lipase to convert soybean oil using stepwise addition of methanol [68]. Inhibition of lipases such as C. rugosa lipase, P. cepacia lipase, R. oryzae lipase, and P. fluorescens lipase was prevented using stepwise addition of methanol and 90% yield was also obtained by converting waste cooking oil into biodiesel [69]. Methanolysis of olive oil increases by 34% using stepwise addition of methanol compared to batch methanolysis [70]. Transesterification of waste cooking oil using Novozym 435 was also reported to yield 93% and 96% conversion for continuous and batch process and lipase did not lose its activity even after 20 cycles [50]. Three-step addition of methanol resulted in 97% conversion of plant oil with 0.25- to 0.4-h intervals. But this method of stepwise addition requires low level maintenance of methanol concentration so it cannot be effectively used for industrial scale. Alcohols as an acyl acceptor other than methanol include high chain primary alcohols, secondary, branched, and linear chain alcohols such as ethanol, isopropanol, t-butanol, and octanol [71].
Choice and selection of appropriate alcohol is important as it can influence some biodiesel properties like lubricity and cold flow properties [75, 76]. Moreover, high chain alcohols cause less lipase inhibition and produce high yield than methanol because lipase show more affinity toward higher chain alcohols than lower chain [12]. The most widely used alcohol as an acyl acceptor after methanol is ethanol as it is less inhibitory, less toxic, and derived from renewable resources [73] unlike methanol which is derived from coal and natural gas. There is minor difference between characteristics of fuels obtained after methanol and ethanol, i.e., FAME and FAEE, respectively. As FAEE has large viscosity and lower pour and cloud points [74, 75]. Hernandez-Martin and Otero [76] showed that, Novozym 435 catalyzed transesterification of sunflower oil, that was performed using methanol and ethanol separately to check which acyl acceptor would perform better. Methanol-mediated transesterification showed more lipase inhibition than ethanol containing reaction. Moreover, ethanol transesterification reaction was faster than methanol reaction. Acyl acceptors other than alcohols can also be used as an alternative such as methyl acetate, ethyl acetate, and dimethyl carbonate (DMC). Methyl acetate was utilized as an acyl acceptor for transesterification of soybean oil catalyzed by Novozym 435. In addition, 92% methyl ester yield was obtained [77]. Similarly, >90% ethyl ester yield was obtained when utilizing ethyl acetate as an acyl acceptor for transesterification catalyzed by Novozym 435 [78].
Use of DMC resulted in over 90% yield even after 10 times reuse of Novozym 435 lipase to convert Chorella sp. KR-1–derived triglyceride [79]. But use of methyl acetate and ethyl acetate is cost expensive and also make the product difficult to separate. Another strategy can be used to reduce methanol inhibition problem, i.e., use of solvents in the reaction mixture [80]. Use of solvents is beneficial for various reasons such as it increases solubility of alcohol and glycerol that results in prevention of lipase denaturation [81]. It increases the rate of reaction because it improves mass transfer rate. Use of solvents do not allow to form new separate phase that hinders enzyme activity because it dissolves most part of alcohol that makes a separate phase if remained undissolved. Moreover, it reduces viscosity and stabilizes lipase [45, 55]. Enzyme stabilization is associated with the presence of water molecules and their activity surrounding the lipase structure. So, use of polar, less hydrophobic solvents is not a good idea because that can lead to distortion of enzyme confirmation [82]. A higher yield of FAME was obtained from microalgae lipids catalyzed by intracellular lipase when non-polar n-hexane solvent was used as compared to polar tert-butanol solvent [83]. Organic solvents such as hexane, petroleum ether, tert-butanol, n-heptane, and ionic liquids are widely used for lipase catalyzed transesterification purpose [88]. Sometimes, it also happens that use of solvents becomes necessary in transesterification reaction if short chain alcohols are being used as an acyl acceptor in order to completely dissolve alcohol and produce maximum output but for the same reaction conditions solvent-free reaction system can be used if higher chain alcohols are used as an acyl acceptor. Iso et al. [85], immobilized P. fluorescence lipase catalyzed transesterification was performed using methanol and ethanol as an acyl acceptor. They also provided 1,4-dioxane solvent to the reaction to carry out effective transesterification reaction. But when they used propanol and butanol as an acyl acceptor, they did not provide any solvent to the reaction because addition of solvents was not necessary required for transesterification. Without solvent reaction worked and appropriate result was obtained. Similar type of findings was also observed in another experiment that in which hexane as solvent was used for methanolysis of various oils or substrates like rapeseed oil, soybean oil, recycled restaurant grease, and tallow. That reaction was catalyzed by C. antartica lipase (SP 435) and M. miehei lipase (lipozyme IM 60) [53]. Solvents stabilized the lipase activity shown by Li et al. [86], where lipase AK did not loss its activity. 1,4-dioxane was used as solvent and gave higher yields. Presence of t-butanol as solvent also resulted in the improvement of methyl ester yield [84].
Effects of various solvents like benzene, tetrahydrofuran, chloroform, and 1-4 dioxane were investigated using different enzymes such as P. cepacia (Lipase PS), C. rugosa (Lipase AY), M. javanicus (Lipase M), P. fluorescens (Lipase AK), and R. niveus (Newlase F) by [85]. Use of solvents definitely increases the reaction rate and solubility of alcohol which is beneficial but it is not economical and environment friendly because to separate organic solvent from reaction mixture a solvent recovery unit is also required that increases the cost of recovery. Its flammability and toxicity is another concerning factor [87]. Choice of lipase according to alcohol is also important as some lipases show more resistance toward different alcohols. Yang et al. [88] found that Photobaterium lipolyticum lipase was more tolerant to methanol inhibition than C. antartica lipase B (Novozym 435) when transesterification was performed using one step methanol addition. Similarly, Pseudomonas lipases were found to be more alcohol tolerant compared to lipases from R. miehei and T. lanuginosus. That is why pseudomonas lipases have higher methanol-to-oil molar ratio. Out of nine lipases, only Pseudomonas cepacia lipase showed high ester yield from soybean oil with 8.2:1 methanol-to-oil molar ratio [93, 94]. A recent and novel approach of countering methanol inhibition is addition of silica gel in the reaction system. Silica gel absorbs methanol and keeps its concentration level below to prevent lipase inhibition. But presence of silica gel makes separation of products difficult.
1.6.4 Effect of Temperature on Enzymatic Biodiesel Production
Use of enzyme in any chemical reaction makes the reaction less energy intensive, but, like every other chemical reaction, increase in temperature enhances reaction speed and rate. Similarly, in case of enzymatic biodiesel production, increase in temperature increases enzyme activity, reaction speed, its rate, and production yield [45]. When Lipozyme TL IM lipase was used to transesterify crude palm oil using methanol then the resulting FAME yield was 96.15% and 85.86% at 40°C and 30°C, respectively [95, 96]. But this effect is limited to certain extent because beyond enzyme optimum temperature, enzyme structure becomes unstable and that leads to enzyme denaturation and reaction becomes slower and yield also decreases. Novozym 435 catalyzed biodiesel production from microalgal lipids, there was 19% decrease in product yield when temperature went from 45°C to 55°C, i.e., higher than optimum temperature [97, 98]. Enzyme temperature should remain below the boiling point of alcohols being used in the reaction system to avoid evaporation of alcohol. In case of methanol and ethanol-mediated transesterification, reaction temperature is 65°C and 78°C, respectively [8]. Free bacterial lipases are considered thermally stable but if they get immobilized thermal stability increases [45]. Optimum enzyme temperature is influenced by lipase thermal stability, type of solvent, alcohol-to-oil molar ratio, and lipase immobilization. Every enzyme has different optimum temperature depending on the source and type of enzyme. Normally, lipases have optimum temperature range that is 20°C–70°C. Optimum temperature for C.antartica lipase is 40°C [99, 100].
1.6.5 Effect of Glycerol on Enzymatic Biodiesel Production
Glycerol is another product obtained along with TAG in transesterification reaction for biodiesel production when alcohols are used as an acyl acceptor. Like methanol inhibition effect, glycerol also hinders enzymatic transesterification. Production of glycerol cause reversal of reaction equilibrium