Biocatalytic desulfurization of dibenzothiophene by Pseudomonas sp. strain KWN5.
Biocatalytic desulfurization of dibenzothiophene by Pseudomonas sp. strain KWN5
IDA BAGUS WAYAN GUNAM*1, I GUSTI AYU LANI TRIANI1, NYOMAN SEMADI
ANTARA1, AGUS SELAMET DUNIAJI2, YOHANES SETIYO3 AND
DEWA ADI SUPATA1
1
Bioindustry Laboratory, Department of Agro-Industrial Technology, 2Department of Food
Science and Technology, 3Department of Agricultural Engineering,
Faculty of Agricultural Technology, Udayana University, Denpasar, Bali, Indonesia
ABSTRACT
Pseudomonas sp. strain KWN5 was tested for the ability to use dibenzothiophene in ntetradecane as the sole of sulfur source. The strain could grow on mineral salt sulfur-free
(MSSF) medium with the n-tetradecane oil phase containing 200 ppm dibenzothiophene
(DBT) and desulfurize this compound. The DBT-desulfurizing ability of KWN5 is high
over a wide temperature range from 27 to 42oC, and the highest at 37oC. This strain could
grow well on incubation period for 4 days at 37 oC, pH 7, and glucose as the carbon source.
In that condition, growing cells of KWN5 could degrade 200 ppm DBT around 75.21%
within 96 h, indicating that this strain was very useful for the removal of DBT from oil.
Keywords: Desulfurization, dibenzothiophene, tetradecane, strain KWN5
INTRODUCTION
Production and world primary energy consumption showed a high increase. Energy
demand increases with increasing income level. The energy of the most widely used today
is still from fossil energy. Use of fossil energy, especially petroleum widely acknowledged
having benefits but also having a negative impact.
The result of incomplete combustion of petroleum and coal produce sulfur oxides
(SOx), which can cause environmental pollution such as air pollution and acid rain (Gunam
et al., 2006). Sources of air pollution in each area are different. Sources of air pollution
may come from motor vehicles, household activities, and industry (Laras 2006).
Petroleum contains sulfur compounds, including aromatic sulfur compounds such as
alkyl dibenzothiophene and benzothiophene. Its compounds cannot be removed by
conventional hydrodesulfurization (HDS) treatment using metallic catalysts (Furuya et al.
2003; Gunam et al. 2006). This proves that the use of HDS require high costs, so many
researchers turned its attention to seeking a more efficient alternative technologies
(Guerinik and Muttawah 2003).
One attempt to reduce the sulfur content of aromatic compounds in petroleum is
biodesulfurization process. In this process, the microbes use sulfur from petroleum as an
energy source for growth. To obtain optimal results in lowering the sulfur content of
petroleum, required certain types of bacteria that have the ability to degrade these
compounds. Efforts to reduce the sulfur content of aromatic compounds in petroleum can
be optimized (Jasrizal, 2009).
Results of previous studies (Gunam et al. 2009), showed that one strain has the highest
ability to degrade aromatic sulfur compounds, known as KWN5 strain (this strain was
isolated from soil samples derived from petroleum-contaminated soil near oil fields
Kawengan, Bojonegoro, East Java). Based on the above, it is necessary to investigate the
optimum conditions (temperature and pH) for the growth of this strain, which can degrade
the highest DBT compounds
MATERIALS AND METHODS
Materials
Dibenzothiophene (DBT) was purchased from Aldrich and Tetradecane was supplied
by Wako Pure Chemical Co., Osaka, Japan. Mineral Salt Sulfur Free (MSSF) Medium was
prepared by previous method (Gunam et al. 2006) and petroleum (light gas oil) was
supplied by Pertamina. All other reagents were of analytical grade and commercially
available.
A concentrated fraction of aromatic compounds (CA) was prepared by fractionation of
commercial light gas oil (Gunam et al. 2006).
*
Correspondence author: Phone: +62 361 222006; Fax: +62 0361 701801;
E-mail: ibwgunam@yahoo.com
Bacterial strains and culture media
The KWN5 strain was grown in a mineral salt sulfur-free (MSSF) medium (pH 7) with
CA as the sole sulfur source, as reported in our previous paper (Gunam et al. 2011). MSSFTD was the standard media for the desulfurization assay, consisted of MSSF: n-tetradecane
= 5:1, with DBT dissolved in n-tetradecane. Bacterial growth was determined by measuring
OD660 of water layer (Gunam et al. 2006).
Seed culture preparation
For seed culture production, KWN5 was cultivated at 30 oC in 500-ml Erlenmeyer
flasks containing 200 ml of MSSF medium with CA as the sole source of sulfur for 4 days.
The cells were harvested by centrifugation at 3500 rpm for 20 min at 4 oC, washed twice
with 0.85% saline solution and suspended in the same solution. The optical density at 660
nm (OD660) of the cell suspension was adjusted to 5.
Bio-desulfurization assay
Six milliliter of MSSF-TD medium containing DBT were inoculated with 0.1 ml of the
seed culture (OD660 = 5) and incubated at various temperature and initial pH for 4 days with
shaking (200 rpm). After incubation, the organic layer of n-tetradecane and water layer
were separated by centrifugation at 3500 rpm for 20 min at 4oC. Un-inoculated medium
served as controls and were treated in the same manner.
Analytical methods
Cells growth was measured turbidimetrically at 660 nm. The cell concentration was
determined from the linear relationship between the optical density at 660 nm (OD660) and
dry cells weight (drying at 105oC for 36 h). Measurements of DBT were performed using
GC with a flame ionization detector (GC-FID). The concentrations of DBT in growth
culture were analyzed by GC-17A (Shimadzu, Kyoto, Japan). Samples for GC analysis
were acidified to pH 2.0 with 1 M HCl and extracted from aqueous cell/DBT suspensions
by liquid–liquid extraction using ethyl acetate in a 2:1 ratio. A portion of the ethyl acetate
layer was removed and centrifuged and 1 µl of the supernatant was injected into a gas
chromatograph. The gas chromatograph was equipped with a fused silica capillary column,
CBPI-m25-025 (25 m 0.22 mm id, df ¼ 0.25 l), packed with silicone OV-1, SE-30
(Shimadzu, Tokyo, Japan). The flow rate of helium carrier was 1 ml/min. The column
temperature was programmed from 140 to 250ºC at 8ºC/min. The injector and detector
temperatures were maintained at 280 and 310ºC, respectively.
RESULTS AND DISCUSSIONS
Temperature-dependent desulfurization of DBT by growing cells of KWN5
The temperature is a major environmental factor that affecting physiological activity of
most prokaryotes. At optimum temperature, microbes perform biological activities at the
maximum rate such as growth and metabolism. The effects of temperature on DBT
degradation by growing cells of KWN5 were examined 96 hours cultivation under different
temperatures. As shown in Fig. 1, KWN5 grew significantly in the MSSF medium
containing 200 ppm DBT in tetradecane as the sole source of sulfur. Moreover, growing
cells of KWN5 exhibited high desulfurizing ability toward 200 ppm DBT in TD over a
wide temperature range from 27 to 47°C, and this was most efficient from 32 to 37°C.
From the results, it was clearly shown that microbial growth of KWN5 strain at 37°C
resulted the highest growth and desulfurizng activity. In that condition showed that the
sulfur content in model oil decreased from 200 to 55.74 ppm DBT (72,13% degradation)
over 4 days incubation. However, the activity suddenly decreased at 42°C. In contrast, no
reduction of sulfur was detected for the un-inoculated samples after treatment under the
same conditions. Kirimura et al., (2001) also reported that B. subtilis WU-S2B also
exhibited DBT-desulfurizing ability over a wide temperature range from 30 to 55°C, and
the activity suddenly decreased at 52°C.
FIG. 1. Effects of temperature on DBT desulfurization by growing cells of KWN5 strain.
KWN5 was cultivated in MSSF medium with 200 ppm DBT as sole source of
sulfur at various temperatures for 96 hours. Symbols: ◆, growth (OD660), and ▲,
DBT degradation.
Effect of various initial pHs on the growth and desulfurization activity
The effect of initial pH towards growth and desulfurization activity of KWN5 was
demonstrated in Figur 2. Growth of KWN5 was studied over a wide range of pH, ranging
from 6.0 until 8.0, and the maximal growth and desulfurization activity were obtained at
pH 7.0. The growth rate (OD660) and desulfurizing activity of KWN5 strain in pH 7.0
medium were 1.2 and 75.21%, respectively. The activity of KWN5 sharply decreased was
observed when the bacteria were grown in the medium with initial pH at lower and higher
then pH 7. However, growth of bacteria was not significantly affected by pH.
The result was almost same with other desulfurizing bacteria such as S. subarctica T7b
(Gunam et al. 2006), Gunam et al. 2011. Gunam et al. (2006) reported that S. subarctica
T7b had optimal growth and desulfurization activities when the value of pH was 7. When
the pH changes from 6.5 to 7.5, the degradation rate of DBt by KWN5 strain was kept at
about 72-75%. Whereas the degradation ability of the suspended cells was only 30-34%.
Meanwhile, the suspended cells lost more than 40% activity when the pH was lower than
5.5.
FIG. 2. Effects of initial pH on DBT degradation by growing cells of KWN5 strain.
KWN5 was cultivated in MSSF medium with 200 ppm DBT as sole source of
sulfur at various pH for 96 hours. Symbols: ◆, growth (OD660), and ▲, DBT
degradation.
CONCLUSIONS
The conclusion that can be drawn from this research were: At 37°C and pH 7 can
provide optimal conditions for growth of strain KWN5 with OD values at a wavelength of
660 was 1.2 and 200 ppm can degrade DBT in TD was 75.21%.
Pseudomonas sp. Strain KWN5 was great potential in degrading aromatic sulfur
compound contained in petroleum, it is necessary to conduct further research especially to
choose an effective method in its application in industry.
REFERENCES
Furuya T, Ishii Y, Ken-ichi Noda, Kino, K and Kirimura K. 2003. Thermophilic
biodesulfurization of hydrodesulfurized light gas oils by Mycobacterium phlei WU-F1,
FEMS Microbiology Letters 221:137-142.
Guerinik K2 and Al-Mutawah Q. 2003. Isolation and characterization of oil-desulfurizing
bacteria. World Journal of Microbiology and Biotechnology 19, 941–945.
Gunam IBW, Yaku Y, Hirano M, Yamamura K, Tomita F, Sone T and Asano K. 2006.
Biodesulfurization of alkylated forms of dibenzothiophene and benzothiophene by
Sphingomonas subarctica T7b. Journal of Bioscience and Bioengineering 101, 322327.
Gunam IBW, Duniaji AS, dan Triani IGAL. 2009. Biodesulfurisasi minyak bumi dengan
menggunakan bakteri pendegradasi sulfur dengan teknik sel terimobilisasi. Laporan
Penelitian Hibah Bersaing Tahap II. Universitas Udayana.
Gunam IBW, Duniaji AS, and Triani IGAL, and Sitepu A. 2011. Biodeslfurization of
dibenzothiophene by growing and immobilized cells of KWN5 strain. 3rd International
Conference on “Maintaining World Prosperity Through Bioscience, Biotechnology and
Revegetation ”, Depasar, Bali, Indonesia, 21-22 September 2011.
Jasrizal DC. 2009. Deskripsi Dokumen: Biodesulfurisasi Minyak bumi Sebagai Upaya
http://www.digilib.
Pengurangan
Pencemaran
Lingkungan.
Tesis
S2.
ui.ac.id//opac/themes/libri2/detail.jsp?id=92772&lokasi=lokal. Diakses Tanggal 28
Februari 2010.
Kirimura K, Furuya T, Nishii Y, Ishii Y, Kino K, and Usami S. 2001. Biodesulfurization of
dibenzothiophene and its derivates through the selective cleavage of carbon-sulfur
bonds by a moderately thermophilic bacterium Bacillus subtilis WU-S2B. J. Biosci.
Bioeng. 91: 262-266.
Laras BK. 2006. Pencemaran Oleh Hujan Asam Dalam Konteks Kebijakan Global.
http://www.rudyct.com/PPS702-ipb/12167/bambang_kl.pdf. Diakses tanggal 30 Juni
2010.
Schiller JE, and Mathiason DR. 1997. Separation method for coal-derived solids and heavy
liquids. Analytical Chemistry 49, 1225-1228.
IDA BAGUS WAYAN GUNAM*1, I GUSTI AYU LANI TRIANI1, NYOMAN SEMADI
ANTARA1, AGUS SELAMET DUNIAJI2, YOHANES SETIYO3 AND
DEWA ADI SUPATA1
1
Bioindustry Laboratory, Department of Agro-Industrial Technology, 2Department of Food
Science and Technology, 3Department of Agricultural Engineering,
Faculty of Agricultural Technology, Udayana University, Denpasar, Bali, Indonesia
ABSTRACT
Pseudomonas sp. strain KWN5 was tested for the ability to use dibenzothiophene in ntetradecane as the sole of sulfur source. The strain could grow on mineral salt sulfur-free
(MSSF) medium with the n-tetradecane oil phase containing 200 ppm dibenzothiophene
(DBT) and desulfurize this compound. The DBT-desulfurizing ability of KWN5 is high
over a wide temperature range from 27 to 42oC, and the highest at 37oC. This strain could
grow well on incubation period for 4 days at 37 oC, pH 7, and glucose as the carbon source.
In that condition, growing cells of KWN5 could degrade 200 ppm DBT around 75.21%
within 96 h, indicating that this strain was very useful for the removal of DBT from oil.
Keywords: Desulfurization, dibenzothiophene, tetradecane, strain KWN5
INTRODUCTION
Production and world primary energy consumption showed a high increase. Energy
demand increases with increasing income level. The energy of the most widely used today
is still from fossil energy. Use of fossil energy, especially petroleum widely acknowledged
having benefits but also having a negative impact.
The result of incomplete combustion of petroleum and coal produce sulfur oxides
(SOx), which can cause environmental pollution such as air pollution and acid rain (Gunam
et al., 2006). Sources of air pollution in each area are different. Sources of air pollution
may come from motor vehicles, household activities, and industry (Laras 2006).
Petroleum contains sulfur compounds, including aromatic sulfur compounds such as
alkyl dibenzothiophene and benzothiophene. Its compounds cannot be removed by
conventional hydrodesulfurization (HDS) treatment using metallic catalysts (Furuya et al.
2003; Gunam et al. 2006). This proves that the use of HDS require high costs, so many
researchers turned its attention to seeking a more efficient alternative technologies
(Guerinik and Muttawah 2003).
One attempt to reduce the sulfur content of aromatic compounds in petroleum is
biodesulfurization process. In this process, the microbes use sulfur from petroleum as an
energy source for growth. To obtain optimal results in lowering the sulfur content of
petroleum, required certain types of bacteria that have the ability to degrade these
compounds. Efforts to reduce the sulfur content of aromatic compounds in petroleum can
be optimized (Jasrizal, 2009).
Results of previous studies (Gunam et al. 2009), showed that one strain has the highest
ability to degrade aromatic sulfur compounds, known as KWN5 strain (this strain was
isolated from soil samples derived from petroleum-contaminated soil near oil fields
Kawengan, Bojonegoro, East Java). Based on the above, it is necessary to investigate the
optimum conditions (temperature and pH) for the growth of this strain, which can degrade
the highest DBT compounds
MATERIALS AND METHODS
Materials
Dibenzothiophene (DBT) was purchased from Aldrich and Tetradecane was supplied
by Wako Pure Chemical Co., Osaka, Japan. Mineral Salt Sulfur Free (MSSF) Medium was
prepared by previous method (Gunam et al. 2006) and petroleum (light gas oil) was
supplied by Pertamina. All other reagents were of analytical grade and commercially
available.
A concentrated fraction of aromatic compounds (CA) was prepared by fractionation of
commercial light gas oil (Gunam et al. 2006).
*
Correspondence author: Phone: +62 361 222006; Fax: +62 0361 701801;
E-mail: ibwgunam@yahoo.com
Bacterial strains and culture media
The KWN5 strain was grown in a mineral salt sulfur-free (MSSF) medium (pH 7) with
CA as the sole sulfur source, as reported in our previous paper (Gunam et al. 2011). MSSFTD was the standard media for the desulfurization assay, consisted of MSSF: n-tetradecane
= 5:1, with DBT dissolved in n-tetradecane. Bacterial growth was determined by measuring
OD660 of water layer (Gunam et al. 2006).
Seed culture preparation
For seed culture production, KWN5 was cultivated at 30 oC in 500-ml Erlenmeyer
flasks containing 200 ml of MSSF medium with CA as the sole source of sulfur for 4 days.
The cells were harvested by centrifugation at 3500 rpm for 20 min at 4 oC, washed twice
with 0.85% saline solution and suspended in the same solution. The optical density at 660
nm (OD660) of the cell suspension was adjusted to 5.
Bio-desulfurization assay
Six milliliter of MSSF-TD medium containing DBT were inoculated with 0.1 ml of the
seed culture (OD660 = 5) and incubated at various temperature and initial pH for 4 days with
shaking (200 rpm). After incubation, the organic layer of n-tetradecane and water layer
were separated by centrifugation at 3500 rpm for 20 min at 4oC. Un-inoculated medium
served as controls and were treated in the same manner.
Analytical methods
Cells growth was measured turbidimetrically at 660 nm. The cell concentration was
determined from the linear relationship between the optical density at 660 nm (OD660) and
dry cells weight (drying at 105oC for 36 h). Measurements of DBT were performed using
GC with a flame ionization detector (GC-FID). The concentrations of DBT in growth
culture were analyzed by GC-17A (Shimadzu, Kyoto, Japan). Samples for GC analysis
were acidified to pH 2.0 with 1 M HCl and extracted from aqueous cell/DBT suspensions
by liquid–liquid extraction using ethyl acetate in a 2:1 ratio. A portion of the ethyl acetate
layer was removed and centrifuged and 1 µl of the supernatant was injected into a gas
chromatograph. The gas chromatograph was equipped with a fused silica capillary column,
CBPI-m25-025 (25 m 0.22 mm id, df ¼ 0.25 l), packed with silicone OV-1, SE-30
(Shimadzu, Tokyo, Japan). The flow rate of helium carrier was 1 ml/min. The column
temperature was programmed from 140 to 250ºC at 8ºC/min. The injector and detector
temperatures were maintained at 280 and 310ºC, respectively.
RESULTS AND DISCUSSIONS
Temperature-dependent desulfurization of DBT by growing cells of KWN5
The temperature is a major environmental factor that affecting physiological activity of
most prokaryotes. At optimum temperature, microbes perform biological activities at the
maximum rate such as growth and metabolism. The effects of temperature on DBT
degradation by growing cells of KWN5 were examined 96 hours cultivation under different
temperatures. As shown in Fig. 1, KWN5 grew significantly in the MSSF medium
containing 200 ppm DBT in tetradecane as the sole source of sulfur. Moreover, growing
cells of KWN5 exhibited high desulfurizing ability toward 200 ppm DBT in TD over a
wide temperature range from 27 to 47°C, and this was most efficient from 32 to 37°C.
From the results, it was clearly shown that microbial growth of KWN5 strain at 37°C
resulted the highest growth and desulfurizng activity. In that condition showed that the
sulfur content in model oil decreased from 200 to 55.74 ppm DBT (72,13% degradation)
over 4 days incubation. However, the activity suddenly decreased at 42°C. In contrast, no
reduction of sulfur was detected for the un-inoculated samples after treatment under the
same conditions. Kirimura et al., (2001) also reported that B. subtilis WU-S2B also
exhibited DBT-desulfurizing ability over a wide temperature range from 30 to 55°C, and
the activity suddenly decreased at 52°C.
FIG. 1. Effects of temperature on DBT desulfurization by growing cells of KWN5 strain.
KWN5 was cultivated in MSSF medium with 200 ppm DBT as sole source of
sulfur at various temperatures for 96 hours. Symbols: ◆, growth (OD660), and ▲,
DBT degradation.
Effect of various initial pHs on the growth and desulfurization activity
The effect of initial pH towards growth and desulfurization activity of KWN5 was
demonstrated in Figur 2. Growth of KWN5 was studied over a wide range of pH, ranging
from 6.0 until 8.0, and the maximal growth and desulfurization activity were obtained at
pH 7.0. The growth rate (OD660) and desulfurizing activity of KWN5 strain in pH 7.0
medium were 1.2 and 75.21%, respectively. The activity of KWN5 sharply decreased was
observed when the bacteria were grown in the medium with initial pH at lower and higher
then pH 7. However, growth of bacteria was not significantly affected by pH.
The result was almost same with other desulfurizing bacteria such as S. subarctica T7b
(Gunam et al. 2006), Gunam et al. 2011. Gunam et al. (2006) reported that S. subarctica
T7b had optimal growth and desulfurization activities when the value of pH was 7. When
the pH changes from 6.5 to 7.5, the degradation rate of DBt by KWN5 strain was kept at
about 72-75%. Whereas the degradation ability of the suspended cells was only 30-34%.
Meanwhile, the suspended cells lost more than 40% activity when the pH was lower than
5.5.
FIG. 2. Effects of initial pH on DBT degradation by growing cells of KWN5 strain.
KWN5 was cultivated in MSSF medium with 200 ppm DBT as sole source of
sulfur at various pH for 96 hours. Symbols: ◆, growth (OD660), and ▲, DBT
degradation.
CONCLUSIONS
The conclusion that can be drawn from this research were: At 37°C and pH 7 can
provide optimal conditions for growth of strain KWN5 with OD values at a wavelength of
660 was 1.2 and 200 ppm can degrade DBT in TD was 75.21%.
Pseudomonas sp. Strain KWN5 was great potential in degrading aromatic sulfur
compound contained in petroleum, it is necessary to conduct further research especially to
choose an effective method in its application in industry.
REFERENCES
Furuya T, Ishii Y, Ken-ichi Noda, Kino, K and Kirimura K. 2003. Thermophilic
biodesulfurization of hydrodesulfurized light gas oils by Mycobacterium phlei WU-F1,
FEMS Microbiology Letters 221:137-142.
Guerinik K2 and Al-Mutawah Q. 2003. Isolation and characterization of oil-desulfurizing
bacteria. World Journal of Microbiology and Biotechnology 19, 941–945.
Gunam IBW, Yaku Y, Hirano M, Yamamura K, Tomita F, Sone T and Asano K. 2006.
Biodesulfurization of alkylated forms of dibenzothiophene and benzothiophene by
Sphingomonas subarctica T7b. Journal of Bioscience and Bioengineering 101, 322327.
Gunam IBW, Duniaji AS, dan Triani IGAL. 2009. Biodesulfurisasi minyak bumi dengan
menggunakan bakteri pendegradasi sulfur dengan teknik sel terimobilisasi. Laporan
Penelitian Hibah Bersaing Tahap II. Universitas Udayana.
Gunam IBW, Duniaji AS, and Triani IGAL, and Sitepu A. 2011. Biodeslfurization of
dibenzothiophene by growing and immobilized cells of KWN5 strain. 3rd International
Conference on “Maintaining World Prosperity Through Bioscience, Biotechnology and
Revegetation ”, Depasar, Bali, Indonesia, 21-22 September 2011.
Jasrizal DC. 2009. Deskripsi Dokumen: Biodesulfurisasi Minyak bumi Sebagai Upaya
http://www.digilib.
Pengurangan
Pencemaran
Lingkungan.
Tesis
S2.
ui.ac.id//opac/themes/libri2/detail.jsp?id=92772&lokasi=lokal. Diakses Tanggal 28
Februari 2010.
Kirimura K, Furuya T, Nishii Y, Ishii Y, Kino K, and Usami S. 2001. Biodesulfurization of
dibenzothiophene and its derivates through the selective cleavage of carbon-sulfur
bonds by a moderately thermophilic bacterium Bacillus subtilis WU-S2B. J. Biosci.
Bioeng. 91: 262-266.
Laras BK. 2006. Pencemaran Oleh Hujan Asam Dalam Konteks Kebijakan Global.
http://www.rudyct.com/PPS702-ipb/12167/bambang_kl.pdf. Diakses tanggal 30 Juni
2010.
Schiller JE, and Mathiason DR. 1997. Separation method for coal-derived solids and heavy
liquids. Analytical Chemistry 49, 1225-1228.