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International Biodeterioration & Biodegradation 84 (2013) 35e43

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International Biodeterioration & Biodegradation

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Carbazole degradation and biosurfactant production by newly isolatedPseudomonas sp. strain GBS.5

Gajendra B. Singh, Sanjay Gupta, Nidhi Gupta*

Department of Biotechnology, Jaypee Institute of Information Technology, Noida, Uttar Pradesh, India

a r t i c l e i n f o

Article history:Received 22 March 2013Received in revised form21 May 2013Accepted 22 May 2013Available online 26 June 2013

Keywords:BiodegradationBiosurfactantCarbazolecar genePseudomonas

* Corresponding author. Department of Biotechnoformation Technology, A-10, Sector-62, Noida 201307,120 2594211; fax: þ91 120 2400986.

E-mail address: nidhi.gupta@jiit.ac.in (N. Gupta).

0964-8305/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.ibiod.2013.05.022

a b s t r a c t

A novel biosurfactant producing bacterium, designated as Pseudomonas sp. strain GBS.5, having theability to degrade carbazole has been isolated. The specific activity of carbazole degradation was found tobe 11.36 mmol min�1 g�1 dry cells. GCeMS analysis revealed that the growth of bacterium on carbazolewas accompanied with the production of biosurfactant. The biosurfactant produced had the maximumemulsification index of 53 � 1.52% with n-hexadecane. Sequence analysis of the carbazole degradinggenes revealed changes in six different amino acids as compared to other well established strains. Studyalso confirmed that in addition to carbazole, bacterium has the ability to degrade other polycyclic aro-matic hydrocarbons such as fluoranthene, fluorene, naphthalene, phenanthrene and pyrene. A highdegradation rate of carbazole and broad substrate range indicates that it has the potential to be used forbioremediation of polycyclic aromatic hydrocarbon contaminated sites.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Carbazole (CAR) is nitrogen containing heterocyclic poly-aromatic hydrocarbon (PAH) and is known to be carcinogenic andmutagenic (US EPA, 1986). It is used as a feedstock in dye, plasticand pharmaceutical industries and is found in soil contaminatedwith crude oil and petrochemicals. Various physicochemicalmethods have been developed to remove PAHs from the environ-ment. However, all these techniques are costly and cause pollution.Bioremediation is a promising technology for cleaning up suchcontaminated sites. Biodegradation processes exploit the naturalpotential of microorganisms to utilize these compounds as theirenergy source. Microorganisms have been reported for the degra-dation of CAR and include both Gram-positive and Gram-negativebacteria like Pseudomonas, Burkholderia, Acinetobacter, Sphingo-monas,Novosphingobium, Klebsiella, Gordonia and others (Castorenaet al. 2006; Santos et al. 2006; Li et al. 2008; Yang et al. 2009; Singhet al. 2011a,b; Nojiri, 2012). The biodegradation pathway of CAR is awell established pathway and it results in the conversion of CAR toanthranilic acid. This pathway was first reported by Ouchiyamaet al. (1993) for Pseudomonas resinovorans strain CA10. The first

logy , Jaypee Institute of In-Uttar Pradesh, India. Tel.: þ91

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step is catalyzed by the enzyme carbazole-1,9a-dioxygenase(encoded by carAaAcAd genes) and results in the formation ofdihydroxylated intermediate which is spontaneously converted to20-aminobiphenyl-2,3-diol. This product is further cleaved by metacleavage enzyme (encoded by carBaBb genes) to form 2-hydroxy-6-oxo-6-(20-aminophenyl)-hexa-2,4-dienoic acid which is then con-verted to anthranilic acid when attacked by hydrolase enzyme(encoded by carC gene). These genes are arranged in the form of anoperon designated as ‘car’ operon.

Owing to the hydrophobic nature of heterocyclic aromatic pol-lutants, microbial degradation studies have been frequently carriedout either with organic solvents or synthetic surfactants. Bio-surfactants, as less toxic, are biodegradable and more eco-friendlythan synthetic surfactant. They are known to enhance the biodeg-radation rate of PAHs by increasing its bioavailability via solubili-zation and emulsification (Lawniczak et al. 2013). Since,bioavailability is a prime limiting factor for the biodegradation ofaromatic pollutants, use of microorganisms with the ability toproduce biosurfactants are preferred in biodegradation techniques.Although Pseudomonas spp. are known to be involved in thedegradation of CAR, there is no report of Pseudomonas sp. pro-ducing biosurfactant during CAR degradation.

In this study, we report the isolation and characterization of anew strain of Pseudomonas sp., designated as GBS.5, capable ofdegrading CAR. Bacterial growth and degradation characteristicswere also analyzed. GCeMS analysis led to the identification ofcertain long chain alkanes that are associated with biosurfactant

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production. Biosurfactant production was also confirmed by otherqualitative and quantitative assays.

2. Materials and methods

2.1. Chemicals and media

CAR (96% purity) was purchased from Acros Organics (NewJersey, USA). Other PAHs (greater than 98% purity) like biphenyl,dibenzofuran (DBF), dibenzothiophene (DBT), fluoranthene, fluo-rene, naphthalene, phenanthrene, pyrene and polymerase chainreaction (PCR) reagents were obtained from SigmaeAldrich(St Louis, MO, USA). Organic solvents and other chemicals were ofHPLC and analytical grade, respectively from Qualigen (Mumbai,India) and Merck (Darmstadt Hesse, Germany). Basal salt medium(BSM) was composed of (per liter of solution) 2.44 g of KH2PO4;5.57 g of Na2HPO4; 2 g of Na2SO4; 2 g of KCl; 0.2 g of MgSO4;0.001 g of FeCl3.6H2O; 0.02 g of MnCl2.4H2O; 0.003 g ofCaCl2.2H2O. The pH of prepared BSM was 7.0 � 0.05. A 15 gl�1 agarwas added to the BSM to make solid BSM plates. During PAHsdegradation study, NH4Cl (2 gl�1) was added to the BSM as ni-trogen source. LuriaeBertani (LB) medium contained (per liter ofsolution) 10.0 g of trypton; 5.0 g of yeast extract and 10.0 g of NaCl.Solid LB medium was prepared by adding 15 gl�1 agar in to aboveliquid media.

2.2. Enrichment and isolation of carbazole degrading bacteria

Various soil and activated sludge samples were collected fromIndian oil refineries (Gujarat, Jaipur, Mathura, Panipat), dye in-dustries (Ahmedabad) and sewage treatment plants (New Delhi,Ghazipur), located across various cities in India. One gram ofsample was added into 250 ml Erlenmeyer flask containing100 ml BSM supplemented with 500 ppm of CAR as carbon andnitrogen source. The media was incubated at 30 �C and 180 rpmin a rotary shaker. After 4 d of incubation, 5% of enriched culturewas transferred to fresh BSM containing CAR and incubated un-der same conditions. This procedure was repeated four times.Later, samples were diluted serially and plated on solid BSMto obtain isolated colonies. Isolates were inoculated in BSMcontaining 500 ppm of CAR and incubated at 30 �C for 7 d. Uti-lization of CAR by isolates was analyzed by quantifying initial andfinal concentrations. Selection of microorganism of interestamong all isolated strains was based on the higher CAR degra-dation activity.

2.3. Phenotypic characterization of isolate GBS.5

Gram staining and spore staining were done using standardprotocol (Gerhardt et al. 1994). Cell motility was determined withan optical microscope using the hanging drop method (Suzukiet al. 2001). Colony morphology was studied after 20 h of incu-bation at 30 �C on LB agar medium. Physiological tests such asgrowth at different temperatures (10e42 �C), pH (6.0e9.0) andtolerance to NaCl (1e4%) were examined on LB medium.Biochemical characterization which included oxidase reaction,catalase reaction, nitrate reduction, IMViC tests, urease tests,phenylalanine deaminase activity, acid or gas production fromcarbohydrates etc. were performed using KB002 HiAssorted�Biochemical test kit according to the manufacturer’s instructions(HiMedia Laboratories Pvt. Ltd., India). The isolated strain GBS.5was identified according to Bergey’s Manual of DeterminativeBacteriology (Holt et al. 1994).

2.4. 16S rRNA sequencing and phylogenetic analysis

The 16S ribosomal RNA identification of strain GBS.5 was per-formed through PCR using primers, forward 518F (50-CCA GCA GCCGCG GTA ATA CG-30) and reverse 800R (50-TAC CAG GGT ATC TAATCC-30). PCR solution consisted of 10X PCR buffer with 25 mMMgCl2 (5 ml), 10 mM dNTP mixture (5 ml), 10 pmol of forward andreverse primers (1.5 ml each), 40 ng of DNA template (2 ml), 1 ml ofTaq DNA polymerase (3Uml�1) and nuclease free water was addedto a total volume of 50 ml. PCR conditions were: 5 min of initialdenaturation at 94 �C followed by 35 amplification cycles (94 �C for60 s, 55 �C for 45 s and 72 �C for 90 s) and a final extension at 72 �Cfor 10 min. Purified PCR products were sequenced using dideox-ynucleotide chain-termination method. Multiple alignment andcomparison with the 16S rRNA sequences available in NCBI Gen-Bank and RDP database was performed using CLUSTAL W andBLAST programs, respectively. Kimura-2 parameter distance modelwas used to calculate pairwise evolutionary distances. Neighbor-joining, maximum likelihood and minimum evolution methodsbased on 1000 resamplings were used to build phylogenetic trees.The MEGA5 software was used to analyze pairwise distance values(Tamura et al. 2011).

2.5. Fatty acid methyl ester (FAME) analysis

Pure culture of strain GBS.5 was cultivated on Trypticase SoyBroth Agar (TSBA) plates at 28 �C for 24 h. Cellular fatty acidmethyl esters of freshly grown culture were obtained by a fourstep method (saponification, methylation, extraction and washing)developed by Microbial I.D. Inc. (MIDI, Newark, Delaware, USA).The samples were injected in to GC (Shimadzu model GC-2010Plus) equipped with a flame ionization detector and 30 m Rtx�-5 (fused silica) capillary column (Restek, Bellefonte, PA). Ultra-highpurity hydrogen was used as a carrier gas and column headpressure was 60 kPa. Injector and detector temperatures were300 �C and 240 �C, respectively. Temperature of oven was pro-grammed to increase from 170 �C to 270 �C at a rate of 5 �C min-1.Fatty acid profiles were identified with Sherlock software version6.0B (RTSBA6 library version 6.00, MIDI).

2.6. Biodegradation studies

Time course of CAR degradation was studied by growing bac-terial cells. Isolate was firstly inoculated in LB and after 16 h incu-bation at 30 �C and 180 rpm, 2% of fresh washed cells (opticaldensity at 600 nm (OD600) w 1.0) were inoculated in 500 mlErlenmeyer flask containing 150 ml BSM supplemented with500 ppm CAR. Culture was incubated under same conditions for72 h in rotary shaker. The samples were collected at regular timeintervals. After ethyl acetate extraction in acidic condition, CAR wasquantified using high performance liquid chromatography (HPLC)while metabolites were identified using gas chromatography massspectrometry (GCeMS). Bacterial growth was analyzed by calcu-lating its colony forming units (CFU).

Specific activity of CAR degradation by resting cells was calcu-lated by harvesting the cells during late log phase by centrifugationat 8000 g for 10 min and washed twice with 50 mM potassiumphosphate buffer (pH 7.0). Finally, cells were resuspended in equalvolumes of same buffer. In this cell suspension (approximately0.38 g dry cells l�1), 500 ppm CAR was added and reaction wasallowed to proceed at 30 �C and 180 rpm for 390min. Samples werecollected at regular intervals and analyzed for residual CAR con-centration. In both the experiments, in addition to test sample, P.resinovorans CA10 was taken as positive control and heat killedGBS.5 acted as a negative control.

G.B. Singh et al. / International Biodeterioration & Biodegradation 84 (2013) 35e43 37

Degradation of PAHs (2e4 rings) with structure similar to CARwas studied. The substrates (1 mM) used were biphenyl, DBT, DBF,fluorene, fluoranthene, phenanthrene, naphthalene and pyrene.Degradation of these substrates was checked by both growing cellsand resting cells. All experiments were conducted in triplicates.

2.7. Effect of environmental factors on carbazole degradation

Various abiotic environmental factors influencing bacterialgrowth and degradation of CAR were examined. These included pH(5, 6, 7, 8, 9 and10), temperature (10, 20, 30, 35,40 and50 �C), salinity(5, 10, 20, 25, 30 and 35 gl�1 of NaCl), initial CAR concentration (50,100, 300, 500,1000, 3000 and5000ppm) andpresence of additionalcarbon substrate (0.05, 0.10, 0.15 and 0.20 gl�1) of yeast extract (YE).CAR degradation was also determined in the presence of varioussurfactants [1% (w/v) or (v/v)] like sodium dodecyl sulphate (SDS),Tween 80, Triton X-100 and cetyl trimethyl ammonium bromide(CTAB). All experiments were conducted in triplicates.

2.8. PCR amplification and sequencing of car genes

To investigate the presence of CAR degrading genes, primerswere designed from the conserved regions of knownCAR degradinggenes. Sequence of the primers, gene amplified and the expectedlength of the amplicons are mentioned in Table 1. For amplification,colonies obtained on LB plate were lysed in 50 ml of nuclease freewater by heating at 95 �C for 10 min. It was then centrifuged at5000 g for 5 min. Aliquot of 30 ml was saved to be used as template.ThePCRwasperformed for ‘car’geneswith94 �C for10min followedby 30 cycles at 94 �C for 30 s, 62 �C for 30 s and 72 �C for 1 min 30 susing primers P1eP2, 30 s with primers P3eP4 and 2 min usingprimers P5eP6, respectively. The final extension was carried out at72 �C for 10 min. The amplified fragments were cloned in TOPO TAcloning vector (Invitrogen, Carlsbad, CA) and sequenced.

2.9. Surface activity tests

The surface activity of strain GBS.5 was determined qualitativelyby microplate assay (Cottingham et al. 2004). A 100 ml of super-natant of cell culture was added into a well of a 96-microwell plate.Presence of surfactant was confirmed by optical distortion of grid.Drop collapsewas performed according to themethod described byJain et al. (1991). Lid of 96-microwell plate was coated with 2 ml ofmineral oil. A 5 ml of supernatant of strain GBS.5 was added to theoil surface. Methylene blue was used to visualize small dropletagainst the transparent surface. The shape of the drop formed onthe oil surface was examined visually.

The emulsification activity of biosurfactant produced by strainGBS.5 was measured according to Chen et al. (2007). A 5 ml of n-hexadecane was mixed with equal volume of supernatant of peri-odically aliquoted grown culture in BSM, supplemented with500 ppm CAR and vortexed at high speed for 2 min. Emulsificationindex (E24) was calculated by measuring the percentage of heightoccupied by the emulsion after 24 h: [%E24¼ (hemulsion/htotal)� 100]

Table 1List of the primers used in this study. F denotes the forward primer and R denotes the reveras calculated from the sequence of Pseudomonas resinovorans CA10 is listed.

Primer Sequence

P1 (carAa F) 50-GTG GCG AAC GTT GAT GAG GC-30

P2 (carAa R) 50-ACG TGC GCT TGG GTC TGA ATA C-30

P3 (carBa F) 50-ATC CAG TAG ACC GCC TGA TTC-30

P4 (carBa R) 50-TGC ATC TGC AGA ACC GGA TG-30

P5 (carBb F) 50-CGA TGG GTG ACA TGG ACA TTC-30

P6 (carAc R) 50-TCC TCC GGC GAC ATA AAC TTC-30

Bacterial cell surface hydrophobicity was measured by bacterialadhesion to hydrocarbon (BATH) assay, a photochemical assaydeveloped by Rosenberg et al. (1980). Samples were harvestedperiodically at different growth stages. The cells were washedfive times with buffer solution containing 2.44 g of KH2PO4and 5.57 g of Na2HPO4 per liter. Finally, cells were resuspended inthe same buffer to give an OD400 of 1.0. Cell suspension (4 ml)was then mixed with n-hexadecane (1 ml) in a screw-top testtube (15 � 100 mm). After vortexing for 120 s, mixture was allowedto phase separation for 30 min. The aqueous phase was removedcarefully for OD400 measurement. Cell surface hydrophobicity(CSH) was calculated as a percentage of adhesion to hexadecane: [%CSH ¼ 1-(OD400 after mixing/OD400 before mixing) � 100]

2.10. Analytical methods

Quantification of CAR and other PAHs was performed usingHPLC (Waters Associates, Milford, MA). Supernatant from bacterialculture was acidified to pH 3 with 2N HCl and extracted with ethylacetate (1:1 v/v). After filtering through 0.2 mm fluoropore mem-brane (Millipore, Billerica, MA), 20 ml of sample was injected foranalysis. Separation was achieved with a reverse-phase C8 column(Waters RP 8; 3.3 mm; 150 � 4.6 mm). Mobile phase used wasacetonitrile:water (80:20 v/v) with a flow rate of 0.5 ml min�1 atroom temperature. Detection was carried out at 254 nm with aphotodiode array detector (PDA 2996; Waters).

Identification of metabolites, formed during CAR degradation,was carried out using GCeMS (Shimadzu model GCeMS QP2010,Japan) equipped with quadrupole mass analyzer. Helium was thecarrier gas with a constant flow rate of 1.21 ml min�1. Extracts foranalysis were prepared as mentioned above. For derivatization,0.5 ml of extract was incubated with 0.2 ml of N,O-bis(-trimethylsilyl)trifluoroacetamide (BSTFA) at 70 �C for 15 min. A 1 mlof derivatized or underivatized sample was injected in splitlessmode. For CAR metabolite identification, 30 m DB-5 MS capillarycolumn (J&W Scientific, Folsom, CA) was used. The oven tempera-ture program started from 60 �C (4 min isothermal hold) and thenwas ramped to 320 �C at a rate of 22 �Cmin-1 and held for 7min. Forthe identification of biosurfactant a 60 m Rtx�-5Sil MS capillarycolumn (Restek, Bellefonte, PA) was used. The oven temperatureprogram started from 80 �C (2 min isothermal hold), then the ovenwas heated to 280 �C at a rate of 5 �Cmin-1, followed by a 10 �Cmin-

1 increment to 320 �C and held for 4 min. Detector and injectortemperatures were 280 �C and 270 �C, respectively. The massspectra were recorded in electron impact mode with electron en-ergy of 70 eV and mass range 40e950 amu. Mass spectrographswere compared with WILEY8 and NIST05 libraries of mass spec-trographs prepared from known pure standards.

2.11. Nucleotide sequence accession numbers

The 16S rRNA and CAR degrading genes sequences in this studyhave been deposited in the GenBank database under the accessionnumbers JX193073 and JX885589eJX885592, respectively.

se primer for every corresponding gene. The expected size of the amplified fragment,

Gene amplified Expected length

carAa 1.15 kb

carBa 0.18 kb

carBb, carC,and carAc

1.71 kb

G.B. Singh et al. / International Biodeterioration & Biodegradation 84 (2013) 35e4338

2.12. Statistical analysis

Statistical analysis was performed using computerized statisti-cal software, OriginPro version 8 (OriginLab Corporation, North-ampton, MA). Statistical significance was accepted at P < 0.05.

3. Results and discussion

3.1. Isolation and identification of CAR degrading strain

Total 141 pure bacterial isolates, which could grow in BSM sup-plemented with CAR as sole source of carbon and nitrogen, wereobtained fromnineteen different soils and activated sludge samples.Among them, bacterium designated as GBS.5 (isolated from the soilof dye industry located in Ahmedabad, Gujarat, India) showedmaximum CAR degradation. This strainwas Gram-negative, motile,catalase and oxidase positive. Biochemical, morphological andphysiological characteristics of the isolate are listed in Table S1.

Analysis of the 16S rRNA sequence and comparative multiplesequence alignment indicated that the strain GBS.5 belonged toPseudomonas sp. (Fig. 1). The sequence of 1457 bases of the 16SrRNA gene of strain GBS.5 had maximum sequence similarity toPseudomonas alcaliphila AL15-21T (99.52%) followed by Pseudo-monas oleovorans subsp. lubricantis RS1T (99.51%), Pseudomonastoyotomiensis HT-3T (99.45%) and Pseudomonas indoloxydans IPL-1T

(99.09%). Nearly all these Pseudomonas spp. are reported to beisolated from hydrocarbon rich soils and involved in the degrada-tion of polyaromatic hydrocarbons. P. toyotomiensis (Hirota et al.2011), a facultatively psychrophilic alkaliphile, was isolated fromsoil contaminated with hydrocarbons and is reported to degradevarious hydrocarbons while P. indoloxydans is involved in theoxidation of indole (Manickam et al. 2008). Although, CAR degra-dation ability of Pseudomonas spp. is not uncommon but interest-ingly none of these homologs are reported to be involved in thedegradation of CAR. Based on the biochemical and phylogeneticanalysis, the isolate was designated as Pseudomonas sp. GBS.5.

Fig. 1. Phylogenetic tree (Neighbor Joining) based on 16S rRNA gene sequence, showing rGenBank accession numbers are given in parentheses. Bar, 0.002 nucleotide substitutions p

Closely related Pseudomonas type strains, P. alcaliphila AL15-21T

(Yumoto et al., 2001), P. oleovorans subsp. lubricantis RS1T (Sahaet al. 2010), P. toyotomiensis HT-3T and P. indoloxydans IPL-1T wereused as a reference strains for the fatty acid profile analysis.Detailed comparative results of cellular fatty acid profile of strainGBS.5 with its close relatives are given in Table S2. FAME analysisrevealed that strain GBS.5 consisted of C12:0 (9.85%), C16:0 (19.65%)and summed feature 3 (C16:1 u6c/C16:1 u7c, 18.96%) with C10:0 3OH(3.65%) and C12:0 3OH (3.61%) as the hydroxyl fatty acids. Among allthe constituents, C18:1 u7c was the major component, comprising36.13% of the total fatty acid. According to FAME analysis, P. oleo-vorans subsp. lubricantis RS1T (GenBank Accession No. DQ842018)was found to be the nearest homolog of strain GBS.5.

3.2. Growth characteristics and resting cell activity

Utilization of CAR as sole source of carbon and nitrogen by thegrowing cells of strain GBS.5 is demonstrated in Fig. 2. The degra-dation was studied for 72 h which showed almost complete (97%)utilization in first 48 h. P. resinovorans CA10 was used as a positivecontrol and it showed only 25% CAR degradation in 72 h. Nodecrease in CAR concentration was observed in heat killed GBS.5.Degradation of CAR increased with the increase in cell countexponentially, both in the test sample and positive control (data notshown) while no degradation was observed during stationaryphase. This is the type of growth pattern desirable during degra-dation of PAHs. It has been suggested that it is possible to increasebioavailability for compounds that show growth related degrada-tion (Calvo et al. 2004).

Specific activity for CAR degradation by the resting cellsof Pseudomonas sp. GBS.5 was found to be 11.36 mmol min�1 g�1

dry cells while there was no significant decrease in CAR concentra-tion in given time frame by the resting cells of P. resinovorans CA10.The specific CAR degradation activity reported for other microor-ganisms is given in Table 2. The results obtained by GBS.5 are

elationship of the isolated strain (GBS.5) with closely related species of Pseudomonas.er position.

Fig. 2. Time course of CAR degradation (---) and growth (-C-) of Pseudomonas sp.GBS.5. CAR degradation pattern of positive control Pseudomonas resinovorans CA10(-,-) and heat killed negative control (-:-). The values are means of three inde-pendent replicates. SD was within the acceptable range.

G.B. Singh et al. / International Biodeterioration & Biodegradation 84 (2013) 35e43 39

comparable with the best results desired for potential bio-remediating strains.

3.3. Abiotic factors affecting carbazole degradation

The degradation efficiency of the microorganisms depends onseveral environmental factors such as temperature, pH, nutrientavailability and bioavailability of the contaminant. The effect ofthese factors on the growth and degradation efficiency of CAR bystrain GBS.5 was examined in the present study. Fig. 3(A) showsCAR degradation at different temperatures. Strain GBS.5 showedcomplete degradation in 72 h for temperature ranging from 20 to40 �C, while 73% CAR was degraded in 216 h at 10 �C. Growthstudies were also conducted along with degradation studies.Approximately equal numbers of cells were present after 48 h tilltemperature 35 �C where as further increase in temperatureresulted in decrease in number of cells (data not shown). This in-dicates that the degradation of CAR is affected by the solubility ofCAR and growth of microorganism (Jacques et al. 2005). The initialincrease in degradation of CAR with increasing temperature can bedue to increase in solubility of CAR with increasing temperaturehowever further rise in temperature affects the viability of micro-organism and thus the degradation. The pH is also an importantfactor that affects the solubility of the pollutant. More than 90% ofCAR was degraded in 48 h when the pH varied from 6 to 9(Fig. 3(B)). However, no degradation was observed when the pHwas reduced below 6 or increased above 9. The growth of micro-organism is affected by acidic or alkaline pH, thus affecting thedegradation ability.

Table 2CAR degrading ability of selected microorganisms.

Microorganisms Media Initial CARconcentration (ppm)

Ef

Acinetobacter sp. Alp6 BSM 500 99Novosphingobium sp. NIY3 MM1a 100 95Pseudomonas sp. XLDN4-9 MM2b 500 98Pseudomonas sp. GBS.5 BSM 500 97Sphingomonas sp. CDH-7 MM3c 500 10Sphingomonas sp. GTIN11 MM4d 283 82

a BSM þ yeast extract and trace elements.b BSM þ trace elements.c BSM þ metal and vitamin mixture.d MM3 þ glucose.

Fig. 3(C) shows the effect of different salt concentration withregard to its application in marine environment. The average NaClconcentration reported inmarine environment is 35 gl�1 (Diaz et al.2000). At salinity 35 gl�1 only 40% degradation was observed in120 h whereas complete degradation was observed with salt con-centrations lesser than 25 gl�1. This indicates that high salinityaffects the growth of the microorganism and the decrease inmetabolic activity of the microorganism is associated with thedecrease in the degradation activity of CAR. Concentration of CARalso affects the degradation ability of the isolate (Fig. 3 (D)).Degraded amount of CAR increased as the initial concentrationincreased from 50 ppm to 1000 ppm. CAR is present in soilcontaminated with effluents of dye industries. The strain coulddegrade 90% CAR in 24 d, when the initial concentration was3000 ppm and get completely degraded in 30 d (data not shown).High concentration CAR degradation activity was also observed bystrain GBS.5, which can degrade 70% of 5000 ppm CAR in 60 d (datanot shown). Despite the presence of substrate concentration highenough to support growth, the decrease in degradation suggeststhat increasing concentration may cause toxic effect or the toxicmetabolites may accumulate in the media. Effect of chemical sur-factants on CAR degradation was also analyzed. In the presence ofnon ionic (Tween 80 and Triton X-100) and anionic (SDS) surfac-tants, complete degradation was achieved in 24 h where asmicroorganisms in the absence of surfactant showed only 58%degradation which is further completely degraded in 48 h(Fig. 3(E)). No degradation was seen in the presence of CTAB. Nonionic detergents are the most preferred surfactants as they are lesstoxic to the microorganism as compared to ionic surfactants. Thepresence of SDS increased the degradation rate by increasing thebioavailability and solubility of the pollutant. No degradation in thepresence of CTAB indicates that it is toxic to cells. Similar result fortoxic effect of cationic detergent is reported for the degradation ofnaphthalene (Pathak et al. 2009) and several other PAH.

Co-substrate containing nitrogen in sufficient quantity is knownto enhance degradation. As an alternate carbon source, YE waschosen as a model compound to predict the effect of co-substrateduring CAR degradation. As shown in Fig. 3(F), addition of YEdelayed the complete degradation of CAR as compared to in itsabsence. The different concentration of YE also did not affect thedegradation rate. The choice of additional substrate is veryimportant in bioremediation studies as they may enhance thedegradation by stimulating the growth of microorganism or mightinhibit bioremediation and result in diauxic growth.

3.4. Biodegradation of other polyaromatic compounds byPseudomonas sp. GBS.5

Biodegradation potential of growing and resting cells of strainGBS.5, for various PAHs, was also investigated (Table 3). Among all

ficiency Specific enzyme activity(mmol min-1 g�1 dry cells)

References

.9% per 216 h 7.96 Singh et al. 2011a% per 72 h 1.7 Ishihara et al. 2008% per 56 h 10.4 Li et al. 2006% per 48 h 11.36 This study0% per 50 h 10.0 Kirimura et al. 1999% per 8 h 8.0 Kilbane et al. 2002

Fig. 3. Factors affecting the degradation of CAR (A) temperature, (B) pH, (C) salinity (NaCl concentration), (D) initial concentration of CAR, (E) presence of surfactants, (F) alternatecarbon source (yeast extract). In all the experiments inoculum concentrationwas 2% and incubation condition: 30 �C, 180 rpm. The values are means of three independent replicates.SD was within the acceptable range.

G.B. Singh et al. / International Biodeterioration & Biodegradation 84 (2013) 35e4340

PAHs, only naphthalene and phenanthrene were utilized as a solecarbon sourcewhile majority of PAHs except biphenyl, DBTand DBFwere degraded by the resting cells of Pseudomonas sp. strain GBS.5,grown in BSM containing CAR. The ability of the resting cells, grownin the presence of CAR, to degrade PAHs but not as the substrate forgrowth indicates that theyhave the requisite enzyme toutilize these

substrates and the enzyme is induced when cells are grown in thepresence of CAR. The inducible character of enzymes responsible forthe conversion of polycyclic and heterocyclic aromatic hydrocarbonis well known (Kanaly and Harayama, 2000). Generally substrate isthe most effective inducer of the enzyme. However, in our studiesCAR is acting as an inducer for the expression of enzymes having

Table 3The range of substrate degradation in BSM by Pseudomonas sp. GBS.5. The utilizationof various substrates was measured by growing cells and resting cells induced withCAR. Incubation condition: 30 �C, 180 rpm, 24 h.

Polyaromatic hydrocarbon Growth Degradation activity (%)(by resting cells)

Fluorene e 92Fluoranthene e 77Pyrene e 56Phenanthrene þ 51Naphthalene þ 42Biphenyl e 0DBT e 0DBF e 0

Fig. 4. Preliminary screening of biosurfactant from 42 h grown culture of GBS.5: (A)Emulsification index (E24), (B) Microplate analysis, (C) Drop-collapse test. Sodiumdodecylsulphate (1%, w/v) was used as positive control and Milli-Q water and unin-oculated BSM were served as negative controls. All experiments were performed intriplicates.

Fig. 5. Emulsification index (E24) (columns) and time course of growth (-,-) ofPseudomonas sp. GBS.5. The values are means of three independent replicates. SD waswithin the acceptable range.

G.B. Singh et al. / International Biodeterioration & Biodegradation 84 (2013) 35e43 41

ability to degrade these compounds. Comparing our results withthose that are reported in literature, it is evident that carbazole-1,9a-dioxygenase (CARDO) has broad substrate specificity. It has theability to carryout wide oxygenation reactions viz angular dioxyge-nation, lateral dioxygenation and monooxygenation (Nojiri, 2012).

3.5. Biosurfactant production during CAR degradation

Bioavailability and hence the biodegradation of PAHs can beincreased with surfactants. The production of biosurfactant by thestrain GBS.5 was primarily screened by microplate and dropcollapse assays (Fig. 4(B), (C)). These qualitative tests are indicativeof surface activity and wetting properties. Further, to quantify thebiosurfactant production, emulsification index was calculated withn-hexadecane (Fig. 4(A)). Biosurfactant production was studied indetail during bacterial growth in BSM with CAR. The biosurfactantproduction started after 12 h and has the highest emulsificationindex of 53 � 1.52% at 48 h (Fig. 5). Increase in biosurfactant con-centration correlated with the increase in degradation of CAR.Emulsions were found to be stable for more than a month at roomtemperature without any change in emulsification index. Bio-surfactants are known to increase the degradation rate either bychanging the hydrophobicity of the cells surface or by increasingthe accessibility of the substrate. The hydrophobicity of bacterialcell surface was tested using BATH assay and it was observed thatGBS.5 possessed a higher cellular hydrophobicity (51%) in BSM.

3.6. Metabolite identification

GCeMS analysis of the culture extract was carried out atdifferent times of incubation. Spectra analysis of the extract at thezero hour (without any incubation) showed the presence of a singlepeak (retention time (Rt)¼ 29.89) (Fig. 6(A)). This peak correspondsto CAR according to the published data (NIST05 and WILEY8database). Over a period of time (42 h) spectra showed a decreasein CAR concentration and emergence of new peaks at differentretention times suggesting that CAR is being utilized during duecourse of time (Fig. 6(B)). By comparing the MS spectra of newpeaks with those of published data it was inferred that peak atretention times 15.74, 20.76, 21.31, 22.39, 30.76 and 33.84 mincorresponded to long chain alkanes viz. n-tetradecane, n-penta-decane, n-hexadecane, n-heptadecane, eicosane and (9Z)-octadec-9-enoic acid, respectively (Table 4). This MS spectra correlates withthe spectra of biosurfactants (glycolipidic), produced by variousmicroorganisms (Monteiro et al. 2009; Patel et al. 2012). Othermajor peaks corresponded to hexadecanamide (Rt ¼ 35.77) andoctadecanamide (Rt ¼ 38.93). It has been proposed that certainmicroorganisms have the ability to convert (9Z)-octadec-9-enoicacid (a major fraction of glycolipid) and its derivatives into theirrespective amides by enzymatic amidation (Kaneshiro et al. 1994;Levinson et al. 2008).

Comparing our results with those that are cited in literature,various Pseudomonas spp. are reported to produce biosurfactantcontaining long chain alkanes in the presence of PAHs but none ofthe CAR degrading Pseudomonas sp. strain is reported to producebiosurfactant during CAR degradation. Interestingly, the closesthomologs of GBS.5 are also not reported to produce biosurfactantduring the degradation of their respective aromatic compound. Thebiosurfactant produced by the strain resulted in the increasedbioavailability of CAR either by mobilization or solubilization.However, further studies are needed to decipher the exact mode ofaction of biosurfactant. During In situ bioremediation the bio-surfactant production by degrading microorganism is preferred ascompared to the addition of exogenous biosurfactant. Thus this

Fig. 6. GCeMS analysis of metabolites formed during CAR degradation by strain GBS.5;(A) 0 h extract, (B) 42 h extract.

Table 4GC retention times and MS data of major compounds formed by Pseudomonas sp.GBS.5.

Retentiontime (min)

Molecular ion fragmentation pattern, m/z Possible product

15.74 198(Mþ), 85, 71, 57, 43 n-Tetradecane20.76 212(Mþ), 99, 85, 71, 57, 43 n-Pentadecane21.31 226(Mþ), 113, 99, 85, 71, 57, 43 n-Hexadecane22.39 240(Mþ), 113, 99, 85, 71, 57, 43, 41 n-Heptadecane30.76 282(Mþ), 141, 113, 99, 85, 71, 57, 43 Eicosane33.84 282(Mþ), 97, 83, 69, 55, 41 (9Z)-octadec-9-enoic

acid35.77 255(Mþ), 212, 128, 114, 86, 72, 59, 41 Hexadecanamide38.93 281(Mþ), 264, 140, 126, 112, 98, 86,

72, 59, 55, 41Octadecanamide

Fig. 7. PCR amplification of the specific fragments of car genes from the Pseudomonassp. GBS.5. M, the molecular size markers; Lane 1, 2 and 3 shows amplification of carAa,carBa and carBbcarCcarAc gene cluster using primers P1eP2, P3eP4 and P5eP6,respectively. The amplicons of the same size were obtained both in Pseudomonasresinovorans CA10 and Pseudomonas sp. GBS.5. Electrophoretic separation was per-formed in 1% agarose gel in TAE buffer.

G.B. Singh et al. / International Biodeterioration & Biodegradation 84 (2013) 35e4342

microorganism will prove to be a beneficial candidate for the fieldbioremediation application.

Along with biosurfactant production no peaks corresponding toCARmetabolites were observed. For metabolite analysis, the isolatewas subjected to high concentration of CAR (1500 ppm) and theextract was derivatized. Accumulation of anthranilic acid at highconcentration of CAR has been reported (Larentis et al. 2011). Thepeak of trimethylsilyl derived anthranilic acid was detected alongwith carbazole (Fig. S1).

3.7. PCR amplification and sequence analysis of car genes

The amplification of known CAR degrading genes was carriedout using primers designed from the conserved sequence. Ampli-cons of expected length of car genes were observed (Fig. 7). The

analysis of nucleotide sequence of the car genes found in Pseudo-monas sp. strain GBS.5 reveals that they are 99% identical to the cargenes of P. resinovorans CA10 (GenBank Accession No. AB088420),Pseudomonas stutzeri OM1 (GenBank Accession No. AB001723) andPseudomonas sp. XLDN4-9 (GenBank Accession No. DQ060076).However, neighbor joining tree of Pseudomonas sp. strain GBS.5with these microbes shows the arrangement of these microbes on aseparate phylogenetic clade (Data not shown). A high identity of cargenes and low phylogenetic relation between the strain GBS.5 andthe selected CAR degraders suggests that car genes are transferredby horizontal gene transfer. Comparison of the sequence with thesequence of CA10 reveals six nucleotide differences, two in carAa,three in carBb and one in carC gene. None of the change was atwobble position and thus all the changes resulted in the change inamino acid (Table 5). It has been reported that changes in fewamino acid can affect the structure of the enzyme, resulting in thechange in their substrate specificity and degradation activity(Vardar and Wood, 2005). These changes may be responsible forthe change in activity and property of the CARDO enzyme.

Comparison of our results with those reported in literatureshows that this isolate has few changes in the amino acids and hasthe biosurfactant producing ability as compared to other microor-ganisms reported for CAR degradation. In future, Pseudomonas sp.strain GBS.5 or the surface active agent, it produces, can be used forbioremediation of soil contaminated with various polyaromaticcompounds. Studies are underway to elucidate the fact that theproduction of biosurfactant by GBS.5 during CAR degradation or the

Table 5Amino acid change in Car proteins of Pseudomonas sp. GBS.5.

Protein Position of amino acid Changes

CarAa 303 Proline (P) replaced with Arginine (R)364 Alanine (A) replaced with Valine (V)

CarBb 201 Methionine (M) replaced with Isolucine (I)205 Valine (V) replaced with Alanine (A)258 Phenylalanine (F) replaced with Valine (V)

CarC 174 Glycine (G) replaced with Arginine (R)

G.B. Singh et al. / International Biodeterioration & Biodegradation 84 (2013) 35e43 43

changes in these amino acids are responsible for the increaseddegradation rate of CAR.

4. Conclusion

Pseudomonas sp. strain GBS.5 isolated and characterized in thiswork showed a potential to degrade CAR at a wide range of pH,temperature, salinityand initial CAR concentrations. Thebroad rangeof PAHs utilized by this microorganism along with biosurfactantproduction makes it an ideal candidate for the remediation of sitescontaminated with mixed PAHs. Further understanding of theunique characteristic of thismicroorganism andCAR degrading geneversus those described is a matter of ongoing investigation.

Acknowledgment

Gajendra B Singh would like to thank Jaypee Institute of Infor-mation Technology, Noida for providing research fellowship andProf. Hideaki Nojiri, University of Tokyo for gifting Pseudomonasresinovorans CA10.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ibiod.2013.05.022.

References

Calvo, C., Toledo, F.L., Gonzalez-Lopez, J., 2004. Surfactant activity of a naphthalenedegrading Bacillus pumilus strain isolated from oil sludge. Journal of Biotech-nology 109, 255e262.

Castorena, G., Mugica, V., Le Borgne, S., Acuna, M.E., Bustos-Jaimes, I., Aburto, J.,2006. Carbazole biodegradation in gas oil/water biphasic media by a new iso-lated bacterium Burkholderia sp. strain IMP5GC. Journal of Applied Microbi-ology 100, 739e745.

Chen, C.Y., Baker, S.C., Darton, R.C., 2007. The application of a high throughputanalysis method for the screening of potential biosurfactants from naturalsources. Journal of Microbiological Methods 70, 503e510.

Cottingham, M.G., Bain, C.D., Vaux, D.J., 2004. Rapid method for measurement ofsurface tension in multiwell plates. Laboratory Investigation 84, 523e529.

Diaz, M.P., Grigson, S.J.W., Peppiatt, C.J., Burgess, J.G., 2000. Isolation and charac-terization of novel hydrocarbon degrading euryhaline consortia from crude oiland mangrove sediments. Marine Biotechnology 2, 522e532.

Gerhardt, P., Murray, R.G.E., Wood, W.A., Krieg, N.R., 1994. Methods for General andMolecular Bacteriology, second ed. ASM Press, Washington, DC.

Hirota, K., Yamahira, K., Nakajima, K., Nodasaka, Y., Okuyama, H., Yumoto, I., 2011.Pseudomonas toyotomiensis sp. nov., a psychrotolerant facultative alkaliphilethat utilizes hydrocarbons. International Journal of Systematic and EvolutionaryMicrobiology 61, 1842e1848.

Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T., Williams, S.T., 1994. Bergey’s Manual ofDeterminative Bacteriology, ninth ed. Williams &Wilkins, Baltimore. (New York).

Ishihara, A., Dumeignil, F., Aoyagi, T., Nishikawa, M., Hosomi, M., Qian, E.W., Kabe, Y.,2008. Degradation of carbazole by Novosphingobium sp. strain NIY3. Journal ofthe Japan Petroleum Institute 51, 174e179.

Jacques, R.J.S., Santos, E.C., Bento, F.M., Peralba, M.C.R., Selbach, P.A., Sa, E.L.S.,Camargo, F.A.O., 2005. Anthracene biodegradation by Pseudomonas sp. isolatedfrom a petrochemical sludge landfarming site. International Biodeteriorationand Biodegradation 56, 143e150.

Jain, D.K., Collins-Thompson, D.L., Lee, H., Trevors, J.T., 1991. A drop-collapsing testfor screening surfactant producing microorganisms. Journal of MicrobiologicalMethods 13, 271e279.

Kanaly, R.A., Harayama, S., 2000. Biodegradation of high molecular weight polycyclicaromatic hydrocarbons by bacteria. Journal of Bacteriology 182, 2059e2067.

Kaneshiro, T., Vesonder, R.F., Peterson, R.E., Weisleder, D., Bagby, M.O., 1994. 9(Z)-Octadecenamide and fatty amides by Bacillus megaterium (8-3437) conversionof oleic acid. Journal of the American Oil Chemists’ Society 71, 491e494.

Kilbane II, J.J., Daram, A., Abbasian, J., Kayser, K.J., 2002. Isolation and character-ization of Sphingomonas sp. GTIN11 capable of carbazole metabolism in pe-troleum. Biochemical and Biophysical Research Communications 297, 242e248.

Kirimura, K., Nakagawa, H., Tsuji, K., Matsuda, K., Kurane, R., Usami, S., 1999. Se-lective and continuous degradation of carbazole contained in petroleum oil byresting cells of Sphingomonas sp. CDH-7. Bioscience, Biotechnology, andBiochemistry 63, 1563e1568.

Larentis, A.L., Sampaio, H.C.C., Carneiro, C.C., Martins, O.B., Alves, T.L.M., 2011.Evaluation of growth, carbazole biodegradation and anthranilic acid productionby Pseudomonas stutzeri. Brazilian Journal of Chemical Engineering 28, 37e44.

Lawniczak, J., Marecik, R., Chrzanowski, L., 2013. Contribution of biosurfactants tonatural or induced bioremediation. Applied Microbiology and Biotechnology 97,2327e2339.

Levinson, W.E., Kuo, T.M., Knothe, G., 2008. Characterization of fatty amides pro-duced by lipase catalyzed amidation of multi-hydroxylated fatty acids. Bio-resource Technology 99, 2706e2709.

Li, L., Li, Q., Li, F., Shi, Q., Yu, B., Liu, F., Xu, P., 2006. Degradation of carbazole and itsderivatives by a Pseudomonas sp. Applied Microbiology and Biotechnology 73,941e948.

Li, Y.G., Li, W.L., Huang, J.X., Xiong, X.C., Gao, H.S., Xing, J.M., Liu, H.Z., 2008.Biodegradation of carbazole in oil/water biphasic system by a newly isolatedbacterium Klebsiella sp. LSSE-H2. Biochemical Engineering Journal 41, 166e170.

Manickam, N., Ghosh, A., Jain, R.K., Mayilraj, S., 2008. Description of a novel indole-oxidizing bacterium Pseudomonas indoloxydans sp. nov., isolated from apesticide-contaminated site. Systematic and Applied Microbiology 31, 101e107.

Monteiro, A.S., Coutinho, J.O., Junior, A.C., Rosa, C.A., Siqueira, E.P., Santos, V.L., 2009.Characterization of new biosurfactant produced by Trichosporon montevideenseCLOA 72 isolated from dairy industry effluents. Journal of Basic Microbiology49, 553e563.

Nojiri, H., 2012. Structural and molecular genetic analyses of the bacterial carbazoledegradation. Bioscience, Biotechnology, and Biochemistry 76, 1e18.

Ouchiyama, N., Zhang, Y., Omori, T., Kodama, T., 1993. Biodegradation of carbazoleby Pseudomonas spp. CA06 and CA10. Bioscience, Biotechnology, andBiochemistry 57, 455e460.

Patel, V., Cheturvedula, S., Madamwar, D., 2012. Phenanthrene degradation by Pseu-doxanthomonas sp. DMVP2 isolated from hydrocarbon contaminated sediment ofAmlakhadi canal, Gujarat, India. Journal of Hazardous Materials 201-202, 43e51.

Pathak, H., Kantharia, D., Malpani, A., Madamwar, D., 2009. Naphthalene degrada-tion by Pseudomonas sp. HOB1: In vitro studies and assessment of naphthalenedegradation efficiency in stimulated microcosms. Journal of Hazardous Mate-rials 166, 1466e1473.

Rosenberg, M., Gutnick, D., Rosenberg, E., 1980. Adherence of bacteria to hydro-carbons: a simple method for measuring cell-surface hydrophobicity. FEMSMicrobiology Letters 9, 29e33.

Saha, R., Sproer, C., Beck, B., Bagley, S., 2010. Pseudomonas oleovorans subsp. lubri-cantis subsp. nov., and reclassification of Pseudomonas pseudoalcaligenes ATCC17440T as later synonym of Pseudomonas oleovorans ATCC 8062T. CurrentMicrobiology 60, 294e300.

Santos, S.C.C., Alviano, D.S., Alviano, C.S., Padula, M., Leitao, A.C., Martins, O.B.,Ribeiro, C.M.S., Sassaki, M.Y.M., Matta, C.P.S., Bevilaqua, J., Sebastian, G.V.,Seldin, L., 2006. Characterization of Gordonia sp. strain F.5.25.8 capable ofdibenzothiophene desulfurization and carbazole utilization. Applied Microbi-ology and Biotechnology 71, 355e362.

Singh, G.B., Gupta, S., Srivastava, S., Gupta, N., 2011a. Biodegradation of carbazole bynewly isolated Acinetobacter spp. Bulletin of Environmental Contamination andToxicology 87, 522e526.

Singh, G.B., Srivastava, A., Saigal, A., Aggarwal, S., Bisht, S., Gupta, S., Srivastava, S.,Gupta, N., 2011b. Biodegradation of carbazole and dibenzothiophene by bacteriaisolated from petroleum contaminated sites. Bioremediation Journal 15, 1e7.

Suzuki, M., Nakagawa, Y., Harayama, S., Yamamoto, S., 2001. Phylogenetic analysisand taxonomic study of marine cytophaga-like bacteria: proposal for Tenaci-baculum gen. nov. with Tenacibaculum maritimum comb. nov. and Tenacibac-ulum ovolyticum comb. nov., and description of Tenacibaculum mesophilum sp.nov. and Tenacibaculum amylolyticum sp. nov. International Journal of System-atic and Evolutionary Microbiology 51, 1639e1652.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5:molecular evolutionary genetics analysis using maximum likelihood, evolu-tionary distance, and maximum parsimony methods. Molecular Biology andEvolution 28, 2731e2739.

US Environmental Protection Agency (EPA), 1986. Health and Environmental EffectsProfile for Carbazole. US Environmental Protection Agency, Washington, DC.EPA/600/X-86/334 (NTIS PB88218789).

Vardar, G.,Wood, T.K., 2005. Protein engineering of toluene-o-xylenemonooxygenasefrom Pseudomonas stutzeriOX1 for enhanced chlorinated ethene degradation ando-xylene oxidation. Applied Microbiology and Biotechnology 68, 510e517.

Yang, M., Li, W., Guo, X., Qu, Z., Zhu, X., Wang, X., 2009. Isolation and identificationof a carbazole degradation gene cluster from Sphingomonas sp. JS1. WorldJournal of Microbiology and Biotechnology 25, 1625e1631.

Yumoto, I., Yamazaki, K., Hishinuma, M., Nodasaka, Y., Suemori, A., Nakajima, K.,Inoue, N., Kawasaki, K., 2001. Pseudomonas alcaliphila sp. nov., a novel faculta-tively psychrophilic alkaliphile isolated from seawater. International Journal ofSystematic and Evolutionary Microbiology 51, 349e355.