Continuous flow biocatalytic conversion of lignin

Lignin represents the most abundant aromatic biopolymer on earth; it exhibits a great potential to replace oil-based products. The molecular structure of lignin is complex, and depends on the botanical species and the employed fractionation process. The depolymerization of lignin can be a promising first and fundamental step towards valorization. The potential of ligninolytic enzymes such as laccases and peroxidases has been extensively investigated for this purpose.1 By generating phenoxy radical species from phenolic endgroups, they can induce oxidative coupling, leading to lignin depolymerization, and lignin oxidation at the C-α position in the β-O-4’ bonding of terminal phenolic units.2 Flow chemistry and continuous processing, initially developed in the context of synthetic chemistry, offers superior control of reaction conditions, mixing, , and potential automation, etc.3 The combination of these two research fields, i.e., the use of enzymes as biocatalysts for lignin depolymerisation and flow chemistry allows for the development of more efficient and environmentally friendly biocatalytic processes in the biorefinery area.4Aim of our research was the development of a continuous flow process for the biocatalytic conversion of lignins using immobilized laccase (EC.1.10.3.2) in combination with a mediator (Fig. 1 and Fig 2). The laccase, immobilised on alumina beads and protected with polyelectrolytes (LbL-technique) and the lignin were packed in two different column reactors, with the lignin being placed between layers of sand and celite (Fig.3). The mediator was dissolved in the aqueous buffer system. Reaction products were analyzed by gas-chromatography and by quantitative 31P NMR spectroscopy, as well as gel permeation chromatography (GPC). For example, corn stover lignin was oxidised by the laccase-mediator system in continuous flow, generating various monomeric compounds from the in the aqueous phase insoluble lignin, such as vanillin and 4-hydroxy benzaldehyde, and 4-hydroxy acetophenone, and, more importantly, also depolymerised, albeit not monomeric fractions that did not undergo repolymerisation. This phenomenon can be attributed to the effective migration of the depolymerized compounds, now water-soluble, towards the sand-celite pad placed at the column exit, where they concentrated while the unactivated, since previously reacted, mediator passed, to be available for renewed activation upon passing the immobilised enzyme before hitting again the lignin. The presentation will highlight further scope and limitations. References 1. Reshmy, R.; Balakumaran, A.B.; Divakar, K.; Philip, E.; Madhavan, A.; Pugazhendhi, A.; Sirohi, R.; Binod, P.; Awasthi, M.K.; Sindhu, R. Bioresour. Technol. 2022 344, 126240-126252. 2. Vignali, E.; Gigli, M.; Cailotto, S.; Pollegioni, L.; Rosini, E.; Crestini, C. ChemSusChem 2022 15, e202201147. 3. Britton, J.; Majumdar, S.; Weiss, G.A. Chem. Soc. Rev. 2018 47, 5891-5918. 4. Coloma, J.; Guinavarc’h, Y.; Hagedoorn, P.; Hanefeld, U. Chem. Commun. 2021 57, 11416-11427.