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.
1.6.2 Effect of Bioreactor
In order to maximize production and benefit of product we need to perform optimized laboratory experimental procedure at a large industrial scale, so, bioreactors are used in this regard. But results should be equivalent to laboratory procedure [45]. There are some complications like production should be cost effective and in good quality. Carefully planned methodologies and objectives should be designed for effectively large-scale production. This also includes bioreactor parameters like fluid flow performance and unexpected environmental variation. In case of industrial transesterification process, the main hurdle is multiphasic nature of lipase catalyzed synthesis and hydrolysis because this does not allow the bioreactor equivalent to laboratory experiment. Many types of bioreactors such as fluid beds, recirculation membrane reactors, expanding beds, static mixers, batch stirred tank reactors (STRs), and packed bed reactors (PBRs) have been used for enzymatic biodiesel production [51, 52]. One of the leading differences between STRs and PBRs is presence of enzyme at specific location in reactor, e.g., in STRs it is dispersed in the reaction mixture but in PBRS it is fixed in a column. STRs are the simplest type of bioreactors containing just reactor and propeller that stirs reaction mixture mechanically. Batch operated STR need to be empty, clean, and again add reactants for the reaction in order to start new batch process and this is main reason of batch process to produce less yield of the product. Solution of this problem is to use STRs with continuous mode. This does not require to remove enzyme and ingredients to start another cycle. There is a filter attached at the reactor outlet that preserves enzyme in the reactor [52]. PBRs can also be used in both batch and continuous mode but later is more advantageous because of its low labor cost, stable and automated controlled operating conditions, high efficiency, protects enzyme from shearing stress, continuous glycerol removal, and ease of maintenance [53–55]. Currently, most of the bioreactors are used in batch mode with STRs but a lot of research has been done on PBRs usage and its optimization for enzymatic biodiesel production to find this PBR method is better than batch mode STR [56–59].
1.6.3 Effect of Acyl Acceptor on Enzymatic Production of Biodiesel
Alcohols are mostly used as an acyl acceptor for biodiesel production. To get maximum economical profit at industrial scale, acyl acceptor (alcohol) should be cheap and readily available and that is why ethanol and especially methanol are widely used for this purpose. Usually, three moles of alcohol are required for each mole of oil and in order to keep the reaction moving forward [56]. By increasing alcohol concentration, yield also increases but up to a certain limit [57]. Methanol as an acyl acceptor is frequently used for biodiesel production [58] because it is less expensive, has low chain length, more volatile, and more reactive, and gives high yield than other alcohols [55]. A lot of research has been done utilizing methanol as acyl acceptor to convert various types of oils such as soybean oil, jatropha oil, and canola oil, in the presence of free or immobilized lipase 96.4% yield of FAME was obtained from microalgal oil using methanol as acyl acceptor in the presence of Candida rugosa lipase immobilized on bio-silica polymer [59, 60]. Different alcohols with different substrates may result in different yield so alcohol-substrate combination should be kept in mind for maximum output. Excess of methanol causes inhibitory effect in the reaction because it changes the stability and configuration of biocatalyst/lipase that can leads to partial or complete inactivation of lipase [60–62]. Moreover, it also causes hindrance in separation of glycerol [61, 62]. Methanol inhibition was observed with Novozym® 435 lipase in transesterification of waste oils [63], microalgae oils, and various vegetable oils [64–67]. Inhibitory effect was also observed with some other lipases such as lipases obtained from Rhizopus oryzae and Burkholderia glumae [65]. Addition of alcohol in each step should be done after considering type of substrate and enzyme to determine alcohol substrate molar ratio [66]. This method of sequential addition of alcohol in reaction system was first performed by [67]. 98% biodiesel yield was obtained utilizing