The enzymatic route is a promising strategy to synthesize biodiesel essentially because of its moderate reaction conditions and easy product separation. Additionally, coupling the biodegradation of agro-industrial raw materials and waste products with the enzyme production has also had a significant impact on the development of industrial biotechnology. This is mainly due to the low-cost and the prevention of pollution they represent. In this work, different kinds of residues and wastes were used for tuning biomass-bound lipolytic biocatalysts from Aspergillus niger MYA 135 by submerged fermentation using the Plackets-Burman design. As a result, by a specific combination of medium components, two distinctive biocatalysts were obtained. Thus, the production of a mycelium-bound esterase was maximized in the presence of (in g l(-1)): CaCl2 0.5, FeCl3 1.0, corn meal 2.0, cerelose 10.0, peptone 10.0, and waste cooking oil 10.0. While, the production of a mycelium-bound lipase was selectively increased in the presence of (in g l(-1)): molasses 5.0, vinasse 5.0, CaCl2 0.5, FeCl3 1.0, milk serum 10.0, cerelose 5.0, peptone 5.0 and waste cooking oil 10.0. Besides, the biomass-bound lipase activity produced in the optimized culture medium was also prepared either free or immobilized in alginate beads, and then individually placed in small-scaled packed-bed reactors to produce biodiesel compounds in a solvent-free system using soybean oil and butanol as substrates. After a three-stepwise addition of butanol, a biodiesel conversion of 57.32 and 95.89% was reached in the presence of free and immobilized biomass, respectively. Biocatalysts morphologies were also analyzed.