Sequential Enzymatic Bioprocessing of Protein-Rich Unhairing-Liming and Lime-Fleshing Tannery Wastewater for the Production of Amino Acid-Based Plant Biostimulants.

The recovery of bioactive compounds from industrial wastewaters is a key strategy for improving resource efficiency and reducing the environmental impact of high-load effluents. In this work, two protein-rich tannery wastewater streams with different origins and compositions, unhairing-liming wastewater and lime-fleshing wastewater, were comparatively evaluated as secondary raw materials for the production of amino acid-based protein hydrolysates with potential biostimulant activity. Both streams were characterised in terms of physicochemical composition, mineral content and amino acid profile, and subsequently subjected to a comparative sequential enzymatic hydrolysis approach using endo- and exo-proteolytic enzymes. Alcalase, followed by Pancreatin, was selected as the most effective enzymatic system for both streams, although different optimal enzyme loadings were required depending on the wastewater composition. For unhairing-liming wastewater, the optimal conditions were 1.5% Alcalase and 1.0% Pancreatin, yielding 70.76 ± 1.95% hydrolysate, 80.87 ± 1.98% protein recovery and 11.42 ± 1.03% free amino acids. For lime-fleshing wastewater, 0.7% Alcalase and 1.0% Pancreatin provided 98.42 ± 1.97% yield, 92.19 ± 1.92% protein recovery and 16.97 ± 0.98% free amino acids. The two hydrolysates showed differentiated amino acid profiles, reflecting the keratin- and collagen-derived nature of the original streams. Germination assays indicated a growth-stimulating effect of the hydrolysates, increasing seed growth by up to 20.7% for the unhairing-liming wastewater hydrolysate at 0.10% (w/v) and by up to 27.1% for the lime-fleshing wastewater hydrolysate at 0.07% (w/v). These results demonstrate that enzymatic hydrolysis can be an effective and environmentally friendly route for converting tannery wastewaters into value-added bio-based products for agricultural applications, while also highlighting the industrial relevance of adapting the enzymatic process to the origin, composition and protein accessibility of each wastewater stream.