Biocatalysts are defined as biological catalysts. They are natural substances, such as enzymes from biological sources or whole cells that are used to accelerate chemical reactions. Enzymes play a critical role in hundreds of reactions, including the production of alcohols. Biocatalysts have become increasingly important in discussions about biodiesel production and, more recently, in most biodiesel manufacturing processes. Again, homogeneous biocatalysts face the same issues as chemical homogeneous catalysts, including reuses, separation, and process cost-effectiveness. Biocatalysts are naturally occurring lipases that were demonstrated to perform the transesterification processes required for production of biodiesel. Pseudomonas fluorescens, Pseudomonas cepacia, Rhizomucor miehei, Rhizopus oryzae, Candida rugosa, Thermomyces lanuginosus, and Candida antarctica have all been found to produce lipases. Lipase can be immobilized using various techniques such as adsorption, covalent bonding, entrapment, encapsulation, and cross-linking. As a result, immobilized or encapsulated biocatalysts play a key role in catalytic reactions. Recent years have seen these immobilization strategies used to improve lipase stability for biodiesel production. Adsorption is still the most common way to keep lipase immobilized. To convert triacylglycerols to their corresponding fatty acid methyl esters, at least stoichiometric amounts of methanol are required (Mamat et al. 2017). For renewable synthesis technologies, biocatalysis has been seen as a trend because of the catalyst's biologic base, selectivity, and potential to recycle agro-industrial wastes for biocatalyst manufacture (Teixeira et al. 2014). Enzymatic catalysis has been utilized to make biodiesel, which is presently being manufactured on a large scale in China (Tan et al. 2010). However, certain considerations affect the conversion of an enzymatic transesterification reaction, such as water content of the reaction medium, solvent type, substrate alcohol type, temperature of the reaction, kind of immobilization, and lipase concentration. In the studies, various lipases have been used to produce biodiesel; nonetheless it's difficult for drawing general conclusions regarding the best conditions of the reaction. This is due to the fact that lipases of different origins respond toward variations in the reaction medium in different ways (Bajaj et al. 2010; Antczak et al. 2009; Abdulla et al. 2013; Ondul et al. 2012). Chemical biodiesel processing costs have remained smaller than those of enzymatic processes; but, when environmental degradation is taken into account, these costs are equivalent. The high enzyme cost has a direct effect on the process viability in enzyme-catalyzed biodiesel processing.
For many reasons, biocatalysts outperform chemical catalysts. Chemical catalysts have a number of drawbacks, in the production of soap in base-catalyzed transesterification, difficulties purifying glycerol, and alkali-catalyzed transesterification involving a lot of energy within downstream biodiesel processing (Madras et al. 2004). When sulfuric acid (H2SO4) is used as a catalyst, the reactor corrodes and a significant amount of wastewater is produced during the neutralization of mineral acid and a large amount of catalyst is required in the base-catalyzed transesterification reaction. Transesterification using an acidic catalyst increases the molar ratio of alcohol and oil (Musa et al. 2016). Chemically catalyzed biodiesel demands a high reaction temperature, and while alkali catalysts (NaOH and KOH) are inexpensive and widely, their operating temperature used is lower (Demirbas et al. 2008). Without a catalyst, the transesterification process can be carried out at a higher temperature, but the biodiesel yield at temperatures below 350oC is undesirable. At temperatures below 400oC, however, thermal degradation occurs (Demirbas et al. 2007). Alcohols in their supercritical state have also been shown to contain higher methyl ester yields of fatty acids (FAME) (Demirbas et al. 2008). Alkali and acid catalysts, as well as enzymatic (lipase) biocatalysts, are required to speed up the process, to minimize energy expenditure. Transesterification with an alkali catalyst is more common in industry than acid-catalyzed transesterification because it is. If the oil contains a large amount of free fatty acids and water (FFA), acid-catalyzed transesterification is a better option because FFA and water obstruct the reaction. While chemical-catalyzed transesterification provides suitable conversion speeds in short reaction cycles, it has many disadvantages, including being energy intensive, difficult to recover glycerol, requires the acidic or alkaline catalyst to be removed from the substance, requiring water treatment, and being affected by FFAs and water. Enzyme-mediated (biocatalysts) transesterification can solve problems that arise during chemical catalysis, and they are becoming most important in biodiesel synthesis as a result of their capacity to replace chemical catalysts. Because of their properties and advantages, enzymes have been identified as possible biocatalysts for biodiesel processing (Fukuda et al. 2001). For example, using an enzyme catalyst throughout the biodiesel synthesis process is a greener choice than processing renewable fuel. Enzymatic catalysts are biologically derived, and utilizing them consumes less energy due to their gentler operating conditions than chemical catalysts (Sendzikiene e al. 2015). As compared to chemical catalysts, enzyme catalysts have many advantages, including being environmentally efficient, having a very rapid reaction, requiring less energy and temperature, no soap is making, and requiring less water for cleaning. Enzymatic methanolysis using lipases as biocatalysts has become more interesting for biodiesel processing because it can overcome several limitations
5. 1. Bio catalysis classification
Lipases have recently been investigated as whole-cell immobilized lipases for biodiesel output. When it comes to lowering the biocatalyst's contribution to the final cost of biodiesel, each kind of biocatalyst has its own set of advantages and disadvantages. Recent research has focused on enhancing the enzyme's catalysis efficiency and stability to save money of lipase in the biodiesel conversion method (Teixeira et al. 2014).
5.1.1. Free biocatalysts
Microbial lipases have grown in importance in industry, accounting for around 5 percent of the enzymes in the world demand afterward carbohydrates and proteases. Plant and animal lipases are less stable than lipases and can be purchased in large quantities at a reduced rate than lipases from other sources. When compared to bacterial lipases, yeast lipases are easier to manage and expand. Candida rugosa is a yeast lipase that has grown in commercial importance. Lipases generated by microbes which formed from a variety of yeast, fungal and bacterial organisms, to produce biodiesel in the presence of biocatalyst are the most widely (Christopher et al. 2014). Immobilized lipases are much more expensive than free enzymes. It can be obtained in an aqueous solution consisting of a solution of enzyme in the presence of a preservative to avoid the growth of microbes such as, benzoate and a stabilizing agent to prevent the enzyme denaturation (for example, glycerin or sorbitol) (Nielsen et al. 2008).
5.1.2. Immobilized biocatalysts
Entrapment, physical adsorption, ion exchange, and crosslinking are all methods used to immobilize lipases. Cellulosic nanofibers, silica, sea beads, and polyurethane foam are examples of carriers for lipase immobilization. According to the requirements for selecting the immobilization system and carrier, the system of reactions (aqueous, organic solution, or two-phase system) and bioreactor type (batch, stirred tank, membrane reactor, column, and plug-flow) can be established depending on lipase's source. Depending on the lipase source, the reaction method (aqueous, organic solution, or two-phase system) and bioreactor form (batch, stirred tank, membrane reactor, panel, and plug-flow) can be adjusted based on the requirements for selecting the immobilization technique and carrier. Lipase-producing bacteria, enzyme immobilization methods, and physical carriers are all mentioned in the literature. The challenges in finding a carrier and immobilization method which allowed for the most lipase operation is stabilization, and stabilization on the oil substrate. The most basic and extensively used method for lipase immobilization is adsorption. The adsorption process involves weak forces as van der Waals or hydrophobic interactions to bind the lipase to the immobilization support board. Due to the low binding strength between the enzyme and the support, the fundamental disadvantage of this technique is enzyme desorption from the support. (Christopher et al. 2014).
5.1.3. Whole-cell biocatalysts
Whole-cell immobilized lipases have been studied for biodiesel synthesis in recent years. This process is less expensive because it avoids the need for the purification of enzymes and the separation of fermentation broth. The transesterification method's performance may be improved with whole-cell biocatalysts made from microbial cells that contain intracellular lipase (Christopher et al. 2014). Filamentous fungi have been discovered must be effective biodiesel processing whole-cell biocatalysts, with Rhizopus and Aspergillus being the greatest commonly used (Gog et al. 2012). Several recent studies have reported the use of yeast, fungi, and bacteria as whole-cell biocatalysts in the biodiesel method (Teixeira et al. 2014).
5.2. Role of Lipase in the transesterification
Long chains of triacylglycerols are hydrolyzed by the lipase enzyme, which serves as a catalyst. The transesterification reaction is catalyzed by lipase in two steps: (a) hydrolyzes fatty acid ester bonds, converting triglycerides to diglycerides. (b) Alcohol serves as an acyl acceptor, resulting in the formation of an ester. The next transesterification reaction is carried out by free lipase (Jegannathan et al. 2008). The presence of water affects lipase production as well. It keeps lipase active by creating an oil–water interface with water, which aids in enzyme activation and active site reconstruction by conformational change. In aqueous conditions, the three-dimensional structure of the lipase enzyme that comprises polar and nonpolar groups, making it distinctive and active. Lipase enzymes, like other enzymes, undergo lipolytic reactions, and Lipolytic processes are complicated by the insolubility of lipids in water (Manurung et al. 2016). Interface characteristics, interfacial nature, and interfacial region can all affect lipase's catalytic activities. The lipase enzyme is activated by an adsorption process and the interface aids in the lipase's catalytic active site is open. As a result, lipases are being used as biocatalysts for biodiesel production has gotten a lot of attention in the last ten years (Fukuda et al. 2008). Many attempts have been made to create an enzymatic pathway using lipase as a biocatalyst, either extracellular or intracellular. Triacylglycerol lipase (also known as Triglyceride lipase) EC 3.1.1.3, is an enzyme that hydrolyzes triglyceride ester linkages) glycerol and free fatty acids (FFAs) produced. Serine, aspartic (or glutamic) acid, and histidine amino acid groups make up the active sites of lipases. Generally, lipases have a unique feature called interfacial activation for their usage in the transesterification of fats and oils, which happens when a lipase active site structure and a substrate are both present. Lipases are being used in a range of fields due to their capability to use mono, di, and triglycerides and FFA, low material inhibition, high activity and produce in non-aqueous media, low reaction time, temp, and alcohol resistance. However, their high cost continues to be a barrier to their industry applications. The enzyme may be immobilized on the appropriate carrier and repeated several times to reduce the cost of the operation. For the immobilization of lipases to manufacture biodiesel, a variety of methods and carriers have been used so far. They've been immobilized on porous kaolinite particles, silica, celite, macroporous resin, biomass support particles, gel-entrapped, and Eupergit C250L (Ondul et al. 2015). Lipases have grown in importance in the enzyme biotechnology world as results of their catalytic reactions are generally chemo-selective and regio-selective, allowing them to be versatile in hydrolysis and synthesis, or enantioselective. Lipases are used in a variety of industries, including food, pharmaceuticals, fine chemicals, oil chemicals, biodiesel, and commercial detergents. Lipases are enzymes that catalyze the hydrolysis of ester carboxylate bonds at the organic-aqueous interface, resulting in the release of fatty acids and organic alcohols. In water-limited conditions, however, the reverse process (esterification) or even alternate transesterification processes can occur, as Pottevin demonstrated for the first time in 1906. Lipases can be of vegetable, fungal, animal (pancreatic, hepatic, and gastric) or microbial (bacterial and yeast) origin, and they have a wide range of catalytic properties. Microbial lipases have been the subject of the most research so far. Microbial lipases account for roughly 58% of all lipase publications, plant lipases for 42%, and latex lipases for only 11%. Despite the wide variety of microbial lipases, industrial usage of these enzymes is currently restricted due to high processing costs, thus other sources of these enzymes are preferred (Mazou et al. 2016).
5.3 Use of immobilized lipase in biodiesel production
In the field of biotechnology, the use of immobilized enzymes is important. Biocatalyst will isolate from the reaction product due to lipase immobilization, and it can be used in the transesterification reaction to increase yield and lower the cost of the operation. An enzyme that has been immobilized has been bound to inert and insoluble substances. Methods for immobilizing lipases enzymes include entrapment, covalent linkage, encapsulation, adsorption, cross-linking, and others (Budzaki et al. 2019; Juna et al. 2019).
Biodiesel has been generated using a variety of immobilized lipases including C. rugosa lipase on Fe3O4 nanocomposite, Lipase from B. cepacia on magnetic nanoparticles, R. oryzae lipase on magnetic graphene oxide, P. fluorescence lipase immobilized on carbon nanomaterials, C. antarctica and R. miehei lipase on silica, C. antarctica lipase on magnetic nanoparticles, Fusarium heterosporum lipase and P. cepacia lipase on bio-support beads (Bhan et al. 2020). Immobilized lipase was used to synthesize biodiesel on a variety of components, including organic polymers, carbon nanotubes, silica nanoparticles, and magnetic nanoparticles, according to Zhong et al. (2020). However, using immobilized lipase in transesterification has the downside of increasing the price of processing. When opposed to chemically catalyzed transesterification, lipase-catalyzed biodiesel processing is considered an environmentally sustainable solution since it takes input with less energy and requires washing in less water. According to Avhad and Marchetti (2019), 2.5 kg of Lipozyme RMIM can convert about 1000 kg of oil to biodiesel, and the enzyme that has been immobilized can be reused. for around 50 cycles. In comparison to traditional acid/alkali catalysis for biodiesel processing, transesterification using enzymes as biocatalysts (Amini et al. 2017) is a relatively new technique. Biocatalysts are enzymes produced naturally by the fermentation of biobased materials. Lipolytic enzymes are essential for convert and lipids, a key element of the earth's biomass, are mobilized from one creature to another. Biosurfactants are lipid solubilizers produced by microorganisms such as bacteria and fungus. Additionally, using enzymes as biocatalysts during algal oil transesterification results in cleaner, more ecologically friendly alternatives, includes a benefit for microalgal biofuels of the third generation. Lipase (triacylglycerol acyl hydrolases, EC3.1.1.3) and esterase (EC 3.1.1.1) are the two primary kinds of lipid hydrolytic enzymes that are being explored as commercial biocatalysts (Jia et al. 2019). Shorter-chain fatty acid esters (C8) are hydrolyzed by the esterase enzyme (Ramnath et al. 2017). Lipases are divided into three groups depending on the substrates they work with: I lipases with regio- or positional specific lipolytic activity, (ii) lipases specific for fatty acids, and (iii) lipases that are very specific to just certain acylglycerols found in oils (Teo et al. 2014). Dairy, fruit, detergents, fats and oils, organic synthesis, biodiesel, agro-chemicals, novel polymeric textiles, paper and pulp, leather, fine chemicals, cosmetics, and medicines are just a few examples. These lipolytic enzymes are in high demand as potential industrial biocatalysts, with applications ranging from soil bioremediation to the biodegradation of environmentally harmful substances such as phenolic chemicals and endocrine disruptors. Although lipases can be found in practically all microorganisms, plants, and humans, microbial lipases generated from bacteria and fungus are the most commonly used lipase sources. (Sreelatha et al. 2017). Microbial lipases have many benefits over other extraction sources, including a shorter cycle time, lower cost, and the ability to expand and immobilize on any inexpensive solid media (substrates). They also produce higher yields and are often convenient for genetic modification. Enzyme catalysts, in comparison to chemical catalyzed reactions, require milder atmospheric conditions for the successful activity, resulting in a significant reduction in energy consumption and hence operating costs. Enzymatic reactions also have high substrate selectivity and can esterify triglycerides and free fatty acids in a single step, resulting in a high-quality byproduct (glycerol) with no waste or recovery expenses. Enzymes are very specific to their substrates, so there are no unintended side reactions and no requirement for post-reaction byproducts. Enzymes are highly specific to substrates, eliminating the need to isolate the byproduct after reaction and unwanted side reactions. Enzymatic reactions are also environmentally safe, presenting no risks during disposal. Over the last decade, immense efforts have been made to focus on lipase enzyme synthesis and characterization for a variety of applications. Lipase as a biocatalyst in biodiesel processing is a relatively new field of research, with specific applications for first- and second-generation biodiesel feedstocks including sunflower oil, Jatropha oil, soybean oil, and palm oil (Saranya et al. 2020). Enzymatic catalysts can be useful in the synthesis of ethyl esters, but the reaction conditions must be optimized (temperature, molar ratio, pH, amount of catalyst). By using a co-solvent or longer reaction durations, ester yields can surpass 80% (Brunschwig et al. 2012). Because oil extraction and transesterification occur concurrently, the enzymatic in situ approach takes longer than standard oil transesterification. The optimal length of the biotechnological phase has been determined to be 19–24 hours (Makareviciene et al. 2020; Gumbyte et al. 2018). The production of biodiesel using an alternative feedstock, date pit oils, has been investigated. These oils provide the same advantages as pure oils. But they are derived from a waste substance. Biodiesel was produced by transesterifying the extracted oils with methanol in the presence of Novozym®435 or Eversa® Transform. Because methanol suppresses lipase activity, the oil and enzyme are introduced first, then the methanol (Al-Zuhair et al. 2017). New enzyme-based catalysts (biocatalysts) have been created in recent years to avoid some problems such as excessive usage of energy, material corrosion, and the difficulty of transesterifying triglycerides as well as post-reaction techniques such as catalyst recovery (Bautista et al. 2015), resulting in improved specificity and selectivity. Transesterification and esterification reactions can be carried out at lower temperatures with immobilized lipases and with less energy, making the catalyst recovery and glycerol purification easier (Guldhe et al. 2015). However, water in the reaction medium which may allow esters to hydrolyze is the major issue with biocatalysts. Furthermore, the usage of some solvents, like methanol, can cause the catalyst to become inactive (Navarro et al. 2016). For the whole procedure to be commercially feasible, heterogeneous biocatalysts must be used. So, they can be reused, owing to the high price of enzymes. Furthermore, immobilizing solid-state enzymes improves thermal and chemical stability while also protecting the denaturation of enzyme molecules. Enzymatic immobilization can be accomplished in a variety of ways, including attachment to a support, cross-linking and containment or encapsulation, and. The most popular method is to covalently or ionically link the enzyme to the support or to use physical adsorption. Materials as carbon nanotubes or mesoporous silicas (FDU-12, MCM-41, SBA-15, SBA-16,) (Rios et al. 2016; Zniszezol et al. 2016) have been used to achieve this immobilization. The enzymes can be housed in huge pores or cavities in the materials mentioned above. However, to obtain improved anchorage, it is normally important to functionalize the support surface with phenol groups, sulfur, chloride, or amine (Da Silva et al. 2018). Lipases' catalytic activity is highly dependent on the source organism that produces the enzyme due to their high specificity for various substrates. Lipases from Thermomyces lanuginosus, Candida rugosa, Pseudomonas cepacia, Candida. antarctica and P. fluorescens, have been used to make the majority of biodiesel from microalgal oil. C. antarctica is commonly in the form of Novozym 4356, a commercial catalyst. Although the majority of studies used single lipases, a few writers found that using a combination of lipases increased Faty Acid Ethyl Ester (FAEE) yield. To enhance biodiesel processing methods, the principle of combi-lipase biocatalysts was recently introduced (Sánchez-Bayo et al. 2019). Because of the simplicity for separation the product and their reusability in subsequent processes, the use of immobilized enzymes will greatly increase fine and specialty chemical production. Biocatalysts have been immobilized on a wide range of surfaces, including alumina, silica composites, silica, alginates, polymers, resins, glass beads, and others. The use of carbonaceous supports in the immobilization of biocatalysts has been reported before, but it has not been fully exploited. The higher cost of biocatalysts, supports production from waste biomass, such as carbon material, is always chosen over the other supports mentioned previously (Dhawane et al. 2019).
5.4. Application of Lipase for processing of biodiesel from a number of sources
Immobilized lipase plays a critical function in the transesterification process. Lipase enzymes are effective catalysts, so their synthesis and usage may be a safer choice than chemical catalysts. Lipase enzyme is primarily generated by microbes in the atmosphere. A number of natural foods produce the lipase enzyme (animals and plants). Maize seeds, developing barley seeds, papaya, babaco latex, and other sources all contain lipase. Lipase enzymes were isolated and immobilized on polypropylene support from P. fluorescens, P. cepacia, and Mucor sp. In the production of biodiesel, lipase produced from fungus, primarily yeast and filamentous mushrooms, is often employed. Lipase from yeasts like Candida rugosa, Candida antarctica, and others is used to make biodiesel in China. Lipase from Aspergillus niger, Thermomyces lanuginosus, Rhizomucor miehei, Rhizopus oryzae, and Penicillium expansum is also used to make biodiesel (Bhan et al. 2020). Kareem et al. (2017) used a solid-state fermentation procedure to manufacture lipase enzyme from A. niger.
Several experiments have established that plant-derived lipase enzymes can also be used to make biodiesel (alkyl ester). Lipase has been tested as a catalyst potential for biodiesel processing using extracts from a variety of plants and their components. Plant lipase, on the other hand, has received less attention for biodiesel production than microbial lipase. Plant parts used to create crude lipase include black cumin seeds, seeds of castor bean, rapeseed, rice bran, lipase of wheat, seed of barley, linseed, and maize seed powder. Lipase, like Babaco and papaya latex, is utilized in oil transesterification. Suwanno et al. (2017), examples, employed palm oil as a substrate and isolated crude lipase from palm fruit. The use of enzyme catalysts will reduce the need for chemical catalysts, making the process more environmentally sustainable. The lack of a large volume of feedstock and the lipase enzyme are the two biggest obstacles to commercial biodiesel processing.