Khiawjan, Sansanee; (2024) Intensification of biocatalytic process for the conversion of lignin derivative ferulic acid to high value chemicals. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Converting lignin-to-high-value products through biocatalytic processes is promising due to its reaction specificity and eco-friendly nature. However, the commercial applicability of this process is limited due to the complex structure of lignin, which makes it difficult to scale up. This research focused on developing an efficient bioprocess to intensify the production of coniferyl alcohol (CA) from lignin-derived ferulic acid (FA). The process involved a two-step enzymatic conversion catalyzed by a carboxylic acid reductase from Norcadia iwensis (NiCAR) and an aldo-keto reductase from Coptotermes gestroi (CgAKR-1). Initially, the study involved determining the kinetics of NiCAR and CgAKR-1 enzymes, revealing that substrate (FA) and product (CA) inhibition negatively affected their activities. To enhance enzyme stability and reusability, NiCAR and CgAKR-1 were immobilized on a Ni-NTA agarose resin, resulting in improved catalytic activities. This was a positive effect of site-specific immobilization and the hydrophilic nature of the agarose resin. Subsequently, the feasibility of using immobilized enzymes or bacterial whole cells to produce CA from either pure FA or lignin-derived FA was demonstrated. The study showed that ratio of NiCAR and CgAKR-1 significantly affected the productivity of CA. The optimum unit ratio of NiCAR to CgAKR-1 was found to be 1:47, representing NiCAR 0.006 U·ml-1 and CgAKR-1 0.29 U·ml-1. To enhance economic feasibility, the oxidation of glucose by the glucose dehydrogenase (GDH) was coupled with NiCAR and CgAKR-1 enzymes for NADPH recycling. This approach is compatible with the conversion system and showed a slight improvement in CA productivity. Performed as a fed-batch bioconversion at the optimum enzyme ratio, immobilized NiCAR and CgAKR-1 exhibited remarkable CA productivity at 95 mgCA·L-1·h-1 with a conversion yield of 0.64 gCA∙ g-1FA. The activity of enzymes was also tested by using FA derived from sugar beet pulp (SBP) and Green Value lignin (GV lignin). The immobilized NiCAR and CgAKR-1 could convert SBP-derived FA into CA at the productivity of 1.5 ± 0.4 mgCA·L-1·h-1, but they were unable to convert GV lignin-derived FA into CA. However, product inhibition was observed in both batch and fed-batch processes, highlighting the need for a continuous bioconversion. The use of bacterial whole cells is promising for the bioconversion of FA into CA since it does not require additional co-factors supplementation. The CA productivities of three recombinant Escherichia coli strains, namely E. coli BL21/ pROB1, E. coli BL21/ Pro B, and E. coli BL21/ Pad R, were examined under different fermentation conditions. Among them, E. coli BL21/ Pro B was the most promising strain. In batch fermentation, it produced 27 mgCA·L-1·h-1 from commercial FA and 8.57 mgCA·L-1·h-1 from SBP-derived FA. Due to the inhibitory effects of FA, CA, and unidentified compounds found in the lignin hydrolysates, the CA productivity was relatively low when the hydrolysate was used as substrate. Subsequently, continuous fermentation using E. coli BL21/ Pro B was demonstrated in a mini-bioreactor system using pure FA as a substrate. The optimum dilution rate was found at 0.2 h-1 with FA feeding rate of 194 mgCA·L-1·h-1. The continuous fermentation exhibited stable CA production for 149 hours with complete conversion of FA and yielded a remarkable CA productivity of 160 mgCA·L-1·h-1, representing a 6-fold increase compared to batch fermentation. The study emphasized that continuous fermentation efficiently intensified the process productivity, highlighting its potential to integrate into existing biorefinery processes to promote a sustainable bioeconomy.

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