Degradation of carbazole and its derivatives by a ... - Springer Link

Appl Microbiol Biotechnol (2006) 73:941–948 DOI 10.1007/s00253-006-0530-3

ENVIRONMENTAL BIOTECHNOLOGY

Degradation of carbazole and its derivatives by a Pseudomonas sp. Li Li & Qinggang Li & Fuli Li & Quan Shi & Bo Yu & Fengrui Liu & Ping Xu

Received: 29 March 2006 / Revised: 31 May 2006 / Accepted: 1 June 2006 / Published online: 9 August 2006 # Springer-Verlag 2006

Abstract Carbazole, carbazoles with monomethyl or dimethyls substituted on different positions (C1-carbazoles or C2-carbazoles), and benzocarbazoles, as toxic and mutagenic components of petroleum and creosote contamination, were biodegradable by an isolated bacterial strain Pseudomonas sp. XLDN4-9. C1-carbazoles were degraded in preference to carbazole and C2-carbazoles. The biodegradation of C1-carbazoles or C2-carbazoles was influenced by the positions of methyl substitutions. Among C1-carbazole isomers, 1-methyl carbazole was the most susceptible. C2-carbazole isomers with substitutions on the same benzonucleus were more susceptible at a concentration of less than 3.4 μg g−1 petroleum, especially when harboring one substitution on position 1. In particular, 1,5-dimethyl carbazole was the most recalcitrant dimethyl isomer. Keywords Biodegradation . Carbazole . Carbazole derivatives . Pseudomonas sp. . Angular dioxygenase

L. Li : Q. Li : F. Li : B. Yu : F. Liu : P. Xu (*) State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People’s Republic of China e-mail: [email protected] L. Li School of Environmental Science and Engineering, Shandong University, Jinan 250100, People’s Republic of China Q. Shi State Key Laboratory of Heavy Oil Processing, China University of Petroleum, Beijing 102200, People’s Republic of China

Introduction Carbazole and its derivatives are common components of fossil fuels and their products. They are also used as chemical feedstocks for the production of dyes, medicines, and plastics. They were detected in atmospheric samples, as well as soils and groundwaters contaminated with petroleum, wood-preserving wastes, and coal-processing residues (Malins et al. 1985; Warshawsky 1992). Carbazole and its derivatives are found to be toxic (O’Brien et al. 2002; Sverdrup et al. 2002) and mutagenic (Reddy and Randerath 1990; Jha and Bharti 2002). Moreover, they readily undergo radical chemistry to generate the more genotoxic hydroxynitrocarbazoles (Benedik et al. 1998). Aerobic biodegradation of carbazole has attracted much attention and as a result, carbazole degrading strains of Pseudomonas (Ouchiyama et al. 1993; Gieg et al. 1996), Sphingomonas (Shepherd and Lloyd-Jones 1998; Kilbane et al. 2002), Ralstonia (Schneider et al. 2000), Bacillus (Kobayashi et al. 1995), Gordonia (Santos et al. 2006), Mycobacterium, and Xanthamonas (Grosser et al. 1991) were isolated. The highly similar pathways of carbazole metabolism were therewith reported in strains P. resinovorans CA10 (Ouchiyama et al. 1993), Pseudomonas sp. LD12 (Gieg et al. 1996), Sphingomonas sp. GTIN11 (Kilbane et al. 2002), and Ralstonia sp. RJGII.123 (Schneider et al. 2000). The genes involved in carbazole degradation were characterized in Sphingomonas CB3 (Shepherd and Lloyd-Jones 1998), Sphingomonas sp. GTIN11 (Kilbane et al. 2002), and P.resinovorans CA10 (Sato et al. 1997a,b). The key enzymes carbazole 1, 9a-dioxygenase (Nam et al. 2002), 2′-aminobiphenyl-2, 3-diol 1,2-dioxygenase (Gibbs et al. 2003; Iwata et al. 2003), and the meta-cleavage compound hydrolase (Nojiri et al. 2003; Riddle et al. 2003a) were purified and characterized.

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Though carbazole and its derivatives are often released into the environment with fossil fuels or their products (Benedik et al. 1998) and are present mostly as alkylated heterocycles (Mao et al. 1994), current studies on the degradation of carbazole are usually carried out in aqueous phase, rarely in oil phase, and little is known about microbial metabolism of alkylcarbazoles. As reported, a mixed culture was capable of degrading most of the carbazoles with monomethyl, dimethyls, and trimethyls substituted on different positions (C1-carbazoles, C2-carbazoles, and C3carbazoles) and one of the carbazoles with tetramethyls substituted on different positions (C4-carbazoles) in crude oil within 192 h (Fedorak and Westlake 1984). However, a pure culture could only degrade carbazole and C1-carbazoles in shale oil (Kilbane et al. 2002). Up to now, there is no other information about the effect of substitutions on the degradation of alkyl carbazoles. We have previously isolated a Pseudomonas sp. XLDN4-9 based on its ability to utilize carbazole as the sole source of carbon and nitrogen. Its enhanced ability to degrade carbazole in the presence of nonaqueous phase liquid has been shown (Li et al. 2004). For the purpose of bioremediation in the environment, in this study, degradation of carbazole and its methyl-substituted derivatives in petroleum by strain XLDN4-9 was carried out. The degradation of carbazole, C1-carbazoles, C2-carbazoles, and benzocarbazoles in diesel and crude oil was investigated. Moreover, the effect of the substitution patterns on the degradation of methyl-substituted carbazoles was examined as well.

Materials and methods Chemicals and oils Analytical grade carbazole, indole, quinoline, anthranilic acid, N-phenylcarbazole, and bis(trimethylsilyl)trifluoroacetamide (BSTFA) and chromatographic grade methanol, tetrahydrofuran, and tert-butylmethyl ether were purchased from Sigma-Aldrich (St. Louis, USA). The fluid catalytic cracking diesel sample, containing carbazole, C1-carbazoles, C2-carbazoles, and C3-carbazoles (about 1,800 μg g−1 diesel in total), was obtained from Petro China Karamay Petrochemical Company. The crude oil sample, containing carbazole, C1-carbazoles, C2-carbazoles, and C3-carbazoles (about 65 μg g−1 crude oil in total), was provided by Xinjiang Dushanzi Petrochemical Factory. Organism and culture condition Pseudomonas sp. XLDN4-9 was isolated by enrichment culture from soil samples collected at a site near a petroleum refinery. This isolated bacterial strain was cultivated in the

Appl Microbiol Biotechnol (2006) 73:941–948

mineral salts medium with 500 mg carbazole l−1 medium. The mineral salts medium contained (l−1 distilled water) 12 g K2HPO4, 11 g KH2PO4, 0.2 g MgSO4·7H2O, 2 g Na2SO4, 2 g KCl, and 1 ml trace metal solution. The pH was adjusted to 7.0. Trace metal solution (l−1 distilled water) contained 0.3 g FeCl2·4H2O, 0.038 g CoCl2·6H2O, 0.02 g MnCl2· 4H2O, 0.014 g ZnCl2, 0.0124 g H3BO3, 0.04 g Na2MoO4· 2H2O, and 0.0034 g CuCl2·2H2O. The strain was identified as Pseudomonas sp. as previously described (Li et al. 2004) and was deposited at the China Center for Type Culture Collection (CCTCC M 205094). Cultures were aerobically incubated at 30°C on a reciprocal shaker at 180 rpm. Degradation of nitrogen containing compounds in aqueous phase The biodegradation of carbazole, anthranilic acid, indole, and quinoline was monitored in growing cell cultures using 50 ml mineral salts medium in 300-ml Erlenmeyer flasks. Carbazole was added to the medium at a concentration of 500 mg l−1 medium. Anthranilic acid, indole, and quinoline were at 300 mg l−1 medium. The initial cell density in the degradation test was adjusted to optical density (OD620) of 0.1 U. In every test, triplicate samples were analyzed and controls without inoculum were monitored. Bacterial growth was monitored by measuring the OD620 of the culture. Cell growth in the medium containing carbazole was measured by plate colony counting. Calibration of the spectrophotometric response against cell dry weight was performed: One OD620 unit (2.75×107 cells ml−1) corresponded to the dry cell weight of 0.495 g l−1. To investigate the influence of indole and quinoline on carbazole degradation, 25 mg of carbazole degradation by the resting cells was performed in the presence of 5, 15, 25, 35, and 45 mg indole or quinoline using 500 ml screw-cap Erlenmeyer flasks containing 50 ml of resting cell suspensions and the resting cell suspension was adjusted to 5 g dry cell weight l−1. Resting cells preparation and specific activity determination were the same as previously described (Li et al. 2004). For every test, triplicate samples were analyzed and controls without cells were also monitored. Degradation of carbazole and its derivatives in petroleum Degradation of carbazole and its derivatives in oil was performed in 300-ml Erlenmeyer flasks. Five grams of oil was mixed with 10 ml of resting cell suspensions, which was 5 g dry cell weight l−1. The flasks were shaken at 30°C, 180 rpm. After incubation for 0, 16, 36, 72, 144, and 216 h, the contents of each flask were transferred to a 50-ml conical tube and centrifuged at 12,000×g for 30 min, then the upper oil layer was removed for analysis.


Specific activity was determined by calculating the degradation rate through the linear portion of the curve. Duplicate samples were analyzed and the allowed tolerance was 0.5%. The controls were prepared and treated identically with potassium phosphate buffer instead of resting cell suspensions. Cloning and sequencing of the car genes Genomic DNA was isolated from Pseudomonas sp. XLDN49 according to the standard protocols (Ausubel et al. 1995). The PCR primers for the amplification of the carbazoledegrading genes were designed based on the DNA sequences from P. resinovorans CA10 (GeneBank accession number D89064). PCR primers for carAaBCAcAd amplification were P 1 , 5′-GCCGACTAGTAAGGAGATGGACG TGGCG-3′, and P2, 5′-GACGAGTACTGCAGCGCCGT CATACGTTGC-3′. Amplification was performed with LA Taq DNA polymerase (TakaRa Biotechnology) according to the manufacturer’s recommendations. The reaction mixture was subjected to 30 cycles of the following procedures: denaturation for 30 s at 94°C, annealing for 30 s at 61°C, and extension for 7 min at 68°C with a final extension at 72°C for 10 min after the cycles were complete. The PCR product without native promoter region was cloned into the vector pMD18-T (TakaRa Biotechnology) with lac promoter to drive its expression, and the recombinant plasmid was introduced into Escherichia coli DH5α (Ausubel et al. 1995). Carbazole-transforming enzyme activity was assayed by testing extradiol dioxygenase activity (Shepherd and Lloyd-Jones 1998) or by visualizing carbazole disappearance (Riddle et al. 2003b). The car genes were sequenced by Shanghai Sangon Biotechnology and the sequence analysis was performed with the BLAST and FASTA programs (Altschul et al. 1990) of the National Center for Biotechnology Information.

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Metabolites of carbazole from growing and resting cells were extracted with tert-butylmethyl ether after acidification by H2SO4 to pH 2. The extracts were dried with Na2SO4 and evaporated by nitrogen gas, and then dissolved in pyridine and derivatized with BSTFA. The derivatives were concentrated under nitrogen gas and analyzed by gas chromatography–mass spectrometry (GC-MS, Hewlett-Packard GCD 1800C) equipped with a 50-m DB-5MS column (J&W Scientific, Folsom, CA, USA). The oven temperature program started at 60°C for 2 min and then was ramped to 320°C at 4°C/min. To analyze carbazole and its derivatives in petroleum, solid phase extraction was adopted to separate nonbasic nitrogen compounds using a 3-ml C18 ISOLUTETM column (Int. Sorbent Tech., UK) following the procedures previously described (Li et al. 1992). Oil samples were deasphaltened by precipitation in n-hexane and eluted from column with n-hexane and dichloromethane. The concentrated fraction was analyzed using a Finnigan Model Trace-DSQ GC-MS system equipped with a HP-5MS fused silica capillary column (30 m, 0.25 mm i.d.) and an Xcalibur data system. The GC oven was initially held at 80°C for 1 min, and was subsequently programmed to 150°C at 7°C/min, then was programmed to 300°C at 3°C/min and held for 15 min with helium as carrier gas. A commercially available internal standard compound, N-phenylcarbazole, was used for the quantification of the carbazoles. Nucleotide sequence accession number The partial carbazole degradation operon nucleotide sequence for XLDN4-9 was submitted to GenBank and the assigned accession number is DQ060076.

Results Analytical methods Bacterial growth and carbazole degradation High-performance liquid chromatography was performed to monitor the degradation of carbazole, anthranilic acid, indole, and quinoline. An Agilent 1100 series (HewlettPackard) instrument equipped with a variable wavelength detector and fitted with a reversed phase C18 column (4.6×150 mm, Hewlett-Packard) was used. Carbazole was analyzed at a flow rate of 0.5 ml min−1 with methanol/ H2O (86:14, v/v) as the mobile phase. Anthranilic acid was analyzed using methanol/H2O/tetrahydrofuran (60:36:4, v/v/v) at a flow rate of 0.5 ml min−1, and indole and quinoline were analyzed using methanol/H2O (70:30, v/v) at a flow rate of 0.3 ml min−1. The effluents were monitored at 254 nm for the samples of carbazole, indole, and quinoline and at 220 nm for anthranilic acid.

Pseudomonas sp. XLDN4-9 grew in mineral salts medium that contained carbazole as the sole source of carbon and nitrogen (Fig. 1a). No increase of biomass was detected when the carbazole-free mineral salts medium was supplied with glucose or NH4Cl with a final concentration of 10 or 0.8 g l−1 medium. Within 56 h, 98% removal of 500 mg l−1 carbazole was achieved by growing cultures of strain XLDN4-9. No significant loss of carbazole was detected in the uninoculated controls. GC-MS analysis was carried out to identify the metabolite of carbazole degradation and anthranilic acid was determined. Anthranilic acid could be utilized by XLDN4-9 as the sole source of carbon and nitrogen (Fig. 1b).

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a

b 0.14 0.22

0.18 0.16

300

0.14 0.12

200

0.10 100

0.08 0.06

Anthranilic acid ( mg L-1)

400

-1

Carbazole (mg L )

-1

Growth ( g dry cell weight L )

0.20

250 0.10 200

100

40

60

80

100

0.06

50 0.04

0

0.04

20

0.08

150

0

0

0.12

300

0

120

24

48

Growth (g dry cell weight L-1)

500

350

72

Time (h)

Time (h) Fig. 1 Utilization of carbazole and its metabolite by Pseudomonas sp. XLDN4-9. a Time course of growth (○), growth in controls without carbon and nitrogen sources (□), degradation of carbazole served as the sole source of carbon and nitrogen ( ), and carbazole in controls without inoculation ( ). b Time course of growth (○), growth in

controls without carbon and nitrogen sources (□), degradation of anthranilic acid served as the sole source of carbon and nitrogen ( ), and anthranilic acid in controls without inoculation ( ). Data are the mean and standard deviation of independent triplicates

To examine the carbazole-degrading genes, a 5.07-kb DNA fragment corresponding to carAaBCAcAd was amplified and sequence blasting showed that it had 99% identity to the 6.9-kb EcoRI DNA fragment of carAaAaBCAcAd from strain CA10 (Sato et al. 1997a,b). It indicated that strain XLDN4-9 might have similar carbazole-degrading steps to that in P. resinovorans strain CA10 (Nojiri et al. 2001).

bazoles. C1-carbazoles were shown to be preferable to that of carbazole and C2-carbazoles. The partial mass chromatograms, representing the distribution of carbazole (m/z 167) and methyl carbazoles (m/z 181) in diesel and crude oil, are shown in Fig. 4a,b. The differences of the peak distribution pattern between the biotreated samples and the untreated controls illustrated that the removal of C1-carbazoles varied

▴

▪

▪

▴

Indole and quinoline are other two main N-heterocyclic aromatic compounds that coexist with carbazole in fossil and its fractions. To show the effect of indole or quinoline on degradation of carbazole, 100 to 900 mg l−1 indole or quinoline were added to the 500 mg l−1 carbazolecontaining resting cell system. As the concentration of indole or quinoline increased, degradation ability of carbazole decreased as shown in Fig. 2, especially when indole was present. Substrate range tests showed that indole and quinoline could serve as the sole source of carbon and nitrogen. Utilization of indole and quinoline with cell growth is shown in Fig. 3.

Specific activity -1 -1 ( mg carbazole min g dry cell )

Effect of indole and quinoline on carbazole degradation 1.8 1.5 1.2 0.9 0.6 0.3 0.0 0

Degradation of carbazole and its derivatives in petroleum Degradation of carbazole and its derivatives by resting cells of strain XLDN4-9 was monitored in diesel and crude oil (Table 1). Significant biodegradation was shown to carbazoles with two or less alkyl substituents and to benzocar-

150

300

450

600

750

900

Coexisting N-heterocycle (mg L-1) Fig. 2 Effect of coexisting N-heterocycle compounds on the degradation of carbazole. Specific activity for degradation of carbazole by Pseudomonas sp. XLDN4-9 in the presence of indole ( ) or quinoline (□). Data are the mean and standard deviation of independent triplicates

▪


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0.14

Discussion

0.12

In pure aqueous phase, the specific activity for carbazole degradation by resting cells of Sphingomonas sp. GTIN11 was 1.34 mg min−1 g−1 dry cells (Kilbane et al. 2002), and it was 1.67 mg min−1 g−1 dry cells by Sphingomonas sp. CDH-7 (Kirimura et al. 1999). Pseudomonas sp. XLDN4-9 showed specific activity toward carbazole at 1.74 mg min−1 g−1 dry cells. Sphingomonas sp. GTIN11 was reported to have degrading abilities to carbazole and C1-carbazoles in shale oil (Kilbane et al. 2002). Degradation of carbazoles with two or more substitutions by a pure culture was not reported until now, though mixed cultures were found to be capable of degrading C1-carbazoles, C2-carbazoles, C3carbazoles, and C4-carbazoles (Fedorak and Westlake 1984). In this study, Pseudomonas sp. XLDN4-9 showed degradation abilities not only to carbazole and C1-carbazoles, but also to C2-carbazoles and benzocarbazoles. The complex chemicals in petroleum may affect the degradation of carbazoles, such as indole and quinoline. Carbazole degraders showed a broad substrate range (Nojiri et al. 1999; Santos et al. 2006). The utilization of indole and quinoline, which are coexisting nitrogen compounds in petroleum, indicated the application potential of strain XLDN4-9. However, the inhibition effect of indole and quinoline on carbazole degradation would be a concern. It was shown that C1-carbazoles were more susceptible than carbazole. Similarly, in the degradation of alkyl dibenzothiophenes, C1-dibenzothiophenes, and C2-dibenzothiophenes were removed in preference to the unalkylated parent (Prince and Grossman 2003) and alternant PAHs could also be degraded more rapidly than nonalternant PAHs (Aitken et al. 1998). Specifically, 1-methyl carbazole was the most quickly degraded isomer of the C1-carbazoles. It is notable that in many aromatic compounds, such as carbazole, the α-position is related to the initial attack by degrading enzymes. Likewise, when microorganism attacked dibenzothiophene isomers, a slight preference for those substituted on α-position was observed (Prince and Grossman 2003; Li et al. 2005). However, there were some differences in the microbial degradation of naphthalene.

280 0.10 240

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Growth ( g dry cell weight L-1)

Degradation of N-heterocycles ( mg L-1)

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0.00 0

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6

Time (d) Fig. 3 Growth (□) of Pseudomonas sp. XLDN4-9 with utilization of indole ( ) and growth (○) of the strain with utilization of quinoline ( ). Control scatter point ( ) showing indole concentrations at the beginning and at the end time of tests and control scatter point (◊) showing quinoline concentrations at the beginning and at the end time of tests. Data are the mean and standard deviation of independent triplicates

▴

▪

•

with the substitution positions of the methyl. Specific activity for 1-methyl carbazole was the highest at 339.8 μg h−1 g−1 dry cells in diesel and 11.6 μg h−1 g−1 dry cells in crude oil, while the specific activity for 3methyl carbazole was the lowest at 221.8 μg h−1 g−1 dry cells in diesel and 5.1 μg h−1 g−1 dry cells in crude oil. In comparison with C1-carbazoles, C2-carbazoles were more recalcitrant. Removal of C2-carbazole isomers within 72 h of treatment and the specific activities for different isomers are shown in Table 2. In diesel, the specific activity for 1,4-dimethyl carbazole was the highest, followed by 2,3dimethyl carbazole and 2,6-dimethyl carbazole. In crude oil, strain XLDN4-9 also showed the highest specific activity for 1,4-dimethyl carbazole, followed by 1,3-dimethyl carbazole and 2,4-dimethyl carbazole. In both of diesel and crude oil, 1,5-dimethyl carbazole showed to be the most recalcitrant isomer.

Table 1 Removal within 72 h of treatment and specific activity for carbazole, C1-carbazoles, and C2-carbazoles Substrates

Carbazole C1-carbazoles C2-carbazoles Benzocarbazoles

Diesel

Crude oil

Controls (μg g−1)

Removal (%)

Specific activity (μg h−1 g−1 dry cells)

Controls (μg g−1)

Removal (%)


90.1 380.8 876.4 2.5

99 71.6 14.2 69.7

550 1,090 410 4.7

3.96 3.9 17.6 0.6

90 97 17.4 64.4

22 23 16 1.2

Data are the mean of independent duplicates

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Fig. 4 Mass chromatograms of carbazole and methyl carbazoles in diesel (a) and in crude oil (b) before and after 72 h of biotreatment. Peak identities: 1 carbazole, 2 1-methyl carbazole, 3 3-methyl carbazole, 4 2-methyl carbazole, and 5 4-methyl carbazole

Naphthalene dioxygenase initiated the biodegradation of naphthalene on the positions 1 and 2, while 1-methylnaphthalene (α-methyl) was degraded more slowly than 2-methylnaphthalene (β-methyl) (Leblond et al. 2001; Wammer and Peters 2005). Another speculated explanation for the susceptibility of 1-methyl carbazole is that the presence of methyl on position 1 led to a favorable orientation of position 8 to the active site, thus, enhanced the initial attack on position 8. This speculation was supported by the degradation of different C2-carbazole isomers. The effect of substitutions on the degradability of C2-carbazole isomers was more intricate in comparison with that of C1-carbazole isomers. It was shown that C2-carbazoles with two substitutions on the same benzonucleus were more susceptible during the degradation at a low concentration (less than 3.4 μg C2-carbazoles g−1 crude

oil). Especially with one of the methyl groups on position 1, C2-carbazoles, such as 1,4-dimethyl carbazole and 1, 3-dimethyl carbazole, were degraded faster than other isomers. This could be because the substitution on one benzo-nucleus made the effective degradation initiate on the opposite nonsubstituted benzo-nucleus, and it seemed that the presence of substitution on position 1 enhanced the enzyme attack on the opposite aromatic nucleus (position 8). In addition, C2-carbazoles with substitutions on both aromatic nuclei, such as 1,7-dimethyl carbazole and 2, 7-dimethyl carbazole, could be degraded at a relatively lower rate. Accordantly, degradation of 10 μg l−1 chlorinated dibenzofurans (DFs) and dibenzo-p-dioxins (DDs) by Pseudomonas resinovorans strain CA10 and Terrabacter sp. strain DBF63 showed that the dioxygenation occurred mainly on the nonsubstituted aromatic nuclei, though chlorine-substituted aromatic rings could also be attacked

Table 2 Removal after 72 h of treatment and specific activity for C2-carbazole isomers C2-carbazole isomers

1,8-Dimethyl 1,3-Dimethyl 1,6-Dimethyl 1,7-Dimethyl 1,4-Dimethyl 1,5-Dimethyl 2,6-Dimethyl 2,7-Dimethyl 1,2-Dimethyl 2,4-Dimethyl 2,5-Dimethyl 2,3-Dimethyl 3,4-Dimethyl

Diesel Controls (μg g−1 oil)

Removal (%)


Controls (μg g−1 oil)

Removal (%)


85.25 85.74 79.08 89.50 95.66 94.37 48.80 104.27 –a 59.45 61.51 20.41 16.27

11.2 8.9 5.3 8.7 13.6 0.1 16.4 4.2 –a 18.2 77.0 40.4 22.8

22.8 45.4 22.1 46.4 76.6 0 48.8 23.2 –a 23.9 26.8 55.3 17.8

3.40 2.11 1.68 1.95 2.26 2.11 0.46 0.94 0.56 1.04 0.73 0.22 0.20

1.3 24.6 0.9 8.2 50.0 0 11.5 13.2 33.4 55.2 7.5 87.2 34.4

0 3.3 0.073 1.0 5.4 0 0.33 0.78 1.2 2.9 0.34 1.1 0.25

Data are the mean of independent duplicates Peak in poor separation

a

Crude oil


(Habe et al. 2001). It was noted that no degradation was detected for 1,5-dimethyl carbazole, and unlike the α-position preference for 4,6-dimethyl dibenzothiophene (Prince and Grossman 2003), no degradation advantage was shown for 1,8-dimethyl carbazole. Effective degradation was detected for 1,4-dimethyl carbazole, 1,3-dimethyl carbazole, and 2,3-dimethyl carbazole within the range of 15 to 105 μg C2-carbazoles g−1 diesel. This result was consistent with the degradation of C2-carbazoles at low concentrations in crude oil, indicating that the effective degradation occurred when two methyls substituted on the same benzo-nucleus. Unlike the occurrence at a lower concentration of C2-carbazoles in crude oil, the degradation of 1,7-dimethyl carbazole and 2,6-dimethyl carbazole in diesel was fast, indicating that the effective degradation also initiated on the substituted aromatic nucleus with a relatively higher concentration. Similarly, during the degradation of monochlorinated DFs and DDs at concentrations from 100 to 1,000 μg l−1, it was shown that angular dioxygenase could attack both substituted and nonsubstituted aromatic nuclei (Nojiri and Omori 2002). However, 1,5-dimethyl carbazole was still the most recalcitrant isomer. Though degradation of 1,5-dimethyl carbazole could be initiated on position 8, the substitution on position 5 might become a block to its further degradation. The results indicated that substitution patterns strongly influenced the degradability of C2-carbazole isomers. In this study, the degradation of carbazole and its derivatives were shown in petroleum, and results indicate the degradability was strongly affected by the coexisting compounds and the patterns of the substitutions. However, for cleanup of carbazole and its derivatives in the environment, the further study on degradation kinetics of individual alkyl carbazole is necessary, and more efforts should address the codegradation mechanism of nitrogen containing aromatic compounds. Acknowledgements The work was supported by the Chinese National Natural Science Foundation (grant nos. 20377026 and 20590368) and Chinese National Programs for High Technology Research and Development (grant no. 2004AA649160).

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