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

International Biodeterioration & Biodegradation 84 (2013) 35e43

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Carbazole degradation and biosurfactant production by newly isolated Pseudomonas 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

a b s t r a c t

Article history: Received 22 March 2013 Received in revised form 21 May 2013 Accepted 22 May 2013 Available online 26 June 2013

A novel biosurfactant producing bacterium, designated as Pseudomonas sp. strain GBS.5, having the ability to degrade carbazole has been isolated. The specific activity of carbazole degradation was found to be 11.36 mmol min
1 g
1 dry cells. GCeMS analysis revealed that the growth of bacterium on carbazole was accompanied with the production of biosurfactant. The biosurfactant produced had the maximum emulsification index of 53  1.52% with n-hexadecane. Sequence analysis of the carbazole degrading genes revealed changes in six different amino acids as compared to other well established strains. Study also confirmed that in addition to carbazole, bacterium has the ability to degrade other polycyclic aromatic hydrocarbons such as fluoranthene, fluorene, naphthalene, phenanthrene and pyrene. A high degradation rate of carbazole and broad substrate range indicates that it has the potential to be used for bioremediation of polycyclic aromatic hydrocarbon contaminated sites. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Biodegradation Biosurfactant Carbazole car gene Pseudomonas

1. Introduction Carbazole (CAR) is nitrogen containing heterocyclic polyaromatic hydrocarbon (PAH) and is known to be carcinogenic and mutagenic (US EPA, 1986). It is used as a feedstock in dye, plastic and pharmaceutical industries and is found in soil contaminated with crude oil and petrochemicals. Various physicochemical methods have been developed to remove PAHs from the environment. However, all these techniques are costly and cause pollution. Bioremediation is a promising technology for cleaning up such contaminated sites. Biodegradation processes exploit the natural potential of microorganisms to utilize these compounds as their energy source. Microorganisms have been reported for the degradation of CAR and include both Gram-positive and Gram-negative bacteria like Pseudomonas, Burkholderia, Acinetobacter, Sphingomonas, Novosphingobium, Klebsiella, Gordonia and others (Castorena et al. 2006; Santos et al. 2006; Li et al. 2008; Yang et al. 2009; Singh et al. 2011a,b; Nojiri, 2012). The biodegradation pathway of CAR is a well established pathway and it results in the conversion of CAR to anthranilic acid. This pathway was first reported by Ouchiyama et al. (1993) for Pseudomonas resinovorans strain CA10. The first

* Corresponding author. Department of Biotechnology , Jaypee Institute of Information Technology, A-10, Sector-62, Noida 201307, Uttar Pradesh, India. Tel.: þ91 120 2594211; fax: þ91 120 2400986. E-mail address: [email protected] (N. Gupta). 0964-8305/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2013.05.022

step is catalyzed by the enzyme carbazole-1,9a-dioxygenase (encoded by carAaAcAd genes) and results in the formation of dihydroxylated intermediate which is spontaneously converted to 20 -aminobiphenyl-2,3-diol. This product is further cleaved by meta cleavage enzyme (encoded by carBaBb genes) to form 2-hydroxy-6oxo-6-(20 -aminophenyl)-hexa-2,4-dienoic acid which is then converted to anthranilic acid when attacked by hydrolase enzyme (encoded by carC gene). These genes are arranged in the form of an operon designated as ‘car’ operon. Owing to the hydrophobic nature of heterocyclic aromatic pollutants, microbial degradation studies have been frequently carried out either with organic solvents or synthetic surfactants. Biosurfactants, as less toxic, are biodegradable and more eco-friendly than synthetic surfactant. They are known to enhance the biodegradation rate of PAHs by increasing its bioavailability via solubilization and emulsification (Lawniczak et al. 2013). Since, bioavailability is a prime limiting factor for the biodegradation of aromatic pollutants, use of microorganisms with the ability to produce biosurfactants are preferred in biodegradation techniques. Although Pseudomonas spp. are known to be involved in the degradation of CAR, there is no report of Pseudomonas sp. producing biosurfactant during CAR degradation. In this study, we report the isolation and characterization of a new strain of Pseudomonas sp., designated as GBS.5, capable of degrading CAR. Bacterial growth and degradation characteristics were also analyzed. GCeMS analysis led to the identification of certain long chain alkanes that are associated with biosurfactant

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production. Biosurfactant production was also confirmed by other qualitative and quantitative assays.

2. Materials and methods 2.1. Chemicals and media CAR (96% purity) was purchased from Acros Organics (New Jersey, USA). Other PAHs (greater than 98% purity) like biphenyl, dibenzofuran (DBF), dibenzothiophene (DBT), fluoranthene, fluorene, naphthalene, phenanthrene, pyrene and polymerase chain reaction (PCR) reagents were obtained from SigmaeAldrich (St Louis, MO, USA). Organic solvents and other chemicals were of HPLC 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 of CaCl2.2H2O. The pH of prepared BSM was 7.0  0.05. A 15 gl
1 agar was added to the BSM to make solid BSM plates. During PAHs degradation study, NH4Cl (2 gl
1) was added to the BSM as nitrogen source. LuriaeBertani (LB) medium contained (per liter of solution) 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 above liquid media. 2.2. Enrichment and isolation of carbazole degrading bacteria Various soil and activated sludge samples were collected from Indian oil refineries (Gujarat, Jaipur, Mathura, Panipat), dye industries (Ahmedabad) and sewage treatment plants (New Delhi, Ghazipur), located across various cities in India. One gram of sample was added into 250 ml Erlenmeyer flask containing 100 ml BSM supplemented with 500 ppm of CAR as carbon and nitrogen source. The media was incubated at 30  C and 180 rpm in a rotary shaker. After 4 d of incubation, 5% of enriched culture was transferred to fresh BSM containing CAR and incubated under same conditions. This procedure was repeated four times. Later, samples were diluted serially and plated on solid BSM to obtain isolated colonies. Isolates were inoculated in BSM containing 500 ppm of CAR and incubated at 30  C for 7 d. Utilization of CAR by isolates was analyzed by quantifying initial and final concentrations. Selection of microorganism of interest among all isolated strains was based on the higher CAR degradation activity.

2.3. Phenotypic characterization of isolate GBS.5 Gram staining and spore staining were done using standard protocol (Gerhardt et al. 1994). Cell motility was determined with an optical microscope using the hanging drop method (Suzuki et al. 2001). Colony morphology was studied after 20 h of incubation at 30  C on LB agar medium. Physiological tests such as growth at different temperatures (10e42  C), pH (6.0e9.0) and tolerance 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 from carbohydrates etc. were performed using KB002 HiAssortedÔ Biochemical test kit according to the manufacturer’s instructions (HiMedia Laboratories Pvt. Ltd., India). The isolated strain GBS.5 was identified according to Bergey’s Manual of Determinative Bacteriology (Holt et al. 1994).

2.4. 16S rRNA sequencing and phylogenetic analysis The 16S ribosomal RNA identification of strain GBS.5 was performed through PCR using primers, forward 518F (50 -CCA GCA GCC GCG GTA ATA CG-30 ) and reverse 800R (50 -TAC CAG GGT ATC TAA TCC-30 ). PCR solution consisted of 10X PCR buffer with 25 mM MgCl2 (5 ml), 10 mM dNTP mixture (5 ml), 10 pmol of forward and reverse primers (1.5 ml each), 40 ng of DNA template (2 ml), 1 ml of Taq DNA polymerase (3Uml
1) and nuclease free water was added to a total volume of 50 ml. PCR conditions were: 5 min of initial denaturation at 94  C followed by 35 amplification cycles (94  C for 60 s, 55  C for 45 s and 72  C for 90 s) and a final extension at 72  C for 10 min. Purified PCR products were sequenced using dideoxynucleotide chain-termination method. Multiple alignment and comparison with the 16S rRNA sequences available in NCBI GenBank and RDP database was performed using CLUSTAL W and BLAST programs, respectively. Kimura-2 parameter distance model was used to calculate pairwise evolutionary distances. Neighborjoining, maximum likelihood and minimum evolution methods based 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 Soy Broth Agar (TSBA) plates at 28  C for 24 h. Cellular fatty acid methyl esters of freshly grown culture were obtained by a four step 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-2010 Plus) equipped with a flame ionization detector and 30 m RtxÒ5 (fused silica) capillary column (Restek, Bellefonte, PA). Ultra-high purity hydrogen was used as a carrier gas and column head pressure was 60 kPa. Injector and detector temperatures were 300  C and 240  C, respectively. Temperature of oven was programmed to increase from 170  C to 270  C at a rate of 5  C min-1. Fatty acid profiles were identified with Sherlock software version 6.0B (RTSBA6 library version 6.00, MIDI). 2.6. Biodegradation studies Time course of CAR degradation was studied by growing bacterial cells. Isolate was firstly inoculated in LB and after 16 h incubation at 30  C and 180 rpm, 2% of fresh washed cells (optical density at 600 nm (OD600) w 1.0) were inoculated in 500 ml Erlenmeyer flask containing 150 ml BSM supplemented with 500 ppm CAR. Culture was incubated under same conditions for 72 h in rotary shaker. The samples were collected at regular time intervals. After ethyl acetate extraction in acidic condition, CAR was quantified using high performance liquid chromatography (HPLC) while metabolites were identified using gas chromatography mass spectrometry (GCeMS). Bacterial growth was analyzed by calculating its colony forming units (CFU). Specific activity of CAR degradation by resting cells was calculated by harvesting the cells during late log phase by centrifugation at 8000 g for 10 min and washed twice with 50 mM potassium phosphate buffer (pH 7.0). Finally, cells were resuspended in equal volumes of same buffer. In this cell suspension (approximately 0.38 g dry cells l
1), 500 ppm CAR was added and reaction was allowed to proceed at 30  C and 180 rpm for 390 min. Samples were collected at regular intervals and analyzed for residual CAR concentration. In both the experiments, in addition to test sample, P. resinovorans CA10 was taken as positive control and heat killed GBS.5 acted as a negative control.

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Degradation of PAHs (2e4 rings) with structure similar to CAR was 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 cells and resting cells. All experiments were conducted in triplicates. 2.7. Effect of environmental factors on carbazole degradation Various abiotic environmental factors influencing bacterial growth and degradation of CAR were examined. These included pH (5, 6, 7, 8, 9 and 10), temperature (10, 20, 30, 35, 40 and 50  C), salinity (5, 10, 20, 25, 30 and 35 gl
1 of NaCl), initial CAR concentration (50, 100, 300, 500,1000, 3000 and 5000 ppm) and presence of additional carbon 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 various surfactants [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, primers were designed from the conserved regions of known CAR degrading genes. Sequence of the primers, gene amplified and the expected length of the amplicons are mentioned in Table 1. For amplification, colonies obtained on LB plate were lysed in 50 ml of nuclease free water by heating at 95  C for 10 min. It was then centrifuged at 5000 g for 5 min. Aliquot of 30 ml was saved to be used as template. The PCR was performed for ‘car’ genes with 94  C for 10 min followed by 30 cycles at 94  C for 30 s, 62  C for 30 s and 72  C for 1 min 30 s using primers P1eP2, 30 s with primers P3eP4 and 2 min using primers P5eP6, respectively. The final extension was carried out at 72  C for 10 min. The amplified fragments were cloned in TOPO TA cloning vector (Invitrogen, Carlsbad, CA) and sequenced. 2.9. Surface activity tests The surface activity of strain GBS.5 was determined qualitatively by microplate assay (Cottingham et al. 2004). A 100 ml of supernatant of cell culture was added into a well of a 96-microwell plate. Presence of surfactant was confirmed by optical distortion of grid. Drop collapse was performed according to the method described by Jain et al. (1991). Lid of 96-microwell plate was coated with 2 ml of mineral oil. A 5 ml of supernatant of strain GBS.5 was added to the oil surface. Methylene blue was used to visualize small droplet against the transparent surface. The shape of the drop formed on the oil surface was examined visually. The emulsification activity of biosurfactant produced by strain GBS.5 was measured according to Chen et al. (2007). A 5 ml of nhexadecane was mixed with equal volume of supernatant of periodically aliquoted grown culture in BSM, supplemented with 500 ppm CAR and vortexed at high speed for 2 min. Emulsification index (E24) was calculated by measuring the percentage of height occupied by the emulsion after 24 h: [%E24 ¼ (hemulsion/htotal)  100]

37

Bacterial cell surface hydrophobicity was measured by bacterial adhesion to hydrocarbon (BATH) assay, a photochemical assay developed by Rosenberg et al. (1980). Samples were harvested periodically at different growth stages. The cells were washed five times with buffer solution containing 2.44 g of KH2PO4 and 5.57 g of Na2HPO4 per liter. Finally, cells were resuspended in the 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 test tube (15  100 mm). After vortexing for 120 s, mixture was allowed to phase separation for 30 min. The aqueous phase was removed carefully 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 using HPLC (Waters Associates, Milford, MA). Supernatant from bacterial culture was acidified to pH 3 with 2N HCl and extracted with ethyl acetate (1:1 v/v). After filtering through 0.2 mm fluoropore membrane (Millipore, Billerica, MA), 20 ml of sample was injected for analysis. Separation was achieved with a reverse-phase C8 column (Waters RP 8; 3.3 mm; 150  4.6 mm). Mobile phase used was acetonitrile:water (80:20 v/v) with a flow rate of 0.5 ml min
1 at room temperature. Detection was carried out at 254 nm with a photodiode 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 the carrier gas with a constant flow rate of 1.21 ml min
1. Extracts for analysis 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 ml of derivatized or underivatized sample was injected in splitless mode. For CAR metabolite identification, 30 m DB-5 MS capillary column (J&W Scientific, Folsom, CA) was used. The oven temperature program started from 60  C (4 min isothermal hold) and then was ramped to 320  C at a rate of 22  C min-1 and held for 7 min. For the identification of biosurfactant a 60 m RtxÒ-5Sil MS capillary column (Restek, Bellefonte, PA) was used. The oven temperature program started from 80  C (2 min isothermal hold), then the oven was heated to 280  C at a rate of 5  C min-1, followed by a 10  C min1 increment to 320  C and held for 4 min. Detector and injector temperatures were 280  C and 270  C, respectively. The mass spectra were recorded in electron impact mode with electron energy of 70 eV and mass range 40e950 amu. Mass spectrographs were compared with WILEY8 and NIST05 libraries of mass spectrographs prepared from known pure standards. 2.11. Nucleotide sequence accession numbers The 16S rRNA and CAR degrading genes sequences in this study have been deposited in the GenBank database under the accession numbers JX193073 and JX885589eJX885592, respectively.

Table 1 List of the primers used in this study. F denotes the forward primer and R denotes the reverse primer for every corresponding gene. The expected size of the amplified fragment, as calculated from the sequence of Pseudomonas resinovorans CA10 is listed. Primer P1 P2 P3 P4 P5 P6

(carAa F) (carAa R) (carBa F) (carBa R) (carBb F) (carAc R)

Sequence 0

0

5 -GTG GCG AAC GTT GAT GAG GC-3 50 -ACG TGC GCT TGG GTC TGA ATA C-30 50 -ATC CAG TAG ACC GCC TGA TTC-30 50 -TGC ATC TGC AGA ACC GGA TG-30 50 -CGA TGG GTG ACA TGG ACA TTC-30 50 -TCC TCC GGC GAC ATA AAC TTC-30

Gene amplified

Expected length

carAa

1.15 kb

carBa

0.18 kb

carBb, carC, and carAc

1.71 kb

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2.12. Statistical analysis Statistical analysis was performed using computerized statistical software, OriginPro version 8 (OriginLab Corporation, Northampton, 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 supplemented with CAR as sole source of carbon and nitrogen, were obtained from nineteen different soils and activated sludge samples. Among them, bacterium designated as GBS.5 (isolated from the soil of dye industry located in Ahmedabad, Gujarat, India) showed maximum CAR degradation. This strain was Gram-negative, motile, catalase and oxidase positive. Biochemical, morphological and physiological characteristics of the isolate are listed in Table S1. Analysis of the 16S rRNA sequence and comparative multiple sequence alignment indicated that the strain GBS.5 belonged to Pseudomonas sp. (Fig. 1). The sequence of 1457 bases of the 16S rRNA gene of strain GBS.5 had maximum sequence similarity to Pseudomonas alcaliphila AL15-21T (99.52%) followed by Pseudomonas oleovorans subsp. lubricantis RS1T (99.51%), Pseudomonas toyotomiensis HT-3T (99.45%) and Pseudomonas indoloxydans IPL-1T (99.09%). Nearly all these Pseudomonas spp. are reported to be isolated from hydrocarbon rich soils and involved in the degradation of polyaromatic hydrocarbons. P. toyotomiensis (Hirota et al. 2011), a facultatively psychrophilic alkaliphile, was isolated from soil contaminated with hydrocarbons and is reported to degrade various hydrocarbons while P. indoloxydans is involved in the oxidation of indole (Manickam et al. 2008). Although, CAR degradation ability of Pseudomonas spp. is not uncommon but interestingly none of these homologs are reported to be involved in the degradation of CAR. Based on the biochemical and phylogenetic analysis, the isolate was designated as Pseudomonas sp. GBS.5.

Closely related Pseudomonas type strains, P. alcaliphila AL15-21T (Yumoto et al., 2001), P. oleovorans subsp. lubricantis RS1T (Saha et al. 2010), P. toyotomiensis HT-3T and P. indoloxydans IPL-1T were used as a reference strains for the fatty acid profile analysis. Detailed comparative results of cellular fatty acid profile of strain GBS.5 with its close relatives are given in Table S2. FAME analysis revealed 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 all the constituents, C18:1 u7c was the major component, comprising 36.13% of the total fatty acid. According to FAME analysis, P. oleovorans 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 the growing cells of strain GBS.5 is demonstrated in Fig. 2. The degradation was studied for 72 h which showed almost complete (97%) utilization in first 48 h. P. resinovorans CA10 was used as a positive control and it showed only 25% CAR degradation in 72 h. No decrease in CAR concentration was observed in heat killed GBS.5. Degradation of CAR increased with the increase in cell count exponentially, both in the test sample and positive control (data not shown) while no degradation was observed during stationary phase. This is the type of growth pattern desirable during degradation of PAHs. It has been suggested that it is possible to increase bioavailability for compounds that show growth related degradation (Calvo et al. 2004). Specific activity for CAR degradation by the resting cells of 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 concentration in given time frame by the resting cells of P. resinovorans CA10. The specific CAR degradation activity reported for other microorganisms is given in Table 2. The results obtained by GBS.5 are

Fig. 1. Phylogenetic tree (Neighbor Joining) based on 16S rRNA gene sequence, showing relationship of the isolated strain (GBS.5) with closely related species of Pseudomonas. GenBank accession numbers are given in parentheses. Bar, 0.002 nucleotide substitutions per position.

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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 independent replicates. SD was within the acceptable range.

comparable with the best results desired for potential bioremediating strains. 3.3. Abiotic factors affecting carbazole degradation The degradation efficiency of the microorganisms depends on several environmental factors such as temperature, pH, nutrient availability and bioavailability of the contaminant. The effect of these factors on the growth and degradation efficiency of CAR by strain GBS.5 was examined in the present study. Fig. 3(A) shows CAR degradation at different temperatures. Strain GBS.5 showed complete degradation in 72 h for temperature ranging from 20 to 40  C, while 73% CAR was degraded in 216 h at 10  C. Growth studies were also conducted along with degradation studies. Approximately equal numbers of cells were present after 48 h till temperature 35  C where as further increase in temperature resulted in decrease in number of cells (data not shown). This indicates that the degradation of CAR is affected by the solubility of CAR and growth of microorganism (Jacques et al. 2005). The initial increase in degradation of CAR with increasing temperature can be due to increase in solubility of CAR with increasing temperature however further rise in temperature affects the viability of microorganism and thus the degradation. The pH is also an important factor that affects the solubility of the pollutant. More than 90% of CAR was degraded in 48 h when the pH varied from 6 to 9 (Fig. 3(B)). However, no degradation was observed when the pH was reduced below 6 or increased above 9. The growth of microorganism is affected by acidic or alkaline pH, thus affecting the degradation ability.

39

Fig. 3(C) shows the effect of different salt concentration with regard to its application in marine environment. The average NaCl concentration reported in marine environment is 35 gl
1 (Diaz et al. 2000). At salinity 35 gl
1 only 40% degradation was observed in 120 h whereas complete degradation was observed with salt concentrations lesser than 25 gl
1. This indicates that high salinity affects the growth of the microorganism and the decrease in metabolic activity of the microorganism is associated with the decrease in the degradation activity of CAR. Concentration of CAR also affects the degradation ability of the isolate (Fig. 3 (D)). Degraded amount of CAR increased as the initial concentration increased from 50 ppm to 1000 ppm. CAR is present in soil contaminated with effluents of dye industries. The strain could degrade 90% CAR in 24 d, when the initial concentration was 3000 ppm and get completely degraded in 30 d (data not shown). High concentration CAR degradation activity was also observed by strain GBS.5, which can degrade 70% of 5000 ppm CAR in 60 d (data not shown). Despite the presence of substrate concentration high enough to support growth, the decrease in degradation suggests that increasing concentration may cause toxic effect or the toxic metabolites may accumulate in the media. Effect of chemical surfactants on CAR degradation was also analyzed. In the presence of non ionic (Tween 80 and Triton X-100) and anionic (SDS) surfactants, complete degradation was achieved in 24 h where as microorganisms 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. Non ionic detergents are the most preferred surfactants as they are less toxic to the microorganism as compared to ionic surfactants. The presence of SDS increased the degradation rate by increasing the bioavailability and solubility of the pollutant. No degradation in the presence of CTAB indicates that it is toxic to cells. Similar result for toxic effect of cationic detergent is reported for the degradation of naphthalene (Pathak et al. 2009) and several other PAH. Co-substrate containing nitrogen in sufficient quantity is known to enhance degradation. As an alternate carbon source, YE was chosen as a model compound to predict the effect of co-substrate during CAR degradation. As shown in Fig. 3(F), addition of YE delayed the complete degradation of CAR as compared to in its absence. The different concentration of YE also did not affect the degradation rate. The choice of additional substrate is very important in bioremediation studies as they may enhance the degradation by stimulating the growth of microorganism or might inhibit bioremediation and result in diauxic growth.

3.4. Biodegradation of other polyaromatic compounds by Pseudomonas sp. GBS.5 Biodegradation potential of growing and resting cells of strain GBS.5, for various PAHs, was also investigated (Table 3). Among all

Table 2 CAR degrading ability of selected microorganisms. Microorganisms

Media

Initial CAR concentration (ppm)

Efficiency

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

References

Acinetobacter sp. Alp6 Novosphingobium sp. NIY3 Pseudomonas sp. XLDN4-9 Pseudomonas sp. GBS.5 Sphingomonas sp. CDH-7 Sphingomonas sp. GTIN11

BSM MM1a MM2b BSM MM3c MM4d

500 100 500 500 500 283

99.9% per 216 h 95% per 72 h 98% per 56 h 97% per 48 h 100% per 50 h 82% per 8 h

7.96 1.7 10.4 11.36 10.0 8.0

Singh et al. 2011a Ishihara et al. 2008 Li et al. 2006 This study Kirimura et al. 1999 Kilbane et al. 2002

a b c d

BSM þ yeast extract and trace elements. BSM þ trace elements. BSM þ metal and vitamin mixture. MM3 þ glucose.

40

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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) alternate carbon source (yeast extract). In all the experiments inoculum concentration was 2% and incubation condition: 30  C, 180 rpm. The values are means of three independent replicates. SD was within the acceptable range.

PAHs, only naphthalene and phenanthrene were utilized as a sole carbon source while majority of PAHs except biphenyl, DBT and DBF were degraded by the resting cells of Pseudomonas sp. strain GBS.5, grown in BSM containing CAR. The ability of the resting cells, grown in the presence of CAR, to degrade PAHs but not as the substrate for growth indicates that they have the requisite enzyme to utilize these

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

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

Growth

Degradation activity (%) (by resting cells)

Fluorene Fluoranthene Pyrene Phenanthrene Naphthalene Biphenyl DBT DBF

e e e þ þ e e e

92 77 56 51 42 0 0 0

ability to degrade these compounds. Comparing our results with those that are reported in literature, it is evident that carbazole-1,9adioxygenase (CARDO) has broad substrate specificity. It has the ability to carryout wide oxygenation reactions viz angular dioxygenation, lateral dioxygenation and monooxygenation (Nojiri, 2012). 3.5. Biosurfactant production during CAR degradation Bioavailability and hence the biodegradation of PAHs can be increased with surfactants. The production of biosurfactant by the strain GBS.5 was primarily screened by microplate and drop collapse assays (Fig. 4(B), (C)). These qualitative tests are indicative of surface activity and wetting properties. Further, to quantify the biosurfactant production, emulsification index was calculated with n-hexadecane (Fig. 4(A)). Biosurfactant production was studied in detail during bacterial growth in BSM with CAR. The biosurfactant production started after 12 h and has the highest emulsification index of 53  1.52% at 48 h (Fig. 5). Increase in biosurfactant concentration correlated with the increase in degradation of CAR. Emulsions were found to be stable for more than a month at room temperature without any change in emulsification index. Biosurfactants are known to increase the degradation rate either by changing the hydrophobicity of the cells surface or by increasing the accessibility of the substrate. The hydrophobicity of bacterial cell surface was tested using BATH assay and it was observed that GBS.5 possessed a higher cellular hydrophobicity (51%) in BSM. 3.6. Metabolite identification GCeMS analysis of the culture extract was carried out at different times of incubation. Spectra analysis of the extract at the zero hour (without any incubation) showed the presence of a single peak (retention time (Rt) ¼ 29.89) (Fig. 6(A)). This peak corresponds to CAR according to the published data (NIST05 and WILEY8 database). Over a period of time (42 h) spectra showed a decrease in CAR concentration and emergence of new peaks at different retention times suggesting that CAR is being utilized during due course of time (Fig. 6(B)). By comparing the MS spectra of new peaks with those of published data it was inferred that peak at retention times 15.74, 20.76, 21.31, 22.39, 30.76 and 33.84 min corresponded to long chain alkanes viz. n-tetradecane, n-pentadecane, n-hexadecane, n-heptadecane, eicosane and (9Z)-octadec9-enoic acid, respectively (Table 4). This MS spectra correlates with the spectra of biosurfactants (glycolipidic), produced by various microorganisms (Monteiro et al. 2009; Patel et al. 2012). Other major peaks corresponded to hexadecanamide (Rt ¼ 35.77) and octadecanamide (Rt ¼ 38.93). It has been proposed that certain microorganisms have the ability to convert (9Z)-octadec-9-enoic acid (a major fraction of glycolipid) and its derivatives into their respective amides by enzymatic amidation (Kaneshiro et al. 1994; Levinson et al. 2008).

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. Sodium dodecylsulphate (1%, w/v) was used as positive control and Milli-Q water and uninoculated BSM were served as negative controls. All experiments were performed in triplicates.

Comparing our results with those that are cited in literature, various Pseudomonas spp. are reported to produce biosurfactant containing long chain alkanes in the presence of PAHs but none of the CAR degrading Pseudomonas sp. strain is reported to produce biosurfactant during CAR degradation. Interestingly, the closest homologs of GBS.5 are also not reported to produce biosurfactant during the degradation of their respective aromatic compound. The biosurfactant produced by the strain resulted in the increased bioavailability of CAR either by mobilization or solubilization. However, further studies are needed to decipher the exact mode of action of biosurfactant. During In situ bioremediation the biosurfactant production by degrading microorganism is preferred as compared to the addition of exogenous biosurfactant. Thus this

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

42

G.B. Singh et al. / International Biodeterioration & Biodegradation 84 (2013) 35e43 Table 4 GC retention times and MS data of major compounds formed by Pseudomonas sp. GBS.5. Retention time (min)

Molecular ion fragmentation pattern, m/z

Possible product

15.74 20.76 21.31 22.39 30.76 33.84

198(Mþ), 212(Mþ), 226(Mþ), 240(Mþ), 282(Mþ), 282(Mþ),

35.77 38.93

255(Mþ), 212, 128, 114, 86, 72, 59, 41 281(Mþ), 264, 140, 126, 112, 98, 86, 72, 59, 55, 41

n-Tetradecane n-Pentadecane n-Hexadecane n-Heptadecane Eicosane (9Z)-octadec-9-enoic acid Hexadecanamide Octadecanamide

85, 71, 57, 43 99, 85, 71, 57, 43 113, 99, 85, 71, 57, 43 113, 99, 85, 71, 57, 43, 41 141, 113, 99, 85, 71, 57, 43 97, 83, 69, 55, 41

analysis of nucleotide sequence of the car genes found in Pseudomonas sp. strain GBS.5 reveals that they are 99% identical to the car genes of P. resinovorans CA10 (GenBank Accession No. AB088420), Pseudomonas stutzeri OM1 (GenBank Accession No. AB001723) and Pseudomonas sp. XLDN4-9 (GenBank Accession No. DQ060076). However, neighbor joining tree of Pseudomonas sp. strain GBS.5 with these microbes shows the arrangement of these microbes on a separate phylogenetic clade (Data not shown). A high identity of car genes and low phylogenetic relation between the strain GBS.5 and the selected CAR degraders suggests that car genes are transferred by horizontal gene transfer. Comparison of the sequence with the sequence of CA10 reveals six nucleotide differences, two in carAa, three in carBb and one in carC gene. None of the change was at wobble position and thus all the changes resulted in the change in amino acid (Table 5). It has been reported that changes in few amino acid can affect the structure of the enzyme, resulting in the change in their substrate specificity and degradation activity (Vardar and Wood, 2005). These changes may be responsible for the change in activity and property of the CARDO enzyme. Comparison of our results with those reported in literature shows that this isolate has few changes in the amino acids and has the biosurfactant producing ability as compared to other microorganisms reported for CAR degradation. In future, Pseudomonas sp. strain GBS.5 or the surface active agent, it produces, can be used for bioremediation of soil contaminated with various polyaromatic compounds. Studies are underway to elucidate the fact that the production of biosurfactant by GBS.5 during CAR degradation or the

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

microorganism will prove to be a beneficial candidate for the field bioremediation application. Along with biosurfactant production no peaks corresponding to CAR metabolites were observed. For metabolite analysis, the isolate was subjected to high concentration of CAR (1500 ppm) and the extract was derivatized. Accumulation of anthranilic acid at high concentration of CAR has been reported (Larentis et al. 2011). The peak of trimethylsilyl derived anthranilic acid was detected along with carbazole (Fig. S1). 3.7. PCR amplification and sequence analysis of car genes The amplification of known CAR degrading genes was carried out using primers designed from the conserved sequence. Amplicons of expected length of car genes were observed (Fig. 7). The

Fig. 7. PCR amplification of the specific fragments of car genes from the Pseudomonas sp. 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 Pseudomonas resinovorans CA10 and Pseudomonas sp. GBS.5. Electrophoretic separation was performed in 1% agarose gel in TAE buffer.

G.B. Singh et al. / International Biodeterioration & Biodegradation 84 (2013) 35e43 Table 5 Amino acid change in Car proteins of Pseudomonas sp. GBS.5. Protein

Position of amino acid

Changes

CarAa

303 364 201 205 258 174

Proline (P) replaced with Arginine (R) Alanine (A) replaced with Valine (V) Methionine (M) replaced with Isolucine (I) Valine (V) replaced with Alanine (A) Phenylalanine (F) replaced with Valine (V) Glycine (G) replaced with Arginine (R)

CarBb

CarC

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