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Effect of light, nutrient, cultivation time and salinity on lipid production of newly isolated strain of the green microalga, Botryococcus braunii KMITL 2
Bioresource Technology 109 (2012) 261–265
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Effect of light, nutrient, cultivation time and salinity on lipid production of newly isolated strain of the green microalga, Botryococcus braunii KMITL 2 Suneerat Ruangsomboon ⇑ Program in Fisheries Science, Division of Animal Production Technology and Fisheries, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand
a r t i c l e
i n f o
Article history: Received 3 February 2011 Received in revised form 28 June 2011 Accepted 11 July 2011 Available online 19 July 2011 Keywords: Botryococcus braunii Light Nutrient Salinity Lipid
a b s t r a c t The green microalga strain, Botryococcus braunii KMITL 2, was isolated from a freshwater reservoir in central Thailand, and the effects of light, nitrogen, phosphorus, iron, cultivation time and salinity on lipid production were studied by varying parameters one at a time. When cultured in Chlorella medium containing 222 mg L 1 phosphorus (PO34 —P) under continuous illumination of 200 lE m 2 s 1 with a salinity of 0 psu, a maximum lipid content of 54.69 ± 3.13% was obtained. Its high lipid content makes strain KMITL 2 a potential source for biodiesel production in tropical regions. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction With a lipid content of 20–50% and even 80%, microalgae are a potential source of biodiesel (Chisti, 2007). Nitrogen deprivation (Illman et al., 2000; Zhila et al., 2005a,b), silicon deficiency (Lynn et al., 2000), phosphate limitation (Reitan et al., 1994), high salinity (Rao et al., 2007; Zhila et al., 2011), optimized light intensities (Kojima and Zhang, 1999), iron content of the medium (Liu et al., 2008), and time of harvest (Zhila et al., 2011) can improve the lipid content. In general, it appears that microalgae maintain lipid synthesis under nutrient starvation or stress condition when the production of other types of cellular constituents is curtailed (Hu, 2004). The green microalga, Botryococcus braunii has a relatively high lipid content (Rao et al., 2007), but higher levels need to be achieved before commercial production can be realized. Most B. braunii strains have an optimal growth temperature of 25–30 °C, and a maximum growth temperature of 32 °C (Kalacheva et al., 2002; Lupi et al., 1991). Thus it is necessary to find locally adapted strains for regions with higher temperatures. For example, the central region of Thailand has average temperatures of around 18–34 °C and may usually reach 40 °C (Thai Meteorological Department, 2011). While B. braunii is usually cultivated in modified Chu 13 medium (Dayananda et al., 2007; Lupi et al., 1991; Rao et al., 2007; Yeesang and Cheirsilp, 2011), Bold basal medium, BG-11
medium (Dayananda et al., 2007), modified BG-11 medium (Ge et al., 2011) or Prat medium (Kalacheva et al., 2001), microalgae are commonly cultured in Chlorella medium in Thailand (Fisheries Department, 2011). Therefore, in this study a B. braunii strain indigenous to Thailand was isolated and conditions for optimal production of lipids in Chlorella medium were established. 2. Methods 2.1. Algal culture B. braunii strain KMITL 2 was isolated from the Klong Boat reservoir, Nakhon Nayok province, Thailand by using the microcapillary pipetted method (Stein, 1973). The isolated microalga was identified as belonging to the species B. braunii according to morphological properties (Philipose, 1967). The strain was cultured in Chlorella medium (Vonshak and Maske, 1982) composed of (g L 1): KNO3, 1.25; KH2PO4, 1.25; MgSO47H2O, 1.00; CaCl2, 0.08; H3BO3, 0.11; FeSO47H2O, 0.05; ZnSO47H2O, 0.08; MnCl24H2O, 0.01; MoO3, 0.007; CuSO45H2O, 0.01; Co(NO3)26H2O, 0.005; EDTA, 0.50 since preliminary experiments indicated that this medium allowed better growth than modified Chu 13, Kratz and Myers or Bold basal medium. 2.2. Effect of light intensity and light cycle on growth and lipid content
⇑ Tel./fax: +66 2 329 8517. E-mail address: [email protected] 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.07.025
B. braunii KMITL 2 was cultured in Chlorella medium in 1-L glass flasks under continuous illumination with daylight fluorescent
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mass with n-hexane (Mexwell et al., 1968), followed by extraction with chloroform and methanol (1:2 v/v) (Bligh and Dyer, 1959). For each extraction the mixture was sonicated at 70 Hz with a sonicator (Transonic model 460/H, Elma, Singen, Germany) at room temperature. Each extraction was repeated (total of two times). The extracts were dried in a rotary evaporator and weighed. The lipid content was calculated based on total lipid from the first and second extraction steps and then expressed as dry weight percentage. The lipid yield was calculated as the average value of lipid content multiplied by the average value of biomass concentration.
tube lamps (Philips, TLD18 W/54) at 0.3 (no bulb), 87.5 (one bulb), 200 (two bulbs) and 538 lE m 2 s 1 (four bulbs) with constant bubbling of air at 25 °C. The light intensity was measured at the center of the flask using a Watch-Dog light meter (model 200). Effect of light and dark cycles on growth and lipid contents were studied by establishing light and dark cycles (L:D) of 12:12, 14:10, 16:8 and 24:0 under illumination of 200 lE m 2 s 1 at 25 °C and with constant bubbling of air. 2.3. Effect of nutrient concentration, cultivation time and salinity in culture medium on growth and lipid content
2.6. Statistical analysis For all experiments the microalgae were grown in Chlorella medium in 1-L glass flasks under continuous illumination of 200 lE m 2 s 1 with constant bubbling of air at 25 °C. The effect of 16.5, 43, 86, 172 and 344 mg N L 1 (using KNO3 as nitrogen source), 22, 55, 111, 222 and 444 mg P L 1 (KH2PO4 as phosphorus source), and 9, 18, 27, 36 and 45 mg Fe L 1 (FeSO4 as iron source) were determined separately. For determination of temporal changes in biomass and lipid content, the microalgae were harvested at 10, 20, 30 and 40 days. The effect of salinity was investigated at 0, 5, 10, 15 and 20 psu (prepared from diluted sea water).
Average values of the biomass concentration and lipid content of four replications and their standard deviations were calculated. Significant differences were determined using analysis of variance (ANOVA) with 95% confidence (probability limit of p < 0.05). Since the lipid yield was calculated as the average value of lipid content multiplied by the average biomass concentration, it is reported as a single value without standard deviation. 3. Results and discussion
2.4. Determination of algal biomass
3.1. Effect of light intensity and light cycle on growth and lipid content
Cultures (10 ml suspensions) were filtered through glass microfiber filter paper (GFC, Whatmann). The paper with attached cells was dried at 105 °C for 24 h, cooled to room temperature in desiccators, and the dry weight was measured.
Light is one of the strongest factors affecting growth and storage products of algae. Low light intensity causes a reduction in dry weight while high intensity causes biochemical damage to the photosynthetic machinery (photoinhibition) (Scott et al., 2010). The effect of light intensity on strain KMITL 2 biomass, lipid content and lipid yield are shown in Fig. 1A–C. The highest lipid yield of 0.45 g L 1 was obtained by cultivation at 538 lE m 2 s 1. The effects of light cycles are shown in Fig. 1D–F. Strain KMITL 2 achieved a biomass of 1.91 ± 0.24 g L 1 under a 24:0 light cycle, which was four time the biomass under 12:12 light/dark cycles.
2.5. Extraction of total lipid
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Except for the time-course experiment, algae were harvested after 30 days of cultivation, dried at 55 °C, ground with a mortar and pestle, and the lipids were extracted from 500 mg of dried bio-
0.3 87.5 200 538
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Light and dark cycle (h:h) Fig. 1. Biomass concentration (A and D), lipid content (B and E) and lipid yield (C and F) of B. braunii KMITL 2 cultured in various light intensities (A–C) and light and dark cycles (D–F). Different small letters on the bars indicate significant difference (p < 0.05). Error bars represent ± S.D. of four replicates. Since the lipid yield was calculated as the average value of lipid content multiplied by the average biomass concentration, it is reported as a single value without standard deviation.
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3.3. Effect of initial phosphorus concentration on growth and lipid content
The same biomass yields were observed at conditions of 24:0, 16:8 and 14:10. The highest lipid content and lipid yield of 33.32 ± 1.97% and 0.59 g L 1 was obtained under 16:8 light conditions. Cultures exposed to low light intensity (87.5 lE m 2 s 1) showed a higher biomass compared to others (200 and 538 lE m 2 s 1). The difference in growth was possibly due to efficient in utilizing low irradiances for inorganic assimilation. High light intensities of 200 and 538 lE m 2 s 1 limited algal growth, but gave the benefit of higher lipid content and yield. This result is similar to that obtained by Niyogi (1999), and could be explained by the production of excessive photoassimilates that can then be stored in the form of lipid, probably as a means to convert excess light to chemical energy in order to avoid photooxidative damage (Solovchenko et al., 2008).
The highest biomass concentration of 1.91 ± 0.03 g L 1 was found under the highest phosphorus concentration of 444 mg L 1. Increasing of the phosphorus concentration, from 22 to 444 mg L 1 led to a 7.3-fold increase in biomass (Fig. 2D) but a decrease in lipid content. A maximum lipid content and yield, 54.69 ± 3.13% and 0.47 g L 1, respectively, were obtained at a phosphorus concentration of 222 mg L 1 (Fig. 2E and F). 3.4. Effect of initial iron concentration on growth and lipid content Hu (2004) reported that iron deficiency reduced growth and biomass of algae, but that excess iron elicits an oxidative stress leading to a physiological changes (Hu, 2004) and negative effects on growth (Yeesang and Cheirsilp, 2011). The effect of iron concentration on KMITL 2 biomass, lipid content and lipid yield are shown in Fig. 2G–I. The biomass concentration was not significantly affected by iron concentration. The highest lipid content of 34.93 ± 1.89% and lipid yield of 0.08 g L 1 was obtained by cultivation with an initial iron concentration of 27 mg L 1. Behrenfeld et al. (2006) had previously demonstrated that iron had a key function in regulating phytoplankton microalgal
3.2. Effect of initial nitrogen concentration on growth and lipid content
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Lack of nitrogen limits protein synthesis and thus increases lipid (Converti et al., 2009) or sometimes, carbohydrate accumulation (Hu, 2004). Biomass of strain KMITL 2 was not significantly affected by the nitrogen concentration after 30 days of cultivation (Fig. 2A). The highest biomass concentration of 0.48 ± 0.02 g L 1, lipid content of 39.42 ± 1.54%, and lipid yield of 0.19 g L 1 were obtained by cultivation with an initial nitrogen concentration of 86 mg L 1 (Fig. 2B and C).
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Iron concentration (mg L-1) Fig. 2. Biomass concentration (A, D, and G), lipid content (B, E, and H) and lipid yield (C, F, and I) of B. braunii KMITL 2 cultured in various nitrogen (A–C), phosphorus (D–F) and iron (G–I) concentrations. Different small letters on the bars indicate significant difference (p < 0.05). Error bars represent ± S.D. of four replicates. Since the lipid yield was calculated as the average value of lipid content multiplied by the average biomass concentration, it is reported as a single value without standard deviation.
S. Ruangsomboon / Bioresource Technology 109 (2012) 261–265
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Salinity (psu) Fig. 3. Biomass concentration (A and D), lipid content (B and E) and lipid yield (C and F) of B. braunii KMITL 2 cultured in various cultivation times (A–C) and salinities (D–F). Different small letters on the bars indicate significant difference (p < 0.05). Error bars represent ± S.D. of four replicates. Since the lipid yield was calculated as the average value of lipid content multiplied by the average biomass concentration, it is reported as a single value without standard deviation.
biomass. Supplementation of the growth media with iron in the late growth phase increased final cell density and only when the iron concentration in the initial medium reached a certain value did substantial amounts of lipid begin to accumulate (Liu et al., 2008).
3.5. Effect of cultivation time on growth and lipid content Total lipid of B. braunii and B. protuberans gradually increases with increasing cultivation time, and increases in stationary phase may be due to the conversion of carbohydrate to lipids (Singh, 2007). B. braunii KMITL 2 cultured for 20–40 days had a significantly higher biomass (1.34–1.42 g L 1) compared to 10 days cultivation, but there was no difference between 20, 30 and 40 days (Fig. 3A). The highest lipid content and lipid yield of 43.01 ± 0.79% and 0.58 g L 1 were found at 20 days (Fig. 3B and C). Lipid content obtained after 20 days cultivation was significantly higher than that at longer cultivation times. Similarly, Zhila et al. (2011) had observed a lower lipid content of B. braunii IPPAS H-252 at 13 days than at 3 days of cultivation. In contrast, Kalacheva et al. (2001) reported that the content of cellular lipids in actively growing B. braunii cells was low and increased in the stationary growth phase.
3.6. Effect of salinity on growth and lipid content Ben-Amotz et al. (1985) and Hu (2004) reported that an increase in salinity may result in a slightly increase in total lipid content of algae; however, B. braunii KMITL 2 cultured in Chlorella medium of salinity 0 psu had a significantly higher lipid content (32.35 ± 1.04%) than cells grown in medium of salinities 5, 10, 15 and 20 psu (Fig. 3D–F). Thus salinity stress did not increase lipid yield in this algal strain as was also observed by Yeesang and Cheirsilp (2011) with a Botryococcus spp. However, Ben-Amotz
Table 1 Lipid content of Botryococcus braunii in the present study and of other reports. Lipid content (% dry wt.)
Reference
12–55 10–33 50 6–17 13–18 25–75 11–18 9–20 6–36 10–13
In present study Okada et al. (1995) Kojima and Zhang (1999) Kalacheva et al. (2001) Dayananda et al. (2007) Chisti (2007) Orpez et al. (2009) Zhila et al. (2011) Yeesang and Cheirsilp (2011) Ge et al. (2011)
et al. (1985) found that the lipid content of B. braunii in salt concentration was higher than that without salt. 3.7. Comparison of lipid content of strain KMITL 2 with those of other algae The lipid contents of various B. braunii isolates is shown in Table 1. Overall, the lipid contents observed with isolate KMITL 2 are competitive with those of the other isolates, but further improvements in yield would be needed for commercial application. Such improvements can potentially be achieved by multifactorial experimental designs that would reveal positive interactions among the parameters. 4. Conclusions B. braunii KMITL 2, a strain adapted to the climate conditions of central Thailand, accumulated as much as 54.69 ± 3.13% of lipid under phosphorus concentration of 222 mg L 1 in Chlorella medium. This value is comparable to those obtained with other B. braunii strains, but further improvements in culture conditions are require to elevate the yield to commercially attractive levels.
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Acknowledgements This research was supported by Grant fund from King Mongkut’s Institute of Technology Ladkrabang (KMITL) and the National Research Council of Thailand (NRTC). References Behrenfeld, M.J., Worthington, K., Sherrell, R.M., Chavez, F.P., Strutton, P., McPhaden, M., Shea, D.M., 2006. Controls on tropical Pacific Ocean productivity revealed through nutrient stress diagnostics. Nature 442, 1025–1028. Ben-Amotz, A., Tomabene, T.G., Thomas, W.H., 1985. Chemical profile of selected species of microalgae with emphasis on lipids. J. Phycol. 21, 72–81. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Converti, A., Casazza, A.A., Ortiz, E.Y., Perego, P., Del Borghi, M., 2009. Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production. Chem. Eng. Process. 48, 1146–1151. Dayananda, C., Sarada, R., Usha Rani, M., Shamala, T.R., Ravishankar, G.A., 2007. Autotrophic cultivation of Botryococcus braunii for the production of hydrocarbons and exopolysaccharides in various media. Biomass Bioenergy 31, 87–93. Fisheries Department, 2011. http://www.fisheries.go.th. Ge, Y., Liu, J., Tian, G., 2011. Growth characteristics of Botryococcus braunii 765 under high CO2 concentration in photobioreactor. Bioresour. Technol. 102, 130– 134. Hu, Q., 2004. Environmental effects on cell composition. In: Richmond, A. (Ed.), Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Blackwell Science, Victoria, pp. 83–93. Illman, A.M., Scragg, A.H., Shales, S.W., 2000. Increase in Chlorella strains calorific values when grown in low nitrogen medium. Enzyme Microb. Technol. 27, 631– 635. Kalacheva, G.S., Zhila, N.O., Volova, T.G., 2001. Lipids of the green alga Botryococcus cultured in a batch mode. Microbiol. 70, 256–262. Kalacheva, G.S., Zhila, N.O., Volova, T.G., Gladyshev, M.I., 2002. The effect of temperature on the lipid composition of the green alga Botryococcus. Microbiol. 71, 286–293. Kojima, E., Zhang, K., 1999. Growth and hydrocarbon production by microalga Botryococcus braunii in bubble column photobioreactor. J. Biosci. Bioeng. 87, 811–817. Liu, Z.Y., Wang, G.C., Zhou, B.C., 2008. Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour. Technol. 99, 4717–4722. Lupi, F.M., Fernandes, H.M.L., Sa-Correia, I., Novais, J.M., 1991. Temperature profiles of cellular growth and exopolysaccharide synthesis by Botryococcus braunii Kutz. UC 58. J. Appl. Phycol 3, 35–42.
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Lynn, S.G., Kilham, S.S., Kreeger, D.A., Interlandi, S.J., 2000. Effect of nutrient availability on the biochemical and elemental stoichiometry in fresh water diatom Stephanodiscus minutulus acillariophyceae. J. Phycol. 36, 510–522. Mexwell, J.R., Douglas, A.G., Eglinton, G., McCormick, A., 1968. The botryococcenes hydrocarbons of novel structure from the alga Botryococcus braunii Kutzing. Phytochemistry 7, 2157–2171. Niyogi, K., 1999. Photoprotection revisited: genetic and molecular approaches. Annu. Rev. Plant Physiol. Mol. Biol. 50, 333–359. Okada, S., Murakami, M., Yamaguchi, K., 1995. Hydrocarbon composition of newly isolated strains of the green microalga Botryococcus braunii. J. Appl. Phycol. 7, 555–559. Orpez, R., Martinez, M.E., Hodaifa, G., ElYousfi, F., Jbari, N., Sanchez, S., 2009. Growth of the microalga Botryococcus braunii in secondarily treated sewage. Desalination 246, 625–630. Philipose, M.T., 1967. Chlorococcales. Indian Council of Agricultural Research, New Delhi. Rao, A.R., Dayananda, C., Sarada, R., Shamala, T.R., Ravishankar, G.A., 2007. Effect of salinity on growth of green alga Botryococcus braunii and its constituents. Bioresour. Technol. 98, 560–564. Reitan, K.I., Rainuzzo, J.R., Olsen, Y., 1994. Effect of nutrient limitation on fatty acid and lipid content of marine microalgae. J. Phycol. 30, 972–979. Scott, S.A., Davey, M.P., Dennis, J.S., Horst, I., Howe, C.J., Lea-Smith, D.J., Smith, A.G., 2010. Biodiesel from algae: challenges and prospects. Curr. Opin. Biotechnol. 21, 277–286. Singh, Y., 2007. Botryococcus: a hydrocarbon producing microalga. In: Gupta, R.K., Pandey, V.D. (Eds.), Advances in Applied Phycology. Daya Publishing House, Delhi, pp. 115–130. Solovchenko, A., Khozin-Goldberg, I., Didi-Cohen, S., Cohen, Z., Merzlyak, M., 2008. Effects of light intensity and nitrogen starvation on growth, total fatty acids and arachidonic acid in the green microalga Parietochloris incise. J. Appl. Phycol. 20, 245–251. Stein, E.D., 1973. Isolation and Culture of Algae, Handbook of Phycological Methods. Culture methods and growth measurements, Cambridge University Press. Thai Meteorological Department, 2011. http://www.tmd.go.th/index.php. Vonshak, A., Maske, H., 1982. Algae: growth techniques and biomass production. In: Coombs, J., Hall, D.O. (Eds.), Techniques in Bioproductivity and Photosynthesis. Pergamon Press, Oxford, pp. 66–77. Yeesang, C., Cheirsilp, B., 2011. Effect of nitrogen, salt, and iron content in the growth medium and light intensity on lipid production by microalgae isolated from freshwater sources in Thailand. Bioresour. Technol. 102, 3034–3040. Zhila, N., Kalacheva, G., Volova, T., 2011. Effect of salinity on the biochemical composition of the alga Botryococcus braunii Kütz IPPAS H-252. J. Appl. Phycol. 23, 47–52. Zhila, N.O., Kalacheva, G.S., Volova, T.G., 2005a. Effect of nitrogen limitation on the growth and lipid composition of the green alga Botryococcus braunii; Kutz IPPAS H-252. Russ. J. Plant Physiol. 52, 311–319. Zhila, N.O., Kalacheva, G.S., Volova, T.G., 2005b. Influence of nitrogen deficiency on biochemical composition of the green alga Botryococcus. J. Appl. Phycol. 17, 309–315.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Effect of light, nutrient, cultivation time and salinity on lipid production of newly isolated strain of the green microalga, Botryococcus braunii KMITL 2 Suneerat Ruangsomboon ⇑ Program in Fisheries Science, Division of Animal Production Technology and Fisheries, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand
a r t i c l e
i n f o
Article history: Received 3 February 2011 Received in revised form 28 June 2011 Accepted 11 July 2011 Available online 19 July 2011 Keywords: Botryococcus braunii Light Nutrient Salinity Lipid
a b s t r a c t The green microalga strain, Botryococcus braunii KMITL 2, was isolated from a freshwater reservoir in central Thailand, and the effects of light, nitrogen, phosphorus, iron, cultivation time and salinity on lipid production were studied by varying parameters one at a time. When cultured in Chlorella medium containing 222 mg L 1 phosphorus (PO34 —P) under continuous illumination of 200 lE m 2 s 1 with a salinity of 0 psu, a maximum lipid content of 54.69 ± 3.13% was obtained. Its high lipid content makes strain KMITL 2 a potential source for biodiesel production in tropical regions. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction With a lipid content of 20–50% and even 80%, microalgae are a potential source of biodiesel (Chisti, 2007). Nitrogen deprivation (Illman et al., 2000; Zhila et al., 2005a,b), silicon deficiency (Lynn et al., 2000), phosphate limitation (Reitan et al., 1994), high salinity (Rao et al., 2007; Zhila et al., 2011), optimized light intensities (Kojima and Zhang, 1999), iron content of the medium (Liu et al., 2008), and time of harvest (Zhila et al., 2011) can improve the lipid content. In general, it appears that microalgae maintain lipid synthesis under nutrient starvation or stress condition when the production of other types of cellular constituents is curtailed (Hu, 2004). The green microalga, Botryococcus braunii has a relatively high lipid content (Rao et al., 2007), but higher levels need to be achieved before commercial production can be realized. Most B. braunii strains have an optimal growth temperature of 25–30 °C, and a maximum growth temperature of 32 °C (Kalacheva et al., 2002; Lupi et al., 1991). Thus it is necessary to find locally adapted strains for regions with higher temperatures. For example, the central region of Thailand has average temperatures of around 18–34 °C and may usually reach 40 °C (Thai Meteorological Department, 2011). While B. braunii is usually cultivated in modified Chu 13 medium (Dayananda et al., 2007; Lupi et al., 1991; Rao et al., 2007; Yeesang and Cheirsilp, 2011), Bold basal medium, BG-11
medium (Dayananda et al., 2007), modified BG-11 medium (Ge et al., 2011) or Prat medium (Kalacheva et al., 2001), microalgae are commonly cultured in Chlorella medium in Thailand (Fisheries Department, 2011). Therefore, in this study a B. braunii strain indigenous to Thailand was isolated and conditions for optimal production of lipids in Chlorella medium were established. 2. Methods 2.1. Algal culture B. braunii strain KMITL 2 was isolated from the Klong Boat reservoir, Nakhon Nayok province, Thailand by using the microcapillary pipetted method (Stein, 1973). The isolated microalga was identified as belonging to the species B. braunii according to morphological properties (Philipose, 1967). The strain was cultured in Chlorella medium (Vonshak and Maske, 1982) composed of (g L 1): KNO3, 1.25; KH2PO4, 1.25; MgSO47H2O, 1.00; CaCl2, 0.08; H3BO3, 0.11; FeSO47H2O, 0.05; ZnSO47H2O, 0.08; MnCl24H2O, 0.01; MoO3, 0.007; CuSO45H2O, 0.01; Co(NO3)26H2O, 0.005; EDTA, 0.50 since preliminary experiments indicated that this medium allowed better growth than modified Chu 13, Kratz and Myers or Bold basal medium. 2.2. Effect of light intensity and light cycle on growth and lipid content
⇑ Tel./fax: +66 2 329 8517. E-mail address: [email protected] 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.07.025
B. braunii KMITL 2 was cultured in Chlorella medium in 1-L glass flasks under continuous illumination with daylight fluorescent
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mass with n-hexane (Mexwell et al., 1968), followed by extraction with chloroform and methanol (1:2 v/v) (Bligh and Dyer, 1959). For each extraction the mixture was sonicated at 70 Hz with a sonicator (Transonic model 460/H, Elma, Singen, Germany) at room temperature. Each extraction was repeated (total of two times). The extracts were dried in a rotary evaporator and weighed. The lipid content was calculated based on total lipid from the first and second extraction steps and then expressed as dry weight percentage. The lipid yield was calculated as the average value of lipid content multiplied by the average value of biomass concentration.
tube lamps (Philips, TLD18 W/54) at 0.3 (no bulb), 87.5 (one bulb), 200 (two bulbs) and 538 lE m 2 s 1 (four bulbs) with constant bubbling of air at 25 °C. The light intensity was measured at the center of the flask using a Watch-Dog light meter (model 200). Effect of light and dark cycles on growth and lipid contents were studied by establishing light and dark cycles (L:D) of 12:12, 14:10, 16:8 and 24:0 under illumination of 200 lE m 2 s 1 at 25 °C and with constant bubbling of air. 2.3. Effect of nutrient concentration, cultivation time and salinity in culture medium on growth and lipid content
2.6. Statistical analysis For all experiments the microalgae were grown in Chlorella medium in 1-L glass flasks under continuous illumination of 200 lE m 2 s 1 with constant bubbling of air at 25 °C. The effect of 16.5, 43, 86, 172 and 344 mg N L 1 (using KNO3 as nitrogen source), 22, 55, 111, 222 and 444 mg P L 1 (KH2PO4 as phosphorus source), and 9, 18, 27, 36 and 45 mg Fe L 1 (FeSO4 as iron source) were determined separately. For determination of temporal changes in biomass and lipid content, the microalgae were harvested at 10, 20, 30 and 40 days. The effect of salinity was investigated at 0, 5, 10, 15 and 20 psu (prepared from diluted sea water).
Average values of the biomass concentration and lipid content of four replications and their standard deviations were calculated. Significant differences were determined using analysis of variance (ANOVA) with 95% confidence (probability limit of p < 0.05). Since the lipid yield was calculated as the average value of lipid content multiplied by the average biomass concentration, it is reported as a single value without standard deviation. 3. Results and discussion
2.4. Determination of algal biomass
3.1. Effect of light intensity and light cycle on growth and lipid content
Cultures (10 ml suspensions) were filtered through glass microfiber filter paper (GFC, Whatmann). The paper with attached cells was dried at 105 °C for 24 h, cooled to room temperature in desiccators, and the dry weight was measured.
Light is one of the strongest factors affecting growth and storage products of algae. Low light intensity causes a reduction in dry weight while high intensity causes biochemical damage to the photosynthetic machinery (photoinhibition) (Scott et al., 2010). The effect of light intensity on strain KMITL 2 biomass, lipid content and lipid yield are shown in Fig. 1A–C. The highest lipid yield of 0.45 g L 1 was obtained by cultivation at 538 lE m 2 s 1. The effects of light cycles are shown in Fig. 1D–F. Strain KMITL 2 achieved a biomass of 1.91 ± 0.24 g L 1 under a 24:0 light cycle, which was four time the biomass under 12:12 light/dark cycles.
2.5. Extraction of total lipid
(A) b
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Lipid yield (g L-1)
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Lipid content (%)
Biomass concentration (g L-1)
Except for the time-course experiment, algae were harvested after 30 days of cultivation, dried at 55 °C, ground with a mortar and pestle, and the lipids were extracted from 500 mg of dried bio-
0.3 87.5 200 538
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Biomass concentration (g L-1)
Light intensity (µE m-2 s-1)
12:12 14:10 16:8 24:0
12:12 14:10 16:8 24:0
Light and dark cycle (h:h) Fig. 1. Biomass concentration (A and D), lipid content (B and E) and lipid yield (C and F) of B. braunii KMITL 2 cultured in various light intensities (A–C) and light and dark cycles (D–F). Different small letters on the bars indicate significant difference (p < 0.05). Error bars represent ± S.D. of four replicates. Since the lipid yield was calculated as the average value of lipid content multiplied by the average biomass concentration, it is reported as a single value without standard deviation.
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3.3. Effect of initial phosphorus concentration on growth and lipid content
The same biomass yields were observed at conditions of 24:0, 16:8 and 14:10. The highest lipid content and lipid yield of 33.32 ± 1.97% and 0.59 g L 1 was obtained under 16:8 light conditions. Cultures exposed to low light intensity (87.5 lE m 2 s 1) showed a higher biomass compared to others (200 and 538 lE m 2 s 1). The difference in growth was possibly due to efficient in utilizing low irradiances for inorganic assimilation. High light intensities of 200 and 538 lE m 2 s 1 limited algal growth, but gave the benefit of higher lipid content and yield. This result is similar to that obtained by Niyogi (1999), and could be explained by the production of excessive photoassimilates that can then be stored in the form of lipid, probably as a means to convert excess light to chemical energy in order to avoid photooxidative damage (Solovchenko et al., 2008).
The highest biomass concentration of 1.91 ± 0.03 g L 1 was found under the highest phosphorus concentration of 444 mg L 1. Increasing of the phosphorus concentration, from 22 to 444 mg L 1 led to a 7.3-fold increase in biomass (Fig. 2D) but a decrease in lipid content. A maximum lipid content and yield, 54.69 ± 3.13% and 0.47 g L 1, respectively, were obtained at a phosphorus concentration of 222 mg L 1 (Fig. 2E and F). 3.4. Effect of initial iron concentration on growth and lipid content Hu (2004) reported that iron deficiency reduced growth and biomass of algae, but that excess iron elicits an oxidative stress leading to a physiological changes (Hu, 2004) and negative effects on growth (Yeesang and Cheirsilp, 2011). The effect of iron concentration on KMITL 2 biomass, lipid content and lipid yield are shown in Fig. 2G–I. The biomass concentration was not significantly affected by iron concentration. The highest lipid content of 34.93 ± 1.89% and lipid yield of 0.08 g L 1 was obtained by cultivation with an initial iron concentration of 27 mg L 1. Behrenfeld et al. (2006) had previously demonstrated that iron had a key function in regulating phytoplankton microalgal
3.2. Effect of initial nitrogen concentration on growth and lipid content
(A)
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a
a
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Lipid content (%)
Biomass concentration (g L-1)
Lack of nitrogen limits protein synthesis and thus increases lipid (Converti et al., 2009) or sometimes, carbohydrate accumulation (Hu, 2004). Biomass of strain KMITL 2 was not significantly affected by the nitrogen concentration after 30 days of cultivation (Fig. 2A). The highest biomass concentration of 0.48 ± 0.02 g L 1, lipid content of 39.42 ± 1.54%, and lipid yield of 0.19 g L 1 were obtained by cultivation with an initial nitrogen concentration of 86 mg L 1 (Fig. 2B and C).
d
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0 22 55 111 222 444
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0.4 0.2
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Iron concentration (mg L-1) Fig. 2. Biomass concentration (A, D, and G), lipid content (B, E, and H) and lipid yield (C, F, and I) of B. braunii KMITL 2 cultured in various nitrogen (A–C), phosphorus (D–F) and iron (G–I) concentrations. Different small letters on the bars indicate significant difference (p < 0.05). Error bars represent ± S.D. of four replicates. Since the lipid yield was calculated as the average value of lipid content multiplied by the average biomass concentration, it is reported as a single value without standard deviation.
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b
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a b
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Salinity (psu) Fig. 3. Biomass concentration (A and D), lipid content (B and E) and lipid yield (C and F) of B. braunii KMITL 2 cultured in various cultivation times (A–C) and salinities (D–F). Different small letters on the bars indicate significant difference (p < 0.05). Error bars represent ± S.D. of four replicates. Since the lipid yield was calculated as the average value of lipid content multiplied by the average biomass concentration, it is reported as a single value without standard deviation.
biomass. Supplementation of the growth media with iron in the late growth phase increased final cell density and only when the iron concentration in the initial medium reached a certain value did substantial amounts of lipid begin to accumulate (Liu et al., 2008).
3.5. Effect of cultivation time on growth and lipid content Total lipid of B. braunii and B. protuberans gradually increases with increasing cultivation time, and increases in stationary phase may be due to the conversion of carbohydrate to lipids (Singh, 2007). B. braunii KMITL 2 cultured for 20–40 days had a significantly higher biomass (1.34–1.42 g L 1) compared to 10 days cultivation, but there was no difference between 20, 30 and 40 days (Fig. 3A). The highest lipid content and lipid yield of 43.01 ± 0.79% and 0.58 g L 1 were found at 20 days (Fig. 3B and C). Lipid content obtained after 20 days cultivation was significantly higher than that at longer cultivation times. Similarly, Zhila et al. (2011) had observed a lower lipid content of B. braunii IPPAS H-252 at 13 days than at 3 days of cultivation. In contrast, Kalacheva et al. (2001) reported that the content of cellular lipids in actively growing B. braunii cells was low and increased in the stationary growth phase.
3.6. Effect of salinity on growth and lipid content Ben-Amotz et al. (1985) and Hu (2004) reported that an increase in salinity may result in a slightly increase in total lipid content of algae; however, B. braunii KMITL 2 cultured in Chlorella medium of salinity 0 psu had a significantly higher lipid content (32.35 ± 1.04%) than cells grown in medium of salinities 5, 10, 15 and 20 psu (Fig. 3D–F). Thus salinity stress did not increase lipid yield in this algal strain as was also observed by Yeesang and Cheirsilp (2011) with a Botryococcus spp. However, Ben-Amotz
Table 1 Lipid content of Botryococcus braunii in the present study and of other reports. Lipid content (% dry wt.)
Reference
12–55 10–33 50 6–17 13–18 25–75 11–18 9–20 6–36 10–13
In present study Okada et al. (1995) Kojima and Zhang (1999) Kalacheva et al. (2001) Dayananda et al. (2007) Chisti (2007) Orpez et al. (2009) Zhila et al. (2011) Yeesang and Cheirsilp (2011) Ge et al. (2011)
et al. (1985) found that the lipid content of B. braunii in salt concentration was higher than that without salt. 3.7. Comparison of lipid content of strain KMITL 2 with those of other algae The lipid contents of various B. braunii isolates is shown in Table 1. Overall, the lipid contents observed with isolate KMITL 2 are competitive with those of the other isolates, but further improvements in yield would be needed for commercial application. Such improvements can potentially be achieved by multifactorial experimental designs that would reveal positive interactions among the parameters. 4. Conclusions B. braunii KMITL 2, a strain adapted to the climate conditions of central Thailand, accumulated as much as 54.69 ± 3.13% of lipid under phosphorus concentration of 222 mg L 1 in Chlorella medium. This value is comparable to those obtained with other B. braunii strains, but further improvements in culture conditions are require to elevate the yield to commercially attractive levels.
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