Exploring the mechanism of biocatalyst inhibition in microbial desulfurization

Abstract

Microbial desulfurization, or biodesulfurization (BDS), of fuels is a promising technology because it can desulfurize compounds that are recalcitrant to the current standard technology in the oil industry. One of the obstacles to the commercialization of BDS is the reduction in biocatalyst activity concomitant with the accumulation of the end product, 2-hydroxybiphenyl (HBP), during the process. BDS experiments were performed by incubating Rhodococcus erythropolis IGTS8 resting-cell suspensions with hexadecane at 0.50 (vol/vol) containing 10 mM dibenzothiophene. The resin Dowex Optipore SD-2 was added to the BDS experiments at resin concentrations of 0, 10, or 50 g resin/liter total volume. The HBP concentration within the cytoplasm was estimated to decrease from 1,100 to 260 μM with increasing resin concentration. Despite this finding, productivity did not increase with the resin concentration. This led us to focus on the susceptibility of the desulfurization enzymes toward HBP. Dose-response experiments were performed to identify major inhibitory interactions in the most common BDS pathway, the 4S pathway. HBP was responsible for three of the four major inhibitory interactions identified. The concentrations of HBP that led to a 50% reduction in the enzymes' activities (IC50s) for DszA, DszB, and DszC were measured to be 60 ± 5 μM, 110 ± 10 μM, and 50 ± 5 μM, respectively. The fact that the IC50s for HBP are all significantly lower than the cytoplasmic HBP concentration suggests that the inhibition of the desulfurization enzymes by HBP is responsible for the observed reduction in biocatalyst activity concomitant with HBP generation.

Fig 1

The four-step biodesulfurization 4S pathway. The first two steps, catalyzed by both DszC and DszD, are the conversion of DBT to DBT-sulfoxide (DBTO) and then to DBT-sulfone (DBTO₂). The third step, catalyzed by both DszA and DszD, is the conversion of DBTO₂ to HBPS. The final step is the conversion of HBPS to HBP by DszB. DBT-MO, DBT monooxygenase.

Fig 2

(A) Specific loading of HBP (white) and DBT (black) by the various resins tested at a resin concentration of 10 g/liter from a 0.50 (vol/vol) hexadecane-water solution initially containing 10 mM DBT and 10 mM HBP. (B) Total amount of HBP produced (white) and cytoplasmic HBP concentration (black) in the four-component BDS experiment at 15.5 g DCW/liter and with an oil fraction of 0.50 (vol/vol) and 10 mM DBT. Specific HBP loadings are shown above the black columns. Data shown are the averages and standard deviations of 3 replicates.

Fig 3

Enzyme kinetics for DszA (A), DszB (B), DszC (C), and DszD (D). Diamonds, the data; solid lines, Michaelis-Menten model fits for DszA, DszB, and DszD and substrate inhibition model for DszC. Data shown are the averages and standard deviations of 3 replicates.

Fig 4

Normalized desulfurization enzyme activity at different inhibitor concentrations for the four major inhibitory interactions identified in the 4S pathway. (A) DszA inhibition by HBP has an IC₅₀ of 60 ± 5 μM; (B) DszB inhibition by HBP has an IC₅₀ of 110 ± 10 μM; (C) DszC inhibition by HBPS has an IC₅₀ of 15 ± 2 μM; (D) DszC inhibition by HBP has an IC₅₀ of 50 ± 5 μM. Data shown are the averages and standard deviations of 3 replicates.

Fig 5

Noncompetitive inhibition of DszC by HBPS and HBP. (A) DszC activity over a range of DBT substrate concentrations from 0 to 5 μM and HBPS concentrations of 0 μM (closed diamonds), 5 μM (closed squares), 25 μM (closed triangles), and 50 μM (crosses). (B) DszC activity over a range of DBT substrate concentrations from 0 to 5 μM and HBP concentrations of 0 μM (closed diamonds), 100 μM (closed squares), and 500 μM (closed triangles). Solid lines are model fits from equation 15.

Fig 6

Enzyme inhibition model predictions for BDS bioreactor experiment. (A) Volumetric desulfurization rate data (circles) and model prediction (line). V_BDS, volumetric desulfurization rate of BDS. (B) Concentration of HBP in the oil phase data (circles) and model (line). (C) Concentration of HBP in the water phase data (circles) and model (line). Data shown are the averages and standard deviations of 3 replicates.