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Toldrá-Reig, F.; Mora, L.; Toldrá, F. Enzymatic Transesterification in Biodiesel Production. Encyclopedia. Available online: (accessed on 18 April 2024).
Toldrá-Reig F, Mora L, Toldrá F. Enzymatic Transesterification in Biodiesel Production. Encyclopedia. Available at: Accessed April 18, 2024.
Toldrá-Reig, Fidel, Leticia Mora, Fidel Toldrá. "Enzymatic Transesterification in Biodiesel Production" Encyclopedia, (accessed April 18, 2024).
Toldrá-Reig, F., Mora, L., & Toldrá, F. (2020, September 08). Enzymatic Transesterification in Biodiesel Production. In Encyclopedia.
Toldrá-Reig, Fidel, et al. "Enzymatic Transesterification in Biodiesel Production." Encyclopedia. Web. 08 September, 2020.
Enzymatic Transesterification in Biodiesel Production

       Biodiesel constitutes an attractive source of energy because it is renewable,  biodegradable, and non-polluting. Up to 20% biodiesel can be blended with fossil diesel and is being produced and used in many countries. Biodiesel is produced through the transesterification reaction of fat waste with a short-chain alcohol, usually methanol, in the presence of a catalyst. Animal fats, usually found as waste from slaughterhouses, meat processing industry, and cooking facilities, constitute an important waste with costly treatment that can be reduced if used as feedstock for biodiesel production. Animal fat waste represents near 6% of total feedstock used to produce biodiesel through alkaline catalysis transesterification after its pretreatment. Lipase transesterification has some advantages such as the requirement of mild conditions, absence of pretreatment, no soap formation, simple downstream purification process and generation of high quality biodiesel. However, it has some disadvantages like the cost of the enzyme, its poor stability, and the enzyme deactivation by alcohol, that can be partly overcome through enzyme immobilization. A few companies are using liquid lipase formulations and, in some cases, immobilized lipases for industrial biodiesel production.

       Biodiesel constitutes an attractive source of energy because it is renewable,  biodegradable, and non-polluting. Up to 20% biodiesel can be blended with fossil diesel and is being produced and used in many countries. Biodiesel is produced through the transesterification reaction of fat waste with a short-chain alcohol, usually methanol, in the presence of a catalyst. Animal fats, usually found as waste from slaughterhouses, meat processing industry, and cooking facilities, constitute an important waste with costly treatment that can be reduced if used as feedstock for biodiesel production. Animal fat waste represents near 6% of total feedstock used to produce biodiesel through alkaline catalysis transesterification after its pretreatment. Lipase transesterification has some advantages such as the requirement of mild conditions, absence of pretreatment, no soap formation, simple downstream purification process and generation of high quality biodiesel. However, it has some disadvantages like the cost of the enzyme, its poor stability, and the enzyme deactivation by alcohol, that can be partly overcome through enzyme immobilization. A few companies are using liquid lipase formulations and, in some cases, immobilized lipases for industrial biodiesel production.

biodiesel fuel energy generation food waste animal waste animal fat enzymatic transesterification

1. Introduction

       Animal byproducts are subjected to rendering where fat such as beef tallow, mutton tallow, pork lard, and chicken fat are obtained [1][2]. Such fat is majorly composed of triacylglycerols with fatty acids of 16 to 18 carbons. The most abundant saturated fatty acids are palmitic (16:0) and stearic (18:0) acids; the major monounsaturated fatty acid is oleic acid (18:1) and the most abundant polyunsaturated fatty acids are linoleic (18:2) and arachidonic (20:4) acids [3][4]. Animal fat waste is also obtained from the meat processing industry and from recycled waste from the cooking business [5][6] and are classified as yellow grease if the content of free fatty acids is lower than 15% by weight and brown grease when it is higher than 15% [7]. In 2019, more than 800 thousand tons of animal fats, equivalent to 6% of total feedstock, were used to produce biodiesel in the European Union [8][9], while 8.4% of total feedstock was used in the case of the US, consisting of mainly 74 tons of poultry fat, 132 tons of tallow and 243 thousand tons of white grease [10].

       Biodiesel produced from animal fats has several advantages in comparison to that produced from vegetable oils. So, it has lower production cost because the feedstock used as raw material for biodiesel production represents up to 80% of the total cost [11], and the fossil CO2 reduction is higher (nearly 80% CO2 reduction may be reached for animal fat in comparison to 30% reduction when using vegetable oil) [12][13][14]. The bioenergy demand is continuously increasing and in 2050 it is expected to reach 30% of the fuel consumed in the world for road transport [14][15]. Research on biodiesel production is trying to maximize the yield and minimize the costs by using better catalysts that can be reused and improve the transesterification efficiency [16][17].

       Total biodiesel world production has been increasing progressively year by year, reaching nearly 45 million tons in 2019 [9]. The European Union has the largest biodiesel production through its 202 plants producing more than 14 million tons of biodiesel in 2019 [8][18]. More than 5.6 million tons of biodiesel were produced in the US in 2019 through its 91 plants [10][19]. Nearly 80% of new diesel vehicles are prepared for B20 use that consists of fossil diesel blended with 20% biodiesel [10].

       Transesterification through alkaline catalysis is the preferred process at industrial biodiesel production plants [20]. However, raw materials like animal fat that contain moisture and free fatty acids are troublesome for alkaline transesterification due to soap formation. Acid catalysis does not have such troubles, but the reaction is much slower than alkaline catalysis, it needs a larger size reactor and requires a higher alcohol to fat molar ratio [21]. Heterogeneous catalysts are not sensitive to the presence of free fatty acids and moisture, can catalyze esterification and transesterification simultaneously, and can be separated from the reaction media. However, such solid catalysts tend to form three phases resulting in a reduced reaction rate and high energy consumption [22]. The simultaneous esterification and transesterification also occur with supercritical technology where high temperature and pressure conditions (i.e., >250 °C and 10 MPa) increase the solubility and reduce the mass transfer limitation resulting in good efficiency but with high energy consumption [23][24][25]. Pseudo catalytic transesterification using biochar as the porous material for the pseudo-catalytic reaction at more than 300 °C has the same advantages as supercritical transesterification, but also has high energy consumption [26][27]. Therefore, an interesting alternative for biodiesel production from animal fats is the enzymatically catalyzed transesterification that is presented in this article.

2. Mechanisms of Action of Lipases

       Lipases, triacylglycerol ester hydrolases (EC, are serine hydrolases with an active site containing an amino-acid triad of serine, histidine and aspartate [28]. Lipases are obtained from a variety of sources such as animal and plant tissues and microorganisms. Lipases show a wide range of pH and temperature for activity and vary from strain to strain regarding specificity and hydrolysis rate [29] Lipases exhibit good stability in non-aqueous mediums and exhibit maximum activity near neutral pH range; lipase stability is increased when the enzyme is immobilized.

       Lipases can catalyze esterification, inter-esterification, and trans-esterification reactions in non-aqueous environments. Lipases catalyze the hydrolysis of triacylglycerols at the aqueous-non aqueous interface but these enzymes can also catalyze the synthesis of esters from alcohols and long chain fatty acids in low moisture environment [29]. Lipases follow a two-step mechanism for the generation of fatty acid methyl esters in transesterification reactions, usually through the Ping-Pong Bi Bi mechanism [30].

       Most triacylglycerol lipases are regiospecific because they can only hydrolyze primary ester bonds at the sn-1 and sn-3 positions, external positions within the triacylglycerol, and can generate either one free fatty acid and diacylglycerol, or two free fatty acids and 2-monoacylglycerol that remain unhydrolyzed. The full process from triacylglycerols into biodiesel and glycerol as end products is shown in Figure 1. Regiospecificity is characteristic of extracellular bacterial lipases from Bacillus sp. [31][32].


Figure 1. Transesterification of animal fat to biodiesel. TGL: triacylglycerol lipase; nsTGL: non specific triacylglycerol lipase; MGL: monoacylglycerol lipase.

       Monoacylglycerol lipases (EC catalyze the hydrolysis at the specific sn-2 position of 2-monoacylglycerol into free fatty acid and glycerol. Such lipases may be present in the enzyme extract and masked when measuring activity with standard activity methods like those based on triolein hydrolysis measurement. Monoacylglycerol lipases have been the object of few studies [33], although they might be present in some microbial enzyme preparations [34]. Other lipases are nonspecific and can act on any of the ester bonds of the triacylglycerol and therefore break down the triacylglycerol to release free fatty acids and glycerol as the final products. This is the case of lipases from Staphylococcus aureus [35]Geotrichum candidum, Corynebacterium acnes, Penicillium cyclopium [21] and Chromobacterium viscosu [36]. Another alternative for the hydrolysis of monoacylglycerols is the acyl migration in the glycerol backbone from the sn-2 position to sn-1 or sn-3 positions [37].

       The specificity of lipases depends on the length of fatty acids, presence of double bonds, branched groups and, consequently, reaction rates may have important variations depending on the composition of triacylglycerols present in the fat waste. Lipases are especially active against medium to long chain fatty acids, which are those more usual in animal fat waste [16].

3. Sources of Lipases

       Most lipases originated from microorganisms are produced in fermenters under controlled conditions (see Table 1). Lipases are produced by a variety of gram-positive and gram-negative bacterial strains, especially from the genera of Pseudomonas [38][39], also by filamentous fungus that are commercially important such as those belonging to the genera of Rhizopus sp. [40]Aspergillus sp. [41]Penicillium sp. [42]Geotrichum sp. [29]Mucor sp. [43] and Thermomyces sp. [44]. Lipases produced from yeasts are also relevant such as those from Candida sp. [45,46].

Table 1. Bacteria, yeasts and filamentous fungi producing lipase and sources of isolation.

       Extracellular lipases are secreted into the production medium and recovered from the microorganism broth. Then, lipases are further separated and purified but downstream processing is costly. Intracellular lipases imply the use of whole cell microorganisms and this fact reduces the costs of enzyme extraction and purification but the efficiency and biodiesel yield is low when catalyzing an oily substrate due to mass transfer limitations for substrate penetration and product release [25,70]. Some whole cell biocatalysts used to produce biodiesel are filamentous fungi like Aspergillus and Rhizopus [45].

4. Industrial Applications of Lipase-Catalyzed Biodiesel

       Even though transesterification through alkaline catalysis is the preferred process in the majority of industrial biodiesel production plants [20], a few lipase-based processes have already been implemented to plant-scale operation. Lipases generate biodiesel under mild reaction conditions through the conversion of free fatty acids and triacylglycerols in the presence of an acyl acceptor [28]. However, it has some disadvantages like the cost of the enzyme, its poor stability, and the enzyme deactivation by alcohol [71], and partly by the generated glycerol [61]. Most of these issues can be partly overcome through enzyme immobilization [72,73] because it increases its stability and efficiency [74,75,76], and also allows an easy downstream separation from the product and decreases costs [77]. The immobilization of lipases consists of the retention of the enzyme at the surface of the support material. In this way, immobilized lipases show an improved efficiency and reduced costs, with longer enzyme stability and better resistance to denaturation by alcohol. There are many available supports of organic, synthetic, and inorganic nature for lipase immobilization and there is a large variety of immobilization procedures such as adsorption, covalent binding, cross-linking, entrapment, or encapsulation [73].

       There are some industrial applications of enzyme transesterification for biodiesel production in different countries. The collaboration of Novozymes (Bagsvaerd, Denmark) with Piedmont Biofuels (Pittsboro, NC, USA) resulted in a patent application to produce fatty acid alkyl esters, by a lipolytic enzyme in a solution containing triacylglycerol, alcohol, water, and glycerol [78,79]. Viesel Fuel (Terrac Stuart, FL, USA) upgraded in 2013 its facility through an enzymatic process developed by Novozymes (Denmark) to use brown grease and waste cooking oil to produce up to 11 million gallons biodiesel per year using Eversa Transform® lipase from Novozymes, a soluble lipase produced by a genetically modified strain of Aspergillus oryzae [80], and an ion exchange resin system for removal of remaining free fatty acids during crude biodiesel refining [81,82]. Viesel Fuel, Novozymes and Tactical Fabrication also collaborated with Buster Biofuels to upgrade its facility in San Diego (CA, USA) to produce up to 5 million gallons per year [83]. Lvming and Environmental Protection Technology Co. Ltd. (Shanghai, China) used lipase of Candida sp. to produce 10,000 tons per year from waste frying oil [84]. A plant in Sumaré (Sao Paulo, Brazil) produces biodiesel from mixed beef tallow and soybean oil using Callera® Trans L lipase in a batch reactor [85]. These companies are using liquid lipase formulations but the efficiency of the process can be improved further by using recent developments in immobilized lipases. So, Hunan Rivers Bioengineering Co. Ltd. (Hunan, China) was reported to use Novozym 435® lipase in a stirred tank reactor to produce 20,000 tons of biodiesel per year. The enzyme is a lipase B from Candida antarctica immobilized on a resin consisting of macroporous support formed by poly(methyl methacrylate) crosslinked with divinylbenzene [86]. New technology protected with patents [87] has been provided by EnzymoCore, a leading global producer company founded in 2007 in Israel and with several active biodiesel plants around the world. This company has developed modified-immobilized enzymes, supported on solid organic resins, with high resistance to methanol and able to produce biodiesel from any type of oil or fat, even those cheap and with very large content of free fatty acids and polar lipids [88].

5. Conclusions

       Animal fat waste, usually resulting from slaughterhouses, the meat processing industry, and cooking facilities, is being increasingly used as feedstock for biodiesel production. Transesterification through alkaline catalysis is the preferred process at industrial biodiesel production plants although some enzymatic transesterification processes have been already implemented to plant-scale operation. Transesterification with lipases has traditional problems including poor enzyme stability, difficulties in reusability, and denaturation by alcohol although they can be partly overcome through enzyme immobilization. An advantage is that lipases are not affected by water and free fatty acids typically found in animal fats. However, some companies have been able to solve such troubles since they are running liquid lipase formulations for producing biodiesel from cooking oil waste at industrial scale although the efficiency of the process can be further improved. Recent developments in immobilized lipases and availability of different types of supports such as mesoporus materials, silica nanoflowers, pickering emulsion, and metal-organic frameworks demonstrate improved efficiency and reduced costs. Immobilization of the enzyme in such materials increases its stability and makes it more resistant to denaturation by alcohol. Magnetic nanomaterials constitute a very good support for enzyme immobilization because they can be recovered when an external magnetic field is applied. These nanoparticles are functionalized on the surface by coating with silica or organic polymers that enhance the efficiency of the process. The entrapment of whole cells with lipase activity, appears to be simple and efficient although more research is needed. Coimmobilization of lipases is an innovative process, but not so attractive for industrial application. It needs further research because of the cost of using different lipases and the steric difficulties for enzymes to hydrolyze triacylglycerols that affects the efficiency of the process.


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