Development of a Sustainable Bioprocess for Kraft Lignin Valorization Using Deep Eutectic Solvents and Adaptive Laboratory Evolution of Pseudomonas putida

The urgent need to mitigate climate change and reduce greenhouse gas emissions necessitates a shift from petroleum-based products to a sustainable bio-based economy. This transition highlights the importance of efficiently valorizing lignocellulosic biomass, particularly lignin, a complex and largely underutilized aromatic polymer. Kraft lignin (KL), an abundant, carbon-rich byproduct from the pulp and paper industry, represents a promising feedstock for microbial bioconversion into value-added products. However, its application is hindered by poor solubility, structural heterogeneity, microbial toxicity, and the limitations of conventional depolymerization methods. Techniques such as oxidative alkaline depolymerization, though effective for lignin solubilization, rely on harsh, energy-intensive conditions and generate hydrolysates requiring extensive detoxification, reducing overall process sustainability.

This thesis presents the development of an integrated and sustainable bioprocess for KL valorization by combining deep eutectic solvent (DES) pretreatment with microbial fermentation. DESs represent a green and tunable alternative to traditional solvents. Among seven DES formulations screened in this thesis, L-arginine:glycerol (L-Arg:Gly) emerged as the most effective, achieving KL solubility of 424.2 ± 26.88 g/L in 50% DES solution and maintaining relatively high solubility of 6.9 ± 1.43 g/L at only 2% DES solution. Optimized depolymerization conditions (2% DES, 95 °C, 1 h) yielded aromatic monomers including syringol, vanillin, and syringaldehyde. Fermentation with Pseudomonas putida KT2440 showed rapid microbial growth, although total lignin conversion was limited by the strain’s inability to fully metabolize syringyl-type compounds. Additionally, bacterial growth was inhibited at DES concentration higher than 0.5%, highlighting the need for strain improvement to enhance DES tolerance.

To address these limitations, adaptive laboratory evolution (ALE) was employed using a low-cost, automated Pioreactor system operated in turbidostat mode. Parallel ALE experiments using traditional tube-based batch culturing were conducted for comparison. The Pioreactor system operated stably for up to 56 days, achieving between 119 and 308 generations depending on the condition. In comparison, the tube-based ALE was conducted for up to 62 days, resulting in approximately 170 to 269 generations. Comparative genomic and phenotypic analyses revealed distinct adaptive strategies. Both evolved populations acquired mutations in c-di-GMP signaling and membrane remodeling, which conferred phenotypes enhancing culture stability and stress tolerance in the Pioreactor-evolved population. In addition, the tube-adapted strains developed further mutations in energy conservation and toxic stress management, resulting in higher growth rates (μ_max = 0.459 h⁻¹) compared to wild type (μmax = 0.181 h⁻¹) and elimination of lag phases under 5% DES.

Finally, a techno-economic analysis (TEA) was conducted to assess the feasibility of producing polyhydroxyalkanoates (PHAs) from DES-treated KL using P. putida KT2440. The analysis, integrating laboratory-scale experimental data with literature-derived process parameters, identified major cost drivers such as, solvent recovery, cost of L-Arg. The strategies for improving process efficiency and scalability were also highlighted.

Collectively, this thesis establishes a sustainable DES-based strategy for KL valorization, validates an accessible ALE platform, delivers evolved microbial strains to advance lignin-based biorefineries, and provides a scalable framework for sustainable lignin valorization within a circular bioeconomy.