Journal of Chemistry, Biochemistry and Molecular Biology

Volume 3, Issue 1, 2011

ISSN 1556-6757

Reduction of Sulphate Released to the Atmosphere by Specific Bacteria

Khalil Y. Mataqi, Associate Research Scientist, KISR, kmataqi@msn.com Fadheelah A. Gholoum, Associate Research Scientist, KISR, batool2t@gmail.com

Abstract Oil compounds release sulphur dioxide into the atmosphere leading to the generation of acid rain. Biodesulphurization technology can be used to reduce the sulphur prior to the decomposition of fossil fuels. The use of microbes to desulphurize fossil fuel provides an improved process of organic sulphur removal before its combustion. In this study the ability of five bacterial strains (3s5a, 3s17d, 3s20d 3s19d and jpn) to desulfurize oil fractions (e.g. gas oil, naphtha and oil) was investigated. Optimization of growth conditions such as ph, temperature; and growth sources (carbon and sulphur) enabled the identification of desulfurization capability in these strains. Keywords: Acid rain, sulphur, biodesulphurization, bacteria. Introduction

Acid rain is rain or any other form of precipitation that is unusually acidic. It has harmful effects on plants, aquatic animals and buildings. Acid rain is mostly caused by human emissions of sulphur from oil compounds which react in the atmosphere to produce acids [1]. To use Biodesulphurization technology to reduce the sulphur prior to the decomposition of fossil fuels bacteria must be isolated that are able to convert organic sulphur compounds to sulphate without degrading the calorific value of the fuel [2, 3]. More than 200 sulphur-containing organic compounds have been identified from crude oils (thiols, thiophenes, substituted benzo- and dibenzothiophenes, and many considerably more complex molecules). Whereas, inorganic sulphur exists in crude oil in five forms (elemental sulphur, sulphates, sulphite, thiosulphate and sulphide). Indeed the major sulphur compounds in crude oils are organic, whereas, the inorganic compounds are negligible [4]. Natural gas from well head contains a number of undesirable components including hydrogen sulphide (H2S], which can be removed by and erotic desulphurization process [5]. Although chemical-physical processes can easily remove inorganic sulphur from crude fuels, organically bound sulphur resists removal [6]. Chemical desulphurization technology is often energy-intensive. Oxidative methods involve oxidation with air; sulphur is eliminated by conversion to volatile products (primarily sulphur dioxide) and soluble sulphates that are removed with subsequent water washes. But they are expensive and, for the most part, they address only post combustion sulphur removal [7]. The use of microbes to desulphurize fossil fuel may provide an improved technology prior to the organic sulphur combustion [8]. However, it is not an easy job and removal of the organic sulphur content continues to be a tenacious problem [9, 10]. Biodesulphurization offers an attractive alternative to hydrodesulphurization as it has the advantages of reduced cost, no requirements of hydrogen gas or pressure, use of ambient temperature, and the possibility of specificity in the sulphur removed. In spite of biodesulphurization is a slow process but scientists were modified the isolated strains (21) genetically for speeding up the process and made it more compatible.

Materials and Methods Bacterial Source. Bacterial strains (3s5a, 3s17d, 3s20d& 3s19d were isolated from the oil field in Kuwait) and jpn strain was isolated from Shimizu-Japan. Growth Media. All media were prepared using deionized water. Thermostable solutions were sterilized o by autoclaving at 121 C for 15-20 min. Thermolabile solutions (glucose and vitamin mixture) were o autoclaved at 110 C for 20-30 min (Table 1). Table 1. Minimal Medium

Content

g l-1

KH2PO4

0.5

K2HPO4

4.0

NH4Cl

1.0

MgCl2.6H2O

0.1

Trace element solution

10 ml

Vitamin solution

1.0 ml

The minimal media pH was adjusted to 7.5 with KOH (2M) in all solutions and then sterilized by o autoclaving at 121 C for 15min. Glucose (22mM) was sterilized and added aseptically to the cooled minimal medium to give a final concentration of 20mM. A 0.5% w/v solution of the sulphur source, dibenzothiophene (DBT), was prepared in ethanol and filter-sterilized using a 0.22 .m Millipore filter. The sterilized DBT (33mM) were transferred aseptically into the medium to give a final concentration of 31mM. Agar plates were prepared similarly with the addition of agar (1.5% w/v) to the above minimal o medium prior to sterilization. After cooling to 45 C in a water bath, the plates were poured and dried o overnight at 37 C (Table 2).

Table 2. Trace Element Solution

Content

g l-1

NaCl

1.00

CaCl2

2.00

FeCl2.6H2O

0.50

ZnCl2

0.50

MnCl2.4H2O

0.50

CuCl2

0.05

NaMO4.2H2O

0.10

Na2WO4.2H2O

0.05

Concentrated HCl

10 ml

The salts were dissolved in the order shown above. After the addition of the first component the pH was adjusted to 6.0 with KOH (2M). This was repeated after each compound was added. The solution o was autoclaved at 121 C for 20-30min. When the sterilized trace element solution had cooled 10ml were transferred aseptically into the medium (Table 3).

Table 3. Vitamin Solution

Content

mgl-1

Ca-pantothenate

400

Inositol

200

Niacin

400

P-aminobenzoate

200

Vitamin B12

0.5

The pH was adjusted to 7.5 with KOH (2M) and autoclaved at 110oC for 20-30 min. When the sterilized vitamin solution shown in Table 3 had cooled, 1.0ml was transferred aseptically into the medium. Preparation of Agar Plates at Acidic pH Values. To make 200ml of the agar-medium a bottle of medium (100ml) containing all the components except agar at concentration required for final 200ml was prepared and the pH of minimal medium adjusted to the pH value required. A second bottle was prepared containing 100ml of deionized water to which agar 3.0% was added. The two bottles were autoclaved at 121oC for 15 minutes and when the contents cooled to 45 C the agar-water was transferred to the medium bottle, mixed and poured immediately.
o

The Modified Minimal Medium. This medium is the same as the one described above but the final concentration in the medium for glucose was lowered to 10mM and the DBT lowered to 6.0mM. Preparation of Pure Cultures. A loopful of culture was streaked onto dry minimal medium agar plates o pH 7.5 and these plates incubated for 2-3 days at 30 C. A stereomicroscope (magnification 50x) was used to identify different colony forms and individual colonies were restreaked on individual plates with the same solid medium. The plates were reincubated as before. The procedure was repeated until plates contained only one colony form and this was assumed to be a pure culture. Determination of Dibenzothiophene (DBT). Use of Gas Chromatograph-Mass Spectroscope (GCMS). Samples (3ml) of culture filtrates were extracted three times with methylene chloride (0.1% and 0.3ml). The mixture was allowed to stand and the supernatants containing DBT were collected carefully and pooled. Gas Chromatograph-Mass Spectroscope (GC-MS) analysis was carried out in a Shimadzu model 6000 with a HP5972 series mass selective detector with a 5890 series II Gas chromatograph. The scanning mass range used was 10.00 to 700 amu. The column (3.6m by 0.3mm diameter) was packed with silicone (0.1.m thickness of the film). The silicone column was washed with methylene chloride (0.1%). The carrier gas (nitrogen) was set at a o flow rate of 1ml min-1 at 250 C. DBT extracted from the samples was injected (0.25ml) in triplicate onto o the column with a temperature programme at 260 C (injector and detector) throughout the experiment. To measure the DBT in the samples, known amounts of DBT (0-10mM) were injected onto the column and the signals recorded and used to produce a standard curve. The peak areas are proportional to concentration of DBT added (see Appendix A). The concentration of the DBT in the steady-state samples was determined using the standard curve.

Use of Gas Chromatograph with an Internal Standard. DBT concentration was measured using a gas chromatograph equipped with a flame ionization detector (Shimadzu GC-14A), coupled to a computing integrator (Shimadzu C R5A). The glass column (1x 2.0m x 5mm o.d. x 3mm i.d.) was packed with 5% Carbowax 20M on Chromosorb W-AW 60-80 o mesh to fit GC-14A and held at 150 C. Helium (2.0kg cm-2) was used as the carrier gas. Benzylphenylsulphide (20mM) dissolved in ethanol was the internal standard. A ratio (Q value) was calculated where Q was defined as the peak area for DBT divided by the peak area for internal standard of constant value benzylphenylsulphide (20mM). A standard curve was produced by preparing known amounts of DBT and plotting the Q values for each value against the concentration of DBT in the solution (see Appendix A). Unknown samples (1.lmM) were added to an equal volume of benzylphenylsulphide (20mM) and subjected to the same procedure. The concentration of DBT was determined from the Q value standard curve. Estimation of Hydroxybiphenyl (HBP). Chemical Assay. Two methods were used (i) microplate titre and (ii) standard assay. In both procedures Gibbs reagent was added to a solution expected to contain hydroxybiphenyl at pH 8.0. After mixing the amount of blue-complex formed was measured at O.D.610 HBP + Gibbs reagent = blue colour pH8.0 Reagents NaHCO3 (38.5mM) was dissolved in 100ml of distilled water. 2,6 dichloroquinone-4-chloramide (Gibbs reagent) (1mM) was dissolved in 100ml of ethanol.

Microtitre Plate Assay. Flasks (250ml) containing minimal medium (50ml) with glucose as carbon source (20mM), DBT as sulphur source (31mM) at pH 7.5 were inoculated with one loopful of a liquid o culture of the strains to be examined and incubated at 30 C in a shaking water bath for 2 days. A sample (1ml) of the culture was transferred into an Eppendorf tube and centrifuged at 1000 g for 5 min. The supernatant (150l) was transferred to the microtitre plates. Sodium bicarbonate (30 l) was added and mixed for 3 min to adjust the pH to 8.0. Gibbs reagent (20 l) was added and after 3 min. mixing, the solution was observed for a blue-coloration. The blue-complex is an indication that HBP was present. Standard Assay. Flasks (250ml) containing minimal medium (50ml) with glucose as carbon source (20mM) and sodium sulphate as sulphur source (0.2mM) were inoculated with one loopful of a liquid o culture of the strain to be examined and incubated at pH 7.5 and at 30 C with shaking for 2 days. The O.D.610 of the overnight culture was measured and the volume required to inoculate a similar culture to give initial O.D.610 of 1.0 was calculated. Flasks (250ml) containing the same medium (50ml) but with DBT as sulphur source (31mM) were inoculated with the volume of inoculum calculated, flasks were o incubated at pH 7.5 and at 30 C in shaking water bath for 1 hour. A sample (2ml) of the culture was transferred into an eppendorf tube and centrifuged at 1500 rpm for 5 min. The supernatant (1ml) was transferred to a test tube, NaHCO3 (200.l) was added and mixed for 3 min. to adjust to pH 8.0. Gibbs reagent (133.l) was added and mixed for 3 min. The O.D.610 was measured using a spectrophotometer at 610nm with distilled water as the blank. The O.D. measured was compared with a standard curve prepared as described above with known amount of HBP over the range of 1 to 10 moles (see appendix B). Use of Gas Chromatograph with an Internal Standard. The procedure was the same as the quantification of DBT. A standard curve was produced by preparing known amounts of DBT and plotting the Q values for each value against the concentration of DBT in the solution (see Appendix A). Unknown samples (1.l) were added to an equal volume of benzylphenylsulphide (20mM) and subjected to the same procedure. The concentration of HBP was determined from the Q value standard curve.

Results and Discussion Measurement of Optimum Growth Conditions Using Agar Plates. The growth conditions of the bacterial strains 3s5a, 3s17d, 3s20d& 3s19d (from the oil field in Kuwait) and jpn a strain isolate (from Shimizu-Japan) were optimized by the use of the agar plates for quick, easy and simple screening [8]. Effect of pH Value on Growth. The pH value of a growth medium is an important parameter which effects the rate of many types of reactions not only the ionisation of acids and bases but also oxidoreduction reactions in the cell [12]. To study the pH which will support optimum growth, the BTD strains were streaked onto solid minimal medium agar plates containing glucose (20mM) as carbon source and DBT (31mM) as sole sulphur source but at 6 different pH values. The plates were o incubated at 30 C for 3 days. The extent of growth was observed daily and the results after 24 and 72 hours incubation are shown in Table 4. All isolates at pH 7.5 were able to grow within 24 hours incubation whereas, at other pH values (5.5, 6.0, 6.5, 7.0, 8.0, and 8.5) growth was only observed after 72 hours incubation. So, the optimum pH value was 7.5 in all strains.

Table 4. The Pure Isolates Grown in Minimal Medium over a pH Range From 5.5 to 8.5

Strain no.

PH

5.5

6.0

6.5

7.0

7.5

8.0

8.5

3s5a

-(+)

-(+)

-(+)

-(+)

+ (++)

-(+)

-(+)

3s17d

-(+)

-(+)

-(+)

-(+)

+ (++)

-(+)

-(+)

3s20d

-(+)

-(+)

-(+)

-(+)

+ (++)

-(+)

-(+)

3s19d

-(+)

-(+)

-(+)

-(+)

+ (++)

-(+)

-(+)

jpn

-(+)

-(+)

-(+)

-(+)

+ (++)

-(+)

-(+)

Cultures (3s5a, 3s17d, 3s20d& 3s19d were isolated from the oil field in Kuwait) and jpn a strain that was isolated from Shimizu-Japan. Cultures were grown on solid minimal medium plates containing o glucose (20mM) as carbon source and DBT (31mM) as sulphur source at 30 C at different pH values. Growth was recorded and the symbols represent -no growth; + growth and ++ good growth. The first symbol represents growth after 24 hours and that in brackets is growth after 72 hours. Effect of Temperature on Growth. The growth temperature has a significant effect not only on the physiology of the cell but also on the changes in the composition of the cell. The most significant changes involve the fatty acid composition of the phospholipids in the bacterial membrane. As the temperature for growth is lowered the proportion of unsaturated fatty acids (those with double bonds) in the phospholipids increases and vice versa [13]. In order to optimize the growth temperature all isolates were streaked onto solid minimal medium agar plates pH 7.5 with glucose (20mM) as carbon source and DBT (31mM) as the sulphur source. The plates were incubated for 3 days at different temperatures. The extent of growth was observed daily and the results after 24 and 72 hours o incubation are shown in Table 5. All isolates could grow within a range 25-37 C and the optimum o growth temperature for all strains was 30 C since it showed good growth in the first 24 hours o incubation. None of the strains grew at 42 or 50 C. Table 5. The Pure Isolates Grown on Minimal Medium over a Temperature Range from 25 to 50oc

Strain No.

Temperature ( C)

25

30

37

42

50

3s5a

+/- (++)

++ (++)

(++)

- (-)

- (-)

3s17d

+/- (++)

++ (++)

(+)

- (-)

- (-)

3s20d

+/- (++)

++ (++)

++ (++)

- (-)

- (-)

3s19d

+/- (++)

++ (++)

(+)

- (-)

- (-)

jpn

+/- (++)

++ (++)

+/- (+/-)

- (-)

- (-)

Cultures were grown on solid minimal medium pH 7.5, plates containing glucose (20mM) as carbon source and DBT (31mM) as sulphur source at different temperatures. Growth was recorded and the symbols represent - no growth; +/- little growth; +growth; and ++ good growth. The first symbol represents growth after 24 hours and that in brackets is growth after 72 hours. Cultures were grown on solid minimal medium pH 7.5 plates containing DBT (31mM) as sulphur source and different carbon o sources at the concentration given and 30 C. Effect of Changing The Carbon Source For Growth Using Agar Plates. When the sole carbon and energy source in the medium is changed the redox-potential within the cell can change and this results in changes in the carbon-flux within the cell [14]. To investigate the range of carbon sources which can support growth when DBT is the sulphur source, the five HBP-producing strains were streaked (purely) onto solid minimal medium pH 7.5 containing DBT (31mM) as sulphur source and various carbon o sources (1.0 or 0.1%). The plates were incubated at 30 C for 3 days. The extent of growth was observed daily. The amount of growth after 24 and 72 hours incubation are shown in Table 6. All isolates showed good growth after 24 hours incubation in medium containing glucose, mannitol and sorbitol (1.0%) but no growth on p-cresol after 24 or 72 hours incubation. Most isolates showed little or no growth after 24 hours incubation but showed good growth after 72 hours incubation in medium containing the other carbon sources tested.

Table 6. The Pure Isolates Grown In Minimal Medium with Different Carbon Sources

Carbon Source 3S5A Tested 3S17D 3S20D 3S19C JPN

Glucose* (1.0%)

++

(++)

++

(++)

++

(++)

++

(++)

++

(++)

Mannitol (1.0%)

++

(++)

++

(++)

++

(++)

++

(++)

++

(++)

Sorbitol (1.0%)

++

(++)

++

(++)

++

(++)

++

(++)

++

(++)

Glycerol (1.0%)

+/-

(+)

(+)

+/-

(+)

+/-

(+)

+/-

(+)

Glucosamine (1.0%)

(++)

(++)

+/-

(+)

(++)

(++)

Inulin (1.0%)

+/-

(++)

(++)

(++)

+/-

(++)

+/-

(++)

Succinate (0.1%)

(++)

+/-

(++)

+/-

(++)

(++)

+/-

(++)

Fumarate (0.1%)

(++)

(++)

(+)

+/-

(++)

(++)

Malate (0.1%)

+/-

(++)

(++)

(++)

(++)

(++)

Lactate (0.1%)

(++)

(++)

(++)

(++)

(++)

Formate (0.1%)

+/-

(++)

(++)

(+)

(++)

(++)

P-Cresol (0.1%)

(-)

(-)

(-)

(-)

(-)

Benzoate (0.1%)

+/-

(++)

+/-

(++)

(++)

+/-

(++)

+/-

(++)

3-Hydroxybenzoate (0.1%)

(++)

(++)

(+)

(++)

(++)

Benzamide (0.1%)

+/-

(++)

(++)

(+/-)

+/-

(++)

(++)

DBT (0.1%)

(++)

(++)

(++)

(++)

(++)

Biphenyl (0.1%)

(++)

(++)

(+)

(++)

(++)

Thiophene (0.1%)

(++)

(++)

(+)

(++)

(++)

1-Benzothiophene (0.1%)

(++)

(++)

(+)

(++)

(++)

Thianthrene (0.1%)

(++)

+/-

(++)

(+)

(++)

(++)

Symbols represents -no growth; +/- little growth; + growth; ++ good growth. * is the control carbon source. The first symbol represents growth after 24 hours incubation and that in the brackets is growth after 72 hours incubation. Effect of Changing the Sulphur Source for Growth Using Agar Plates. The effect of different organic sulphur sources on the growth of the BTD isolated strains was studied by streaking the colonies onto solid minimal medium pH 7.5 with glucose (20mM) as a carbon source and various o sulphur sources (0.1%). The plates were incubated at 30 C for 3 days. The extent of growth was observed daily. The amount of growth after 24 and 72 hours incubation are shown in Table 4. Strain JPN showed good growth on most sulphur sources after 24 hours. Only strains JPN, 3S20D and 3S5A showed good growth after 24 hours in medium containing 1,3-dithiane or n,benzylidenebenzensulphonamide as sole source of sulphur whereas, the rest showed little or no growth. Strain 3S19D showed growth or good growth with all sulphur sources except thianthrene and 2-methyl-1,3-dithiane when little or no growth was observed after 24 hours incubation. All strains showed good growth after 72 hours with all the compounds containing sulphur no matter where the position of that sulphur was in the ring or as a side chain. Surprisingly, all the strains could grow on 1,3dithiane and 2methyl-1,3-dithane, which are compounds where the methyl group is next to the sulphur (see Table 7). Rate of Production of Hydroxybiphenyl Using Pure Isolates. To detect the strains with the highest rate of HBP production an inoculum must be produced with no HBP present. This inoculum is then used to inoculate the experimental flasks. To produce cells free of HBP the individual isolated strains were inoculated into modified standard minimal medium containing glucose (20mM) as carbon source but sodium sulphate (2.0mM) as sole source of sulphur. These cultures were used to inoculate flasks (250ml) containing standard minimal medium glucose (20mM) and DBT (31mM) as sole source of sulphur, but without a nitrogen source. These cultures are resting cells. The initial O.D. 600 was measured to make sure the culture density is high enough to ensure cells could produce detectable o amounts of HBP (see Table 8). The flasks were incubated at 30 C with shaking for 1 hour. The -1 biomass of samples were calculated from the final O.D.600 value (O.D.600. of 1.0 = dry wt of 0.35 mg l ). Sample (2ml) of each culture was collected, the O.D.600 measured and then centrifuged at 1500 rpm for 5 min. Supernatants (1ml) were placed in test tubes and the Gibbs assay was used to measure HBP produced. When Gibbs assay was completed and the blue colour formed the O.D.610 was measured -1 and compared with a standard curve (see Appendix B) to calculate the HBP formed in mol l . The -1 -1 HBP formed mol mg dry wt h was recorded (see Table 8). Strains 3S19c and 3S20d showed the highest HBP production whereas, strain 3S5a showed the lowest (13.05, 12.22 and 6.07 mol mg-1dry -1 wt h respectively).

10

Table 7. The Pure Isolates Grown On Minimal Medium With Different Sulphur Sources

Sulphur Source 3S5A Tested (0.1%) 3S17D 3S20D 3S19C JPN

DBT*

+/-

(++)

+/-

(++)

(++)

(++)

++

(++)

Thiophene

+/-

(++)

+/-

(++)

(++)

++

(++)

(++)

1-Benzothiophene

+/-

(++)

+/-

(++)

(++)

++

(++)

++

(++)

Thianthrene

+/-

(++)

+/-

(++)

(++)

+/-

(++)

(++)

1,3-Dithiane

++

(++)

(++)

++

(++)

++

(++)

++

(++)

2-methyl-1,3-dithiane

+/-

(++)

(++)

(++)

(++)

++

(++)

1,3,5-trithiane

(++)

(++)

++

(++)

++

(++)

++

(++)

4-Sulphamoylbenzoic acid

(++)

(++)

(++)

++

(++)

++

(++)

5,5-Dithiobis +/(2-nitrobenzoic acid) (++) +/(++) + (++) ++ (++) ++ (++)

6-Amino-1-naphthol3sulphonic acid

(++)

(++)

(++)

++

(++)

++

(++)

Phenyldisulphide

(++)

+/-

(++)

(++)

(++)

(++)

Benzylphenylsulphide

+/-

(++)

+/-

(++)

++

(++)

++

(++)

++

(++)

Thiamine Hydrochloride

(++)

(++)

(++)

++

(++)

++

(++)

N,Benzylidenebenzenes ulphon -amide

++

(++)

(++)

(++)

++

(++)

++

(++)

11

Phenol red**

(++)

+/-

(++)

++

(++)

++

(++)

++

(++)

Thymol blue

+/-

(++)

+/-

(++)

+/-

(++)

++

(++)

++

(++)

Sulphanilamide

(++)

(++)

++

(++)

++

(++)

(++)

L-Methionine

(++)

(++)

(++)

++

(++)

++

(++)

L-Cysteine

(++)

(++)

(++)

++

(++)

++

(++)

Cultures were grown on solid minimal medium pH 7.5 plates containing glucose (20mM) as carbon o source and different sulphur sources at the concentration given at 30 C. Symbol represents - no growth; +/- little growth; + growth; and ++ good growth. The first symbol represents growth after 24 hours incubation and that in the brackets is growth after 72 hours incubation. * is the control sulphur source ** Colonies JPN and 3S19C produced yellow background with the sulphur source phenol red, which is most probably due to acid production.

12

Table 8. Hydroxybiphenyl Produced by the Pure Isolates

Strain no.

O.D.600 n.m Initial

O.D.600 n.m After 1 hour

Dry wt mg l
-1

HBP Initial Gibbs

O.D.610 Gibbs After 1 hour

HBP mol l-1

1

HBP mol mg-1 dry wt h

3S20d

0.50

0.50

0.18

0.00

0.35

2.20

12.22

3S17d

0.50

0.50

0.18

0.00

0.24

1.40

7.80

JPN

0.50

0.50

0.18

0.00

0.30

1.80

10.00

3S5a

0.80

0.80

0.28

0.00

0.28

1.70

6.07

3S19c

0.50

0.50

0.18

0.00

0.38

2.35

13.05

Blank

0.00

0.00

0.00

0.00

0.00

0.00

0.00

Cultures were resting cells in minimal medium without nitrogen source pH 7.5 at 30oC.

Further Studies on the Bacteria (3s19c) in Reducing the Percentage of Oil Fractions. To investigate the effect of the strain (3s19c) on further reducing the percentage of the sulphur in oil fractions (gas oil, naphtha, oil ready for export KEC and hydrodesulphurised oil) an inoculum must be produced to inoculate the experimental flasks. To produce cells free of HBP the individual isolated strain was inoculated into modified standard minimal medium containing glucose (20mM) as carbon source but sodium sulphate (2.0mM) as sole source of sulphur. The culture was used to inoculate flasks (250ml) containing standard minimal medium glucose (20mM) and (naphtha, KEC, gas oil or desulphurised oil) as sole source of sulphur (0.1, 2.5, 1.3 or 0.5%) but without a nitrogen source. This culture is resting cells. The initial O.D.600 was measured to make sure the culture density is high enough (O.D.600 = 3.2) to ensure cells could produce detectable amounts of HBP. The flasks were o incubated at 30 C with shaking for 48 hours. The biomass of sample was calculated from the final -1 O.D.600 value (O.D.600. of 1.0 = dry wt of 0.35 mg l ). Sample (2ml) of the culture was collected at 30 min time intervals from the flask and analyzed for oil fractions (gas oil, naphtha, oil ready for export KEC or hydrodesulphurised oil) and HBP using GC. The results are shown in Table 9.

Table 9. The Percentage of Sulphur Reduction from Oil Fractions by the 3s19c Strain

Oil Fraction

Percentage of sulphur

13

Before bidesulphurisation

After bidesulphurisation

KEC

2.5

1.88

Hydrodesulphurised Oil

0.5

0.16

Gas oil

1.3

0.75

Naphtha

0.1

0.05

Cultures were resting cells in minimal medium without nitrogen source pH 7.5 at 30 C for 48 hours. Conclusion When the bacterial strains were grown on plates, good growth was observed over a large range of carbon sources. These results have been reported not only with the same strains (Rhodococcus erythropolis and Agrobacterium sp.) by Kirk & Crawford [15], but also with other strains (Pseudomonas acidovorans and Thiobacillus) by Chang, et al., [16]. In this study R. erythropolis and Agrobacterium sp. showed little or no growth with 3hydroxybenzoate or DBT as sole source of carbon source and this contradicts other reports of good growth when genetically engineered R. erythropolis KA-2-5-10 was exposed to the same compounds as sole carbon sources in the same medium [17, 6]. Perhaps the most interesting data are those reported in Table 7 & 9 when all the isolated strains showed good growth after 72 hours of incubation with different sulphur containing compounds and, in particular, when the compound had a methyl group next to the sulphur atom (e.g. 2-methyl-1,3dithiane). No growth has been recorded when other studies ([18] (Sulpholobus acidocaldarius); [19] (Corynebacterium sp. SY1); [13] (Nocardia sp. CYKS2) and [20] (Mycobacterium sp. G3 and Gordona sp. CYKS1) used the same compounds in similar conditions. These results were explained as the inhibition of the methyl group on the reactivity of the sulphur atom [20]. However, at least in the case of 2-methyl-1,3-dithene the enzymes of R. erythropolis and Agrobacterium sp. did not exhibit this inhibitory effect. Many studies [15, 2, 9] have reported that when R. erythropolis was grown in minimal medium in the presence of sodium sulphate together with DBT the sulphate ion led to the inhibition of DBT desulphurisation as judged by the failure to detect HBP production. This was observed in this study when the BTD strains were grown under the same conditions. This suggests that sulphate is the preferred sulphur source for growth. Therefore, this study demonstrated an improved system to desulphurize fossil fuel prior to organic sulphur combustion, through the use of unique desulphurizing bacterial stains.

14

APPENDIX A

The Relationship between DBT Peak Area Ratio (Q-value) and DBT Concentration

15

APPENDIX B

Standard Curve of Hydroxybiphenyl Production.

16

References

[1] Will, N. (2004). Acid Rain Limits Global Warming. Journal of New Scientist, Proceedings of an National Academy of Sciences (Dol:10.1073/pnas.04044/2/01. [2] Omori, T., Monna, L., Saiki, Y., and Kodama, T. (1992). Desulphurisation of Dibenzothiophene by Corynebacterium sp. Strain SY1. Applied and Environmental Microbiology.58: 911-915. [3] Shennan, J. L. (1996). Microbial Attack on Sulphur-Containing Hydrocarbons: Implications for the Biodesulphurisation of Oils and Coals. Journal of Technology and Biotechnology. 67: 109-123. [4] Monticello, D.J., Bakker, D., and Finnerty, W.R. (1985). Microbial Desulphurization of Fossil Fuels. Applied Environmental Microbiology 39:756-760. [5] Raju, H., Hossain, S.K., Anatharaman, N., Das, M. (2009). Biodesulphurization of Natural Gas in a Three-Phase Fludized Bed Bioreactor Using Thio bacillus denitrificans. Journal of Scientific and Industrial Research 68: 406-411. [6] Yan, H., Kishimoto, M., Omasa, T., Katakura, Y., Suga, K., Okumura, K. and Yoshikawa, O. (2000). Increase in Desulphurisation Activity of Rhodococcus erythropolis KA2-5-1 using Ethanol Feeding. Journal of Bioscience and Bioengineering.89: 361-366. [7] Kilbane, J. J. and Bielaga, B. A. (1988). Toward Sulphur-Free Fuels. American Chemical Society.69: 317-330. [8] Tessa, V., Ellen, M., Marius, M. (2006). Patterns of Technology Dynamics in Dutch Biotechnology Firms towards a New Conceptualization. Department of Innovation and Environmental Sciences. Utrecht University Heidel-Bergluan 2, 3508 TC Utrecht, The Netherlands. [9] Christopher, S.P., Brain, R.K., and Ramosek, J. (1995). Sequence and Molecular Characterization of a DNA Region Encoding the Dibenzothiophene Desulphurisation Operon of Rhodococcus sp. Strain IGTS8. Applied and Environmental Microbiology. 61: 468-475. [10] Wang, P. and Krawiec, S. (1994). Desulphurisation of Dibenzothiophene to 2 Hydroxybiphenyl by some newly Isolated Bacterial Strains. Archives Microbiology.161: 266-271. [11] Gerhardt, P., Murry, R.G.E., Costilow, R.N., Nester, E.W., Wood, W.A., Krieg, N.R., and Phillips, G.B. (1981). In manual and methods for general bacteriology. Chapter 6-12, American Society for Micorbiology. Washington D.C. Karen, J. and Slaght. [12] Gale, E. F. and Epps, H. M. R. (1942). The Chemical Activities of Bacteria. Biochemical Journal. 36: 600-618. [13] Neidhardt, F. C., Ingraham, J. L. and Schaechter, M. (1990). In Physiology of the Bacterial Cell, Neidhart, F. C. pp: 30-41. (ed), Sinaur Associates. [14] Snoep, J. L., de Graef, M. R., Teixeira de Mattos, M. J. and Neijssel, O.M. (1992). Pyruvate Catabolism during Transient State Conditions in Chemostat Cultures of Enterococcus facealis NCTC 775-Importance of Internal Pyruvate Concentrations and NADH/NAD+ Ratios. Journal of General Microbiology. 138: 2015-2020. [15] Kirk, T. O. and Crawford, R. L. (1989). Kinetics of p-Cresol Degradation by an Immobilised Pseudomonas sp. Applied and Environmental Microbiology. 55: 866-870.

17

[16] Chang, J. H., Rhee, S. K., Chang, Y. K. and Chang, H. N. (1999). Desulphurisation of Diesel Oils by a Newly Isolated Dibenzothiophene-Degrading Nocardia sp. Strain CYKS2. Biotechnol. Prog. 14: 851-855. [17] Oldfield, C., Wood, N. T., Gilbert, S. C., Murray, F. D. and Faure, F. R. (1997). Desulphurisation of Benzothiophene and Dibenzothiophene by Actinomycete Organisms belonging to the genus Rhodococcus and Related Taxa. Microbiology. 74: 119-132. [18] Murphy, J., Riestenberg, E., Mohler, R., Marek, D., Beck, B. and Skidmore, D. (1984). Coal Desulphurisation by Microbial Processing. Biotechnology and Bioengineering. 26: 643-652. [19] Lee, M. K., Senius, J. D. and Grossman, M. J. (1995). Sulphur-Specific Microbial Desulphurisation of Stearically Hindered Analogs of Dibenzothiophene. Applied Environmental Microbiology.61: 4362-4366. [20]Kirimura, K., Furuya, T., Nishi, Y., Ishii, Y., Kino, Kane Usami, S. (2001). Biodesulphurisation of Dibenzothiophene and its Derivatives through the Selective Cleavage of Carbon-Sulphur Bonds by a Moderately Thermophilic Bacterium Bacillus subtilis WU-S2B. Journal of Bioscience and Bioengineering. 91(3): 262-266. [21]Wei, L., Miao, D., Wang, H., Jian, C., Yao, S. (2006). Biodesulfurization of dibenzothiophene by growing cells of Gordonia sp. in batch cultures. Journal of Biotechnology letters. 28: 1175-1179.

18