Improved biodesulfurization of hydrodesulfurized diesel oil using

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May 31, 2008 - of heterocyclic sulfur compounds found in petroleum that are resistant to desulfurization via the traditional hydrodesulfurization method, but can ...
Biotechnol Lett (2008) 30:1759–1764 DOI 10.1007/s10529-008-9748-8

ORIGINAL RESEARCH PAPER

Improved biodesulfurization of hydrodesulfurized diesel oil using Rhodococcus erythropolis and Gordonia sp. Guo-Qiang Li Æ Shan-Shan Li Æ Shi-Wei Qu Æ Qing-Kun Liu Æ Ting Ma Æ Lin Zhu Æ Feng-Lai Liang Æ Ru-Lin Liu

Received: 5 February 2008 / Revised: 6 May 2008 / Accepted: 6 May 2008 / Published online: 31 May 2008 Ó Springer Science+Business Media B.V. 2008

Abstract Substituted benzothiophenes (BTs) and dibenzothiophenes (DBTs) remain in diesel oil following conventional desulfurization by hydrodesulfurization. A mixture of washed cells (13.6 g dry cell wt l-1) of Rhodococcus erythropolis DS-3 and Gordonia sp. C-6 were employed to desulfurize hydrodesulfurized diesel oil; its sulfur content was reduced from 1.26 g l-1 to 180 mg l-1, approx 86% (w/w) of the total sulfur was removed from diesel oil after three cycles of biodesulfurization. The average desulfurization rate was 0.22 mg sulfur (g dry cell wt)-1 h-1. A bacterial mixture is therefore efficient for the practical biodesulfurization of diesel oil.

G.-Q. Li  L. Zhu College of Environment Sciences and Engineering, Nankai University, Weijin Road 94, Tianjin 300071, China G.-Q. Li  S.-S. Li  Q.-K. Liu  T. Ma (&)  F.-L. Liang  R.-L. Liu Tianjin Key Laboratory for Microbial Functional Genomics, Key Laboratory of Molecular Microbiology and Technology Ministry of Education, College of Life Sciences, Nankai University, Weijin Road 94, Tianjin 300071, China e-mail: [email protected] S.-W. Qu College of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China

Keywords Biodesulfurization  Gordonia sp.  Hydrodesulfurized diesel oil  Mixed bacteria  Rhodococcus erythropolis

Introduction The combustion of sulfur-containing fossil fuels contributes to environmental pollution in the form of SO2 emissions. A biodesulfurization process could remove sulfur from hydrodesulfurized diesel oil, yielding a product with a sulfur content low enough to meet current environmental regulations (Kilbane 2006). Dibenzothiophene (DBT), benzothiophene (BT) and their derivatives represent a broad range of heterocyclic sulfur compounds found in petroleum that are resistant to desulfurization via the traditional hydrodesulfurization method, but can easily be desulfurized using the biodesulfurization process (Gupta et al. 2005). A successful commercial process for biodesulfurization requires a biocatalyst capable of desulfurizing a broad range of sulfur-containing compounds. Most microorganisms can remove sulfur from only one type of compounds. Examples of desulfurizing organisms include Rhodococcus erythropolis IGTS8 (Denome et al. 1993), which removes organic sulfur from DBT and its derivatives, and Gordonia sp. 213E (Gilbert et al. 1998), which removes sulfur from BT and its derivatives. A few organisms such as Rhodococcus sp. KT462 (Tanaka et al. 2002), Mycobacterium goodii

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X7B (Li et al. 2003), M. phlei WU-0103 (Ishii et al. 2005), Rhodococcus erythropolis (Kobayashi et al. 2000, Zhang et al. 2007), Paenibacillus sp. A11-2 (Konishi et al. 2000), Sphingomonas subarctica T7b (Gunam et al. 2006), and Rhodococcus sp. ECRD-1 (Grossman et al. 2001) have the ability to desulfurize both DBT and BT. However their efficiency of desulfurization of BT and DBT is not equal, and the effectiveness and throughput of the process are limited by the lowest efficiency rate. In the present study mixtures of R. erythropolis DS-3 and Gordonia sp. C-6 were employed as catalysts for the biodesulfurization of hydrodesulfurized diesel oil. The structural diversity of potential substrates and effectiveness in desulfurizing hydrodesulfurized diesel oil were determined.

Materials and methods

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Desulfurization diesel oil by washed cells Rhodococcus erythropolis DS-3 and Gordonia sp. C-6 were separately cultured at 30°C in BSM medium with shaking. DBT was used as the sole sulfur source for culturing R. erythropolis DS-3, and BT was used as the sole sulfur source for culturing Gordonia sp. C-6. After cultivation, each culture was separately centrifuged (4 9 103g for 10 min at 4°C), washed twice with 100 mM potassium phosphate buffer (pH 7.5) and then suspended them in the potassium phosphate buffer at proper proportion to 17 g [dry cell wt (DCW)] l-1 with 2% (w/v)glucose as an energy source. The model diesel oil was prepared with 0.5 mM of each heterocyclic sulfur compound in n-hexadecane (Li et al. 2008). Desulfurization of model diesel oil or diesel oil was performed as follows: 10 ml hydrodesulfurized diesel oil and 40 ml bacterial suspension were shaken in 250 ml baffled flasks at 30°C.

Chemicals Substrate and product analysis Benzothiophene, 2-methyl-benzothiophene, 3-methylbenzothiophene, 5-methyl-benzothiophene, thianaphthene-2-carboxylic acid, dibenzothiophene, 1-methyldibenzothiophene, 2-methyl-dibenzothiophene, 4methyl-dibenzothiophene, 4,6-dimethyl-dibenzothiophene, thiophene, 2,20 -bithiophene, thioanisole and diphenyl sulfide were purchased from Acros. The hydrodesulfurized diesel oil was obtained from Fushun Research Institute of Petroleum & Petrochemicals (SINOPEC, China). All other reagents were of analytical grade and were obtained from various commercial sources. Bacterial strains and medium Rhodococcus erythropolis DS-3, a strain capable of desulfurizing DBT and its derivatives (Ma et al. 2002), and Gordonia sp. C-6, which is capable of desulfurizing BT and its derivatives (Li et al. 2006), were isolated from oil-contaminated soil of GuDao Oil Field. They were grown in basal salts medium (BSM) supplemented with 0.5 mM of various organic sulfur compounds to provide a sulfur source. BSM contained (per liter) 4 g glycerol, 2.44 g KH2PO4, 14.04 g Na2HPO412H2O, 2 g NH4Cl, 0.4 g MgCl26H2O, 0.01 g FeCl3, 0.02 g CaCl2, and 200 ll vitamin mixture (Li et al. 2003).

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Culture samples (0.5 ml) were withdrawn and centrifuged (9 9 103g, 10 min). The oil phase was separated for the subsequential analysis, and the aqueous phase was extracted with an equal volume of ethyl acetate. The extracts analyzed by HPLC according to the method described by Li et al. (2007). Hydrocarbons were detected by GC equipped with flame ionization detection (GC-FID). The organic sulfur compounds and desulfurization products in diesel oil were determined by GC equipped with atomic emission detection (GC-AED). The gas chromatogram equipped with a HP-5MS column (0.32 mm i.d. 9 30 m length) and operated with helium as carrier gas. The degree of sulfur removal from the diesel oil was determined by comparing the difference in sulfur content between treated and untreated oil.

Results and discussion Heterocyclic sulfur compounds in hydrodesulfurized diesel oil The content of inhomogeneous heterocyclic sulfur compounds in hydrodesulfurized diesel oil used in this

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1761

160

-1

Content of sulfides(mg l )

140 120 100 80 60 40 20 0

1,4,7-TMDBT

1,3,7-TMDBT

2,4,8-TMDBT

2,4,6-TMDBT

1,9-DMDBT

1,7-DMDBT

1,2-DMDBT

1,3-DMDBT

2,4-DMDBT

2,6-DMDBT

2,3-DMDBT

3,4-DMDBT

4,6-DMDBT

4-EDBT

C4DBT

C2DBT

C3DBT

DBT

C1DBT

C5BT

C4BT

C2BT

C3BT

BT

C1BT

C4 or C5T

Fig. 1 Sulfur components of the hydrodesulfurized diesel oil. Organic sulfur-containing compounds were determined by GCAED. The total sulfur content of the oil was 1.26 g l-1, containing 418 mg BTs l-1 and 840 mg DBTs l-1. * Benzothiophene (BT), dibenzothiophene (DBT), ethyldibenzothiophene (EDBT), dimethyldibenzothiophene (DMDBT), trimethyldibenzothiophene

(TMDBT), Cx- thiophene, Cx-benzothiophene and Cx-dibenzothiophene (Cx-T, Cx-BT, and Cx-DBT, x = 1, 2, 3, 4 & 5, x indicated the carbon number of the alkyl substituent group of BT and DBT derivatives), the numbers in 4-EDBT, 4, 6-DMDBT, or 2,4,6-TMDBT indicated the position of alkyl substituent group in EDBT, DMDBT, or TMDBT.

study was determined using GC-AED (Fig. 1). The total sulfur content of the oil was 1.26 g l-1, including 418 mg BTs l-1 and 840 mg DBTs l-1. The concentrations of C3-BT, C5-BT, C1-DBT, and C2-DBT were greater than 100 mg l-1. Dimethyl-DBTs and trimethyl-DBTs accounted for approximately 15% (w/w) of the total sulfur content. These results indicated that the substituted BTs and DBTs are still the main component of sulfur compounds in hydrodesulfurized diesel oil. It is important to determine the composition and concentration of these sulfur compounds for selecting an appropriate desulfurization biocatalyst.

Table 1 Degradation of heterocyclic sulfur compounds by the mixed cells for 24 h

Substrate specificity and desulfurization activity of bacterial mixture The employed desulfurization biocatalyst was the mixture of washed cells of R. erythropolis DS-3 and Gordonia sp. C-6. Bacterial degradation of heterocyclic sulfur compounds was investigated using the mixed cells of the two bacteria at 1:1 (w/w) in an oil/water reaction system with model diesel oil as the substrates. Results listed in Table 1 demonstrated that the mixed bacteria were capable of desulfurizing various heterocyclic sulfur compounds commonly found in diesel oil, including BT, 2-Methyl-BT, 5-Methyl-BT, DBT, 4-Methyl-DBT, and 4,6-Dimethyl-DBT, with a degradation ratio above 90%

Substratea

Degradationb (%)

Benzothiophene

98

2-Methyl-benzothiophene

86

3-Methyl-benzothiophene

33

5-Methyl-benzothiophene

97

Dibenzothiophene

98

1-Methyl-dibenzothiophene 2-Methyl-dibenzothiophene

5 31

4-Methyl-dibenzothiophene

97

4,6-Dimethyl-dibenzothiophene

96

2,20 -Bithiophene

10

Dimethyl sulphoxide

98

Diphenyl sulfide

9

Phenyl sulfide

8

Thianaphthene-2-carboxylic acid

23

Thianthrene

24

Thioanisole

6

Thiophene

22

a

Each compound was at 0.5 mM in the model diesel oil

b

The total cells of R. erythropolis DS-3 and Gordonia sp. C-6 were 13.6 g DCW l-1 at 1:1(w/w) in the reaction system

(w/w) within 24 h. The DBT desulfurization activity of R. erythropolis DS-3 was 0.83 mg (S) (g DCW)-1 h-1 and the BT desulfurization activity of Gordonia sp.

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C-6 was 0.91 mg (S) (g DCW)-1 h-1. These results demonstrated that application of mixed bacteria to desulfurization increases the number of targeted substrates and enhances the desulfurization efficiency toward a variety of sulfur compounds. Desulfurization of hydrodesulfurized diesel oil Biodesulfurization of the hydrodesulfurized diesel oil was performed three cycles in an oil/water reaction system with the mixed cells of R. erythropolis DS-3 and Gordonia sp. C-6 at 2.2:1 (w/w) as the catalyst. This proportion was determined by desulfurization activities of the two bacteria and the content of sulfur compounds in hydrodesulfurized diesel oil. The products (substituted 2-HBP and o-hydroxystyrene) were separated after each desulfurization cycle. Fig. 2 GC-AED chromatogram of sulfur components of the hydrodesulfurized diesel oil before (a) and after (b) three cycles biodesulfurization at 30°C by the mixed cells. Cx-BT and Cx-DBTs were classified with standard substrates and the reference of previous report (Ishii et al. 2005). * Benzothiophene (BT), dibenzothiophene (DBT), dibenzothiophenes (DBTs), Cx-benzothiophene and Cx-dibenzothiophenes (CxBT and Cx-DBTs, x = 1, 2, 3, 4 & 5, x indicated the carbon number of the alkyl substituent group of BT and DBT derivatives)

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GC-FID analysis revealed that diesel oil samples from the reaction mixtures showed little change in the resolvable peaks, demonstrating that the alkane structure and content of the diesel oil was not significantly altered during this bioprocess. The desulfurization effect was evaluated by GC-AED (Fig. 2). Total sulfur content of the hydrodesulfurized diesel oil was reduced from 1.26 g l-1 to 180 mg l-1 (Table 2), corresponding to a sulfur reduction of 86% (w/w). The average desulfurization rate of the mixed bacteria was 0.22 mg (S) (g DCW)-1 h-1. Previously reported sulfur reduction rates for benzothiophene-dibenzothiophene biodesulfurizing strains were 0.14 mg (S) (g DCW)-1 h-1 for Mycobacterium sp. X7B (Li et al. 2003), 0.12 mg (S) (g DCW)-1 h-1 for R. erythropolis XP (Yu et al. 2006).

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Table 2 Biodesulfurization of hydrodesulfurized diesel oil by the mixed cells Sample

Diesel oil

Total sulfur content (g l-1)

1.26

Sulfur content after each cycle (mg l-1) Cycle 1a

Cycle 2a

Cycle 3a

590 ± 20

320 ± 17

180 ± 13

a

Each desulfurization cycle was performed for 24 h at 30°C with shaking at 160 rpm. The total cells of R. erythropolis DS-3 and Gordonia sp. C-6 were 13.6 g DCW l-1 at 2.2:1 (w/w) used as catalyst in the reaction system

Conclusion Most organisms cannot efficiently desulfurize both BTs and DBTs which are present in hydrodesulfurized diesel oil. In our study, the mixture of washed cells of R. erythropolis DS-3 and Gordonia sp. C-6 were employed as catalyst for the biodesulfurization of hydrodesulfurized diesel oil. Rhodococcus erythropolis DS-3 could break the C–S bond of DBT and converting it into 2-hydrobenzophene (2-HBP) by the ‘‘4S’’ pathway (Ma et al. 2002, Piddington et al. 1995). Gordonia sp. C-6 could degrade BT to o-hydroxystyrene or o-hydroxyhyacinthin through a mechanism analogous to the ‘‘4S’’ pathway (Li et al. 2006). The desulfurization results indicated that the mixed bacteria could efficiently desulfurize most of the heterocyclic sulfur compounds in the hydrodesulfurized diesel oil. The desulfurization rate was 0.22 mg (S) (g DCW)-1 h-1, which was about 1.6fold of Mycobacterium sp. X7B and 1.8-fold of R. erythropolis XP. Acknowledgments This work was supported by China Postdoctoral Science Foundation (No. 20060400689) and National Natural Science Foundation of China (No. 50674058).

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