Isolation and characterization of hydrocarbon producing green alga Botryococcus braunii from Indian freshwater bodies
support: Department of Biotechnology, Government of India,
Keywords: biomass, carbon dioxide, carotenoids, fatty acids, inter simple sequence repeats, saturated hydrocarbons.
of green colonial unicellular microalga Botryococcus braunii
were collected from
The green colonial hydrocarbon rich unicellular microalgae Botryococcus braunii (Banerjee et al. 2002; Metzger and Largeau, 2005) is widespread in freshwater, brackish lakes, reservoirs and ponds. It is also widely distributed in reservoirs at temperate, tropical and arctic latitudes (Tyson, 1995). It is recognized as one of the potent renewable resource for production of liquid hydrocarbons. B. braunii is classified into A, B and L races based on the type of hydrocarbons produced (Metzger and Largeau, 2005). Race - A produces C21 to C33 odd numbered n-alkadienes, mono-, tri-, tetra-, and pentaenes and they are derived from fatty acids (Banerjee et al. 2002; Metzger et al. 2005). The L race yields a single C40 isoprenoid hydrocarbon, lycopa -14(E),18(E)-diene (Metzger et al. 1990). The B race produces two types of triterpenes called botryococcenes of C30 - C37 of general formula Cn H2n-10 as major hydrocarbons and small amounts of methyl branched squalene. Certain strains of the B race also biosynthesise cyclobotryococcenes (David et al. 1988; Achitouv et al. 2004). Also a feature common to all three races is the presence of a highly aliphatic, non-hydrolysable and insoluble biomacromolecule (algaenan) found in their outer cell walls (Audino et al. 2002). The highly resistant nature of the B. braunii algaenan to degradation allows it to be selectively preserved during fossilisation, leading to fossil B. braunii remains, a major contributor to a number of high oil potential sediments (Simpson et al. 2003). The alga B. braunii produces hydrocarbon in the range of 2-86% (on dry weight basis). This variation in the content of hydrocarbon is due to the differences in the strains, the race it belongs and also depends on cultural and physiological conditions (Dayananda et al. 2005).
oils extracted from B. braunii, when hydrocracked, produce
a distillate comprising of 67% gasoline fraction, 15% aviation turbine
fuel, 15% diesel fuel fraction and 3% residual oil (Banerjee
et al. 2002; Dayananda et al. 2006), these fuels
were free from oxides of sulphur and nitrogen (SOX and NOX) after
their combustion. Being a photosynthetic organism, it can reduce CO2
emissions by 1.5 x 105 tons/yr
and 8.4 X 103 ha of microalgal cultivation area would be necessary
et al. 1999). The uptake of some toxic metals like chromium, cadmium
and arsenic is also been reported (Sawayama et al. 1995).
There is a need for isolation and identification of newer species or
strains of Botryococcus which are efficient in hydrocarbon
synthesis and could be adopted for the mass cultivation. In the present
study focus was on isolation of indigenous strain of Botryococcus
strain of Botryococcus braunii (N-836) was obtained from the
National Institute for Environmental Studies,
samples were collected from different water bodies of Kodaikanal (latitude
10.31 N and longitude 77.32 E), (
time course study was carried out on B. braunii growth. The
experiment was carried out in Erlenmeyer flasks of 150 ml capacity,
containing 40 ml modified
effect of pH on growth of the alga and hydrocarbon yields was studied
two- tier Erlenmeyer flask (Tripathi et al. 2001)
was used for photoautotrophic growth experiments. The medium in upper
chamber was inoculated with 25% (v/v) of two week old B. braunii
culture. The mouth of the upper and lower compartments was sealed
tightly with cotton plug and parafilm. The mixture of carbonate (3M)
and bicarbonate (3M) solutions were added (100 mL) to the lower compartments
to get a CO2 partial pressure of 0.5, 1.0 and 2.0% (v/v)
respectively as given by Tripathi et al. (2001).
The culture flasks were incubated for 3 weeks under 16:8 hrs light
dark cycle with 1.2 ± 0.2 klux light intensity at 25 ±
The cultures were harvested and the cells were washed with distilled water after centrifugation at 5000 rpm. Then the pellet was freeze dried. The dry weight of algal biomass was determined gravimetrically and growth was expressed in terms of dry weight.
known volume of culture was centrifuged (8000 rpm) for 10 min and
the pellet was treated with known volume of methanol and kept in water
bath for 30 min at
A known quantity of algal dry biomass was homogenized and extracted repeatedly with acetone. The pooled extracts absorbance was read at 470 nm and total carotenoid contents were quantified according to Lichtenthaler (Lichtenthaler, 1987).
The cells were harvested by centrifugation at 8000 rpm and the supernatant was analyzed for phosphate content by Fiske-Subbarao’s method (Fiske and Subbarow, 1925).
content in the cell free medium was analyzed by
Hydrocarbon was extracted in hexane after homogenizing the dry biomass in a mortar and pestle in the presence of glass powder and the supernatant recovered after centrifugation was evaporated to complete dryness under the stream of nitrogen. Hydrocarbon content was measured gravimetrically and expressed as dry weight percentage (Dayananda et al. 2005; Dayananda et al. 2006).
extract was purified by column chromatography on silica gel. The hydrocarbon
samples were analyzed on SPB-1 column (
were extracted with chloroform - methanol (2:1) and quantified gravimetrically.
The fatty acid methyl esters (FAME) were prepared as per the procedure
of Christie (1982). FAME were analyzed by GC-MS (PerkinElmer, Turbomass Gold, Mass spectrometer)
equipped with FID using SPB-1 (poly(dimethysiloxane)) capillary column
acetone extract of the alga B. braunii (CFTRI-1) was analysed
by HPLC using a reversed phase C18 column (
cells were processed for scanning electron microscopy (SEM) according
to Fowke et al. (1994). The samples were fixed in
2% glutaraldehyde in
DNA was isolated using plant genomic DNA isolation kit (Sigma). Three inter
simple sequence repeats (ISSR) primers (AC)
has been reported that, Botryococcus braunii exists in the
form of blooms in fresh water bodies like ponds, lakes and reservoirs
(Metzger and Largeau, 2005). The samples collected
morphological heterogeneity of the alga makes the identification difficult.
So in the present study we used ISSR as a tool to identify the alga
by comparing with the known strain and also the nature of hydrocarbons
it synthesize. ISSR finger printing (Figure 3)
revealed a very close genomic similarity among the known B. braunii (B.
braunii N-836, National Institute for Environmental Studies,
liquid culture developed from the single colonies was established
early stationary phase culture was harvested, and the hydrocarbons
were extracted from the dry biomass with hexane. Hydrocarbons were
identified by their M+ ions and comparison of the mass
spectra with those of the standards (Sigma) and also with
the NIST library. The types of hydrocarbons produced by the alga were
identified as saturated hydrocarbons in the range of C21
to C33 (peak 1 to peak 13) by GCMS (Table
1; Figure 6 and Figure
7). Among the saturated hydrocarbons produced by the alga, tetracosane
(peak 4) and octacosane (peak 8) were found to be the major and constituted
17.6% and 14.8% respectively, and their mass spectra and gas chromatograms
have been presented in Figure 6 and Figure
7. Yang et al. (2004) reported that the alga
B. braunii (obtained from Culture Collection of Algae,
An increase of 1.1 to 1.3 fold in biomass yield was observed in B. braunii culture supplemented with carbon dioxide over the control (Figure 8). Similarly hydrocarbon accumulation also increased with the supplementation of carbon dioxide (Figure 8). Increase in chlorophyll and carotenoids contents was observed with increase in carbon dioxide supplementation up to 2% (v/v) (Figure 9). This shows its effective utilization of carbon dioxide through photosynthesis. Tripathi et al. (2001) reported photoautotrophic growth of different microalgae for higher growth and carotenoid production. It was evident that different algae require different levels of CO2 for their photoautotrophic adaptability (Tripathi et al. 2001). In the present study 2% of CO2 supplementation was found to be better for growth and hydrocarbon production.
The fat content of the organism was found to be 22% (w/w) while palmitic and oleic acids as the major fatty acids constituting 40.6 and 22.3% respectively (Table 2). Similar observations were made by Fang et al. (2004) and Dayananda et al. (2006) and they reported that palmitic acid and oleic acids as the major components in the B. braunii.
Lutein (64.1%) and β-carotene (25.1%) were found to be the major carotenoids (Figure 10) during the early stationary phase of the culture. In CO2 supplemented cultures, there was a 3.2 to 3.6 fold increase in β-carotene content (Figure 10) and total carotenoids content of the organism which enhanced (up to 1.75 mg g-1) with increasing carbon dioxide concentration (Figure 9).
From Figure 11 it is evident that there is no significant effect of pH on the biomass yield and production of hydrocarbons since they varied in the range of 0.75 gL-1 to 0.86 gL-1 and 13 to 15% (w/w) respectively. However at pH 7.5 the algae showed maximum growth and production of hydrocarbons (Figure 11).
In conclusion, the alga B. braunii (CFTRI-Bb1) can be of use in production of hydrocarbons. The supplementation of CO2 enhanced both biomass and hydrocarbon production. The organism exhibited wide range of pH adaptability. With further understanding on the influence of cultural conditions on hydrocarbon production, the alga can be exploited for outdoor cultivation.
thank Dr. V. Prakash, Director, CFTRI for his encouragement in carrying
out this study and Dr. Narasimha Rao, Regional Director,
Indira Gandhi National Open University,
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