A novel biorefinery route towards a next-generation biofuel feedstock from cellulosic primary sewage sludge

This study proposes an integrated biorefinery approach for exploiting the cellulosic primary sludge, a cellulose-rich byproduct of urban wastewater treatment, to obtain single cell oils (SCOs) for biodiesel production. This process employs enzymatic hydrolysis and oleaginous yeast fermentation as a biocatalytic cascade strategy. Enzymatic hydrolysis was performed using Cellic® CTec 2, producing a fermentable sugar-rich suspension. The liquid phase, containing a high concentration of glucose (44.1 g/L) and xylose (9.3 g/L), was adopted as fermentation substrate. The commercial oleaginous yeast Trichosporon oleaginosus was employed to convert the undetoxified hydrolyzate into lipids, evaluating three carbon-to-nitrogen (C/N) weight ratios (80, 100 and 120 g/g) for optimal lipid accumulation. The yeast efficiently consumed nearly all sugars when the 80 g/g C/N ratio was adopted, achieving the best balance between biomass growth and lipid accumulation, reaching the promising 45.6 wt% SCO content. Three extraction protocols were assessed as alternative downstream processing strategies: two chemical approaches, based on acid-assisted and surfactant-assisted extractions, respectively, and a mechanical one using bead milling. These methods are based on different cell disruption mechanisms: acid hydrolysis with HCl, alteration of membrane permeability with Triton X-100 and mechanical rupture through bead milling. Furthermore, the effect of the heating system and the extraction solvent was investigated. The resulting fatty acid methyl esters (FAMEs) profile closely resembled commercial vegetable oils used in biodiesel production. Overall, the proposed biorefinery approach demonstrates the potential of cellulosic primary sewage sludge as a feedstock for microbial lipid production, contributing to resource recovery within a circular bioeconomy framework.