Vol. 45 No. 5 SCIENCE IN CHINA (Series B) October 2002

Isolation and identification of nondestructive desulfurization bacterium

GOU Zhongxuan (缑仲轩), LIU Huizhou (刘会洲), LUO Mingfang (罗明芳), LI Shan (李 珊), XING Jianmin (邢建民) & CHEN Jiayong (陈家镛)

Young Scientist Laboratory of Separation Science and Engineering, State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080, China Correspondence should be addressed to Liu Huizhou (email: hzliu@home.ipe.ac.cn)

Received May 17, 2002

Abstract A nondestructive desulfurization microorganism has been isolated. The metabolism product analyses show that the strain can be a kind of biocatalyst to oxidize dibenzothiophene (DBT) into 2-hydroxydiphenyl (HBP), therefore the sulfur in DBT is removed selectively. The 16SrRNA information, cell wall analysis, physical, biochemical properties and morphological prop-erties suggest that the isolated strain is Rhodococcus erythropolis. The strain can grow in the basal salts medium (BSM) that DBT concentration is no more than 10 mmol/L, and the optimal DBT concentration for growth is 1 mmol/L, however, the optimal DBT concentration for desulfuriza-tion is 0.5 mmol/L. The further research shows that the strain can also desulfur some other or-ganosulfur-containing compounds such as thianaphthene, phenyl sulfide and 4,6-dimethyldiben- zothiophene (4,6-DMDBT). Keywords: dibenzothiophene, isolation, identification, biodesulfurization, Rhodococcus erythropolis.

The sulfur-containing compounds in fossil oils have many negative effects such as they can deactivate the catalysts used in oil refinery and cause the corrosion of equipment and pipes during transportation and storage. As it is even more serious in combustion of the oil, the sulfur will be oxidized into the poisonous and corrosive SO2 or SOx, which leads to the air pollution and acid rain. The current commercial method to remove the sulfur compounds is hydrodesulfurization (HDS). Under high temperature and high pressure, catalyzed by rare metals, the hydrogen can react with many sulfur-containing compounds, and reduce them into hydrogen sulfide that can be separated from the petroleum. However, there are also numbers of other organic sulfur compounds in the petroleum and its distillates, which are rather recalcitrant to the HDS process. One example is dibenzothiophene (DBT), which is commonly used as a model of organic sulfur-containing het- erocyclic compound for biodesulfurization (BDS) studies. The BDS process differs from the con-ventional HDS processes in several important aspects. Compared with HDS, BDS process oper-ates at ambient temperature and pressure and uses air instead of hydrogen used by HDS. Most of BDS work has shown good desulfurization results starting with DBT or its alkyl groups substi-tuted compounds. The analysis of results reported on BDS shows that BDS is very promising with potential commercial value, and it is also both inexpensive and metabolically specific[1]. BDS fol-

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lows two different pathways. One is to remove sulfur by the “Kodama pathway”(fig. 1), which attacks the C-C bond of DBT molecule. The sulfur compounds change to one kind of wa-ter-soluble compound that can be removed easily from the oil phase during reaction[2]. The other way is to remove sulfur by the so-called “4S pathway”(fig.2), which is a breakthrough in BDS achieved by Institute of Gas Technology (“IGT”) in 1988. They isolated two strains of bacteria which can remove sulfur from DBT selectively. The further research reveals that the strain can oxidize the DBT into dibenzothiophene sulfoxide first, then to dibenzothiophene sulfone, 2-hydroxydiphenyl or 2,2′-biphenol and sulfate, while the carbon structure is intact[3—5]. Because selectively attacking C-S bond does not affect the combustion value of petroleum, this method has attracted the attention of many investigators. Several species of bacteria have been isolated and reported in the world, such as Corynebacterium sp.[6], Gordona Strain [7], Bacillus sp.[8], Pseudo-monas sp.[8], and Rhodococcus sp.[9]. The relevant desulfurization (dsz) genes from the bacteria were cloned and sequenced in 1993[10]. Three genes, named dszA, dszB and dszC, encode BDS enzymes, named DszA, DszB and DszC respectively that are sufficient for the conversion of DBT to HBP. The dsz promoter and associated regulatory regions were also analyzed [11]. In 2001, a

Fig. 1. “Kodama pathway” of biodesulfurization.

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Fig. 2. “4S pathway” of biodesulfurization.

pilot-scale BDS plant for 5000 barrel per day was set up successfully, which testifies the feasibil-ity of BDS.

The domestic BDS research work begins relatively later, however, several breakthroughs have been achieved in bacteria screen and bench-scale BDS research[8,12]. The soil samples in Northern China have been collected to screen the biodesulfurization bacteria, and a few strains have been screened out, which can remove the sulfur from DBT nondestructively, such as Bacillus brevie R-6，Bacillus sphaericus R-16，Nocardia globerula R-9, and Pseudomonas delafieldii R-8[8]. The natural environment is very different between the South and North of China, which might affect the behavior of the bacteria. In order to screen out more effective bacteria, the soil samples of Guizhou Province, located in Southern China, were collected to isolate the nondestruc-tive biodesulfurization bacteria.

1 Experimental

1.1 Materials The purity of phenyl sulfide, DBT and dibenzothiophene sulfone (DBTO2) is 99%; the purity

of thianaphthene is 97% and that of 4,6-dimethyldibenzothiophene (4,6-DMDBT) is 95%. Sigma company provided the above reagents. The 2-hydroxydiphenyl (2-HBP) was purchased from TCI, Japan. The hexane and methyl alcohol are HPLC grade and all other reagents are analytical grade.

1.2 Methods

1.2.1 Isolation methods. The soil samples contaminated with petroleum were collected around the gas well in Chishui, Guizhou Province of China. The samples were mashed and 5 g samples

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were transferred into the asepsis water and agitated. Then 5 mL of this solution was taken and in-cubated in the 100 mL selective medium for 5 d (28—31℃, 150 r/min). The selective medium

contains 2.44 g KH2PO4, 14.03 g Na2HPO4·12H2O, 0.3589 g MgCl2·6H2O, 0.001 g CaCl2·

2H2O, 0.001 g FeCl3·6H2O, 0.004 g MnCl2·4H2O, 1.45 mL of glycerol and 2.00 g NH4Cl per liter of deionized water. DBT were taken as the sole sulfur source in the culture and the concentra-tion of DBT was 0.1 mmol/L. The solid agar plates were prepared with 15 g agar per liter.

Serial dilutions (up to 10−5) of culture were prepared, and 0.1 mL of the 10−3 — 10−5 dilutions

were plated on nutrient agar plates. Then the plates were incubated at 28—30℃ until colonies

were visible (approx. 2—3 d). The bacterial colonies were selected and incubated into 50 mL of

0.1 mmol/L DBT selective cultures until the bacteria grew well (28—30℃, 150 r/min, approx.

5—7 d). 2 mL culture was taken and acidified with 1 mmol/L HCl solution to pH 2.0, then ex-tracted with 2 mL hexane for 2 min with agitation. The extracted solution was analyzed by HPLC (Agilient 1100 with DAD detector) to determine if HBP or DBTO2 had been accumulated. The HPLC separation column was Zorbax SB-C18 (4.6 mm × 25 cm); the flow phase used was of 90% methane and 10% water. The flow rate was 1 mL/min.

1.2.2 Identification. (i) Morphological Properties. The microorganism was incubated by cover slip technique in the agar of glucose-asparagines culture medium under the condition of 30℃ for 12 and 72 h, then properties of mycelia were examined and studied both in the culture and in the air with the conventional methods. The bacteria were dyed with Gram’s method and acid-fast staining method. (ii) Culture characteristics. The strain was incubated in the glucose-asparagine culture agar, nutriment agar, Sauton’s agar, TY agar and tomato extraction agar respectively under 28℃ for 3—7 d, then the mycelium colors were examined. (iii) Cell wall analysis. By Hase-gawa’s TLC (thin layer chromatography) method, the types of sugars and amino acids in the whole cell hydrolysate were studied. (iv) Aycolic acid analysis. Aycolic acid was analyzed by Lechevalier’s method. (v) The physical and biochemical properties. The physical and biochemical properties of the strain were studied according to Bergey’s Manual of Systematic Bacteriology (vol. IV). (vi) Sequence analysis of 16SrRNA. The strain was incubated and the total DNA were isolated and picked up with lysozyme method. The polymerase chain reaction (PCR) was done to amplify the DNA by the universal primers, and then the PCR products were purified. The RNA sequence was analyzed directly by Taq Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems). The electrophoresis and data gathering was done automatically by Applied Biosys-tems DNA sequencer (model 377A). The sequence was compared with that of the correlative strains in GeneBank to ascertain the suspending strain.

1.2.3 Biodesulfurization properties of the strain. The DBT, 4,6-dimethyl-dibenzothiophene (4,6-DMDBT), phenyl sulfide and thianaphthene were added respectively to 200 mL basic culture in 500 mL flasks to study the biodesulfurization properties. The BSM used contains 2.44 g

No. 5 ISOLATION & IDENTIFICATION OF dsz BACTERIUM 525

KH2PO4, 14.03 g Na2HPO4· 12H2O, 2 g NH4Cl, 0.5 g CH3COONH4, 0.3589 g MgCl2·6H2O,

0.001 g CaCl2·2H2O, 0.001 g FeCl3·H2O, 0.004 g MnCl2 and 5 g glycerol per liter of deionized

water. The inoculating amount was 4% (volume percent). The cultures were incubated at 30℃ with agitating 200 r/min. 1 mL samples were taken each time to detect the substrate concentration and 2—3 mL samples were taken to detect the absorbance values at the wavelength of 600 nm, that were measured by Lambda Bio40 UV/Vis spectrophotometer. The contents of DBT, HBP, 4,6-DMDBT, thianaphthene and phenyl sulfide were detected by HPLC.

2 Results and discussion

2.1 Isolation of microorganisms Under certain separation conditions, the retention time of DBT, HBP and DBTO2 is 5.77,

3.37 and 2.88 min respectively. The extraction of culture with hexane was analyzed by HPLC. By retention time and the ultraviolet-spectra, the metabolic products were analyzed and data show that most of the chosen colonies do not indicate the accumulation of HBP or DBTO2, except the strain LSSE8-1. The metabolic product of the strain is shown in fig. 3. Peaks of the spectrum in 3.37 min was compared with that of HBP standard sample by the Chemstation, and found that they are the same. However, no DBTO2 can be found. It was reported that there are four enzymes in the “4S pathway” reactions, they are DszA, DszB, DszC and oxidize-reducing enzyme DszD. By DszC and DszD, DBT can be oxidized to DBT-sulfoxide and DBT-sulfone, and by DszA and DszD, it can be oxidized further to HBP-sulfonate, followed by DszB, it can be oxidized further to HBP and sulfite; the sulfite will then be oxidized to sulfate [13]. The accumulation of the metabolite

Fig. 3. HPLC chromatography of the culture extraction.

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products depends on activities of these four kinds of enzymes. If the activities of the DszA and DszB are higher than that of DszC, the DBT-sulfone will be oxidized to HBP-sulfonate, then to HBP and sulfate immediately. In consequence, there will be no DBT-sulfone and DBT-sulfide ac-cumulation in the culture at all. If DszB or DszA is not active, then either DBT-sulfone or HBP-sulfonate will be found in the culture. The result reveals that activities of both DszB and DszA in the cell are higher than that of DszC, and DszC catalyzing reaction is the rate-limiting step. The property of the strain is consistent with other bacteria.

2.2 Identification

2.2.1 Morphological properties. When microorganisms were incubated in the agar of glucose-

asparagines culture medium under 30℃ for 14 h, the mycelia of strain began to rupture from the

center of the colony; and for 24 h, it would produce mycelium with ball shape or rod-shape body (fig. 4). After 72 h, the brim of colony kept on growing, and there was no aerial mycelium found. The strain is Gram positive and partly acid-fast.

Fig. 4. Pictures of the mycelium and the ruptured little body. (a) 750×, (b) 10000×.

2.2.2 Culture properties. The strain was incubated in 5 kinds of solid cultures to detect proper-ties in different cultures, and the result is shown in table 1. It reveals that the strain produces no aerial mycelium and soluble pigment; the substrate mycelium of the strain has different colors in different cultures.

Table 1 The culture properties

Culture Aerial mycelium Substrate mycelium Soluble pigment Glucose-asparagine agar none rose pink none

Nutriment agar none flesh-color none Sauton’s agar none baby pink none

Tomato extraction agar none brown none TY agar none rose pink none

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2.2.3 Cell wall analysis. By Hasegawa’s TLC (thin layer chromatography) method, the meso- DAP (diaminopimelic acid), arabinose and galactose were found in the cell wall of the strain. They are the typical components in the cell wall of type IV of the actinomyces.

2.2.4 Aycolic acid analysis. The aycolic acid analysis shows that the strain LSSE8-1 contains nocardomycolic acid in the cell wall.

2.2.5 Physical and biochemical properties. The physical and biochemical properties of the strain are shown in table 2.

Table 2 The physical and biochemical properties of LSSE8-1

Biochemical reactions Result Utilize the carbon sources Result

Liquefy glutin − glucose +

Solidify milk − L-arabinose −

Peptonize milk + D-xylose −

Hydrolyze starch − D-fructose +

Reduce nitrate − D-galactose −

Grown on cellulose − D-manitose +

Produce H2S − fucose +

Produce melanin − maltose −

Hydrolyze urea + rhamnose −

Hydrolyze tyrosine + D-sorbtose +

Decompose adenine + manitol +

Decompose esculin + inositol −

Hydrolyze Tween 60 + paraffin +

Hydrolyze Tween 40 + sodium citrate +

sodium acetate +

2.2.6 Sequence analysis of 16SrRNA. The sequence of 16SrRNA of strain studied is shown in fig. 5. The sequence was compared with the other sequences in Genebank by BLAST (basic local alignment search tool). The result (table 3) shows that the strain is most similar to that of Rhodococcus erythropolis and the identity is 99.77%.

Fig. 5. Sequence of 16SrRNA in the strain.

528 SCIENCE IN CHINA (Series B) Vol. 45

Table 3 A list of strains producing significant 16SrRNA sequence alignments

Date base sources Accession number Species Identities gb|AF235012.1 AF235012 Rhodococcus sp. UFZ-B528 99.77% emb|AJ011328.1 RER011328 Rhodococcus erythropolis 99.77%

gb|U82667.1 NSU82667 Nocardioides simplex 99.77% gb|U82666.1 NSU82666 Nocardioides simplex 99.77% gb|U87968.1 RSU87968 Rhodococcus sp. X309 99.77%

emb|AJ131637.1 RER131637 Rhodococcus erythropolis DCL14 99.77% emb|X79289.1 RE43066RR R.erythreus (DSM 43066) 99.54% emb|X81929.1 RE16RNA1 R.erythropolis(ATCC 4277T) 99.30%

dbj|AB046362.1 AB046362 Rhodococcus erythropolis 98.38%

In all, the strain LSSE8-1 belongs to the evolution branch of Rhodococcus, and the substrate

mycelium grows well. The mycelia have primary ramification and septa, which can rupture into the little body of rod or ball shape and the surface is smooth. The strain does not have aerial my-celium. The cell wall contains aycolic acid, acid-fast partly, which belongs to the cell wall of type IV. In consequence, the strain is named Rhodococcus erythropolis LSSE8-1.

By now, there have been dozens of bacteria isolated that can selectively remove the sulfur in the DBT molecule. But the main strains are Rhodococcus and considered to be the most promising strain for biodesulfurization. The cell or enzyme and/or membrane extracts of Rhodococcus rhodochrous cultures have been suggested in processes for the cleavage of organic C-S bonds in oil or coal[14—16]. It can be used in continuous process involving oxygenation and regeneration steps[17], and in combination with hydrodesulfurization for the “deep desulfurization” of liquid fossil fuels[18], as well as in a process for the desulfurization and desalting of fossil fuels[19]. The pilot-plant scale of biodesulfurization factory works well with Rhodococcus rhodochrous in Amer-ica. The isolated strain will be very useful for the further study on biodesulfurization.

2.3 Biodesulfurization properties Since the substrate concentration will affect

the growth and the activity for biodesulfurization, the relationship between concentration of the model compound DBT in the culture and the growth of the strain as well as the desulfurization activity of the strain was analyzed. Fig. 6 shows the growth curve of the strain in different con-centrations of DBT cultures. Data show that the strain cannot grow in the culture where DBT concentration is higher than 10 mmol/L, because excessive DBT will damage the strain and affect the cell growth. At the same time the strain can-not grow well in the culture where DBT concen-

Fig. 6. Growth curve of the strain in different concentra-tions of DBT cultures. 1, 1 mmol/L DBT; 2, 0.5 mmol/L DBT; 3, 5 mmol/L DBT; 4, 0.1 mmol/L DBT; 5, 0.01 mmol/L DBT; 6, 10 mmol/L DBT.

No. 5 ISOLATION & IDENTIFICATION OF dsz BACTERIUM 529

tration is 0.01 mmol/L. The reason is that DBT is the only sulfur source in the culture, and the extremely low concentration of DBT will affect the growth of the cell. The optimal DBT concen-tration for the strain growth is 1 mmol/L. The income of the dry cells (table 4) accords with the above conclusion. Accompanied by the cell growth, the sulfur in DBT is usually removed out and then the product HBP accumulates. The HBP content in different cultures is shown in fig. 7. The data reveals that the excess DBT would affect the desulfurization activity of the cells. Despite that there are more cells in the 1 mmol/L DBT culture, the HBP content is nearly the same as that in 0.5 mmol/L DBT culture, and it suggests that the rate of desulfurization activity in 1 mmol/L is lower than that in 0.5 mmol/L.

Table 4 Income of drying cell in different DBT concentration cultures

DBT concentration / mmol·L−1 0.1 0.5 1 5 Income of drying cells / g·L−1 2.04 3.06 3.46 2.88

There are many other sulfur-containing or-

ganic compounds in petroleum besides DBT, such as thianaphthene, 4,6-DMDBT and phenyl sulfide. The strain may show different activities in different substrates. Fig. 8 shows the desulfu-rization curve of 0.2 mmol/L thianaphthene and phenyl sulfide respectively. Data reveal that the strain can remove the sulfur rapidly in these two compounds. Within 1 d, the strain can remove the sulfur in phenyl sulfide totally, and can deal with about 80% of sulfur of thianaphthene. Het-erocyclic sulfur compounds such as alkyl dibenzothiophenes and alkyl benzothiophenes are major sulfur compounds in the hydrodesulfurized oil fractions because these sulfur compounds

are highly recalcitrant to HDS treatment. 4,6-DMDBT is taken as a model compound of alkyl dibenzothiophenes to study the desulfurization properties. Fig. 9 shows the desulfurization properties of the growing cells in 0.5 mmol/L 4,6-DMDBT. Data reveal that the strain can remove the sulfur in 4,6- DMDBT. The property is similar to that of the Rhodococcus erythroplis KA2-5-1, which can desulfur alkyl thianaphthene, DBT and the alkyl DBT[20], however, the strain IGTS8

Fig. 7. HBP accumulation in different cultures. 1, 0.5 mmol/L DBT; 2, 1 mmol/L DBT; 3, 5 mmol/L DBT; 4, 0.1 mmol/L DBT; 5, 0.01 mmol/L DBT; 6, 10 mmol/L DBT.

Fig. 8. Biodesulfurization curve of thianaphthene and phenyl sulfide. 1, Thianaphthene; 2, phenyl sulfide.

530 SCIENCE IN CHINA (Series B) Vol. 45

cannot deal with sulfur in thianaphthene and al-kyl thianaphthene at all because the desulfuriza-tion enzymes may be different for thianaphthene and DBT.

3 Conclusions

A strain of nondestructive desulfurization bacterium has been screened out, which can re-move the sulfur in dibenzothiophene (DBT) se-lectively and the metabolic product is 2-hy- droxydiphenyl. There is no dibenzothiophene

sulfone (DBTO2) accumulated. The 16SrRNA, cell wall analysis, physical, biochemical properties and morphological properties show that the strain LSSE8-1 is Rhodococcus erythropolis and it is named Rhodococcus erythropolis LSSE8-1. The strain can grow in the basal salts medium when DBT is not more than 10 mmol/L, and the optimal DBT concentration for growth is 1 mmol/L with the optimal concentration for desulfurization of 0.5 mmol/L. The strain can also be used to cope with the sulfur in thianaphthene, phenyl sulfide and 4,6-DMDBT.

Fig. 9. Result of desulfurization of DMDBT by strain LSSE8-1.

Acknowledgements This work was supported by the State Major Basic Research Development Program of China (Grant No. G2000048004) and SINO Petrochemical Corporation Ltd. (SINOPEC) (Grant No. X599009).

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