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Bio degrading ability of organo-sulphur compound of a newly isolated microbe Bacillus sp. KS1 from the oil contaminated Soil

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darkys devia

on 26 July 2013

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Transcript of Bio degrading ability of organo-sulphur compound of a newly isolated microbe Bacillus sp. KS1 from the oil contaminated Soil

Bio degrading ability of organo-sulphur
compound of a newly isolated microbe
Bacillus sp. KS1 from th.e oil contaminated Soil

Kalyani Rath, Bishwambhar Mishra and Suneetha Vuppu
School of Bio Sciences and Technology, VIT University, Vellore, India

INTRODUCTION◙
DESULFURIZATION
Hydrodesulphurization

I
s carried out within a high temperature(450ºC) and high hydrogen pressure(150-200 atm).CoMo and NiMo are used as metal catalysts for this process.

High costs


Not completely eliminate organo-sulfur compounds such as dibenzothiophene (DBT).


Reduces the octane


Biodesulfurization
Normal temperature and pressure.


Microbial catalysts.


Not using hydrogen.


It not affects the octane number.


Does not produce undesirable compounds.
MATERIALS AND METHODS
Isolation and cultivation of DBT desulphurizing microorganism
Sample: various oil contaminated soils from the different site of Chennai Petroleum Corporation Limited's refinery in Manali, near Chennai, Tamilnadu state, India.

Medium A :

-Samples were incubated in 250 ml flasks containing 50 ml media, on an orbital shaker (180 rpm, 45 °C). DBT was added after the sterilization of the medium.

- After five times sub cultivations of the initial culture, repeated streaking on the same media with 17 g/litre agar were carried out to obtain isolated colonies. The growth and cultivation of the purified and isolated microorganism with DBT as a sulphur source was detected in liquid media.

- The selection of strain among the isolated strains was based on the high desulfurization activity and the higher production rate of 2-hydroxyl biphenyl from DBT.







Estimation of DBT and 2-HBP
2-HBP :

centrifugation (2000 rpm for 10 min) from bacterial cultures .

The supernatant (250 pl) was taken and mixed with 30 pl of 1 M NaHCO3 (pH 8.0).

25 pl of Gibbs reagent (0.1% in ethanol) was added and the culture broth was gently agitated in the room temperature for a period of 20 min .

The absorbance was then measured at 595 nm

DBT degraded:

HPLC (Dual X Absorbance Detector) in specific time intervals with mobile phase acetonitrile-water (1:1, v/v) ) and the flow rate was 1.5 ml/min

2 ml of culture broth was acidified with 6 M HCl to make the final pH of the reaction mixture is equal to 2.

Extraction with 2 ml of ethyl acetate

After the separation of compounds in the HPLC column the absorbance of the effluent mixture was measured at 280 nm.


Study of desulfurization activity in aqueous and Bi-phasic systems
system aqueous:


-buffer :potassium phosphate (0.1M, pH 7.2) with 3 mM of DBT as sulfur source.
Bi-phasic system:
-25 ml of potassium phosphate buffer (0.1 M, pH 7.2) along with 25 ml of n-tetradecane (The hydrocarbons present in petroleum) was employed.

-T: 30 'C
-200 r.p.m.
-estimating DBT and 2-HBP by HPLC every two hours
Various organo-sulphur compound specificity of strain
Mediums:

1. Benzothiphene

2. DBT

3. Thiophene

4. Thiophene-2-carboxyllic acid

5.Dimethyle sulfoxide;

6. 5,5'-di thio bis(2-nitro benzoic acid)

7. Designed medium (mixture of all the above organo-sulphur compounds).
RESULTS AND DISCUSSION
Isolation and identification of DBT-desulphurizing microorganism
Growth and DBT degradation study of strain KS1
Various organo-sulphur compound specificity of strain KSI
Desulphurization assay in aqueous and Biphasic systems
conclusions
GRACIAS
-K2HPO4; 4 gm,

-KH2PO4 0,5 g,

-NH4Cl, 1 g,

-MgCl2.6H20; 0,2 g,

- CaCl20.02gm,

-NaCl; 0.01gm

-solución de metal; 10 ml ((Na2Mo0; 0.1 gm, FeCl2.4H20; 0.5 gm, ZnCl2; 0.5 gm, CuCl2; 0.05 gm, Na2W04.2H20; 0.05gm).

- mezcla de vitamina; 1 ml. (Calcium pantothenate; 400 mg, Inositol; 200 mg, Niacin; 400 mg, Pyridoxine hydrochloride; 400 mg, P- Aminobenzoic acid; 200 mg, Cyanocobalamin; 0.5 mg ) .

- DBT (25Mm/Litre -en etanol)

- 6 g / litro de glucosa

Summary

The fossil fuels like the oil have sulphured compounds, for what during his combustion there are generated oxides of sulphur that contaminate the air. The Desulfurization is a way to desulfurize dibenzothiophene( is an sulphured aromatic hydrocarbon), is currently used a method called hydrodesulfurization, but not completely eliminate the organo-sulfur and further reduces the octane to hydrogenate the olefins, therefore has been considered biodesulfuration as an alternative since they do not present this problem and do no produces undesirable.


The microorganisms used in this project were isolated from soil contaminated with crude oil refinery regions Petroleun chenai corporation limited in India. Then cultured using DBT as sulfur source. And strain was selected that had the most activity to the increased rate of production of 2-hydroxy biphenyl from DBT.


Was measured 2-HBP produced from DBT degradation using the Gibbs reagent and the amount of DBT using HPLC (x dual absorbance detector).


Two trials were conducted more, comparing the activity of the strain in an aqueous medium and in biphasic medium and studied the ability of the strain to degrade other organic compounds sulfur.


The following results were obtained:

- A selected strain was called ks1 and identified as belonging to the genus Bacillus.

-There was a maximum growth of the bacteria at 168 days and a decrease of 68.75% of DBT concentration accompanied by the increase in the concentration of 2-HBP.

-The desulfurization is greater in a biphasic medium in an aqueous one.

-And, in the was very good growth DBT and benzohifeno but otherwise no carboxylic acid was very considerable.

From this it can be said that the Bacillus sp can be used in the desulfurization of DBT, but requires genetic improvements to increase the rate of desulfurization other organic sulfur compounds

-50 ml cells (10 gm dry cells/litre) .
Miguel Ramírez Salcedo
Darkys Devia Torres
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