STUDIES OF BIOENERGY COMPOUNDS AND MOLECULAR CHARACTERIZATION OF BOTRYOCOCCENE SYNTHASE GENE FROM BOTRYOCOCCUS BRAUNII KUTZING

T. Sangeetha 1 , G.K. Saravanan 2 , *S. Elumalai 3 and D. Bhuvana 4 . 1. Department of Chemistry, Presidency College (Autonomous), Chennai – 600005. 2. Department of Plant Biology and Plant Biotechnology, Presidency College (Autonomous), Chennai – 600005. 3. Department of Biotechnology, University of Madras, Guindy campus, Chennai600025. 4. Department of Biotechnology, Valliammal College for Women, Chennai600 102. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 4 (8), 979-991 981 Extraction and estimation of carbohydrate:-The total carbohydrate of green algae B. braunii was determined based on the method well defined by Dubois et al. in the year 1956 (Phenol Sulphuric acid method). About 100 µl of the crude sample was subjected to treat with 5% phenol and 2.5 mL of Conc. Sulphuric acid. Then the whole content was incubated for 10-15 min. at room temperature. Then the optical absorbance values were measured at 490 nm using a UV-Vis. Spectrophotometer. The total carbohydrate content was determined by comparing the absorbance values with the standard graph using Dglucose as a known carbohydrate sample.

Extraction and estimation of protein:-
The estimation of protein of green algae B. braunii, in this present study was done based on the method described by Bradford (1976). The protein content of the unknown sample was determined in comparison with the standard graph of the known sample. The standard graph for protein was constructed using Bovine serum albumin as a standard known protein. About 1 mL of the crude sample used to determine the total protein content with 5 mL of the Bradford reagent. After incubation for 15 min. the content was subjected to determine the absorbance values at 595 nm in a UV-Vis. Spectrophotometer.

Extraction and estimation of lipid:-
The total lipid constituent of green algae B. braunii was determined based on the method described by Folch et al. in 1957. A 6 mL of chloroform and methanol (2:1 ratio) was mixed with the extract and vortexed for 3 min. at room temperature. The extract was then centrifuged at 8000 rpm for 5 min. The resultant liquid phase was washed with 6 mL of 0.9% of NaCl and vortexed for 5 min. The entire content was then centrifuged at 2000 rpm for 5 min. which resulted to form two distinct phases. The upper phase conserved due to its richness in lipid and the lower aqueous phase was discarded. The total lipid content was determined gravimetrically after the complete evaporation of the solvent (Upper phase).
Extraction and analysis of fatty acid methyl esters (FAME) by gas chromatography:-About 20 mL of green algae B. braunii sample was subjected to direct Transesterification. About 1 mL of reagent 1 was poured to the fresh algal samples and vortexed for 5-10 sec. using a cyclomixer. The mixture was incubated in a water bath at 100 o C for 5 min. and again vortexed for 5-10 sec. Then after incubated at 100 o C for 25 min. reagent 2 was added and vortexed for 5-10 min. followed by thermal incubation at 80 o C and rapidly cooled down to 4 o C. Reagent 3 was added the mixture and mixed gently up to 10 min. Two phases were seen blatantly from which, the lower phase was conserved. To the content 3 mL of reagent 4 was added followed by mixing for 5 min. and obtained upper phase was removed. The lower phase rich in fatty acid methyl esters were stored in a vial at 4 o C in a refrigerator.
The tranesterified fatty methyl esters were analyzed with the help of a gas chromatography equipped with flame ionization detector (FID) (Perkin Elmer, USA). A SP-2560 column (100 m × 0.25 mm I. D., 0.20 µm) (Sigma, Germany) along with standard fatty acid Supelco 37 Component FAME mix from Supelco (Bellefonte, PA, USA) was employed. About 5 µl of the sample was injected and the GC conditions were injector temperature: 260 o C; Column temperature: 140 o C and detector temperature: 260 o C. Helium was used as a carrier gas with the flow rate of 1 mL/min. The unknown FAMEs were determined in comparison with the retention times of the standard FAMEs (Supelco) using a mass spectra from NIST library.

Fourier transforms infra red spectrometric (FT-IR) analysis of FAME:-
The FAME sample were analyzed under infra red (Perkin Elmer model spectrum -I PC). The FT-IR spectra with the resolution of 4 cm -1 , Scan Number: 3 were performed after the evaporation of the lipid fraction on Thalium bromide tablets. The FT-IR spectrums of all the FAME samples were obtained as a percentage of transmission ranged from 450 cm -1 to 4000 cm -1 .

Isolation of RNA:-
About 300 µl of the culture was ground into fine powder in liquid nitrogen using a mortar and pestle. A 2 mL of solution I was added while grinding to make homogenous mixture and allowed to thaw completely with intermittent grinding. Then 800 µl of nuclease free water was added by mixing and grinding. The whole content was transferred to 2 mL microcentrifuge tubes and kept undisturbed place for 5 min. at room temperature. Chloroform (200 µl) was added to each tube and vortexed briefly for less than 10 seconds and kept undisturbed for 10 min. at room 982 temperature. Then the whole mixture was allowed to centrifuge for 10 min. at 4 o C and the upper aqueous phase was transferred into to fresh microcentrifuge tubes. Then about 0.6 volumes of isopropanol were added and vortexed briefly less than 10 seconds and left for 10 min. at room temperature. Then after centrifugation at 13,000 rpm for 10 min. at 4 o C the supernatant was discarded and the obtained pellet with RNA was washed with 70 % ethanol, air dried and dissolved in 50 µl of DEPC-treated water. The extracted RNA was separated and analyzed using 1.5 % Agarose in submarine gel electrophoresis. cDNA synthesis:-The first strand of cDNA was synthesized using 3 µl of RNA sample with SMART IV oligonucleotide and CDS/3' PCR primer 1 µl each. After thorough mixing the content was incubated at 72 o C for 2 min. and cooled on ice for 2 min. After spinning briefly, 5X first strand buffer (2 µl), DTT (20mM) (1µl), dNTP mix (10mM) (1µl) and Power script RT (1µl) were added and mixed. After incubation for 1 hr at 42 o C, the content was stored at -80 o C in an ice.

PCR amplification:-
A PCR was performed in a total volume of 30μl containing 15μl master mixture, 1μl of μM each of primer and 1 μl of cDNA template. PCR conditions were as follows: denaturation at 94°C for 5 min; 35 cycles of 94°C for 1 min, primer-specific annealing temperature at 45 sec and extension at 72°C for 1 min, and a final extension at 72°C for 5 min. The PCR products were resolved by electrophoresis in a 1% agarose gel in 1 × TAE buffer. The gels were prestained with 10 mg/mL ethidium bromide.
Gene sequence and submission to NCBI GenBank:-Amplified PCR product purified using Qiaquick PCR purification kit (QIAGEN, USA). Sequencing reactions were carried out in both directions using same forward and reverse primers used for amplification with BigDye Version 3.1 kit (Applied Bio-systems) on an ABI-PRISM 3730 DNA Sequencer (Applied Bio-systems). Ambiguous sequences from the base called sequences were corrected with Chromas (Version 2.01) and the sequences were assembled with Bio-Edit (Version 7.0.9.0). The search for sequence homolog of potential gene product was made using the BLASTn and BLASTx program (NCBI).

Microscopical identification of Botryococcus braunii:-
In the present investigation, Botryococcus braunii was microscopically identified from the collected fresh water samples. The identified Botryococcus braunii was presented in Fig.1. The identification of microalgae was based on their distinctive characteristics (Butcher, 1959;Carmelo and Grethe, 1997) and the instruction for microscopical identification was suggested by the Krishnamurthy Institute of Algology, Chennai.

Growth kinetics of the microalga Botryococcus braunii:-
The growth curve has blatantly shown that the log phase was initiated from the second day and attained stationary phase from third day till the sixteenth day (Graph 1). The death stage was not occurred up to sixteen days instead all 983 the biomass was settled at the bottom of culture flask. The microalga Botryococcus braunii attained the growth kinetic (K) of 0.099 and the generation time (G) was 3.09.

Estimation of pigments:-
The important photosynthetic pigments of green microalgae B. braunii including chlorophyll a and b were found to be 7.09 µg ml -1 and 10.51 µg ml -1 respectively. The total carotene was found high with 18.27 µg ml -1 , when compared with the chlorophyll pigments. The production of pigments seems to be correlated with the proliferation of the microalga Botryococcus braunii. Where, hike in the pigment production starts from the third day and found approximately stable in the stationary phase and propelled further during the last 14 th , 15 th and 16 th days (Graph 2).

Graph 2:-The figure shows the production of the three important pigments by Botryococcus braunii
Biochemical constituents:-After in vitro mass cultivation, the harvested biomass was dried and the dried biomass estimated in an electronic weighing balance which was about 236mg ml -1 . From the obtained biomass, total carbohydrate, total protein and total lipid content was estimated. The total carbohydrate content was 62.57 mg ml -1 which engulfs about 43.93 % of total biochemical composition. The total protein comprises 10.11 % of the total biochemical constituents with 14.4 mg ml -1 . The total lipid content was 65.46 mg ml-1 and holds 45.95 % of the total biochemical component (Graph 3). On the whole, it is evident that the lipid and carbohydrate content was produced high by the green microalga Botryococcus braunii, but the protein content was approximately and comparatively half when compared with the total lipid content.

Fourier transforms infra red spectrometric (FT-IR) analysis of FAME:-
The alkane (C-H) and alkyne (-C=C-) groups were determined in the FAME samples of Botryococcus braunii at 2972, 2932, 2887, 1467, 1380 cm -1 and 2194 cm -1 respectively. The presence of alkenes (=C-H) were determined by the occurrence of peak at 952 cm -1 . The esters were also reported at 1308 cm -1 and as a result the FAME is rich in long chain hydrocarbon compounds (Table 2) (Graph. 5).

Isolation of RNA and cDNA synthesis:-
The total RNA was extracted from the green microalga Botryococcus braunii was then separated and analyzed under submarine agarose gel electrophoresis. Two different bands in the gel shows mixed of RNA where the major RNAs are 28S and 18S rRNA (Fig. 2). The cDNA was synthesized using the SSL-3 primer (Table 3) already mentioned above and were qualitatively confirmed by 1.5 % agarose gel electrophoresis. Where, the single band shows the cDNA synthesized and were allowed to sequence based on the Sanger's method of dideoxy sequencing (Fig. 3). The SSL-3 gene sequence was submitted to NCBI through BankIt, GenBank and the temporary BankIt submission number was retrieved (BankIt submission no. 1897142) and the accession number yet to be received.  Phylogenetic analysis:-The dendrogram represents the phylogenetic relationship of the microalga Botryococcus braunii with other group of organisms using the gene SSL-3 (Squalene Synthase like -3 or Botryococcene synthase) (Fig. 4). In which, the SP-2 is the sequenced SSL-3 gene of Botryococcus braunii placed in a separate clade along with the green microalgae. Another phylogenetic tree was constructed using the NCBI BLAST Pair wise alignment of Neighbour joining method with maximum sequence difference of 0.005. Where, a unique clade was formed (1Cl|Query_224699) between the similar strains of Botryococcus braunii and which molecularly confirms that the gene belongs to SSL-3 and the strain belongs to Botryococcus braunii (Fig. 5).
The evolutionary history was inferred using the UPGMA method. The optimal tree with the sum of branch length = 2.34483239 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown above the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [3] and are in the units of the number of amino acid substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 1). The analysis involved 12 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 408 positions in the final dataset. Evolutionary analyses were conducted in MEGA5.

Discussion:-
Mogilevskii and his associates investigated the microbial Petroleum prospecting incited the interest of petroleum geochemists worldwide. Initially proposed and applied the microbial surveys in the USSR (Mogilevskii, 1940). It has been shown that out of 20 microbial anomalies, 16 were proved successful drilling. These investigations initiated the interest of petroleum geochemists worldwide. In USA microbial prospecting surveys showed positive correlation about 85.7% respectively (Sealy, 1974). In oil field of microbial survey at USA indicated that good contrast nearby dry area (Miller, 1976).
Microalgae are the major sources of lipids in lacustrine and marine environments. Volkman et al. (1989) provides a review of some recent advances in our knowledge of the wide variety of lipid types that have been isolated from microalgae with an emphasis on those likely to be useful biomarkers for identifying sources of organic matter in sediments. In many numbers of microalgae species were found among these Botryococcus sp. is a remarkable due to its capacity for synthesizing major amount of unsaturated hydrocarbons (up to 75 % from dry biomass). Similar to those from oil deposits (Moldowan and Seifert, 1980), physical and chemical analysis of kerogen from boghead coals identified in different geographical regions, presented a similar composition with B. braunii hydrocarbons.
Over recent years, many new compounds have been identified in sediments deposited in marine and lacustrine environments. Despite the fact that our knowledge of algal lipids is still far from comprehensive, microalgal sources have now been identified for many of the lipids that are widely distributed in recent sediments (Volkman et al., 1995;Conte et al., 1994). Volkman et al. (1998) provides a review of some recent advances in our knowledge of the wide variety of lipid types that have been isolated from microalgae with an emphasis on those likely to be useful biomarkers for identifying sources of organic matter in sediments. From the obtained biomass, total carbohydrate, total protein and total lipid content was estimated. The total carbohydrate content was 62.57 mg ml -1 which engulfs about 43.93 % of total biochemical composition. The total protein comprises 10.11 % of the total biochemical constituents with 14.4 mg ml -1 . The total lipid content was 65.46 mg ml-1 and holds 45.95 % of the total biochemical component (Fig. 4.3). On the whole, it is evident that the lipid and carbohydrate content was produced 990 high by the green microalga Botryococcus braunii, but the protein content was approximately and comparatively half when compared with the total lipid content.
Microalgae are major source of fatty acids in most sedimentary environments. The contribution from different microalgal classes can often be discerned from characteristic differences between the distributions, especially if the positions of double bonds in polyunsaturated fatty acids are considered (Volkman and Johns, 1977). Some microalgae contain high concentrations of certain long-chain essential polyunsaturated fatty acids such as 20:5n-3 and 22:6n-3 (Volkman et al., 1989). For example, marine eustigmatophytes such as Nannochloropsis spp. contain 20:5n-3 but little 22:6n-3, whereas haptophytes (prymnesiophytes) such as Pavlova spp. contain both 20:5n-3 and 22:6n-3. Chlorophytes rarely contain significant amounts of these fatty acids, but instead have a predominance of C 18 polyunsaturated fatty acids such as 18:2n-6 and 18:3n-3. Dinoflagellates have high levels of 20:5n-3 and 22:6n-3 and many contain the unusual fatty acid 18:5n-3. However, the latter is not unique to dinoflagellates and has been found in algae as diverse as haptophytes, raphidophytes and some prasinophytes (Volkman et al., 1989). The total fatty acid methyl esters obtained in the Botryococcus braunii strain was about 64.26 % of the total lipid content. The fatty acid methyl esters include Propanoic acid C27:0 (22.60 %); Propanoic acid C27:0 (15.65 %); 9-octadecenoic acid C38:0 (15.21 %) and Carotenoic acid C27:0 (10.8 %) ( Table 3) (Fig.7 to 12). The presence of high cetane number compounds in the FAME of Botryococcus braunii represents the occurrence of long chain fatty acids and hydrocarbon. Fourier transform infra red spectroscopy (FT-IR) was studied in B. braunii . The presence of esters, alkanes and alkenes confirms the presence of hydrocarbons from the FAME.
Botryococcene represents a particularly diagnostic biomarker for the assessment of paleoenvironments. This C 34 isoprenoidal alkane is considered to be derived from botryococcene, a compound known to exist only in Botryococcus braunii, which is widely distributed in freshwater lakes. Thus the identification of botryococcene in oils and sediments indicates the contribution of B. braunii to the depositional environment. However despite the quite widespread presence of Botryococcus remains in type I kerogen, this specific biomarker was only detected in a few extracts and oils, such as Sumatran oils (Seifert and Moldowan, 1985), Australian coastal bitumen (McKirdy et al., 1985) and Maoming oil shale (Brassell et al., 1986). This observation may be explained, at least in part, by the fact that of the three modern chemical races of B. braunii (A, B and L; for a review see Metzger et al., 1991), only race B produces botryococcene. Indeed, the fully hydrogenated derivatives, botryococcanes and tetrametylsqualane, are specific markers of B. braunii which are found in petroleum and oil shales, sometimes in high amounts (McKirdy et al., 1986;Summons et al., 2002). The SSL-3 gene sequence of Botryococcus braunii was translated into amino acids and the molecular structure of the protein Botryococcene synthase (SSL-3) was predicted and illustrated. The predicted molecular structure of botryococcene synthase has shown about 14 helical structures with the absence of β-pleated sheets. The superimposed study on the obtained molecular structure of botryococcene synthase protein of Botryococcus braunii with the Human Squalene synthase protein resulted that the former protein has more conserved amino acid sequence, thus the structure resembles with the human squalene synthase protein.
But there are some conformational differences in their quaternary structure.

Conclusion:-
The green microalga Botryococcus braunii has been already reported widely to be the efficient candidate for the production of biodiesel due to the high accumulation of lipid molecules. The biodiesel production from the microalga Botryococcus braunii is a cost effective, ecofriendly, efficient and intensive method for the oil production. In this present study, a new micro algal strain Botryococcus braunii was isolated from the temple tanks. The biochemical constituents and occurrence of different fatty acid methyl esters (FAME) proves the present microalgal strain Botryococcus braunii a potent candidate for the production of biodiesel.