Rate-limiting step analysis of the microbial desulfurization of dibenzothiophene in a model oil system

Abstract

A mechanistic analysis of the various mass transport and kinetic steps in the microbial desulfurization of dibenzothiophene (DBT) by Rhodococcus erythropolis IGTS8 in a model biphasic (oil-water), small-scale system was performed. The biocatalyst was distributed into three populations, free cells in the aqueous phase, cell aggregates and oil-adhered cells, and the fraction of cells in each population was measured. The power input per volume (P/V) and the impeller tip speed (vtip ) were identified as key operating parameters in determining whether the system is mass transport controlled or kinetically controlled. Oil-water DBT mass transport was found to not be limiting under the conditions tested. Experimental results at both the 100 mL and 4 L (bioreactor) scales suggest that agitation leading to P/V greater than 10,000 W/ m(3) and/or vtip greater than 0.67 m/s is sufficient to overcome the major mass transport limitation in the system, which was the diffusion of DBT within the biocatalyst aggregates.

Keywords: Rhodococcus erythropolis IGTS8; aggregation; biodesulfurization; dibenzothiophene; power input per volume; rate-limiting step.

Figure 1

Mechanistic steps in a BDS system at high cell density. Biocatalyst may be present in one of three populations: free cells in aqueous phase, oil-adhered cells and cells in aggregates. Oxygen transport and uptake is necessary because the 4S pathway is an oxidative pathway that requires 3 moles of O₂ per mole of DBT desulfurized.

Figure 2

Volumetric mass transport coefficient (k_wa) for the oil-to-water mass transport of DBT from hexadecane to water at mixing speeds from 300-500 RPM (A) and oil fractions from 0.05 to 0.25 (B). Experiments were done in the small-scale system. Solid and dashed lines are model predictions from equation 9.

Figure 3

(A) - Total area covered by the cells at three different cell densities in aqueous-phase only system was measured using CellProfiler. (B) – Cells at a density of 15.5 g DCW/L were agitated at 200 to 1000 RPM, which led to decreasing mean aggregate sizes (right to left along the x-axis). Fraction of cell in aggregates (squares) and total area covered by cells (diamonds) were also measured.

Figure 4

Initial DBT desulfurization rate data (diamonds) and Michaelis-Menten fit (line) for R. erythropolis IGTS8 cells in aqueous-phase-only system with a cell density of 0.05 g DCW/L. The line plotted corresponds to values of k_cat of 13.2 ± 1.6 µmole DBT/g DCW/h and K_m of 10 nM.

Figure 5

Desulfurization rate by a 15.5 g DCW/L resting cell suspension as a function of HBP accumulated in the aqueous media. Initial DBT concentration was 1 mM. No HBP was added exogenously. Diamonds represents data and the model fit corresponding to equation 10 is shown by the line.

Figure 6

Small-scale, three-component BDS experiments at a cell density of 15.5 g DCW/L, oil fraction 0.25, initial DBT in oil of 10 mM and mixing speeds of 500, 800 and 1000 RPM. (A) - Effect of mixing speed on Sauter mean aggregate size (open diamonds) and on effectiveness factor (filled squares); (B) - Effect of mixing speed on the fraction of cells in each biocatalyst population: free cells (open diamonds), oil-adhered cells (filled squares), cells in aggregates (filled triangles); (C) - Increasing mixing speed led to a clear increase in the contribution to total specific desulfurization rate by cells in all three populations: free cells (black), oil-adhered cells (gray), aggregate cells (white).

Figure 7

(A) – Effect of power input per volume (P/V) on the fraction of the maximum desulfurization rate attained (R_total/R_max). (B) – Effect of impeller tip speed (v_tip) on the fraction of the maximum desulfurization rate attained (R_total/R_max). Diamonds represent data and line corresponds to least-squares correlation equation shown on plot.