INTRODUCTION:

The need to fulfill the ever increasing global energy demand caused the intensive use of fossil fuels like coal, petroleum and natural gas during the last century and nowadays they represent more than the 80% of the energetic resources. Owing to their exhaustibility and unsustainable environmental impact, both generated by their fossil origin, growing attention has emerged about renewable energy, with the aim of diversifying the energy resources to reduce the utilization of fossil fuels and hence limiting their negative effect. In this context one of the most important renewable energy resources is biodiesel, which is produced from triglycerides by tranesterification reactions¹. Commercial production of microalgae has been practiced since the 1950s, commencing with the production of Chlorella vulgaris in Japan and Taiwan, and later with the production of Spirulina.

The past decades have witnessed great strides in the production and utilization of algae through the efforts of many scientists in several countries².Against a backdrop of rising crude oil prices, depletion of resources, political instability in producing countries and environmental challenges, besides efficiency and intelligent use, only biomass has the potential to replace the supply of an energy hungry civilization. Actually, one of the most promising feedstock for biodiesel production are unicellular algae ^{3, 4}.

In fact, when compared with superior plants microalgae show higher photosynthetic efficiency, higher biomass productivities and faster growth rates. Microalgae are very diverse⁵. They can create a range of useful products, and there is a variety of ways in which they can be cultivated, manipulated, harvested and utilized ⁶. Most importantly for the interests of this study, they have the ability to produce large amounts of lipids, including triacylglycerides (TAGs), a high energy density storage molecule⁷, which can be converted into biodiesel via transesterification. Natural oil levels vary between microalgae species; a review of 14 microalgae genera reported oil contents of 15-77% total dry weight⁸. Lipid production can be increased by manipulating the environment of the microalgae, also known as biochemical engineering ⁷. As lipid content is affected by many environmental factors, there exist a variety of ways of doing this in different algal species, including nitrogen deprivation, temperature and light, pH stress, CO₂ aeration⁹, and osmotic stress¹⁰. It has been well-reported that biodiesel obtained from canola and soybean, palm, sunflower oil, algal oil as a diesel fuel substitute¹¹. Some algal species are halo tolerant and produce lipids as compatible solutes to cope with high or fluctuating salinities.

Photosynthetic microorganisms use inorganic carbon for growth and hence can convert CO₂ from a point source into biomass. CO₂ bio fixation method is the most important and the most effective carbon sequestration method on earth. Microalgae have higher photosynthetic efficiency, higher biomass production and faster growth compared to other energy crops ^12-14. Photosynthetic CO₂ fixation by microalgae is thought to be a feasible technology with energy-saving and environment friendly¹⁵. Chlorella sp.¹⁶ and Nannochloris oculata¹⁷ showed an optimal growth potential with 2% CO₂, the growth of microalgae were completely inhibited with 5%, 10%, and 15% CO₂. The highest values of maximum biomass concentration about 1.2 g /L of Chlorella sp.¹⁶ and Nannochloris oculata ¹⁷ were obtained in the presence of 2% CO₂. The main fatty acid component of Chlorella Pyrenoidosa was fatty acids with C₁₆–C₁₈ under different CO₂ concentrations. High CO₂ levels (30–50%) were favourable for the accumulation of total lipids and polyunsaturated fatty acids¹⁸.

MATERIALS & METHODS:

Medium:

Bolds basal medium was used as a growth medium. It comprises of KH₂PO₄(0.7g/L), K₂HPO₄(0.3g/L), MgSO₄. 7H₂O (0.3g/L), Glycine (0.1g/L), FeSO₄.7H₂O (3mg/L), itamin B₁(0.01mg/L).

Inoculum preparation:

The micro alga, Chlorella Pyrenoidosa (NO.2738) obtained from national collection of industrial micro organisms (NCIM) was inoculated in Bold Basal Medium (BBM) incorporated with 30 g/L of glucose and 4 g/L of yeast with a pH of 6.6 and the culture was incubated for 4 days at 24±1ºC in a thermo-statically controlled chamber and illuminated with cool inflorescence lamps at an intensity of 60 µmol/ (m² s) (14 WTL5 tungsten filament lamps; Philips Co.,).

Experimental design:

The growth and lipid accumulation of Chlorella pyrenoidosa was subjected to Bolds Basal medium with differing carbon sources at the autotrophic levels say 0%, 10%, 20% of CO₂and heterotrophic levels such as glucose, fructose and sucrose as carbon sources individually and determination the lipid content in the algal cells by means of Rapid Colorimetric Determination, also to find out the carbon source at which maximum lipid accumulation of the algae is achieved during the growth period.

Cultivation in the photo bioreactor:

The culture medium is prepared and adjusted to pH 6.6 sterilized with autoclave sterilizer at 112 ºC, 120kPa for 30 min. Chlorella Pyrenoidosa in logarithmic phase was inoculated into the culture medium. The reactor is subjected to continuous illumination at an intensity of 60 µmol/ (m² s) (14 WTL5 tungsten filament lamps; Philips Co.,) [19]. The process is subjected to different carbon sources along with the basal medium composition. The CO₂supply to the medium is been supplemented at a low flow rate of 0.003 vvm¹⁹.

Estimation of cell mass concentration:

The spectral analysis was carried out (540 nm) for monitoring the cell growth using ELICO SL 150. The biomass concentration was recorded by collecting each day sample centrifuged at 14,000 ×g for 10 min. The supernatant was discarded. The cell pellets were lyophilized to constant weight and it was measured in terms of dry weight.

Lipid estimation:

The lipid content in the algal cells was estimated by using rapid calorimetric determination¹⁹. Laurie acid equivalent is used as a standard for the determination of the fatty acid content in each day analysis. 10mM stock solution of lauric acid is prepared in 1M NaOH. Longer chain fatty acids had to be solubilised by heating the standard to 60 ºC. Dilutions were prepared in pre-warmed 1N NaOH. 2ml of standard and dilutions thereof were added to the centrifuge tubes.

Estimation of lipid content in algal cells:

The standard curve was drawn for the corresponding dilutions by means of the optical density from which the amount of lipid in the algal cells are determined for their corresponding optical density.

Extraction and determination of total lipids:

The lipid fraction was extracted from the biomass by the Bligh and Dyer method²⁰ obtaining an immiscible system consisting of the sample water content and a mixture of chloroform and water. The total lipid concentration was determined gravimetrically from the chloroform extract by evaporating the chloroform in an atmosphere of nitrogen and subsequently drying to constant weight in a vacuum oven.

Preparation of the fatty acid methyl esters:

The method of Hartman and Lago²² was used to saponify and esterify (methylation reaction) the dried lipid extract to obtain the fatty acid methyl esters (biodiesel). An amount of 250 mg of oil was added to 5.0 mL of NaOH 0.50 mol L⁻¹ in methanol. The mixture was then heated under reflux for 5 min. After adding 15.0mL of the esterification reagent (prepared from a mixture of 2.0 g of ammonia chloride, 60.0 mL of methanol, and 3.0 mL of concentrated sulphuric acid for about 15 min), the mixture was heated under reflux for another 3 min and subsequently transferred to a separation funnel containing 25.0mL of petroleum ether and 50.0mL of deionized water. After stirring the mixture and separating the phases, the aqueous phase was discarded. Then 25.0mL of deionized water was added to the organic phase. This mixture was stirred and, after phase separation, the aqueous phase was discarded. This procedure was repeated. The organic phase was collected, the solvent was evaporated in a rotary evaporator and the residue was removed under nitrogen flow. The methyl esters were solubilised in n-heptane before injection in the gas chromatograph.

RESULTS:

Effect of different carbon sources on algal growth:

Chlorella pyrenoidosa is said to be subjected to different carbon sources individually such as glucose, sucrose, fructose and also differing CO₂ concentrations as carbon sources in concentrations of 0%, 10%, 20% and determined for growth. It has been observed that the biomass concentration is said to be higher with glucose as the carbon source and said to be 7.3 g/L. It consists of a lipid content of 0.1622 g/L.

Fig. 1 Standard result for Lipid using Lauric acid equivalent

Fig.2. Growth rate of different carbon sources

In the observed study it has been found that the supply of CO₂to the medium deprivates the growth of the algal cells. At the heterotrophic condition, a maximum of 7.3g/L of biomass content was obtained. With sucrose as the carbon source, a maximum biomass content of 6.03g/L is obtained. Fructose achieved a maximum biomass content of 6.15g/L of biomass concentration. At the autotrophic level, 0% CO₂obtained a maximum biomass content of 0.76g/L. Considering 10% CO₂supply, the maximum biomass content at this level is said to be 1.63g/L. At 20% of CO₂supply it is said to be 1.44g/L. Hence from the obtained results it is clear that maximum growth is said to be achieved with heterotrophic carbon supply with glucose as the carbon source compared with that of sucrose and fructose.

Table 1 Obtained Biomass concentration for different carbon sources

Time (days)	Biomass Concentration (g/L)
Time (days)	Glucose	Sucrose	Fructose	0% CO₂	10% CO₂	20% CO₂
0	0.2	0.2	0.2	0.2	0.2	0.2
1	3.41	3.1	3.23	0.21	0.27	0.23
2	4.44	4.21	4.35	0.21	0.3	0.28
3	5.23	5.05	5.2	0.24	0.34	0.31
4	5.7	5.13	5.37	0.28	0.49	0.42
5	6.01	5.17	5.49	0.33	0.52	0.47
6	6.22	5.23	5.53	0.38	0.67	0.61
7	6.41	5.34	5.64	0.44	0.76	0.7
8	6.63	5.49	5.73	0.49	0.82	0.78
9	6.82	5.54	5.8	0.53	0.98	0.84
10	6.94	5.66	6.13	0.58	1.08	0.92
11	7.1	5.74	6.14	0.64	1.26	0.98
12	7.2	5.83	6.15	0.71	1.37	1.06
13	7.3	5.93	6.15	0.74	1.52	1.12
14	7.3	6.01	6.15	0.76	1.6	1.14
15 16	7.3 7.2	6.03 6.03	6.13 6.12	0.75 0.75	1.63 1.63	1.44 1.13

Effect of different CO₂concentrations:

The Effect of CO₂towards the algal growth is said influence a negative effect the growth of the algal cells. Increasing CO₂concentrations led to decrease in the biomass content of Chlorella pyrenoidosa. Here the CO₂supply as the carbon source has showed an increased effect towards the production of lipid content in the cell. Compared to that of the heterotrophic sources of carbon the autotrophic supply of carbon source is said to increase the lipid content in the cell. A higher yield of 0.3391g/L of lipid is obtained at 20% of CO₂. Whereas with glucose as the carbon source, a maximum lipid content of 0.1622g/L is obtained at the twelfth day. With respect to sucrose, a maximum lipid content of 0.136g/L was obtained at fifteenth day. Considering fructose, a maximum lipid content of 0.1462g/L is obtained at the twelfth day of the study. This shows that in the heterotrophic carbon source comparison, glucose is said to yield considerably higher amount of lipid than fructose and sucrose. Observation towards autotrophic carbon sources, for 0% CO₂condition, a maximum lipid content of 0.0324g/L is obtained at the fourteenth day of the study. A maximum lipid content of 0.3191g/L was achieved at sixteenth day for 10% CO₂supply. Considering 20% CO₂supply, the highest of all the obtained lipid content of 0.3391g/L is obtained at the fourteenth day. Hence from the observed results it has been concluded that higher accumulation of lipid content in the algal cells is achieved at the autotrophic carbon source supplement in the increasing levels of CO₂.

In this study it has been observed that the lipid content increases with the increasing amount of CO₂ levels and is said to be higher than that of the lipid content in the glucose medium. A maximum lipid content of 0.3391g/L was obtained with 20% of CO₂ supply with the corresponding biomass concentration of 1.54g/L.

DISCUSSION:

The study has been implemented for observing the effect of algal growth with different carbon sources. Differing the carbon sources with respect of the same components of the medium has enabled to determine the carbon source at which maximum growth of the algal cells is achieved. Glucose is said to give maximum algal growth with a concentration of 10g/L along with the components of the basal medium. The study is preceded with different carbon sources such as fructose and sucrose also with respect of the CO₂ concentrations. Here yeast extract is added to the inoculum preparation for increased growth rate. The study is being carried out according to the observation of the growth rate of the cells in the reactor.

The lipid content in the cells is said to increase with the increase in the supply of the C0₂ to the medium. Hence considering the growth of the algal cells with concentrations of 0%, 10% and 20%, maximum lipid content of 0.339 g/L is obtained at 20% of CO₂. This depicts that the lipid content does not depend upon the growth rate of the cells. Here the supply of CO₂ at varied concentrations indicates that the lipid content increases with the increase in CO₂ levels to the growth medium. The supply of CO₂ is said to be the carbon source for the medium similarly replacing glucose, sucrose and fructose which play the role of carbon sources along with the components of basal medium. According to the observations recorded it is clear that the maximum growth rate was acquired with glucose as the carbon source said to be 7.3g/L of biomass concentration. This study was observed for each of sucrose and fructose. Next to glucose, fructose yields a biomass concentration of 6.15 g/L. Thirdly sucrose yields a maximum biomass concentration of 6.03%. The CO₂concentrations 0%, 10% and 20% achieved a biomass concentration of 0.76 g/L, 1.63 g/L and 1.54 g/L respectively. This shows that the growth rate of the algal cells were maximum in the presence of glucose as the carbon source.

Table 2 Obtained Lipid content on different carbon sources

Time (days )	Lipid Content (g/L)
Time (days )	Glucose	Sucrose	Fructose	0% CO₂	10% CO₂	20% CO₂
0	0.001	0.001	0.001	0.001	0.001	0.001
1	0.024	0.0195	0.021	0.0014	0.0024	0.00276
2	0.03	0.024	0.029	0.0023	0.0039	0.00476
3	0.048	0.031	0.042	0.0031	0.0061	0.00868
4	0.05	0.04	0.049	0.00476	0.01176	0.0197
5	0.054	0.048	0.05	0.0066	0.01404	0.0277
6	0.06	0.053	0.058	0.0087	0.0214	0.0445
7	0.0681	0.06	0.065	0.0114	0.0317	0.0686
8	0.0721	0.062	0.069	0.0132	0.0591	0.0875
9	0.0841	0.071	0.079	0.0153	0.0948	0.1149
10	0.1081	0.092	0.099	0.0179	0.11005	0.1406
11	0.1261	0.103	0.113	0.023	0.168	0.1746
12	0.1622	0.124	0.1462	0.0276	0.1989	0.202
13	0.1522	0.128	0.146	0.031	0.244	0.308
14	0.098	0.136	0.146	0.0324	0.273	0.3391
15	0.089	0.13	0.144	0.0285	0.297	0.34
16	0.086		0.142	0.0282	0.3191	0.309

The estimation of fatty acid content present in the cells was determined by using the calorimetric determination. The use of standards helps to determine the corresponding amount of the fatty acid present in the algal cells for the respective optical density of the sample. From this the appropriate fatty acid content can be determined for each day sample. The maximum lipid content of 22% was obtained with 20% CO₂ supply to the medium.

CONCLUSIONS:

As a result of this study, the effect of different carbon sources over the algae Chlorella pyrenoidosa has been understood over the growth period. The heterotrophic carbon supplement is said to increase the algal growth at a rapid extent considering glucose as the carbon source. In the case of autotrophic carbon supplement, the growth is said to be lower than that of the heterotrophic level but leads to higher accumulation of lipid content in the algal cells. Hence supply of CO₂ is said to be stress supplement as algae is said to accumulate high amounts of lipid at stress induced conditions. Therefore this study concludes that the rate of lipid accumulation in the algal cells increases with increasing CO₂ concentrations as the autotrophic carbon supplement to the medium. Also higher yield to biomass is obtained with glucose as the carbon source under heterotrophic condition.

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Received on 27.08.2013 Accepted on 01.09.2013

Research J. Engineering and Tech. 4(4): Oct.-Dec., 2013 page 152-156