Unveiling Ruminal Fluid as an Enzymatic Catalyst for Biodiesel Synthesis from Waste Fat

Enzymatic biocatalysis offers a promising route for converting highly acidic and water-rich feedstocks into biodiesel, but its large-scale application is constrained by the high cost of commercial lipases. Here, we propose an unprecedented and low-cost strategy employing bovine ruminal fluid (RF), an abundant agro-industrial residue as a natural biocatalyst for the esterification of waste fat (WF) from a grease-trap stream. The RF was comprehensively characterized, including 16S rRNA metagenomic sequencing, which revealed a functionally diverse microbial consortium enriched in lipolytic enzymes, supporting its catalytic potential. A 2 5−1 fractional factorial design identified the lipid/methanol ratio and RF loading as the dominant variables, which were subsequently optimized using a face-centered composite design. Under optimal conditions (9% RF, 1:6 lipid/methanol), a conversion of 39.5% ± 0.5% was achieved. GC–MS and FTIR analyses confirmed the formation of fatty acid methyl esters. This study demonstrates, for the first time, the feasibility of using crude ruminal fluid as an effective and low-cost biocatalyst for biodiesel synthesis, adding value to slaughterhouse residues and expanding bio-based catalytic routes for renewable fuel production.