Magnetic Iron Oxide Nanomaterials for Lipase Immobilization: Promising Industrial Catalysts for Biodiesel Production

: Biodiesel is a mixture of fatty acid alkyl esters (FAAEs) mainly produced via transes-terification reactions among triglycerides and short-chain alcohols catalyzed by chemical catalysts (e.g., KOH, NaOH). Lipase-assisted enzymatic transesterification has been proposed to overcome the drawbacks of chemical synthesis, such as high energy consumption, expensive separation of the catalyst from the reaction mixture and production of large amounts of wastewater during product separation and purification. However, one of the main drawbacks of this process is the enzyme cost. In recent years, nano-immobilized lipases have received extensive attention in the design of robust industrial biocatalysts for biodiesel production. To improve lipase catalytic efficiency, magnetic nanoparticles (MNPs) have attracted growing interest as versatile lipase carriers, owing to their unique properties, such as high surface-to-volume ratio and high enzyme loading capacity, low cost and inertness against chemical and microbial degradation, biocompatibility and eco-friendliness, standard synthetic methods for large-scale production and, most importantly, magnetic properties, which provide the possibility for the immobilized lipase to be easily separated at the end of the process by applying an external magnetic field. For the preparation of such effective magnetic nano-supports, various surface functionalization approaches have been developed to immobilize a broad range of industrially important lipases. Immobilization generally improves lipase chemical-thermal stability in a wide pH and temperature range and may also modify its catalytic performance. Additionally, different lipases can be co-immobilized onto the same nano-carrier, which is a highly effective strategy to enhance biodiesel yield, specifically for those feedstocks containing heterogeneous free fatty acids (FFAs). This review will present an update on the use of magnetic iron oxide nanostructures (MNPs) for lipase immobilization to catalyze transesterification reactions for biodiesel production. The following aspects will be covered: (1) common organic modifiers for magnetic nanoparticle support and (2) recent studies on modified MNPs-lipase catalysts for biodiesel production. Aspects concerning immobilization procedures and surface functionalization of the nano-supports will be highlighted. Additionally, the main features that characterize these nano-biocatalysts, such as enzymatic activity, reusability, resistance to heat and pH, will be discussed. Perspectives and key considerations for optimizing biodiesel production in terms of sustainability are also provided for future studies.


Introduction
The rapid growth of industrialization and modernization has progressively increased the consumption of fossil fuels (e.g., coal, oil, gas), causing tremendous environmental, economic and ecological impacts [1,2].Currently, non-renewable energy resources account for 70-80% of the total energy demand, and their use has generated uncontrolled greenhouse gas emissions that are considered the main cause of the warming up of our planet [3].Even if some countries, including European countries, Japan and the USA, are reducing their oil consumption (Table 1), it is projected that the worldwide need for petroleum liquid fuels will experience a 38% increase by 2040, reaching 119 million barrels/day.Such growth in fuel demand is already occurring mainly in developing countries, such as Middle Eastern nations, India, and China [4,5].As an alternative to limited fossil resources, renewable energy sources are attracting growing attention, as they may offer environmental and energy sustainability benefits [6].Vegetable oils and animal fats are widely available feedstocks that are rich in long-chain triglycerides, which can be converted into biodiesel (fatty acid alkyl esters), a biodegradable and eco-friendly fuel.Compared to conventional fossil fuels, biodiesel provides higher combustion efficiency and lower sulfur and aromatic content, with a significant decrease in the emission of toxic compounds and greenhouse gases [7].Another benefit of biodiesel is that it can be easily blended with petroleum diesel (e.g., B20 and B5, indicating the percentage of biodiesel).Such blends have high flash points and cetane numbers, and they may be used without any modifications to current engines [8].From another point of view, biodiesel can be obtained from locally available natural resources, which improves the economy of local communities [9].
Chemical production of biodiesel includes the transesterification of triglycerides (from the feedstocks) with short-chain alcohols (e.g., methanol, ethanol, propanol), in which methanol is the most preferred due to its low cost [10].There are many types of feedstocks for biodiesel production, and among them, edible vegetable oils and discarded oils are commonly employed because of their potential economic feasibility [11].Of course, every country has its own suitable oil feedstock because of the unique regional ecosystems.The global manufacturing of biodiesel is steadily increasing to reach, as projected, about 41.4 billion litres in 2025 [12].Considering the main biodiesel producers, the USA is ranked first (18%), followed by Indonesia (17%), Brazil (13%), Germany (8%) and Thailand (4%).In the USA and Brazil, the main feedstock for biodiesel production is soybean biomass, while in Europe, Indonesia, and Thailand, biodiesel production mostly depends on rapeseed, palm and coconut oils, respectively [13].Compared to these pure oils, waste oils (e.g., waste cooking oils) are cost-effective alternatives for biodiesel production [14].
Currently, different countries are focusing on finding more effective catalysts to enhance reaction efficiencies, maximize profits and minimize waste production [15,16].In fact, commonly used basic catalysts (e.g., KOH and NaOH) have some disadvantages, including high environmental pollution, difficult catalyst recovery and high reaction temperature, which promotes the undesirable hydrolysis of triglycerides, producing large amounts of free fatty acids (FFAs) [17], decreasing the reaction efficiency.To address such issues connected with the use of chemical catalysts, lipases (triacylglycerol hydrolases, EC 3.1.1.3-IUPAC)have been introduced as highly effective biocatalysts for the enzymatic transesterification of triglycerides for biodiesel production, as shown in Figure 1 [18].
Thailand (4%).In the USA and Brazil, the main feedstock for biodiesel production is soy bean biomass, while in Europe, Indonesia, and Thailand, biodiesel production mostly de pends on rapeseed, palm and coconut oils, respectively [13].Compared to these pure oils waste oils (e.g., waste cooking oils) are cost-effective alternatives for biodiesel production [14].
Currently, different countries are focusing on finding more effective catalysts to en hance reaction efficiencies, maximize profits and minimize waste production [15,16].In fact, commonly used basic catalysts (e.g., KOH and NaOH) have some disadvantages including high environmental pollution, difficult catalyst recovery and high reaction tem perature, which promotes the undesirable hydrolysis of triglycerides, producing large amounts of free fatty acids (FFAs) [17], decreasing the reaction efficiency.To address such issues connected with the use of chemical catalysts, lipases (triacylglycerol hydrolases, EC 3.1.1.3-IUPAC)have been introduced as highly effective biocatalysts for the enzymatic transesterification of triglycerides for biodiesel production, as shown in Figure 1 [18].Compared with alkaline catalysis, the use of lipolytic enzymes displays several ad vantages, including easy glycerol recovery and product separation, minimal by-produc formation and mild reaction conditions [19,20].Unlike alkaline catalysts, lipases can effi ciently catalyze biodiesel production from feedstocks containing high amounts of FFAs mixed with triglycerides in mild reaction conditions [21,22].However, for scaling up li pase-catalyzed approaches, three main challenges should be addressed: (1) high enzyme cost, (2) non-separability of enzymes due to their water solubility, and (3) low biodiese production yield.All these factors increase the costs of the final biodiesel product [23,24] Cheap alcohols, e.g., ethanol and methanol, are used to optimize production costs.As shown in Figure 2, a 3:1 methanol/triglyceride ratio allows the use of the methanol excess increasing the transesterification yield.However, such alcohol excess can also inhibit the enzyme, particularly for those alcohols which are insoluble in the reaction mixture [25,26] Therefore, the alcohol:triglyceride molar ratio must be optimized for each alcohol-fat-li pase system.To minimize this inhibition effect, stepwise alcohol addition was successfully introduced [27,28].However, maintaining low alcohol concentrations is not an appropri ate approach for large-scale biodiesel production.So, alternative strategies have been de veloped to overcome the methanol deactivation of lipases, including (1) using organic sol vents to improve methanol solubility, (2) replacing methanol with methyl acetate, and (3 applying salt-saturated solutions and silica-gel based controlled release systems for meth anol.However, organic solvents (e.g., hexane) decrease the methanolysis compared with aqueous-based mediums.Although a significant increase in reaction rate using tert-buta nol has been reported, a large molar excess of methanol (6:1 methanol to oil ratio) is re quired to obtain higher biodiesel yields.Replacing methanol with methyl acetate pro duces the by-product triacylglycerol, which complicates product purification.Further more, methyl acetate increases the costs of the process.Finally, salt-saturated solutions and silica-gel methods cause difficulties in downstream separation.Compared with alkaline catalysis, the use of lipolytic enzymes displays several advantages, including easy glycerol recovery and product separation, minimal byproduct formation and mild reaction conditions [19,20].Unlike alkaline catalysts, lipases can efficiently catalyze biodiesel production from feedstocks containing high amounts of FFAs mixed with triglycerides in mild reaction conditions [21,22].However, for scaling up lipase-catalyzed approaches, three main challenges should be addressed: (1) high enzyme cost, (2) non-separability of enzymes due to their water solubility, and (3) low biodiesel production yield.All these factors increase the costs of the final biodiesel product [23,24].Cheap alcohols, e.g., ethanol and methanol, are used to optimize production costs.As shown in Figure 2, a 3:1 methanol/triglyceride ratio allows the use of the methanol excess, increasing the transesterification yield.However, such alcohol excess can also inhibit the enzyme, particularly for those alcohols which are insoluble in the reaction mixture [25,26].Therefore, the alcohol:triglyceride molar ratio must be optimized for each alcohol-fat-lipase system.To minimize this inhibition effect, stepwise alcohol addition was successfully introduced [27,28].However, maintaining low alcohol concentrations is not an appropriate approach for large-scale biodiesel production.So, alternative strategies have been developed to overcome the methanol deactivation of lipases, including (1) using organic solvents to improve methanol solubility, (2) replacing methanol with methyl acetate, and (3) applying salt-saturated solutions and silicagel based controlled release systems for methanol.However, organic solvents (e.g., hexane) decrease the methanolysis compared with aqueous-based mediums.Although a significant increase in reaction rate using tert-butanol has been reported, a large molar excess of methanol (6:1 methanol to oil ratio) is required to obtain higher biodiesel yields.Replacing methanol with methyl acetate produces the by-product triacylglycerol, which complicates product purification.Furthermore, methyl acetate increases the costs of the process.Finally, salt-saturated solutions and silica-gel methods cause difficulties in downstream separation.In order to maximize lipase efficiency while minimizing operational costs, enzymes can be immobilized on different micro-or nano-materials to provide lipase stability, higher efficiency and easy purification.Moreover, enzyme immobilization strategies enhance enzyme dispersity in low-water content media, resulting in higher catalytic activity compared to reactions catalyzed by free lipases [30].Regarding the supporting materials, nano-based immobilization is particularly promising because of the unique surface properties of nanomaterials, which provide large specific surface areas, low mass transfer resistance and superior loading capacity [31].
For lipase immobilization, an ideal nano-carrier should possess mechanical and chemical stability, be resistant to microbial attack, and have a low cost for industrial purposes and development [32,33].Currently, several organic and inorganic nanostructures are used for lipase immobilization [34], such as: carbon nanotubes [35,36], nanofibers [37,38], graphene oxide nanostructures [39,40], chitosan [41,42], niobium oxide [43], magnetic nanoparticles [44], silica [45], agarose [46], metallic nanoparticles (Ag- [47] and AuNPs [48]) and TiO2 nanoparticles [49].Among them, magnetic iron oxide nanoparticles have stood out as superior enzyme immobilization matrices due to their unique superparamagnetic properties, which allow easy lipase recovery at the end of the process and controlled stabilization in packed and fluidized bed reactors using an external magnetic field [50,51].Magnetite (Fe3O4) and maghemite (γ-Fe2O3) nanostructures are the most common magnetic nano-carriers due to their high magnetizations, well-known chemistry and ease of synthesis [52,53].Surface modification of these magnetic nanostructures is an effective strategy to increase the stability of the magnetic core against oxidation and agglomeration, as well as its flexibility for the effective interaction with enzymes in order to achieve high loading and enhancement of lipase effectiveness [54].
This review presents an update on biodiesel biotechnological production using magnetic-based immobilization of lipolytic enzymes.Currently used nanostructures include both magnetite (Fe3O4) and maghemite (γ-Fe2O3), used in different nanocomposites containing other organic and inorganic components.First, a brief discussion of influencing factors on biodiesel production using immobilized lipases will be presented, followed by the review and critical analysis of the magnetic nanomaterials used for lipase immobilization based on recent publications.

Feedstocks
The main feedstocks suitable for biodiesel production are vegetable oils, animal fats, and microbial and algae oils.Regarding the production price of biodiesel, the cost of the raw materials is the most important parameter [55].Based on their availability and source, feedstocks are classified into three main groups: edible, non-edible, and waste oils [56,57].Edible vegetable oils, e.g., palm and soybean oils, are the most commonly used feedstocks In order to maximize lipase efficiency while minimizing operational costs, enzymes can be immobilized on different micro-or nano-materials to provide lipase stability, higher efficiency and easy purification.Moreover, enzyme immobilization strategies enhance enzyme dispersity in low-water content media, resulting in higher catalytic activity compared to reactions catalyzed by free lipases [30].Regarding the supporting materials, nano-based immobilization is particularly promising because of the unique surface properties of nanomaterials, which provide large specific surface areas, low mass transfer resistance and superior loading capacity [31].
For lipase immobilization, an ideal nano-carrier should possess mechanical and chemical stability, be resistant to microbial attack, and have a low cost for industrial purposes and development [32,33].Currently, several organic and inorganic nanostructures are used for lipase immobilization [34], such as: carbon nanotubes [35,36], nanofibers [37,38], graphene oxide nanostructures [39,40], chitosan [41,42], niobium oxide [43], magnetic nanoparticles [44], silica [45], agarose [46], metallic nanoparticles (Ag- [47] and AuNPs [48]) and TiO 2 nanoparticles [49].Among them, magnetic iron oxide nanoparticles have stood out as superior enzyme immobilization matrices due to their unique superparamagnetic properties, which allow easy lipase recovery at the end of the process and controlled stabilization in packed and fluidized bed reactors using an external magnetic field [50,51].Magnetite (Fe 3 O 4 ) and maghemite (γ-Fe 2 O 3 ) nanostructures are the most common magnetic nano-carriers due to their high magnetizations, well-known chemistry and ease of synthesis [52,53].Surface modification of these magnetic nanostructures is an effective strategy to increase the stability of the magnetic core against oxidation and agglomeration, as well as its flexibility for the effective interaction with enzymes in order to achieve high loading and enhancement of lipase effectiveness [54].
This review presents an update on biodiesel biotechnological production using magnetic-based immobilization of lipolytic enzymes.Currently used nanostructures include both magnetite (Fe 3 O 4 ) and maghemite (γ-Fe 2 O 3 ), used in different nanocomposites containing other organic and inorganic components.First, a brief discussion of influencing factors on biodiesel production using immobilized lipases will be presented, followed by the review and critical analysis of the magnetic nanomaterials used for lipase immobilization based on recent publications.

Feedstocks
The main feedstocks suitable for biodiesel production are vegetable oils, animal fats, and microbial and algae oils.Regarding the production price of biodiesel, the cost of the raw materials is the most important parameter [55].Based on their availability and source, feedstocks are classified into three main groups: edible, non-edible, and waste oils [56,57].Edible vegetable oils, e.g., palm and soybean oils, are the most commonly used feedstocks for biodiesel production [58,59]; however, the supply of this type of raw materials strongly depends on the food market, which is not completely predictable [60].Conversely, non-edible oils are cheaper and not influenced by the food industry; hence, they are better candidates as biodiesel feedstocks [61].Among them, Jatropha plants are particularly interesting since they can grow on wasteland with little demand for water and fertilizers, producing seeds with high oil content (30-50% of plant weight) [62].Jatropha food applications are restricted by the presence of toxic compounds, e.g., phorbol esters, but present no limitation for biodiesel production.The general disadvantages of non-edible oils are their high FFAs and water content, which limit their use in alkaline-catalyzed transesterification processes.It is worth mentioning that both edible and some non-edible oils are agricultural feedstocks, requiring large amounts of land and other resources (water, fertilizers, etc.) for their production, which still compete with the food market [63] and cause environmental problems (i.e., deforestation) that are not compatible with current sustainable development goals.For these reasons, non-edible microalgae oil, not requiring land for its production, is a promising alternative source for biodiesel [64]; however, current technologies for algae farming, oil recovery and separation are still expensive; therefore, further research is required for large-scale biodiesel production from algal oil.
Another important feedstock is waste oil that includes frying oil, waste cooking oil and other waste products, including waste animal fat (major wastes of the leather industry, the meat industry and fish processing), tall oil (a by-product of the paper industry) and tobacco seed oil (a by-product of tobacco leaf manufacturing) [29].Every day, a large amount of waste cooking and frying oils are ubiquitously produced by the cooking and food industries and require to be properly disposed of.Obviously, the use of these waste oils as a feedstock brings environmental and economic benefits and is perfectly in line with the principles of circular economy.Compared to non-edible and edible oils, the acquisition of waste oil is much cheaper because it does not require additional land use or other resources, and, more importantly, it can reduce the environmental pollution caused by their disposal in nature [65].Waste animal fats contain more FFAs and saturated fatty acids.The traditional alkaline-catalytic biodiesel process promotes the hydrolysis of triglycerides, which increases the FFA content when waste frying oils or cooking oils are used as raw materials [10].Moreover, these waste frying oils have high water content and include other animal or vegetable oils, depending on what food was cooked with them.However, high FFAs and water contents are not compatible with the traditional alkaline-based transesterification technology in which the FFAs form soap in the presence of water (catalyzed by the basic catalyst), which decreases catalyst efficiency, leading to a difficult downstream separation of glycerol [66].Therefore, waste oils are not suitable for alkaline-catalyzed biodiesel production.Conversely, lipase-catalyzed transesterification is compatible with the high FFAs and water contents of waste oil feedstocks [67].

Immobilization Methods
There are four general strategies for lipase immobilization, including (1) physical surface adsorption, (2) chemical surface attachment, (3) encapsulation and (4) cross-linking (Figure 2).Via physical adsorption, the enzyme is immobilized on the surface of the carrier material through non-covalent interactions such as hydrogen bonding, electrostatic attraction, van der Waals forces and hydrophobic interactions.Despite the simplicity and popularity of this method, the weak interactions between lipase and carrier usually result in enzyme leakage issues, especially in complex media having high ionic strengths.This kind of immobilization is commonly used for porous supports [68], which can efficiently trap lipase molecules inside their pores and protect them from external denaturing conditions and subsequent leakage.Lipases have a natural affinity for hydrophobic supports, so physical adsorption occurs fast by hydrophobic interactions [69,70] to form a shell of immobilized enzyme molecules on the carrier surface, in which the lipase assumes a monomeric, open and catalytically active conformation.The so-called "open" conformation of the lipase can be maintained by the support and protected from other external interfaces [71,72].
In this scenario, hydrophobic triacylglycerols and fatty acid esters (precursors) strongly interact with the hydrophobic support, but the hydrophilic glycerol (product) shows no affinity for it.Both factors favour reaction efficiency [73].
In covalent immobilization, the lipase is chemically attached to the functional groups of the support to guarantee strong enzyme adhesion with negligible leakage during the catalytic process.Both lipase and support usually require a pre-activation of their functional groups before covalent attachment.Bifunctional organic molecules having suitable functional groups (e.g., glutaraldehyde), are commonly used for the covalent conjugation of lipases onto support surfaces.For instance, the aldehyde moiety of glutaraldehyde can be easily condensed with the amine groups of both lipase and support under mild conditions [74,75], which also enhances the hydrophobicity of the surface [76].There are also other conjugating reagents, such as EDC (N-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), that drive the formation of amide bonds between lipase amine groups and carboxyl groups of the support or vice versa [77,78].There are also epoxy functional groups for activating the supports, which are suitable for covalent immobilization by direct mixing of these activated supports with lipases [79].In some supports, multi-point covalent binding may occur due to the surface properties of the support and lipase molecular structure, which provides enhanced stability to the immobilized enzyme [80].This multiple covalent attachment fixes the native lipase structure on the surface of the support, which protects it against external denaturing conditions such as high ionic strength, heat and organic solvents.However, covalent conjugation should be designed accurately in order to preserve the catalytically active conformation of lipase molecules [81].
In the third method, lipases are encapsulated into porous supports by a simultaneous co-precipitation method, which rarely has leakage problems.The other advantages of this method are the high compatibility with the enzymes and a fast immobilization process [82]; however, high mass transfer resistance limits this type of immobilization.
Regarding the fourth immobilization strategy, cross-linking, lipases are interconnected by crosslinking agents, e.g., glutaraldehyde, to form multiple covalent bonds within or among enzyme molecules.These multi-point attachments enhance the rigidity of the lipase and protect the immobilized enzyme against external agents (heat, organic solvents, etc.).Additionally, there can be a combination of these four immobilization methods to enhance enzyme loading and stability [83], which eventually results in improving catalytic performance.Finally, it is worth mentioning that for all these immobilization methods, the reaction conditions should be optimized in terms of pH, immobilization time, temperature, lipase concentration and enzyme/support ratio.

Lipase Specificity
In transesterification reactions, lipases follow a two-step mechanism for the synthesis of fatty acid methyl esters, usually through the Ping-Pong Bi Bi mechanism [84].Most lipases are regiospecific due to their specificity to only hydrolyze primary ester bonds at the sn-1 and sn-3 sites, external positions within the triacylglycerol, generating either one free fatty acid and diacylglycerol or two free fatty acids and 2-monoacylglycerol (Figure 3) [85].
Monoacylglycerol lipases (EC 3.1.1.23)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 methods.Monoacylglycerol lipases have been the object of only a few studies [86], although they might be present in microbial enzyme preparations [87].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 and hyicus [88], Geotrichum candidum, Corynebacterium acnes, Penicillium cyclopium [24] and Chromobacterium viscosum [89].Another alternative for the hydrolysis of monoacylglycerols is the acyl migration in the glycerol backbone from the sn-2 position to the sn-1 or sn-3 positions [90].Monoacylglycerol lipases (EC 3.1.1.23)catalyze the hydrolys sition of 2-monoacylglycerol into free fatty acid and glycerol.Such in the enzyme extract and masked when measuring activity with s oacylglycerol lipases have been the object of only a few studies [8 be present in microbial enzyme preparations [87].Other lipases act on any of the ester bonds of the triacylglycerol and, therefore, glycerol to release free fatty acids and glycerol as the final prod lipases from Staphylococcus aureus and hyicus [88], Geotrichum can acnes, Penicillium cyclopium [24] and Chromobacterium viscosum [89] the hydrolysis of monoacylglycerols is the acyl migration in the the sn-2 position to the sn-1 or sn-3 positions [90].
Lipase specificity depends on the length of fatty acids, the pr and branched groups.Consequently, reaction rates may have im pending on the triacylglycerol composition of the fat waste.Lipa on medium to long-chain fatty acids, which are more common waste [91].

Lipase Source
As triacylglycerol hydrolases, lipases are commonly used for ysis of long-chain insoluble glycerides in aqueous media.Lipases Lipase specificity depends on the length of fatty acids, the presence of double bonds and branched groups.Consequently, reaction rates may have important variations depending on the triacylglycerol composition of the fat waste.Lipases are especially active on medium to long-chain fatty acids, which are more commonly present in animal fat waste [91].

Lipase Source
As triacylglycerol hydrolases, lipases are commonly used for the biocatalytic hydrolysis of long-chain insoluble glycerides in aqueous media.Lipases belong to the serine hydrolase class and have a catalytic centre composed of three amino acids: a histidine, a nucleophilic serine and an aspartic acid or glutamic acid [92,93].As mentioned above, lipases can also catalyze the multi-step transesterification of triglycerides with short-chain alcohols with high selectivity [94], which makes them promising biocatalysts in different industries, e.g., food, cosmetics, textile, pharmaceutical, as well as biodiesel technology [95].Lipases are enzymes working at water/lipid interfaces [96].In more detail, lipases possess amphiphilic polypeptide chains that are able to form an interface between water and the substrate.In this way, the hydrophobic substrate can selectively interact with the active centre of the lipase due to the amphiphilic nature of the lipase and its unique stereo-specificity.Lipases obtained from different sources have different molecular structures and, therefore, show different substrate selectivity.In fact, the reaction rate strongly depends on the type of lipase as well as on the molecular structure of the substrate [97,98].For biodiesel production, the transesterification ability of lipases can convert long-chain triglycerides into FAAEs and simultaneously catalyze the esterification of alcohol with FFAs, which is valuable when using feedstocks with high FFA content.Biodiesel production does not require the use of stereo-specific lipases, so all tri-, di-, monoglycerides and FFAs with different molecular weights can be converted into FAAE [99].The feedstocks generally contain heterogeneous mixtures of triglycerides and FFAs; hence, using a mixture of different lipases (includ-ing selective and nonselective enzymes) can be useful to enhance catalytic activity [100].In order to reduce lipase specificity and improve reaction efficiency, the stereo-chemical structure of lipases can also be modified by immobilization [101,102].Lipases may be obtained from different natural sources, including microorganisms, plants and animals.Hence, different lipases show different catalytic performance and physicochemical properties due to their diverse origin, resulting in differences in production processes and immobilization methods.Similarly to other catalysts, lipases should have high diversity, low price and long-term chemical stability.In this regard, microbial-derived lipases (e.g., from fungi, yeast and bacteria) fulfil all requirements.For industrial production, lipase-producing microorganisms should have high specificity and be amenable to genetic modification [103].There are several suitable microorganisms, e.g., Rhizopus oryzae [104], Pseudomonas cepacia [105], Aspergillus niger [106], Candida antarctica [107], Pseudomonas fluorescens [108], Burkholderia cepacia [109], Candida rugosa [110], Rhizomucor miehei [111], and Thermomyces lanuginosus [112].Regarding animal-derived lipases, porcine pancreatic lipase (PPL) can be manufactured by transgenic recombinant bacteria with high yield [113] and has received much attention also for applications in biodiesel manufacturing.

Operation Mode
The operational process of biodiesel manufacturing has a significant impact on the versatility, feasibility and final cost of the product.Lipase-based catalysis can make the process more simple, environmentally valuable and cost-effective.Figure 4 presents the schemes of alkaline-catalyzed and lipase-catalyzed biodiesel production.However, chemical catalysis still requires additional subsequent steps, which are not shown in the figure.For instance, the produced glycerol needs to be separated from biodiesel by, for example, methanol evaporation and removal of saponification products, and, more importantly, the alkaline process produces large amounts of toxic wastewater.As shown in Figure 1, the transesterification reaction employed for biodiesel production is reversible, and in the alkaline process, methanol is usually used in excess to push the reaction forward, based on Le Chatelier's principle [114].In contrast, this excess addition of methanol is not compatible with enzyme-catalyzed reactions because of its inactivating effect on lipases; hence, a lower methanol-to-oil ratio is used, and therefore longer reaction times are needed.Additionally, in lipase-based processes, there are no saponification by-products or large amounts of wastewater, which is another benefit of this method since, after the separation of the immobilized enzyme, highly pure biodiesel (and glycerol) is obtained [115].Other benefits of batch reactions are the simple equipment required, easy process control and high mixing of the substrate.However, the separation of nano-based immobilized lipases in these kinds of reactors requires additional purification steps such as nano-filtration and ultracentrifugation, which are expensive and may lead to lipase inactivation, so these simple reactors are not beneficial for large-scale biodiesel production.For the scale-up of lipase-based methods, reactors are another important factor to take into consideration.In this regard, there are two general types of production processes: batch reactions and continuous reactions.As shown in Figure 5, different reactors have been used with immobilized lipases for biodiesel production, including fluidized-bed reactors (FBRs), stirred-tank reactors (STRs) and packed-bed reactors (PBRs) [116].Table 2 summarizes some studies on reactor-scale biodiesel production employing nano-based lipase processes.In 2019, Tabatabaei et al. published a comprehensive review on reactor technologies for biodiesel production and processing, so we will not discuss this topic further in this context [117].The commonly used batch reactions are usually carried out in STRs, which show simplicity of operation and are suitable for laboratory-scale research.The FBR system is a hybrid of both STR and PBR, in which catalysts with smaller particles than PBRs can be used because there is no need to control the pressure.However, traditional FBRs still have problems, such as catalyst settling, because nano-carriers are easily carried away in the reaction mixture, even at very low flow rates.This problem can be solved by applying circulating fluidized-bed bioreactors, as previously reported [131].However, separation devices in FBRs are still necessary to recover the nano-catalysts, and the cost of nano-filtration remains high.Again, an alternative strategy could be the use of magnetic nanoparticles for lipase immobilization, but it has not been reported yet in largescale biodiesel production processes [132].
In general, the scale-up of biodiesel production using nano-based biocatalysts re- Other benefits of batch reactions are the simple equipment required, easy process control and high mixing of the substrate.However, the separation of nano-based immobilized lipases in these kinds of reactors requires additional purification steps such as nano-filtration and ultracentrifugation, which are expensive and may lead to lipase inactivation, so these simple reactors are not beneficial for large-scale biodiesel production.This disadvantage can be handled by using magnetic nanomaterials as lipase carriers that can be easily separated from the reaction products, reducing both the cost of recovery and the loss of enzyme activity.Batch processing usually suffers from long reaction times, which require high volume-reactors for industrial production.High-capacity reactors increase the time and operational costs of unloading, cleaning and reloading of products and raw materials [118].Another challenging factor is the high shear force of the stirrer, which might impact the activity of the immobilized lipase [119].From these points of view, continuous flow reactions have higher efficiencies than batch reactions for large-scale production [120].Continuous reactors are superior to batch reactors in terms of long-term stability of processing maintaining lipase productivity and activity.There are many kinds of continuous flow reactors and among them, PBRs are the most popular ones, in which the immobilized lipase is loaded into the column, followed by continuous pumping of the feedstock into the column.To control the pressure, the size of the immobilized biocatalyst should not be too small [121], so large nanofibers are more effective and practical for PBRs.

Nano-Support
Reactor Process Ref.
magnetic nanoparticles STR batch [122] snowman-like Fe 3 O 4 /Au nanoparticles STR batch [123] Fe 3 O 4 coated with poly(styrene-methacrylic acid) STR batch [124] electrospun poly(acrylonitrile) PBR continuous [125] nano silicon FBR batch [126] nano silicon reverse fluidized-bed reactor continuous [127] nano silicon PBR continuous [128] Fe The FBR system is a hybrid of both STR and PBR, in which catalysts with smaller particles than PBRs can be used because there is no need to control the pressure.However, traditional FBRs still have problems, such as catalyst settling, because nano-carriers are easily carried away in the reaction mixture, even at very low flow rates.This problem can be solved by applying circulating fluidized-bed bioreactors, as previously reported [131].However, separation devices in FBRs are still necessary to recover the nano-catalysts, and the cost of nano-filtration remains high.Again, an alternative strategy could be the use of magnetic nanoparticles for lipase immobilization, but it has not been reported yet in large-scale biodiesel production processes [132].
In general, the scale-up of biodiesel production using nano-based biocatalysts requires optimizing the reactor/process, and in this field, there has been much research for STRs, PBRs and FBRs using nano-biocatalysts, which proves their feasibility; however, the high cost of nano-biocatalyst separation and enzyme deactivation should be taken into account.For industrial biodiesel production, more reactor-scale studies are still required to gather the advantages of batch and continuous processing in a single platform, reducing overall costs and improving the feasibility and versatility of the process.
Regarding the current reactor technology, other types of transesterification reactors were comprehensively reviewed by Tabatabaei et al. [117].The authors discussed in detail the effects of the main parameters on the transesterification reaction in order to better understand the mechanisms behind each reactor technology.Different transesterification reactors, including rotating, tubular/plug-flow, cavitational, simultaneous reaction-separation and microwave reactors, were then described from scientific and practical viewpoints.The merits and drawbacks of each reactor technology for biodiesel production were highlighted to guide future R&D in this field.

Glycerol
Glycerol is the major by-product of biodiesel production, and it is a valuable material for other industries.Glycerol accumulates on the surface of catalysts and changes the reaction direction, reducing the yield.More importantly, the surface accumulation of glycerol leads to the formation of a hydrophilic environment around the catalyst, which hinders its catalytic performance [133,134].To solve this inhibition effect, hydrophobic carriers are used for catalyst immobilization, while the use of organic solvents (hexane, petroleum ether, n-heptane and tert-butanol) is another option to decrease the reaction medium viscosity and increase the solubility of the organic phase, avoiding phase separation and improving mass transfer efficiency [135,136].Among suitable organic solvents, tert-butanol has the advantage of providing steric hindrance through its tert-butyl group, which inhibits its participation in the transesterification reaction.However, the environmental concerns, toxicity and flammability of organic solvents limit the large-scale application of this strategy due to safety and economic aspects.Therefore, solvent-free systems and green solvents such as supercritical carbon dioxide and ionic liquids have been studied as alternatives to organic solvents [137,138].

Inorganic Nanocarriers Used to Develop Lipase-Based Nano-Biocatalysts
To date, some noteworthy nanocatalysts have been developed for biodiesel production, as comprehensively reviewed by Pandit et al. in 2023 [139].In the case of inorganic nanocatalysts, CaO, ZnO, zeolites, and hydrotalcites have attracted significant research interest [140], however magnetic nanostructures are a unique class of nanomaterials that are able to allow the easy recovery and recycling of the catalyst.

Magnetic Iron Oxide Nanoparticles
After catalyzing biodiesel synthesis, the biocatalyst should be separated from the mixture and reused again for the next catalytic cycles [141].As mentioned above, this recovery process is considered one of the main challenges in the biodiesel industry, so recyclable catalysts are urgently required to replace non-recyclable ones.A promising alternative is the immobilization of lipases onto magnetic nanoparticles (MNPs), which allows the separation of the biocatalyst by simply applying an external magnetic field.Magnetic nanostructures have been extensively applied as nanocarriers for different pharmaceutical and biological species to improve the effectiveness of the immobilized molecules and, more importantly, to provide magnetic properties to the resultant nanosystem.In fact, magnetic nanostructures benefit from both their nano-and magnetic aspects and have several advantages for industrial applications, such as ease of synthesis, low cost, biocompatibility, superparamagnetism and magnetic separability, high loading capacity for the loading of different molecules and standardized methods for their large-scale production [142,143].For biodiesel technology, considerable efforts have been devoted to developing efficient magnetic supports for lipase immobilization, simplifying the post-treatment process and catalyst recovery, and decreasing operation costs [144,145].Among MNPs, Fe 3 O 4 (magnetite) nanoparticles are the most widely used systems for different technologies [146,147].In this section, the most recent publications on the application of this type of nano-support for lipase immobilization will be discussed in detail.

Importance of Surface Modification of Magnetic Nanoparticles
The main methods to fabricate Fe 3 O 4 nanoparticles (Table 3) [148] include chemical co-precipitation, micro-emulsion, thermal decomposition and solvothermal methods [149].For large-scale applications, MNPs should first be modified/functionalized to prevent their aggregation, oxidation of the magnetic core, loss of magnetism and dispersibility.Further, surface modification facilitates lipase loading by providing suitable functional groups on the surface of MNPs [150,151].
The functionalizing molecules can attach to Fe 3 O 4 NP surfaces by either physical or chemical interactions.Chemical functionalization is much more favourable than physical functionalization, especially when the long-term stability of the modified Fe 3 O 4 NPs is required.In the following sections, recent publications on biodiesel production will be divided into two groups: (1) those performed using silane-functionalized MNPs and (2) nanocarriers functionalized with non-silane organic molecules.In the structure of silane molecules, there are active functional groups (e.g., hydrolysable alkoxys) which are suitable for the condensation with -OH surface groups of MNPs.Conversely, physical modification occurs via electrostatic, hydrogen bonds, van der Waals, and hydrophobic interactions [152].In a recent research, João Brandão Júnior et al. studied biodiesel production from vegetable oils obtained from two common Brazilian trees: tucuman and babassu, whose composition is dominated by petroleum acid [153,154].The authors first synthesized Fe 3 O 4 NPs by the co-precipitation method and then functionalized them with APTES ((3-aminopropyl)triethoxysilane) to introduce-NH 2 groups on the magnetic core.Afterwards, Fe 3 O 4 NPs-APTES were activated with glutaraldehyde to convert their amine groups into -CHO groups in mild conditions and at 20 • C. Lipase immobilization was then performed in the last step by introducing a low-cost commercial lipase (Eversa ® Transform 2.0 (Novozymes, Bagsvaerd, Denmark), derived from genetically modified Aspergillus oryzae) into the Fe 3 O 4 NPs-CHO NPs to chemically link the lipase to the magnetic nanocarrier [155].Biodiesel production from two vegetable oils was studied using both free and immobilized lipase (enzymatic load: 80 UpNPB g −1 ) through esterification of the FFAs with both ethanol (E) and methanol (M).Upon the selected reaction conditions (1:1 oil:alcohol molar ratio; temperature: 37 • C; reaction time: 2-8 h for free enzyme and 8 h for the immobilized biocatalyst), the free Eversa ® Transform 2.0 lipase showed higher conversion efficiency for the babassu oil (96.7% (M) and 93.4% (E)) than tucuman (52.6% (M) and 57.5% (E)).The free lipase worked very well on babassu oil with both methanol and ethanol.However, for the heterogeneous esterification reactions using the immobilized enzyme, the best results were obtained with ethanol for both tucuman and babassu oils, with 86.0% and 82.2% yields, respectively.Furthermore, the immobilization resulted in a dramatic enhancement of the conversion efficiency in methanol for tucuman, from 52.6% (free lipase) to 68.7% for immobilized lipase.After each catalytic cycle, the immobilized biocatalyst was magnetically separated, washed with hexane and reused for three cycles.The results indicated a subtle decrease in catalytic activity after the third cycle, which might be due to the residual hexane (not evaporated) in the enzymatic fraction, interfering with the enzyme's behaviour by changing its 3D structure and, consequently, its catalytic activity.
In 2022, Parandi et al. studied biodiesel production from waste cooking oil (WCO) catalyzed by immobilized Candida antarctica Lipase B (CALB) [156].To this aim, the authors synthesized Fe 3 O 4 NPs (by the co-precipitation method) and then functionalized them using two silane reagents, tetraethyl orthosilicate (TEOS) and N- [3-(trimethoxysilyl)propyl] ethylenediamine (TSD), to improve their colloidal stability and provide -NH 2 groups onto the surface of functionalized Fe 3 O 4 NPs to facilitate and enhance lipase immobilization (Figure 6).After optimizing lipase immobilization (2.5 mg mL −1 , 5 h, 35 • C, pH 7), they studied operating parameters such as temperature (30-70 • C), methanol:oil molar ratio (1:1 to 5:1), contact time (12-36 h) and catalyst dosage (0.2-1 g) to maximize biodiesel production yield.The highest yield (96%) was obtained using 1 g catalyst and 4:1 methanol:oil molar ratio at 40 • C for 30 h.The reusability of this nano-biocatalyst was performed by magnetic separation, washing with hexane-acetone (1:1), and the results showed the maintaining of the catalytic activity until the 6th cycle (>70%), then it dropped to <50% which can be due to the detachment of lipase from the nano-support (due to weak physical interactions among lipase molecules and MNPs) and lipase denaturation due to sequential washing steps with organic solvents.For the long-term sustainable development of lipase-based biodiesel technology, the organic solvents commonly used in the production process should be changed with green solvents that are easy to recycle and recover.Ionic liquids (ILs) [157], supercritical carbon dioxide (scCO2) [158] and deep eutectic solvent (DES) [159] have been proposed as ecofriendly candidates for biodiesel manufacturing.ILs can act as non-aqueous reaction media due to their unique characteristics, such as low toxicity, low volatility, low flammability and high solubility in both inorganic and organic materials [160,161].Furthermore, ILs exhibit a stabilizing effect on enzymes and have negligible vapour pressures [162].For instance, Patel et al. used [Hmim][PF6] as the reaction medium to use Candida rugosa lipase (CRL) for biodiesel production from Chinese tallow kernel oil [163].In this sense, the application of MNPs for this type of solvent-free enzymatic biodiesel production is rare.In a recent study in 2022, Xing's group [164] immobilized Rhizomucor miehei lipase (RML) onto Fe3O4NPs-APTES using glutaraldehyde and studied Jatropha oil transesterification for biodiesel production in ILs (Figures 7 and 8).Fifteen kinds of ILs were assessed as the reaction medium, and several reaction parameters were also optimized in this study, e.g., IL:substrate weight ratio, methanol:oil molar ratio, amount of catalyst, oil feedstock, reaction temperature and time.For the long-term sustainable development of lipase-based biodiesel technology, the organic solvents commonly used in the production process should be changed with green solvents that are easy to recycle and recover.Ionic liquids (ILs) [157], supercritical carbon dioxide (scCO 2 ) [158] and deep eutectic solvent (DES) [159] have been proposed as ecofriendly candidates for biodiesel manufacturing.ILs can act as non-aqueous reaction media due to their unique characteristics, such as low toxicity, low volatility, low flammability and high solubility in both inorganic and organic materials [160,161].Furthermore, ILs exhibit a stabilizing effect on enzymes and have negligible vapour pressures [162].For instance, Patel et al. used [Hmim][PF 6 ] as the reaction medium to use Candida rugosa lipase (CRL) for biodiesel production from Chinese tallow kernel oil [163].In this sense, the application of MNPs for this type of solvent-free enzymatic biodiesel production is rare.In a recent study in 2022, Xing's group [164] immobilized Rhizomucor miehei lipase (RML) onto Fe 3 O 4 NPs-APTES using glutaraldehyde and studied Jatropha oil transesterification for biodiesel production in ILs (Figures 7 and 8).Fifteen kinds of ILs were assessed as the reaction medium, and several reaction parameters were also optimized in this study, e.g., IL:substrate weight ratio, methanol:oil molar ratio, amount of catalyst, oil feedstock, reaction temperature and time.
plication of MNPs for this type of solvent-free enzymatic biodiesel production is rare.In a recent study in 2022, Xing's group [164] immobilized Rhizomucor miehei lipase (RML) onto Fe3O4NPs-APTES using glutaraldehyde and studied Jatropha oil transesterification for biodiesel production in ILs (Figures 7 and 8).Fifteen kinds of ILs were assessed as the reaction medium, and several reaction parameters were also optimized in this study, e.g., IL:substrate weight ratio, methanol:oil molar ratio, amount of catalyst, oil feedstock, reaction temperature and time.[BMIM][PF6] demonstrated to be the best medium for immobilized RML, showing five times higher catalytic activity than the free enzyme and maintaining 60% of its initial performance after five cycles in a 48 h reaction.Moreover, the immobilized RML showed high storage stability, maintaining its catalytic activity after a 98-day storage at −20 °C.
As mentioned in Section 2.4, there are two general types of production processes for scaling up lipase-based methods: continuous and batch operations.In this regard, continuous reactions are more economical when compared to batch systems [165].In lab-scale studies, continuous systems can be made by glass columns with a packed-bed structure in which there is an upward or downward flow of starting materials and feedstock, and the products are gathered from the outlet of the column.Despite the low flow rates of packed-bed reactors (PBR), they show increased contact time between the oil feedstock and biocatalyst, leading to high conversion rates [166], so the application of continuoustype compacted-bed reactors for immobilized lipases is increasing [167].In this regard, Bento et al. immobilized a commercial Burkholderia cepacia lipase onto MNPs-APTES (Fe3O4/γ-Fe2O3) using glutaraldehyde as a coupling agent [168].The immobilized biocatalyst was used for producing biodiesel from kernel oil in a continuous-operating fixedbed reactor operating in a flow with ethanol at an oil:ethanol molar ratio of 1:12, 50 °C and 16 h reaction time.The enhanced stability of the immobilized lipase allowed operating up to 47 days with high productivity of 38.7 ± 0.7 mg g −1 h −1 , ester content of >96.5% and kinematic viscosity value of 5.32 ± 0.4 mm 2 s −1 at 40 °C, meeting the requirements of the ASTM (D6751) and ANP standards for biodiesel fuel.These results indicate that this magnetic biocatalyst can be further studied for the continuous development of different industrial sectors.
Lipase-producing microorganisms are important biotools for preparing lipases due to their unique features such as rapid reproduction, large variety, short production cycle, adaptability and easy scale production.Biodiesel production from microbial oils and plants requires two steps: (1) oil extraction and (2) transesterification; however, during oil [BMIM][PF 6 ] demonstrated to be the best medium for immobilized RML, showing five times higher catalytic activity than the free enzyme and maintaining 60% of its initial performance after five cycles in a 48 h reaction.Moreover, the immobilized RML showed high storage stability, maintaining its catalytic activity after a 98-day storage at −20 • C.
As mentioned in Section 2.4, there are two general types of production processes for scaling up lipase-based methods: continuous and batch operations.In this regard, continuous reactions are more economical when compared to batch systems [165].In lab-scale studies, continuous systems can be made by glass columns with a packed-bed structure in which there is an upward or downward flow of starting materials and feedstock, and the products are gathered from the outlet of the column.Despite the low flow rates of packed-bed reactors (PBR), they show increased contact time between the oil feedstock and biocatalyst, leading to high conversion rates [166], so the application of continuous-type compacted-bed reactors for immobilized lipases is increasing [167].In this regard, Bento et al. immobilized a commercial Burkholderia cepacia lipase onto MNPs-APTES (Fe 3 O 4 /γ-Fe 2 O 3 ) using glutaraldehyde as a coupling agent [168].The immobilized biocatalyst was used for producing biodiesel from kernel oil in a continuous-operating fixed-bed reactor operating in a flow with ethanol at an oil:ethanol molar ratio of 1:12, 50 • C and 16 h reaction time.The enhanced stability of the immobilized lipase allowed operating up to 47 days with high productivity of 38.7 ± 0.7 mg g −1 h −1 , ester content of >96.5% and kinematic viscosity value of 5.32 ± 0.4 mm 2 s −1 at 40 • C, meeting the requirements of the ASTM (D6751) and ANP standards for biodiesel fuel.These results indicate that this magnetic biocatalyst can be further studied for the continuous development of different industrial sectors.
Lipase-producing microorganisms are important biotools for preparing lipases due to their unique features such as rapid reproduction, large variety, short production cycle, adaptability and easy scale production.Biodiesel production from microbial oils and plants requires two steps: (1) oil extraction and (2) transesterification; however, during oil extraction, some unsaturated fatty acids are lost due to their oxidation, decreasing biodiesel yield.Hence, the one-step synthesis of microorganism-derived biodiesel has attracted growing interest in recent years.For instance, Cao et al. used a lipase-producing Burkholderia pyrrolica WZ10-3, isolated from the soil of Mt.Emei in China, possessing high lipase activity (up to 280.0 U/mL) and good tolerance to pH and temperature (with maximum transesterification rate of 97%) [169].To enhance lipase efficiency, the authors developed a one-step transesterification method of wet yeast using the immobilized lipase, the oleaginous yeast Saitozyma podzolica Zwy-2-3 and the lipase-producing Burkholderia pyrrolica WZ10-3.To achieve this, Fe 3 O 4 NPs were first functionalized with APTES, followed by glutaraldehyde activation and condensation with the primary amine of WZ10-3 lipase to realize the covalent immobilization of the enzyme.The APTES-glutaraldehyde modification can change the surface property from hydrophilic to hydrophobic, and during this immobilization process, the hydrophobic interface activated the lipase conformation and its "open" form on the Fe 3 O 4 NPs, improving its dispersion stability, surface hydrophobicity and biocompatibility.Furthermore, compared with direct immobilization, the use of APTES-glutaraldehyde extends the spacer arm, which results in lower steric hindrance and higher activity of the immobilized enzyme.In the optimized lipase immobilization conditions (enzyme dosage: 30.22 mL, glutaraldehyde concentration: 2.0%, T: 40 • C and time: 4 h), the immobilized lipase showed a 50% enhanced temperature tolerance.Additionally, immobilization dramatically improved lipase stability for up to 48 days when stored at 4 • C.Besides enhancing the transesterification rates, the immobilized enzyme still showed 90% catalytic activity after ten cycles.
Carbon-based stabilizing agents have been extensively used for Fe 3 O 4 NP functionalization because of their biodegradable nature and thermo-chemical stability, which makes them suitable for the physical adsorption and covalent binding of the enzyme to the support.Graphene oxide (GO) has been used in many enzyme immobilization studies because of its thermal stability, large surface area with suitable functional groups for high enzyme loading and easy modification [170,171].In 2020, Nematian et al. studied the immobilization of Rhizopus oryzae lipase (ROL) on GO-functionalized MNPs (MGO) and used it for producing biodiesel from Chlorella vulgaris microalgae oil [172].Additionally, the authors studied the immobilization of MGO-APTES and MGO-APTES-GA (where both APTES and glutaraldehyde were used).Rhizopus oryzae lipase was immobilized on MGO and MGO-APTES through electrostatic interactions, while the MGO-APTES-GA support immobilized lipase through covalent bonding.The maximum loading of ROL (70.2%) and the highest biodiesel conversion (71.19%) was achieved by MGO-APTES-GA-ROL, which maintained 58.77% of its initial catalytic performance after five cycles of biodiesel production and showed the best catalyst reusability.The authors reported an enhanced loading capacity and thermal and storage stability due to the covalent linkage of the enzyme.
In 2020, Nematian et al. compared the physical and chemical loading of Rhizopus oryzae lipase (ROL) on MNPs for producing biodiesel to study the effect of enzyme covalent bonding on catalytic performance [173].For non-covalent immobilization, ROL was loaded onto bare MNPs and APTES-functionalized MNPs, while for covalent attachment, glutaraldehyde (GA) was used to chemically link ROL to MNPs-APTES (Figure 9).The authors compared these three nano-biocatalysts for their esterification productivity, hydrolytic activity, kinetic parameters and reusability.For all the immobilized nanosystems, rapid magnetic separation was achieved and successfully repeated for five cycles.The results showed an increased conversion in the presence of MNPs-APTES-ROL, compared to MNPs-ROL, which can be due to a larger polar area around the support surface provided by APTES amine groups.It results in dipolar interactions between ROL and the support surface, providing less steric hindrance compared with MNPs-ROL.The results showed that the covalent attachment enhanced lipase loading from 36.35% (in MNPs-ROL) to 51.75 wt% (in MNPs-APTES-GA-ROL).The covalent bonding of this lipase resulted in an improved transesterification reaction with the highest conversion (69.8%) among the immobilized biocatalysts, minimized catalyst wasting and maximum recyclability.The formation of covalent bonds promotes lipase rigidity, protects it from leakage, and maintains its activity for longer periods.Palm fatty acid distillate (PFAD) is a by-product of palm oil refining, which can be a promising feedstock for biodiesel production, reducing by 60-70% biofuel cost [174].PFAD is considered a low-grade feedstock and can be used instead of coconut oil as a food ingredient, solving disposal-related problems.Cho et al. studied the non-catalytic esterification process of PFAD and obtained >85% conversion efficiency [175].The produced biodiesel was in accordance with European standards, but the whole process required long times and high temperatures, increasing the production cost.Hidayat et al. studied biodiesel production from PFAD from coconut shells using activated carbon catalysts at 60 °C with a conversion efficiency of 91% [176].
For this type of oil feedstock, biocompatible composites such as carbon-based materials (e.g., activated carbon, graphene oxide and chitosan) can increase the catalytic activity of the lipase.
Buchori et al. reported PFAD-derived biodiesel production using MNPs-APTES-GAlipase impregnated with activated carbon oxide [177].The transesterification reaction was carried out at 56 °C for 6 h, with a 16:1 methanol to PFAD molar ratio.A 94.915% yield for biodiesel production was obtained in such conditions.Biodiesel yield decreased with repeated use of the catalyst after three cycles due to lipase morphological changes.

Silane Functionalizion Providing Epoxy Groups
In 2023, Wang et al. studied biodiesel production from waste oil using the co-immobilization of two different lipases, non-specific Burkholderia cepacia lipase (BCL) and 1,3specific Thermomyces lanuginosus lipase (TLL), on Fe3O4 MNPs functionalized with 3-glycidyloxypropyltrimethoxysilane (3-GPTMS) (co-BCL-TLL@Fe3O4NPs) [178].The 3-GPTMS silane acts as both a stabilizer and coupling agent, linking the magnetic core to the lipases.Fe3O4 NPs were synthesized using the well-known co-precipitation method, which is suitable for large-scale production.In the next step, they were functionalized with 3-GPTMS silane in a typical sol-gel reaction.In detail, -OCH3 groups of the silane were hydrolyzed and condensed with the surface -OH groups of bare MNPs to form a silica network around the magnetic core.More importantly, this silane provided epoxy groups on the surface of functionalized Fe3O4 NPs for the next step, lipase immobilization, which was achieved by a multicomponent reaction between epoxy group, cyclohexyl isocyanate and carboxy residues of lipase, accelerated by nucleophilic attack of isocyanate to epoxy functional groups of modified Fe3O4 NPs.The co-immobilization process was optimized by tuning the main factors affecting the catalytic activity of co-BCL-TLL@Fe3O4NPs in biodiesel production: e.g., amount of carrier (functionalized Fe3O4 NPs), TLL:BCL immobilization ratio and cyclohexyl isocyanate dosage for the coupling reaction.The co-immobilized lipases exhibited an enhanced catalytic activity (92.9% yield) after 6 h, compared with individually immobilized-TLL (63.3% yield), -BCL (74.2% yield) and their combineduse of free forms (70.6%).Under optimal reaction conditions, six various feedstocks were Palm fatty acid distillate (PFAD) is a by-product of palm oil refining, which can be a promising feedstock for biodiesel production, reducing by 60-70% biofuel cost [174].PFAD is considered a low-grade feedstock and can be used instead of coconut oil as a food ingredient, solving disposal-related problems.Cho et al. studied the non-catalytic esterification process of PFAD and obtained >85% conversion efficiency [175].The produced biodiesel was in accordance with European standards, but the whole process required long times and high temperatures, increasing the production cost.Hidayat et al. studied biodiesel production from PFAD from coconut shells using activated carbon catalysts at 60 • C with a conversion efficiency of 91% [176].
For this type of oil feedstock, biocompatible composites such as carbon-based materials (e.g., activated carbon, graphene oxide and chitosan) can increase the catalytic activity of the lipase.
Buchori et al. reported PFAD-derived biodiesel production using MNPs-APTES-GAlipase impregnated with activated carbon oxide [177].The transesterification reaction was carried out at 56 • C for 6 h, with a 16:1 methanol to PFAD molar ratio.A 94.915% yield for biodiesel production was obtained in such conditions.Biodiesel yield decreased with repeated use of the catalyst after three cycles due to lipase morphological changes.

Silane Functionalizion Providing Epoxy Groups
In 2023, Wang et al. studied biodiesel production from waste oil using the coimmobilization of two different lipases, non-specific Burkholderia cepacia lipase (BCL) and 1,3-specific Thermomyces lanuginosus lipase (TLL), on Fe 3 O 4 MNPs functionalized with 3-glycidyloxypropyltrimethoxysilane (3-GPTMS) (co-BCL-TLL@Fe 3 O 4 NPs) [178].The 3-GPTMS silane acts as both a stabilizer and coupling agent, linking the magnetic core to the lipases.Fe 3 O 4 NPs were synthesized using the well-known co-precipitation method, which is suitable for large-scale production.In the next step, they were functionalized with 3-GPTMS silane in a typical sol-gel reaction.In detail, -OCH 3 groups of the silane were hydrolyzed and condensed with the surface -OH groups of bare MNPs to form a silica network around the magnetic core.More importantly, this silane provided epoxy groups on the surface of functionalized Fe 3 O 4 NPs for the next step, lipase immobilization, which was achieved by a multicomponent reaction between epoxy group, cyclohexyl isocyanate and carboxy residues of lipase, accelerated by nucleophilic attack of isocyanate to epoxy functional groups of modified Fe 3 O 4 NPs.The co-immobilization process was optimized by tuning the main factors affecting the catalytic activity of co-BCL-TLL@Fe 3 O 4 NPs in biodiesel production: e.g., amount of carrier (functionalized Fe 3 O 4 NPs), TLL:BCL immobilization ratio and cyclohexyl isocyanate dosage for the coupling reaction.The co-immobilized lipases exhibited an enhanced catalytic activity (92.9% yield) after 6 h, compared with individually immobilized-TLL (63.3% yield), -BCL (74.2% yield) and their combined-use of free forms (70.6%).Under optimal reaction conditions, six various feedstocks were used for biodiesel production, catalyzed by co-BCL-TLL@Fe 3 O 4 NPs, including waste oil (waste cooking oil and rancid oil), edible vegetable oil (soybean oil), animal oil (chicken oil) and non-edible vegetable oil (cottonseed oil and jatropha oil).
co-BCL-TLL@Fe 3 O 4 NPs showed superior catalytic activity (above 90%) for all tested oils after 12 h, compared to the mono-lipase immobilization and combined use of free lipases.Both free lipases showed an oil-dependent catalytic activity, indicating that they possessed different typoselectivity and regioselectivity for the transesterification with triacylglycerols.Furthermore, the recycling operation of co-BCL-TLL@Fe 3 O 4 NPs was tested by rapid magnetic separation, and results showed that this nano-enzyme could maintain 77% of its initial activity after nine cycles.The reusability, high catalytic efficiency and wide substrate adaptability of this co-BCL-TLL@Fe 3 O 4 NPs catalyst introduce it as an economical biocatalyst for further studies on biodiesel production.
In another study, two lipases from Rhizomucor miehei (RML) and Thermomyces lanuginosa (TLL) were immobilized on silica-modified magnetic nanoparticles (Fe 3 O 4 NPs@SiO 2 ) [179].Tetraethoxysilane (TEOS) was used as the silane coupling agent, followed by the introduction of epoxy groups on their surface by using a second silane, 3-glycidyloxypropyl trimethoxysilane.As the third step, the epoxy groups were oxidized to activate the surface and provide aldehyde functionalities, which can react with different nucleophilic amine groups of the lipase surface, leading to its covalent binding and high stability against conformational changes by external stimuli (e.g., pH, heat, organic solvents) (Figure 10).These sequential functionalizations also benefit high loading capacity for the RML (81 mg) and TLL (97 mg) with enhanced thermal stability (54 and 97% initial activity retention at 65 • C for RML and TLL, respectively).The immobilized lipases were used for biodiesel production from waste cooking oil with yields of 93.1% (immobilized TLL) and 57.5% (immobilized RML) under optimal conditions.The results showed good reusability of both nano-catalysts at 50 • C with the retention of 80% (for immobilized RML) and 95% (for immobilized TLL) of the initial catalytic activities after five cycles.This can be a good indication of the covalent binding of lipases onto the support, which prevents enzyme leakage under mechanical stirring during FAME production.
Catalysts 2024, 14, x FOR PEER REVIEW 18 of 36 used for biodiesel production, catalyzed by co-BCL-TLL@Fe3O4 NPs, including waste oil (waste cooking oil and rancid oil), edible vegetable oil (soybean oil), animal oil (chicken oil) and non-edible vegetable oil (cottonseed oil and jatropha oil).co-BCL-TLL@Fe3O4 NPs showed superior catalytic activity (above 90%) for all tested oils after 12 h, compared to the mono-lipase immobilization and combined use of free lipases.Both free lipases showed an oil-dependent catalytic activity, indicating that they possessed different typoselectivity and regioselectivity for the transesterification with triacylglycerols.Furthermore, the recycling operation of co-BCL-TLL@Fe3O4NPs was tested by rapid magnetic separation, and results showed that this nano-enzyme could maintain 77% of its initial activity after nine cycles.The reusability, high catalytic efficiency and wide substrate adaptability of this co-BCL-TLL@Fe3O4NPs catalyst introduce it as an economical biocatalyst for further studies on biodiesel production.
In another study, two lipases from Rhizomucor miehei (RML) and Thermomyces lanuginosa (TLL) were immobilized on silica-modified magnetic nanoparticles (Fe3O4NPs@SiO2) [179].Tetraethoxysilane (TEOS) was used as the silane coupling agent, followed by the introduction of epoxy groups on their surface by using a second silane, 3-glycidyloxypropyl trimethoxysilane.As the third step, the epoxy groups were oxidized to activate the surface and provide aldehyde functionalities, which can react with different nucleophilic amine groups of the lipase surface, leading to its covalent binding and high stability against conformational changes by external stimuli (e.g., pH, heat, organic solvents) (Figure 10).These sequential functionalizations also benefit high loading capacity for the RML (81 mg) and TLL (97 mg) with enhanced thermal stability (54 and 97% initial activity retention at 65 °C for RML and TLL, respectively).The immobilized lipases were used for biodiesel production from waste cooking oil with yields of 93.1% (immobilized TLL) and 57.5% (immobilized RML) under optimal conditions.The results showed good reusability of both nano-catalysts at 50 °C with the retention of 80% (for immobilized RML) and 95% (for immobilized TLL) of the initial catalytic activities after five cycles.This can be a good indication of the covalent binding of lipases onto the support, which prevents enzyme leakage under mechanical stirring during FAME production.11) [180].The presence of TiO 2 and GO layers enhanced the stability of the magnetic core against oxidation and aggregation.Furthermore, GO contains some hydrophilic functional groups which facilitate the accessibility of oil to alcohol to convert FFA into esters.The GO layer also improved the charge transfer via the interface, providing a synergic catalytic activity [181,182].The hydrophobic TiO 2 layer, on the other side, enhanced the immobilization rate of the lipase via hydrophobic interactions [183].Fe 3 O 4 NPs-Ti-GO improved lipase stability, lifetime and catalyst recovery.The authors studied the effect of different parameters on the immobilization efficiency and immobilized lipase activity, including lipase concentration, pH, temperature and time.For lipase immobilization, the optimum condition was found to be: 2 mg/mL of lipase, 4 h immobilization time at pH 7 and 35 • C. Beyond each of these parameters, there is a diminishing trend for both immobilization efficiency and catalytic activity due to the blocking of lipase active sites (increasing lipase concentration) or lipase denaturation (pH and temperature parameters).The authors also evaluated the thermal stability of both free and immobilized lipase in the range of 30-80 • C. The complete inactivation of free lipase was observed at 80 • C; however, the immobilized lipase maintained 64% of its initial activity at the same temperature, attributed to the rigidity of the immobilized lipase skeleton as a result of the binding to the magnetic support.The results also showed an enhanced stability of immobilized lipase in methanolic solutions.The highest biodiesel yield of 92% was observed with 4 wt% of catalyst, the methanol:oil ratio of 5:1, at 45 • C and for 40 h.The reusability of the magnetic nanocatalyst was tested by separation of Fe 3 O 4 NPs-Ti-GO-CALB using a magnet, followed by washing with hexane.Biocatalyst activity was maintained until the 5th cycle and then reduced due to lipase denaturation owing to continuous washing and recycling.

Magnetic Nanocarriers Functionalized with Small Molecules and Other Functionalizing Agents
In 2023, Parandi et al. studied the non-covalent immobilization of Candida antarctica Lipase B (CALB) on the surface of a magnetic nanocomposite containing a Fe3O4NPs core coated with TiO2 and a secondary layer of graphene oxide (GO) to form Fe3O4NPs-Ti-GO (Figure 11) [180].The presence of TiO2 and GO layers enhanced the stability of the magnetic core against oxidation and aggregation.Furthermore, GO contains some hydrophilic functional groups which facilitate the accessibility of oil to alcohol to convert FFA into esters.The GO layer also improved the charge transfer via the interface, providing a synergic catalytic activity [181,182].The hydrophobic TiO2 layer, on the other side, enhanced the immobilization rate of the lipase via hydrophobic interactions [183].Fe3O4NPs-Ti-GO improved lipase stability, lifetime and catalyst recovery.The authors studied the effect of different parameters on the immobilization efficiency and immobilized lipase activity, including lipase concentration, pH, temperature and time.For lipase immobilization, the optimum condition was found to be: 2 mg/mL of lipase, 4 h immobilization time at pH 7 and 35 °C.Beyond each of these parameters, there is a diminishing trend for both immobilization efficiency and catalytic activity due to the blocking of lipase active sites (increasing lipase concentration) or lipase denaturation (pH and temperature parameters).The authors also evaluated the thermal stability of both free and immobilized lipase in the range of 30-80 °C.The complete inactivation of free lipase was observed at 80 °C; however, the immobilized lipase maintained 64% of its initial activity at the same temperature, attributed to the rigidity of the immobilized lipase skeleton as a result of the binding to the magnetic support.The results also showed an enhanced stability of immobilized lipase in methanolic solutions.The highest biodiesel yield of 92% was observed with 4 wt% of catalyst, the methanol:oil ratio of 5:1, at 45 °C and for 40 h.The reusability of the magnetic nanocatalyst was tested by separation of Fe3O4NPs-Ti-GO-CALB using a magnet, followed by washing with hexane.Biocatalyst activity was maintained until the 5th cycle and then reduced due to lipase denaturation owing to continuous washing and recycling.Zhong et al. synthesized a hybrid nanoflower through the self-assembly of Cu3(PO4)2 and loaded it with enzyme molecules, obtaining low diffusion limits and high enzyme loading (Figure 12) [184].In the enzyme-hybrid nanoflowers, the favourable interactions Zhong et al. synthesized a hybrid nanoflower through the self-assembly of Cu 3 (PO 4 ) 2 and loaded it with enzyme molecules, obtaining low diffusion limits and high enzyme loading (Figure 12) [184].In the enzyme-hybrid nanoflowers, the favourable interactions among enzymes and metal ions afford high enzyme activity and stability.The hybrid nanoflowers can be synthesized in mild conditions and are suitable for preparing hybrid nanocatalysts for biodiesel production.Additionally, the combination of specific surfactants can activate lipases that maintain a higher activity even after immobilization.Despite the unique features of hybrid nanoflowers, they have low mechanical properties and reusability due to their fragile nanostructures, which can be improved by their combination with MNPs to achieve magnetic recoverability of the catalyst.To this aim, the authors integrated two types of Fe 3 O 4 NPs into the surfactant-activated lipase hybrid nanoflowers by coprecipitation (Fe 3 O 4 NPs embedded-MhNF) and covalent cross-linking (Fe 3 O 4 NPs-APTES-GA cross-linked-MhNF) to obtain recyclable and magnetic activated lipase-inorganic hybrid nanoflowers (MhNF).In biodiesel production from soybean oil, a dramatic enhancement of activity recovery was observed for both MhNF (190%) and cross-linked-MhNF (174%), compared to low activity recovery of non-magnetic lipase hybrid nanoflowers (77%).Compared with activated free lipase, the MhNF showed high resistance against methanol and longer storage stability; they could be easily recovered by applying a magnet and reused with no significant activity loss even after ten cycles (84% of initial activity), unlike the activated hNF which retained only 26% of their initial activity.In repeated batch biodiesel production, the yield obtained with MhNF catalysis reached 88%, while the free enzyme afforded only 69%.More importantly, the yield of the MhNF-catalyzed process was maintained at 76% even after six cycles.
Catalysts 2024, 14, x FOR PEER REVIEW 20 of 36 among enzymes and metal ions afford high enzyme activity and stability.The hybrid nanoflowers can be synthesized in mild conditions and are suitable for preparing hybrid nanocatalysts for biodiesel production.Additionally, the combination of specific surfactants can activate lipases that maintain a higher activity even after immobilization.Despite the unique features of hybrid nanoflowers, they have low mechanical properties and reusability due to their fragile nanostructures, which can be improved by their combination with MNPs to achieve magnetic recoverability of the catalyst.To this aim, the authors integrated two types of Fe3O4NPs into the surfactant-activated lipase hybrid nanoflowers by co-precipitation (Fe3O4NPs embedded-MhNF) and covalent cross-linking (Fe3O4NPs-APTES-GA cross-linked-MhNF) to obtain recyclable and magnetic activated lipase-inorganic hybrid nanoflowers (MhNF).In biodiesel production from soybean oil, a dramatic enhancement of activity recovery was observed for both MhNF (190%) and cross-linked-MhNF (174%), compared to low activity recovery of non-magnetic lipase hybrid nanoflowers (77%).Compared with activated free lipase, the MhNF showed high resistance against methanol and longer storage stability; they could be easily recovered by applying a magnet and reused with no significant activity loss even after ten cycles (84% of initial activity), unlike the activated hNF which retained only 26% of their initial activity.In repeated batch biodiesel production, the yield obtained with MhNF catalysis reached 88%, while the free enzyme afforded only 69%.More importantly, the yield of the MhNF-catalyzed process was maintained at 76% even after six cycles.As mentioned previously, agricultural wastes are available worldwide, and their exploitation may provide significant economic, social and environmental benefits.Approximately 38-52% of solid wastes in wine industries are grape seeds, which account for 2-5% of the grape weight.Grape seeds generally contain about 10% proteins, 10-20% lipids, 40% fibres, sugars, minerals, complex phenolics and indigestible components (mainly pectins and cellulose).Particularly, grape seed oil comprises high amounts of unsaturated fatty acids, e.g., linoleic acid (72-76% w/w), so this agricultural waste may be considered as a new potential feedstock for large-scale biofuel production.In a first study aimed at exploring the potential of such feedstock, Sarno et al. synthesized Fe3O4NPS/AgNPs made of 9 nm-Fe3O4NPs supporting AgNPs (~6 nm), coated with tartaric acid and used for the electrostatic immobilization of Thermomyces lanuginosus (TLL) lipase [185].The activity recovery and immobilization efficiency of this Fe3O4NPS/AgNPs support have an indirect relationship with the initial enzyme concentration due to the saturation of binding sites of support and diffusion restrictions.With a lipase concentration of 0.1 mg/mL, activity recovery and immobilized efficiency of 92% and ~90% were obtained, respectively, after 3 h.Regarding the AgNPs' function, they facilitate the transfer of electrons (acting as conduction centres), inducing enzymes to adopt an advantageous alignment, leading to high conversion yields (94%).The analysis of the biodiesel showed that linoleic acid methyl ester and oleic acid methyl ester account for ~89% of the total biodiesel, consistent with the oil composition.Using an oil:methanol ratio of 1:6, lipase concentration of 10%, and As mentioned previously, agricultural wastes are available worldwide, and their exploitation may provide significant economic, social and environmental benefits.Approximately 38-52% of solid wastes in wine industries are grape seeds, which account for 2-5% of the grape weight.Grape seeds generally contain about 10% proteins, 10-20% lipids, 40% fibres, sugars, minerals, complex phenolics and indigestible components (mainly pectins and cellulose).Particularly, grape seed oil comprises high amounts of unsaturated fatty acids, e.g., linoleic acid (72-76% w/w), so this agricultural waste may be considered as a new potential feedstock for large-scale biofuel production.In a first study aimed at exploring the potential of such feedstock, Sarno et al. synthesized Fe 3 O 4 NP S /AgNPs made of 9 nm-Fe 3 O 4 NPs supporting AgNPs (~6 nm), coated with tartaric acid and used for the electrostatic immobilization of Thermomyces lanuginosus (TLL) lipase [185].The activity recovery and immobilization efficiency of this Fe 3 O 4 NPS/AgNPs support have an indirect relationship with the initial enzyme concentration due to the saturation of binding sites of support and diffusion restrictions.With a lipase concentration of 0.1 mg/mL, activity recovery and immobilized efficiency of 92% and ~90% were obtained, respectively, after 3 h.Regarding the AgNPs' function, they facilitate the transfer of electrons (acting as conduction centres), inducing enzymes to adopt an advantageous alignment, leading to high conversion yields (94%).The analysis of the biodiesel showed that linoleic acid methyl ester and oleic acid methyl ester account for ~89% of the total biodiesel, consistent with the oil composition.Using an oil:methanol ratio of 1:6, lipase concentration of 10%, and 45 • C for 24 h, a total FAME of 96.54 ± 0.26% was obtained, in accordance with the requirements reported in European EN14124 standard for biodiesel [186].
Sarno et al. reported the synthesis of MNPs-tartaric acid (TA) through a solvothermal method and subsequently used for direct physical immobilization of Thermomyces lanuginous (TLL) lipase (via interaction between support functional groups and the enzyme) [187].MNPs-tartaric acid-TLL was used for biodiesel production from DWSO in a solvent-free system.With an alcohol/oil molar ratio of 6:1, the immobilized lipase afforded a 90% yield (with excellent reusability), higher than the yield of free lipase (76% with a molar ratio of 3:1).DWSO-derived biodiesel presented a total FAMEs content of about 96.6 ± 0.02% with a linolenic methyl ester content of 0.62 ± 0.07%, in agreement with the EN14214 standard, indicating the feasibility of DWSO as feedstock for biodiesel production.
In 2020, Sarno et al. studied biodiesel production from WCO using Thermomyces lanuginosus (TL) lipase (E.C.3.1.1.3)directly immobilized on citric acid/oleic acid modified Fe 3 O 4 /Au nanocomposite, without requiring complex procedures and reagents, through physical interactions [188].The snowman-like Fe 3 O 4 /Au nanocomposite was synthesized by a hydrothermal reaction of Fe(acac) 3 , HauCl 4 and oleic acid precursors at high temperatures (>200 • C) for 2 h, followed by a ligand exchange of oleic acid with citric acid at room temperature, to obtain hydrophilic Fe 3 O 4 /Au nanocomposite.This immobilized lipase afforded a remarkable biodiesel yield of ∼90% without any pre-treatment under the selected conditions (lipase concentration of 20%, 45 • C, 24 h, 1:6 oil/methanol molar ratio), maintaining its catalytic performance above 74% after the first three cycles of use.The produced biodiesel was analyzed according to the European biodiesel specification, and the results obtained (ester content of 97.8 ± 0.21% and a linolenic methyl ester content of 0.53 ± 0.03%) were in agreement with the EN14214 requirements, demonstrating the feasibility of WCOs-derived biodiesel as a fuel.Regarding the role of Fe 3 O 4 /Au nanocomposite, they reported that the citric acid and the presence of residual oleic acid help the lipase to expose its polar tail to the medium.This not only favours interfacial activation but also good activity in the presence of water.The comparison between different AuNP sizes containing Fe 3 O 4 /Au highlights that AuNPs help favourable lipase orientation and thus increase enzyme loading and activity.
In 2020, Li et al. selected Thermomyces lanuginosus lipase (TLL) due to its relatively low cost and covalently immobilized it onto Fe 3 O 4 NPs, obtaining a magnetic responsive biocatalyst [189].Aqueous monodisperse Fe 3 O 4 NPs with surface carboxyl group were synthesized by thermal decomposition of iron carboxylate salts.These carboxylate functional groups were activated using EDC/NHS coupling agents.Then, TLL amine groups were covalently linked with MNPs-activated groups to form amide bonds.The effects of pH and temperature on the enzymatic activity of both Fe 3 O 4 NPs-TLL and free TLL were studied.The same optimal pH (8.5) and temperature (40 • C) values were obtained for both free and immobilized TLL, but the latter showed a higher tolerance to both pH and temperature variations [190,191].The improved tolerance of immobilized lipase might be due to a stabilization of the covalent immobilization.The optimized parameters for biodiesel synthesis were the following: methanol:oil molar ratio = 4.0, water content = 1.5% of oil weight, immobilized lipase to oil = 9.0% (w/w) at 41 • C for 28 h, which resulted in an 82.20% yield.The immobilized biocatalyst could be recycled and reused up to 10 cycles with a 10.97% yield decrease.

Magnetic Nanocarriers Functionalized with Polymers
Chitosan (CS) is another biocompatible and cost-effective functionalizing molecule for MNPs to enhance lipase loading and prevent the surface oxidation of the magnetic core [192,193].The polycationic CS can form microaggregates in the presence of multivalent anions, such as phosphate or citrate, to mediate the self-assembly of anionic MNPs and form a surrounding shell.Then, this template can be disassembled, leaving a microcapsule structure suitable for the encapsulation of different negatively charged lipases by electrostatic interaction with the polycationic chains of CS [194,195].The microcapsule structure protects the confined lipases against the external environment, simultaneously allowing the transport of substrates and products across the shell [196].For instance, Wei et al. developed a one-pot, cost-effective and fast co-immobilization of Aspergillus oryzae (AOL) and Rhizomucor miehei (RML) lipases in magnetic chitosan microcapsules to obtain highly efficient biocatalysts for WCO (waste cooking oil)-derived biodiesel production (Figure 13) [197].For heterogeneous oils such as WCO, the combination or co-immobilization of different lipases with various selectivities is considered an efficient strategy for increasing biodiesel yield.In previous studies, AOL exhibited high resistance to FFA and methanol, while RML showed improved acyl migration efficiency and prominent tolerance under anhydrous conditions, showing selectivity for oleic/elainic acids-containing substrates, which are present in WCOs.
zation of different lipases with various selectivities is considered an efficient strategy for increasing biodiesel yield.In previous studies, AOL exhibited high resistance to FFA and methanol, while RML showed improved acyl migration efficiency and prominent tolerance under anhydrous conditions, showing selectivity for oleic/elainic acids-containing substrates, which are present in WCOs.
Lipase encapsulation was performed by self-assembly of negatively-charged Fe3O4NPs-citrate onto cationic cross-linked chitosan-citrate aggregates containing lipases.The co-immobilization of AOL@RML achieved a dramatic increase in biodiesel yield under optimum conditions (up to 98.5%).The presence of thermally stable RML and methanol-tolerant AOL in a single nanoplatform provided a synergic catalytic performance under excess methanol and variable temperatures, which was superior to the individual lipase counterparts.The co-immobilized AOL@RML maintained 96% of its initial activity after three cycles without inactivation.This research was introduced as the first study of the synergic effect between co-immobilized RML and AOL for WCO-derived biodiesel production, and this easy and cost-effective biocatalyst preparation suggests a promising pathway for the preparation of highly efficient industrial co-immobilized lipases.AOL: free Aspergillus oryzae lipase; RML: free Rhizomucor miehei lipase; Tween-AOL: Aspergillus oryzae lipase bio-imprinted using Tween 80; Tween-RML: Rhizomucor miehei lipase bio-imprinted using Tween 80; co-im Tween-AOL@RML: co-immobilized Tween-AOL and Tween-RML).Adapted from Ref. [197], copyright 2022, with permission from Elsevier.
Both natural and synthetic polymers have been used as coatings for MNPs, providing active surface functionalities, such as epoxy and amine groups, suitable for lipase immobilization through interaction with amine groups.Despite the popularity of this type of surface modification, it still requires the activation of amine groups using aldehydes (e.g., glutaraldehyde), which adds operational costs and time to the whole process, prompting the search for alternative strategies.
As a versatile coating for MNPs, polydopamine contains both amine and catechol groups, which efficiently modify the surface of NPs and enhance the colloidal stability [198,199].For one-step surface modification of MNPs with polydopamine, a common method is alkaline self-polymerization of dopamine in the presence of MNPs, in which the catechol group of dopamine converts into the indo-5, 6-quinone, followed by a series of inter/intra molecular reactions to form a polydopamine layer on MNPs surface.After polymerization, there will be residual catechol and quinone surface groups, which can react with nucleophiles such as amine and thiol groups through Schiff base formation and Michael addition reaction.AOL: free Aspergillus oryzae lipase; RML: free Rhizomucor miehei lipase; Tween-AOL: Aspergillus oryzae lipase bio-imprinted using Tween 80; Tween-RML: Rhizomucor miehei lipase bio-imprinted using Tween 80; co-im Tween-AOL@RML: co-immobilized Tween-AOL and Tween-RML).Adapted from Ref. [197], copyright 2022, with permission from Elsevier.
Lipase encapsulation was performed by self-assembly of negatively-charged Fe 3 O 4 NPscitrate onto cationic cross-linked chitosan-citrate aggregates containing lipases.The coimmobilization of AOL@RML achieved a dramatic increase in biodiesel yield under optimum conditions (up to 98.5%).The presence of thermally stable RML and methanol-tolerant AOL in a single nanoplatform provided a synergic catalytic performance under excess methanol and variable temperatures, which was superior to the individual lipase counterparts.The co-immobilized AOL@RML maintained 96% of its initial activity after three cycles without inactivation.This research was introduced as the first study of the synergic effect between co-immobilized RML and AOL for WCO-derived biodiesel production, and this easy and cost-effective biocatalyst preparation suggests a promising pathway for the preparation of highly efficient industrial co-immobilized lipases.
Both natural and synthetic polymers have been used as coatings for MNPs, providing active surface functionalities, such as epoxy and amine groups, suitable for lipase immobilization through interaction with amine groups.Despite the popularity of this type of surface modification, it still requires the activation of amine groups using aldehydes (e.g., glutaraldehyde), which adds operational costs and time to the whole process, prompting the search for alternative strategies.
As a versatile coating for MNPs, polydopamine contains both amine and catechol groups, which efficiently modify the surface of NPs and enhance the colloidal stability [198,199].For one-step surface modification of MNPs with polydopamine, a common method is alkaline self-polymerization of dopamine in the presence of MNPs, in which the catechol group of dopamine converts into the indo-5, 6-quinone, followed by a series of inter/intra molecular reactions to form a polydopamine layer on MNPs surface.After polymerization, there will be residual catechol and quinone surface groups, which can react with nucleophiles such as amine and thiol groups through Schiff base formation and Michael addition reaction.
Touqeer et al. synthesized Fe 3 O 4 NPs by solvothermal method and functionalized them with polydopamine through the self-polymerization of dopamine in alkaline conditions in the presence of MNPs [200].The thiol and amine nucleophilic groups of Aspergillus terreus AH-F2 lipase reacted with the surface quinone and catechol groups of Fe 3 O 4 NPs-PDA by Michael addition and Schiff base mechanisms without the need for any activating or coupling agents (Figure 14).The synthesized (Fe 3 O 4 NPs-PDA-Lipase) biocatalyst was used for biodiesel preparation from WCO, achieving a maximum activity of 17.82 U/mg/min (equal to 97.27% of free lipase activity.Moreover, the immobilized biocatalyst revealed better tolerance to higher temperatures/pHs compared with the free lipase.Under optimum conditions (10% biocatalyst percentage concentration, 6:1 CH 3 OH:oil ratio and 0.6% water content, at 37 • C and 30 h), the highest yield of 92% was achieved and maintained for the first four cycles, then dropping to 25.79% after seven cycles.The properties of the produced biofuel were in agreement with the ASTM D standard, qualifying this system as a cost-effective and sustainable biocatalyst for biodiesel production.
Catalysts 2024, 14, x FOR PEER REVIEW 23 of 36 Touqeer et al. synthesized Fe3O4NPs by solvothermal method and functionalized them with polydopamine through the self-polymerization of dopamine in alkaline conditions in the presence of MNPs [200].The thiol and amine nucleophilic groups of Aspergillus terreus AH-F2 lipase reacted with the surface quinone and catechol groups of Fe3O4NPs-PDA by Michael addition and Schiff base mechanisms without the need for any activating or coupling agents (Figure 14).The synthesized (Fe3O4NPs-PDA-Lipase) biocatalyst was used for biodiesel preparation from WCO, achieving a maximum activity of 17.82 U/mg/min (equal to 97.27% of free lipase activity.Moreover, the immobilized biocatalyst revealed better tolerance to higher temperatures/pHs compared with the free lipase.Under optimum conditions (10% biocatalyst percentage concentration, 6:1 CH3OH:oil ratio and 0.6% water content, at 37 °C and 30 h), the highest yield of 92% was achieved and maintained for the first four cycles, then dropping to 25.79% after seven cycles.The properties of the produced biofuel were in agreement with the ASTM D standard, qualifying this system as a cost-effective and sustainable biocatalyst for biodiesel production.Waste chicken fat is another alternative feedstock for biodiesel production due to its high-fat content (about 10% w/w) and low cost.Commercial broiler chicken meat has a relatively higher polyunsaturated lipid content than organic chicken, and it is known that chicken fat comprises about 40-75% unsaturated-and 25-35% saturated fatty acids [200].Oleic acid, stearic acid, palmitic acid and linoleic acid are the main fatty acids in chicken fat [201].Regarding the biocatalytic transesterification of chicken fat oil, there are few reports; for instance, Shafiq et al. used waste chicken fat oil for biodiesel production through both enzymatic and chemical (using KOH and sodium methoxide) approaches [202].The process of enzymatic transesterification was performed by using free and immobilized forms of Aspergillus terreus lipase on Fe3O4NPs-PDA-Lipase.Under the reaction conditions of methanol:oil ratio of 6:1, 42 °C for 36 h, the maximum biodiesel yield (90.6%) was achieved from waste chicken fat oil by 6% w/w Fe3O4NPs-PDA-Lipase nano-biocatalyst.The measured fuel properties (kinematic viscosity, flash point, pour point, cloud point, fire point) met ASTM standard biodiesel specifications, suggesting the compatibility of waste chicken fat with biodiesel production.
Regarding the non-silane-based approach for the chemical attachment of lipase on MNPs, Xie et al. coated Fe3O4NPs with a synthetic poly(glycidyl methacrylate-co-methacrylic acid) in an in situ reaction, providing reactive epoxy and carbonyl groups on the surface of NPs [203].Then, Candida rugosa lipase was immobilized onto the magnetic core through coupling reactions between the functional groups of the modified MNPs and lipase amine groups via EDC/NHS chemistry (1-ethyl-3-(3-dimethylaminopropyl)car- Waste chicken fat is another alternative feedstock for biodiesel production due to its high-fat content (about 10% w/w) and low cost.Commercial broiler chicken meat has a relatively higher polyunsaturated lipid content than organic chicken, and it is known that chicken fat comprises about 40-75% unsaturated-and 25-35% saturated fatty acids [200].Oleic acid, stearic acid, palmitic acid and linoleic acid are the main fatty acids in chicken fat [201].Regarding the biocatalytic transesterification of chicken fat oil, there are few reports; for instance, Shafiq et al. used waste chicken fat oil for biodiesel production through both enzymatic and chemical (using KOH and sodium methoxide) approaches [202].The process of enzymatic transesterification was performed by using free and immobilized forms of Aspergillus terreus lipase on Fe 3 O 4 NPs-PDA-Lipase.Under the reaction conditions of methanol:oil ratio of 6:1, 42 • C for 36 h, the maximum biodiesel yield (90.6%) was achieved from waste chicken fat oil by 6% w/w Fe 3 O 4 NPs-PDA-Lipase nano-biocatalyst.The measured fuel properties (kinematic viscosity, flash point, pour point, cloud point, fire point) met ASTM standard biodiesel specifications, suggesting the compatibility of waste chicken fat with biodiesel production.
Regarding the non-silane-based approach for the chemical attachment of lipase on MNPs, Xie et al. coated Fe 3 O 4 NPs with a synthetic poly(glycidyl methacrylate-co-methacrylic acid) in an in situ reaction, providing reactive epoxy and carbonyl groups on the surface of NPs [203].Then, Candida rugosa lipase was immobilized onto the magnetic core through coupling reactions between the functional groups of the modified MNPs and lipase amine groups via EDC/NHS chemistry (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/ hydroxysulfosuccinimide) (Figure 15).This covalent attachment led to increased stability of the immobilized lipase by preventing its leakage and reducing its conformational changes [204,205].As mentioned in Section 2.1, unmodified MNPs aggregate easily due to their high surface energy and their attractive magneto-dipole forces, so the authors encapsulated MNPs into a synthetic copolymer as a reliable strategy to improve their long-term colloidal stability.They selected glycidyl methacrylate (GMA) and methacrylic acid (MAA) monomers for their in situ co-polymerization onto MNPs because they provide both epoxy and carboxylate groups on the surface of modified MNPs, resulting in multipoint covalent attachments between the lipase and the nano-carrier [206].Before the co-polymerization step, the authors synthesized MNPs through a solvothermal method, using sodium polyacrylate (PAAS), FeCl 3 •6H 2 O, urea and sodium citrate precursors in a two-step reaction.The use of urea and citrate in the synthesis of Fe 3 O 4 NPs provided surface hydroxyl and carboxyl groups, which can form hydrogen bonds with the functional groups of the co-polymer to encapsulate the magnetic core into the polymeric layer.Candida rugosa lipase can form covalent bonds with such groups, including (1) the carboxylic groups of the modified-Fe 3 O 4 NPs, activated by EDC/NHS reagents to convert the surface carboxylic groups to active NHS esters, which can react with the primary amine of lipase forming amide bonds, as shown in Figure 15, (2) the epoxy groups also can take part in nucleophilic substitution reactions with the N-terminal amino groups of the lipase.This dual modality of covalent attachment guarantees the stability of the immobilized lipase, preventing its leakage.The authors also tested the magnetic support (without immobilized lipase), as well as Fe 3 O 4 NPs-immobilized lipase in biodiesel production from soybean oil.The Fe 3 O 4 -poly(GMA-co-MAA) displayed no catalytic activity, whereas the free lipase afforded a biodiesel yield of 91.8%, obtained by the three-step addition of methanol at 40 • C. On the other hand, the use of Fe 3 O 4 -poly(GMA-co-MAA)-lipase not only maintained enzymatic activity in the transesterification reaction (yield of 92.8%) but also showed good long-term stability.Thanks to the magnetic carrier, the immobilized lipase was recovered and reused for five cycles, still retaining 79.4% of biodiesel yield.
Catalysts 2024, 14, x FOR PEER REVIEW 24 of 36 bodiimide/hydroxysulfosuccinimide) (Figure 15).This covalent attachment led to increased stability of the immobilized lipase by preventing its leakage and reducing its conformational changes [204,205].As mentioned in Section 2.1, unmodified MNPs aggregate easily due to their high surface energy and their attractive magneto-dipole forces, so the authors encapsulated MNPs into a synthetic copolymer as a reliable strategy to improve their long-term colloidal stability.They selected glycidyl methacrylate (GMA) and methacrylic acid (MAA) monomers for their in situ co-polymerization onto MNPs because they provide both epoxy and carboxylate groups on the surface of modified MNPs, resulting in multipoint covalent attachments between the lipase and the nano-carrier [206].Before the co-polymerization step, the authors synthesized MNPs through a solvothermal method, using sodium polyacrylate (PAAS), FeCl3•6H2O, urea and sodium citrate precursors in a two-step reaction.The use of urea and citrate in the synthesis of Fe3O4NPs provided surface hydroxyl and carboxyl groups, which can form hydrogen bonds with the functional groups of the co-polymer to encapsulate the magnetic core into the polymeric layer.Candida rugosa lipase can form covalent bonds with such groups, including (1) the carboxylic groups of the modified-Fe3O4NPs, activated by EDC/NHS reagents to convert the surface carboxylic groups to active NHS esters, which can react with the primary amine of lipase forming amide bonds, as shown in Figure 15, (2) the epoxy groups also can take part in nucleophilic substitution reactions with the N-terminal amino groups of the lipase.This dual modality of covalent attachment guarantees the stability of the immobilized lipase, preventing its leakage.The authors also tested the magnetic support (without immobilized lipase), as well as Fe3O4NPs-immobilized lipase in biodiesel production from soybean oil.The Fe3O4-poly(GMA-co-MAA) displayed no catalytic activity, whereas the free lipase afforded a biodiesel yield of 91.8%, obtained by the three-step addition of methanol at 40 °C.On the other hand, the use of Fe3O4-poly(GMA-co-MAA)lipase not only maintained enzymatic activity in the transesterification reaction (yield of 92.8%) but also showed good long-term stability.Thanks to the magnetic carrier, the immobilized lipase was recovered and reused for five cycles, still retaining 79.4% of biodiesel yield.Table 4 shows other magnetic nano-supports for lipase immobilization used in biodiesel production.A few real-world applications of lipases immobilized on magnetic nanosupports for biodiesel production have been reported [219].These feature enzyme immobilization via glutaraldehyde crosslinking and provide high yields as well as excellent reusability, paving the way for further future industrial applications.

Conclusions: Current Challenges and Future Trends
Nano-biocatalysts have attracted a growing interest in the field of biodiesel production due to the rapid development of both lipase-and nano-technologies.Such interest will undoubtedly continue to grow; however, these technologies still require improvements in the preparation of robust nano-biocatalysts to ensure higher enzyme activity and stability on the nano-surface.Enzyme immobilization via physical methods (i.e., physical adsorption) benefits from maintaining lipase conformation but is prone to lipase desorption, affected by external stimuli such as pH, temperature, light and mechanical forces.Conversely, with chemical attachment, the enzyme is irreversibly linked to the support, although the altered enzymatic structure may affect catalytic properties.So, it is vitally required to find a good balance among the different structural and biological properties of nanobased immobilization systems while having the highest economic efficiency.To date, most research on biocatalyzed biodiesel preparation has been performed on a bench scale with outstanding results; however, there are few reports on the scale-up of biocatalyzed biodiesel production.Thus, the transfer of knowledge from lab to industry is considered another important challenge in this field.The development of engineered reactors is expected to provide promising advancements in large-scale biodiesel production.Optimizing the structure-function interactions of nano-immobilized lipases and lowering the operational costs of their manufacturing is of profound relevance to achieving long-term sustainability and development.
Feedstocks are another important factor for the advancement of biodiesel technology.As the major feedstock of biodiesel (95%), vegetable oils need agricultural resources (e.g., water, land, fertilizers) and indirectly increase biodiesel carbon emissions due to their life cycle.Furthermore, vegetable oil accounts for 75-80% of biodiesel production costs.Research should, therefore, be oriented towards the use of cost-effective and regionally available feedstocks in order to address energy security and economic viability challenges.Waste cooking oils (WCO) are easily accessible raw materials with an increasing production trend due to the global development of the catering trade.Furthermore, using WCO for biodiesel production prevents relevant environmental pollution caused by their disposal.Due to their local availability, WCO may replace part of the current feedstocks based on vegetable oils.Further, other waste oils, such as dairy waste scum and waste chicken fat oil, can also be considered a potential feedstock for biodiesel production, in perfect agreement with the principles of a circular economy.
The interference of alcohol with lipase activity can be solved by using solvent-free systems (e.g., ionic liquids) that also allow the avoidance of large amounts of organic solvents.Like any other technology, lab-scale research in this field is developing faster than large-scale manufacturing, and, with an organized and multidisciplinary collaboration between industry and academy, the bench-scale knowledge can be transferred to industry, for example by developing engineered reactors providing high operational speed and efficiency to maximize the production yield and minimize the cost of the process.
Although there have been admirable efforts to develop efficient and robust nanobiocatalysts, the involvement of conformational changes in the immobilization process, nanoparticles-enzyme binding sites, and structure-function relationships still require to be fully addressed.From a perspective point of view, magnetic immobilization can be a successful strategy for the industrial development of biodiesel technology.However, there are still some issues that need to be overcome.Although the reusability allowed by these materials is one of their most appealing advantages, the complexity of the synthesis and subsequent lipase immobilization process still requires research and optimization.To date, several reactors have been developed to enhance the possibility of using magnetic biocatalysts in large-scale applications.Companies working in the field have shown a growing interest in these smart materials, with an increasing number of new patents year by year.Future perspectives related to magnetic biocatalysts are connected to developing immobilization technologies to guarantee the stability and catalytic activity of the enzyme while resulting in low production costs.The evolving regulations on the use of fossil fuels will most probably influence the market, making biodiesel more sought after.However, in order to become more advantageous than chemical catalysis, enzymatic catalysis for biodiesel production will necessarily require further research and technological evolutions.

Figure 6 .
Figure 6.Schematic routes for the preparation of Fe 3 O 4 NPs@TEOS-TSD nanocomposite and immobilization of CALB onto the magnetic support.Adapted from Ref. [156], copyright (2021), with permission from Elsevier.

Figure 8 .
Figure 8. Schematic representation of the cyclic process for the biocatalytic synthesis of biodiesel in ILs.Adapted from Ref. [164], copyright (2022), with permission from Springer.

Figure 9 .
Figure 9. Schematic routes for the preparation of biocatalysts to produce biodiesel.Redrawn from Ref. [173], copyright 2020, with permission from Elsevier.

3. 4 .
Magnetic Nanocarriers Functionalized with Non-Silane Linkers 3.4.1.Magnetic Nanocarriers Functionalized with Small Molecules and Other Functionalizing Agents In 2023, Parandi et al. studied the non-covalent immobilization of Candida antarctica Lipase B (CALB) on the surface of a magnetic nanocomposite containing a Fe 3 O 4 NPs core coated with TiO 2 and a secondary layer of graphene oxide (GO) to form Fe 3 O 4 NPs-Ti-GO (Figure

Figure 14 .
Figure 14.Schematic representation of lipase immobilization on dopamine-coated MNPs.Redrawn from Ref. [200] under the terms and conditions of the Creative Commons Attribution (CC BY) license, MDPI.

Figure 14 .
Figure 14.Schematic representation of lipase immobilization on dopamine-coated MNPs.Redrawn from Ref. [200] under the terms and conditions of the Creative Commons Attribution (CC BY) license, MDPI.
* OECD: Organization for Economic Cooperation and Development.

Table 3 .
[150]ds for the synthesis of MNPs, their advantages and disadvantages.Reprinted with permission from an open-access article distributed under the terms and conditions of the Creative Commons Attribution license, MDPI[150].

Table 4 .
Other magnetic nano-supports for lipase immobilization used in biodiesel production.