Conversion of aromatic compounds from fractionated industrial hydrolysis lignin by Pseudomonas putida and environmental microbial strains

Background The utilization of Pseudomonas putida was explored in this study as a promising approach for lignin valorization. To this end, dry hydrolysis lignin was used as a feedstock for the first time. Hydrolysis lignin is a product of the enzymatic hydrolysis and separation of cellulose and hemicellulose from the lignin backbone in diverse lignocellulosic sources. Various fractionation techniques were applied to obtain lignin monomers and multimers in solution for use as a growth medium for P. putida , whose tolerance of inhibitory phenolic compounds distinguishes it from most bacteria. Results Physiological evaluations revealed that Pseudomonas putida strains KT2440 and PaW85 exhibited broad pH tolerance ranges, with robust growth observed at elevated pH levels. Batch fermentations using hydrolysis lignin (HL) solutions showed complete consumption of sugars within 24 hours, demonstrating the viability of fractionated HL as a substrate for P. putida cultivation. HPLC analysis of HL monomer concentrations during simulated fed-batch fermentation revealed rapid catabolism of catechol and increased CCMA concentration, followed by stabilization, indicating that CCMA is synthesized more quickly than degraded when the initial catechol concentration is high. Filtered alkaline HL fractionations yielded more than twice as much catechol as unfiltered fractionations. Screening of indigenous bacterial strains isolated from various soil and water samples (CELMS Collection, website ) identified five new candidate strains for CCMA production, two for PCA production, and three for vanillic acid production. Conclusions The novel use of fractionated hydrolysis lignin as a growth medium shows potential for lignin valorization and chemical production. Filtered alkaline fractionation yields more catechol and is superior for cis,cis -muconic acid production; however, unfiltered fractionations may be more suitable for other compounds and upscaling. Further investigation of screened strains could reveal more efficient enzymes, which could be optimized and transformed into P. putida in future research. ### Competing Interest Statement The authors have declared no competing interest. * (HL) : Hydrolysis lignin (CCMA) : cis,cis -muconic acid (GHG) : greenhouse gas (BDP) : benzoate degradation pathway (LB) : Luria broth (PCA) : protocatechuic acid (MMPAM) : modified minimal media formulation (SIM) : Selected Ion Monitoring (GC-MS) : gas chromatography-mass spectrometry (HPLC) : high-performance liquid chromatography