Introduction

Combustion of petroleum-derived fuels leads to the release of vast amount of sulfur dioxide (SO₂) into the atmosphere, which is a principle source of acid rain and air pollution. Thus, most countries have imposed strict regulations to control these releases mainly by enforcing stringent restrictions on the levels of sulfur in transportation fuels. However, a common problem, which petroleum refineries are facing around the world is that crude oil reserves being used as feedstock for refining process are becoming heavier day after day with elevated sulfur contents (Bhatia and Sharma, 2012 and Liand Jiang, 2013).

Hydrodesulfurization (HDS) process has been routinely applied in refineries worldwide. HDS involves the use of chemical catalysts containing metals at high pressures and temperatures to remove sulfur compounds. However, with the different classes of sulfur compounds found in the middle-distillate fraction, Cx-BTHs and Cx-DBTs with alkyl substitutions in positions four and six on the DBT ring are more resistant to HDS treatment than mercaptans and sulfides (Kabe et al., 1992). Compared with HDS process, the biodesulfurization (BDS) process using microorganisms and/or enzymes could be carried out more safely, under mild conditions (Chen et al., 2009).

BDS of petroleum and its fractions offers an attractive alternative to HDS process. For this process to be commercial, microorganisms with high activity and selectivity are required. DBT is widely used as a model sulfur compound for isolation and enrichment of suitable strains (Konishi et al., 1997 and Maghsoudi et al., 2000).

Research on BDS using DBT has resulted in the elucidation of two different biochemical pathways, named Kodama (Kodama et al., 1973)  and 4S(Kilbane and Bielaga, 1989). Kodama pathway is considered unsuitable because in this pathway watersoluble sulfur compounds are produced which are then unavailable for burning and are therefore forfeited. Through 4S pathway, DBT is first oxidized to DBT sulfoxide (DBTO), then transformed to DBT sulfone (DBTO₂) which in turn to2-hydroxyphenyl benzene sulfinate (HBPSi) by monooxygenase leading to the cleavage of thiophene ring. Finally HBPSi is reduced to 2-hydroxybiphenyl (2-HBP) by hydrolase enzyme leading to the subsequent release of sulfite or sulfate. In this pathway, sulfur of DBT is selectively removed without destroying the hydrocarbon skeleton so that thermal value of fuels is not decreased.

Various types of bacteria have been recognized to desulfurize DBT via 4S pathway; Rhodococcus erythropolis IGTS8 (Kilbane, 1990).Other DBT desulfurizing microorganisms, mostly mesophilic and a few thermophilic, have been isolated; Rhodococcus  erythropolisH-2 (Ohshiro et al., 1996);Mycobacterium sp. G3 (Nekodzuka et al., 1997);Gordonia sp. CYKS1 (Rhee et al., 1998); Pseudomonas delafieldii R-8(Luo et al., 2003);Microbacterium sp. ZD-M2 (Zhang et al., 2005);Bacillus subtilis WU-S28 (Kirimura et al., 2001)andMycobacteriumpheli WU-F1 (Furuya  et al., 2001);Gordonia alkanivoransRIPI90A (Mohebali et al., 2007);Pantoea agglomerans D23W3 (Bhatia and Sharma, 2010);Sphingomonas subarctica T7b (Gunam et al., 2013).

This study aims to isolateand characterizebiodesulfurizimg microorganism(s) (BDSM) capable of utilizing DBT in a sulfur specific manner (4S-pathway)relevant to Rhodococcus erythropolis IGTS8.

Materials and Methods

Standard Bacterium

Rhodococcus erythropolis IGTS8 (ATCC 53968) was used as a standard and well-characterized strain for comparison and control.

Chemicals

Dibenzothiophene (DBT) (99%), Benzothiophene (BTH) (97%) and Thiophene (TH) (99%) are products of Merck, Germany. 2-hydroxybipheyl (2-HBP) (98%) and 2,2′-Dihydroxybiphenyl (2,2′-DHBP) (99%) were purchased from Aldrich, United Kingdom.Gibb’s reagent (2,6-dichloroquinone-4-chlorimide) is product of Fluka. Potassium phosphate monobasic (99%), Glycerol (99%), Sodium chloride (99%), Ferric chloride (97%), Ammonium chloride, Magnesium sulfate and Ethanol (99.8%) were purchased from Honeywell, Germany. Tryptone, Agar-Agar bacteriological and Yeast extracts were obtained from, Oxoid, United Kingdom.Diethyl ether (99.8%) and Ethyl acetate (99.8%) were purchased from Sigma-Aldrich, Spain.Acetonitrile and Water (HPLC grade) were obtained from POCH, Poland.Dibenzothiophene sulfoxide (DBTO) and Dibenzothiophene sulfone (DBTO₂) were not commercial compounds, it was necessary to perform the synthesis of both in our laboratory using previously described methods (Gilman and Esmay, 1953 and Oldfield et al., 1997). All other chemicals were of analytical grade, commercially available and used without further purification.

Media

Basal salts medium (BSM) containing 1000 ppm DBT was prepared according to Piddington et al. (1995) with some modification, as follows; In order to avoid precipitation and turbidity of the medium, the preparation of BSM was performed in 2 parts separately and then these two parts were mixed together to get one liter of BSM (Ansari, 2008).

The chemicals listed in part (a) and part (b);were dissolved in 850 mL and 150 mL of deionized water, respectively.After mixing these two parts, the pH was adjusted to 7.0. Then sterilized by autoclaving at 121°C and 1.2 barfor 20 min.

Composition of BSM Part (a)

Component	Amount
KH2PO4	2.44 g
Na2HPO4	5.57 g
NH4Cl	0.5 g
Glycerol	6.4 ml

Composition of BSM Part (b)

Component	Amount
MgCl2.6H2O	2.44 g
CaCl2.2H2O	5.57 g
FeCl3.6H2O	2.00 g
Yeast extract	0.1g

Tryptone Glucose Yeast Extract Medium (TGY) is a complex medium used for the inocula build up and was prepared according to Benson, (1994).

Experimental Procedures

Enrichment and Isolation of DBT-BDSM

Coke (C1) with sulfur content of 3.8% was used in this study to isolate BDSM and was collected from El-Nasr Company for coke and chemicals, Tebeen, Helwan, Egypt.

100g of C1 was supplemented with 0.1g DBT. The mixture was carefully homogenized using a sterile spatula. Then placed in sterile plastic pot, and then incubated at 30^oC for one month. Sterile distilled water was added regularly and then mixed well with sterile spatula to keep the humidity constant and to guaranteegood aeration respectively. All the above steps were done under aseptic conditions.

Enrichment(En) was done for detecting and assessing the size of indigenous DBT/BDM in the samples used for isolation. This was done according to Duarte et al. (2001) and Bhatia and Sharma, (2010), but with some modification; 10g of sample C1 before contamination and after contamination (BC and AC, respectively) were mixed with 100 mL En medium. Then, the flasks were incubated at 30^oC for 168 h on a rotary shaker (150 rpm). One mL from each flask was transferred separately to fresh flasks containing 100 mL of En media and the procedure was repeated in a total of three transfers. Serial dilutions (10^-1) of each transfer were inoculated on TGY agar plates to enumerate TCFU and onto BSM-DBT agar plates to count BDSM. The Plates were incubated at 30^oC, and colonies were enumerated after 48 h on TGY plates and after 168 h on BSM-DBT plates. Separate colonies from BSM-DBT plates were picked and purified on BSM-DBT plates.

The bacteria were maintained by sub-culturing into a liquid medium or plating on a solid medium (BSM-DBT plates) weekly. For long-term storage, 7 mL of culture was transferred to 3 mLl of sterile glycerol in a screw cap tube according to Ishii et al. (2005). All tubes were mixed by vortex to ensure that the glycerol was evenly dispersed and kept at -20°C.

2.4.2. Testing the ability of isolates for BDS of DBT:

I- DBT-Spray plate Assay:

This test was performed as described by Denome et al. (1994) with minor modifications as follows; Cells from individual colonies were transferred separately to TGY agar plates and were incubated at 30^oC for 24 h.

500 μL of 1000 ppm DBT solution in ethylether were sprayed individually on BSM/agar plates. Large amounts of cells from these batches were transferred onto BSM-DBT plates. The BSM-DBT plates were incubated at 30^oC for 7 dand monitored periodically every day to detect the initial growth.

Clear zones or colored products around the colonies were detected under day light (Krawiec, 1990 and Denome et al., 1994). Fluorescent products around the batches were detected under short-wave (254nm) UV illumination (Denome et al., 1993).  R. erythropolis IGTS8 was used as a positive control for 4S-pathway.

DBT-Bioavailability/ BiodegradabilityAssay:

Desulfurization assays were also performed in liquid culture to examine the ability of isolated microorganisms to utilize DBT as a sole-sulfur. According to Denome et al. (1994), this assay was done as follows; Cells were incubated at 30oC in TGY broth for 24 h in a shaking incubator (150 rpm).

Cells were pelleted by centrifugation at 5000 rpm for 15 min and then washed three times with BSM. Washed cells were inoculated into BSM that contained DBT as a sole-sulfur source (dissolved in ethylether and added to BSM in a final concentration of 1000 ppm DBT before sterilization).The inocula were adjusted so that the initial absorbance was (A₆₀₀ 0.3). The cultures were incubated at 30^oC for 7 d, in a shaking incubator (150 rpm). The growth was monitored by measuring optical density at λ_600nm andnon-inoculated BSM was used as a blank. The pH of the cultures was determined using pH-meter (DIGMED DM-22, Brazil). DBT removal was estimated by HPLC and the production of hydroxybiphenyl (a dead end product of 4S-pathway) was evaluated by Gibb’s assay and HPLC analysis.

DBT-Biodegradability assay was done according to Kayser et al. (1993) and Frassinetti et al. (1998), applying the same proceduresas mentioned above, but with using BSM free of any C-source other than DBT.

erythropolis IGTS8 was used as a positive control for 4S-pathway.

Identification of the Selected DBT-BDSM:

Colony morphology was visualized by growth on TGY agar plates. The purity of the most promising isolated bacterium was checked microscopically (Axiolab Carl Zeiss Microscope, USA).The morphology of selected isolate was determined by transmission electron microscope (TEM) (Jeol Jem 2100F, 80 to 200 kV, Japan).

Identification of the most promising isolated bacterial strain was done using Biolog system model; Biolog GEN III Omnilog II system 2012, USA, at Holding Company for Biological Products and Vaccins, Cairo, Egypt.

Genomic DNA of the selected isolatewas extracted using GeneJET Genomic DNA Purification Kit K0729 (Fermentas, USA), according to the manufacturer’s instructions at National Research Center, Cairo, Egypt.A region of approximately 375 bp from the 16S rDNA gene was amplified using the forward primer5¢-AACTGGAGGAAGGTGGGGAT-3¢and the reverse primer5¢-AACTGGAGGAAGGTGGGGAT-3¢(Greisen et al.,1994).

PCR condition: The reaction was prepared with 0.5 µL of Dream-Taq 5U/µL (fermentas, USA), 5 µL of Dream-Taq Buffer 10x, 5 µL of target DNA, 1 µL of dNTP each 20 mM, 1 µL of each appropriate primer 10 pmol/µL and 36.5 µL dH₂O were added. The final reaction volume was 50 µL.

After PCR program was completed, for visualizing PCR products, 5 μL of the suspension was electrophoresed on 1% agarose gels in 1X Tris-Acetate EDTA (TAE) buffer, which were then stained with ethidium bromide and examined under UV light. Bands were excised, and DNA was purified from gel slices using QIAquick Gel Extraction Kit, Cat. No. 28704 (Qiagen, USA) at National Research Center, Cairo, Egypt.

The purified PCR products sequenced with the same primer that have been used in amplification of the target sequence. Sequencing was done by an ABI 3730 XL automatic DNA sequencer (Macrogen Inc., South Korea).

The 16S rDNA sequences (Query sequence) were initially analyzed at NCBI server (http://www.ncbi.nlm.nih.org) using BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and corresponding sequences from database were downloaded. Evolutionary history was inferred using the Neighbor-joining method (Patil et al., 2008). The tree was drawn to the scale, with branch lengths in the same units as those of the evolutionary distance used to infer the phylogenetic tree (Dhanve et al., 2009).

Studying of BDS Pathway of the Most Efficient BDSM

To identify the pathway of DBT utilization by the most efficient DBT-BDSM, the following steps were done; Cells were incubated at 30^oC in TGY medium for 24 h in a shaking incubator (150 rpm). Cells were pelleted by centrifugation at 5000 rpm for 15 min and then washed three times with BSM, resuspended in fresh BSM.Washed cells were inoculated into BSM that contained 1000 ppm DBT as a sole sulfur source. The cultures were incubated at 30^oC for 7 d, in a shaking incubator (150 rpm). Culture broth was acidified to pH 2 using 1M HCl and then extracted with equal volume of ethylacetate, dehydrated over anhydrous Na₂SO₄, concentrated by evaporation at 60 ^oC and then subjected to GC-MS analysis.R. erythropolis IGTS8 was used as a positive control for 4S-pathway.

Biodesulfurization of different thiophenic compounds by the mostefficient BDSM

Two groups of seven 250 mL capacity Erlenmeyer flasks each with 100 mL of BSM containing TH, BTH, DBTO, DBTO₂, DMSO, 4-MDBT and 4,6-DMDBT as sole source of sulfur in a final concentration of 1000 ppmwere incubated at 30^oC for 7 d. Preparation of fresh inoculum was done as mentioned before. Another set of flasks were the negative-control group without inoculation.

Non-inoculated flasks of each sulfur compound were used as negative control and run in parallel with the inoculated flasks through all the experimental procedure. Rodococcus erythropolis IGTS8 was used as a positive control for 4S-pathway.

Analytical Procedures

The optical densities (O.D.) of cell suspensions weremeasured using a spectrophotometer (JASCO, V570, USA).

Gibb’s assay was used to screen the conversion of DBT to phenolic compounds by the isolates (Oldfield et al., 1997). For this culture supernatant (5 mL) was taken and incubated with 50 μL of Gibb’s reagent (10 mM ethanol) at 30°C. Positive reactions developed blue to purple color after 30 min of incubation at room temperature and was also monitored at λ₆₁₀against a blank containing no DBT.

Quantitative and qualitative estimation of DBT removal was performed using ethyl acetate as the extractant. After extraction, ethyl acetate layer was analyzed using high performance liquid chromatography (HPLC) model waters 600E equipped with a UV detector model waters 2487 (set at 254 nm) and C18 reversed column (4.6X250 mm, 300^oA, 5µL).

To study the pathway of DBT utilization by the new isolated BDSM;GC-MS analysis,were performed using a Clarus 500 MS System (USA). Compounds were separated on DB-5 column (5%-phenyl) methyl polysiloxane (30 m X 0.25 (mm) I.D X 0.25 (µm) Film) using helium with flow rate; 2 mL/min. The oven temperature was held isothermally at 50°C for 3 min andthen programmed to increase to 250°C at a rate of 7^oC/min. Injector was set at 200°C.

All the experiments and measurements were done in duplicates and arithmetic averages were taken throughout the data analysis and calculations.

 Results and discussion

Collection and preparation of sample used for isolating biodesulfurizing microorganisms (BDSM):

All organisms require sulfur for growth. In bacteria, sulfur makes up to 0.5-1% of the cell dry weight, and is needed primarily as a component of the amino acids cystine and methionine. Sulfur also plays an essential role in a variety of enzyme cofactors including biotin, coenzyme A, coenzyme M, thiamine and lipoic acid, and is critical in many redox processes, both as a building block for iron-sulfur centers and a redox-active component of disulfide bonds. Sulfur for biosynthetic processes is derived from the assimilation of inorganic sulfate by bacteria, yeasts and filamentous fungi (Kertesz, 1999).

More than 60% of the sulfur in the higher boiling petroleum fractions is present as DBT and substituted DBTs (Kropp and Fedorak, 1998). DBT has accordingly been used as a model compound for most of the performed BDS studies.

Total sulfur content in coke varies from 0.5% to 11%, depending on the geographical location of the coke source (Gogoi and Bezbaruah, 2002). Organic sulfur in coke consist predominantly of dibenzothiophene (DBT) and benzothiophene (BT) with lower amounts of disulfide joining cyclic structures, sulfide linked to alkyl- and thio-groups attached either to an aromatic ring or to an alkyl chain(Calkins, 1994). Thatwas why, a sample of coke with sulfur content of 3.8% was used in this study to isolate different BDSM.

Microbial removal of sulfur from organic compounds, especially heterocyclic ones, is a matter of both theoretical and commercial interest. To study such an ability; C1 was collected for isolating BDSM. In addition, an En-technique described above was applied to determine the effect of the presence of sulfur compounds as a pollutant on the indigenous microbial communities as well as to study the prevalence and microbial diversity of BDSM. This En-technique would help in isolating BDSM with good BDS efficienciesandhigh tolerance to DBT and its metabolites.

Many researchers have managed to isolate different BDSM from different environmental samples; oily soil, coke and water contaminated with crude oil (Furuya et al., 2001; Kirimura et al., 2001; Castorena et al., 2002; Chengying et al., 2002; Xiaojuan et al,. 2008; Bhatia and Sharma, 2010 and Bahuguna et al., 2011).

In the present study; total colony forming unit (TCFU) as well as the culturable microorganisms able to grow on DBT as a sole source of sulfur (BDSM) were enumerated on TGY plates and BSM-DBT plates respectively, cultivation was carried out directly after collection and after three weeks of enrichment on En broth medium containing 1000 ppm DBT as a sole-sulfur source.

Enumeration of total colony forming units and BDSM

The total viable count on TGY plates (CFU/mL) and the count of BDSM (cells/mL) on BSM-DBT plates directly after collection showed good microbial population in the collected sample. According to Madigan et al. (1998); contaminants are often potential energy sources for microorganisms, and according to Ilyina et al. (2003); microorganisms could survive in contaminated habitat because they are metabolically capable of utilizing its resources and can occupy a suitable niche.

In general, artificial contamination with 0.1% (w/w) DBT recorded an increase in both BDSM (cells/mL) and TCFU (CFU/mL) either before or after En. This might be due to the adaptation of the indigenous microbial populations in the samples used for isolation to DBT as a model compound for organic sulfur compounds found in crude oil and its distillates. Similar observation was reported by Abed et al. (2002).

Carman et al. (1995); Abed et al. (2002) and Margesin et al. (2003) reported that, the chronic exposure to relatively high levels of DBT in contaminated environments, have resulted in the higher concentrations of DBT utilizing microorganisms that would be well adapted to DBT.

Generally, either before or after contamination (BC and AC, respectively); the microbial count increase with 1^st En cycle then decreased thereafter up to the 3^rd En cycle as shown in fig. (1).

Chen et al. (2008) reported that; repeated exposure to a compound usually increases the adaptive capabilities of microorganisms. In addition, the longer En period and the transfer of microorganisms after first En to a fresh medium resulted in decantation of toxic co-metabolites and apparently enhanced the proliferation of bacteria capable of utilization of a certain compound.

The obvious increase in the count of BDSM and TCFU after En for one week in En medium,illustrated in Fig. (1), might be due to the adaptation of the indigenous microbial populations in the C1 sample used for isolation to organic sulfur compound (DBT) and/or due to the production of the sulfate starvation-induced proteins (SSI proteins), as according to the concept of sulfur starvation and the theory of enrichment culture; microorganisms capable of desulfurizing DBT (BDSM) can be selected by depriving the microbial populations of all sources of sulfur except DBT where only the organism(s) with the necessary DBT utilizing abilities would grow significantly under these conditions and these organisms would outgrow the very large number of other organisms found at the start of the experiment (Krawiec, 1990; Soto et al., 1998 and Abbad-Andaloussi et al., 2003). Under sulfate-limited conditions a set of extra proteins are synthesized by several species of bacteria, yeasts and fungi. In this study, also the presence of yeast extract in the En medium could accelerate the adaptation of the bacterial strains with high rate and capacity of DBT utilization. Konishi et al. (1997); Kishimoto et al. (2000) and Nassar, (2009),reported similar observation during isolating different BDSMs using different substrates for En.

While, the  viable  counts  of  BDSM either before  or after En were less than TCFU indicating that; not all the indigenous microorganisms have the enzymatic system capable to degrade DBT and only microorganisms that have the required enzymatic system to metabolize DBT would grow on the BSM/DBT plates.

Fig. (1): Effect of enrichment on enumeration of TCFU and BDSM before and after artificial contamination (BC and AC, respectively).

Purification and selection of the tolerant DBT-BDM:

From the local environmental sample (C1), 28 different aerobic culturable microorganisms, able to grow on BSM-DBT plates using DBT as sole source of sulfur were isolated. About 32.14% (10.71% BC and 21.42% AC) of the isolates were obtained directly after sample collection (before En) and more than 67.86% (10.71% BC and 57.14% AC) obtained throughout the successive En cycles in En medium.This indicates an increase of the microbial diversity after artificial contamination either before or after En.

This mirrored succession in the adaptation of the isolated BDSM for the continuous exposure to DBT which is in agreement with the results obtained by Duarte et al. (2001); El-Gendy, (2004) and Xiaojuan et al. (2008).

Selection of the most promising DBT-BDM

DBT-spray plate assay

Spray plate assay for rapid screening of organisms able to degrade water-insoluble hydrocarbons, was originally described by Kiyohara et al. (1982). This assay had been modified by Denome et al. (1994), in order to allow a rapid screening of organisms with the presumptive ability to transform DBT either through Kodama or 4S pathway.

Separate different colonies were incubated on BSM agar plates sprayed with 1000 ppm DBT solution in ethylether (BSM-DBT plates). The test substrate precipitated out as a white film immediately when sprayed onto the plate surface and fluoresced golden yellow under short wave (254 nm) UV-illumination, as mentioned in Table (1).

Table 1: Fluorescence of the standard desulfurization products of DBT under UV- illumination(Cooper et al., 2010).

Compounds	Fluorescence
DBT DBTO DBTO2 2,2′-BHBP 2-HBP Benzoic acid	Golden yellow V.V. weak purple Weak purple Strong  Purple Bright white blue V.V. bright green-blue

After the 7^th day of incubation at 30^oC, BSM-DBT plates were viewed in a day light and under (254 nm) UV-illumination for the presumptive detection of the obtained products.

Colonies of R. erythropolis IGTS8 surrounded with clear zones appeared on BSM-DBT plates after 2 d incubation.

Four isolates (C1, C7, C10 and C15) appeared on BSM-DBT plates with clear zones around their colonies after only 1 d of incubation period, which might indicate a shorter lag phase than that of R. erythropolis IGTS8. Shorter lag phase, may indicate that these isolates would be well adapted to DBT.

Five isolates (C2, C3, C4, C9 andC26) appeared on BSM-DBT plates with clear zones surrounding their colonies after 2 d of incubation period, which might indicate that they could have nearly the same lag phase as R.erythropolis IGTS8.

The rest of the isolates appeared on BSM-DBT plates with clear zones surrounding their colonies after 3 d of incubation period, which might indicate that they require longer lag period than that of R. erythropolis IGTS8.

Generally, for all the 28 isolates and IGTS8, as shown in Table (2); the substrate sprayed on the BSM-DBT plates completely disappeared, beginning as a clear zone around the colonies after their appearance on the BSM-DBT plates and spreading out across the entire plate throughout the incubation period up to the 7^th day. The clear zones around the colonies and the disappearance of DBT indicated that the isolated microorganisms were capable of metabolizing DBT but with different rates (Fought and Westlake, 1990; Denome et al., 1993; Rhee et al., 1998;Castorena et al., 2002 and Arulazhagan et al., 2010).

Only one isolate(C24) showed clear zones without colored products in day light but showed green-purple fluorescent product around their colonies which spread out across the entire plate when viewed under (254 nm) UV-illumination.

Four isolates; C1, C8, C13 and C14 showed a blue UV-Fluoresence product under (245nm) UV-illumination and orange-red product when viewed in day light. Similar observations had been reported by Krawiec, (1990) and Denome et al. (1993). These results might indicate that these isolates utilize DBT through Kodama pathway (Fought and Westlake, 1990 and Nassar, 2009).

According to Krawiec, (1988); colonies of organisms which transform DBT by the sulfoxide/sulfone/sulfonate/sulfate pathway (4S-pathway) are surrounded by fluorescent rings which are clearly blue when illuminated with ultraviolet light. According to the fluorescence of the tested standard compounds listed in Table (1) the bright blue fluorescence observed around the colonies of C3, C10, C19 and C20 which spread out across the entire plate when viewed under (254nm) UV illumination might indicate the production of 2-HBP. Similar observations had been reported by Krawiec, (1990); Li et al. (1996) and El-Gendy, (2004).

Table 2: Results of DBT-Spray Plate Assay.

Isolate	I.G.	Fluorescence under 254nm UV-illumination	Day Light
			Clear Zone	Colored Products
IGTS8	2 days	Blue	+ve	——
C1	1 days	Blue	+ve	Orange-red
C2	2days	—–	+ve	—–
C3	2 days	Blue	+ve	——
C4	2days	—–	+ve	—–
C5	2days	—–	+ve	—–
C6	3 days	—–	+ve	—–
C7	1 days	—–	+ve	—–
C8	3 days	Blue	+ve	Orange-red
C9	2 days	—–	+ve	—–
C10	1 day	Blue	+ve	——
C11	3 days	Strong Purple	+ve
C12	3 days	—–	+ve	—–
C13	3 days	Bright Blue	+ve	Orange-red
C14	3 days	Blue	+ve	Orange-red
C15	1 days	Blue	+ve	—–
C16	3 days	——	+ve	——
C17	3 days	Bright Blue	+ve	—–
C18	3 days	——	+ve	——
C19	1 day	Blue	+ve	——
C20	1 day	Blue	+ve	——
C21	3 days	—–	+ve	—–
C22	3 days	—–	+ve	—–
C23	3 days	—–	+ve	—–
C24	3 days	Green-Purple	+ve	——
C25	3 days	—–	+ve	—–
C26	2 days	—–	+ve	—–
C27	3 days	—–	+ve	—–
C28	3 days	—–	+ve	—–

The very weak purple fluorescent product and the clear zone observed closely around the colonies of C11 which did not spread out across the entire plate might indicate the ability of isolates to oxidize DBT to DBTO and/or DBTO₂ without further metabolism to 2-HBP or 2,2′-BHBP. A similar observation was reported by Denome et al. (1994).

The rest of the isolates produced neither color nor fluorescence but only clear zones around the colonies were observed. Similar results were obtained by Krawiec, (1988) and El-Gendy, (2004).

Briefly, four different distinct results were illustrated in Table. (2), 14.28% of the isolates (C1, C8, C13 and C14) produced both colour and fluorescence which might indicate Kodama pathway. 25% (C3, C10, C11, C15, C17, C19 and C20) produced only blue or purple fluorescence which might indicate 4S-pathway. While only one isolate (C24) produced green/purple fluorescence which might indicate production of benzoic acid. All other isolates produced neither color nor fluorescence but only cleared zones.

In conclusion, DBT-spray plate assay demonstrated the presence of fluorescence but it didnot establish whether the fluorescence arose from 2-HBP and/or 2,2′-BHBP or other metabolites. Further tests were carried out in order to confirm these results.

DBT-bioavailability assay

This assay was done according to Lu et al. (1999) to examine the ability of the isolated microorganisms to utilize DBT as a sole sulfur source for the growth in a liquid culture.

Growth of the 28 tested microorganisms revealed their abilities to utilize DBT as a sole sulfur source to a varying extent. These results did not exclude that DBT might also serve as a carbon source (Soleimani et al., 2007).

The pH of the different cultures showed slight decrease from the initial pH 7 as it ranged for all the isolates between pH 6.06-pH 6.98, except in case of C3 (pH4.86) and C13 (pH5.07). This decrease in pH value could be explained by the production of intermediates or dead end products which might cause the decline in the pH of the media.

erythropolis IGTS8gave positive blue results with Gibb’s assay which might indicate the production of 2-HBP and/or 2,2′-BHBP. These results were in agreement with those obtained by Gilbert et al. (1998); Wang and Krawiec, (1994); Ansari, (2008); Bhatia and Sharma,(2010) and Rath et al. (2012).

Nine isolates showed positive blue results with Gibb’s assay (C3, C7, C10, C11, C18, C19, C20, C25 and C26), while all the rest of the isolates gave negative results.

HPLC analysis using system managed to separate DBT, intermediates (DBTO and DBTO₂) and the end products of  the 4S-pathway (2-HBP and/or 2,2′-BHBP), as illustrated in HPLC chromatogram Fig. (2).

Fig. 2: HPLC chromatogram of DBT, DBTO, DBTO₂, 2,2-DHBP and 2-HBP in sterile medium.

The ability of the 28 isolates and R. erythropolis IGTS8 to utilize DBT as a sole source of sulfur was additionally monitored by HPLC analysis after 7 d of incubation as shown in histogram (Fig. 3).

The results confirmed that 9 isolates (C3, C7, C10, C11, C18, C19, C20, C25 and C26) had the ability to utilize DBT as a sole-sulfur source to a varying extent. The difference in the conversion of DBT by different isolates might be due to different enzyme systems utilized by each strain to desulfurize DBT and/or might be due to other factors other than kinetics, physical and chemical parameters required by each isolate to utilize DBT. Several factors could also be responsible for this behavior, such as substrate mass transfer through the cell wall, the pathway through which they could utilize DBT and/or inhibition of activity by the intermediates and products produced from DBT metabolism (Monticello, 2000; Caro et al., 2007 and Irani et al., 2011).

Losses of DBT due to abiotic processes were calculated, which represented 3.47%. Any observed loss exceeding this value in the inoculated flasks can be attributed to BDS processes.

HPLC analysis constituted a more exacting tool for the identification of the intermediates and the final product as indicated from the results showed in Fig. (2). The presence of DBT intermediates (DBTO and DBTO₂) and the final product (2-HBP and/or 2,2′-BHBP) of the 4S-pathway had been confirmed with this analytical technique after comparing the retention time of the obtained separated compounds from the different cultures extract to that of the available standard compounds of the 4S-pathway (DBT, DBTO, DBTO₂, 2-HBP and 2,2′-BHBP). A further advantage of the HPLC analysis; the amount of DBT was also quantified to determine the BDS abilities of the isolated BDSM comparing them with the ability of the standard bacterium; R. erythropolis IGTS8 to desulfurize DBT.

erythropolis IGTS8 produced DBTO, DBTO₂ and 2-HBP and not 2,2′-BHBP. Similar results were obtained by Gallagher et al. (1993); Monticello, (1998); Kobayashi et al. (2001); Castorena et al. (2002); Raheb et al. (2009) andAmin, (2011).

All the isolates (C3, C7, C10, C11, C18, C25 and C26) which showed lower BDS abilities than that of R. erythropolisIGTS8 were excluded from further BDS studies (Fig. 3).

In conclusion, only one isolate; C19 showed higher abilities to desulfurize DBT relative toR. erythropolis IGTS8. They removed up to 66.85% and 50% of the initial 1000 ppm DBT and produced 31.98 ppm and 31.34 ppm 2-HBP, respectively (Fig.  3). C19 was chosen for further BDS studies.

HPLC chromatograms (fig. 4) illustrate the identified peaks of DBT and its metabolites for IGTS8 and C19 cultures.

Fig. (3): BDS efficiencies and production of 2-HBP of the most potent nine bacterial isolates.

Fig. (4): HPLC chromatogram of DBT-BDS metabolites by R. erythropolis IGTS8 and Brevibacillus invocatus C19.

Testing the ability of selected BDSM to utilize DBT as a sole carbon and sulfur source:

DBT-spray plate assay and DBT-bioavailability assay lacking glycerol were performed to determine if the selected BDSM could utilize DBT as a sole carbon and sulfur source.

erythropolis IGTS8 was used as a negative control since IGTS8 is reported to be unable to use any of the organosulfur compounds as carbon sources (Kayser et al., 1993) i.e.; DBT is not degraded but only transformed into 2-HBP and sulfate (Olson et al., 1993; Oldfield et al., 1997; Ohshiro and Izumi, 1999 and Castorena et al., 2002).

C19 and R. erythropolis IGTS8 showed no growth on DBT-spray plate assay or DBT-bioavailability assay in absence of glycerol, and gave negative results with Gibb’s assay.Moreover, the pH of their media did not undergo any significant change from the initial pH7.

In conclusion, these results confirmed the ability of the isolate C19 to utilize DBT as a sole sulfur source without altering its hydrocarbon skeleton.

Identification of the most efficient BDSM

Morphological and Physiological Characteristics

Identification of the most promising biodesulfurizing isolate (C19) was first done on the basis of their morphological and physiological properties.

The isolate C19 on the TGY plate is white round colony, edge entire, raised and transparent.The strain is Gram-positive, long bacilli. The cell dimensions of C19 are (1.9 x 0.4 μm) as determined using TEM (Fig. 5).

The results listed inTable (3), are the output of BIOLOG OmniLog GEN III system and identified C19 as Bacillus funiculus.

Fig. (5): Gram stain and transmission electron micrograph of isolated bacteria.

Table 3: Physiological and biochemical properties of C19.

Test	Result          Test			Result
Negative control		-ve	L-Histidine	-ve
Dextrin		+/-	L-Pyroglutamic acid	+/-
D-Maltose		+ve	L-Serine	+ve
D-Trehalose		+/-	Pectin	+ve
D-Cellobiose		+ve	D-Galacturonic acid	+/-
Gentiobiose		+ve	L-Galactonic acid lactone	+/-
Sucrose		+/-	D-Gluconic acid	+ve
D-Turanose		+ve	D-Glucoronic acid	+ve
Stachyose		-ve	Glucoronamide	+ve
D-Raffinose		-ve	Mucic acid	-ve
α-D-Lactose		-ve	Quinic acid	-ve
D-Melibiose		-ve	D-Saccharic acid	-ve
B-Methyl-D-glucoside		+/-	P-hydroxy-phenyl acetic acid	-ve
D-Salicine		+/-	Methyl pyruvate	+/-
N-Acetyl-D-glucosamine		+/-	D-Lactic acid methyl ester	+/-
N-Acetyl-β-D-mannosamine		+/-	L-Lactic acid	+ve
N-Acetyl-D-galactosamine		+ve	Citric acid	-ve
N-Acetyl-nuraminic acid		+ve	α-Keto-glutaric acid	+/-
α-D-Glucose		+ve	D-Malic acid	+/-
D-Mannose		+ve	L-Malic acid	+/-
D-Fructose		+ve	Bromo-succinic acid	-ve
D-Galactose		+/-	Tween 40	+/-
3-Methyl glucose		-ve	γ-Amino-Butyric acid	-ve
D-Fucose		+ve	α-Hydroxy-butyric acid	+/-
L-Fucose		+ve	β-Hydroxy-D,L-Buyric acid	+/-
L-Rhamnose		+ve	α-Keto-butyric acid	+/-
Enosine		-ve	Acetoacetic acid	-ve
D-Sorbitole		+/-	Propionic acid	-ve
D-Mannitol		+ve	Acetic acid	-ve
D-Arabitol		-ve	Formic acid	-ve
Myo-Enositol		-ve	pH 5	-ve
Glycrol		+ve	pH6	+ve
D-Glucose 6-PO4		-ve	1% NaCl	+ve
D-Fructose 6-PO4		+ve	4% NaCl	-ve
D-Aspartic acid		-ve	8% NaCl	-ve
D-Serine		-ve	1% Sodium lactate	+ve
Gelatin		+/-	Fusidic acid	-ve
Glycyl-L-Proline		-ve	D-Serine	-ve
L-Alanin		+/-	Troleandomycin	-ve
L-Arginine		+/-	Rifamycin SV	-ve
L-Aspartic acid		-ve	Mynocycline	-ve
L-Glutamic acid		-ve	Lincomycin	-ve
Niaprouf 4		-ve	Guanidine HCl	+ve
Vincomycin		-ve	Azireonam	+ve
Tetrazolium violet		-ve	Sodium butyrate	-ve
Tetrazolium blue		-ve	Sodium bromate	+/-
Nalidixic acid		+ve	Positive control	+ve
Lithium chloride		+/-
Potassium teliurite		+/-

 16S rDNA sequencing and phylogenetic analysis of C19

Agarose gel electrophoresis of PCR product indicated a sharp band in 350bp area as shown in Fig. (6).

For analysis of sequencing result, the sequence of C19 (Fig. 7) was first compared with others in a non-redundant sequence database at NCBI server (http://www.ncbi.nlm.nih.org) by using the BLAST program. The BLAST results of the 16S rDNA gene sequence indicate the strain is closely related to Brevibacillusinvocatus LMG 18962 by 99.05% (Table4).

Fig. (8) illustrates the phylogenetic tree reconstructed by the Neighbor-joining method of 16rDNA gene from isolate C19 and closely related bacteria. In this tree, isolate C19 and Brevibacillus invocatus LMG 18962 (GenBank Accession no. NR041836) constituted a branch of phylogenetic. Many Brevibacillus strains were found to have identity with the C19 sequence. The homology levels for the 16 rDNA genes of the strain C19 and Brevibacillus invocatus LMG 18962 was 99.05%.

The Genbank accession number for its 16S rDNA gene sequence is KC999852.

Fig. (6): DNA agarose gel electrophoresis of PCR amplified 16S rDNA.

Table 4: Closest bacteria to strain C19 according to 16S rDNA sequencing.

Identities		Species	Accession number
Percent	Match
99.05%	316/319	Brevibacillus invocatus LMG 18962	NR041836
98.43%	314/319	Brevibacillus limnophilus DSM 6472	NR024822
98.43%	314/319	Brevibacillus choshinensis DSM 8552	NR040980
97.49%	311/319	Brevibacillus reuszeri DSM 9887	NR040982
97.41%	302/310	Brevibacillus ginsengisoli Gsoil 3088	NR041376
96.86%	309/319	Brevibacillus borstelensis DSM 6347	NR040984
95.06%	308/324	Brevibacillus thermoruber BT2	NR026514

 

Forward

1    CGGATATATG   TCCCNTTATG    ACCTGGGCTA   CACACGTGCT  AAATGGTTGG

51   TACAACGGGA   TGCTACCTCG   CGAGAGGATG   CCAATCTCTT   AAAACCAATC

101   TCAGTTCGGA    TTGTAGGCTG   CAACTCGCCT   ACATGAAGTC  GGAATCGCTA

151  GTAATCGCGG   ATCAGCATGC   CGCGGTGAAT   ACGTTCCCGG   GCCTTGTACA

201   CACCGCCCGT   CACACCACGG   GAGTTTGCAA   CACCCGAAGT  CGGTGAGGTA

251ACCGCAAGGA  GCCAGCCGCC   GAAGGTGGGG  TAGATGACTG  GGGTGAAGTC

301   GTAACAAGGT   ATCCGTACCG   GAAGGTGCGG  TTGGATCACA   CTCCTACAGT

Reverse

1  GCGGCAGATA   CTTGGTTCGA   CTTCCCCCAG    TCATCTACCC   CACCTTCGGC

51  GGCTGGCTCC   TTGCGGTTAC   CTCACCGACT    TCGGGTGTTG   CAAACTCCCG

101  TGGTGTGACG   GGCGGTGTGT   ACAAGGCCCG  GGAACGTATT   CACCGCGGCA

151  TGCTGATCCG    CGATTACTAG   CGATTCCCAC   TTCATGTAGG   CGAGTTGCAG

201  CCTACAATCC    GAACTGAGAT   TGGTTTTAAG   AGATTGGCAT   CCTCTCGCGA

251  GGTAGCATCC    CGTTGTACCA   ACCATTGTAG   CACGTGTGTA   GCCCAGGTCA

301  TAAGGGGCAT    GATGATTTGA   CGTCATCCCC   ACCTCCCTCC    AGTTTATATA

Fig. (7): Forward and reverse nucleotide sequence of the 350 bp fragment containing the Brevibacillus invocatus C19 16S rDNA structural genes.

Fig.(8): Phylogenetic tree reconstructing by neighbor joining method of 16S rDNA gene from C19 and closely related bacteria.

Jiang et al. (2002) reported the ability of Bacillus brevis R6, Bacillus sphaericus R16 to metabolize DBT to DBTO₂ and 2-HBP.

A biosurfactant-producing bacterium was isolated from petrochemical contaminated site, identified as Brevibacillus sp. PDM-3 has the ability to grow on the expense of phenanthrene, anthracene and DBT. For DBT desulfurization after the optimization of different growth parameters, bacteria showed 93% degradation in six days (Reddy et al., 2010).

Studying of BDS pathway of Brevibacillus invocatus C19 and Rhodococcus erythropolisIGTS8

Fig. (9) showsthat 6 major peaks other than DBT (1) were detected in the GC chromatogram at different retention times as listed in Table (5).

DBT is first oxidized through “4S”-pathway to DBT-sulfoxide (2), DBT-sulfone (3) then to 2′-HBP-2-sulfinic acid (4) and sultine (5) which led to the production of 2-HBP (6). These results confirmed those of the DBT-spray plate assay and also explained the blue coloration developed in Gibbs assay and confirmed results of HPLC analysis in DBT bio-availability assay.

Table 5: GC-MS mass spectral data of DBT-BDS metabolites by R. erythropolis IGTS8 and Brevibacillus invocatus C19.

ID	Chemical name	RT(min)	MS Fragmentation pattern (m/z)
1 2 3 4 5 6	Dibenzothiophene Dibenzothiophene sulfoxide Dibenzothiophene sulfone 2-HBP-2- sulfinic acid Sultine 2-HBP	32.71 38.73 39.81 30.96 35.75 27.24	184,152,139,113 200,184,171,152,139,118 216,187,171,160,144,136,116,104 234,216,126,111,97 232,168,152,139,113 186,139,129,78

 

Fig. (9): GC of significant metabolites detected by GC-MS analysis.

The presence of sultine may have been formed by the cyclization of 2′-HBP-2-sulfinic acid under the acidic conditions used in the work up and preparation of samples for analysis (Oldfield et al., 1997; Gilbert et al., 1998; Acero et al., 2003 and Mohebali  and Ball, 2008).

In case of Brevibacillus invocatus C19, the accumulation of   2′-HBP-2-sulfinic acid is higher than 2-HBP. This may suggestthat the desulfination step which is catalyzed by DszB (Piddington et al., 1995 and Zhongxuan et al., 2002) is the rate limiting step. Other studies by McFarland et al., (1998)and Nakayama et al., (2002) supported this conclusion.

Data obtained from GC/MS analysis of ethyl acetate extract of DBT culture with Brevibacillus invocatus C19 and Rhodococcus erythropolis IGTS8 (Fig. 9) and its mass spectra (Fig.10) suggested the same desulfurization pathway as illustrated in Fig. (11).

In conclusion,R. erythropolis IGST8 and Brevibacillus invocatusC19 have the ability to desulfurize DBT while conserving its hydrocarbon skeleton, and producing 2-HBP as dead end product through the 4S-pathway.

DBT

DBT-sulfoxide

DBT-sulfone

2-HBP-2′- sulfinic acid

Sultine

2-HBP

Fig. 10: Mass spectra of significant metabolites produced by Brevibacillus invocatus C19 and Rhodococcus erythropolis IGTS8 and detected by GC-MS analysis.

Fig. (11): Postulated pathway of DBT desulfurization by R. erythropolis IGTS8 and Brevibacillus invocatus C19.

Biodesulfurization of Different Thiophenic Compounds by Brevibacillus invocatus C19

This experiment was done to investigate the ability of C19 to grow on different thiophenic compounds as the sole-sulfur source as compared toR. erythropolis IGTS8.

The BDS efficiency of C19 decreased in the following order; DMSO> DBTO₂> BTH> DBTO > TH> 4,6-DMDBT > 4-MDBT, where 99.79%, 98.14%, 96.94%, 96.89%, 93.25%, 90.05% and 69.22% were obtained, respectively.While the BDS efficiency of IGTS8 decreased in the following order; TH> DBTO₂> BTH> DBTO> DMSO> 4-MDBT > 4,6-DMDBT, recording;91.65%, 85.06%, 80.88%, 78.10%, 74.56%, 70.96% and 69.64%, respectively as shown in fig. (12).

Several bacteria have been isolated for the desulfurization of DBT, BT, and their alkylated derivatives (Xu et al., 2009).

Kirkwood et al. (2007) reported that methylation decreases aqueous solubility and increases toxicity. Lower aqueous concentrations due to reduced aqueous solubility, and lower reaction rates due to mass transfer limitations and possibly steric hindrance for the more hydrophobic 4-MDBT and 4,6-DMDBT, would limit the efficiency of detoxification and could therefore be responsible for the retardation in growth and BDS of these compounds.

Fedorak and Westlake, (1983 and 1984) showed that the susceptibility of DBTs in prodhoe Bay crude oil to BDS decreased with increasing alkyl substitution.

The results from this study are consistent with the results reported by Kropp et al. (1997) and Zhang et al. (2013); C₁-DBTs are more susceptible to BDS than C₂-DBTs. Thus, in environments contaminated with crude oil, BT and methyl-DBT will be depleted before the isomers of dimethyl-DBTs.

In conclusion, the relatively broad range of specificity for organic sulfur compounds showed by strain Brevibacillus invocatus C19 compared to R. erythropolis IGTS8suggests its potentiality to be used in the BDS of petroleum and its fractions.

Fig. (12): Biodesulfurization of different thiophenic compounds by Brevibacillus invocatus C19 and R. erythropolis IGTS8.

Conclusions

The suggested enrichment technique has proven to be effective for isolation of biodesulfurizing microorganisms (BDSMs) with capabilities to remove sulfur from dibenzothiophene (DBT) without altering its hydrocarbon skelton.

The higher BDS abilities of the isolated Gram +ve, long bacilli, Brevibacillus invocatus C19 relevant to the standard BDS strain R. erythropolis IGTS8, would suggest its potentiality to be used in the BDS of different sulfur compounds in petroleum and its fractions.

Further work is undertaken now in EPRI Biotechnology Lab., to enhance the BDS efficiency of C19 throughout immobilization and applying nano-technology.

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