Biocatalytic desulfurization of thiophenic compounds and crude oil by newly isolated bacteria

Abstract

Microorganisms possess enormous highly specific metabolic activities, which enable them to utilize and transform nearly every known chemical class present in crude oil. In this context, one of the most studied biocatalytic processes is the biodesulfurization (BDS) of thiophenic sulfur-containing compounds such as benzothiophene (BT) and dibenzothiophene (DBT) in crude oils and refinery streams. Three newly isolated bacterial strains, which were affiliated as Rhodococcus sp. strain SA11, Stenotrophomonas sp. strain SA21, and Rhodococcus sp. strain SA31, were enriched from oil contaminated soil in the presence of DBT as the sole S source. GC-FID analysis of DBT-grown cultures showed consumption of DBT, transient formation of DBT sulfone (DBTO2) and accumulation of 2-hydroxybiphenyl (2-HBP). Molecular detection of the plasmid-borne dsz operon, which codes for the DBT desulfurization activity, revealed the presence of dszA, dszB, and dszC genes. These results point to the operation of the known 4S pathway in the BDS of DBT. The maximum consumption rate of DBT was 11 μmol/g dry cell weight (DCW)/h and the maximum formation rate of 2-HBP formation was 4 μmol/g DCW/h. Inhibition of both cell growth and DBT consumption by 2-HBP was observed for all isolates but SA11 isolate was the least affected. The isolated biocatalysts desulfurized other model DBT alkylated homologs. SA11 isolate was capable of desulfurizing BT as well. Resting cells of SA11 exhibited 10% reduction in total sulfur present in heavy crude oil and 18% reduction in total sulfur present in the hexane-soluble fraction of the heavy crude oil. The capabilities of the isolated bacteria to survive and desulfurize a wide range of S compounds present in crude oil are desirable traits for the development of a robust BDS biocatalyst to upgrade crude oils and refinery streams.

Keywords: 4S pathway; bacteria; biodesulfurization; crude oil; thiophenic compounds.

FIGURE 1

Growth curves of bacterial isolates SA11 (A), SA21 (B), and SA31 (C) in CDM containing glycerol as the sole C-source and different S-sources (DBT▪, DMSO▴, and SO₄•).

FIGURE 2

Growth (▴), time course of DBT consumption (▪), and 2-HBP formation (•) by bacterial isolates SA11 (A), SA21 (B), and SA31 (C).

FIGURE 3

Viability of SA11 cells in crude oil environment compared to control cultures containing DBT. Viability is expressed as a % of cells recovered (CFU after 48 h on DBT-agar plates) from assays containing different crude oil concentrations (10–50% vol/vol) at different time intervals.

FIGURE 4

(A) Imaging of DNA fragments obtained from PCR assays of SA11 designed to amplify dszA (1.45 kb), dszC (1.25 kb), dszAB (2.5 kb), and dszAC (3.7 kb) genes, which code for the desulfurization of DBT via the 4 S pathway, (B) Organizational order of the dszABC genes in one operon in the newly isolated SA11. P, promoter.

FIGURE 5

Neighbor-joining consensus tree based on 16S rRNA gene sequence analysis showing the relationship between the strains SA11 and SA 31 and SA21 to the closest relatives in GenBank data base. The bar represents 0.1 substitutions per site and bootstrap values (indicated at the nodes) were calculated from 1000 trees.

FIGURE 6

The 4S pathway involved in desulfurization of DBT into 2-HBP and sulfite mediated by the activity of two monooxygenases (DszC, DszA), a desulfinase (DszB) and an oxidoreductase (DszD). DszC oxidizes DBT in two sequential steps forming DBTO and DBTO₂, DszA catalyzes the oxidative C-S bond cleavage in DBTO₂ forming HBPS, DszB catalyzes the conversion of HPBS into 2-HBP and sulfite (HSO₃^-). DszD delivers the reducing equivalent (FMNH₂) required for the function of DszC and DszA.