Bacterial conversion of depolymerized Kraft lignin

Abstract

Background: Lignin is a potential feedstock for microbial conversion into various chemicals. However, the degradation rate of native or technical lignin is low, and depolymerization is needed to obtain reasonable conversion rates. In the current study, base-catalyzed depolymerization-using NaOH (5 wt%)-of softwood Kraft lignin was conducted in a continuous-flow reactor system at temperatures in the range 190-240 °C and residence times of 1 or 2 min. The ability of growth of nine bacterial strains belonging to the genera Pseudomonas and Rhodococcus was tested using the alkaline-treated lignin as a sole carbon source.

Results: Pseudomonas fluorescens and Rhodococcus opacus showed the best growth of the tested species on plates with lignin. Further evaluation of P. fluorescens and R. opacus was made in liquid cultivations with depolymerized lignin (DL) at a concentration of 1 g/L. Size exclusion chromatography (SEC) showed that R. opacus consumed most of the available lower molecular weight compounds (approximately 0.1-0.4 kDa) in the DL, but the weight distribution of larger fractions was almost unaffected. Importantly, the consumed compounds included guaiacol-one of the main monomers in the DL. SEC analysis of P. fluorescens culture broth, in contrast, did not show a large conversion of low molecular weight compounds, and guaiacol remained unconsumed. However, a significant shift in molecular weight distribution towards lower average weights was seen.

Conclusions: Rhodococcus opacus and P. fluorescens were identified as two potential microbial candidates for the conversion/consumption of base-catalyzed depolymerized lignin, acting on low and high molecular weight lignin fragments, respectively. These findings will be of relevance for designing bioconversion of softwood Kraft lignin.

Keywords: Base-catalyzed depolymerization; Biorefineries; Guaiacol; Indulin AT; Microbial conversion; Pseudomonas; Rhodococcus.

Fig. 1

Molecular weight distributions of Indulin Kraft AT and depolymerized lignin products, measured as UV absorbance at 280 nm at two different residence times. The depolymerization reaction sets were carried out at temperatures 190, 220 and 240 °C with the flowrates of a 10 mL/min (residence time of 1 min) and b 5 mL/min (residence time of 2 min)

Fig. 2

Growth (OD) of R. opacus, P. fluorescens and P. putida EM42 on 1 g/L a depolymerized (at 220 °C with 2 min residence time) lignin b non-depolymerized lignin as the only carbon source in M9 medium. A non-inoculated control is shown with a black line. Same scale on the Y-axis is maintained for easy comparison between the graphs. All experiments were performed in duplicates

Fig. 3

SEC (a–c) and UHPLC (d–f) chromatograms of 1 g/L depolymerization lignin on day 0, 4 and 14 after bacterial conversion by R. opacus, P. fluorescens and P. putida EM42. The bacterial cultures, which were inoculated with a single colony, were grown using depolymerized lignin as the only carbon source in M9 medium. In the UHPLC chromatograms (d–f), the peaks at retention time 1.6, 2.4, 3.5, 4.6 and 4.7 min were identified as 4-HBA, vanillate, vanillin, guaiacol and acetovanillone, respectively. The consumption of low molecular weight fraction (0.1–0.4 kDa) by R. opacus within 4 days is highlighted with a green dashed circle. The consumption of monomers (vanillin and guaiacol) by R. opacus is highlighted with green arrows

Fig. 4

a Growth (OD) of R. opacus, P. fluorescens and P. putida EM42 on 1 g/L depolymerized (at 220 °C with 2 min residence time) lignin supplemented with 5 g/L glucose in M9 medium. A non-inoculated control is shown with a black line. b Change in media color on day 14 for 1-non-inoculated control; 2-R. opacus; 3-P. fluorescens; 4-P. putida EM42

Fig. 5

SEC chromatograms of 1 g/L depolymerized lignin on day 0 and 14 after high cell density bacterial conversion by a R. opacus b P. fluorescens c P. putida EM42 and d non-inoculated control in M9 medium. The consumption of low molecular weight fraction (0.1–0.4 kDa) by R. opacus and breakdown of high molecular weight fraction (1–10 kDa) by P. fluorescens are highlighted with green dashed circles

Fig. 6

UHPLC chromatograms of 1 g/L depolymerized lignin on day 0, 4 and 14 after high cell density bacterial conversion by a R. opacus b, c P. fluorescens d P. putida EM42 e non-inoculated control in M9 medium. The peaks at retention time 1.6, 2.4, 3.5, 4.6 and 4.7 min were identified as 4-HBA, vanillate, vanillin, guaiacol and acetovanillone, respectively. The occurrence of new large peaks at retention times 0.4–0.5 min for P. fluorescens on day 14 are highlighted with a green arrow. For a better visibility of the newly formed peaks on day 14, the chromatograms are arranged in reverse order (bottom to top) for b, c

Fig. 7

Measured growth (OD) and substrate concentrations for cultures of R. opacus grown on guaiacol as the only source of carbon in M9 medium. The standard deviation of duplicate experiments is indicated with error bars

Fig. 8

Growth (OD) and consumption of model compounds by a P. putida EM42 b P. fluorescens c R. opacus in M9 medium. 3 mM each of guaiacol, 4-HBA and vanillin were provided as the only source of carbon. The standard deviation of duplicate experiments is indicated with error bars