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Biomass and Bioenergy 31 (2007) 8793 www.elsevier.com/locate/biombioe

Autotrophic cultivation of Botryococcus braunii for the production of hydrocarbons and exopolysaccharides in various media
C. Dayanandaa, R. Saradaa,, M. Usha Ranib, T.R. Shamalab, G.A. Ravishankara
a

Plant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore 570 020, India b Food Microbiology Department, Central Food Technological Research Institute, Mysore 570 020, India Received 18 July 2005; received in revised form 28 April 2006; accepted 6 May 2006 Available online 12 September 2006

Abstract Growth of Botryococcus braunii was studied using different autotrophic media such as bold basal medium (BBM), and bold basal with ammonium carbonate (BBMa), BG11, modied Chu 13 medium. Among the different autotrophic media used, BG11 was found to be the best medium for biomass and hydrocarbon production, although B. braunii showed appreciable level of growth and biomass production in all the tested media. The culture maintained at 16:8 h light and dark cycle with 1.270.2 klux light intensity at 2571 1C temperature was found to be the best for growth (2.0 and 2.8 g L1 of biomass was produced by the B. braunii strains SAG 30.81 and LB572, respectively) and hydrocarbon production (46% and 33%, respectively, by SAG 30.81 and LB 572 strains on dry weight basis) whereas continuous illumination with agitation at 90 rpm had maximum inuence for the production of exopolysaccharides. The results of the present study indicate that the organism can acclimatize to different culture conditions and to a wide range of culture media with production of more than one metabolite. r 2006 Published by Elsevier Ltd.
Keywords: Botryococcus braunii; Microalgae; Autotrophic media; Hydrocarbon; Biomass; Exopolysaccharide

1. Introduction The development of alternative source for energy and chemicals, particularly by utilizing renewable energy resources like algae and other plants has recently received much attention. Botryococcus braunii is a green colonial, slow growing microalga and it is widespread in freshwater and brackish lakes, reservoirs, ponds and it is recognized as one of the potent renewable resources for production of liquid hydrocarbons. B. braunii is classied into A, B and L races depending on the type of hydrocarbons synthesized. Race-A produces C23C33 odd numbered n-alkadienes, mono-, tri-, tetra-, and pentaenes, which are derived from fatty acids [13]. These linear olens can constitute up to 61% of the dry cell mass of the green active state colonies [4]. The L race produces a single tetraterpene hydrocarbon known as lycopadiene (C40H78) and it constitutes up to 28% of the dry biomass. The B race produces polyCorresponding author. Tel.: +91 821 516501; fax: +91 821 517233.

E-mail address: pcbt@cftri.res.in (R. Sarada). 0961-9534/$ - see front matter r 2006 Published by Elsevier Ltd. doi:10.1016/j.biombioe.2006.05.001

unsaturated and branched C30C37 terpenoid hydrocarbons referred to as polymethylated botryococcenes. These compounds are promising as a renewable energy source as they accumulate to very high levels (2686% on dry weight) in the algae [1,2,5,6]. Hydrocarbons are extracted from the total lipids as the hexane-soluble component and can be converted into useful fuels such as gasoline by catalytic cracking [7]. Apart from hydrocarbons B. braunii is also capable of producing exopolysaccharides. Race A and B strains of B. braunii can produce exopolysaccharides up to 250 g m3 where as for the L race 1 kg m3 [8]. However the amount of exopolysaccharides production varies with the strains, the race it belongs and physiological and cultural conditions. Different strengths of modied Chu 13 medium has been used for B. brauniii cultivation (Largeau et al. [9] used fourfold strength of modied Chu 13 medium; Brown et al. [5] used twofold strength of modied Chu 13 medium). Therefore, a systematic study was carried out on B. braunii strains to study the effect of different strengths of modied

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Chu 13 medium and other media on their growth, hydrocarbon and exopolysaccharide production under the experimental conditions. B. braunii is identied as an untapped resource for production of hydrocarbons. Successful use of this organism as an alternate source of energy depends on its growth rate, hydrocarbon productivity and their fuel efciency. Therefore increasing its growth rate in terms of biomass yields is an important parameter. Hence the present study focused on its growth in different media so as to get higher biomass yields with high hydrocarbon content. Incidentally, the organisms were found to produce exopolysaccharides and this was compared in different media. 2. Materials and methods 2.1. Algal culture B. braunii (LB 572) and (SAG 30.81) were obtained from the university of Texas, USA and Sammlung von AlgenKulturen, panzenphysiologisches Institut, Universitat Gottingen, Germany, respectively. Stock cultures of B. braunii were maintained routinely on both liquid and agar slants of modied Chu 13 medium [9] by regular subculturing at 2-week intervals. Cultures were maintained at 2571 1C temperature with 1.270.2 klux light intensity under 16:8 light dark cycle. 2.2. Media and culture conditions As shown in Table 1, different autotrophic media differed mainly in their nitrogen source and concentration. BG 11 contains high amount of sodium nitrate while modied Chu 13 contained potassium nitrate and modied BBM contained ammonium carbonate. A 2-week-old modied Chu 13 (1X) media culture of both B. braunii LB 572 and SAG 30.81 were used as inoculum at 25% for

all experiments. Cultures were grown autotrophically in bold basal (BBM) [10] and modied BBM, BG11 [11], modied Chu13 media [9] (Table 1). Cultures of B. braunii were incubated in three different culture conditions. One set was incubated at 2571 1C temperature with 1.270.2 klux light intensity and 16:8 light dark cycle; second set at 2571 1C temperature with continuous light intensity of 1.270.2 klux and third set at 2571 1C temperature with continuous light intensity of 1.270.2 klux on shaker with 90 rotations min1. All the experiments were carried out in triplicate. All the cultures were incubated for 6 weeks. 2.3. Biomass estimation The cultures were harvested and the cells were washed with distilled water after centrifugation at 5000 rpm for 10 min. Then the pellet was freeze dried. The dry weight of algal biomass was determined gravimetrically and growth was expressed in terms of dry weight. 2.4. Carbohydrate estimation The occurrence of dissolved polysaccharides in the spent medium was checked by their precipitation in 45% ethanol [12]. Cell-free medium was analysed for total carbohydrate by phenol-sulphuric acid method [13]. 2.5. Protein estimation Protein content in the cell-free medium was analysed by Bradford protein assay [14]. 2.6. Hydrocarbon extraction Dried algal biomass was homogenized in mortar and pestle with n-hexane for 15 min and centrifuged. The

Table 1 Composition of autotrophic culture media Composition (g L1) Modied Chu 13 0.25X KNO3 NaNO3 K2HPO4 KH2PO4 CaCl2 2H2O MgSO4 7H2O Na2CO3 NaCl FeSO4 EDTA Citric acid Ferric ammonium citrate Ferric citrate Ammonium carbonate 0.05 0.01 0.02 0.025 0.025 0.0025 0.5X 0.1 0.02 0.04 0.05 0.05 0.005 0.75X 0.15 0.03 0.06 0.075 0.075 0.0075 1X 0.2 0.04 0.08 0.1 0.1 0.01 2X 0.4 0.08 0.16 0.2 0.2 0.02 1.5 0.04 0.036 0.075 0.02 0.001 0.006 0.006 0.25 0.074 0.0175 0.024 0.073 0.025 0.005 0.045 0.074 0.0175 0.024 0.073 0.025 0.005 0.045 0.157 BG11 BBM BBMa

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supernatants were taken into pre-weighed glass vial. The extraction process was repeated two more times and the solvents were pooled and evaporated under the stream of nitrogen to complete dryness at room temperature. The quantity of residue was measured gravimetrically [15].

electron impact ionization at 70 eV electron energy with a mass range from 40 to 600 at a rate of one scan per second. Mass spectra were identied by matching their fragmentation pattern with literature data [17].

3. Results and discussion 2.7. Hydrocarbon analysis by GC/GCMS The crude extracts were puried by column chromatography on silica gel with n-hexane as eluent. GC analysis was done on BP-5 capillary column as described by Dayananda et al. [16]. Hydrocarbons analysed by GC were grouped into two categories as less than C30 and higher than C30 with reference to their elution with that of the retention time of the internal standard triacontane. GC-MS analysis was carried out using ELITE 5 (30 m 0.25 id) capillary column as described by Dayananda et al. [16]. The mass spectra were recorded under
2.4 Continuous light 16:8 light and dark cycle 2.0 Biomass (gL-1) Continuous light with shaking

Growth proles of B. braunii LB 572 and SAG 30.81 in different media under three culture conditions are shown in Fig. 1. The biomass yields of SAG 30.81 were less than LB 572 under all the conditions. The biomass yields were comparatively high under media shaking conditions with continuous light in SAG 30.81 while stationary culture with 16:8 h light and dark cycle was favourable for LB 572. In both the cultures, maximum biomass was obtained in BG 11 followed by BBM. However modied BBMa (with ammonium carbonate) resulted in lower biomass yields compared to BBM. There was no considerable increase in

1.6

1.2

0.8

0.4

0.0 0.25X (A) 3.6 Continuous light 3.2 16:8 light and dark cycle 2.8 Continuous light with shaking Biomass (gL-1) 2.4 2 1.6 1.2 0.8 0.4 0 0.25X (B) 0.5X 0.75X 1X Media 2X BG 11 BBM BBMa 0.5X 0.75X 1X Media 2X BG 11 BBM BBMa

Fig. 1. Biomass yields of B. braunii cultured in various media, (A) B. braunii SAG 30.81, (B) B. braunii LB 572.

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Continuous light 16:8 light and dark cycle Continuous light with shaking 50

Hydrocarbon %(w/w)

40

30

20

10

0 0.25X 0.5X 0.75X 1X Media 40 35 30 Hydrocarbon % (w/w) 25 20 15 10 5 0 0.25X 0.5X 0.75X 1X Media 2X BG11 BBM BBMa Continuous light 16:8 light and dark cycle Continuous light with shaking 2X BG11 BBM BBMa

(A)

(B)

Fig. 2. Hydrocarbon yields of B. braunii cultured in various media, (A) B. braunii SAG 30.81, (B) B. braunii LB 572.

Table 2 Hydrocarbon prole of B. braunii (SAG 30.81) grown in different media and culture conditions Media B. braunii (SAG 30.81) 16:8 Light dark cycle Less than C30 0.25X 0.50X 0.75X 1X BG 11 BBM BBMa 68.8371.66 66.5971.36 65.9371.47 54.2273.19 59.8972.26 67.3771.76 59.9072.60 Higher than C30 31.1771.27 33.4172.02 34.0772.11 45.7871.24 40.1171.82 32.6370.72 40.1070.32 Continuous light Less than C30 64.4771.45 61.0371.93 57.1770.78 56.2071.45 52.8272.57 59.8171.20 67.4376.31 Higher than C30 35.5373.48 38.9773.02 42.8374.76 43.8071.32 47.1873.57 40.1974.01 32.5770.87 Continuous light with shaking Less than C30 61.8770.99 57.3670.96 55.6372.19 53.3772.64 47.8771.14 60.4971.63 54.8571.45 Higher than C30 38.1373.51 42.6474.64 44.3775.55 46.6371.44 52.1376.04 39.5174.18 43.1576.57

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the biomass yields of both the strains with increase in strength of modied Chu 13 medium. Hydrocarbon content was more under constant agitation with continuous light especially at low strength of modied

Chu 13 medium whereas under light dark cycle it was 0.75 to 1X strength medium, while 2X media had resulted in least production of hydrocarbon in both the strains in the tested conditions. In other media there was no signicant

Table 3 Hydrocarbon prole of B. braunii (LB 572) grown in different media and culture conditions Media B. braunii (LB 572) 16:8 Light dark cycle Less than C30 0.25X 0.50X 0.75X 1X BG 11 BBM BBMa 80.6374.51 76.6874.87 71.5074.99 64.2673.56 70.8876.43 73.6975.48 68.9074.60 Higher than C30 19.3775.29 23.3273.51 28.5073.24 35.7474.16 29.1273.17 26.3174.13 31.1074.59 Continuous light Less than C30 60.9476.13 58.6874.01 59.3472.13 50.2774.52 51.6576.89 63.0271.79 61.8470.52 Higher than C30 39.0675.12 41.3277.31 40.6675.31 49.7374.07 48.3575.91 36.9873.04 38.1674.71 Continuous light with shaking Less than C30 72.1073.03 67.8474.83 63.4671.11 57.9177.38 62.1176.08 66.1372.94 70.4971.93 Higher than C30 27.971.23 32.1671.64 36.5472.65 42.0972.95 37. 8973.77 33.8772.78 29.5173.30

0.9 Continuous light 0.8 16:8 light and dark cycle 0.7 Carbohydrate (gL-1) Continuous light with shaking 0.6 0.5 0.4 0.3 0.2 0.1 0 0.25X 0.5X 0.75X 1X Media 2 1.8 1.6 Carbohydrate (gL-1) 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0.25X 0.5X 0.75X 1X Media 2X BG11 BBM BBMa Continuoust light 16:8 light and dark cycle Continuous light with shaking 2X BG11 BBM BBMa

(A)

(B)

Fig. 3. Concentrations of carbohydrate in the cell free medium of B. braunii cultured in various media, (A) B. braunii SAG 30.81, (B) B. braunii LB 572.

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(po0.05) difference in the hydrocarbon contents between light: dark cycle and continuous light cycle (Fig. 2). Hydrocarbon productivity of B. braunii SAG 30.81 was in the range of 3040% and LB 572 in the range of 2030% on dry weight basis irrespective of the tested media and culture conditions, although they represent the same race. As analysed by GC, hydrocarbons were grouped into two categories as less than C30 and higher than C30 with reference to their elution with that of the retention time of the internal standard triacontane. As shown in Tables 2 and 3, the hydrocarbon prole of B. braunii varies with the strains, culture conditions and culture media. Higher chain hydrocarbons (4C30) content was considerably higher in both the strains of B. braunii especially under continuous light and continuous light with shaking. Higher chain hydrocarbon content (4C30) was relatively more in case of SAG 30.81 strain than the strain LB 572. In all the tested media and culture conditions the lower chain hydrocarbons (oC30) contents were proportionately higher than the other (4C30). However there are variations in the individual hydrocarbon proportion in all the tested media and culture conditions. It is evident from Tables 2 and 3 that both the strains have shown to produce similar proportions of hydrocarbon proles under all the tested
160 Continuous light 140 16:8 light and dark cycle 120 Protien(mgL- 1) Continuous light with shaking 100 80 60 40 20 0 0.25X 0.5X 0.75X 1X

conditions. So it can be concluded that the alga B. braunii can be cultivated in varied range of culture conditions and in various media. Production of exopolysaccharides by both the strains of B. braunii was also monitored in the spent medium of all the tested media and conditions. As indicated in Fig. 3, continuous light inuenced an increased production of exopolysaccharides with shaking compared to other two tested conditions. B. braunii LB 572 has recorded maximum production (1.6 g L1) of exopolysaccharides than the strain SAG 30.81(0.7 g L1) even though both the strains belong to A race as identied by their characteristic hydrocarbons by GC-MS. Production of exopolysaccharides by B. braunii was reported for the A race by Casadevall et al. [12] whereas Fernandes et al. [18] reported poor production of hydrocarbons in contrast to the high yield of exopolysaccharides production and this was reected in the strains LB 572 and SAG 30.81. However the amount of exopolysaccharides production varies with the strains, the race it belongs and physiological and cultural conditions [8]. From Fig. 4, it can be seen that lesser amount of protein was recorded in light: dark cycle, whereas in other two culture conditions there is a signicant increase in the

2X Media

BG11

BBM

BBMa

(A)
160 140 120 Protien(mgL-1) 100 80 60 40 20 0 0.25X 0.5X 0.75X 1X

Continuous light 16:8 light and dark cycle Continuous light with shaking

2X Media

BG11

BBM

BBMa

(B)

Fig. 4. Concentrations of protein in the cell free medium of B. braunii cultured in various media, (A) B. braunii SAG 30.81, (B) B. braunii LB 572.

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protein content in the cell-free medium; this protein may be due to the lyses of the cells. It was also observed that the cells were pale yellowish and a few colonies degenerated in all the tested autotrophic media and under all the tested cultural conditions. Casadevall et al. [12] reported that degeneration of the cells is associated with considerable decrease in chlorophyll content. In contrast, degenerated cells and colonies were minimal in the cultures of BG11 and BBM media and in light dark cycle with stationary conditions. However protein content of the cell-free medium was very minimal in all the tested conditions. From Figs. 1 and 4, we can observe that lesser amount of protein was recorded wherever the good growth was achieved and vice versa. 4. Conclusions

[4] [5]

[6]

[7]

[8]

[9]

[10]

From the present study it can be concluded that the organism is highly capable of adapting to wider growth conditions and culture media. The resistance of B. braunii to various stress conditions (anaerobic conditions, continuous illumination, and prolonged darkness) have also been reported [12,19]. This property is very important particularly for out door cultivation of B. braunii. The study also found out two important culture conditions such as cultivation of B. braunii in 16:8 h light dark cycle yields higher hydrocarbon whereas continuous illumination with agitation yields higher amounts of exopolysaccharides. Acknowledgements

[11]

[12]

[13]

[14]

[15]

Authors thank Department of Biotechnology, Government of India for their nancial support and Dr. V. Prakash, Director, CFTRI for his encouragement in carrying out this study. References
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