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Biomass and Bioenergy 31 (2007) 87–93 Autotrophic cultivation of Botryococcus braunii for the production of hydrocarbons and exopolysaccharides in various media C. Dayananda a , R. Sarada a, , M. Usha Rani b , T.R. Shamala b , G.A. Ravishankar a 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, modified 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 L À1 of biomass was produced by the B. braunii strains SAG 30.81 and LB- 572, 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 influence 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 classified into A, B and L races depending on the type of hydrocarbons synthesized. Race-A produces C 23 –C 33 odd numbered n-alkadienes, mono-, tri-, tetra-, and pentaenes, which are derived from fatty acids [1–3]. These linear olefins 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 (C 40 H 78 ) and it constitutes up to 2–8% of the dry biomass. The B race produces poly- unsaturated and branched C 30 –C 37 terpenoid hydrocar- bons referred to as polymethylated botryococcenes. These compounds are promising as a renewable energy source as they accumulate to very high levels (26–86% 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 m À3 where as for the L race 1 kg m À3 [8]. However the amount of exopolysaccharides production varies with the strains, the race it belongs and physiological and cultural conditions. Different strengths of modified Chu 13 medium has been used for B. brauniii cultivation (Largeau et al. [9] used fourfold strength of modified Chu 13 medium; Brown et al. [5] used twofold strength of modified Chu 13 medium). Therefore, a systematic study was carried out on B. braunii strains to study the effect of different strengths of modified ARTICLE IN PRESS www.elsevier.com/locate/biombioe 0961-9534/$ - see front matter r 2006 Published by Elsevier Ltd. doi:10.1016/j.biombioe.2006.05.001 Corresponding author. Tel.: +91 821 516501; fax: +91 821 517233. E-mail address: [email protected] (R. Sarada).

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Biomass and Bioenergy 31 (2007) 87–93

www.elsevier.com/locate/biombioe

Autotrophic cultivation of Botryococcus braunii for the production ofhydrocarbons and exopolysaccharides in various media

C. Dayanandaa, R. Saradaa,�, M. Usha Ranib, T.R. Shamalab, G.A. Ravishankara

aPlant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore 570 020, IndiabFood 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, modified 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 gL�1 of biomass was produced by the B. braunii strains SAG 30.81 and LB-

572, 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 influence 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 andchemicals, particularly by utilizing renewable energyresources like algae and other plants has recently receivedmuch attention. Botryococcus braunii is a green colonial,slow growing microalga and it is widespread in freshwaterand brackish lakes, reservoirs, ponds and it is recognized asone of the potent renewable resources for production ofliquid hydrocarbons. B. braunii is classified into A, B and Lraces depending on the type of hydrocarbons synthesized.Race-A produces C23–C33 odd numbered n-alkadienes,mono-, tri-, tetra-, and pentaenes, which are derived fromfatty acids [1–3]. These linear olefins can constitute up to61% of the dry cell mass of the green active state colonies[4]. The L race produces a single tetraterpene hydrocarbonknown as lycopadiene (C40H78) and it constitutes up to2–8% of the dry biomass. The B race produces poly-

e front matter r 2006 Published by Elsevier Ltd.

ombioe.2006.05.001

ing author. Tel.: +91821 516501; fax: +91 821 517233.

ess: [email protected] (R. Sarada).

unsaturated and branched C30–C37 terpenoid hydrocar-bons referred to as polymethylated botryococcenes. Thesecompounds are promising as a renewable energy source asthey accumulate to very high levels (26–86% on dryweight) in the algae [1,2,5,6].Hydrocarbons are extracted from the total lipids as the

hexane-soluble component and can be converted intouseful fuels such as gasoline by catalytic cracking [7].Apart from hydrocarbons B. braunii is also capable ofproducing exopolysaccharides. Race A and B strains of B.

braunii can produce exopolysaccharides up to 250 gm�3

where as for the L race 1 kgm�3 [8]. However the amountof exopolysaccharides production varies with thestrains, the race it belongs and physiological and culturalconditions.Different strengths of modified Chu 13 medium has been

used for B. brauniii cultivation (Largeau et al. [9] usedfourfold strength of modified Chu 13 medium; Brown et al.[5] used twofold strength of modified Chu 13 medium).Therefore, a systematic study was carried out on B. braunii

strains to study the effect of different strengths of modified

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Chu 13 medium and other media on their growth,hydrocarbon and exopolysaccharide production underthe experimental conditions.

B. braunii is identified as an untapped resource forproduction of hydrocarbons. Successful use of this organ-ism as an alternate source of energy depends on its growthrate, hydrocarbon productivity and their fuel efficiency.Therefore increasing its growth rate in terms of biomassyields is an important parameter. Hence the present studyfocused on its growth in different media so as to get higherbiomass yields with high hydrocarbon content. Inciden-tally, the organisms were found to produce exopolysac-charides 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 fromthe university of Texas, USA and Sammlung vonAlgenKulturen, pflanzenphysiologisches Institut, Universi-tat Gottingen, Germany, respectively. Stock cultures of B.

braunii were maintained routinely on both liquid and agarslants of modified Chu 13 medium [9] by regular sub-culturing at 2-week intervals. Cultures were maintained at2571 1C temperature with 1.270.2 klux light intensityunder 16:8 light dark cycle.

2.2. Media and culture conditions

As shown in Table 1, different autotrophic mediadiffered mainly in their nitrogen source and concentration.BG 11 contains high amount of sodium nitrate whilemodified Chu 13 contained potassium nitrate and modifiedBBM contained ammonium carbonate. A 2-week-oldmodified Chu 13 (1X) media culture of both B. braunii

LB 572 and SAG 30.81 were used as inoculum at 25% for

Table 1

Composition of autotrophic culture media

Composition (gL�1) Modified Chu 13

0.25X 0.5X 0.75X

KNO3 0.05 0.1 0.15

NaNO3 — — —

K2HPO4 0.01 0.02 0.03

KH2PO4 — — —

CaCl2 � 2H2O 0.02 0.04 0.06

MgSO4 � 7H2O 0.025 0.05 0.075

Na2CO3 — — —

NaCl — — —

FeSO4 — — —

EDTA — — —

Citric acid 0.025 0.05 0.075

Ferric ammonium citrate — — —

Ferric citrate 0.0025 0.005 0.0075

Ammonium carbonate — — —

all experiments. Cultures were grown autotrophically inbold basal (BBM) [10] and modified BBM, BG11 [11],modified Chu13 media [9] (Table 1). Cultures of B. braunii

were incubated in three different culture conditions. Oneset was incubated at 2571 1C temperature with1.270.2 klux light intensity and 16:8 light dark cycle;second set at 2571 1C temperature with continuous lightintensity of 1.270.2 klux and third set at 2571 1Ctemperature with continuous light intensity of1.270.2 klux on shaker with 90 rotationsmin�1. All theexperiments were carried out in triplicate. All the cultureswere incubated for 6 weeks.

2.3. Biomass estimation

The cultures were harvested and the cells were washedwith distilled water after centrifugation at 5000 rpm for10min. Then the pellet was freeze dried. The dry weight ofalgal biomass was determined gravimetrically and growthwas expressed in terms of dry weight.

2.4. Carbohydrate estimation

The occurrence of dissolved polysaccharides in the spentmedium was checked by their precipitation in 45% ethanol[12]. Cell-free medium was analysed for total carbohydrateby phenol-sulphuric acid method [13].

2.5. Protein estimation

Protein content in the cell-free medium was analysed byBradford protein assay [14].

2.6. Hydrocarbon extraction

Dried algal biomass was homogenized in mortar andpestle with n-hexane for 15min and centrifuged. The

BG11 BBM BBMa

1X 2X

0.2 0.4 — — —

— — 1.5 0.25 —

0.04 0.08 0.04 0.074 0.074

— — — 0.0175 0.0175

0.08 0.16 0.036 0.024 0.024

0.1 0.2 0.075 0.073 0.073

— — 0.02 — —

— — — 0.025 0.025

— — — 0.005 0.005

— — 0.001 0.045 0.045

0.1 0.2 0.006 — —

— — 0.006 — —

0.01 0.02 — — —

— — — — 0.157

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supernatants were taken into pre-weighed glass vial. Theextraction process was repeated two more times and thesolvents were pooled and evaporated under the stream ofnitrogen to complete dryness at room temperature. Thequantity of residue was measured gravimetrically [15].

2.7. Hydrocarbon analysis by GC/GCMS

The crude extracts were purified by column chromato-graphy on silica gel with n-hexane as eluent. GC analysiswas done on BP-5 capillary column as described byDayananda et al. [16]. Hydrocarbons analysed by GCwere grouped into two categories as less than C30 andhigher than C30 with reference to their elution with that ofthe retention time of the internal standard triacontane.GC-MS analysis was carried out using ELITE 5(30m� 0.25 id) capillary column as described by Day-ananda et al. [16]. The mass spectra were recorded under

(A)

0.0

0.4

0.8

1.2

1.6

2.0

2.4

Bio

mas

s (g

L-1

)B

iom

ass

(gL

-1)

Continuous light

16:8 light and dark cycle

Continuous light with shaking

(B)

0

0.4

0.8

1.2

1.6

2

2.4

2.8

3.2

3.6

0.25X 0.5X 0.75X 1X

0.25X 0.5X 0.75X 1X

Continuous light

16:8 light and dark cycle

Continuous light with shaking

Fig. 1. Biomass yields of B. braunii cultured in various me

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

3. Results and discussion

Growth profiles of B. braunii LB 572 and SAG 30.81 indifferent media under three culture conditions are shown inFig. 1. The biomass yields of SAG 30.81 were less than LB572 under all the conditions. The biomass yields werecomparatively high under media shaking conditions withcontinuous light in SAG 30.81 while stationary culturewith 16:8 h light and dark cycle was favourable for LB 572.In both the cultures, maximum biomass was obtained inBG 11 followed by BBM. However modified BBMa (withammonium carbonate) resulted in lower biomass yieldscompared to BBM. There was no considerable increase in

Media

Media

2X BG 11 BBM BBMa

2X BG 11 BBM BBMa

dia, (A) B. braunii SAG 30.81, (B) B. braunii LB 572.

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(A)

0

10

20

30

40

50

60

Hyd

roca

rbo

n %

(w/w

)

Continuous light

16:8 light and dark cycle

Continuous light with shaking

Continuous light

16:8 light and dark cycle

Continuous light with shaking

(B)

0

5

10

15

20

25

30

35

40

0.25X 0.5X 0.75X 1X 2X BG11 BBM

Media

Hyd

roca

rbo

n %

(w

/w)

BBMa

0.25X 0.5X 0.75X 1X 2X BG11 BBM

Media

BBMa

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 profile of B. braunii (SAG 30.81) grown in different media and culture conditions

Media B. braunii (SAG 30.81)

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

Less than C30 Higher than C30 Less than C30 Higher than C30 Less than C30 Higher than C30

0.25X 68.8371.66 31.1771.27 64.4771.45 35.5373.48 61.8770.99 38.1373.51

0.50X 66.5971.36 33.4172.02 61.0371.93 38.9773.02 57.3670.96 42.6474.64

0.75X 65.9371.47 34.0772.11 57.1770.78 42.8374.76 55.6372.19 44.3775.55

1X 54.2273.19 45.7871.24 56.2071.45 43.8071.32 53.3772.64 46.6371.44

BG 11 59.8972.26 40.1171.82 52.8272.57 47.1873.57 47.8771.14 52.1376.04

BBM 67.3771.76 32.6370.72 59.8171.20 40.1974.01 60.4971.63 39.5174.18

BBMa 59.9072.60 40.1070.32 67.4376.31 32.5770.87 54.8571.45 43.1576.57

C. Dayananda et al. / Biomass and Bioenergy 31 (2007) 87–9390

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the biomass yields of both the strains with increase instrength of modified Chu 13 medium.

Hydrocarbon content was more under constant agitationwith continuous light especially at low strength of modified

Table 3

Hydrocarbon profile of B. braunii (LB 572) grown in different media and cult

Media B. braunii (LB 572)

16:8 Light dark cycle Continuous lig

Less than C30 Higher than C30 Less than C30

0.25X 80.6374.51 19.3775.29 60.9476.13

0.50X 76.6874.87 23.3273.51 58.6874.01

0.75X 71.5074.99 28.5073.24 59.3472.13

1X 64.2673.56 35.7474.16 50.2774.52

BG 11 70.8876.43 29.1273.17 51.6576.89

BBM 73.6975.48 26.3174.13 63.0271.79

BBMa 68.9074.60 31.1074.59 61.8470.52

(A)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0.25X 0.5X 0.75X 1X

0.25X 0.5X 0.75X 1X

M

Car

bo

hyd

rate

(g

L-1

)C

arb

ohy

dra

te (

gL

-1)

Continuous light

16:8 light and dark cycle

Continuous light with shaking

(B)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

M

Continuoust light

16:8 light and dark cycle

Continuous light with shaking

Fig. 3. Concentrations of carbohydrate in the cell free medium of B. braunii cu

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

ure conditions

ht Continuous light with shaking

Higher than C30 Less than C30 Higher than C30

39.0675.12 72.1073.03 27.971.23

41.3277.31 67.8474.83 32.1671.64

40.6675.31 63.4671.11 36.5472.65

49.7374.07 57.9177.38 42.0972.95

48.3575.91 62.1176.08 37. 8973.77

36.9873.04 66.1372.94 33.8772.78

38.1674.71 70.4971.93 29.5173.30

2X BG11 BBM BBMa

2X BG11 BBM BBMa

edia

edia

ltured 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 betweenlight: dark cycle and continuous light cycle (Fig. 2).Hydrocarbon productivity of B. braunii SAG 30.81 wasin the range of 30–40% and LB 572 in the range of 20–30%on dry weight basis irrespective of the tested media andculture conditions, although they represent the same race.

As analysed by GC, hydrocarbons were grouped intotwo categories as less than C30 and higher than C30 withreference to their elution with that of the retention time ofthe internal standard triacontane. As shown in Tables 2and 3, the hydrocarbon profile of B. braunii varies with thestrains, culture conditions and culture media. Higher chainhydrocarbons (4C30) content was considerably higher inboth the strains of B. braunii especially under continuouslight and continuous light with shaking. Higher chainhydrocarbon content (4C30) was relatively more in case ofSAG 30.81 strain than the strain LB 572. In all the testedmedia and culture conditions the lower chain hydrocarbons(oC30) contents were proportionately higher than theother (4C30). However there are variations in theindividual hydrocarbon proportion in all the tested mediaand culture conditions. It is evident from Tables 2 and 3that both the strains have shown to produce similarproportions of hydrocarbon profiles under all the tested

(A)

0

20

40

60

80

100

120

140

160

0.25X 0.5X 0.75X 1X

Me

Pro

tien

(mg

L-

1 )

Continuous light

16:8 light and dark cycle

Continuous light with shaking

(B)

0

20

40

60

80

100

120

140

160

Pro

tien

(mg

L-1

)

0.25X 0.5X 0.75X 1X

Me

Fig. 4. Concentrations of protein in the cell free medium of B. braunii cultu

conditions. So it can be concluded that the alga B. braunii

can be cultivated in varied range of culture conditions andin various media.Production of exopolysaccharides by both the strains of

B. braunii was also monitored in the spent medium of allthe tested media and conditions. As indicated in Fig. 3,continuous light influenced an increased production ofexopolysaccharides with shaking compared to other twotested conditions. B. braunii LB 572 has recordedmaximum production (1.6 gL�1) of exopolysaccharidesthan the strain SAG 30.81(0.7 gL�1) even though both thestrains belong to ‘A’ race as identified by their character-istic hydrocarbons by GC-MS. Production of exopolysac-charides by B. braunii was reported for the A race byCasadevall et al. [12] whereas Fernandes et al. [18] reportedpoor production of hydrocarbons in contrast to the highyield of exopolysaccharides production and this wasreflected in the strains LB 572 and SAG 30.81. Howeverthe amount of exopolysaccharides production varies withthe strains, the race it belongs and physiological andcultural conditions [8].From Fig. 4, it can be seen that lesser amount of protein

was recorded in light: dark cycle, whereas in other twoculture conditions there is a significant increase in the

2X BG11 BBM

dia

Continuous light16:8 light and dark cycle

Continuous light with shaking

BBMa

2X BG11 BBM

dia

BBMa

red 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 bedue to the lyses of the cells. It was also observed that thecells were pale yellowish and a few colonies degenerated inall the tested autotrophic media and under all the testedcultural conditions. Casadevall et al. [12] reported thatdegeneration of the cells is associated with considerabledecrease in chlorophyll content. In contrast, degeneratedcells and colonies were minimal in the cultures of BG11and BBM media and in light dark cycle with stationaryconditions. However protein content of the cell-freemedium was very minimal in all the tested conditions.From Figs. 1 and 4, we can observe that lesser amount ofprotein was recorded wherever the good growth wasachieved and vice versa.

4. Conclusions

From the present study it can be concluded that theorganism is highly capable of adapting to wider growthconditions and culture media. The resistance of B. braunii

to various stress conditions (anaerobic conditions, contin-uous illumination, and prolonged darkness) have also beenreported [12,19]. This property is very important particu-larly for out door cultivation of B. braunii. The study alsofound out two important culture conditions such ascultivation of B. braunii in 16:8 h light dark cycle yieldshigher hydrocarbon whereas continuous illumination withagitation yields higher amounts of exopolysaccharides.

Acknowledgements

Authors thank Department of Biotechnology, Govern-ment of India for their financial support and Dr. V.Prakash, Director, CFTRI for his encouragement incarrying out this study.

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