Chlorococcum humicola ( Nageli ) Rabenhorst as a Renewable Source of Bioproducts and

Among the diverse new generation biomass yielding species, green algae are the most promising organisms. Compared to biomass production of other organisms, production of algae is less laborious, quite fast, and more economical. Moreover, eutrophicated waters get naturally purified in the cultivation process of algae. Algal biomass from monoculture of specific species, which are rich in carbohydrates, proteins and lipids, is considered a good source of diverse bio-products and feed-stock for food, feeds and bio-fuels. Quantity and quality of algal biomass for specific products depend on the species and strains as well as environmental conditions of cultivation. In this connection, biomass productivity and oil-yield of a local strain of Chlorococcum humicola (Nageli) Rabenhorst was assessed in Bold’s Basal Medium. Long-term storage capacity of the alga was tried by entrapping the algal cells in sodium alginate beads, which showed viability up to 14 months. Estimation of total carbohydrate, protein, lipid and chemical characterization of oil as well as the feasibility of its conversion to biodiesel revealed the industrial potential of this local strain as a source of food and biofuel. Fatty acid profiling of the extracted oil showed that 70% are mono-saturated and 12.2 % are nutritionally important polyunsaturated fatty acids. The oil could be effectively trans-esterified to methyl esters and the conversion was confirmed by FTIR spectroscopy. Further standardization of the mass production of the alga in natural environmental conditions for biomass and oil is progressing to optimize its value as globally competent food, nutraceutical and biofuel resource.


Introduction
Green algae are significant new biomass resource for the production of natural bioactive compounds and renewable energy.They have unique nutritional quality (Becker, 2007) that can add value to conventional food preparations for humans and animals.Because of the high protein content, biomass from many species of microalgae is generally considered as the potential source of proteins (Spolaore, Joannis-Cassan, Duran, & Isambert, 2006) for the future.In addition to proteins, green algae are good source of carbohydrates and lipids for food and fuels.Among the known commercially and industrially amenable green resources, algae can provide the highest and cheapest biomass per unit light and area.Simultaneously they can remediate nutrient load or degrade other toxic pollutants in water (Chiu et al., 2015) and reducing atmospheric CO 2 level through carbon fixation process (Morais & Costa, 2007).Moreover, several pharmaceutical products are derived from algal biomass (Yamaguchi, 1997), especially from that of Chlorophytes.Biopharmaceutical industries are in search of low cost biomaterials for production of therapeutics in a sustainable manner (Johnson, 2008).Chlorococcum humicola (Nageli) Rabenhorst (Figure 1) is a freshwater unicellular green alga coming under the class Chlorophyta.Even though C. humicola is proved to be a rich source of structurally novel and biologically active metabolites (Bhagavathy, Sumathi & Jancy Sherene Bell , 2011), biomass-productivity of its specific strains are not well known.
Long-term storage of algal stock in sodium alginate beads in pure culture form is useful for stock culture management (Gaudin, Lebeau, & Robert , 2006) as well as long term storage of the algal seeds (Faafeng, Donk, & Källqvist, 1994) for biomass production and production of secondary metabolites (Moreno-Garrido, 2008).Immobilized algae can also be used in wastewater treatment (Travieso et al., 1996)

Extraction of Algal Oil
Total lipids were extracted as per the method of Bligh and Dyer (1959) using Soxhlet; 10 gm of freeze dried biomass was taken into a round bottom flask and added 100 mL of chloroform: methanol (2:1 v/v) mixture into the biomass.The biomass was then kept soaked in the organic solvents for 4 hrs under continuous shaking in a rotary shaker at 750 rpm; afterwards the mixture was centrifuged at 6000 rpm for 5 minutes at room temperature (27 0 C -30 0 C).Residual biomass was separated from the extract and then the oil along with the solvent was transferred in to a separating funnel.About 40 mL of distilled water was added to this mixture to separate the oil from the solvent.The oil got separated as an organic phase in bottom layers; this was then collected into a bottle.The separated biomass and the oil were made free of the solvent by using rotary evaporator.
The air dried residual biomass free of the solvent was further subjected to hot method of extraction for collection of the remaining neutral lipids.The biomass was taken in to Soxhlet extractor with 75 mL of hexane, refluxed under 70 0 C for 2 hours.The extracted oil components were collected and the oil was made-free of the solvent by using rotary evaporator.Finally, the two extracted oil samples were mixed together to get the total oil.

Chemical Characterization of Algal Oil
Chemical characterization of the oil was carried out using the advanced Government of India analytical facility at CARE Kerala, Chalakkudy.Exactly 50 mg of algal oil was saponified with 1 mL of saturated KOH-CH 3 OH solution at 50 0 C for 10 minutes and then followed by methanolysis with 5% HCl in methanol at 60 0 C for another 10 minutes in screw capped test tubes.The methyl fatty acids were separated by adding 2 mL of water into it and fatty acid phase was recovered.GC-MS (Agilent make 7890A-5975C) instrument was used for the fatty acid profiling.1 mL of methyl fatty acid sample was injected to the GC column.Helium was used as carrier gas at flow rate of 54 mL/min.Chromatographic data was recorded and compared using Agilent data analysis software.

Transesterification of Algal Oil and the Production of Biodiesel
400 mg of algal oil extracted were taken into a round bottom flask and mixed with 15 mL of methanolic sulphuric acid containing 2% sulphuric acid in methanol (v/v) and refluxed at 60 0 C for 4 hours with continuous shaking.The reaction was monitored by thin layer chromatography (TLC) with the solvent system, Hexane: Ethyl acetate/ hexane: Toluene at the ratio of 9:1.The reaction was continued till the oil spot was disappeared on TLC plate.After the completion of reaction (2-4 hr), the contents were transferred to separating funnel and 25 mL water was added to it.The aqueous layer was extracted twice with ethyl acetate (25 mL each) and pooled the ethyl acetate layer.The extract was dried over anhydrous Na 2 SO 4 and concentrated under vacuum.

FTIR Analysis of Algal biodiesel
FTIR characterization for 'biodiesel' samples produced was carried out (IS10 FTIR, Thermo Scientific) in transmission mode in 400-4000 cm -1 wave number range.

Fatty acid composition of algal oil
Percentage of oil in algae (%) was calculated using the formula (Abubakar, Mutie, & Muhoho, 2012

Biomass Productivity of Alga in Vitro Culture Media
Since BBM is known to enhance maximum production of protein and chlorophyll in green algae (Sankar & Ramasubramanian, 2012), the same medium was used for the assessment of biomass productivity of C. humicola (Table 1) in the current experimentation.Productivity of 73.8% mg/ L/ day obtained suggests the alga to be a suitable candidate for high yield of biomass and other derivatives, easily amenable to industrial trials.

Experi
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