Efficient glycerol transformation by resting Gluconobacter cells

Abstract

In the present work, glycerol biotransformation using Gluconobacter strains was studied with a process intensification perspective that facilitated the development of a cleaner and more efficient technology from those previously reported. Starting from the industrial by-product, crude glycerol, resting cells of Gluconobacter frateurii and Gluconobacter oxydans were able to convert glycerol under batch reactor conditions in water with no other additive but for the substrate. The study of strains, biomass:solution ratio, pH, growth stage, and simplification of media composition in crude glycerol bioconversions facilitated productivities of glyceric acid of 0.03 g/L.h and 2.07 g/L.h (71.5 g/g % pure by NMR) of dihydroxyacetone (DHA). Productivities surmounted recent reported fermentative bioconversions of crude glycerol and were unprecedented for the use of cell suspended solely in water. This work proposes a novel approach that allows higher productivities, cleaner production, and reduction in water and energy consumption, and demonstrates the applicability of the proposed approach.

Keywords: Gluconobacter; biotransformation; dihydroxyacetone; glyceric acid; glycerol.

Figure 1

Pure glycerol bioconversion with Gluconobacter strains. GA (dots) and DHA (stripes) production after 20 hr by resting cells of Gluconobacter frateurii (a) and Gluconobacter oxydans (b) collected during different growth stages. Kinetics of DHA and GA production from pure glycerol with resting cells of G. frateurii (c) and G. oxydans (d). DHA (●), GA (■),pH (▲). Reactions were performed in 30 ml total volume at 30°C using 2 mg dry cell weight and 50 g/L initial glycerol concentration. Further conditions are described in Methods

Figure 2

DHA and GA production after 45 hr starting from different glycerol concentrations. Gluconobacter frateurii (a), Gluconobacter oxydans (b). % Conversion (dotted line), DHA (stripes), GA (dots). Reactions were performed in 30 ml total volume at 30°C using 20 mg dry cell weight. Further conditions are described in Methods

Figure 3

DHA and GA production after 45 hr starting from crude and pure glycerol. (a) (Gluconobacter frateurii), (b) (Gluconobacter oxydans). Products from pure glycerol (full line), products from crude glycerol (dotted line). DHA (upper circles), GA (lower circles) [Correction added on 26 November 2019 after first online publication: Figure 3 caption has been corrected]

Figure 4

Reuses of resting cells of Gluconobacter frateurii (a) and Gluconobacter oxydans (b) in the conversion of crude glycerol to DHA and GA. DHA (stripes), GA (dots). Reactions were performed in 30 ml total volume at 30°C using 20 mg dry cell weight. Each use cycle was after 20 hr. Further conditions are described in Methods

Figure 5

Fed‐batch conversion of crude glycerol using resting cells of Gluconobacter frateurii (a) and Gluconobacter oxydans (b). DHA (full line), glycerol (dashed line), and glycerol applications (arrows) were 25 g/L in (a) and 50 g/L in (b)

Figure 6

DHA production intensification using Gluconobacter oxydans as a catalyst and crude glycerol as substrate. (1) Starting conditions (glycerol 50 g/L, KH₂PO₄ 0.9 g/L, K₂HPO₄ 0.1 g/L, MgSO₄.7H₂O 1 g/L, pH 6 in distilled water, 2 mg of dry cell weight), (2) 10‐fold inoculum increase (20 mg of dry cell weight) conditions as in 1, (3) reaction media without salts, (4) use of regular water as reaction media, and (5) decrease in final total volume (20 mg of dry cell weight, 100 g/L glycerol in 15 ml regular water). All the reactions were performed at 30°C

Figure A1

Effect of initial pH of the reaction medium in the conversion of glycerol to GA (dots) and DHA (stripes) by resting cells of Gluconobacter frateurii (a) and Gluconobacter oxydans (b) [Correction added on 26 November 2019 after first online publication: Figure A1 caption has been corrected]

Figure A2

¹H NMR spectroscopy analysis of bioconversion endpoint (24 h). (a) Spectra of glycerol conversion with Gluconobacter oxydans. (b) Spectra of glycerol conversion with Gluconobacter frateurii. NMR spectral assignments: DHA: 4.405 (s, 1H); 3.563 (s, 4H). Glycerol: 3.763 (tt, 4.4, 9.9Hz, 1H); 3.642 (dd, 4.4, 11.7Hz, 2H); 3.547 (dd, 6.5, 11.7Hz, 2H). Glyceric acid: 4.139 (dd, 3.1, 5.7Hz, 1H); 3.823 (dd, 3.2, 11.9Hz, 1H); 3.734 (dd, 5.5, 11.7Hz, 1H). Methanol: 3.35 (s, 3H). Acetate: 2.05 (s, 3H)

Figure A3

DHA production using resting cells of Gluconobacter frateurii (a) and Gluconobacter oxydans (b), starting from crude glycerol (stripes) and crude glycerol in the presence of DHA (dots). Reactions for G. frateurii were started with 25 g/L glycerol or 25 g/L DHA and 25 g/L de glycerol. In the case of G. oxydans, reactions were started with 50 g/L of glycerol or 50 g/L of DHA and 50 g/L of glycerol [Correction added on 26 November 2019 after first online publication: Figure A3 caption has been corrected]

Figure A4

DHA production using resting cells of Gluconobacter oxydans in different reaction media. DHA (stripes), glycerol (dots). Control (distilled water, glycerol, KH₂PO₄, K₂HPO₄, MgSO₄.7H₂O), 1 (distilled water and glycerol), 2 (distilled water, glycerol, MgSO₄.7H₂O), 3 (distilled water, glycerol, KH₂PO₄, K₂HPO₄). Reactions were started with 50 g/L and analyzed after 20 hr [Correction added on XX November 2019 after first online publication: Figure A4 caption has been corrected]