Sequential sludge solubilization and hydrolysate conditioning of pulp and paper mill activated sludge for enhanced polyhydroxyalkanoates production.

Pulp and paper mill activated sludge (PPMAS) contains microbial biomass, lignocellulosic residues, and nutrients that make it a promising feedstock for the production of biodegradable polyhydroxyalkanoates (PHAs). However, alkaline pre-treatment required to solubilize sludge solids and increase substrate accessibility can also generate pre-treatment-derived inhibitory compounds that impair microbial carbon conversion. In this study, the formation of inhibitory compounds during alkaline pre-treatment of PPMAS was investigated, and a sequential detoxification strategy was developed to improve hydrolysate fermentability for PHA production by Cupriavidus necator NCIMB 11599. Simultaneous addition of calcium hydroxide with sodium hydroxide or ammonium hydroxide before sterilization showed limited sludge solubilization, with suspended solids decreasing from 15.0 to 11.9 g/L. In contrast, a sequential strategy consisting of NaOH pre-treatment (0.133 g/g suspended solids) followed by post-sterilization Ca(OH)₂ conditioning reduced suspended solids from 15.0 to 8.89 g/L. GC-MS and LC-MS/MS analyses showed that NaOH-treated hydrolysates contained several lignin- and pre-treatment-derived compounds, including vanillic acid, vanillin, syringaldehyde, benzene ethanamine, and fatty-acid derivatives, whereas concentrations of several detected compounds were lower following Ca(OH)₂ conditioning. Fermentation of the sequentially treated hydrolysate supplemented with glucose and minerals resulted in a biomass increase of 10.44 g/L, PHA content of 35.5 wt %, and PHA concentration of 6.86 g/L (0.095 g/L/h PHA productivity), representing a 2.4-fold increase in PHA content compared with NaOH pre-treatment alone. These findings demonstrate that maximizing sludge solubilization alone does not necessarily maximize subsequent biological resource recovery. Instead, hydrolysate quality should be considered together with organic-matter solubilization when designing pre-treatment strategies for sludge valorization.