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Cultivation of green alga Botryococcus braunii in raceway, circular ponds under outdoor conditions and its growth, hydrocarbon production
Bioresource Technology 123 (2012) 528–533
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Cultivation of green alga Botryococcus braunii in raceway, circular ponds under outdoor conditions and its growth, hydrocarbon production A. Ranga Rao 1, G.A. Ravishankar, R. Sarada ⇑ Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Institute, Mysore 570 020, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" B. braunii (LB-572 and N-836) strains
Cultivation of Botryococcus braunii in raceway and circular ponds.
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were used for the current study. Cultivated LB-572 and N-836 in both raceway & circular ponds. Evaluated biomass yield, hydrocarbon content and fatty acid profile. Biomass and hydrocarbon content were observed in various seasons. Effect of NaHCO3 on biomass and hydrocarbon production in N-836 were evaluated.
a r t i c l e
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Article history: Received 16 April 2012 Received in revised form 6 July 2012 Accepted 7 July 2012 Available online 26 July 2012 Keywords: Microalgae Botryococcus braunii Outdoor cultivation Hydrocarbon Fatty acids
a b s t r a c t The present study focused on cultivation, seasonal variation in growth, hydrocarbon production, fatty acids profiles of Botryococcus braunii (LB-572 and N-836) in raceway & circular ponds under outdoor conditions. After 18 days of cultivation the biomass yield and hydrocarbon contents were increased in both raceway and circular ponds. The fat content was found to be around 24% (w/w) with palmitic and oleic acids as prominent fatty acids. Hydrocarbons of C20–C30 carbon chain length were higher in raceway and circular ponds. Maximum biomass yield (2 g L 1) and hydrocarbon content (28%) were observed in Nov– Dec. In case of B. braunii (N-836) after 25 days of cultivation the biomass yield was 1 g L 1 and hydrocarbon content was 27%. Supplementation of 0.1% NaHCO3 in the medium resulted in biomass yield of 1.5 g L 1 and hydrocarbon content of 30% compared to control. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Botryococcus braunii is a green colonial fresh water micro alga which produces hydrocarbons. It is recognized as one of the renewable resource for the production of hydrocarbons. Three races of B. braunii have been documented, and they are differentiated on the basis of the characteristic hydrocarbons they produce. The ‘A’ race produces odd numbered C25 to C31, n-alkadienes and trienes. The ⇑ Corresponding author. Tel.: +91 821 2516501; fax: +91 821 2517233. E-mail addresses: [email protected], [email protected] (R. Sarada). Present address: Department of Applied Sciences and Mathematics, Arizona State University, 7001 E. Williams Field Road, Mesa, AZ 85212, USA. 1
0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.07.009
‘B’ race produces triterpenoid hydrocarbons known as botryococcenes and ‘L’ race produces lycopadiene, a C40 tetraterpene. Another difference among the races is the colony color in the stationary phase. Race ‘A’ and ‘B’ strains are known to produce exopolysaccharides up to 250 g m3, whereas ‘L’ race produced up to 1 kg m3 (Banerjee et al., 2002; Niehaus et al., 2011). However the amount of exopolysaccharides production varies with the strains and the culture conditions. B. braunii is a promising renewable resource for the production of hydrocarbons and it has been reported that on hydrocracking, the distillate yields 67% gasoline, 15% aviation turbine fuel, 15% diesel fuel and 3% residual oil (Hillen et al., 1982; Samori et al., 2010). B. braunii is also known to produce large amounts of fatty
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acids. The quantity and composition of fatty acids varies with species and also among the races (Grice et al., 1998; Metzger et al., 1990; Niitsu et al., 2011; Weiss et al., 2010). Being a photosynthetic organism, it can reduce CO2 emissions by 1.5 105 tons y 1 per 8.4 103 ha of micro algal cultivation area would be necessary (Sawayama et al., 1999; Tanoi et al., 2011). The alga B. braunii produces hydrocarbons in the range of 2–86% (on dry weight basis). This variation in the content of hydrocarbon is due to the differences among different strains/races in the production of hydrocarbons and changes in cultural and physiological conditions (Barupal et al., 2010; Samori et al., 2010). Dayananda et al. (2005) reported optimization of media constituents for growth and hydrocarbon production in B. braunii (SAG 30.81). In the changing energy scenario it is necessary to exploit the potential of this microalga as a source of hydrocarbons. One of the important strategies may be adaptation of the organism for outdoor cultivation. The present study focused on the growth and hydrocarbon production of B. braunii in both raceway and circular ponds under outdoor culture conditions. 2. Methods 2.1. Micro algal strains B. braunii strains were obtained from various culture collection centers such as B. braunii (LB-572, ‘A’ race) from UTEX culture collection, USA and B. braunii (N-836, ‘B’ race) from National Institute for Environmental Studies, Tsukuba, Japan respectively. The stock cultures were maintained both in agar slants and liquid medium of modified Chu 13 (Largeau et al., 1980). 2.2. Experimental design 2.2.1. Cultivation of B. braunii in raceway and circular ponds Forty litres medium was inoculated with 25% (v/v) of B. braunii culture and grown in raceway pond (Length 1.13 m; Width 0.6 m; Depth 0.3 m) under outdoor conditions with 15 rpm agitation. The culture volume was increased gradually to the pond capacity (80 L) and then continued cultivation for a period of 18 days. Forty litres medium was inoculated with 25% (v/v) of B. braunii culture and grown in circular ponds (Diameter 1.21 m; Depth 0. 25 m) under outdoor conditions without agitation. The cultures were mixed twice a day manually. One batch culture was run for a period of 25 days and the yields were estimated. 2.2.2. Effect of NaHCO3 on B. braunii (N-836) A set of 500 mL Erlenmeyer conical flasks were taken and 200 mL of modified Chu 13 medium was distributed and sodium bicarbonate was added in the range of 0.05–0.1% to the flasks. Two weeks old culture of B. braunii (N-836) grown in modified Chu 13 was used as inoculum at 25% (v/v). The culture flasks were incubated for 25 days at 26 ± 1 °C temperature under 20 lmol photons m 2 s 1 light intensity and 16:8 h light dark cycle. All the experiments were carried out in triplicates.
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content in the pooled extract was estimated by reading absorbance at 645 and 661.5 nm using spectrophotometer and quantified by the method of Lichtenthaler (1987). 2.3.3. Carbohydrate estimation A known quantity of cell free (spent) medium was taken and analyzed for total carbohydrate using phenol–sulfuric acid method (Dubois et al., 1956). 2.3.4. Hydrocarbon extraction The dry biomass was homogenized in mortar and pestle with nhexane for 15 min and centrifuged. The extraction process was repeated twice and supernatant was transferred to pre-weighed glass vial and evaporated under the stream of nitrogen to complete dryness. The quantity of residue was measured gravimetrically (Sawayama et al., 1992) and hydrocarbon content was expressed as percent of dry weight. 2.3.5. Hydrocarbon analysis Hydrocarbon extract was purified by column chromatography on silica gel. The hydrocarbon sample was analyzed using ELITE 5 capillary column. The conditions used were as per Dayananda et al. (2005). The initial temperature of oven was at 130 °C for 5 min which was increased to 200 °C at the rate of 8 °C per minute. After maintaining at 200 °C for 2 min, the temperature was increased to 280 °C at the rate of 5 °C/min and maintained for 15 min. The injector port and the detector temperatures were 240 °C and 250 °C respectively. Hydrocarbons were grouped into three categories as less than C20, higher than C30 and in between C20 and C30 with reference to their elution with that of the retention times of the internal standard. 2.3.6. Fatty acid analysis The lipids were extracted with chloroform–methanol (2:1, v/v) and quantified gravimetrically. The lipid sample was dissolved in benzene and 5% methanolic hydrogen chloride (95 mL chilled methanol + 5 mL of acetyl chloride). The mixture was refluxed for 2 h and then 5% sodium chloride solution was added and the fatty acid methyl esters (FAME) were extracted with hexane. The hexane layer was washed with 2% potassium bicarbonate solution and dried over anhydrous sodium sulphate (Christie, 1982). FAME were analyzed by GC–MS (PerkinElmer, Turbomass Gold, Mass spectrometer) equipped with FID using SPB-1 (poly(dimethysiloxane)) capillary column (30 m 0.32 mm ID 0.25 lm film thickness) with a temperature programming 150–280 °C at a rate of 5 °C min 1. The FAME were identified by comparing their fragmentation pattern with authentic standards (Sigma) and also with NIST library. 2.4. Statistical analysis Results were expressed as the mean ± SD of three replicates. Difference between the groups were statistically analyzed by using one-way ANOVA.
2.3. Analytical methods 3. Results and discussion 2.3.1. Biomass estimation The cultures were harvested by centrifugation at 5000 rpm and the cells were washed with distilled water. The pellet was freeze dried. The dry weight of algal biomass was determined gravimetrically and growth was expressed in terms of dry weight (g L 1). 2.3.2. Chlorophyll estimation A known volume of B. braunii culture was centrifuged and the residue was extracted with methanol repeatedly. The chlorophyll
3.1. Growth and hydrocarbon production of B. braunii (LB-572) in raceway and circular ponds under outdoor conditions B. braunii (LB-572) was able to grow in raceway and circular ponds under outdoor conditions. The biomass yields were observed at different time intervals in raceway and circular ponds. Marginally higher chlorophyll (26 lg mL 1) and biomass (1.8 g L 1) contents were achieved in raceway pond at the end of
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the 18th day as shown in Fig. 1A and B. Variation in pH (7.5–9.5) was observed during the growth of alga. As B. braunii (LB-572) strain is known to produce hydrocarbons and polysaccharides, these were estimated during cultivation of alga. Hydrocarbon content in B. braunii was found to be higher in raceway pond (Fig. 1C). After 18 days of cultivation, hydrocarbon content was found to be 24% and 19% in raceway and circular ponds respectively. Maximum hydrocarbon content was observed in raceway pond. However the hydrocarbon profile as analyzed by GC indicated that relative proportion of hydrocarbons of less than C20, between C20–C30 and higher than C30 was not changed much between the alga grown in raceway pond and that grown in circular pond (Table 1). Fig. 1D shows that the carbohydrate content in the medium as analyzed in terms of total sugars increased with the growth of the alga. The maximum carbohydrate content was observed in race-
Table 1 Hydrocarbon profile of B. braunii (LB-572) in raceway and circular ponds under outdoor conditions as analyzed by GC. B. braunii (Race ‘A’)
Less than C20 (%)
Between C20–C30 (%)
Higher than C30 (%)
Raceway pond Circular pond
11.20 ± 1.54 28.23 ± 3.38
60.39 ± 4.26 51.45 ± 2.13
28.41 ± 3.19 20.32 ± 2.81
Data represents mean ± SD of three replicates. Data recorded for 18 day old culture.
Table 2 Fatty acid profile of B. braunii (LB-572) in raceway and circular ponds under outdoor conditions as analyzed by GC. Fatty acid
Raceway pond (%)
Circular pond (%)
16:0 16:1 18:0 18:1 18:2 22:0 22:1 24:0
16.52 ± 0.15 15.45 ± 0.07 8.21 ± 0.01 34.23 ± 0.13 12.26 ± 0.11 3.87 ± 0.05 5.27 ± 0.09 1.69 ± 0.01
22.13 ± 0.04 12.5 ± 0.08 5.19 ± 0.11 28.35 ± 0.07 16.24 ± 0.10 2.31 ± 0.09 8.42 ± 0.06 Trace
Data represents mean ± SD of three replicates. Data recorded for 18 day old culture.
way pond at the end of the 18 days of cultivation. The total fat content of the alga was found in the range of 20–24% (w/w). The fatty acid profile is shown in Table 2, which indicated the presence of C16:0, C16:1, C18:0, C18:1, C18:2 and C22:0 fatty acids in both the ponds with variation in their relative proportion. Palmitic and oleic acids were the major fatty acids in both raceway and circular ponds. 3.2. Seasonal variation on growth, biomass yield and hydrocarbon production of B. braunii (LB-572) in raceway pond The seasonal variation on growth, biomass yield and hydrocarbon production of B. braunii in raceway and circular ponds were evaluated. During Jan–Dec period the hydrocarbon content was estimated in raceway pond. May onwards due to seasonal continuous rains the outdoor culture got diluted frequently which resulted in lower biomass yields. As shown in Fig. 2 the biomass yields were lower during rainy season (Jun–Aug). Maximum biomass (2 g L 1) was obtained during winter (Oct–Dec). Hydrocarbon production correlated with biomass yields and maximum hydrocarbon content 28% (w/w) was observed during winter season (Nov–Dec). The hydrocarbon profile was analyzed by GC data (Table 3). During Nov–Dec month higher than C30 hydrocarbons increased compared to other seasons.
Fig. 1. Biomass yield (A), chlorophyll (B), hydrocarbon (C) and carbohydrate (D) of B. braunii (LB-572) in raceway and circular ponds under outdoor conditions. Data represents mean ± SD of three replicates. Data recorded for 18 day old culture.
Fig. 2. Biomass yield and hydrocarbon production of B. braunii (LB-572) in raceway pond under outdoor conditions during different months in a year. Data represents mean ± SD of three replicates. Data recorded for 18 day old culture.
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A. Ranga Rao et al. / Bioresource Technology 123 (2012) 528–533 Table 3 Hydrocarbon profile of B. braunii (LB-572) in raceway pond during different seasons in a year as analyzed by GC. Season
Table 4 Hydrocarbon profile of B. braunii (N-836) in raceway and circular ponds as analyzed by GC.
Max temp (°C)
Less than C20 (%)
Between C20–C30 (%)
Higher than C30 (%)
Time in (days)
Winter Oct Nov Dec Jan Feb
25 ± 1 27 ± 1 29 ± 1 28 ± 1 31 ± 1
10.00 ± 0.98 11.31 ± 1.65 10.25 ± 1.23 17.48 ± 2.31 15.12 ± 3.21
33.36 ± 2.76 22.40 ± 3.45 21.13 ± 2.01 33.10 ± 3.65 32.72 ± 3.01
56.24 ± 5.23 66.22 ± 5.81 68.22 ± 4.96 49.32 ± 5.67 52.34 ± 4.31
Summer Mar Apr May
32 ± 1 31 ± 1 29 ± 1
13.22 ± 0.95 13.79 ± 1.53 11.25 ± 2.45
37.18 ± 4.25 31.18 ± 2.86 41.51 ± 3.09
49.60 ± 4.98 55.03 ± 5.96 47.24 ± 3.78
Rainy Jun Jul Aug Sep
24 ± 1 23 ± 1 23 ± 1 25 ± 1
14.20 ± 1.97 18.20 ± 2.35 15.90 ± 1.14 10.25 ± 0.76
51.38 ± 4.32 52.08 ± 3.92 59.55 ± 4.41 34.06 ± 2.38
34.41 ± 4.25 29.83 ± 3.48 24.55 ± 2.05 55.59 ± 6.72
Data represents mean ± SD of three replicates. Data recorded for 18 day old culture.
3.3. Growth and hydrocarbon production in B. braunii (N-836) culture under outdoor conditions Since B. braunii (LB-572) was found to be of ‘A’ race producing saturated higher hydrocarbons, N-836 which is ‘B’ race was selected for outdoor cultivation to compare the hydrocarbon yields in A & B races and their adaptability to outdoor conditions. Growth and hydrocarbon yields of B. braunii (N-836) grown in both circular and raceway ponds are shown in Fig. 3A–B. The biomass yield and
Less than C20 (%)
Between C20–C30 (%)
Higher than C30 (%)
Raceway 5 10 15 20 25
25.92 ± 2.65 19.34 ± 2.54 12.22 ± 1.98 10.46 ± 1.06 9.34 ± 2.38
54.32 ± 3.24 42.80 ± 3.98 45.24 ± 2.16 43.28 ± 3.76 38.62 ± 3.98
19.76 ± 2.06 37.86 ± 3.75 42.54 ± 5.11 46.22 ± 3.83 52.03 ± 4.65
Circular 5 10 15 20 25
22.81 ± 1.23 19.25 ± 3.02 15.03 ± 2.87 12.61 ± 1.53 6.78 ± 0.54
55.06 ± 2.87 48.93 ± 2.91 49.79 ± 3.20 47.02 ± 4.52 46.06 ± 2.10
21.69 ± 2.50 32.41 ± 2.17 35.13 ± 4.28 40.19 ± 5.13 47.16 ± 4.09
Data represents mean ± SD of three replicates. Data recorded on 25 day of old culture.
hydrocarbon content was estimated at five day intervals. After 25 days of cultivation in outdoor pond maximum biomass yield of 1 g L 1 with 27% hydrocarbon (w/w) content was obtained. The hydrocarbon profile as analyzed by GC indicated that major proportion of hydrocarbons after 25 days of outdoor cultivation were of higher than C30 chain length (Table 4). 3.4. Effect of NaHCO3 on growth and hydrocarbon production in B. braunii strain (N-836) B. braunii (N-836) was evaluated for growth and metabolite production at different levels of NaHCO3. It was found that 0.1% (w/v) NaHCO3 favored rapid growth resulting in increased biomass accumulation and hydrocarbon production at the end of the experimental period. B. braunii strain (N-836) was able to grow at all the concentrations of NaHCO3 (0.01%, 0.020%, 0.050% and 0.1%) tested. The biomass yields were analyzed after 25 days. The biomass yields were found to increase with increasing concentrations of NaHCO3 and maximum biomass was achieved at 0.1% NaHCO3 concentration (Fig. 3C). Hydrocarbon content in B. braunii was found to be similar to growth pattern as shown in Fig. 3C. Hydrocarbon content varied in the range of 16–30% at different NaHCO3 levels and maximum hydrocarbon content was found at 0.1% NaHCO3. The hydrocarbon profile as analyzed by GC, indicated that C30 category hydrocarbon level increased up to NaHCO3 concentration of 0.05% while C20 category hydrocarbons decreased (Table 5). The unicellular photosynthetic micro alga B. braunii is a member of the chlorophyceae which produces hydrocarbons. To date only a limited number of micro algae such as Dunaliella (high salinity), Spirulina (high alkalinity), and Chlorella (high nutrient) have been maintained as monocultures and successfully cultivated in open raceway ponds for using commercially. These micro algae were mass cultured in custom made raceway and circular ponds for using as a source of biomass and biomolecules. Raceway ponds are most widely utilized at the industrial level of algal biomass production in many countries like Israel, United
Table 5 Effect of NaHCO3 on hydrocarbon profile in B. braunii (N-836) as analyzed by GC.
Fig. 3. Biomass yield (A) and hydrocarbon content (B) of B. braunii (N-836) in raceway and circular ponds under outdoor conditions (C). Effect of NaHCO3 on biomass and hydrocarbon content. Data represents mean ± SD of three replicates. Data recorded for 25 day old culture.
NaHCO3 (%)
Less than C20 (%)
Between C20–C24 (%)
Higher than C30 (%)
Control 0.025 0.05 0.1
24.23 ± 2.81 14.64 ± 0.98 11.38 ± 2.33 9.76 ± 1.27
29.44 ± 3.29 34.10 ± 2.11 22.40 ± 2.14 22.97 ± 2.37
46.32 ± 5.32 51.26 ± 4.87 66.22 ± 5.76 67.27 ± 4.30
Data represents mean ± SD of three replicates. Data recorded for 25 day old culture.
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States of America, China, Japan, Taiwan and Indonesia. The present study attempted to cultivate B. braunii in open raceway and circular ponds. In our earlier studies on B. braunii sp. under indoor (controlled) conditions we reported maximum biomass yield of 2.0 and 2.8 g L 1 and hydrocarbon content of 46% and 33% on dry weight in B. braunii SAG 30.81 and LB-572 respectively under 16:8 h light and dark cycle with 1.2 ± 0.2 klux light intensity at 25 ± 1 °C temperature (Dayananda et al., 2005, 2007). It was also reported that the hydrocarbon content varied in the range of 14–28% at different CO2 levels and the fat content in the range of 25–30% in different species of B. braunii (Ranga Rao et al., 2007a). Various algae are reported to accumulate high levels of secondary metabolites under various stress conditions (Banerjee et al., 2002; Ranga Rao et al., 2010). Fang et al., 2004 reported palmitic acid and oleic acids as major components in the Botryococcus sp. Ashok Kumar and Rengasamy (2012) reported that oleic, linolenic and palmitic fatty acids were the major fatty acids in B. braunii Kutz (AP-103). B braunii (B. mahabali) was scaled up in open raceway ponds in batch mode and the biomass yields were found to be 2 g L 1 (w/w) (Dayananda et al., 2010). Present results showed that both the B. braunii strains (LB-572 and N-836) were successfully grown under outdoor conditions although they differed in biomass yields and hydrocarbon content. Interestingly these strains differed in the type of hydrocarbons they produce. B. braunii (LB-572) belong to ‘A’ race producing saturated hydrocarbons and alkadienes while B. braunii (N-836) belongs to race ‘B’, which produces triterpenoid hydrocarbons known as botryococcenes (Dayananda et al., 2006). The yields in terms of biomass and hydrocarbon are found to be more with ‘A’ race compared to ‘B’ race. The B. braunii (LB-572) culture also exhibited seasonal variations in growth and hydrocarbon profiles (Fig. 3 and Table 3). Surprisingly growth and hydrocarbon yields in both the strains were considerably less under outdoor conditions when compared to that obtained under controlled conditions (Sakamoto et al., 2012). This may be possibly due to the exposure of the culture to different light intensities and temperatures during the day night cycles unlike in controlled cultures. Hu and Richmond (1996) also suggested that optimum biomass concentration was difficult to achieve in raceway ponds at high and low irradiances of day/night. Thus, it is practically impossible to operate at optimum cell concentration for the whole range of irradiance, which changes throughout the day. Moreover Guterman et al. (1989) suggested that the biosyntheses of outdoor micro algal cultures lag behind photosynthesis, which responds to rapidly changing irradiance in the day. Although the biomass yields were improved by bicarbonate addition, occasional CO2 bubbling and mode of cultivation (Yaming et al., 2011), increasing the metabolite production under outdoor condition is a challenging aspect. Since hydrocarbons are accumulated in the intercellular spaces of the cells the shear forces under outdoor conditions might be disrupting the colony morphology thereby the accumulation sites or the cultures are amenable to hydrocarbon degrading micro flora under outdoor conditions. Selective adaptation of B. braunii to extreme conditions would be an alternative as monoculture of algae is usually achieved by maintaining an extreme culture environment, such as high salinity, high alkalinity and high nutritional status (Lee, 1986). Further Ranga Rao et al. (2007b) reported that the biomass yields increased with increasing concentration of sodium chloride and maximum biomass yield was achieved in 17 mM and 34 mM NaCl and the hydrocarbon content varied in the range of 12–28% in different salinities and maximum hydrocarbon content was observed in 51 mM and 68 mM NaCl. Detailed studies are necessary on B. braunii to make its outdoor cultivation a commercial viability for obtaining high content of hydrocarbons.
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Cultivation of green alga Botryococcus braunii in raceway, circular ponds under outdoor conditions and its growth, hydrocarbon production A. Ranga Rao 1, G.A. Ravishankar, R. Sarada ⇑ Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Institute, Mysore 570 020, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" B. braunii (LB-572 and N-836) strains
Cultivation of Botryococcus braunii in raceway and circular ponds.
" "
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were used for the current study. Cultivated LB-572 and N-836 in both raceway & circular ponds. Evaluated biomass yield, hydrocarbon content and fatty acid profile. Biomass and hydrocarbon content were observed in various seasons. Effect of NaHCO3 on biomass and hydrocarbon production in N-836 were evaluated.
a r t i c l e
i n f o
Article history: Received 16 April 2012 Received in revised form 6 July 2012 Accepted 7 July 2012 Available online 26 July 2012 Keywords: Microalgae Botryococcus braunii Outdoor cultivation Hydrocarbon Fatty acids
a b s t r a c t The present study focused on cultivation, seasonal variation in growth, hydrocarbon production, fatty acids profiles of Botryococcus braunii (LB-572 and N-836) in raceway & circular ponds under outdoor conditions. After 18 days of cultivation the biomass yield and hydrocarbon contents were increased in both raceway and circular ponds. The fat content was found to be around 24% (w/w) with palmitic and oleic acids as prominent fatty acids. Hydrocarbons of C20–C30 carbon chain length were higher in raceway and circular ponds. Maximum biomass yield (2 g L 1) and hydrocarbon content (28%) were observed in Nov– Dec. In case of B. braunii (N-836) after 25 days of cultivation the biomass yield was 1 g L 1 and hydrocarbon content was 27%. Supplementation of 0.1% NaHCO3 in the medium resulted in biomass yield of 1.5 g L 1 and hydrocarbon content of 30% compared to control. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Botryococcus braunii is a green colonial fresh water micro alga which produces hydrocarbons. It is recognized as one of the renewable resource for the production of hydrocarbons. Three races of B. braunii have been documented, and they are differentiated on the basis of the characteristic hydrocarbons they produce. The ‘A’ race produces odd numbered C25 to C31, n-alkadienes and trienes. The ⇑ Corresponding author. Tel.: +91 821 2516501; fax: +91 821 2517233. E-mail addresses: [email protected], [email protected] (R. Sarada). Present address: Department of Applied Sciences and Mathematics, Arizona State University, 7001 E. Williams Field Road, Mesa, AZ 85212, USA. 1
0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.07.009
‘B’ race produces triterpenoid hydrocarbons known as botryococcenes and ‘L’ race produces lycopadiene, a C40 tetraterpene. Another difference among the races is the colony color in the stationary phase. Race ‘A’ and ‘B’ strains are known to produce exopolysaccharides up to 250 g m3, whereas ‘L’ race produced up to 1 kg m3 (Banerjee et al., 2002; Niehaus et al., 2011). However the amount of exopolysaccharides production varies with the strains and the culture conditions. B. braunii is a promising renewable resource for the production of hydrocarbons and it has been reported that on hydrocracking, the distillate yields 67% gasoline, 15% aviation turbine fuel, 15% diesel fuel and 3% residual oil (Hillen et al., 1982; Samori et al., 2010). B. braunii is also known to produce large amounts of fatty
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acids. The quantity and composition of fatty acids varies with species and also among the races (Grice et al., 1998; Metzger et al., 1990; Niitsu et al., 2011; Weiss et al., 2010). Being a photosynthetic organism, it can reduce CO2 emissions by 1.5 105 tons y 1 per 8.4 103 ha of micro algal cultivation area would be necessary (Sawayama et al., 1999; Tanoi et al., 2011). The alga B. braunii produces hydrocarbons in the range of 2–86% (on dry weight basis). This variation in the content of hydrocarbon is due to the differences among different strains/races in the production of hydrocarbons and changes in cultural and physiological conditions (Barupal et al., 2010; Samori et al., 2010). Dayananda et al. (2005) reported optimization of media constituents for growth and hydrocarbon production in B. braunii (SAG 30.81). In the changing energy scenario it is necessary to exploit the potential of this microalga as a source of hydrocarbons. One of the important strategies may be adaptation of the organism for outdoor cultivation. The present study focused on the growth and hydrocarbon production of B. braunii in both raceway and circular ponds under outdoor culture conditions. 2. Methods 2.1. Micro algal strains B. braunii strains were obtained from various culture collection centers such as B. braunii (LB-572, ‘A’ race) from UTEX culture collection, USA and B. braunii (N-836, ‘B’ race) from National Institute for Environmental Studies, Tsukuba, Japan respectively. The stock cultures were maintained both in agar slants and liquid medium of modified Chu 13 (Largeau et al., 1980). 2.2. Experimental design 2.2.1. Cultivation of B. braunii in raceway and circular ponds Forty litres medium was inoculated with 25% (v/v) of B. braunii culture and grown in raceway pond (Length 1.13 m; Width 0.6 m; Depth 0.3 m) under outdoor conditions with 15 rpm agitation. The culture volume was increased gradually to the pond capacity (80 L) and then continued cultivation for a period of 18 days. Forty litres medium was inoculated with 25% (v/v) of B. braunii culture and grown in circular ponds (Diameter 1.21 m; Depth 0. 25 m) under outdoor conditions without agitation. The cultures were mixed twice a day manually. One batch culture was run for a period of 25 days and the yields were estimated. 2.2.2. Effect of NaHCO3 on B. braunii (N-836) A set of 500 mL Erlenmeyer conical flasks were taken and 200 mL of modified Chu 13 medium was distributed and sodium bicarbonate was added in the range of 0.05–0.1% to the flasks. Two weeks old culture of B. braunii (N-836) grown in modified Chu 13 was used as inoculum at 25% (v/v). The culture flasks were incubated for 25 days at 26 ± 1 °C temperature under 20 lmol photons m 2 s 1 light intensity and 16:8 h light dark cycle. All the experiments were carried out in triplicates.
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content in the pooled extract was estimated by reading absorbance at 645 and 661.5 nm using spectrophotometer and quantified by the method of Lichtenthaler (1987). 2.3.3. Carbohydrate estimation A known quantity of cell free (spent) medium was taken and analyzed for total carbohydrate using phenol–sulfuric acid method (Dubois et al., 1956). 2.3.4. Hydrocarbon extraction The dry biomass was homogenized in mortar and pestle with nhexane for 15 min and centrifuged. The extraction process was repeated twice and supernatant was transferred to pre-weighed glass vial and evaporated under the stream of nitrogen to complete dryness. The quantity of residue was measured gravimetrically (Sawayama et al., 1992) and hydrocarbon content was expressed as percent of dry weight. 2.3.5. Hydrocarbon analysis Hydrocarbon extract was purified by column chromatography on silica gel. The hydrocarbon sample was analyzed using ELITE 5 capillary column. The conditions used were as per Dayananda et al. (2005). The initial temperature of oven was at 130 °C for 5 min which was increased to 200 °C at the rate of 8 °C per minute. After maintaining at 200 °C for 2 min, the temperature was increased to 280 °C at the rate of 5 °C/min and maintained for 15 min. The injector port and the detector temperatures were 240 °C and 250 °C respectively. Hydrocarbons were grouped into three categories as less than C20, higher than C30 and in between C20 and C30 with reference to their elution with that of the retention times of the internal standard. 2.3.6. Fatty acid analysis The lipids were extracted with chloroform–methanol (2:1, v/v) and quantified gravimetrically. The lipid sample was dissolved in benzene and 5% methanolic hydrogen chloride (95 mL chilled methanol + 5 mL of acetyl chloride). The mixture was refluxed for 2 h and then 5% sodium chloride solution was added and the fatty acid methyl esters (FAME) were extracted with hexane. The hexane layer was washed with 2% potassium bicarbonate solution and dried over anhydrous sodium sulphate (Christie, 1982). FAME were analyzed by GC–MS (PerkinElmer, Turbomass Gold, Mass spectrometer) equipped with FID using SPB-1 (poly(dimethysiloxane)) capillary column (30 m 0.32 mm ID 0.25 lm film thickness) with a temperature programming 150–280 °C at a rate of 5 °C min 1. The FAME were identified by comparing their fragmentation pattern with authentic standards (Sigma) and also with NIST library. 2.4. Statistical analysis Results were expressed as the mean ± SD of three replicates. Difference between the groups were statistically analyzed by using one-way ANOVA.
2.3. Analytical methods 3. Results and discussion 2.3.1. Biomass estimation The cultures were harvested by centrifugation at 5000 rpm and the cells were washed with distilled water. The pellet was freeze dried. The dry weight of algal biomass was determined gravimetrically and growth was expressed in terms of dry weight (g L 1). 2.3.2. Chlorophyll estimation A known volume of B. braunii culture was centrifuged and the residue was extracted with methanol repeatedly. The chlorophyll
3.1. Growth and hydrocarbon production of B. braunii (LB-572) in raceway and circular ponds under outdoor conditions B. braunii (LB-572) was able to grow in raceway and circular ponds under outdoor conditions. The biomass yields were observed at different time intervals in raceway and circular ponds. Marginally higher chlorophyll (26 lg mL 1) and biomass (1.8 g L 1) contents were achieved in raceway pond at the end of
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the 18th day as shown in Fig. 1A and B. Variation in pH (7.5–9.5) was observed during the growth of alga. As B. braunii (LB-572) strain is known to produce hydrocarbons and polysaccharides, these were estimated during cultivation of alga. Hydrocarbon content in B. braunii was found to be higher in raceway pond (Fig. 1C). After 18 days of cultivation, hydrocarbon content was found to be 24% and 19% in raceway and circular ponds respectively. Maximum hydrocarbon content was observed in raceway pond. However the hydrocarbon profile as analyzed by GC indicated that relative proportion of hydrocarbons of less than C20, between C20–C30 and higher than C30 was not changed much between the alga grown in raceway pond and that grown in circular pond (Table 1). Fig. 1D shows that the carbohydrate content in the medium as analyzed in terms of total sugars increased with the growth of the alga. The maximum carbohydrate content was observed in race-
Table 1 Hydrocarbon profile of B. braunii (LB-572) in raceway and circular ponds under outdoor conditions as analyzed by GC. B. braunii (Race ‘A’)
Less than C20 (%)
Between C20–C30 (%)
Higher than C30 (%)
Raceway pond Circular pond
11.20 ± 1.54 28.23 ± 3.38
60.39 ± 4.26 51.45 ± 2.13
28.41 ± 3.19 20.32 ± 2.81
Data represents mean ± SD of three replicates. Data recorded for 18 day old culture.
Table 2 Fatty acid profile of B. braunii (LB-572) in raceway and circular ponds under outdoor conditions as analyzed by GC. Fatty acid
Raceway pond (%)
Circular pond (%)
16:0 16:1 18:0 18:1 18:2 22:0 22:1 24:0
16.52 ± 0.15 15.45 ± 0.07 8.21 ± 0.01 34.23 ± 0.13 12.26 ± 0.11 3.87 ± 0.05 5.27 ± 0.09 1.69 ± 0.01
22.13 ± 0.04 12.5 ± 0.08 5.19 ± 0.11 28.35 ± 0.07 16.24 ± 0.10 2.31 ± 0.09 8.42 ± 0.06 Trace
Data represents mean ± SD of three replicates. Data recorded for 18 day old culture.
way pond at the end of the 18 days of cultivation. The total fat content of the alga was found in the range of 20–24% (w/w). The fatty acid profile is shown in Table 2, which indicated the presence of C16:0, C16:1, C18:0, C18:1, C18:2 and C22:0 fatty acids in both the ponds with variation in their relative proportion. Palmitic and oleic acids were the major fatty acids in both raceway and circular ponds. 3.2. Seasonal variation on growth, biomass yield and hydrocarbon production of B. braunii (LB-572) in raceway pond The seasonal variation on growth, biomass yield and hydrocarbon production of B. braunii in raceway and circular ponds were evaluated. During Jan–Dec period the hydrocarbon content was estimated in raceway pond. May onwards due to seasonal continuous rains the outdoor culture got diluted frequently which resulted in lower biomass yields. As shown in Fig. 2 the biomass yields were lower during rainy season (Jun–Aug). Maximum biomass (2 g L 1) was obtained during winter (Oct–Dec). Hydrocarbon production correlated with biomass yields and maximum hydrocarbon content 28% (w/w) was observed during winter season (Nov–Dec). The hydrocarbon profile was analyzed by GC data (Table 3). During Nov–Dec month higher than C30 hydrocarbons increased compared to other seasons.
Fig. 1. Biomass yield (A), chlorophyll (B), hydrocarbon (C) and carbohydrate (D) of B. braunii (LB-572) in raceway and circular ponds under outdoor conditions. Data represents mean ± SD of three replicates. Data recorded for 18 day old culture.
Fig. 2. Biomass yield and hydrocarbon production of B. braunii (LB-572) in raceway pond under outdoor conditions during different months in a year. Data represents mean ± SD of three replicates. Data recorded for 18 day old culture.
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A. Ranga Rao et al. / Bioresource Technology 123 (2012) 528–533 Table 3 Hydrocarbon profile of B. braunii (LB-572) in raceway pond during different seasons in a year as analyzed by GC. Season
Table 4 Hydrocarbon profile of B. braunii (N-836) in raceway and circular ponds as analyzed by GC.
Max temp (°C)
Less than C20 (%)
Between C20–C30 (%)
Higher than C30 (%)
Time in (days)
Winter Oct Nov Dec Jan Feb
25 ± 1 27 ± 1 29 ± 1 28 ± 1 31 ± 1
10.00 ± 0.98 11.31 ± 1.65 10.25 ± 1.23 17.48 ± 2.31 15.12 ± 3.21
33.36 ± 2.76 22.40 ± 3.45 21.13 ± 2.01 33.10 ± 3.65 32.72 ± 3.01
56.24 ± 5.23 66.22 ± 5.81 68.22 ± 4.96 49.32 ± 5.67 52.34 ± 4.31
Summer Mar Apr May
32 ± 1 31 ± 1 29 ± 1
13.22 ± 0.95 13.79 ± 1.53 11.25 ± 2.45
37.18 ± 4.25 31.18 ± 2.86 41.51 ± 3.09
49.60 ± 4.98 55.03 ± 5.96 47.24 ± 3.78
Rainy Jun Jul Aug Sep
24 ± 1 23 ± 1 23 ± 1 25 ± 1
14.20 ± 1.97 18.20 ± 2.35 15.90 ± 1.14 10.25 ± 0.76
51.38 ± 4.32 52.08 ± 3.92 59.55 ± 4.41 34.06 ± 2.38
34.41 ± 4.25 29.83 ± 3.48 24.55 ± 2.05 55.59 ± 6.72
Data represents mean ± SD of three replicates. Data recorded for 18 day old culture.
3.3. Growth and hydrocarbon production in B. braunii (N-836) culture under outdoor conditions Since B. braunii (LB-572) was found to be of ‘A’ race producing saturated higher hydrocarbons, N-836 which is ‘B’ race was selected for outdoor cultivation to compare the hydrocarbon yields in A & B races and their adaptability to outdoor conditions. Growth and hydrocarbon yields of B. braunii (N-836) grown in both circular and raceway ponds are shown in Fig. 3A–B. The biomass yield and
Less than C20 (%)
Between C20–C30 (%)
Higher than C30 (%)
Raceway 5 10 15 20 25
25.92 ± 2.65 19.34 ± 2.54 12.22 ± 1.98 10.46 ± 1.06 9.34 ± 2.38
54.32 ± 3.24 42.80 ± 3.98 45.24 ± 2.16 43.28 ± 3.76 38.62 ± 3.98
19.76 ± 2.06 37.86 ± 3.75 42.54 ± 5.11 46.22 ± 3.83 52.03 ± 4.65
Circular 5 10 15 20 25
22.81 ± 1.23 19.25 ± 3.02 15.03 ± 2.87 12.61 ± 1.53 6.78 ± 0.54
55.06 ± 2.87 48.93 ± 2.91 49.79 ± 3.20 47.02 ± 4.52 46.06 ± 2.10
21.69 ± 2.50 32.41 ± 2.17 35.13 ± 4.28 40.19 ± 5.13 47.16 ± 4.09
Data represents mean ± SD of three replicates. Data recorded on 25 day of old culture.
hydrocarbon content was estimated at five day intervals. After 25 days of cultivation in outdoor pond maximum biomass yield of 1 g L 1 with 27% hydrocarbon (w/w) content was obtained. The hydrocarbon profile as analyzed by GC indicated that major proportion of hydrocarbons after 25 days of outdoor cultivation were of higher than C30 chain length (Table 4). 3.4. Effect of NaHCO3 on growth and hydrocarbon production in B. braunii strain (N-836) B. braunii (N-836) was evaluated for growth and metabolite production at different levels of NaHCO3. It was found that 0.1% (w/v) NaHCO3 favored rapid growth resulting in increased biomass accumulation and hydrocarbon production at the end of the experimental period. B. braunii strain (N-836) was able to grow at all the concentrations of NaHCO3 (0.01%, 0.020%, 0.050% and 0.1%) tested. The biomass yields were analyzed after 25 days. The biomass yields were found to increase with increasing concentrations of NaHCO3 and maximum biomass was achieved at 0.1% NaHCO3 concentration (Fig. 3C). Hydrocarbon content in B. braunii was found to be similar to growth pattern as shown in Fig. 3C. Hydrocarbon content varied in the range of 16–30% at different NaHCO3 levels and maximum hydrocarbon content was found at 0.1% NaHCO3. The hydrocarbon profile as analyzed by GC, indicated that C30 category hydrocarbon level increased up to NaHCO3 concentration of 0.05% while C20 category hydrocarbons decreased (Table 5). The unicellular photosynthetic micro alga B. braunii is a member of the chlorophyceae which produces hydrocarbons. To date only a limited number of micro algae such as Dunaliella (high salinity), Spirulina (high alkalinity), and Chlorella (high nutrient) have been maintained as monocultures and successfully cultivated in open raceway ponds for using commercially. These micro algae were mass cultured in custom made raceway and circular ponds for using as a source of biomass and biomolecules. Raceway ponds are most widely utilized at the industrial level of algal biomass production in many countries like Israel, United
Table 5 Effect of NaHCO3 on hydrocarbon profile in B. braunii (N-836) as analyzed by GC.
Fig. 3. Biomass yield (A) and hydrocarbon content (B) of B. braunii (N-836) in raceway and circular ponds under outdoor conditions (C). Effect of NaHCO3 on biomass and hydrocarbon content. Data represents mean ± SD of three replicates. Data recorded for 25 day old culture.
NaHCO3 (%)
Less than C20 (%)
Between C20–C24 (%)
Higher than C30 (%)
Control 0.025 0.05 0.1
24.23 ± 2.81 14.64 ± 0.98 11.38 ± 2.33 9.76 ± 1.27
29.44 ± 3.29 34.10 ± 2.11 22.40 ± 2.14 22.97 ± 2.37
46.32 ± 5.32 51.26 ± 4.87 66.22 ± 5.76 67.27 ± 4.30
Data represents mean ± SD of three replicates. Data recorded for 25 day old culture.
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States of America, China, Japan, Taiwan and Indonesia. The present study attempted to cultivate B. braunii in open raceway and circular ponds. In our earlier studies on B. braunii sp. under indoor (controlled) conditions we reported maximum biomass yield of 2.0 and 2.8 g L 1 and hydrocarbon content of 46% and 33% on dry weight in B. braunii SAG 30.81 and LB-572 respectively under 16:8 h light and dark cycle with 1.2 ± 0.2 klux light intensity at 25 ± 1 °C temperature (Dayananda et al., 2005, 2007). It was also reported that the hydrocarbon content varied in the range of 14–28% at different CO2 levels and the fat content in the range of 25–30% in different species of B. braunii (Ranga Rao et al., 2007a). Various algae are reported to accumulate high levels of secondary metabolites under various stress conditions (Banerjee et al., 2002; Ranga Rao et al., 2010). Fang et al., 2004 reported palmitic acid and oleic acids as major components in the Botryococcus sp. Ashok Kumar and Rengasamy (2012) reported that oleic, linolenic and palmitic fatty acids were the major fatty acids in B. braunii Kutz (AP-103). B braunii (B. mahabali) was scaled up in open raceway ponds in batch mode and the biomass yields were found to be 2 g L 1 (w/w) (Dayananda et al., 2010). Present results showed that both the B. braunii strains (LB-572 and N-836) were successfully grown under outdoor conditions although they differed in biomass yields and hydrocarbon content. Interestingly these strains differed in the type of hydrocarbons they produce. B. braunii (LB-572) belong to ‘A’ race producing saturated hydrocarbons and alkadienes while B. braunii (N-836) belongs to race ‘B’, which produces triterpenoid hydrocarbons known as botryococcenes (Dayananda et al., 2006). The yields in terms of biomass and hydrocarbon are found to be more with ‘A’ race compared to ‘B’ race. The B. braunii (LB-572) culture also exhibited seasonal variations in growth and hydrocarbon profiles (Fig. 3 and Table 3). Surprisingly growth and hydrocarbon yields in both the strains were considerably less under outdoor conditions when compared to that obtained under controlled conditions (Sakamoto et al., 2012). This may be possibly due to the exposure of the culture to different light intensities and temperatures during the day night cycles unlike in controlled cultures. Hu and Richmond (1996) also suggested that optimum biomass concentration was difficult to achieve in raceway ponds at high and low irradiances of day/night. Thus, it is practically impossible to operate at optimum cell concentration for the whole range of irradiance, which changes throughout the day. Moreover Guterman et al. (1989) suggested that the biosyntheses of outdoor micro algal cultures lag behind photosynthesis, which responds to rapidly changing irradiance in the day. Although the biomass yields were improved by bicarbonate addition, occasional CO2 bubbling and mode of cultivation (Yaming et al., 2011), increasing the metabolite production under outdoor condition is a challenging aspect. Since hydrocarbons are accumulated in the intercellular spaces of the cells the shear forces under outdoor conditions might be disrupting the colony morphology thereby the accumulation sites or the cultures are amenable to hydrocarbon degrading micro flora under outdoor conditions. Selective adaptation of B. braunii to extreme conditions would be an alternative as monoculture of algae is usually achieved by maintaining an extreme culture environment, such as high salinity, high alkalinity and high nutritional status (Lee, 1986). Further Ranga Rao et al. (2007b) reported that the biomass yields increased with increasing concentration of sodium chloride and maximum biomass yield was achieved in 17 mM and 34 mM NaCl and the hydrocarbon content varied in the range of 12–28% in different salinities and maximum hydrocarbon content was observed in 51 mM and 68 mM NaCl. Detailed studies are necessary on B. braunii to make its outdoor cultivation a commercial viability for obtaining high content of hydrocarbons.
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