| Abstract | In this study, four microalgae strains were cultivated with synthetic media for production of biomass following lipid and carbohydrates extraction for biofuel production. Four microalgal strains i.e. Chlorococcum sp. TISTR 8583, Scenedesmus obliquus TISTR 8522, C. humicola TISTR 8461 and Haematococcus sp. TISTR 8627 were evaluated for the set research objectives. The selected strains were studied to analyze various parameters for their effects on biomass yield and accumulation of starch and lipid contents in microalgal strains. Initially, the Chlorococcum sp. TISTR 8583 and Scenedesmus obliquus TISTR 8522 were subjected to three different light intensities such as low irradiance (LI), medium irradiance (MI) and high irradiance (HI) with 3.25 klx, 4.34 klx and 6.05 klx respectively for their effect on biomass yield. As a result, the medium irradiance of 4.34 Klx was found to be optimum for both the strains with maximum biomass yield of 1.0 g/L from Chlorococcum sp. TISTR 8583 and 0.8 g/L from Scenedesmus obliquus TISTR 8522.
Further study was conducted to enhance the accumulation of sugars and lipids in algal cells by limiting the nitrogen contents (NL) in cultivation media (BG-11). The limitation of nitrogen in growth media is proven to be an efficient way to manipulate the algal systems to obtain desired product in large quantity. The Chlorococcum sp. TISTR 8583 and Scenedesmus obliquus TISTR 8522 were cultivated on two different nitrogen concentrations such as nitrogen supplemented (NS) and nitrogen limited (NL) BG-11 nutrient media. Consequently, the nitrogen limitation (NL) enhanced the accumulation of both carbohydrate and lipid contents in both the strains especially Chlorococcum sp. TISTR 8583, while decreasing in biomass yield in case of both strains. As a result, enhanced carbohydrates (34.02% w/w) accumulation was observed from NL strategy as compared to the carbohydrates yield (22.57% w/w) from nitrogen supplemented (NS) strategy in Chlorococcum sp. TISTR 8583. Similarly, the nitrogen supplemented (NS) media produced 29.59% (w/w) lipids from NL condition as compared to 17.05% (w/w) lipids of NS condition in the same strain. In case of Scenedesmus obliquus TISTR 8522, the NS and NL sample produced 12.49% (w/w) and 17.14% (w/w) lipids respectively with about 5% increase in accumulation. However, the biomass yield was decreased from 1.0 g/L (NS) to 0.66 g/L in NL strategy in Chlorococcom sp. TISTR 8583. Similar scenario was observed in Scenedesmus obliquus TISTR 8522 with 0.8 g/L from NS and 0.4 g/L biomass from NL strategy. The biomass produced from Chlorococcum sp. TISTR 8583 and Scenedesmus obliquus TISTR 8522 was further investigated for lipid extraction, types of fatty acids and biodiesel production. The most abundant fatty acid produced by both the strains was 9-Octadecenoic acid with the concentration of 26.17% and 34.403% in NS samples of Chlorococcum sp. TISTR 8583 and Scenedemus obliquus TISTR 8522 respectively. The concentration of C-16, C-18 and saturated fatty acids (SFA) was higher in Scenedesmus obliquus TISTR 8522 than that of Chlorococcum sp. TISTR 8533 which on the whole, makes Scenedesmus obliquus TISTR 8522 far efficient strain in terms of biodiesel quality than Chlorococcum sp. TISTR 8583 (with higher levels of mono unsaturated fatty acids and poly unsaturated fatty acids) but in case of lipid yield, the Chlorococcum sp. TISTR 8522 was found more efficient than Scenedesmus obliquus TISTR 8522. In a seperate experiment, the NL biomass samples of Chlorococcum sp. TISTR 8583 were further evaluated for the release of sugar contents using acid, alkaline and enzyme pretreatment following the fermentation for bioethanol production. The alkaline pretreatment was found to be the most efficient strategy (23.67% sugars/g algal biomass with 1.2% (w/v) alkali concentration at 140 ⁰C of pretreatment temperature for 30 min) while the acid pretreatment (1% v/v; 140 ⁰C) was least effective pretreatment strategy with the yield of 14.83% sugars/g algal biomass.
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Furthermore, all the four strains (Chlorococcum sp. TISTR 8583, Scenedesmus obliquus TISTR 8522, C. humicola TISTR 8461 and Haematococcus sp. TISTR 8627) were cultivated for evaluation of the impact of varying size culture reactors on the biomass yield, calculated as g/L. Among them, the higher biomass was produced by Chlorococcum sp. TISTR 8583 with 1.0 g/L followed by Scenedesmus obliquus TISTR 8522 with biomass yield of 0.8 g/L while the C. humicola TISTR 8461 and Haematococcus sp. TISTR 8627 produced average biomass of 0.96 g/L and 0.51 g/L from 1 L capacity culture reactor respectively. Similarly in case of 5 liter reactor vessel, the Chlorococcum sp. TISTR 8583 produced 0.64 g/L biomass while Scenedesmus obliquus TISTR 8522 produced 0.49 g/L. The C. humicola TISTR 8461 yielded 0.60 g/L almost equal to that of Chlorococcum sp. TISTR 8583 followed by Haematococcus sp. TISTR 8627 with least biomass of 0.32 g/L. As the culture vessel was upsclaed to 30 L, the Chlorococcum sp. TISTR 8583 produced almost 3 times less biomass (0.33 g/L) as compared to 1 L reactor vessel. In case of 30 L reactor, the least biomass (0.16 g/L) was produced by Haematococcus sp. TISTR 8627, which makes it one of the least suitable strains for biofuel purpose. The decrease in biomass concentration was the effect of large size in which light penetration was impeded coupled with gaseous exchange problem.
Moreover, in comparison to synthetic media, being costly, the wastewater as nutrient source for algal cultivation seems feasible for its production. For this purpose, all strains were further cultivated in municipal wastewater for algal biomass production following lipid extraction and conversion to fatty acid methyl esters (FAME: biodiesel). As a result of cultivation in wastewater, the Chlorococcum sp. TISTR 8583 produced 0.63 g/L biomass being the highest followed by Scenedesmus obliquus TISTR 8522 with a biomass yield of 0.44 g/L wastewater media. The least biomass was produced from Chlorococcum humicola TISTR 8461 with a yield of 0.281 g/L. The lipid content was the highest as 21.21% (w/w) from Chlorococcum sp. TISTR 8583 following Scenedesmus obliquus TISTR 8522 with a lipid yield of 16.61% (w/w) while other two strains were found low in lipid yield. Both the strains were investigated for fatty acid methyl ester (FAME) composition in order to evaluate the quality of biodiesel. The scenario of fatty acid composition of two strains (Scenedesmus obliquus TISTR 8522 and Chlorococcum sp. TISTR 8583) was almost consistent with NL and NS samples. However, in present scenario, wastewater seems having exposed to nanoparticles, such as silver nanoparticles, zinc oxide (ZnO) nanoparticles other than other antifactor chemicals which damage the potential of wastewater as good nutrient source for algal cultivation. It was aimed to visualize the effect of ZnO nanoparticles on the aquatic life and potential of wastewater as a nutrient choice for algal cultivation. For this purpose, all four selected strains were exposed to different concentrations (20, 40 and 60 mg/L) of ZnO nanoparticles for their NPs tolerance, survival capacity, cellular damage and cellular death rate. The Scenedesmus obliquus TISTR 8522 was found to having better survival capacity due to 8 days survival in high nanoparticles environment releasing extracellular polymeric substances (EPS) for entrapment of nanoparticles while Chlorococcum sp. TISTR 8583 survived 6 days in NPs rich environment. The rest two strains were found to be less tolerant to nanoparticles load especially the Haematococcus sp. TISTR 8627 with only 3 days (72 h) survival to all three concentrations of ZnO nanoparticles which indicates the presence of NPs may shrink the potential of wastewater as nutrient source for algal cultivation. |