capacity to produce oil yield 250 times greater than soybean water free oil [35]. Some examples of microalgae used for biodiesel production are Botryococcus sp., Cylindrotheca sp., Schizochytrium sp., Chlorella sp., and Nitzschia sp.
1.4 Methods in Biodiesel Production
There can be many ways for biodiesel production but esterification and transesterification are the two most widely used methods. Esterification is the reaction of FFAs and alcohol to make FAAEs and water is released, while transesterification is the reaction of triglycerides or triacylglycerols (TAGs) with alcohol to make FAAE and glycerol is produced as by-product [9]. Transesterification is slower than esterification process because of its multiple steps or reactions. It is a three-step process to convert TAGs into FAAE. In the first step, TAG reacts with one molecule of alcohol to produce one molecule of FAAE and diacylglycerol (DAG). In second step, DAG further reacts again with one molecule of alcohol to produce one molecule of FAAE and monoacylglycerol and in the last step monoacylglycerol is converted into one molecule of glycerol and FAAE after reacting with an alcohol molecule. In each of these three steps, FAAEs are produced and in total one molecule of TAG and three molecules of alcohol are consumed to produce three molecules of FAAE and one molecule of glycerol [6–10]. Transesterification is a reversible reaction, and in order to make the reaction go forward to produce more biodiesel, we have to supply alcohol in large excess so that the reaction equilibrium shifts toward the product [36, 37].
1.5 Types of Catalysts Involved in Biodiesel Production
Biodiesel production process is carried by either catalytic or non-catalytic methods. Non-catalytic methods include use of alcohols or supercritical fluids or ionic liquids in the reaction system to produce biodiesel but mostly catalytic methods have been used for last 2 or 3 decades because of their advantages over non-catalytic methods [38]. Catalytic methods can be categorized into chemical homogenous catalysts, solid heterogenous catalysts, and biocatalysts.
1.5.1 Chemical Homogenous Catalysts
Chemical homogenous catalysts include combination of base and acid catalysts. NaOH, KOH, and methoxides are the base catalysts while HCl and H2SO4 are the acid catalysts [39]. Acid catalysts are mostly used to overcome the problem of FFAs in the reaction system but the rate of trans esterification by acid catalyst is slower than alkaline or base catalysts [8]. Chemical catalytic processes either alkaline or acid catalysis both have several disadvantages. Alkaline catalysis provides high conversion of triacyl glycerol into the alkyl esters in a very short time but it has many drawbacks. Alkaline catalysis is very prone to FFA concentration (>2.5%) in the reaction system because high FFA concentration results in saponification reaction producing soaps and leads to loss in enzymatic activity and makes difficult to separate transesterification by-product, i.e., glycerol from bio-diesel. Hence, biodiesel yield decreases. Moreover, it needs high energy requirement [40]. To counter FFA problem, acid catalysts are used, e.g., sulfuric acid but it also causes some technical problems regarding separation and purification of glycerol. Moreover, acid catalysis is a slow process compared to alkaline process. Reactors, pipelines, and other equipment are badly affected by acid catalysts because of their corrosive nature that can increase the cost of biodiesel production [41].
1.5.2 Solid Heterogeneous Catalysts
Solid heterogeneous catalysts include acid heterogenous catalysts and base heterogenous catalysts. Solid acid heterogenous catalysts include heteropolyacid catalysts (HPAs), mineral salts, acids, and cationic exchange resins. Among these, titanium oxide, sulfonic ion exchange resin, tin oxide, sulfonated carbon-based catalysts, zirconium oxide, zeolites, and sulfonic modified mesostructured silica are the main acid heterogeneous catalysts. Solid base heterogeneous catalysts have been categorized as mixed metal oxides, supported alkaline earth metals, single metal oxides, and nano-oxides. Among these, the most studied are magnesium oxide, calcium oxide, and strontium oxide [44, 45].
1.5.3 Biocatalysts
Biocatalysts include enzymes especially lipases which are very popular in bio-diesel production [43]. Enzymatic biodiesel production method diminishes problems associated with alkali and acid catalyzed methods. Use of enzyme catalysts has several economic and environmental advantages over chemical biodiesel production processes. Advantages of enzyme catalysis include production of pure and high market value glycerol, minor, or no waste water generation that is why treatment of waste water is not required, mild reaction conditions are required, no soap formation because enzymes can esterify low quality feedstock having high concentration of FFA that is why this method is insensitive to feedstock concentration. Enzymatic biodiesel production is simple so energy consumption is very low, enzymes can be reused because of their easy separation from the reaction mixture, and overall chance of contamination is lower than other transesterification methods [13].
1.6 Factors Affecting Enzymatic Transesterification Reaction
There are a lot of factors effecting enzymatic transesterification reaction such as source of enzyme, its type, preparation method, applying technique, its dosage, activity, and life time. Apart from these enzymes related factors, there are also some other factors which affect transesterification reaction, e.g., feedstock type and its quality, type of alcohol as acyl acceptor, reaction pH, presence or absence of solvent, type of solvent, reaction temperature, alcohol-to-oil molar ratio [46].
1.6.1 Effect of Water in Enzyme Catalyzed Transesterification
Presence of water is not only required for chemically catalyzed biodiesel production but also very much required for enzymatic biodiesel production. It helps in maintaining enzyme structural confirmation and stability so it directly affects activity of enzyme. Oil-water interface is required for enzyme-substrate complex to proceed and water helps to increase this interfacial area [44]. So, without water, transesterification is not possible and absence of water can lead to permanent or temporal changes in protein (enzyme) structure. If water content is minimal, then increase in water concentration moves the reaction equilibrium toward more hydrolysis. Thus, it enhances reaction rate by providing greater stability to enzyme [45]. Excess of water content also has some negative effects on the reaction as well as on enzyme. Excess water content can be accumulated in the reaction medium and within enzyme active site, that leads to decrease the reaction rate as well as its alkyl ester yield [46]. So, concentration of water should be optimally perfect in order to gain maximum benefit from it. Every enzyme has its specific water content requirement, i.e., optimal water requirement, at which that particular enzyme performs its best [47, 48]. Optimal water content not only provides great support, flexibility, and stability to the enzyme but also maximizes transesterification yield by diluting methanol that has an inhibitory effect on enzyme. Factors that determine optimal water content include feedstock and type of solvent used, enzyme, and its immobilization technique used [48]. Chaudhary et al. [49] studied the effect of water content in lipase catalyzed transesterification. At low water activity (aw = 0.33), synthetic activity of enzyme was increased and at high water activity (aw = 0.96) enzyme became more hydrolytically active. They tested various enzymes/lipases at different water activity to check transesterification rates. The lipase from Aspergillus niger was found more prominent to give maximum transesterification rate of 0.341 mmolmin−1 mg−1 at aw = 0.75. Measuring water content as weight percentage is a better choice and more convenient to use than water activity (aw), measured by Karl-Fischer method [50]. Maximum methyl ester yield was at water concentration of 10-15% while increasing water content from 0% to 40% to study the effect of water in conversion of salad oil into methyl ester. But after much increased water concentration, methyl ester yield became very low. So, for maximum transesterification yield, optimum water concentration is required.
