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 T. lanuginosus 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.
