World J Microbiol Biotechnol (2012) 28:3075–3080 DOI 10.1007/s11274-012-1105-3

SHORT COMMUNICATION

Isolation and characterization of a new Achromobacter sp. strain CAR1389 as a carbazole-degrading bacterium Zahra Farajzadeh • Hamid Reza Karbalaei-Heidari

Received: 24 December 2011 / Accepted: 11 June 2012 / Published online: 20 June 2012 Ó Springer Science+Business Media B.V. 2012

Abstract In this work, a bacterial strain with suitable capability to metabolize carbazole (CAR) as a main nitrogen containing compound of petroleum was isolated and characterized. 16S rDNA gene analysis and morphological characteristics of the strain showed that the isolate belonged to the genus Achromobacter and was tentatively named as Achromobacter sp. strain CAR1389. The growth monitoring and biodegradation rate measurements of carbazole in minimal medium supplemented by 6 mM CAR revealed that the strain CAR1389 is able to remove more than 90 % of this compound at 25, 30, and 37 °C during 7 days. The effect of higher concentrations of the carbazole on growth rate and metabolizing activity of the strain exhibited the Achromobacter sp. strain CAR1389 can tolerate increasing levels of CAR concentration up to 21 mM in culture media and degrade 43 % of this toxic material. According to these results and high tolerance of this bacterium in regards to higher concentrations of CAR, we suggest the strain CAR1389 as a suitable isolate to do biorefining of crude oil and also bioremediation processes in highly contaminated area of carbazole. Keywords Achromobacter sp.  Biodegradation  Carbazole  Nitrogen heterocyclic compound  Polycyclic aromatic hydrocarbons Electronic supplementary material The online version of this article (doi:10.1007/s11274-012-1105-3) contains supplementary material, which is available to authorized users. Z. Farajzadeh  H. R. Karbalaei-Heidari (&) Molecular Biotechnology Laboratory, Department of Biology, Faculty of Sciences, Shiraz University, P. O. Box: 71467-13565, Shiraz, Iran e-mail: [email protected] H. R. Karbalaei-Heidari Institute of Biotechnology, Shiraz University, Shiraz, Iran

Introduction Among the major environmental pollutants, polycyclic aromatic hydrocarbons (PAHs) are of great concern because of their toxicity, carcinogenic potential, and various hazardous effects on natural environment and humans. These compounds occur naturally in the environment and are also produced in large quantities as a consequence of industrial activities (Cao et al. 2009). Investigation on biodegradation of these xenobiotic by harnessing diverse metabolic pathways of microbes can achieve complete removal of the aromatic pollutants and could have an important role in environmental cleanup (Philp et al. 2005). Carbazole, a typical nitrogen heterocyclic compound (NHC), is one of the main nitrogen components in coal tars, shale oil, crude oil, petroleum products and also used as chemical feedstock for the production of dyes, pharmaceuticals, insecticides and plastics (Padoley et al. 2008). The wide utilization of carbazole in chemical industry and its difficult elimination from petroleum has led to appearance of this recalcitrant compound in atmospheric samples, as well as soil, ground waters, and river sediments (Kuehl et al. 1984). Therefore, it is necessary to find effective biological methods to remediate sites contaminated with carbazole and its derivatives. In this regards, researchers have reported some bacterial strains as carbazole (CAR)-utilizing bacterium. A number of CARdegraders have been isolated and characterized so far, including Pseudomonas sp. CA06 (Ouchiyama et al. 1993); Sphingomonas sp. GTIN11 (Kilbane et al. 2002); Nocardioides aromaticivorans IC177 (Inoue et al. 2006) and Klebsiella sp. LSSE-H2 (Li et al. 2008) which indicate CAR-degrading bacteria can be found in different genera. Most of the isolates degrade carbazole by following a

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similar metabolic pathway in which carbazole is first degraded to 20 -aminobiphenyl-2,3-diol through angular dioxygenation by carbazole 1,9a-dioxygenase (CARDO) (Sato et al. 1997). Subsequent biodegradation of carbazole by all characterized cultures involve the conversion of it to 2-aminobenzoate (anthranilic acid) as an intermediate which is then completely mineralized. Moreover, Guo et al. (2008) reported that a microbial consortium consisting of Chryseobacterium sp. NCY and Achromobacter sp. NCW which is able to utilize carbazole as the sole source of carbon, nitrogen, and energy; although neither the pure strain NCY nor NCW could degrade carbazole after domestication for several times. In this study, we describe the isolation and characterization of a novel CAR-metabolizing bacterium, Achromobacter sp. CAR1389 from the oil-contaminated area in Fars province in Iran, which has outstanding potential to degrade carbazole even in culture media supplemented by the high concentrations of carbazole.

Materials and methods Chemicals and media Analytical grade carbazole was obtained from BDH Company. Bacterial culture media and dimethyl sulfoxide (DMSO) was purchased from Merck (Darmstadt, Germany). Solvents for HPLC and GC/MS analysis were chromatographic grade. All other chemicals used in this study were also analytical grade. Enrichment and isolation of CAR-utilizing bacteria The minimal medium (MM) containing final concentration of 6 mM carbazole (CAR) was used for enrichment culture and isolation of CAR-degrading strains. Several samples from different oil contaminated area in Fars province in Iran were collected. Each of the soil samples was suspended in 0.9 % NaCl solution and filtered through Wathman paper No.1. Then the filtrate was put on a cellulose acetate filter with a pore size of 0.2 lm to collect bacteria. The membrane filters were suspended in 50 ml of MM supplemented by 6 mM CAR and incubated at 30 °C and 180 rpm. The MM had the following composition (g/liter): Na2HPO412H2O, 2.2; KH2PO4, 0.8; FeSO4 7H2O, 0.015; CaCl22H2O, 0.015; MgSO47H2O, 0.015; yeast extract, 0.025. The pH was adjusted to 7.0 and carbazole was supplied as the source of C and N of the medium. After 2 weeks of cultivation, each of test flasks which showed carbazole utilization was transferred to fresh medium. Pure cultures were obtained by streaking the enrichment culture on MM plus carbazole agar plates to

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select colonies that were capable of using carbazole. The dispersion of water-insoluble CAR with dimethyl sulfoxide enhanced mineralization of CAR by bacterial strain (Ouchiyama et al. 1993). One percent concentration of DMSO was applied in all experiments. Phenotypic characteristics and molecular identification Bacterial species identification was done by microscopic observation of colony morphology, Gram staining, measuring some biochemical parameters (Smibert and Krieg 1994) and by determining the DNA sequences of partial 16S rDNA gene. Total DNA of strain CAR1389 was extracted by using QIAamp DNA Mini kit (Qiagen). The partial 16S rDNA gene sequence of the selected isolate was amplified by PCR using the primers: HRK1 (50 -ACTCCTACGGGAGGCAGCAG-30 ) as the forward and HRK2 (50 -TGACGGGCGGTGTGTACAAG-30 ) as the reverse primer. The amplification was carried out in 50 ll of reaction mixtures as follows: 3 min at 94 °C; 35 cycles of 1 min at 93 °C, 45 s at 58 °C, and 1:30 min at 72 °C, and a final extension at 72 °C for 10 min. The purified PCR product was sequenced in both directions using an automated sequencer by SeqLab Company, Germany. The phylogenic relationship of the isolate was determined by MEGA 4.0 software (Tamura et al. 2007). Carbazole degradation experiments The biodegradation of carbazole was monitored in growing cell cultures using 50 ml minimal medium containing 6 mM carbazole in 250 ml Erlenmeyer flasks. The isolated bacterium cultures were incubated at 25, 30, 37 °C, and samples were analyzed every 24 h during 7 days. The samples were centrifuged (4,000 g, 10 min) to remove the cells and residual substrate. The supernatants were adjusted to pH 3.0 using 6 N HCL and then extracted twice with 15 ml ethyl acetate. Water was removed from ethyl acetate extracts with sodium sulfate anhydrous (Na2SO4) and then each samples was evaporated to dry at below 40 °C. After evaporation of ethyl acetate, the samples were resuspended in 1 ml acetonitrile and analyzed by HPLC (Kilbane et al. 2002). For resting cell experiment, Achromobacter sp. strain CAR1389 was cultivated in 100 ml MM ? 6 mM of CAR and incubated under same incubation conditions described above. The cells from 3 days culture were harvested by centrifugation at 3,000g for 10 min at 4 °C, and the pellet washed twice with 50 mM potassium phosphate buffer, pH 7.0 and aliquoted into several new small scale fresh medium containing the initial concentration of CAR (6 mM in 10 ml culture medium). Then, the new cultures were allowed to grow at 30 °C and 180 rpm for 6 h intervals,

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each culture was removed and the sample extract was analyzed for residual CAR concentration by HPLC. In addition to test samples, a culture medium without cell was taken as a negative control. Test samples and controls studies were conducted in triplicates. In order to study the effect of carbazole concentration on growth and biodegradation activity of the strain CAR1389, minimal medium supplemented by 1, 5, 9, 13, 17, and 21 mM carbazole were prepared and inoculated with 1 % of an overnight isolate culture. After 7 days cultivation, similar mentioned method was used to ready the samples for HPLC analysis.

Analytical methods High pressure liquid chromatography equipment (HPLC) (model Jasco, Japan) with a reverse phase C18 column (250 9 4.6 mm) was used. Analysis was carried out at room temperature. The mobile phase was acetonitrile and 10 mM phosphate buffer (pH 6.0) with the ratio of 60:40. The flow rate of mobile phase was 1.0 ml/min. The effluent was monitored at an absorbance of 230 nm. In order to identify the products formed during carbazole degradation by the isolated strain, thin layer chromatography (TLC) and GC– MS analysis were also conducted. The dried ethyl acetate extract from a 3 days culture medium was developed on a silica gel plate with a solvent system of toluene–dioxane– acetate (30:8:16, by volume) (Inoue et al. 2005). In addition to the TLC analysis, a portion of each culture extract was directly analyzed by GC–MS. The gas chromatography/ mass spectrometry (GC–MS) analysis were carried out using a DB-5 capillary column (Phenyl-methylpolysiloxane,

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25 m 9 0.25 mm). Mass spectrographs were compared with various libraries of mass spectrograph data prepared from known standard compounds.

Results Isolation and characterization of strain CAR1389 Several petroleum contaminated soil samples were collected from different area of Fars province in Iran and isolation of potent CAR-utilizing bacterial strains was performed by enrichment method in liquid culture using MM supplemented by 6 mM carbazole as the sole source of carbon and nitrogen. Initially, a mixed culture was obtained; however, after streaking onto MM plus carbazole agar plates, individual colonies were isolated and, eventually, a pure culture of strain CAR1389 with good ability to grow in the presence of carbazole (6 mM) was obtained. Microscopic analysis showed the strain CAR1389 is a Gram-negative, rod-shaped bacterium. It is an aerobic, nonmotile, non-sporulating, and catalase-positive strain. The strain was able to use urea and citrate, but not glucose, lactose and sucrose. Production of H2S, gas and indole was also measured negative. A part of 16S rDNA (1,006-bp) gene sequence of the present strain was amplified and has been deposited in the GenBank database under accession number HQ832894.1. Based on BLAST search analysis performed, the strain CAR1389 showed 99 % sequence identity in 16S rRNA with the genus Achromobacter. Figure 1 exhibits the phylogenic tree constructed by neighbor-joining method based on 16S rDNA gene sequences of the strain.

Fig. 1 Neighbor-joining tree based on 16S rDNA gene sequences showing the position of strain CAR1389 relative to some other validly published species from the genus Achromobacter. The bootstrap consensus tree inferred from 1,000 replicates are shown next to the branches (Bootstrap values [50 % are indicated). The accession numbers of all 16S rDNA sequence data are shown in parentheses. Phylogenetic analyses were conducted in MEGA4

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Fig. 2 Time course of CAR degradation (filled square) and growth rate (filled triangle) of the Achromobacter sp. strain CAR1389 at 30 °C and 180 rpm. The carbazole concentration values are means of three independent replicates. The average standard for all data point was 5 % or less

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Fig. 3 The percentage of CAR biodegradation in different carbazole concentrations by the strain CAR1389

Growth of strain CAR1389 in different concentrations of carbazole Growth and characterization of carbazole degradation The growth and carbazole utilization of the strain CAR1389 was monitored for 7 days in MM containing 6 mM carbazole. As shown in Fig. 2, incubation of the inoculated flasks at 30 °C and 180 rpm, revealed the growth of CAR1389 reached to stationary phase during 2 days and 55 % of the existing carbazole was metabolized, decreasing its amount from 6 to 2.7 mM. After third day, more than 75 % of carbazole was degraded and mineralized. Moreover, the resting cell growth of strain CAR1389 showed 56 % removal of CAR after 12 h and reached up to 95 % degradation within 36 h (Fig. S1). To confirm that the similar carbazole degradation pathway was used by the strain CAR1389, GC-Mass analysis and TLC of the culture extract after 3 days were conducted. The results showed that the anthranilic acid and 20 -aminobiphenyl-2,3-diol, as the main intermediate metabolites, produced during CAR mineralization by this strain (data not shown). Effect of temperature on carbazole biodegradation In order to study the effect of incubation temperature on carbazole biodegradation, the isolate was incubated at 25, 30, and 37 °C and other culture conditions were similar. Actually, similar to the metabolic activities of the strain at 30 °C, the isolate was able to grow up and metabolize carbazole at both 25 and 37 °C. Kinetics of CAR-utilization is also almost similar with a little modification. In all three tested temperatures, the Achromobacter sp. strain CAR1389 was able to mineralize 75 % of CAR within 3 days (Fig. S2).

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The strain CAR1389 can grow up in high concentration levels of carbazole in comparison with previously reported strains. To investigate the amount of carbazole degradation in different concentration of CAR, several inoculated flasks supplemented by various CAR concentrations from 1 mM up to 21 mM were incubated at 30 °C and 180 rpm. As shown in Fig. 3, increasing of CAR concentration up to 21 mM did not have serious toxicity effect on strain CAR1389 and the isolate grew well and could degrade 43 % of 21 mM of carbazole. Although the consumption rate of substrate was slower in higher concentrations of carbazole when compared with those of lower CAR concentrations.

Discussion Based on screening program designed for carbazoledegrading bacteria, a new bacterial strain CAR1389 with suitable ability to remediate carbazole from culture media was isolated. According to morphological and physiological characteristics and comparative sequence analysis of the 16S rDNA gene of strain CAR1389 and other closely related microorganisms showing maximum sequence similarity with the Achromobacter species in the GenBank database, the isolate was placed in the Achromobacter spp. and designated as ‘‘Achromobacter sp. strain CAR1389’’. The phylogenic tree constructed by neighbor-joining method based on 16S rDNA gene sequences, indicated that the strain CAR1389 is formed a monophyletic clade with Achromobacter piechaudii and Achromobacter spanius strains with a high bootstrap value.

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As shown in Fig. 2, Carbazole biodegradation activity of the strain CAR1389 reveals the CAR-utilizing ability by an Achromobacter spp. Until now, nearly most reports in regard to biodegradation rate of carbazole exhibited the degradation of CAR by bacteria in 3 mM concentration or lower in culture media (Kilbane et al. 2002). Recently, Singh et al. (2011) reported that an Acinetobacter sp. degraded 84 % of the 3 mM CAR during the first 3 days of incubation. Also, Zhao et al. (2011) showed 97 % biodegradation of carbazole by seven Pseudomonas sp. strains when cultured in 3 mM of carbazole and according to Kirimura et al. (1999) the Sphingomonas sp. CDH-7 could completely degrade 3 mM of carbazole within 50 h (1999). In comparison with these reports, our strain shows relatively lower CAR-mineralization rate. However, the isolate CAR1389 studied in higher initial concentration of CAR to survey the growth and biodegradation ability of carbazole and showed that it can live and thrive in this harsh condition. The TLC and GC-Mass analysis results showed the same findings to those obtained by Ouchiyama et al. (1993) which proposed a CAR degradation pathway for Pseudomonas spp. Based on previous reports, the color change of culture media to yellow as a result of bacterial growth in the presence of carbazole also confirm that a meta-cleavage of 2-aminobiphenyl-2,3-diol was occurred during CARmineralization pathway. It is obvious that the conversion of CAR to anthranilic acid, as an easily degradable and harmless substrate, is essential for the tryptophan biosynthesis pathway in minimal medium by the isolate (Gibson and Pittard 1968). Ability of a bacterial strain to remediate carbazole in various temperatures around average environmental temperature (25, 30 and 37 °C) at the similar level could be one of its advantages to apply as a suitable bioremediation candidate strain. A few reports exists which examine the effect of temperature in CAR-utilizing activity of a microorganism. In 2006, Santos et al. (2006) reported a Gordonia sp. strain F.5.25.8 with capability to grow at 42 °C, although the number of cells was lower than that at 30 °C after 10 days incubation. In the case of strain CAR1389, the cell growth rate and carbazole removal quantities showed the same level in all three tested temperatures. Moreover, the potent capability of the strain CAR1389 to growth and mineralization activity in media with high concentrations of carbazole revealed excellent remediation potential of the strain CAR1389 to environmental cleanup of area contaminated by high levels of PAHs. There are a few reports about the ability of a strain to degrade high concentrations of carbazole (Larentis et al. 2011). Li and colleagues reported carbazole degradation activity up to 19 mM of CAR by Klebsiella sp. LSSE-H2 (2008). To the best of our knowledge, this is the first report

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from an Achromobacter sp. which is able to grow and have a degradation activity in the medium consisting of high concentration of carbazole up to 21 mM concentration. Acknowledgments The authors are indebted to Dr. Younes Ghasemi for GC-Mass analysis. This study was supported by Shiraz University. Conflict of interest of interest.

The authors declare that they have no conflict

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