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Methodology
3.1. Collection of Algal Sample
Seaweeds are collected from Aghnashini estuary and washed thoroughly with tap water to remove salts, epiphytes and debris. Shade dried or oven dried to a constant weight at temperature of 50° C. After drying, the seaweed samples are powdered using grinder for chemical composition analysis.
3.2. Chemical Composition of Seaweed
Seaweeds chemical composition analysisincludes the determination of protein, carbohydrate, lipid, cellulose content and reducing sugars following the method as per standard protocol (Lowry et al. 1951; Dubois et al. 1956; Folch et al. 1957; Updegroff 1969; Miller 1959).
3.3. Pre-treatment Method
Seaweed carbohydrates are broken down to simple sugars for bioethanol production. These sugars are firstly released from the biomass using pre-treatment methods such as dilute acid and enzyme hydrolysis. In dilute acid pre-treatment method complex carbohydrates of seaweed is broken down to simple sugars (disaccharides), enzyme hydrolysis further breaks down these sugars to fermentable sugars. Mineral acids such as H2 SO4 and HCL of various concentration are used for acid hydrolysis. Enzyme hydrolysis is carried out using different enzymes as they are highly substrate specific. Since major portion of seaweed cell wall is composed of cellulose, to break down cellulose cellulase enzyme is employed at different dosage and incubation period for the maximum yield of fermentable sugars. Similarly, agarases for agar, alginases for alginate, carrageenase for carrageenan, laminarases for laminarin. Compounds with rigid structures are hydrolyses using bioengineered genes.
3.4. Fermentation
Fermentable sugars are utilised by yeast mi-croorganisms and ethanol is obtained as byproduct. Favourable thermal conditions to convert fermentable sugars into ethanol is around 35-40°C. Most common organism such as Saccharomyces cerevisiae (ethanol tolerant species) are used for fermentation process toconvert fermentable sugars into ethanol, these microorganisms are highly specific to hexose sugars. In case of certain pentose sugars, specific organisms like Pichia angophorae, Zymobacter palmae, Pichia stipitis are employed for fermentation (Horn et al. 2000; Yeon et al. 2011; Lee et al. 2013; Cho et al. 2013, Trivedi et al. 2015).
Above equation shows the basic biological reaction in the conversion by fermentation of one kilogram of glucose to ethanol, carbon dioxide, and heat. Theoretically, the maximum conversion efficiency of glucose to ethanol is 51 percent on the weight basis. However, some glucose is used by the yeast for the production of cell mass and for metabolic products other than ethanol. Therefore, only 40 to 48 percent of glucose is converted to ethanol with a 45-percent fermentation efficiency, 1,000 kilograms of fermentable sugar produce bout 570 Lof pure ethanol. Economicallyfeasible ethanol concentration for distillation should be around 4-5 percent.
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