석유탈황을 위한 생물촉매의 개발

장 제환, 이 범환, 장 용근, 장 호남

한국과학기술원 화학공학과 및 생물공정연구센터

Development of Biocatalysts for Petroleum Desulfurization

Je Hwan Chang,  Bum Hwan Lee, Yong Keun Chang, Ho Nam Chang

Dept. of Chemical Engineering and BioProcess Engineering Research Center,

Korea Advanced Institute of Science and Technology

Introduction

Sulfur dioxide released from combustion of oils brings detrimental effects on environment. As concerns about environment increases, more stringent regulations of sulfur content in petroleum are being implemented. But the availability of low-sulfur crude oil has decreased over the last ten years as a consequence of the increasing reserves of heavy oil. Therefore, refiners are increasingly requested to desulfurize high-sulfur crude oils. However, there are some limitations in complying with the more stringent regulations in employing current chemical processes so called hydrodesulfurization(HDS). Desulfurization with biocatalysts, called biodesulfurization (BDS) is expected to be a complement or a alternative to hydrodesulfurization10). BDS is expected to help refiners cost-effectively meet the restrictions and improve the value of high-crude oil reserves. A number of microorganisms are known to have the ability of desulfurizing organic sulfur compounds that exist in petroleum. Some microorganisms are reported to be able to degrade thiols and sulfides. But, the microorganisms capable of degrading thiols and sulfides are of little interest because thiols and sulfides are readily desulfurizable in HDS process. Dibenzothiophene(DBT) is widely accepted as a model hetelocycle compound that is recalcitrant to HDS process. Selective desulfurization, that is removal of sulfur atom without breaking carbon to carbon bonds, of DBT is possible by some strains. It was reported that Rhodococcus rhodochrous IGTS8, Rhodococcus erythropolis D-1, Corynebacterium sp. SY1 could selectively remove sulfur from DBT under aerobic conditions, and convert DBT to 2-hydroxybiphenyl (2-HBP)1),2),6) or 2,2-dihydroxybiphenyl (2,2-DHBP)3). The selective removal of sulfur from organic compounds by these strains is quite desirable in practical viewpoint, for valuable combustible carbons are retained without being lost as water soluble forms.

New DBT-degrading bacteria, tentatively identified as Gordona sp. CYKS1 and Nocardia sp. CYKS2 were isolated in this study. These strains were found to selectively remove sulfur from DBT. Growth and DBT-degradation characteristics and applications to desulfurization of a model oil(hexadecane  containing DBT) and a real diesel oil were investigated.

Experiments

The minimal salt medium(MSM) used in this study was a sulfur-free medium containing 10 g of glucose, 4.5 g of K2HPO4, 1.5 g of NaH2PO4, 2 g of NH4Cl, 0.2 g of MgCl2, 0.02 g of CaCl2, 1 mL of vitamin and 1 mL of sulfur-free trace metal solution per 1 L of deionized water. The relatively high concentrations of K2HPO4 and NaH2PO4 were to provide a good buffering capability to maintain pH around 7.0. DBT was dissolved in ethanol (100 mM) and added to a sterilized MSM unless otherwise indicated(DBT/ethanol system). Flask cultures of isolated strains were performed at 30 C, and 180 strokes per minute in a reciprocal shaking incubator. Fermentor-scale cultures were carried out in a 7 L jar fermentor with 5 L of working volume. Initial DBT concentration was set to 0.2 mM. In treating a model oil (DBT/hexadecane hereafter) and a real diesel oil with growing cells, the DBT concentration in model oil was set to 10 mM and volumetric phase ratios of oil to water were set to 1/10 or 1/20. In case of DBT/ethanol system, DBT and its metabolites was extracted using ethyl acetate. and analyzed using a reverse-phase HPLC as described previously7). In case of DBT/hexadecane or diesel oil was used, a dispersion sample was taken and centrifuged. The supernatant oil phase was analyzed by gas chromatography(GC) equipped with a sulfur chemiluminescence detector.

Results and discussion

Some amounts(about 0.05 mM) of dibenzothiophene sulfone (DBT-O2) and 2-HBP were detected as metabolites of DBT in primary enrichment culture. This meant some bacterial strains capable of transforming DBT to DBT-O2 and 2-HBP existed. Subsequent transfer cultures were carried out on DBT-coated solid medium. After 10 days of incubation, individual colonies that showed clear zones due to DBT degradation were selected and cultured in MSM with supplying DBT as a sole sulfur source. Eight strains were found to be capable of degrading DBT. Among them, three strains including CYKS1 and CYKS2 were found to be selectively removed sulfur atom from DBT. Isolated strains were identified on the basis of their morphological, physiological, and chemotaxonomical properties. The strains CYKS1 and CYKS2 were aerobe, nonmotile, nonsporeforming, and Gram-positive bacterium. The cell morphology were rod when observed by light microscopy. The strains had mycolic acid and meso-form of diaminopimelic acid(DAP) in cell wall. The major cell wall sugars were arabinose and galactose. The strains CYKS1 and CYKS2 had MK-9 and MK-8(H4) as menaquinone, respcetively. On the basis of these chemotaxonomic characteristics, CYKS1 and CYKS2 were tentatively identified as Gordona sp. and Nocardia sp., respectively.

From GC-EI/MS analysis, one metabolite that had a molecular weight of 170 and trace amount of two other metabolites that had a molecular weight of 216 were detected . The metabolite with a molecular weight of 170 was identified as 2-HBP and one of two metabolites whose molecular weight were 216 was identified as DBT-O2 by comparing its mass spectra with that of authentic samples. However, the other metabolite with a molecular weight of 216 was not identified.. Since 2-HBP concentration did not decrease after DBT and glucose were depleted, 2-HBP was thought to be the dead-end metabolite of DBT. Several pathways which sulfur in DBT is selectively removed have been proposed. The dead-end metabolites are biphenyl, 2-HBP or 2,2-DHBP. It has been reported that another metabolite called 2-(2-hydroxyphenyl) benzenesulfinate(HPBS) exists between DBT-O2 and 2-HBP in case of Rhodococcus rhodochrous IGTS84). Although it was not confirmed yet, the above mentioned unidentified metabolite with a molecular of 216 was considered to be HPBS. Therefore the metabolic pathway of DBT degradation was considered to be similar to that of Rhodococcus rhodochrous IGTS8. The proposed DBT degradation pathway is shown in Figure 1.

Figure 2 shows the time course of DBT utilization by the strain CYKS1 in a MSM supplemented with 0.3 mM of DBT as a sole sulfur source. The strain showed a maximum growth (2.7 g/L in DCW) at 80 hours of cultivation. The pH decreased from 7.4 to 5.9. Desulfurization activity and cell growth decreased about 23 % according to this change of pH. A major product accumulated was identified as 2-HBP by GC-MS. The conversion of DBT to 2-HBP was about 95 % and 2-HBP concentration did not decreased during the stationary phase. 2-HBP could be accumulated up to 0.25 mM in the culture medium and was not used as a carbon source for cell growth. No other metabolites except 2-HBP were detected during the batch cultivation.

Fig. 1 Metabolic pathway of DBT	Fig. 2 Batch culture of CYKS1; ●, cell;

■, glucose; ▲, DBT; ◆, 2-HBP

Studies on the isolation of DBT-degrading strains and characteristics of isolated strains have been carried out in DBT/ethanol system in most cases. But, such a system has some limitations in practical viewpoints. In actual situation, oil desulfurization is a two-phase process with organic sulfur compounds like DBT in the oil phase and cells in aqueous phase. Therefore, the DBT/ethanol system is far from a real situation of oil treatment. In addition, influence of the oil itself on the microorganism could not be examined in such a system. Hexadecane containing DBT(DBT/hexadecane) was chosen as a model oil system for real oil treatment as a previous report9). As shown in Figure 3, 10 mM of DBT in hexadecane was completely desulfurized in 72 hours. Ten millimoles of DBT is equal to 0.03 wt.% of sulfur. The cell concentration reached 3.2 g-dry cell weight/L in the meanwhile. In this case, the desulfurization rate per unit volume of oil/water dispersion was 0.449 mg-sulfur/L-dispersion/h. The cell showed better growth in DBT/hexadecane system. It was previously found that the desulfurization rate significantly increased when a hydrocarbon was added to DBT/ethanol system5),8). They explained it by using the term carrier effect. We speculate that hydrocarbons with the capability of dissolving DBT increase the chance for the cells adhered to them to contact DBT, and that this may explain the higher desulfurization rate for the case of DBT/hexadecane system.

The feasibility of diesel oil desulfurization was investigated. Light gas oil(LGO) was used in this series of experiments. The LGO used was not treated with hydrodesulfurization process and had 0.3 wt% of sulfur. One milliliters of LGO was added to 20 mL of MSM and then Gordona sp. CYKS1 was inoculated. Total sulfur content decreased from 0.30 to about 0.23 wt% in 48 hours and no further decrease was observed thereafter. The average desulfurization rate during the first 48 hours was 1.121 mg-sulfur/L-dispersion/h. Figure 4 shows that sulfur compounds which have a longer retention time in GC analysis were more readily desulfurized than those having a shorter retention time. Considering that compounds with a longer retention time than DBT are generally recalcitrant to HDS processes, BDS processes have a potential to be a complement or alternative to HDS processes.

Fig. 3 Desulfurization of DBT/hexadecane	Fig. 4 GC-SCD chromatogram of LGO	before (A) and after (B) treatment

Acknowledgment

This work was supported by grant of the R & D Management Center for Energy and Resources as part of the Clean Energy Program.

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