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 and thus opposes biodiesel production. Glycerol insolubility in the reaction system makes it to accumulate in the system and increases the viscosity of the reaction mixture. Not only it just increases viscosity but because of its hydrophilic nature, it surrounds the lipase and hence prevents substrate to interact with enzyme and binding at enzyme catalytic site. This effect is more prominent when enzyme is in immobilized state. These things all together impacts negatively on the transesterification reaction [93]. According to a research study, rapeseed oil was transesterified using ethanol as an acyl acceptor with different immobilized enzymes such as Novozym 435, Lipozyme TL HC (immobilized on polymeric resin) and Lipozyme TL IM (immobilized on silica). Among all reactions, glycerol became more accumulated and hindered reaction when silica was used for immobilization purpose because it had large number of micropores that helped in accumulation [94].
A lot of methods have been devised to overcome and tolerate glycerol inhibition problem such as continuous removal of glycerol, use of solvents, and use of acyl acceptors other than alcohols. Biodiesel production in PBR is effective for continuous production and also to tolerate glycerol inhibition because it allows continuous removal of glycerol from it [95]. Bélafi-Bakó [96] showed that 97% conversion yield was obtained by methanolysis due to continuous removal of glycerol. Use of solvents is another strategy to resolve glycerol inhibition problem. Solvents, for example, tert-butanol and ionic liquids dissolve glycerol and thus reduce the glycerol inhibition problem. Moreover, lipases also perform better in the presence of solvents [97]. Azócar et al. [4] inferred that inhibition effect was eliminated when tert-butanol was used as solvent to convert soybean oil into biodiesel production in a continuous way because it is an excellent solvent for methanol and glycerol to dissolve in it and, thus, reduces inhibitory effects of both methanol and glycerol. These methods have shown promising work but these are not as good for industrial scale production.
There is another novel method in which instead of alcohols other compounds like methyl acetate, ethyl acetate, and DMC are in use. Use of methyl acetate or ethyl acetate does not produce glycerol as a product along with the main product instead it leads to produce triacetylglycerol that does not inhibit any enzymatic or reaction activity and further downstream processes are not halted [98]. According to Zhang et al. [99], transesterification of palm oil was done using Novozyme 435 as catalyst and DMC as acyl acceptor with reaction conditions were 10:1 DMC to oil ratio, 55°C reaction temperature and 20% lipase in a solvent-free system. In addition, 90.5% conversion yield, i.e., FAME was obtained, and after eight reaction cycles, no reduction in enzyme activity and loss of yield was observed. It was just because glycerol was not produced instead glycerol dicarbonate was formed because of DMC as acyl acceptor for the reaction. After discussing all negativity about glycerol, it seems glycerol does not have any positive effect but it’s not true. Glycerol if purified absolutely from the transesterification, as mostly huge biodiesel producing companies do, has vast number of uses in diverse industrial fields. Moreover, 99.7% purified glycerol can be used as a raw material for various types of fields such as paints, toiletries, animal feed, emulsifiers, pharmaceuticals, textiles, drugs, tobacco, cosmetics, toothpaste, leather, plasticizers, paper, food, and for different chemicals production [100, 101].
1.6.6 Effect of Solvent on Biodiesel Production
One of the major problems in enzymatic biodiesel production is enzyme inhibition by short chain alcohols such as methanol and ethanol that are used in the reaction. These alcohols’ insolubility in the reaction system denatures the enzyme and hence reduces yield of biodiesel. So, solvent application plays its role in this regard. Organic solvents are used to solubilize these excessive alcohols so that enzyme denaturation can be prevented. Hence, it stabilizes the enzyme. Solubility of oils and alcohols become increased due to presence of organic solvents, this provides the required environment for substrate to interact with enzyme at its active site. Organic solvents also reduce viscosity of the reaction mixture and enhances mass transfer toward the enzyme that leads to improved reaction rate [101].
Organic solvents
