Real-time NMR monitoring of HMF biotransformation by Ectopseudomonas oleovorans CECT 5344 R1D.

This research identifies Ectopseudomonas oleovorans CECT 5344 R1D as a novel and highly efficient biocatalyst for the sustainable production of 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). By employing benchtop Nuclear Magnetic Resonance (NMR) spectroscopy with a recirculating flow system, we achieved real-time monitoring and a detailed stoichiometric characterisation of the 5-hydroxymethylfurfural (HMF) detoxification pathway. This work contributes to the specialised field of real-time metabolic tracking of living biocatalysts, a niche where in situ NMR data is still scarce compared to end-point analytical methods. Our findings demonstrate that this strain, while not utilising HMF for biomass growth, maintains exceptional metabolic stability and quantitative molar yields over 125 h of operation, requiring significantly lower cell densities than other reported Pseudomonas strains. The in-line NMR setup enabled the definitive identification of 2,5-bis(hydroxymethyl)furan (BHMF) as a transient intermediate, clarifying previous metabolic misidentifications and revealing a highly specific, non-consuming "cell factory" behaviour. Furthermore, the system allowed for the assessment of bacterial tolerance thresholds and the exploration of the enzymatic substrate scope against other environmentally relevant aldehydes. This work underscores the synergy between a robust, living biocatalyst and a precise monitoring platform, providing an optimised workflow for bioprocess development in sustainable aldehyde valorisation. KEY POINTS: • Real-time monitoring of a microbial process via in-line benchtop NMR. • Versatile biodetoxification of various natural and synthetic aldehydes. • Green chemistry alternative to petrochemicals using renewable feedstocks.