The hydrolysis of triglycerides to free fatty acids and glycerol is catalyzed by lipases (triacylglycerol acylhydrolases; EC 188.8.131.52) . Besides hydrolysis, lipases catalyze various synthetic reactions, such as transesterification, in low water activity media .
It is widely known that severe environmental pollution can result from oil-contaminated wastewater. The diffusion of oxygen from air into water is prevented by the formation of oil film on water surfaces, causing the death of numerous aquatic lives . Also, particles in wastewater, especially aggregates formed by oil droplets, can also cause blockage in drainage lines . Asides the aforementioned environmental problems, there are also operational problems, which may arise from the non-treatment of wastewater with high fat contents, such as granular biomass flotation , fat scum layers forming at the surface of the reactor as well as fats solidifying at lower temperatures leading to clogging and unpleasant odour .
Employment of specific enzymes such as lipases has gained wide attention due to the clean and friendly nature of enzyme applications, as well as the firm environmental regulations . Previous studies have shown the feasibility of enzymatic treatment of lipid-rich wastewater which aids in increasing its anaerobic biodegradability .
Free enzymes can only be utilized once in solutions because they are generally soluble and unstable. They are also susceptible to and frequently inactivated by various environmental conditions including inhibitors, ionic strength and pH . Most importantly, they are too expensive to be used in wastewater treatment. These challenges can be overcome by immobilization which has its unique benefits.
Immobilization confers certain advantages on the enzymes such as increased stability at different temperature, pH and ionic strength, as well as recyclability from the reaction mixture . Different methods have been employed for lipase immobilization over the years, including cross-linking, adsorption, multipoint covalent attachment and physical entrapment , with each having its advantages, disadvantages and peculiarities. The cross-linking of enzyme molecules' physical aggregates results in the formation of cross-linked enzyme aggregates (CLEAs) . Some advantages over carrier-bound immobilized enzyme is the important savings on the cost of the manufactured superstructure due to the lack of support, the simplicity and broad applicability of the technique  and the reality that no previous purification of the enzyme is required [10, 12]. The study aimed to immobilize Rhizopus oryzae lipase by cross-linking and evaluate the application of the immobilized enzyme in wastewater hydrolysis.