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Antioxidant activity of CO2 fixing microalgae
T. Inui, M. Anpo, K. Izui, S. Yanagida, T. Yamaguchi (Editors) Advances in Chemical Conversions for Mitigating Carbon Dioxide Studies in Surface Science and Catalysis, Vol. 114 9 1998 Elsevier Science B.V. All rights reserved.
641
A n t i o x i d a n t activity of CO2 fixing microalgae* R. Matsukawaa, b, Y. WadaC,d, N. TanC,d, N. SakaiC,d, M. Chiharaa, e and I. Karube a aResearch Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, 153 Tokyo, Japan bNew Energy and Industrial Technology Development Organization, Sunshine 60, 21F, 3-1-1, Higashi-Ikebukuro, Toshima-ku, Tokyo 170, Japan CResearch Institute of Innovative Technology for the Earth, 2-8-11, Nishishinbashi, Minato-ku, Tokyo 105, Japan dToyo Engineering Corporation, Research Engineering, Biotechnology, 1818 Fujimi, Togo, Mobara-shi, Chiba 297, Japan ejapanese Red Cross College of Nursing, 4-1-3, Hiro-o, Shibuya-ku, Tokyo 150, Japan Microalgae from hot spring and ponds in Japan were screened for antioxidant activity using a lipoxygenase inhibition test. The ethanolic extracts of some microalgae showed high lipoxygenase inhibition activity. The ethanolic extracts of isolated Chlorella species with tolerance to high temperatures and high concentrations of CO2 possessed high radical scavenging activity. These results suggest that CO2 fixing microalgae may be potential sources of natural antioxidants. 1. INTRODUCTION Carbon dioxide (CO2) is the principle greenhouse effect gas and the increase in the atmospheric CO2 concentration is suspected to be causing global warming via the greenhouse effect [1]. In order to decrease the CO2 concentration in the atmosphere, the development of CO2 fixation and removal systems, in conjunction with a system which converts the fixed CO2 to useable material, is desirable. "This work was supported by the New Energy and Industrial Technology Development Organization (NEDO) in Japan.
642 It is well known that photosynthetic plants convert CO2 to organic matter using solar energy. Photosynthesis is more efficient in microalgae than in terrestrial higher plants and CO2 from industrial exhaust could be a useful carbon source for growing algae. In order to fix excessive atmospheric CO2, microalgae could be cultured and then be exploited as a useful resource. Photosynthesizing plant cells, including microalgae, are exposed to a combination of intense light and high oxygen levels [2, 3], leading to the formation of free radicals and other strong oxidizing agents. The ability of microalgae to thrive under such extremely oxidizing conditions led us to focus our investigation on identifying potential antioxidant activity in microalgae. In this study, we report the screening of microalgae for antioxidant activity and the radical scavenging activity of microalgae with tolerance to high temperatures and high concentrations of CO2. 2. MATERIALS A N D METHODS 2.1 Preparation of microalgal extract Over 100 species of microalgae were collected from hot springs and ponds in Japan. Some of these were isolated and cultured in suitable media. The microalgae were separated from the medium by centrifugation at 3,000 rpm for 10 min. After washing the solid algal pellets with distilled water, a volume of solvent (water, ethanol or methanol) weighing ten times the sample weight was added to the pellets. The sample was then homogenized in a sonicator for 30 rain in 30 sec intervals. The homogenate was then centrifuged at 3,000 rpm for 10 rain, and the supernatant was used as the algal extract. 2.2 Determination of antioxidant activity As an assay for antioxidant activity, we examined the inhibition of lipoxygenase activity and radical scavenging activity using the o~, cz-diphenyl-~picrylhydrazyl (DPPH)decolorization test. Aqueous, ethanolic and methanolic extracts of microalgae were used in the assays. The assay of lipoxygenase activity was carried out using the method of BenAziz et al. [4]. The enzyme reaction was carried out in the cuvette of a spectrophotometer monitored at 234 nm until the reaction rate reached a steady state. That wavelength is the peak of absorption for the hydroperoxides generated by the action of lipoxygenase on linoleic acid, with the uptake of oxygen. The extent of inhibition was defined as the ratio of the rate of increase in OD234 in the absence of algal extract, to that measured in the presence of the sample. The assay of radical scavenging activity was determined using of a stable free radical, DPPH, according to the method of Blois [5]. The decrease in absorbance due to DPPH was measured at 540 nm using a microplate reader. The activity was defined as the ratio of OD540 in the absence of algal extract, to that measured in the presence of the sample.
643 3. RESULTS AND DISCUSSION
3.1 Screening of microalgae for antioxidants Several microalgae samples from hot springs and ponds showed lipoxygenase inhibition activity in both aqueous and ethanolic extracts. The ethanolic extracts of Chlorella sp. were observed to be more effective than aqueous extracts. The methanolic extract of another Chloretla sp. showed lipoxygenase inhibition. The inhibition of lipoxygenase by the extracts of microalgae suggested the existence of a cellular mechanism protecting them from oxidation in vivo. 3.2 Effects of temperature on cell growth Our previous paper reported the isolation and growth characteristics of Chtoretla strains H-84 and A-2 with tolerance to high temperature and high concentrations of CO2 [6]. Figure 1 shows the effects of temperature on the growth of Chlorella sp. H-84. The strains showed a high growth rate at 40~ but the growth rate was significantly decreased at 30~ and above 45~ The algae was unable to grow at temperatures higher than 50~ Thus, the optimum temperature for this alga seemed to be around 40~ The isolated Chlorella spp. were identified as Chlorella sorokiniana. Previously, the thermophilic Chloretla species known was only Chlorella sorokiniana [7]. 3.3 Effects of CO2 concentration on cell growth Growth of Clorella sp. H-84 was examined under different CO2 concentrations as shown in Figure 2. The alga grew well at concentrations of CO2 from 5 to 40%. It was able to grow in the culture medium with CO2 concentrations up to 60% (data not shown). The optimum concentration of CO2 is about 20%. Similar results were obtained for Clorella sp. A-2. These results suggest that these microalgae may be suitable for the biological fixation of CO2 from
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Figure 2. Effects of CO2 concentration on cell growth.
644 industrial flue gases.
3.4 Radical scavenging activity The radical scavenging activities of aqueous, ethanolic and methanolic extracts of microalgae with tolerance to high temperatures and high concentrations of CO2 are shown in Table 1. The stable free radical DPPH was oxidized and decolorized in the presence of extracts of the microalgae. The ethanolic extracts of Chlorella sp. A-2 and Chlorella sorokiniana UTEX-1230 showed high radical scavenging activity. Ethanolic extracs of Chlorella sp. H-84 showed a radical scavenging activity of about 53%. These species of microalgae have antioxidant activity and may be used for purposes such as food, animal feed, pharmaceuticals and cosmetics.
Table 1 Antioxidant activity of extracts of Chlorella species Chlorella sp.
Activity (%) aqueous
ethanolic
methanolic
H-84
39.2
52.8
43.9
A-2
32.4
98.5
77.1
52.7
98.3
97.5
sorokiniana
REFERENCES 1. S.H. Scheider, Science, 243 (1989) 771. 2. A. Abeliovich and M. Shilo, J. Bacteriol., 111 (1972) 682. 3. R.R. Bidigrare, M.E. Ondrusek, M.C.II., Kennicutt, R. Iturriaga, H.R. Harvey, R.W. Hoham and S.A. Macko, J. Phycol., 29 (1993) 427. 4. A. Ben-Aziz, S. Grossman, I. Ascarelli, P. Budowski, Anal. Biochem., 34 (1970) 88. 5. M.S. Blois, Nature, 181 (1958) 1199. 6. N. Sakai, Y. Sakamoto, N. Kishimoto, M. Chihara and I. Karube, Energy Convers. Mgmt., 36 (1995) 693. 7. C. Sorokin and J. Myers, Science, 117 (1953) 330.
641
A n t i o x i d a n t activity of CO2 fixing microalgae* R. Matsukawaa, b, Y. WadaC,d, N. TanC,d, N. SakaiC,d, M. Chiharaa, e and I. Karube a aResearch Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, 153 Tokyo, Japan bNew Energy and Industrial Technology Development Organization, Sunshine 60, 21F, 3-1-1, Higashi-Ikebukuro, Toshima-ku, Tokyo 170, Japan CResearch Institute of Innovative Technology for the Earth, 2-8-11, Nishishinbashi, Minato-ku, Tokyo 105, Japan dToyo Engineering Corporation, Research Engineering, Biotechnology, 1818 Fujimi, Togo, Mobara-shi, Chiba 297, Japan ejapanese Red Cross College of Nursing, 4-1-3, Hiro-o, Shibuya-ku, Tokyo 150, Japan Microalgae from hot spring and ponds in Japan were screened for antioxidant activity using a lipoxygenase inhibition test. The ethanolic extracts of some microalgae showed high lipoxygenase inhibition activity. The ethanolic extracts of isolated Chlorella species with tolerance to high temperatures and high concentrations of CO2 possessed high radical scavenging activity. These results suggest that CO2 fixing microalgae may be potential sources of natural antioxidants. 1. INTRODUCTION Carbon dioxide (CO2) is the principle greenhouse effect gas and the increase in the atmospheric CO2 concentration is suspected to be causing global warming via the greenhouse effect [1]. In order to decrease the CO2 concentration in the atmosphere, the development of CO2 fixation and removal systems, in conjunction with a system which converts the fixed CO2 to useable material, is desirable. "This work was supported by the New Energy and Industrial Technology Development Organization (NEDO) in Japan.
642 It is well known that photosynthetic plants convert CO2 to organic matter using solar energy. Photosynthesis is more efficient in microalgae than in terrestrial higher plants and CO2 from industrial exhaust could be a useful carbon source for growing algae. In order to fix excessive atmospheric CO2, microalgae could be cultured and then be exploited as a useful resource. Photosynthesizing plant cells, including microalgae, are exposed to a combination of intense light and high oxygen levels [2, 3], leading to the formation of free radicals and other strong oxidizing agents. The ability of microalgae to thrive under such extremely oxidizing conditions led us to focus our investigation on identifying potential antioxidant activity in microalgae. In this study, we report the screening of microalgae for antioxidant activity and the radical scavenging activity of microalgae with tolerance to high temperatures and high concentrations of CO2. 2. MATERIALS A N D METHODS 2.1 Preparation of microalgal extract Over 100 species of microalgae were collected from hot springs and ponds in Japan. Some of these were isolated and cultured in suitable media. The microalgae were separated from the medium by centrifugation at 3,000 rpm for 10 min. After washing the solid algal pellets with distilled water, a volume of solvent (water, ethanol or methanol) weighing ten times the sample weight was added to the pellets. The sample was then homogenized in a sonicator for 30 rain in 30 sec intervals. The homogenate was then centrifuged at 3,000 rpm for 10 rain, and the supernatant was used as the algal extract. 2.2 Determination of antioxidant activity As an assay for antioxidant activity, we examined the inhibition of lipoxygenase activity and radical scavenging activity using the o~, cz-diphenyl-~picrylhydrazyl (DPPH)decolorization test. Aqueous, ethanolic and methanolic extracts of microalgae were used in the assays. The assay of lipoxygenase activity was carried out using the method of BenAziz et al. [4]. The enzyme reaction was carried out in the cuvette of a spectrophotometer monitored at 234 nm until the reaction rate reached a steady state. That wavelength is the peak of absorption for the hydroperoxides generated by the action of lipoxygenase on linoleic acid, with the uptake of oxygen. The extent of inhibition was defined as the ratio of the rate of increase in OD234 in the absence of algal extract, to that measured in the presence of the sample. The assay of radical scavenging activity was determined using of a stable free radical, DPPH, according to the method of Blois [5]. The decrease in absorbance due to DPPH was measured at 540 nm using a microplate reader. The activity was defined as the ratio of OD540 in the absence of algal extract, to that measured in the presence of the sample.
643 3. RESULTS AND DISCUSSION
3.1 Screening of microalgae for antioxidants Several microalgae samples from hot springs and ponds showed lipoxygenase inhibition activity in both aqueous and ethanolic extracts. The ethanolic extracts of Chlorella sp. were observed to be more effective than aqueous extracts. The methanolic extract of another Chloretla sp. showed lipoxygenase inhibition. The inhibition of lipoxygenase by the extracts of microalgae suggested the existence of a cellular mechanism protecting them from oxidation in vivo. 3.2 Effects of temperature on cell growth Our previous paper reported the isolation and growth characteristics of Chtoretla strains H-84 and A-2 with tolerance to high temperature and high concentrations of CO2 [6]. Figure 1 shows the effects of temperature on the growth of Chlorella sp. H-84. The strains showed a high growth rate at 40~ but the growth rate was significantly decreased at 30~ and above 45~ The algae was unable to grow at temperatures higher than 50~ Thus, the optimum temperature for this alga seemed to be around 40~ The isolated Chlorella spp. were identified as Chlorella sorokiniana. Previously, the thermophilic Chloretla species known was only Chlorella sorokiniana [7]. 3.3 Effects of CO2 concentration on cell growth Growth of Clorella sp. H-84 was examined under different CO2 concentrations as shown in Figure 2. The alga grew well at concentrations of CO2 from 5 to 40%. It was able to grow in the culture medium with CO2 concentrations up to 60% (data not shown). The optimum concentration of CO2 is about 20%. Similar results were obtained for Clorella sp. A-2. These results suggest that these microalgae may be suitable for the biological fixation of CO2 from
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F i g u r e 1. Effects o f t e m p e r a t u r e
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Figure 2. Effects of CO2 concentration on cell growth.
644 industrial flue gases.
3.4 Radical scavenging activity The radical scavenging activities of aqueous, ethanolic and methanolic extracts of microalgae with tolerance to high temperatures and high concentrations of CO2 are shown in Table 1. The stable free radical DPPH was oxidized and decolorized in the presence of extracts of the microalgae. The ethanolic extracts of Chlorella sp. A-2 and Chlorella sorokiniana UTEX-1230 showed high radical scavenging activity. Ethanolic extracs of Chlorella sp. H-84 showed a radical scavenging activity of about 53%. These species of microalgae have antioxidant activity and may be used for purposes such as food, animal feed, pharmaceuticals and cosmetics.
Table 1 Antioxidant activity of extracts of Chlorella species Chlorella sp.
Activity (%) aqueous
ethanolic
methanolic
H-84
39.2
52.8
43.9
A-2
32.4
98.5
77.1
52.7
98.3
97.5
sorokiniana
REFERENCES 1. S.H. Scheider, Science, 243 (1989) 771. 2. A. Abeliovich and M. Shilo, J. Bacteriol., 111 (1972) 682. 3. R.R. Bidigrare, M.E. Ondrusek, M.C.II., Kennicutt, R. Iturriaga, H.R. Harvey, R.W. Hoham and S.A. Macko, J. Phycol., 29 (1993) 427. 4. A. Ben-Aziz, S. Grossman, I. Ascarelli, P. Budowski, Anal. Biochem., 34 (1970) 88. 5. M.S. Blois, Nature, 181 (1958) 1199. 6. N. Sakai, Y. Sakamoto, N. Kishimoto, M. Chihara and I. Karube, Energy Convers. Mgmt., 36 (1995) 693. 7. C. Sorokin and J. Myers, Science, 117 (1953) 330.