Global fossil fuel consumption has increased over the years. An increase in fossil fuel consumption by 5.8% in 2021 was reported to account for 82% of the primary energy consumption [1]. Therefore, the depletion of this finite energy resource is alarming. Moreover, the increased combustion of fossil fuels has caused a startling increase in greenhouse gas emissions. These challenges have driven governments, environmentalists, and scientists to seek sources of renewable and sustainable fuels. Biodiesel produced from microalgae is a promising alternative that has been proposed to meet this global challenge [2]. The ability of many microalgal strains to grow in saline water can reduce the freshwater demand. Additionally, microalgae can utilize atmospheric CO2, which can further help in mitigating the rising CO2 levels [3]. In addition, microalgae can grow under harsh conditions and produce up to 10 times more oil than other oil crops [4]. This makes microalgae the preferred feedstock for biodiesel production. There are approximately 300,000 microalgal strains [5], and among them, Chlorella sp. has been the most studied [4] owing to their high lipid productivity of 33–45 mg/L/d [6].

The microalgae-to-biodiesel process is a multi-step process consisting of cultivation, harvesting, drying, cell disruption, lipid extraction, transesterification, and product separation [7]. Drying and cell disruption are essential for the efficient operation of conventional oil extraction processes. However, these steps are time-consuming and/or energy-intensive, accounting for almost 90% of the overall energy required for all the process [8], [9], [10]. Therefore, several recent studies have focused on eliminating these steps.

Conventional cell disruption methods include bead milling, high-pressure homogenization, ultrasonication, and microwave treatment [9]. Recently, it has been reported that efficient cell disruption can be achieved using hydrophilic ionic liquids. However, these solvents require the use of an additional separation unit before extraction, which increases the total overall process cost.

Biodiesel is formed through transesterification, in which triglycerides react in the presence of a catalyst with short-chain alcohols to form fatty acid methyl esters (FAMEs) and glycerol as a by-product [10]. Recently, there has been a shift towards the use of biocatalysts because they can overcome the challenges involved with conventional chemical catalysts, and particularly mitigates soap formation and high energy demand [2].

Lipases are enzymes that can be found in bacteria, fungi, and yeast, as well as in the pancreas of pigs and in plants, such as castor bean and rapeseed [11]. The preferable lipases for industrial applications are those from microbial origin, owing to the ease of their manipulation for better yields and purity [12]. Lipases belong to the triacylglycerol ester hydrolase family, and can catalyze the hydrolysis and transesterification of long-chain triglycerides [13]. The lipase molecule has two main regions; a larger amino-terminal domain and a smaller carboxy-terminal domain that are linked by a flexible peptide. It is made up of the catalytic triad, an oxyanion hole, and a polypeptide chain (like a lid). The catalytic triad is primarily composed of nucleophilic serine, glutamic, or aspartic acid and histidine residues that catalyze the reaction. The hole serves primarily to stabilize the carbonyl group of the substrate during intermediate reactions, whereas the lid protects the enzyme active site [14]. The water-soluble lipase acts on hydrophobic oil substrates, in which a configuration change in the enzyme molecules occur at the water/oil interface. In this change, also referred to as interfacial activation, the amphiphilic peptidic loop covering the active sites of the enzyme is unfolded, rendering the active sites accessible to the substrate [15].

To allow the repeated use of enzymes and enhance their stability, enzymes must be used in an immobilized form [11], [12]. In immobilization, the enzyme is attached to a surface, while maintaining its catalytic activity [18]. The most commonly used supports include silica-based carriers, acrylic resins, synthetic polymers, silica gels, microporous resins, celite, and zeolite porous kaolinite [14], [15]. Immobilization is achieved through physical adsorption, covalent boning, encapsulation, or crosslinking [21]. In encapsulation, the enzyme molecules are trapped inside the support matrix during synthesis, unlike other techniques, which are all post-synthesis attachments. This adds stability to the enzyme, but simultaneously increases the mass transfer limitation of the reactants and products [21]. To overcome this problem, Metal Organic Frameworks (MOFs), which are characterized by their high porosity have been proposed in this work. To allow encapsulation of the enzyme, a MOF that cures at temperatures lower than that of the denaturation of the enzyme need to be used. In that regard, ZIF-8, which is prepared at room temperatures has been selected in this work. This would allow encapsulation of the enzyme, coupled within a matrix known for its high surface area and high porosity[22], [23], [24]. In our previous study [24], we successfully used lipase immobilized via surface attachment and encapsulation on a zeolitic imidazole framework (ZIF)-8 for biodiesel production. Our study showed that the adsorbed system produced a higher FAME yield in the first cycle, which was approximately 58%, compared to only 10% from the encapsulated system, at 40 ℃ and a methanol-to-oil ratio of 6:1. The reusability of the two immobilized enzyme systems was tested, and the encapsulated system had maintained its residual activity by up to 83% after five cycles, compared to the 34% residual activity in the adsorbed system [24]. The higher activity of the adsorbed system was due to the easier accessibility of the surface-attached enzyme, compared to the encapsulated system. In contrast, the lesser stability of the adsorbed system was due to its susceptibility to leaching.

Recently, in situ microalgae-to-biodiesel transesterification from wet microalgal paste has gained considerable interest. In this process, the cost-intensive drying step was eliminated and cell disruption-oil extraction-transesterification was performed in a single pot, simplifying the overall process. Non-catalyzed in situ direct transesterification was successfully achieved using supercritical methanol and ethanol from wet biomass. A biodiesel yield of 46% was achieved for supercritical methanol used with wet Nannochloropsis sp. (containing 75% moisture) at a methanol-to-dry algae ratio of 10:1 (v/w %), temperature of 255–265 ℃, and pressure of 21 MPa, [25]. A better yield (64.4%) was obtained using supercritical ethanol from Chlorella pyrenoidosa, at 350 ℃ with an ethanol-to-microalgae ratio of 24:5 [26],[26]. Despite the desirable results, operations under these conditions are energy-intensive, rendering the overall process uneconomical. Another interesting approach involves a simultaneous extraction process using immobilized lipase as a catalyst and supercritical (SC)-CO2 as the extraction solvent. The process was tested on Scendesmus sp. at 35 ℃, a methanol-to-oil molar ratio of 8:1, and a pressure of 40 MPa. A biodiesel conversion of 19.3% was achieved [27]. It should be noted, however, that in this study, lyophilized biomass was used and not untreated wet biomass. In addition, despite operating at low temperatures, owing to the low critical temperature of CO2, the high critical pressure required eventually results in a high energy input.

CO2-triggered switchable solvents (SSs) switch their hydrophilicity by changing their conditions. By bubbling with CO2, the solvents become hydrophilic, and when CO2 is removed by purging with N2, they return to their original hydrophobic form [28]. In their hydrophilic state, SSs can disrupt microalgae cell walls, while in their hydrophobic form, they can dissolve and extract the oil. SSs like n,n-dimethylcyclohexylamine (DMCHA) and n-ethylbutylamine (EBA) have been used for simultaneous cell disruption and oil extraction from wet, untreated microalgal paste. Using these two solvents for 1 h for each of the cell disruption, extraction/reaction, and product separation steps, biodiesel yields of 47.5 and 25.4% were achieved using DMCHA and EBA, respectively [28]. Although these results are promising, the use of gases and the need for a reflux condenser to minimize methanol evaporation complicates the overall process [28]. To simplify this process, CO2-triggered SSs were replaced with thermoresponsive switchable solvents (TSSs), which alter their hydrophobicity based on the temperature. TSS was prepared by mixing an IL (n,n-diethyl-n-methylammonium methane sulfonate) with polypropylene glycol (PPG) 400 and water. The solvent was hydrophilic at 25 ℃ and became hydrophobic at temperatures above 45 ℃. TSS was tested on wet, untreated Chlorella Sp paste for the production of biodiesel using the commercially available enzyme Novozyme 435. The solvent was maintained at its hydrophilic state for 1.5 h to disrupt the cell wall and then changed to its hydrophobic state by raising the temperature to 45 ℃. Then 0.1 mL methanol and the enzyme were added to start the reaction, which was left undisturbed for 1.5 h. The solvent was then changed back to its hydrophilic form to separate the product. Under these conditions, a biodiesel yield of 50.5% was obtained. The feasibility of this process was affected by the reusability of the enzyme-TSS system, which measures the overall stability and difficulty in recovery [29]. In our previous study, a significant decrease in the enzyme activity with its repeated reuse was noticed, wherein the biodiesel yield dropped to 20% in the fourth cycle [30]. This decrease was attributed to the leaching of the enzyme from the support through its exposure to the TSS system. Deposition of glycerol, a by-product, on the support was also suggested as a reason for the loss of activity [30]. Unless the reusability is improved, commercialization of the process will not be possible.

The use TSS with immobilized lipase for the simultaneous cell wall disruption, oils extraction-reaction and product separation process from wet, undisrupted microalgae biomass is a promising process that results in significant simplification of the microalgae-to-biodiesel process. However, the low stability of commercially available immobilized lipases remains the main disadvantage that hinders the commercialization of the process. The main objective of this study is to evaluate the use of ZIF-8 as an alternative support for lipase encapsulation for enhanced activity and stability. The use of encapsulated lipase in ZIF-8 in a process similar to the one described in this paper has never been studied before. The successful enhancement of the immobilized enzyme stability in the process may bring the process closer to industrial application.