SciELO - Scientific Electronic Library Online

Home Pagelista alfabética de revistas  

Servicios Personalizados




Links relacionados


Journal of the Chilean Chemical Society

versión On-line ISSN 0717-9707

J. Chil. Chem. Soc. v.52 n.4 Concepción  2007 


J. Chil. Chem. Soc, 52, N° 4 (2007), págs: 1299-1301





1 Institute of Chemical Sciences, University of Peshawar, N.W.F.P, 25120, Pakistan
2 Department of Chemical Engineering, YIC, Yanbu AL Sinayah, KSA


Removal of benzothiophene was studied with pseudomonas putida. Crude oil distillates were inoculated with p. putida and desulphurization activity was studied as a function of inoculation time. Significant amount of benzothiophene has been depleted in reaction time of 12 hours in all the three distillates studied. The depletion observed was 58.38 %, 60.90 % and 59.29 %, in case of light, middle and heavy fractions, respectively. Benzothiophene was concentrated mostly in the heavy fraction (570 ppm) followed by medium fraction (440 ppm), followed by light fraction (334 ppm). Benzothiophene in virgin and variously inoculated samples was determined spectrometrically by using GC-MS technique.

Key words: Petroleum, environment, sulphur, microbes, bacterial strain


Crude petroleum and coal derived liquids particularly heavy oils are laden with aromatic configurations containing sulphur as heteroatom (1). Among sulphur containing compounds, benzothiophene are the major sulphur-containing compounds of environmental concern. Emanation of sulphur oxides upon combustion of such products, creating a nuisance to the public as well as harm to various environmental media particularly air. Reduction or minimizing such emanations is a need of the day in order to meet with strict environmental legislations.

Various methods are being viewed in this direction. Removal prior combustion is gaining importance and various processes are being studied. Biodesulphurization proved to be very effective particularly for degradation of aromatic sulphurous compounds (2). Aerobic naphthalene degradation by pseudomonas (3-5) and the conversion of sulphur atom to sulfone (6-8) are researched in recent years. Many of the environmental studies have been undertaken with the inferences that heterocyclic compounds have been used to facilitate the aerobic and anaerobic degradation of such compounds (9-14). Degradation of benzothiophene and other aromatic compounds in crude oils has been extensively reported (15-20).

Anaerobic degradation may convert benzothiophene and di benzothiophene to hydrogen sulfide (16, 17) while inoculating with bacterium Desulfovibrio desulphuricans strain M6. Biphenyl has been found to be the major product of dibenzothiophene degradation (18, 19). In methanogenic degradation, mononuclear aromatic and alicyclic transformation products of benzothiophene, such as p-hydroxybenzene sulfonic acid, phenylacetic acid, benzoic acid, phenol, cyclohexane carboxylic acid, 2-hydroxythiophene, and many others analogues have been reported (20).

The present study is focused on the removal of benzothiophene sulphur analogue with p. putida from the crude oil distillate fractions in order to deplete them in this analogue of sulphur and to make their use environmentally friendly. We report on the influence of inoculation time on the depletion of sulphur in light, middle and heavy distillate fractions.


Collection of sample

Crude oil: Crude sample was obtained from Attock Oil Refinery, Morgah, Rawalpindi, Pakistan in a well closed ten made gallon, and was subsequently fractionated into light ( 200 °C), medium ( 300 °C) and heavy ( 360 °C) distillate fractions using laboratory distillation apparatus. All chemicals used were of analytical grade. Solvents used were distilled before use for rectification. Reference compounds and internal standards for GC-MS analyses used were of 100 % purity.

Coalmine water: Coalmine water was sampled from Much coalmine (Baluchistan, Pakistan) in a dry clean polythene bottle and incubated at 28 °C. Slides (100 NOS) were prepared and microscopically studied. From the structural and physiological appearances, p. putida presence was confirmed.

Culture of p. putida

The culture was performed according to the method described elsewhere (21, 22). The organism was grown in 100-ml serum bottles half-filled with bicarbonate buffered mineral medium (pH 7.3).The medium was reduced with 1 mM sodium sulfide, and then 10 mM sodium sulfate was added as an electron acceptor. The headspace was flushed with N2-C02 (80:20), and the bottles were closed with butyl rubber stoppers. The substrate benzothiophene (2 ml) was injected with a syringe through the stoppers as a source of energy and nutrient. The culturedp. putida was incubated at 28 °C in the dark, and microbial activity was monitored by measuring benzotiophene concentration.

Sample Inoculation

Cultured strain was inoculated to the oil samples i,e. light (b. pt 200 °C). medium (b. pt 300 °C) and heavy (b. pt 360 °C) distillate fractions . The mix was kept in contact for time duration selected from 12 hours to one-month (720 hr).The other conditions maintained were:

Reactions temperature: 30 °C

Reactions pressure: Atmospheric

Mass of oil and amount of bacteria: 3:1 volumetric water-to-oil ratio (WOR)

Type of reactor used: 2-liter stirred vessel (Applikon Inc.) equipped with temperature,  agitation, pH control, and dissolved oxygen (DO) monitor.

Atmosphere used: Air flow (400 ml min—-1)

After being kept in contact for specified duration of time, samples were removed with a syringe through the stoppers and injected to GC-MS for quantitative determination of benzopthiophene. After their testing, biological activity was stopped with adding 0.1 M NaOH in the samples.

GC-MS Analyses

GC-MS analyses were performed for benzothiophene using selective ion monitor (SIM) mode. Between 2 and 10 ml of the testing oil was filtered through a 0.22-µm-pore-size Millex-GP filter (Millipore, Bedford, Mass) in order to remove residual cellular particles. The instrument was calibrated with the standard benzothiophene solutions of known concentration before and after the original and bacterially treated oil samples. For quantification of benzopthiophene (BTH), an HP 5890 II plus GC with a 5972 mass spectrometer (MS) (Hewlett-Packard) operated in selective ion monitoring (SIM) mode was applied. A 1-µl filtered oil sample was injected onto a 0.25-mm-interior-diameter, 30-m column with a 0.5-µm film RTX-1 column (Restek) operated at 50.7-cms-1 linear velocity of helium carrier gas. The injector and detector temperatures were maintained at 90 and 220°C, for light fraction (200 °C), 195 and 305 °C for medium fraction (300 °C) whereas 295 °C and 370 °C for heavy fraction (360 °C), respectively. GC analyses with a sulphur-specific detector (FPD) verified the presence of the sulphur heteroatomic compound in the distillates under study.


Light fraction (200 °C)

Analysis of benzothiophene in original light fraction was performed by GC-MS. the value of BTH determined was 334 ppm. The same sample was inoculated with the pre cultured p. putida for 12 hr and benzothiophene content determined was 180 ppm. Comparing with the value obtained in case of original sample, considerable depletion can be seen, which shows that a significant amount of sulphur has been depleted during this initial inoculation time. In order to optimize the inoculation time, sample was kept in contact with the same microorganism beyond 12 hr. The inoculated time selected were; 24 h, 48 h, one week and one month. The data of sulphur depletion is provided in Table-1.The values of BTH determined in these samples were 162, 153, 144 and 139 ppm, respectively.

It can be seen from the data that depletion is quite significant and rapid in case of 12 hr inoculation time. The reason for this significant reduction of BTH in the initial inoculation time is that sulphur heterocyclic compounds can serve as a sulphur source for growth of microorganism under anoxic conditions as reported elsewhere (23) which release its desulphurizing enzymes (dzs) for the rapid consumption of benzopthiophene in testing distillates, and thus reproduce its self very rapidly forming colonies.

It can also be seen from the data compiled in Table 1 that when the time was extended beyond 12 hrs, sulphur depletion was in route in case of each inoculation. However, the extent is small compared to 12 hr inoculation. This dormant desulphurization is due to the fact that upon initial metabolism of sulphur configurations by the miocrorganism, aromatic acids are produced and hence released in to the solution medium leading to change in pH. This change in pH alters the affinity of microorganism and hence its capacity for uptake or binding with benzothiophene thereby minimizes the sulphur depletion. Aromatic acids are well-known by-products of anaerobic degradation of unsubstituted or alkylated aromatic hydrocarbons (24-26). The release of such acids in to the solution medium alters the pH, which lead to non availability of sulphur induced proteins hence the organism loses its ability to oxidize sulphur further.

Table-1 also shows the percent reduction obtained in case of original and variously inoculated samples. In case of 00 hr (original sample) to 12 hr inoculated time, 46.10 % reduction has being achieved, which seems to be a significant reduction percentage when compared to reduction percentages obtained for the rest of periods. In case of 00 h-24h, it was 51.49 % and in case of 00 hr-48 hr; 54.19 %, in case of 00 hr-one week; 54.88 % and in case of 00 hr-one month; 58.38 %. It is evident from the data that the reduction is considerable in case of 00 hr-one month. However, the difference with 00 hr-12 hr is only 12.28 % which is not a significant one. Therefore, the inoculation time of 12 hr is suggested to be appropriate time for maximum sulphur removal in sample under study.

Medium fraction (300 °C)

Medium fraction was also tested for benzothiophene depletion by the same strain. Same inoculation times were selected. BTH was determined in original sample was 440 ppm, & in case of 12 hr inoculation time was 238 ppm. Similarly, in case of 24 hr inoculation time, the amount determined was 209 ppm, in case of 48 hr, wasl93 ppm, in case of one-week was 185 ppm, and in case of one month was 172 ppm. The data has been assembled in Table-2. It is apparent from the data compiled that depletion of sulphur is very significant in the initial inoculation time. Depletion is observed in other inoculations; however, the amount depleted is minimal with respect to initial inoculation time. The reason for the insignificant reduction in sulphur in case of 24 hr, 48 hr, one week and one month inoculation times is the poor starvation survival of bacterium as a consequence of several stresses; alteration of pH of medium, nutrients deprivation & residual metabolites accumulation. Under these circumstances, the cell viability is affected. The organisms are denourished, rendered the chances of invasion on target configurations inactive and eventually die due to starvation. Bacterial starvation can affect the performance and cellular culturabilities; cell growth and cell survival and hence leading to cell death which stops the biological activities. Dead cells can also affect cell adhesion and biofilm attachment, hence leading to poor activity both in natural and engineering applications. In addition, formation of complex bacterial communities and some chemo repellents results in phenotypic changes in microorganisms there by rendering them inactive (27). The effect of organic solvents, pH of solution medium and nutrient deprivation on the activity of p. putida is reported elsewhere (28-30).

Reduction percentages have also been assembled in Table 2. It is evident that 12 hr inoculation time has caused maximum depletion when compared with original sample i.e. 45.90 %. An enhancement of 6.6 % can be seen in case of 24 hr inoculation time, the reduction is increased by 10.60 % in case of one week. Similarly, when the time was extended to one month, the increase in sulphur depletion was 15.00 %. Inoculation time beyond 12 hr caused depletion but not too significant, hence, 12 hr reaction time is suggested to be an optimum time.

Heavy fraction was also tested in a similar way as the light and medium fractions were tested. First of all, original heavy distillate was analyzed for BTH and the value was found to be 570 ppm. Distillate was then fed with p. putida BTH analysis was again done for various inoculation times. Benzothiophhene noted was 302, 278, 263, 244, and 232 ppm for 12 h, 24 h, 48 h, one week and one month reaction periods, respectively as shown in Table 3. Reduction % age of the depleted benzopthiophene sulphur versus the original and variously inoculated samples was also determined, which was 47.01, 51.22, 55.61, 57.19, and 59.2 % for 12 h, 24 h, 48 h, one week and a month reaction times, respectively (Table 3).

The depletion obtained in our study is quite significant compared to the previously reported microbial desulphurization particularly for middle and heavy oil fractions (31-32).


Sulphur removal with cultured p. putida in coal mine water was very significant in case of 12 h inoculation time. Sulphur depletion is in route further if time is extended beyond 12 hr, however the extent of desulphurization is not rapid and meaningful and hence costly in term of attendant requirement Concentration of benzopthiophene was found to be increased across the boiling point. Benzopthiophene BTH has been found to be concentrated in heavy distillation fraction compared to medium and light fractions.



1.     J. G. Mueller, P. J. Chapman, and P. H. Pritchard. Environ. Sci. Technol. 23, 1197(1989).        [ Links ]

2.     D.C. Bressler, J. A. Norman, and P. M. Fedorak.. Biodegradation, 8, 297 (1998)        [ Links ]

3.     S.A. Denome, D. C. Stanley, E. S. Olson, and K. D. J. Bacterial. 175, 6890(1993)        [ Links ]

4.     R.W. Eaton, and J. D. Nitterauer. J. Bacterial. 176, 3992 (1994)        [ Links ]

5.     M.F. Fedorak, and D. Grbic-Galic. Appl. Environ. Microbiol. 57, 932 (1991)        [ Links ]

6.     S.C. Gilbert, J. Morton, S. Buchanan, C. Oldfield, and A. McRoberts. Microbiology, 144, 2545 (1998)        [ Links ]

7.     M. J. Grossman, M. K. Lee, R. C. Prince, K. K. Garrett, G. N. George, and I. J. Pickering. Appl. Environ. Microbiol. 65, 181 (1999)        [ Links ]

8.     C. Oldfield, N. T. Wood, S. C. Gilbert, F. D. Murray, and F. R. Faure. Antonie Leeuwenhoek; 74,119( 1998)        [ Links ]

9.     E. Arvin, B. K. Jensen, and A. T. Gunderson. Appl. Environ. Microbiol. 55, 3221 (1989)        [ Links ]

10.   S. Dyreborg, E. Arvin, and K. Broholm. Biodegradation; 7,191 (1996)        [ Links ]

11.   S. Dyreborg, E. Arvin, and K. Broholm. Biodegradation; 7, 97 (1996)        [ Links ]

12.   D. Licht, B. K. Ahring, and E. Arvin. Biodegradation; 7, 83 (1996)        [ Links ]

13.   K.L. Londry, and J. M. Suflita. Environ. Toxicol. Chem. 17, 1199 (1998)        [ Links ]

14.   S. Meyer, and H. Steinhart. Chemosphere, 40, 359(2000)        [ Links ]

15.   K. Broholm, B. Nilsson, R. C. Sidle, and E. Arvin. J. Contain. Hydrol. 41, 239(2000)        [ Links ]

16.   S. Kurita, T. Endo, H. Nakamura, T. Yagi, and N. Tamiya. J. Gen. Appl. Microbiol. 17, 185 (1971)        [ Links ]

17.   S. Liu, M., W. J. Jones, and J. E. Rogers. Appl. Microbiol. Biotechnol. 41, 717(1994).        [ Links ]

18.   H. Kim, T. S. Kim, and B. H. Kim. Biotechnol. Lett. 10, 761 (1990)        [ Links ]

19.   H. Laue, K. Denger, and A. Cook. Appl. Environ. Microbiol. 63, 2016 (1997)        [ Links ]

20.   D. Grbic-Galic. Dev. Ind. Microbiol. 30, 237 (1989)        [ Links ]

21.   F. Widdel, and F. Bak Theprokaryotes, 4, 3352 (1992)        [ Links ]

22.   J.D. Cline. Limnol. Oceanogr. 14, 454 (1969)        [ Links ]

23.   F. Bak, and F. Widdel. Arch. Microbiol. 146, 170 (1986)        [ Links ]

24.   I.M. Cozarelli, R. P. Eganhouse, and M. J. Baedecker. Environ. Geol. 16, 135(1990)        [ Links ]

25.   J. Heider, A. M. Spormann, H. R. Beller, and F. Widdel. FEMS Microbiol. Rev. 22, 459 (1999).        [ Links ]

26.   X. Zhang and L. Y. Young. Appl. Environ. Microbiol. 63, 4759 (1997).        [ Links ]

27.   K. Sauer and A.K. Camper. Journal of Bacteriology, 183, 6579 ( 2001).        [ Links ]

28.   S. Isken, A. Derks, P.F.P. Wolffs, J.A.M. de Bont. Appl. Environ. Microbiol. 65, 2631(1999).        [ Links ]

29.   R. O. Jenkins and S. C. Heald. Applied Microbiolo and Biotechnolo 46, 388 (1996).        [ Links ]

30.   K. O'Connor, W. Duetz, B. Wind and A.D. Dobson. Appl. Environ. Microbiol, 62, 3594 (1996).        [ Links ]

31.   S. M. Armstrong, B. M. Sankey, G. Voordouw. Fuel, 76 (3), 223 (1997).        [ Links ]

32.   M.J. Grossman , M.K. Lee , R.C. Prince, K.K. Garrett, G.N. George, I.J. Pickering. Appl. Environ Microbiol. 65(1)181 (1999).        [ Links ]




Creative Commons License Todo el contenido de esta revista, excepto dónde está identificado, está bajo una Licencia Creative Commons