A facile process for adipic acid production in high yield by oxidation of 1,6-hexanediol using the resting cells of Gluconobacter oxydans

Abstract

Background: Adipic acid (AA) is one of the most important industrial chemicals used mainly for the production of Nylon 6,6 but also for making polyurethanes, plasticizers, and unsaturated polyester resins, and more recently as a component in the biodegradable polyester poly(butylene adipate terephthalate) (PBAT). The main route for AA production utilizes benzene as feedstock and generates copious amounts of the greenhouse gas NO₂. Hence, alternative clean production routes for AA from renewable bio-based feedstock are drawing increasing attention. We have earlier reported the potential of Gluconobacter oxydans cells to oxidize 1,6-hexanediol, a potentially biobased diol to AA.

Results: The present report involves a study on the effect of different parameters on the microbial transformation of 1,6-hexanediol to adipic acid, and subsequently testing the process on a larger lab scale for achieving maximal conversion and yield. Comparison of three wild-type strains of G. oxydans DSM50049, DSM2003, and DSM2343 for the whole-cell biotransformation of 10 g/L 1,6-hexanediol to adipic acid in batch mode at pH 7 and 30 °C led to the selection of G. oxydans DSM50049, which showed 100% conversion of the substrate with over 99% yield of adipic acid in 30 h. An increase in the concentrations of the substrate decreased the degree of conversion, while the product up to 25 g/L in batch and 40 g/L in fed-batch showed no inhibition on the conversion. Moreover, controlling the pH of the reaction at 5-5.5 was required for the cascade oxidation reactions to work. Cell recycling for the biotransformation resulted in a significant decrease in activity during the third cycle. Meanwhile, the fed-batch mode of transformation by intermittent addition of 1,6-hexanediol (30 g in total) in 1 L scale resulted in complete conversion with over 99% yield of adipic acid (approximately 37 g/L). The product was recovered in a pure form using downstream steps without the use of any solvent.

Conclusion: A facile, efficient microbial process for oxidation of 1,6-hexanediol to adipic acid, having potential for scale up was demonstrated. The entire process is performed in aqueous medium at ambient temperatures with minimal greenhouse gas emissions. The enzymes involved in catalyzing the oxidation steps are currently being identified.

Keywords: 1,6-hexanediol; Adipic acid; Gluconobacter oxidation; Polymer building block; Whole cell oxidation.

Scheme 1

Synthesis of adipic acid by A conventional catalytic process by oxidation of KA-oil (ketone-alcohol oil) obtained from cyclohexane derived from fossil benzene, and B biocatalytic oxidation of 1,6-hexanediol (potentially produced from biobased 5-HMF) via 6-hydroxyhexanoic acid

Fig. 1

Oxidation of 10 g/L 1,6-HD to adipic acid via 6-HHA by A G. oxydans 50049, B G. oxydans 2003, C G. oxydans 2343 in 1 mL of 100 mM sodium phosphate buffer, pH 7 at 30 °C. The symbols represent conversion (%) of 1,6-HD (filled diamond), and content (%) of 6-HHA (filled square), aldehydes (x) and AA (filled triangle) in the reaction. D Conversion (%) of 1,6-HD and content (%) of products formed at 12 h during the oxidation of 10 g/L 1,6-HD by G. oxydans strains

Fig. 2

Effect of cell amounts on the oxidation of 10 g/L 1,6-HD to AA via 6-HHA at 30 °C in 1 mL for 12 h by 2 mL (1.6 mg CDW), 4 mL (3.2 mg CDW) and 6 mL (4.8 mg CDW) of G. oxydans 50049 in 1 mL of 100 mM sodium phosphate buffer, pH 7 at 30 °C. A Effect of cell amount of G. oxydans DSM50049, and B initial pH on the oxidation of 10 g/L 1,6-HD to AA via 6-HHA in 1 mL at 30 °C. The cell amounts used were 1.6 mg, 3.2 mg and 4.8 mg cell dry weight from 2, 4 and 6 mL culture broth, respectively, in (A), and 3.2 mg CDW in (B). AA, 6-HHA, and residual 1,6-HD were measured after 12 h in (A) and after 24 h in (B). pH was not controlled during the reaction

Fig. 3

Recycling of G. oxydans DSM50049 cells (3.2 mg CDW) for the oxidation of 10 g/L 1,6-HD to AA via 6-HHA in 1 mL of 100 mM sodium phosphate buffer, pH 7 at 30 °C for 24 h (1st and 2nd run)

Fig. 4

A Effect of initial 1,6-hexanediol and adipic acid concentrations on the oxidation of 1,6-HD to AA via 6-HHA in 1 mL at 30 °C for 30 h without pH control by 3.2 mg CDW (obtained from 4 mL cell suspension) of G. oxydans 50049. B Effect of different starting concentrations of 1,6-HD and AA on the cell viability after 0, 3 and 24 h of oxidation at 30 °C for 30 h without pH control. The initial concentrations of 1,6-HD: AA in g/L were: A 0:0, B 5:0, C 10:0, D 15:0, E 20:0, F 10:10, G 10:30, and H 10:50

Fig. 5

Fed-batch oxidation of 1,6-HD to AA by the resting cells (5.4 g dry weight) of G. oxydans DSM 50049 in 1 L scale in 3 L bioreactor at 30 °C, 70% DO (aeration control), and pH controlled at pH 5–5.5. Symbols: accumulated AA concentration (g/L, filled triangle), 6-HHA concentration (g/L, filled square), and 1,6-HD concentration (g/L, filled diamond), and pH (filled circle)