(66h) Bioconversion of Rich Containing ?-O-4 Linkage Lignin to Medium Chain Length-Polyhydroxyalkanoates

Agave tequilana bagasse (ATB) is one of the main residues of the Mexican agro-industry. During the last year by itself more than 800,000 tons of ATB were generated by the Tequila industry according to data from the Tequila Regulatory Council and estimations of the conversion of Agave plant to bagasse (⁓40%). With tequila demand growing every year, the disposal of this lignocellulosic residue has become an economic and environmental issue for the tequila industry. Although several papers have already been published on the use of the holocellulose fraction of this residue for the production of biofuels and chemicals, scarce information is available on the lignin fraction. Herein, we proposed a sequential pretreatment based on chemical/enzymatic hydrolysis to release the holocellulose and lignin fractions from ATB in different liquid streams. Our work focused on the unexplored lignin fraction, which was fully characterized by gravimetric, 2D ¹H–¹³C HSQC and ³¹P nuclear magnetic resonance (NMR) and gas chromatography mass spectrometry (GC-MS) analyses. Then, the novel application of this type of lignin for PHA production using engineered P. putida strains was initially evaluated in batch and fed-batch fermentation. Thereafter, the fermentation conditions were optimized using a central composite design and optimal conditions were evaluated using three P. putida strains. Finally, further insights into the mechanisms of ATB lignin bioconversion were gained by analyzing the chemical composition and structure of lignin before and after fermentation. Results of the chemical structure of the native ATB lignin by NMR analyses revealed that this lignocellulosic residue contains 63% of β-O-4 inter-unit linkages and this percentage only slightly decreased to 50% during our systematic pretreatment, making this lignin an attractive substrate for PHA bioconversion. Exploratory fermentation experiments in batch and fed-batch showed an increase on the PHA titer from 0.09 g/L to 0.39 g/L by using the fed-batch strategy and an engineered P. putida strain. Further improvement to 0.76 g/L was possible by using the central composite design to optimize the inoculum, substrate and Nitrogen contents. Finally, the PHA depolymerase gene phaZ was knocked out from P. putida and evaluated at optimal conditions, achieving a PHA titer of 0.97 g/L. Analysis by NMR and GC-MS confirmed that lignin derivatives like vanillin were totally consumed during fermentation and the β-O-4 inter-unit linkages decreased 30%. Overall, this is the first work reported on the valorization of ATB lignin and sets the precedent for future design of chemical and biological valorization methods for this and other rich containing β-O-4 linkage residues.