Biotechnology Letters 23: 899–901, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
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Biodegradation of dibenzothiophene by thermophilic bacteria A. Bahrami1 , S.A. Shojaosadati1,∗ & G. Mohebali2 1 Biotechnology Group,
Chemical Engineering Department, Tarbiat Modarres University, Tehran, Iran Institute of Petroleum Industry, Tehran, Iran ∗ Author for correspondence (Fax: +98 21/8005040; E-mail: shoja
[email protected]) 2 Research
Received after revisions 27 March 2001; Accepted 27 March 2001
Key words: biodegradation, crude oil, dibenzothiophene, thermophilic bacteria
Abstract Anaerobic microbial biodegradation of dibenzothiophene (DBT) was studied using thermophilic bacteria obtained from crude oil. A mixed culture was obtained that degraded 98% of DBT at 0.5 mg ml−1 at 65 ◦ C over 15 days both in the presence and in the absence of Methyl Viologen.
Introduction
Microorganisms and culture conditions
Elimination of pollutants such as sulfur compounds, from the heavy oil fractions, is a major goal of the petroleum industry. Currently, petroleum is treated thermo-chemically to decrease its sulfur content. This process is energy intensive due to its requirement for high pressures and high temperature. In addition, the process is unsuitable for certain sulfur compounds in oil, particulary polyaromatic sulfur hetrocyclics found in the heavier fractions. Over the past 50 years, attempts have been made to develop superior alternative processes, including microbial desulfurization, utilizing both aerobic and anaerobic consortia (Armstrong et al. 1995, Lizama et al. 1995, TS Kim et al. 1990, HY Kim et al. 1990, Eckart et al. 1986, Kohler et al. 1984), in which sulfate reducing bacteria are capable of reducing organic sulfur to hydrogen sulfide. In this paper we describe the anaerobic biodegradation of dibenzothiophene by a mixed culture of thermophilic bacteria obtained from crude oil.
Crude oil samples were taken from the Southern Iranian oil field facilities. Microorganisms were enriched by adding 70 ml crude oil to 700 ml of sterile media in 1000 ml sterile bottles under H2 at 65 ◦ C. Growth medium consisted of KH2 PO4 0.5 g, NH4 Cl 1 g, CaSO4 1 g, MgSO4 · 7H2 O 2 g, yeast extract 1 g, FeSO4 · 7H2 O 0.5 g, NaCl 10 g, ascorbic acid 0.1 g, thioglycollic acid 0.1 g, and 3.5 ml 50% (w/v) sodium lactate in 1000 ml distilled water. Final pH was between 7 and 7.5. Rezazurine indicator was added at 1 mg/1000 ml media to create a medium Eh of less than −100 mV. To enrich the samples, the headspace of vial was filled with H2 and cultures were incubated at 65 ◦ C for 30 days.
Matherials and methods Chemicals All chemicals used were of reagent grade.
Biodegradation experiments At the end of enrichment, 5 ml culture was centrifuged anaerobically (10 000 g, 20 min), and suspended in 10 ml medium without sulfate. This medium was as above but without MgSO4 and FeSO4 which were replaced by the corresponding amounts of MgCl2 and FeCl2 , then 0.1 ml of 2.5% (w/v) Methyl Viologen and 0.1 ml 5% (w/v) dibenzothiophene in dimethylformamide were added to the cell suspension. The headspace of the vial was filled with H2 and the vial was incubated at 65 ◦ C for 15 days. Biodegradation
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Fig. 1. HPLC of dibenzothiophene biodegradation by a mixed culture of bacteria. Detection: UV 280 nm; sample: 5 µl of ethyl acetate extract.
experiments were carried out in 20 ml anaerobic serum bottles.
Analyses Degraded samples were acidified to pH 2 with 1 M HCl, extracted with 3 vol ethyl acetate and then the organic fraction evaporated and redissolved in ethyl acetate. The final samples were analysed for dibenzothiophene or biphenyl by GC-MS and also HPLC. The latter used a Nova Pak phenyl column (3.9 × 300 mm) with UV detector using acetonitrile/water (7:3, v/v) at 1 ml min−1 under isocratic conditions. The ethyl acetate extract was also used for GC-MS analyses on a Varian 3400 gas chromatograph using DB-5 column (25 m × 0.2 mm i.d.), Incos 50 series, mass selective detector. A temperature program starting at 50 ◦ C for 1 min, and then increasing to 280 ◦ C at 5 ◦ C min−1 was employed. The injector and detector temperature were 280 ◦ C.
Results and discussion The end points of biodegradation experiments were determined by production of H2 S which is easily visualized by the formation of black iron sulfide precipitates, generated from the reaction of H2 S with
Fig. 2. GC/MS analysis of dibenzothiophene biodegradation by a mixed culture of bacteria. Sample: 5 µl of ethyl acetate extract.
soluble ferrous ion in the growth medium. HPLC analyses showed that the thermophilic mixed culture had degraded 98% of dibenzothiophene (DBT) both in the presence and in the absence of Methyl Viologen. Methyl Viologen was used in these experiments as an electron mediator, which is essential for microbial desulfurization (Kurita et al. 1971). Based on these results, thermophilic bacteria can reduce DBT in absence of Methyl Viologen. In our experiments the concentration of biphenyl detected in growing cultures was very low (
