakadze, S.; Khizanishvili, A.; Majagaladze, G.; Monaselidze, J. Microcalorometric study of iodized and noniodized cells and C-Phycocyanin of Spirulina Platensis. Biomacromolecules, in press. (12) Topchishvili L.; Tananashvili, D.; Kikvilashvili, Z. Device for Microorganizm Cultivation. Certificate No. 1323042, Geo. 1997. (13) Zarrouk, C. Ph.D. Thesis, University of Paris, 1966. (14) Majagaladze, G.; Chikvashvili, R.; Monaselidze, J. Scanning Calorimetry. Copyright. Ru. Certificate No. 1267175. 1986.
Monaselidze et al. (15) Monaselidze, J.; Kalandadze, Ya. Thermodynamic properties of serum and plasma of patients sick with cancer. High Temp.-High Pressures 1997, 29, 677-681. (16) Takahashi, K. Thermochemical characterization of Saccharomyces cereVisiae under nonaerobic conditions. Agric. Biol. Chem. 1973, 12, 2743-2747. (17) Tomaselli, Luisa Spirulina: Morphology, Ultrastructure and Taxony of Arthrospira (Spirulina) maxima and Arthrospira (Spirulina) platensis. In Spirulina platensis (Arthrospira): Physiology, CellBiology and Biotechnology; Vonshak, A., Ed.; Taylor and Francis Ltd.: Basingstoke, U.K., 1997. (18) Lu, C.; Vonshak, A. Photoinhibition in outdoor Spirulina platensis cultures assessed by polyphasic chlorophyll fluorescence transients. J. Appl. Phycol., in press.
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