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 also eliminate the need of stepwise addition of alcohol. All these things in combination increase production of biodiesel. Organic solvents that are commonly used include tert-butanol, petroleum ether, hexane, and n-heptane [102]. Some other organic solvents that are used are 2-butanol, cyclohexane, isooctane, acetone, 1,4-dioxane, and chloroform. While considering nature of organic solvents, hydrophobic organic solvents are majorly used. Hydrophobicity of the organic solvents helps in accumulating water molecules around enzyme which is important for enzyme structural stability [103]. Polar or hydrophilic solvents work opposite to hydrophobic organic solvents by playing role in distortion of enzymatic structure. But solvent with little polarity can be beneficial to dissolve oil and alcohol. For example, hydrophilic 1,4-dioxane and tert-butanol have produced some good results by producing high enzymatic transesterification yield [104]. Tert-butanol, having moderate polarity, eliminates glycerol and methanol inhibition problem for enzyme because it can dissolve both in itself. This makes the enzyme more stable and active and then ultimately produce better reaction yield [105]. Tertbutanol is the most common solvent that proved its effectiveness in various cases. According to Royon et al. [84], cottonseed oil was transesterified in the presence of Candida antartica lipase. Methanol was found to be the cause of enzyme inhibition in the reaction but when tert-butanol was used as solvent, reaction yield goes up to 97% with minimal enzyme inhibition. Similarly, in another research experiment, tert-butanol was tested for its effectiveness when rapeseed oil was used as substrate for biodiesel production. In solvent-free system, methyl ester yield was 10% but after utilizing tert-butanol yield was 75%. But under optimum conditions having Lipozyme TL IM and Novozyme 435 both in the reaction system, biodiesel yield reached 95% and the reaction was so stable that enzymes did not lose their activity even after 200 cycles. Reaction was favored and well supported by tert-butanol [86].
Use of solvents provide many benefits but they also come with some disadvantages such as organic solvents do not completely dissolve glycerol, by-product of the reaction, that causes the enzyme to lose its activity and become unstable. Use of solvents also make the process very costly because there is a need of extra purification step to separate out solvent and product from the reaction mixture. Organic solvents are mostly toxic and highly flammable so there are also environmental and health concerns while using them [11]. In order to tackle problems of conventional organic solvents, researchers have suggested some alternatives. Diesel oil was found to be an interesting alternative but the most recent, beneficial, and popular alternatives are super critical carbon dioxide (SC-CO2) and ionic liquids (ILs). Researchers have also confirmed the positive effect of using SC-CO2 and ILs in the enzymatic transesterification [106, 107].
1.7 Lipases as Biocatalysts for Biodiesel Production
Transesterification of oils for biodiesel production is done using either chemical or enzymatic catalyst [108]. An enzymatic catalyst is used at first place due to their normal reaction conditions, reusability, easy products separation, and production of high-quality product. There is less energy consumption in enzyme catalysis as it occurs at a low temperature as compared to chemical catalysis requiring high energy consumption [109, 110]. Further, enzymatic catalysis is environment-friendly as there is no wastewater production and produces pure biodiesel as compared to chemical catalysis [107]. Among enzymatic catalysts, lipase with excellent biochemical and physiological properties is most commonly used to catalyze the transesterification process. Lipases play their role in several industrial processes like alcoholysis, acidolysis, amynolysis, and hydrolysis reactions but their leading role in biodiesel production is considered very important [108–111]. The use of lipase in biodiesel production is proved to be beneficial due to its characteristics like high efficiency, convert FFAs completely into methyl/ethyl esters, reaction specificity, require low temperature, minimum energy consumption, and fewer side products [109]. Lipases belong to class “hydrolases” as they carry out hydrolyses of triglycerides producing glycerol and fatty acids from it in an oil-water interface [110]. A general reaction for biodiesel production using lipase is as follows:
Lipases work on