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In: BiocataIyticTechnologyandNanotechnology ISBN 1-59454-117-5 Editor:G E. Zaikov, pp. 51-58 C92004 Nova Science Publishers, Inc.

Chapter4

SCREENING OF RHODOCOCCUS SPECIES REVEALING DESULFURIZATION ACTIVITY WITH REGARD TO DIBENZOTIDOPHENE

A. A. Zakharyants, ~ P. Murygina andS. ~ Kalyuzhnyi
Chemical Enzymology Dept., Chemistry Faculty, Lomonosov Moscow State University, Vorobiovy Gory 1-11, Moscow, 119899, Russia, Tel. +7(095)939-5083, <1>aKc:+7 (095) 939-5417; e-mail: arpesha@inbox.ru

ABSTRACT
The screening Rhodococcus capableof utilizing dibenzothiophene of sp., (DBT) asa sole sulfur source, was perfonned. After multiple consecutivepassages the selective on medium, Rhodococcus erythropolis Ac-1514D and Rhodococcus ruber Ac-1513D were adaptedto DBT. As a result, the lag periods of thesecultures decreased 120 to 20-22 hours.The kinetic parameters, suchas the specific growth ratesand specificactivities of DBT desulfurizationby both strains,were calculated.DBT at 0.43mM conversionby R. erythropolis was 78-80% to fonD typical productsfor 4S-pathway. the first time, R. For ruber was also shownto convertDBT (63-65%). Key words: biodesulfurization, dibenzothiophene, Rhodococcussp., specific growth rate, specific activity.

INTRODUCTION
To date, more than 200 sulfur-containing organic compoundsare identified in crude oil. In general, they are presentedas sulfides, mercaptanesand thiophenes. The crude oil from different reservoirs contains 0.03 to 8 % (wt.) sulfur compounds [1]. The content of derivatives of thiophenes in oil depends on their maturity degrees. The high-molecular alkylated derivatives of benzo- and dibenzothiophenein mature oils [2] are most persistent


52

A. A. Zakharyants,V. P. Murygina and S. V. Kalyuzhnyi

for physical-chemical industrial methods of desulfurization (e.g., hydrodesulfurization). During combustion of sulfur compounds,various gaseoussulfur oxides, such as SO2and SO) (general name is Sax), are formed, if not treated they end up as acid rains in atmosphere[3]. This and other facts place stringent requirementsfor oil fuel quality. Various technologiesof oil desulfurization including biological methods are worldwide developed. A number of microorganisms including Rhodococcussp., able to desulfurize DBT and its derivatives, are described [4, 5, 6, 7, 8, 9, 10, II]. This process proceedsvia so-called 4S-pathway to form sulfite/sulfate and 2-hydroxybiphenyl (2-HBP) derivatives as final products [12, 13, 14, 15, 16,17] (Fig. I):

~"MS

I~---~

DBT

OH-

"

OH-

HO+ 3

()

11:~) ~OH OH
2,2'-OHBP

---""SO:"

(

SO;-

/5,0 0/ '0 HBPSo
HO+ 3
~

()
S024

2-HBP

1I::) OH

~---~ v~ OH 2-HBP
Fig. I. Scheme the suggested of DBT-metabolizing pathwayby Rhodococcus DBTO - dibenzothiophene sp. sulfoxide; DBTO2 - dibenzothiophene sulfone; HBPSi - 2-(2'-hydroxy-phenyl)benzene sulfinate;HBPSo- 2. (2'-hydroxyphenyl)benzene sulfonate;2-HBP - 2-hydroxybiphenyl;2,2'-DHBP - 2,2'-dihydroxybiphenyl [12,16].


Screeningof RhodococcusSpeciesRevealing Desulfurization Activity

53

This paper is concerned with the screening of RhodococcusSF. from our laboratory collection, which can desulfurize DBT, and with the investigation of kinetics of these
nTocesses.

MATERIALS AND METHODS

Chemicals
DBT (Sigma-Aldrich, USA) was used as a sole sulfur source for growth of microorganismson diesel fuel "Z" grade (Russian State Standard No. 305-62) or sodium succinate carbon source.2-HBP was obtained from Acros (Germany) and used as standard as compoundin the Gibbs assaywith 2,6-dichloroquinone-4-chloroimide (Acros, Germany) to determine concentrationin culture medium [16]. Other chemicals used were reagentgrade its andwere supplied by "Reahim" and "Laverna" (Russia).

Microorganismsand their Cultivation
Rhodococcuserythropo/is Ac-15l4D, Rhodococcus ruber Ac-15l3D and Rhodococcus rhodochrous were cultivated in modified Raymond's medium (RM) supplementedwith succinate % w/v) or diesel oil (0.5% v/v) as carbon source. The medium has the following (1 composition (g/l): Na2CO3 - 0,1, CaCI2*6H2O - 0,01, MnCI2*4H2O - 0,007,
Na2HPO4*12H2O

-

35,8, KH2 PO4

-

13,6, MgC12*6H20

-

0,16, NH4Cl

-

2,0, NaCI

-

5,0; the

final pH was 6,7. DBT was added in acetonesolution after autoclaving (I atm, 30 min) as a solesulfur source, the final concentrationsin vials were 0.86 mM and 0.43 mM. The strains werealso cultured on RM agar plates (20 g/l) containing 200 I.1MDBT and succinate1% w/v. The inoculated vials (total volume of 120 ml) containing 40 ml of RM medium were incubated in an orbital shaker (180 rpm, 27 °C) to the end of exponential phase (approximately90 hours).

Bradford Assay for Intracellular Protein
The growth of microorganisms was monitored by determination of intracellular protein concentration.An aliquot (1 ml) of culture broth was taken - every 4 hours. The samples were centrifuged for 15 min at 12000 rpm to separatecells from the supernatant.The cells were disrupted in 1 ml NaOH (3 M) by boiling in water bath for 45 min. Then the samples werecentrifuged (12000 rpm, 10 min.) again to separatedebris. Bradford reagent(3 ml) (100 mg Coomassie brilliant blue G-250/50 ml in 95% ethanol/lOOml concentratedphosphoric acid and distilled water was added to get 1 1 solution) were addedto 100 ~l of the prepared samples.The optical density at 595 nm was measuredafter 10 min of colour development.


54

Phased-Reversed HPLC Assay for DBT
To detennine DBT concentrations, 0.5 ml methanol was added to 0.5 ml aliquots of culture broth or else previously, the equal volume (40 ml) of ethyl acetate was added to culture broth and left to stir for 4 h to extract DBT [16]. The preparedprobes were analyzed using a Gilson model 302 liquid chromatograph equipped with Du Pont Instruments UV detector (:>-=240 nm) and fitted with a phased-reversed Diasorb 130-CI6T, 6 ~m and loop 20 ~m. The column was eluted with vacuum-degassed methanol/water (85: I 5, v/v); the rate of eluting was I ml/min, the pressurewas 130 barrel. Approximate retention time of DBT was 7.9 min.

Standard Gibbs Assay for 2-HBP
The reaction with Gibbs reagent was used to determine the concentration of biodesulfurization product - 2-HBP [16]. An aliquots (5 ml) of culture broth supernatantwere put into Eppendorftubes and centrifuged (1200 rpm, 10 min) to remove cells. Supernatant (2 ml) of was transferredto Eppendorf tube, and 20 J!l 10 mM (1,985 g/l) Gibbs reagentsolution in acetonewas added. The assayswere incubated over night at 30 °C to complete the colour development. The optical density at 610 nm was measured on a Shimadzu UV-1202 spectrophotometer.

Titration Assay for Sulphite
The filtrated culture broth was titrated with standardiodine solution to determine sulphite [18] according to the following reaction:

SO32- H2O+

SO4Z- 2M' +

21-

An aliquot (20 ml) of culture broth was filtered to remove cells and then transferredto a 100 ml Erlenmeyer flask. Five drops of 5% starch solution and concentratedhydrochloric acid to adjust pH to -2 were added. The assayswere titrated with 0.001 N standard iodine solution to get a violet tint.

Turbidometric Assay for Sulphate
The sulfate concentrationwas determinedturbidometrically [18]. An aliquot (20 ml) was filtered through membranefilter (pore diameter 0.4 ~) to remove all cells. The filtrate was transferred to a volumetric flask (100 ml) contaning 20 ml electrolyte solution (60 g NaCl, 5.125 ml HCl/250 ml of the solution) and 15 ml BaClz solution (30 g BaClz*2HzO/250 ml distilled water). Then, distilled water was added to 100 ml; in 5 min, the optical density was measuredat 405 nm.


Screeningof RhodococcusSpeciesRevealing Desulfurization Activity

55

RESULTS AND DISCUSSION
Three collection cultures of Rodococcussp. were checked for their ability to grow with DBT as a sole sulfur source. It is known [12, 19,20] that R. erythropolis and R. rhodochrous aremost active in DBT desulfurization. However, in our studies, the desulfurization activity of R. rhodochrous for DBT was not undoubtedly established.The duration of lag phasefor R. erythropolisand R. ruber was around 120-122hours, and the desulfurization activity for DBT waslow. Therefore, the two cultures in the selective liquid medium containing DBT 0.43 mM wasenriched. As a result, the enriched cultures of R. erythropolis Ac-1514D and R. ruber Ac-1513D wereobtained by meansof three serial subinoculations in the above liquid medium. The lag phases were reducedto 20-22 hours for both cultures (Fig. 2 and Fig. 3).

Fig. 2. Time course ofDBT

desulfurization by R. erythropolis Ac-1514D. Ac-1514D was cultivated at 27 °C

in RM with 0.43 roM DBT as a sole sulfur source. 8, DBT (roM); 8, protein (gil); sulphite (roM); ... pH.

.

,2-HBP (roM);

"

The typical curves of DBT conversion, biomass growth and accumulation of biodesulfurizationproducts are presented (Fig. 2 and Fig. 3). DBT biodesulfurization data wereprocessedusing the Monod equation as described[64]. The specific growth rates (~) of two enriched strains were obtained by the analysis of the linear ranges of the growth curves (cell protein vs time) in the semi-logarithmic coordinates.The ~ values of R. erythropolis Ac15l4D and R. ruber Ac-15l3D were actually identical: 0,080 :t 0,006 h:1H 0,086 :t 0,004 h-1, respectively.The obtained values for Rodococcussp. are 2.5 times less than known so far in literaturefor R. erythropolis [9,21]. The yield coefficients for our cultures were 0,86 :t 0,04 and 5,85 :t 0,3 g protein/mmol DBT for R. erythropolis and R. rubey, respectively. Using


56

A. A. Zakharyants,V. P. Murygina and S. V. Kalyuzhnyi

these data, the specific activities were determined for both cultures: 185 mmol DBT (kg dry wt)-lh-I and 30 mmol DBT (kg dry wt)-lh-J forR. erythropolis and R. rober, respectively.

0.9

7,5

0,75

E Im c

~

S
0 ... c..

7

:::: 0,6
6,5
:I: c..

.5(),45 Q)
0.3 0,15 0 0 7 21 25 29 33 45 50 53 57 70 75 89 106 Time (h)

6

A---t.I-L:s=t="i~:i!~:C: I I

I

I

I

-

I

I

I

I

--:J

5.5

5

Fig. 3. Time courseofDBT conversion R. ruber Ac-1513D.Ac-1513D was cultivatedat 27.C in RM by
with 0.43 mM DBT as a sole sulfur source. ., DBT (mM); ~, protein (gil); , 2-HBP (mM);

(mM); ~ pH.

.

" sulphite

The specific activity of R. erythropolis Ac-1514D characterizingits ability to desulfurize DBT to 2-HBP was 80 mmol 2-HBP (kg dry wt)-lh-l. The obtained data are in accordance with thosedeterminedfor non-mutantRhodococcus [4,9,20]. SF. Since R. ruber Ac-1513D was never characterizedas desulfurizing microorganism, this paper representsthe first report indicating DBT desulfurization by this bacterium. The DBI conversions, about 63-65 %, were observed after 80 hours of growth with the initial concentrationofDBT 0.43 mM (Fig. 3). Earlier, it was establishedthat biodesulfurization of DBT by RhodococcusSF.proceeded via so-called 4S-pathway [12, 13, 16, 17,22]. The dead products were sulfite and 2-HBP. Other biodesulfurization paths were not proposed. The analysis of data obtained for R. erythropolis (Fig. 2) and R. ruber (Fig. 3) suggestthat 4S-pathwayoccurred in our case.

CONCLUSIONS
Thus, the desulfurizing ability and activity of R. erythropolis and R. ruber were establishedand investigated. The consecutivepassageson the selective medium of both strains allowed us to obtain active cultures with regard to DBT desulfurization. A six-fold decreaseof the lag phase durations was reached. However, we did not succeedin our attempts to adapt to DBT R. rhodochrous, which is known as the bacteria showing the desulfurizing activity.


The conversion of DBT (initial concentration 0.43 mM) according to 4S-pathway by R. erythropolis and R. ruber was investigated. Thus, some kinetic parameters,such as specific growthrates and specific activities ofDBT desulfurization, were determined.

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[13] P. Wang, S. Krawiec: Desulfurization Dibenzothiophene 2-hydroxybiphenyl of to by
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