Studies on Growth and Biochemical Analysis of Three Microalgal Strains on Different Molar Concentration of Sodium Bicarbonate

In this present investigation an attempt was made to study the Bicarbonate tolerance of selected micro algal strains (Scenedesmus sp. Botryococcus sp. and Euglena sp.). The three microalgal strains were collected from the Presidency College, Chennai, India. These strains were grown in different molar concentrations of NaHCO 3 in two dif- ferent medium BBM and BG11 without any changes in other substances of media. The Optical density (OD) was measured at regular interval time of 24hrs at 680nm employing UV-Vis Spectrophotometer. Growth rate and Biochemical composition were investigated in this study. All the three microalgal strains were grown well in the 0.1 molar concentration of NaHCO 3 in both BBM and BG11 media but the best growth rate was showed in BG11 media when compared to BBM. The highest growth rate was seen in Euglena sp. and lipid content was found high in Botryococcus sp. at 0.1 molar concentration of NaHCO 3 .


Introduction
Microalgae are aquatic unicellular organisms in both fresh and marine water having unique potential to synthesize valuable natural products (Plaza et al., 2009;Plaza et al., 2010). In recent years, lipid storage in microalgal cells turns the focus on biodiesel production, Triglycerides present in storage lipid used as a source for biodiesel and as a sustainable alternative for petroleum fuels (Mata et al,. 2010). Microalgae promises to be a suitable and efficient energy resource that can convert atmospheric carbon dioxide into biomass, fatty acid and lipids. Microalgae are low cost effective to culture, characterized by rapid growth and high biomass production, which utilizes bicarbonate as the external source of carbon for photosynthesis (Munoz and Beer, 1994). Few algae are capable of uptake of carbon dioxide directly (Raven, 1991) while others convert bicarbonate to carbon dioxide either inside the Plasmalemma (Dixon et al., 1987) or externally allowing only bicarbonate to diffuse into the cell (Badger et al., 1980). Microalgae can fix CO 2 from different sources which can be categorized as CO 2 from atmosphere, CO 2 from industrial flue gases, and fixed CO 2 in the form of soluble carbonates (NaHCO 3 /Na 2 CO 3 ). Carbon dioxide (CO 2 ) could be directly fed into microalgae culture for biofixation but sometimes it is difficult to obtain a stable and consistent supply of CO 2 unless the location of microalgae cultivation system is very close to a factory or a power plant. The major objective of this study was to optimize the growth rate of three different microalgal strains in different molar concentrations of NaHCO 3 to compare BBM and BG11 media, and analysis of biochemical composition from obtained microalgal biomass.

Materials and Methods
Three green microalgae, Scenedesmus sp., Botryococcus sp., and Euglena sp., were selected for this experiments. The strains were obtained from Culture Collection of Presidency College, Chennai, India. The aforementioned strains were explored in terms of Growth and biochemical study in different molar concentration of NaHCO 3 in two different medium (BBM and BG11). BBM and BG11 culture media were selected and prepared for the growth of microalgae strains, in which BG11 consists of carbonate source in it. Then different molar concentrations (0.1, 0.5, 1.0, 1.5, and 2.0) of NaHCO 3 salt was weighed and allowed to dissolve in BBM and BG11 media for carbon source and Control were prepared having normal BBM and BG11 without any strain inoculation. The pH was adjusted to 7.5 in each of the flasks using pH tutor meter (Eutech instruments).

Light condition
For our analysis, white inflorescence lamps (Crompton 40W, Cool day light 6500 K) at an intensity of 2000 lux in a 12:12 light and dark regime were employed and the temperature was adjusted to 25°C for all the flasks.

Analytical method
The Optical Density of the three microalgal cultures Scenedesmus sp. Botryococcus sp. and Euglena sp. were measured at regular interval time of 24 hrs by absorbance at 680 nm using spectrophotometer (Hitachi U-2900) till 15 th day. At the end of the experiment, all the culture flasks were centrifuged and filtered. The pellet obtained biomass were oven dried, weighed and stored for further biochemical analysis.
Biochemical composition and analysis on microalgal biomass Total Protein, Carbohydrate and Lipid was analyzed from biomass, based on Lowry et al., 1951, Dubois et al., 1956and Ronald, 2001 methods respectively.

Effect of Bicarbonate tolerance and utilization
Three microalgae strains Scenedesmus sp. Botryococcus sp. and Euglena sp. were investigated against different molar concentrations of NaHCO 3 in their media. Primary and suitable media for microalgae BG11 and BBM were selected and analyzed for this study. The three microalgal strains Scenedesmus sp. Botryococcus sp. and Euglena sp. showed simultaneous growth initially on both the media, but the rate was deviated after further growth based on the utilization and tolerance of sodium bicarbonate on each organism. The biomass and growth rate was higher at the 12 th and 13 th day after inoculation. Scenedesmus sp. showed slightly deviated growth rate in both the media but the growth rate was higher at 0.1 M concentration of NaHCO 3 in both BBM and BG11 media when compared to other strains. Botryococcus sp. gained growth rate at the same molar concentration as in RESEARCH PAPER Scenedesmus sp. but the rate was deviated between BBM and BG11 hence, BG11 showed the best growth rate when compared to BBM. Euglena sp. shows the highest growth rate when compared to both the microalgal strains. In overall analysis, 0.1 M concentration of bicarbonate and BG11 media was the feasible and suitable for uptake of carbonate and high growth biomass production of microalgal species. The growth rate of the three microalgal species in both BBM and BG11 are expressed in Fig. 1 -6.

Total microalgal biomass
The total biomass obtained from three microalgal strains was 1.522 gl -1 , 1.684 gl -1 and 1.886 gl -1 , from Scenedesmus sp., Botryococcus sp. and Euglena sp. respectively. In which, Euglena sp. showed high biomass than the other two micro algal strains.

Biochemical composition Total Protein
The total protein content of the three microalgal strains was estimated from the obtained algal biomass and expressed in mg g -1 . Euglena sp. gained 17.93 mg g -1 ; Botryococcus sp. showed 14.57 mg g -1 and Scenedesmus sp. obtained 18.21 mg g -1 . Scenedesmus sp. secured the highest protein among the three microalgal strains (Fig.7).

Total Carbohydrate
The total carbohydrate was estimated from the biomass, Scenedesmus sp. obtained high amount of carbohydrate when compared to others. Scenedesmus sp. 36.74 mg g -1 , Botryococcus sp. 31.42 mg g -1 and Euglena sp. 33.24 mg g -1 of carbohydrate present in it (Fig.7), follows less than Scenedesmus sp.

Total Lipid
As we expect, the total lipid content of Botryococcus sp. was the highest when compared to Scenedesmus sp. and Euglena sp. 50.62 mg g -1 , 41.24 mg g -1 and 45.5 mg g -1 respectively. The overall biochemical composition Carbohydrate contributed 45%, Protein 33% and Lipid 17% in terms of percentage from the three microalgal strains (Fig.7).

Discussion
The microalgae have their own distinct applications in the field of algal biotechnology. Botryococcus a green microalga considered as a potent resource for fuel due its ability on high production of hydrocarbons. Botryococcus also involved in extraction of various carotenoids form it. Free fatty acids such as C 18 and C 16 fatty acids were isolated and studied form Botryococcus braunii and showed high amount of lipid content thus, may equip in third generation biofuel (Dragone et al., 2010). The lipid content of all the microalgal strains increased when they were grown in media supplemented with bicarbonate salt, Scenedesmus strain showed highest accumulation of lipid along with highest growth response (Devgoswami et al., 2011). Scenedesmus sp. could convert approximately 15-25% of atmospheric CO 2 into biodisel (Ho et al., 2010), (Mandal and Mallick, 2009). From the present investigation, Botryococcus sp. found as the high lipid producer than the other strains as we expect, 50.62 mg of lipid form 1g of algal biomass. It did not show any increase in biomass when compared to other microalgal strains, but compared to different concentrations of NaHCO 3 0.1 M was the best in high yielding of biomass. Freshwater Scenedesmus obliquus was one of best lipid producer in large scale level due to high lipid production (da silva et al., 2008). Algal biomass will potentially increased by optimizing the culture medium, Scenedesmus sp. strain JPCC GA0024 showed high lipid content equivalent to highest lipid producers but not high than Botryococcus braunii (Matsunaga et al., 2009). Euglena sp. accumulates somewhat high amount of lipid than Scenedesmus sp. and increase in its biomass than other two strains. Thus, Euglena sp. is viable in production of high biomass at 0.1 M concentrations of NaHCO 3. Scenedesmus sp. acquired less biomass than the other strains but produce high biomass at 0.1 M concentration by comparing different concentrations of NaHCO 3 . Addition of sodium bicarbonate is a feasible strategy to control cellular abundance and concentrations of pigments and lipids.

Conclusion
An attempt was made to study the Bicarbonate tolerance by three different Micro algae viz., Scenedesmus sp, Botryococcus sp. and Euglena sp. at different concentration of NaHCO 3 dissolved in BBM and BG11 under In-vitro condition. At 0.1M concentration of NaHCO 3 , the different micro algal strains Scenedesmus sp., Botryococcus sp. and Euglena sp. showed highest accumulation of lipid with highest growth rate. The technical data presented in this study are of relevance and value for further development and will generate additional interest in the field of algal technology. In future these algae can be grown in different concentrations of sodium bicarbonate (NaHCO 3 ) and the optimized concentration can be employed in biofuel production and Phycoremediation processes.