**1. Introduction**

Overpopulation, urbanization, industrialization and augmentation of personal transport have increased the petroleum consumption immensely. It is predicted that petroleum reserves will become depleted soon if the rate of its consumption continue to increase with the same rate [1]. Depletion of non-renewable fuel reserves, elevation in price of fossil fuels and pollution caused by usage of petroleum products has spurred the production of eco-friendly, inexpensive alternative energy source that can reduce petroleum consumption and that is biodiesel [2]. Biodiesel has emerged as an alternative of petroleum due to its renewable nature, biodegradability, and interest of consumers in nature friendly products [3].

Feedstock selection for biodiesel production is of chief concern because of high prices associated with various feedstock. Edible and non-edible feedstocks have been used by many researchers for the biodiesel synthesis, edible oils including; sunflower, corn, rapeseed and soybean oil, while non edible oils including; Jatropha, *Eruca sativa*, Caster, Jojoba and other oils. Utilizing edible oils as feedstock will cause conflict with food production and food price so it is more desirable to use non edible oil [4]. A very economical feedstock is used cooking oil. Excess of waste frying/cooking oil is produced every day in restaurants and fast food shops. The use of edible oil for excessive frying/cooking at high temperature destroys the structure of triglyceride and produce the free fatty acids in oil, changing its pH, which is harmful for human health [5,6]. The oil is therefore being dumped o ff through drainage, which causes water pollution. This oil is useless and causes hazards to aquatic life. Using this waste oil to make something valuable is highly appreciated, because it is almost free feedstock for biodiesel production [7,8].

Transesterification is the preferred method for biodiesel production as compared to other techniques [9]. Various strategies of transesterification have been adopted to achieve better yield, purity and fast reaction rates. Catalytic transesterification is frequently used as, the presence of catalyst enhances the solubility of oil in alcohol and increases the reaction rate [10]. Catalysts of transesterification can be homogeneous or heterogeneous. Homogenous catalysts include; acid and alkaline catalyst. Alkaline catalysts are commonly used because of their low cost and higher catalytic activity at ambient conditions, while alkaline catalysts do not work e fficiently under higher FFA conditions [11]. Besides that, there are many drawbacks of using these catalysts in excess which includes; formation of toxic waste that need to be neutralized by several washings, hence it causes environmental pollution, contaminated glycerol is produced, purification of this glycerol increases the production cost, partial saponification can lead to the production of soap which makes it di fficult to separate glycerol and alkyl esters hence decreases the yield of biodiesel [12]. Furthermore, another major drawback is that these catalysts cannot be reused or recycled.

Heterogenous catalysts include; enzymes (lipase), alkaline earth metals, zirconias of potassium and silicates of titanium etc. The enzyme used for transesterification is usually lipase. Lipase has several advantages over convention alkali catalysts; e.g., there is no need of purification of biodiesel after transesterification thus no toxic waste is produced—especially in case of waste cooking oil, where there are lot of free fatty acids, lipase reduces the chance of saponification [13]. However, there are some reasons that hampered its utilization, which include; high cost, di fficulty in its recovery and instability at high temperature and pH. The best strategy to tackle these problems is the immobilization of enzyme on some support. Previously, lipases have been immobilized on several surfaces such as; ceramics, calcium alginate beads and other inorganic matrixes, which increase the thermal stability of the enzyme. However, the activity of the enzyme is revealed to be reduced due to a decrease in conformational flexibility and capability of adsorption on support surface [14]. Another e ffective way of using bio-catalyst is to immobilize it on nano-support. Di fferent techniques such as; entrapment, adsorption and covalent immobilization are being used for this purpose. Enzymes immobilized on nanoparticles provide the advantage of greater enzymatic activity, better selectivity along with thermal stability, adaptability towards wider pH range, easy recovery and purification [15]. If the nanoparticles used for immobilization are metal oxides, then these particles provide the advantage of low pricing, and higher stability even in harsh conditions, another advantageous attribute of metal oxide nanoparticles (NPs) is their magnetic property. Nanoparticles of iron, nickel, cobalt, chromium, manganese and their oxides show enhanced magnetic moment as compared to other metals [16]. Among these metal oxides, iron oxides—especially magnetite Fe3O4—shows very strong magnetism and it is also less toxic when compared to nickel and cobalt. Enzyme was first immobilized on magnetic nanoparticles surface by Matsunaga and Kamiya [17]. Immobilizing lipase on magnetite will generate a nano-biocatalyst, which will gran<sup>t</sup> the edge of better activity in harsh conditions and reusability [18].

To reduce the risk of toxicity and make them more biocompatible, the surface of nanoparticles can be modified with di fferent polymers using "graft to and graft from methods". Polymers (natural/synthetic) are used as coatings that also provide active groups at the surface to a fford immobilization of lipase on nanoparticles. These polymers mostly contain epoxy and amine groups that react with active groups of lipases. However, the drawback of using polymers with amine group is the need of activation of amine group using some aldehydes, most commonly, amine group activation is done using gutaraldehyde [19]. This activation step increases the cost and preparation time of catalyst.

A versatile coating for the nanoparticles is polydopamine (formed by self-polymerization of dopamine monomers through oxidation in slightly alkaline conditions). Dopamine contains catechol as well as amine group, which e fficiently interacts with metal oxide nanoparticles [20]. In an alkaline medium on oxidation, the catechol group of dopamine converts to the indo-5, 6-quinone, which further undergoes series of inter/intra molecular reactions, forming polydopamine grafted on the NPs surface. The residual quinone and catechol groups present on the surface after polymerization are reactive towards nucleophiles such as thiol and amine groups. These groups of lipases covalently immobilize the lipase on polydopamine through Schi ff base formation and Michael addition reaction. Lipase immobilized on polydopamine containing nanoparticles show higher e fficiency and higher enzyme loading as compared to the immobilization at naked nanoparticles [21]. The present work was therefore planned to prepare magnetic metal oxide nanoparticles by solvothermal method followed by polydopamine grafting and immobilization of *Aspergillus terrus* lipase for development of nano-biocatalyst. The synthesized nano-biocatalyst was then employed for the synthesis of biodiesel.

## **2. Materials and Methods**

All the chemicals/reagents used, i.e., FeCl3·6H2O, ethylene glycol, ethylene diamine, sodium acetate, methanol, ethanol, potassium iodide, iodine, n-hexane, chloroform, toluene, acetic acid, distilled water, potassium hydroxide, hydrochloric acid, phenolphthalein and isopropanol etc., were of analytical research grade obtained from Sigma-Aldrich (St. Louis, MO, USA). Lipase was produced from *Aspergillus terreus* AH-F2. The feedstock waste cooking oil was obtained from local restaurant situated in district Gujrat Pakistan.
