Degradation of 4-Nitrophenol, 2-Chloro-4-nitrophenol, and 2, 4

Jan 5, 2010 - Institute of Microbial Technology, Sector-39A,. Chandigarh-160036, India., Division of Material Engineering,. Graduate Institute of Engi...
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Environ. Sci. Technol. 2010, 44, 1069–1077

Degradation of 4-Nitrophenol, 2-Chloro-4-nitrophenol, and 2,4-Dinitrophenol by Rhodococcus imtechensis Strain RKJ300 ANURADHA GHOSH,† MEENU KHURANA,† ARCHANA CHAUHAN,† MASAHIRO TAKEO,‡ ASIT K. CHAKRABORTI,§ AND R A K E S H K . J A I N * ,† Institute of Microbial Technology, Sector-39A, Chandigarh-160036, India., Division of Material Engineering, Graduate Institute of Engineering, Himeji Institute of Technology, 2167 Shosha, Himeji, Hyogo 671-2201, Japan, and Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Sector 67, Phase X, SAS Nagar (Punjab)-160062, India

Received June 1, 2009. Accepted December 14, 2009.

A bacterial strain Rhodococcus imtechensis RKJ300 () MTCC 7085T ) JCM 13270T) was isolated from pesticidecontaminated soil of Punjab by the enrichment technique on minimal medium containing 4-nitrophenol. Strain RKJ300 is capable of utilizing 4-nitrophenol, 2-chloro-4-nitrophenol, and 2,4dinitrophenol as sole sources of carbon and energy. The strain involved both oxidative and reductive catabolic mechanisms for initial transformation of these compounds. In the case of 2-chloro-4-nitrophenol, colorimetric analysis indicated that nitrite release was followed by stoichiometric elimination of chloride ions. Experiments using whole cells and cell-free extracts showed chlorohydroquinone and hydroquinone as the intermediates of 2-chloro-4-nitrophenol degradation. This is the first report of degradation on 2-chloro-4-nitrophenol by a bacterium under aerobic condition to the best of our knowledge. However, pathways for degradation of 4-nitrophenol and 2,4dinitrophenol were similar to those reported in other strains of Rhodococcus. Laboratory-scale soil microcosm studies demonstrated that the organism was capable of degrading a mixture of nitrophenols simultaneously, indicating its applicability toward in situ bioremediation of contaminated sites. The fate of the augmented strain as monitored by the plate-counting method and hybridization technique was found to be fairly stable throughout the period of microcosm experiments.

Introduction Nitroaromatic compounds are frequently used as building blocks for dyes, plastics, explosives, herbicides, and pesticides. As a consequence, they are abundantly present in nature and, in particular, found as contaminants in wastewaters, rivers, and herbicide- or pesticide-treated soils (1, 2). The U.S. Environmental Protection Agency listed mononitro* Corresponding author address: Institute of Microbial Technology, Sector-39A, Chandigarh-160036, India; phone: 0091 172 2690694; fax: 0091 172 2690632; e-mail: [email protected]. † Institute of Microbial Technology. ‡ Himeji Institute of Technology. § National Institute of Pharmaceutical Education and Research. 10.1021/es9034123

 2010 American Chemical Society

Published on Web 01/05/2010

phenols and dinitrophenols on its “Priority Pollutant List” as these are highly toxic to all forms of living organisms (http://www.epa.gov/waterscience/methods/pollutants.htm). However, despite their hazardous nature some microorganisms that are able to transform or degrade nitrophenols have been found to exist in nature (3-5). Studies have revealed that nonspecific reduction of nitro group(s) in nitrophenols by reductases often lead to the formation of more toxic and recalcitrant nitroso and hydroxylamino derivatives (6, 7). However, microbes, in particular, belonging to the order Actinomycetales, are widely distributed in soil and have evolved the metabolic enzymes which selectively remove nitro groups from parent compounds by oxidative or reductive means, resulting in productive catabolism of nitroaromatics (2, 3, 8). These organisms have the potential for bioremediation since they can degrade a number of xenobiotic compounds. Among them rhodococci are of great interest because of their remarkable metabolic diversity (9), as exemplified by their ability to transform nitriles, nitroaromatic and chloroaromatic compounds, etc. (10, 11). Research on rhodococcal metabolism has revealed that they harbor a unique spectrum of enzyme activities which help them to degrade diverse classes of xenobiotic compounds (9, 12). Degradation pathways of few nitroaromatic compounds have been well characterized for certain bacteria. For example, in the case of 4-nitrophenol (4-NP) degradation two major initial pathways have been characterized. The degradation pathway in which 4-NP is converted to maleylacetate (MA) via hydroquinone (HQ) has been preferentially reported in Gram-negative bacteria such as Burkholderia spp. and Moraxella spp., and the other where 4-NP degradation occurs via 4-nitrocatechol (4-NC) and benzenetriol (BT) has been mostly reported in Gram-positive bacteria such as Arthrobacter spp., Bacillus spp., and Rhodococcus spp (11, 13-16). On the other hand, it has been shown that hydride Meisenheimer complex and 4,6-dinitrohexanoate are the intermediates in 2,4-dinitrophenol (2,4-DNP) metabolism in Rhodococcus erythropolis (17, 18). A very recent study demonstrated 4-NP as the initial intermediate of 2,4DNP degradation in a Burkholderia sp. strain KU-46 (19). Interestingly, to date, there is only one report indicating the presence of both 4-NP and 2,4-DNP degradation pathways in a single strain of Rhodococcus sp. PN1; however, these pathways have not been thoroughly characterized (20). Although structurally related to nitroaromatic compounds, the recently introduced chlorinated nitroaromatic compounds have been found to be more resistant to microbial degradation due to the simultaneous existence of two electron-withdrawing chlorine and nitro moieties. Therefore, knowledge of their biodegradation is very limited (21). Rhodococcus imtechensis strain RKJ300 isolated in our laboratory is capable of utilizing 4-NP, 2-chloro-4-nitrophenol (2-C-4-NP), and 2,4-DNP as sole sources of carbon and energy (22). The major objectives of the present study are to elucidate the nitrophenols degradation pathways in strain RKJ300 and carry out microcosm studies to determine their potential for exploitation as a bioaugmented strain for remediation of the contaminated sites.

Experimental Section Isolation of Strain RKJ300 and Growth Studies. Strain RKJ300 was isolated on 4-NP using the enrichment culture technique from a pesticide-contaminated agricultural field of Punjab, India, and it was characterized following the polyphasic taxonomic approach (22). A number of substituted nitrophenols were checked for degradation by supplementing VOL. 44, NO. 3, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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the minimal medium (MM; 13) with or without an additional carbon source. The organism was cultivated at 30 °C under shaking conditions, and OD600 was measured using a UV-vis spectrophotometer (Perkin-Elmer Lambda EZ 201, Waltham, MA). Release of chloride ions was detected and quantified following a colorimetric method described by Bergmann and Sainik (23). Briefly, to the diluted culture supernatant 200 µL each of 250 mM ferric ammonium sulfate (prepared in 9 N nitric acid) and saturated solution of mercuric thiocyanate (prepared in ethanol) were added, mixed, and incubated at room temperature for 10-15 min. Quantification of the released chloride ions was performed by spectrophotometric measurement of absorbance at 460 nm based on the standard curve for chloride ions generated with known concentrations of NaCl. Release of nitrite ions was detected spectrophotometrically in the culture supernatants using the method described by White et al. (24). To the culture supernatant an equal volume of reagent A [0.1% (w/v) N-(1-naphthyl)ethylenediamine dihydrochloride in 30% (v/v) acetic acid] was added. After 2 min, an equal volume of reagent B [0.1% (w/v) sulfanilic acid in 30% (v/v) acetic acid] was added and incubated at room temperature for 25-30 min. The presence of nitrite ions in the sample was shown by the appearance of a purple color and quantified by calculating the absorbance at 540 nm. Concentration of nitrite ion released into the growth medium was determined with a standard calibration curve of NaNO2. Preparation of Cell-Free Extract. Nitrophenol(s) degrading cells of strain RKJ300 were collected at different time points from incubations grown on MM supplemented with 0.3 mM of appropriate nitrophenol(s) and frozen at -70 °C for abolishing all the metabolic activities. Cell-free extract was later prepared by cell lysis by ultrasonication (Vibra-Cell VCX 130) (Sonics & Materials, Inc., Newtown, CT) with 2 cycles of 5 min of sonication with intermittent pulse on and off for 20 and 10 s, respectively, at 65 W. Sonicated samples were centrifuged at 4 °C. The cell-free supernatant was collected and later subjected to extraction of aromatic compounds over neutral and acidic pH. Analytical Methods. Metabolites were extracted from culture supernatants using ethyl acetate, evaporated to dryness under a gentle stream of nitrogen at 40 °C using a Turbovap II concentration workstation (Caliper lifesciences, Hopkinton, MA), dissolved in an appropriate volume of ethyl acetate, and subjected to thin layer chromatography (TLC), gas chromatography (GC), and/or gas chromatography-mass spectrometry (GC-MS) analyses. TLC was performed using precoated silica gel 60 F254 plates (20 × 20 cm, 0.25 mm; Merck) in either solvent A (toluene:ethyl acetate:acetic acid (60:30:5)) (25), or solvent B (benzene:dioxane:acetic acid (60: 36:4)) (26). GC studies were carried out using an AutoSystemXL gas chromatograph equipped with a DB-1 (100% dimethyl polysiloxane) capillary column (30 m × 0.25 mm) and flameionization detector (Perkin-Elmer UV/vis spectrometer lambda EZ 201, Waltham, MA). Temperatures for the injector, oven, and detector were kept constant at 280, 200, and 250 °C, respectively. GC-MS analysis was carried out using a GCMS instrument equipped with quadrupole mass filter and DB-1 capillary column with an ionization of 70 eV, scan interval of 1.5 s, and mass range of 40-700 (Shimadzu GCMS-QP5000, Columbia, MD). For direct-MS analysis the metabolites were purified by preparative TLC (precoated silica gel 60 F254 plates, 20 × 20 cm, 2 mm; Merck) using the above-mentioned solvent systems. Resting cell studies were carried out as described by Takeo et al. (15) with little modification. Briefly, cells were grown on 10 mM sodium succinate until mid log phase (O.D. 600 nm ∼0.5) and induced with 0.3 mM 4-NP/2-C-4-NP/2,41070

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DNP. After 2 h, induced cells were harvested, washed twice, and resuspended in the ice cold phosphate buffer (10 mM, pH 7.0). 4-NP/2-C-4-NP/2,4-DNP were added as filtersterilized solution at a final concentration of 0.3 mM to each flask, and 1.5 mL aliquots were collected every 5 min. The collected samples were immediately centrifuged at 15 000 rpm at 4 °C for 2 min, and supernatant was filtered through a 0.2 µm filter. The formation of metabolites in 2-C-4-NP and 2,4-DNP degradation pathways was measured by analyzing samples of the supernatant collected at regular time intervals (10 min) using a high-performance liquid chromatography (HPLC, Waters 600 model, Wein, Austria) instrument equipped with a Waters 996 photodiode array detector operating at 215-600 nm and C18 reversed phase (5 µm, 4.6 × 250 mm, Waters Spherisorb ODS2) column. For detection of 4-NP and 2-C-4-NP degradation intermediates, the mobile phase consisted of 1% acetic acid in methanol: 1% acetic acid in water with a gradient from 35:65 to 68:32 over 10 min at a flow rate of 1 mL min-1. On the other hand, for detection of 2,4-DNP degradation intermediates the mobile phase consisted of methanol:50 mM phosphate buffer (pH 8.0) in a ratio of 40:60 under isocratic condition at a flow rate of 1 mL min-1. 2,2′-Dipyridyl, a chelating chemical, was added to the culture media whenever required in order to detect the intermediate(s) formed before aromatic ring cleavage (27). Intermediates were identified and quantified by comparison of retention times and UV-vis spectra with those of standards and their calibration curves. To identify the products formed during enzymatic reactions, aliquots were taken out at regular time points for HPLC analysis. Enzyme Assays. In order to further strengthen the results of biochemical characterization of the nitrophenols degradation and demonstrate the induction of enzymes involved, different enzymatic assays were performed with the induced cells of strain RKJ300. 4-NP monooxygenase assay was performed as described previously (28). The reaction mixtures (final volume, 1.0 mL) contained 0.05 mM substrate, 0.1 mM NADPH, 0.03 mM flavin adenine dinucleotide (FAD), 0.6-10/g of protein, and 20 mM phosphate buffer (pH 7.0). The reference cuvette contained all of these compounds except the substrate, and the assay was initiated by addition of substrate. The molar extinction coefficient for NAD(P)H was 6220 M-1 cm-1, and the molar extinction coefficient for 4-NP was 7000 M-1 cm-1 at 420 nm and pH 7.0 (28). For time course assays, monooxygenase-catalyzed reactions were carried out using 30 mL reaction mixtures containing 75 µM 4-NP, 300 µM NADPH, 30 µM FAD, and 20 mM phosphate buffer (pH 7.0). The reaction was initiated by addition of crude extract. One millilliter samples were withdrawn from the reaction mixture and extracted with equal volumes of ethyl acetate after acidification with HCl. The ethyl acetate layer was collected by centrifugation prior to HPLC analysis. For GC-MS analysis, the reaction mixtures were extracted with ether after acidification, and the extract was dried over sodium sulfate. As an important part of the elucidation of the degradation pathway, the enzyme activity of the ring hydroxylating dioxygenase (RHDO) functional in this degradation pathway was tested. According to the results obtained so far the most probable RHDO involved in this degradation pathway would be ‘1,2,4-benzenetriol dioxygenase’ (BtD) in the case of 4-NP and hydroquinone in the case of 2-C-4-NP. Therefore, ring cleavage enzyme activity assays were performed with the crude extracts of strain RKJ300 induced for the degradation of 4-NP and 2-C-4-NP. BtD assay was performed with a ‘wavelength scan’ analysis over a range of 210-360 nm as described by Paul et al. (29). BtD was assayed by observing the depletion of BT at 283 nm and formation of MA at 243 nm. One milliliter of the reaction mixture contained 50 mM sodium phosphate buffer (pH 6.8),

∼60 µg of crude extract of 4-NP grown cells, and 0.2 mM BT as substrate. One unit of activity was defined as the amount of enzyme required to catalyze the oxidation of 1 µmol BT in 1 min. Hydroquinone oxidation by the cell extracts of strain RKJ300 was assayed as described previously by Spain et al. (30). Reaction mixtures contained 0.53 mg of protein (crude cell extract), 8.5 × 10-5 M hydroquinone, and 20 mM phosphate buffer (pH 7.0) in a final volume of 1.0 mL. The reaction was initiated by the addition of cell extract. UV spectra were determined before extract was added (spectrum 0) and after 1.0 min (spectrum 1), 5.0 min (spectrum 2), 8.5 min (spectrum 3), 12.0 min (spectrum 4), and 15 min (spectrum 5). Cell lysate of strain RKJ300 grown on MM with 10 mM sodium succinate was used as the negative control of the enzyme assay. This control also served for determination of the nonconstitutive nature of enzymes. Hydrolase enzyme is required to cleave the ring of the aromatic intermediate of the 2,4-DNP degradation pathway (18). The hydrolase activity was detected by monitoring the formation of hydrolyzed product at 210 nm by HPLC (18). The reaction was performed in 50 mM Tris-HCl (pH 8.0) containing ∼80 µg of crude extract of 2,4-DNP-grown cells and 0.15 mM 2-NCH. Microcosm Studies. For microcosm studies soil was collected from the campus of the Institute of Microbial Technology, Chandigarh, India. The soil contained 37% clay, 28% silt, 35% sand, 0.16% organic carbon, 2.5 ppm phosphorus, 120 ppm potassium, and 59 ppm nitrogen with a pH of 8.8. Strain RKJ300 was grown in the presence of 20 mM sodium succinate at 250 rpm at 30 °C followed by induction with 0.3 mM of each of 4-NP, 2-C-4-NP, and 2,4-DNP separately at midlog phase and grown for another 6 h. Harvested washed cells were resuspended in MM and added to the spiked sterile and nonsterile soils in microcosms to obtain 2 × 106 colony-forming units (CFUs) g-1 soil. The microcosms were prepared as described by Labana et al. (31) with some modifications. Each microcosm containing 20 g of soil was spiked with 70 ppm each of 4-NP, 2-C-4-NP, and 2,4-DNP separately or mixed together. Uninoculated soil microcosms and nonspiked microcosms inoculated with wild-type strain were prepared as controls, and all treatments were carried out in triplicate. Soil samples (1 g) were removed at 0, 1, 2, 3, 5, 7, 10, 15, 20, and 30 days. The method described by Labana et al. (31) was followed to extract 4-NP, 2-C-4-NP, and 2,4-DNP from the soil samples, and the residual compounds in the samples were quantified using HPLC as described above. The various factors such as inoculum size, pH, temperature, substrate concentration, etc., affecting nitrophenols degradation in spiked soil were optimized prior to the study. Monitoring the Bioaugmented Bacteria in Microcosm. In order to monitor the fate of the bioaugmented strain in soil microcosm (both sterile and nonsterile) the unique properties of strain RKJ300, such as utilization of different nitrophenols as the sole source of carbon and energy and resistance to ampicillin (50 µg mL-1), were exploited. Soil samples (1 g) removed at regular intervals were suspended in normal saline, serially diluted, and plated on nutrient agar. Suspected colonies of strain RKJ300 were picked and patched on selective plates of 4-NP (0.5 mM) or 2,4-DNP (0.5 mM) or both containing ampicillin (50 µg mL-1). Colonies that grew on these selective plates were calculated and compared with the control sets. To quantify strain RKJ300 in soil using the hybridization technique two specific probes were designed that hybridized specifically to genomic DNA of strain RKJ300. These probes, a 620 bp segment of the “benzenetriol dioxygenase gene (btd)” and 680 bp segment of the “NADPHdependent F420 reductase gene (npdG)” involved in 4-NP and 2,4-DNP degradation, respectively, were PCR amplified with

FIGURE 1. (A) Degradation of 4-NP by cells of strain RKJ300: (O) OD of culture, (9) 4-NP depletion, ()) nitrite released in the medium. (B) Degradation of 2-C-4-NP by cells of strain RKJ300: (O) OD of culture, (9) 2-C-4-NP depletion, ()) nitrite release, (2) chloride release. (C) Degradation of 2,4-DNP by cells of strain RKJ300: (O) OD of culture, (9) 2,4-DNP depletion, ()) nitrite release. two sets of primers by using an Eppendorf Mastercycler gradient. The reaction mixture contained ∼70 ng genomic DNA, 1 U Deep Vent DNA polymerase, 1 × Thermopol reaction buffer, 200 µM of each deoxynucleoside triphosphate (New England Biolabs, Ipswich, MA), and 20 pmol of each primer (BioBasic Inc., Canada). The sequences of the primer sets used for btd were (forward, 5′-CAGGAGTTCATCCTGCTCTCC-3′; reverse, 5′-CACGAAGTCCTTGATCAGCG-3′) and for npdG were (forward, 5′-ATGAAGAGCAGCAAGATCGCSGTC-3′; reverse, 5′-CGCMGCYCGCGGATCGTGRAC-3′) (11, 32). PCR cycling parameters included an initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at 48 or 51 °C (for btd or npdG, respectively) for 1 min, extension at 75 °C for 1 min, and a final extension for 5 min at 75 °C. These amplicons were cloned at the SmaI restriction site of pBluescript II KS (+) (Stratagene, CA) and sequenced using VOL. 44, NO. 3, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Mass Fragmentation Pattern and Identification of Metabolites of 4-NP and 2-C-4-NP Degradation As Determined by GC-MS Analysisa compound

retention time (min)

m/z of observed fragment ions +

I

5.3

II III IV

3.0 2.6 2.4

+

identification of compound

+

155* [M ], 108 [M - NO2], 97 [M - 58], 80 [97 - OH], 53 [base peak] 126* [M+], 97 [M+ - H - C2H4], 80 [97 - OH], 52 [base peak] 144* [M+], 108 [M+ - Cl], 80 [108 - C2H4], 52 [base peak] 110* [M+], 81 [M+ - C2H4], 52 [base peak]

4-NC BT CHQ HQ

a Samples analyzed were extracted at 5 (compound I) and 10 h (compound II) for 4-NP degradation and 10 (compound III) and 20 h (compound IV) for 2-C-4-NP degradation.

vector-specific primers KS and SK with an automated DNA sequencer (ABI PRISM, 3100 Genetic Analyzer, Applied Biosystems, Foster City, CA). The amplicons were radiolabeled with 32P using the Megaprime DNA Labeling System (GE Healthcare, NJ) following the manufacturer’s instruction. For colony hybridization 100 mg of soil was removed from each soil sample and suspended in 1 mL of MM by vortexing. After serial dilutions 50 µL was plated on nutrient agar and incubated at 30 °C overnight. The colonies were transferred on to a Hybond N+ nylon membrane, treated, and hybridized using both the probes viz. btd and npdG sequentially following the protocol described by Sambrook and Russel (33). Chemicals. All the nitroaromatic compounds and standards of the intermediates were purchased from SigmaAldrich, Fluka, and Merck. All other chemicals used were of the highest analytical grade available. Hydride Meisenheimer complex of 2,4-DNP (H--DNP) was synthesized as described by Behrend and Heesche-Wagner (34). Nucleotide Sequence Accession Number. The nucleotide sequences determined in this study have been deposited in the GenBank database under the accession numbers GQ153939 (for npd gene cluster) and GQ15340 (btd).

Results and Discussion Growth on Different Nitrophenols. Growth profiles on 4-NP, 2-C-4-NP, and 2,4-DNP indicated that strain RKJ300 utilized these substrates as sole sources of carbon and energy at a concentration of 0.3 mM; nitrite ions were released into the culture medium concomitant with the degradation of these compounds (Figure 1). In the case of 2-C-4-NP, degradation time course studies revealed that there was a difference of approximately 2 h between the release of nitrite and chloride ions in the medium (Figure 1). Degradation experiments were also carried out with preinduced cells of strain RKJ300. The results clearly demonstrated that the rate of degradation was significantly increased by preinduction; 0.3 mM substrate (4-NP, 2-C-4NP, and 2,4-DNP) was completely degraded within 10-12 h by induced cells as compared to 18-20 h by uninduced cells (Figure S1, Supporting Information). Similarly, preinduction also allowed degradation of nitrophenols at a slightly higher concentration (0.5 mM) that was not degraded by uninduced cells (Figure S1, Supporting Information). The ability of preinduced cells to degrade higher concentration of the substrate could be a useful feature for further application of strain to decontaminate the nitrophenols-polluted samples/environments. Identification of Intermediates in the 4-NP Degradation Pathway. Strain RKJ300 was grown on MM supplemented with 4-NP, and samples were prepared for various biochemical analyses. Preliminary results obtained from TLC using ‘solvent A’ detected an intermediate (compound I) with an Rf value of 0.65. This was further purified using preparative TLC and subjected to HPLC and direct-MS. The HPLC retention time (7.44 min), UV-vis spectra (Amax 347), and molecular ion at m/z 155 corresponded to the molecular mass of 4-NC (Table 1; Figure S2, Supporting Information). 1072

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Further, upon iron chelation with 2,2′-dipyridyl (i.e., conditions under which ring cleavage enzyme is inhibited), a metabolite (compound II) was accumulated in the medium. TLC analysis of the extracted sample using ‘solvent B’ detected the metabolite with an Rf value of 0.27. This was identified as BT based on the GC retention time (3.0 min), molecular ion at m/z 126, and fragmentation pattern obtained from GC-MS (Table 1; Figure S2, Supporting Information). These results therefore clearly indicated that 4-NC and BT are the intermediates of 4-NP metabolism in strain RKJ300. To further support the proposed 4-NP degradation pathway enzyme assays for 4-NP monooxygenase and BtD were performed. Crude extracts prepared from 4-NP-grown cells catalyzed a NADPH-dependent oxidation of 4-NP with concomitant stoichiometric release of nitrite, indicating the activity of 4-NP monooxygenase. Rapid degradation of 4-NP (max 400 nm) occurred together with consumption of NADPH (max, 340 nm; Figure 2A). Further, 4-NC was identified as a product of 4-NP monooxygenation in the system by HPLC analysis by comparison with standards (Figure S3A, Supporting Information). Similarly, crude extract of 4-NP-grown cells of strain RKJ300 exhibited activity for BtD (1.4 U mg-1), suggesting BT as a substrate for ring cleavage in the 4-NP catabolic pathway (wavelength spectra for depletion of BT at 283 nm and formation of MA at 243 nm; Figure 2B). Results of our study corroborate with the reported 4-NP degradation pathway in other strains of Gram-positive Rhodococcus spp (11, 15). Identification of Intermediates in the 2-C-4-NP Degradation Pathway. To characterize the metabolites of the 2-C-4-NP degradative pathway TLC and GC-MS studies were performed on the extracted samples following growth of strain RKJ300 on 2-C-4-NP. These studies showed the presence of two major compounds in the degradative pathway. A TLC study using ‘solvent A’ separated compound III and compound IV with Rf values of 0.73 and 0.60, respectively. GC retention times (2.6 and 2.4 min) and mass fragmentation patterns obtained from GC-MS analysis identified these intermediates as CHQ and HQ, respectively, which corresponded well with the authentic standards (Table 1; Figure S2, Supporting Information). It is interesting to note that the 2-C-4-NP degradation pathway followed a route different to that of 4-NP degradation because of the presence of an additional substitution to the aromatic ring. The compound is first attacked by an oxygenase, resulting in denitrification and formation of CHQ followed by a reductive dechlorination to form HQ. This is also supported by the results obtained from time course assays for detection of nitrite and chloride ions released in the media (Figure 1). A resting cell study using 2,2′-dipyridyl could accumulate an intermediate having an HPLC retention time of 1.29 min and Amax ) 290 nm, which was identified as HQ based on the retention time and absorption maxima of authentic standard (Figure S3B, Supporting Information). This suggested that HQ is the probable substrate for ring cleavage in the 2-C-4-NP deg-

FIGURE 2. (A) 4-NP monooxygenase activity with the crude extract of 4-NP-grown cells of strain RKJ300: (V) depletion of 4-NP and NADH at 400 and 340 nm, respectively. (B) Conversion of BT into MA catalyzed by crude extract of 4-NP-grown cells of strain RKJ300: (v) formation of MA at 243 nm; (V) depletion of BT at 283 nm. (C) Conversion of hydroquinone (V, depletion at 288 nm) to transient peak absorbing at 290-320 nm. radation pathway, upon which hydroquinone dioxygenase acts for further metabolism of the compound. To support the above findings, enzyme assay was performed to show the activity of hydroquinone ring cleavage enzyme. When the reaction was carried out in the spectrophotometer with crude extracts, the low absorbance at 288 nm due to hydroquinone was replaced by a broad, transient peak absorbing at 290-320 nm (Figure 2C). After 30 min the reaction mixture showed no significant absorption in the UV range. The results indicated that the hydroquinone was converted to an intermediate which was further metabolized by the enzymes in the crude extract. There is only scarce information available regarding the degradation of chloronitrophenolic compounds. For example, the 2-chloro-5-

nitrophenol degradation pathway has been characterized in a Gram-negative strain Ralstonia eutropha JMP134 where the nitro group was initially reduced to an amino group followed by reductive dechlorination. This subsequently resulted in the formation of aminohydroquinone followed by the release of ammonia prior to ring cleavage (35). Interestingly, strain RKJ300 degraded 2-C-4-NP via an oxygenase attack to form CHQ as the initial intermediate, which was subjected to reductive dechlorination. This indicates that the position of the substituent group is decisive in either the initial oxidative or reductive mechanism to occur. Lenke and Knackmuss (36) reported complete degradation of 2-chloro-4,6-dinitrophenol (2-C-4,6-DNP) in a Rhodococcus erythropolis strain HL 24-1 with subsequent release of chloride and nitrite ions in the medium; however, no intermediates could be detected. Mineralization of 4-chloro2-nitrophenol (4-C-2-NP) by a mixed culture in a coupled anaerobic-aerobic process has also been reported (37). Strain RKJ300 was not able to degrade either 2-C-4,6-DNP or 4-C2-NP. However, this is the first report of complete aerobic degradation of 2-C-4-NP in a Gram-positive bacterium. Identification of Intermediates in the 2,4-DNP Degradation Pathway. To investigate the intermediates of the 2,4DNP degradation pathway initially TLC and GC analyses of the extracted samples were carried out. Although chromatograms could not detect any intermediate, depletion of the parent compound was monitored with time. Thereafter, resting cell studies were performed with induced cells of strain RKJ300 and samples were analyzed by HPLC. Formation of H--DNP from 2,4-DNP could be monitored by the appearance of a peak with a retention time of 2.26 min and characteristic spectral absorbance (Amax ) 305 and 442 nm) corresponding to the spectrum of chemically synthesized H--DNP in the laboratory (Figure S4, Supporting Information). It is known in the literature that the hydrogenated product of H--DNP, i.e., 2,4-dinitrocyclohexanone (2,4DNCH), is an unstable compound (18). A commercially available compound 2-nitrocyclohexanone (2-NCH), an analogue of 2,4-DNCH, was used to further elucidate the pathway. HPLC analysis of the product formed during enzyme assay revealed the formation of an intermediate with a retention time of 1.10 min and exhibiting UV-vis spectra (Amax at 203 and 255 nm) in agreement with the formation of 6-nitrohexanoate (6-NH; an analogue of 4,6-dinitrohexanoate) as described previously (18). The hydrolase activity for 2-NCH was found to be 0.53 U mg-1 of crude extract. Furthermore, a growth study showed release of nitrite ions in the medium, suggesting that the ring cleavage product was completely metabolized by the elimination of nitrite ions. Therefore, degradation of 2,4-DNP in R. imtechensis followed the pathway similar to that reported in other Rhodococcus spp (17, 18). On the basis of the above results the proposed pathways for degradation of 4-NP, 2-C-4-NP, and 2,4-DNP by strain RKJ300 have been summarized in Figure 3. With reference to the members of genus Rhodococcus there is only one report suggesting the presence of two independent degradation pathways for 4-NP and 2,4-DNP in strain PN1, but the pathways were not studied in detail (20). However, to the best of our knowledge, this is the first report for elucidation of the 2-C-4-NP degradation pathway in a bacterium. Therefore, the findings of this study claim novelty as three independent degradation pathways have been thoroughly characterized in R. imtechensis RKJ300. Microcosm Studies Using Strain RKJ300. In order to determine the capability of strain RKJ300 to degrade a mixture of nitrophenols in soil, we set up a model system in the form of microcosm studies using both the sterile and the nonsterile soils to simulate the complex in situ environment. Conditions for growth of strain RKJ300 and other factors such as inoculum VOL. 44, NO. 3, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Proposed nitrophenols degradation pathways in strain RKJ300: compound 1, 4-NP; compound 2, 4-NC; compound 3, BT; compound 4, MA; compound 5, 2-C-4-NP; compound 6, CHQ; compound 7, HQ; compound 8, γ-hydroxymuconic semialdehyde; compound 9, β-ketoadipate; compound 10, 2,4-DNP; compound 11, H--DNP; compound 12, 2,4-DNCH; compound 13, 4,6-DNH. size, pH, temperature, and substrate concentration affecting nitrophenols degradation in spiked soil were optimized. The optimum cell density for rapid depletion of the compound(s) was determined by inoculating spiked soil at final concentrations of 2 × 105, 2 × 106, 2 × 107, and 2 × 108 CFU g-1 soil followed by sample analysis by HPLC. Temperature and pH concentrations for maximum degradation of the compound(s) were tested over a range of 10-60 °C and pH 1.5-11.5. Optimum concentration of the compound(s) to be spiked in the soil was determined in a range of 1.4, 14, 70, 140, and 210 ppm using induced and optimized inoculum concentrations. The optimized parameters were as follows: inoculum size 2 × 106 CFU g-1 soil, pH 7.0, temperature 30 °C, and substrate concentration 70 ppm of each compound. Soil microcosm studies were performed under optimized conditions using cells of strain RKJ300 grown on 20 mM sodium succinate and induction with appropriate compound(s). In the 4-NP-spiked microcosm of sterile soil bioaugmented with cells of strain RKJ300 there was complete removal of the compound within 7 days (Figure 4A). Although the initial rate of degradation was quite slow (∼30% depletion in 3 days), on fifth day there was an increase in the rate of 4-NP degradation from the initial concentration of 70 to 10 ppm (∼80% depletion). The residual amount, i.e., 10 ppm, was also completely degraded by 7 days. In the case of 4-NPspiked microcosm of nonsterile soil bioaugmented with strain RKJ300, 40% 4-NP depletion occurred in the first 3 days. After the third day cells were able to degrade 7 ppm of 4-NP (∼ 85% depletion). Within 6 days 4-NP was completely degraded by strain RKJ300 (Figure 4B). Earlier, Labana et al. (31) performed a microcosm study using Arthrobacter protophormiae RKJ100 and demonstrated successful bioremediation of 4-NP-contaminated soil under optimized conditions within 5 days. Hence, the 4-NP degradation efficiency shown by strain RKJ300 in soil is comparable. A similar pattern of slow degradation was also observed in the case of 2-Cl-4-NP and 2,4-DNP. In 2-C-4-NP-treated microcosm this compound was not detected in the 10 days sample, indicating its complete degradation (Figure 4A and 4B). Similarly, results showed that the strain was able to completely degrade 2,4-DNP within 15 days in soil microcosm (Figure 4A and 4B). To date, there is no information available indicating degradation of 2,4-DNP in soil. In contrast, in the control microcosms, i.e,. uninoculated sterile and nonsterile soils, nitrophenol(s) degradation was observed to the extent of only 5-8% in the case of the sterile soil and 20-25% in 1074

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the case of the nonsterile soil after 30 days (Figure 4A and 4B). The later could be due to either the presence of indigenous but efficient degraders or some abiotic factors. Samples collected from microcosm spiked with all three compounds together showed complete degradation of each of the compounds within 20 and 18 days for bioaugmented sterile and nonsterile soil, respectively, as revealed by HPLC analysis (Figure 4C and 4D), indicating no significant inhibition was imparted by any of the compounds in the mixture. Microcosm studies have been conducted to estimate biodegradation rates of a mixture of five polyaromatic hydrocarbons and a combination of chlorinated aliphatic compounds using subsurface microbiota (38, 39). Very few reports are available in regard to degradation of a mixture of nitroaromatic compounds in soil. Blasco et al. (40) reported that the presence of mononitrophenols (2-NP or 4-NP) strongly inhibited 2,4-DNP degradation. A recent report has demonstrated that there was a negative environmental effect if nitrate and 2,4-DNP were released together to natural habitats since 2,4-DNP uptake was probably repressed by nitrate at the transport level (41). In another study Lors et al. (5) reported that the presence of dinitro-o-cresol adversely affected the degradation of 2,4-D in soil microcosms. This evidence suggests that degradation of a single compound may be competitively inhibited by the presence of other compounds in soil. However, strain RKJ300 showed robustness since it was capable of simultaneous degradation of substituted nitrophenols, as evident from the above results. Monitoring the Fate of Strain RKJ300 in Soil. To determine the survival of the introduced bacteria and influence of the soil ecosystem it is important to monitor the fate of the inoculant population. In this work we exploited certain unique properties of strain RKJ300 to monitor its fate in the soil by viable plate count. To the control microcosm set (sterile soil not spiked with compound) an innoculum of 2 × 106 CFU g-1 of soil was added. Results of the plate count revealed that cell survival was slightly reduced (2 × 105 CFU g-1 of soil) followed by a gradual steady decrease in the survival of the cells on 10th (1.8 × 103 CFU g-1 of soil) and 15th days (1.3 × 102 CFU g-1 soil). By the 20th day of the experiment only a negligible amount of cells was present in the control set. A similar but comparatively fast decline in the cell population was observed in the case of control microcosm with nonsterile soil. Therefore, cells of strain RKJ300 in the control microcosm set showed a steady decline in their survival, indicating that the sterile soil could not

FIGURE 4. (A) Degradation of nitrophenols by strain RKJ300 in (A) sterile microcosms spiked with 4-NP, 2-C-4-NP, or 2,4-DNP separately along with uninoculated controls. (B) Nonsterile microcosms spiked with 4-NP, 2-C-4-NP, or 2,4-DNP separately along with uninoculated controls. (C) Sterile microcosms spiked with the above three compounds together along with uninoculated control. (D) Nonsterile microcosms spiked with the above three compounds together along with uninoculated control. (b) 4-NP depletion in test; (O) 4-NP depletion in uninoculated control; (9) 2-C-4-NP depletion in tests; (0) 2-C-4-NP depletion in uninoculated controls; (1) 2,4-DNP depletion in test; (3) 2,4-DNP depletion in uninoculated control. supply any nutrient for their growth. Furthermore, the situation might have been more challenging in the case of the nonsterile soil where strain RKJ300 had to compete with indigenous microbial population. However, the soil spiked with compound(s) supported the growth of the induced cells. Tables S1 and S2, Supporting Information, show the population of strain RKJ300 in sterile and nonsterile soil, respectively, spiked with individual or a mixture of nitrophenols. The CFUs remained quite stable up to 15-20 days; however, within 20-30 days there was a steady decrease of ∼6-7 fold of CFUs in the experimental sets of microcosms spiked with compounds separately or together in the case of sterile microcosms, whereas a comparatively faster decline in the cell population was observed in the case of nonsterile soil. This decrease may be explained as a consequence of the unavailability of carbon sources as within 20 days the spiked compounds were completely utilized by the microorganism. Keeping in mind the bias of various culturing techniques, monitoring of strain RKJ300 was also performed using colony hybridization experiments. The strain-specific radiolabeled probes involved in the 4-NP and 2,4-DNP degradation pathways were used for this purpose (Figure S5, Supporting Information; also see Experimental Procedures). Results obtained from the catabolic gene-based hybridization technique were found to be in agreement with that of plate count

data and explicitly indicated survival of strain RKJ300 in the microcosm spiked with a mixture of compounds (both sterile and non sterile, Tables S1 and S2, Supporting Information). In control microcosms in which strain RKJ300 was not added there were no hybridization signals, indicating the specificity of the probe. Use of catabolic genes as molecular markers for detection of bioaugmented strain has been exploited successfully in order to monitor survival of naphthalene degrader strain Rhodococcus sp. 1BN in soil microcosm along with viable plate counting method (42). This study would further help in the design of small-scale field experiments and subsequently an in situ bioremediation system for field applications. From this study it can be concluded that R. imtechensis RKJ300 is a metabolically versatile organism since it has evolved pathways for degradation of 4-NP, 2-C-4-NP, and 2,4-DNP. This is the first report to the best of our knowledge demonstrating complete aerobic degradation of 2-C-4NP by a Gram-positive bacterium. Biochemical characterization of the 4-NP, 2-C-4-NP, and 2,4-DNP degradation pathways indicated that the initial reactions involved in degradation were independent of each other. It could also be postulated that addition of a single halo group, e.g., chloride group, may perhaps direct the pathway in a route different from that of 4-NP degradation. Furthermore, VOL. 44, NO. 3, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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microcosm studies demonstrated complete degradation of a mixture of nitrophenols with a fairly stable population of the bioaugmented strain. Consequently, the versatility and stability of strain RKJ300 make it an attractive organism for its use in the treatment of soils and industrial wastewaters containing a mixture of substituted nitrophenolic compounds. In future studies molecular characterization of these degradation pathways may shed light on their regulation and evolution.

Acknowledgments We thank Abhineet Goyal for technical assistance. We are grateful to Narinder K. Sharma and Janmejay Pandey for helpful discussion. This work was supported, in part, by the Council of Scientific and Industrial Research, India, Department of Science and Technology, India, and Japan Science and Technology agency. A.G. acknowledges a research fellowship awarded by the CSIR.

Supporting Information Available Plate counts and colony hybridization of sterile spiked microcosm in Table S1, plate counts and colony hybridization of non-sterile spiked microcosm in Table S2, degradation of nitrophenols by strain RKJ300 in Figure S1, GC-MS spectra of degradation metabolites in Figure S2, HPLC chromatogram showing 4-NC and HQ in Figure S3, HPLC chromatogram showing formation of Hydride-Meisenheimer complex in Figure S4, and Gel photograph of btd and npdG genes in Figure S5. This material is available free of charge via the Internet at http://pubs.acs.org.

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