Journal of Hazardous Materials 284 (2015) 261–268
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Biodegradation of 4-chloroindole by Exiguobacterium sp. PMA Pankaj Kumar Arora ∗ , Hanhong Bae ∗ School of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk 712-749, Republic of Korea
h i g h l i g h t s • • • •
Exiguobacterium sp. PMA utilized 4-chloroindole as its sole source of carbon and energy. Strain PMA degraded 4-chloroindole via formation of indole, isatin, anthranilic acid, and salicylic acid. Strain PMA also degraded 4-chloroindole in the soil. This is the first report of the bacterial degradation of 4-chloroindole.
a r t i c l e
i n f o
Article history: Received 11 August 2014 Received in revised form 5 November 2014 Accepted 14 November 2014 Available online 20 November 2014 Keywords: Biodegradation 4-Chloroindole Isatin Anthranilic acid Salicylic acid
a b s t r a c t Exiguobacterium sp. PMA utilized 4-chloroindole as its sole source of carbon and energy. The effect of initial concentrations of substrate on the 4-chloroindole degradation was studied and observed that strain PMA was capable of degrading 4-chloroindole up to concentration of 0.5 mM. The degradation pathway of 4-chloroindole was studied for Exiguobacterium sp. PMA based on metabolites identified by gas chromatography–mass spectrometry. 4-Chloroindole was initially dehalogenated to indole that was further degraded via isatin, anthranilic acid, and salicylic acid. The potential of strain PMA to degrade 4-chloroindole in soil was monitored using soil microcosms, and it was observed that the cells of strain PMA efficiently degraded 4-chloroindole in the soil. The results of microcosm studies show that strain PMA may be used for bioremediation of 4-chloroindole-contaminated sites. This is the first report of the bacterial degradation of 4-chloroindole. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Indole and its derivatives constitute a major group of Nheterocyclic compounds which are widely used for synthesis of pharmaceuticals, cosmetics, pesticides, disinfectants, agrochemicals, and dyestuffs [1–3]. These compounds have been considered as environmental pollutants due to their toxicity and worldwide occurrence. The high concentrations of indole and its derivatives are toxic to cells due to perturbations in membrane potential [4]. Kim et al. [4] showed that indole inhibits adenosine triphosphate production and protein folding in Pseudomonas putida. Indole inhibits bacterial quorum sensing signal transmission by interfering with quorum sensing regulator folding [5]. Indole may cause low pigmentation in plant tissues by inhibition of anthraquinone biosynthesis [6]. Furthermore, indole may produce toxic chlorinated aromatic compounds when dilute aqueous solution of
∗ Corresponding authors. Tel.: +82 538103031. E-mail addresses: [email protected] (P.K. Arora), [email protected] (H. Bae). http://dx.doi.org/10.1016/j.jhazmat.2014.11.021 0304-3894/© 2014 Elsevier B.V. All rights reserved.
chlorine or chlorine dioxide are used for the chemical disinfection of indole-containing wastewater [7]. Metabolism of indole has been extensively studied in microbes under aerobic as well as anaerobic conditions [8–11]. Several aerobic degradation pathways have been studied for metabolism of indole [8,9]. One of them is known as the isatin pathway in which indole was degraded via the formation of indoxyl, 2,3dihydroxyindole, isatin, formylanthranilic acid, anthranilic acid, salicylic acid, and catechol [8]. Another pathway is the gentisate pathway in which indole was degraded via indoxyl, isatin, anthranilic acid, and gentisate [9]. Under anaerobic conditions, indole degraded to methane and carbon dioxide [10]. Bak and Widdel [3] reported that a sulfate-reducing bacterium, Desulfobacterium indolicum utilized indole as an electron donor and its carbon source. Under anaerobic and denitrifying conditions, indole was degraded via oxindole and isatin by the bacterial consortia [11–13]. An indole-degrading methanogenic consortium enriched from sewage sludge degraded indole and 3-methylindole via oxindole and 3-methyloxindole [14]. Hong et al. [15] studied anaerobic bacterial communities in indole-degrading bioreactors under denitrifying and sulfate-reducing conditions. In the denitrifying
262
P.K. Arora, H. Bae / Journal of Hazardous Materials 284 (2015) 261–268
bioreactor, -proteobacteria including bacteria from genera Alicycliphilus, Acaligenes, and Thauera were dominant and responsible for indole degradation whereas in the sulfate-reducing bioreactor, Clostridia, and Actinobacteria were dominant bacteria [15]. Literature studies show that there are several reports of bacterial degradation of indole [8–15]. However, only few reports are available for degradation of chloro derivatives of indole [16], which are highly toxic to living beings. For example, 4-chloroindole causes eye and skin irritation, and may also cause irritation of the lungs and respiratory system. The antimicrobial activity of 4-chloroindole has also been investigated against several grampositive and gram-negative bacteria [17]. Tiedink et al. [18] studied the genotoxic effects of nitrosated 4-chloroindole and 4-chloro-6methoxyindole, and reported that both of the chloroindoles were highly mutagenic to Salmonella typhimurium TA100 as well as potent inducers of sister chromatid exchanges. Despite the fact that 4-chloroindole is a toxic compound, there is no report of its degradation. In this communication, we report the aerobic degradation of 4-chloroindole by a previously isolated bacterium, Exiguobacterium sp. PMA [19]. 2. Material and methods 2.1. Bacterial strain The bacterial strain used in this study was Exiguobacterium sp. PMA isolated previously from a chemically-contaminated site of India by enrichment method which utilized 4-chloro2-nitrophenol, 2-nitrophenol, 4-chloro-2-aminophenol, and 2aminophenol as its sole carbon and energy sources [17]. 2.2. Screening for degradation of indole derivatives Exiguobacterium sp. PMA was screened for its ability to degrade indole derivatives. For screening, Exiguobacterium sp. PMA was streaked on minimal agar plates containing 0.2 mM test compound as its sole carbon and energy source. Minimal agar plates were prepared as described previously [19]. 4-Chloroindole, indole-3-acetic acid, indole-3-methyl, indole-3-carboxylic acid were used as test compounds. The growth of Exiguobacterium sp. PMA on minimal agar plate containing an indole derivative was considered positive result. 2.3. Degradation and growth studies For degradation studies, strain PMA was grown on minimal medium containing different concentrations of 4-chloroindole (0.2–0.6 mM). Minimal medium was prepared by dissolving the following compounds in 100 ml of double distilled water: 0.4 g Na2 HPO4 , 0.2 g KH2 PO4 , 0.08 g NaNO3 , 0.08 g MgSO4 ·7H2 O, 0.1 ml trace element solution [19]. The composition of trace element solution was exactly same as described previously [19–22]. 4Chloroindole was added into medium at before the autoclave [19–22]. The media was autoclaved at 15 lbs for 20 min. The 2% inoculums of overnight grown cells of strain PMA (2 × 108 CFU/ml) were added into the medium and the flasks were incubated at 30 ◦ C under shaking conditions (200 rpm). The samples were collected at regular intervals and centrifuged. 4-Chloroindole was analyzed by spectrophotometrically or by high performance liquid chromatography using a method as described by Yin et al. [23]. For growth studies, strain PMA was grown on minimal media containing 0.5 mM 4-chloroindole, samples (2 ml) were collected at regular intervals (0 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, 48 h, 52 h, and 56 h) and the bacterial growth was measured by taking optical density at 600 nm using a spectrophotometer.
2.4. Detection of ammonia and chloride ions Chloride ions were analyzed using QuantiChromTM Chloride assay kit (DICL-250) from BioAssay Systems, Hayward, CA. Ammonia ions were detected with the ‘Ammonia Assay Kit’ from Sigma–Aldrich (GmbH, Germany) according to the manufacturer’s instructions [19–22].
2.5. Effect of different inoculum sizes on 4-chloroindole degradation Exiguobacterium sp. PMA was grown on 250 ml nutrient broth at 30 ◦ C under shaking condition. When the culture reached the late logarithmic phase of growth, the cells were harvested by centrifugation at 10 000 × g for 20 min at 4 ◦ C, washed with minimal medium [19–22]. The resultant pellets were re-suspended in double distilled water [19–22]. To study the effect of different inoculum sizes on degradation, different quantities of cells suspension were added to 200 ml minimal media containing 0.5 mM 4-chloroindole as its sole source of carbon and energy. At different time intervals, the 4-chloroindole degradation was monitored. The final concentrations of the inoculum used in this study were: 2.0 × 106 , 2 × 107 , and 2 × 108 CFU/ml which were confirmed at the start of the experiment by the plate count method.
2.6. Identification of metabolites Strain PMA was grown on minimal medium containing 0.5 mM 4-chloroindole. Samples were collected at regular intervals (0, 12, 24, 36, and 48 h), centrifuged and extracted with ethyl acetate. The extracted samples were dissolved into 100 l methanol, and analyzed by gas chromatography–mass spectrometry (GC–MS). Agilent gas chromatography system mconnected to model high throughput time-of-flight mass spectrometer (HT-TOFMS) was used with a HP-5 (30 m × 0.320 mm × 0.25 m) column for detection of the metabolites [21]. Separation was performed under the following temperature program on the low-polarity columns (HP5ms) with helium as a carrier gas at 1.5 ml/min; 50 ◦ C held for 1 min, and the temperature was increased at 25◦ /min to 280 ◦ C and held for 5 min [21]. The samples (1 l) were injected in splitless mode. The temperatures of the transfer line and ion source (electron ionisation mode, EI, 70 eV) were 225 ◦ C and 250 ◦ C, respectively [21].
2.7. Enzyme assay for 4-chloroindole dehalogenase The activity for 4-chloroindole dehalogenase was monitored by the detection of the total released chloride ions at room temperature in a reaction mixture containing 100 mM Tris-Acetate buffer (pH 7.5), 0.2 mM NADPH, 200 mg of cell-free lysate, and 500 M of 4-chloroindole. The final volume of the reaction mixture was 5 ml and the reaction mixture without crude extract was used as a control [19]. Samples were collected at regular intervals and assayed for chloride ions. Standard curve was prepared using NaCl as standard to quantify the chloride ions [19]. Samples were also analyzed by GC–MS to detect the product of the reaction [19]. The cell free lysate was prepared as described previously [20]. The overnight grown cells of strain PMA (1%, v/v) were inoculated into 500 ml minimal media containing 20 mM sodium succinate and 0.5 mM 4-chloroindole [20]. The bacterial cells were harvested upon the incubation of 24 h, washed with phosphate buffer (50 mM, pH 7.5) and re-dissolved in the same buffer. The cells were sonicated as described previously [20] and were centrifuged at 4 ◦ C for 15 min. The supernatant was used for enzyme assays.
P.K. Arora, H. Bae / Journal of Hazardous Materials 284 (2015) 261–268
2.8. Enzyme assay for anthranilic acid deaminase The activity for anthranilic acid deaminase was monitored by the detection of the total released ammonium ions at room temperature in a reaction mixture containing 100 mM Tris-Acetate buffer (pH 7.5), 0.2 mM NADPH, 100 mg of cell-free lysate, and 500 M of anthranilic acid. The final volume of the reaction mixture was 2 ml. After 10 min, the reaction mixture was centrifuged and supernatant was divided into two parts. One part (0.5 ml) was used to detect the ammonium ions and the second part (1.5 ml) was extracted with ethyl acetate and analyzed by the GC–MS for identification of the product. 2.9. Microcosm studies Soil used in microcosm studies was collected from outside the campus. Microcosm studies were performed as described previously [19–22]. The soil contained 54% sand, 30% silt, 16.0% clay, 2.4% organic matters, 1.8 ppm phosphorus, 107 ppm potassium, 80 ppm nitrogen, and had a pH 7.2. Microcosms were prepared using 250 ml glass beakers and each backer contained 50 g of soil
263
spiked with 100 ppm 4-chloroindole. Four types of microcosms were prepared (a) test microcosm with sterile soil, (b) test microcosms with non-sterile soil, (c) control microcosm with sterile soil, and (d) control microcosm with non-sterile soil [19–22]. Test microcosms with non-sterile and sterile soils were inoculated with pre-grown and 4-chloroindole induced cells of Exiguobacterium sp. PMA at ∼2 × 108 cells colony-forming units (CFUs) g−1 soil, whereas the control microcosms with sterile and non sterile soils were left non-bioaugmented. The bioaugmentation was performed by thorough mixing of the pre-grown cells of Exiguobacterium sp. PMA with the soil samples [19–22]. All the microcosms were covered with perforated aluminum foil and incubated at 30 ◦ C for 15 days. During the incubation period all the microcosms were sprinkled with distilled water at regular intervals to compensate the loss of water via evaporation [19–22]. Soil samples were removed at regular intervals, and extracted for analysis as described previously [19–22]. The various factors such as inoculum size, pH, temperature, and substrate concentration, affecting 4-chloroindole degradation in microcosm were optimized prior to the study as described previously [19–22]. All experiments were carried out in triplicate.
Fig. 1. Degradation and Growth Studies. (a) Degradation of various concentrations of 4-chloroindole by Exiguobacterium sp. PMA. (b) Growth of Exiguobacterium sp. PMA on the minimal media containing 4-chloroindole as its sole source of carbon and energy. (c) Analysis of chloride and ammonia releases from 4-chloroindole by Exiguobacterium sp. PMA. (d) Degradation of 4-chloroindole by different inoculum sizes.
264
P.K. Arora, H. Bae / Journal of Hazardous Materials 284 (2015) 261–268
Fig. 2. GC–MS profiles of various samples of degradation of 4-chloroindole by Exiguobacterium sp. PMA.
P.K. Arora, H. Bae / Journal of Hazardous Materials 284 (2015) 261–268
3. Results 3.1. Screening for indole degradation Screening results show that Exiguobacterium sp. PMA grow on minimal agar plates containing 0.2 mM 4-chloroindole as its sole source of carbon energy, however strain PMA did not grow on the minimal agar plates containing any other indole derivative including indole-3-aceticacid, indole-3-methyl, indole-3-carboxylic acid. This data suggests that strain PMA utilized only 4-chloroindole as its sole source of carbon and energy. 4-Chloroindole was selected for its degradation study by strain PMA.
265
degradation was observed when the concentration of 4chloroindole was above 0.5 mM. The optimum concentration for degradation of 4-chloroindole was determined as 0.5 mM. All experiments were performed using the optimum concentration. For growth study, strain PMA was grown on minimal media containing 0.5 mM 4-chloroindole. At the initial 12 h, no growth was observed. The exponential growth of strain PMA was observed between 16 and 36 h (Fig. 1(b)). After 36 h, bacterial growth became slow due to utilization of most of the 4-chloroindole (about 0.4 mM). After 48 h, no more growth was observed due to complete consumption of 4-chloroindole. 3.3. Detection of ammonia and chloride ions
3.2. Degradation and growth studies Degradation studies showed that strain PMA degraded 4chloroindole up to concentration of 0.5 mM (Fig. 1(a)). No
4-Chloroindole was degraded via the removal of the chloride and ammonium ions. At the initial 12 h, neither chloride nor ammonium ions were detected. The detection of chloride ions was
Fig. 3. Mass spectra of metabolites and authentic standards.
266
P.K. Arora, H. Bae / Journal of Hazardous Materials 284 (2015) 261–268
initiated from the sample of the 16 h whereas the detection of the ammonium ions was initiated from the sample of 20 h. The stoichiometric amounts of chloride and ammonium ions were detected at the sample of 44 h and 48 h, respectively (Fig. 1c). These data suggest that chloride release occurred before the ammonia release. 3.4. Effect of different inoculum sizes We have also monitored the effects of the various inoculum sizes on the 4-chloroindole degradation by Exiguobacterium sp. PMA. It was observed that inoculum sizes significantly affected the degradation of 4-chloroindole. The larger inoculum sizes degraded 4-chloroindole more rapidly as compared to lower inoculum sizes (Fig. 1(d)). The culture with highest cell densities (2 × 108 ) degraded 4-chloroindole completely within 44 h, whereas the cultures with the cell densities of 2 × 107 and 2 × 106 degraded 4-chloroindole within 48 and 52 h, respectively. 3.5. Identification of metabolites GC–MS analysis of the samples collected from regular intervals confirmed the complete degradation of 4-chloroindole (Fig. 2). In the 4-chloroindole degradation, no metabolite was detected in the samples of 0 h and 12 h. Only 4-chloroindole was detected in the samples of 0 h and 12 h. In the sample of 24 h, metabolites I and II were detected along with 4-chloroindole. The molecular ions of metabolites I and II were observed at m/z 117 and m/z 147, respectively (Fig. 3(a) and (b)). Apart from 4-chloroindole, metabolite III, and metabolite IV were detected in the sample of 36 h with molecular ions at m/z 137 and m/z 138, respectively (Fig. 3(c) and (d)). In the sample of 48 h, neither 4-chloroindole nor any metabolite was detected. The mass spectra of these four metabolites were exactly matched to the authentic standards of indole, isatin, anthranilic acid, and salicylic acid (Fig. 3(e–h). 3.6. 4-Chloroindole dehalogenase activity 4-Chloroindole dehalogenase catalyzed the conversion of 4-chloroindole to indole with removal of chloride ion. The stoichiometric amounts of chloride ions were detected during the enzyme assay and indole was detected as a product. In the control, neither chloride release was observed nor indole was detected. 3.7. Anthranilic acid deaminase activity This enzyme catalyzed the conversion of anthranilic acid to salicylic acid with release of ammonium ions. The stoichiometric amounts of ammonium ions were detected during the enzyme assay and salicylic acid was detected as a product. 3.8. Microcosm studies In order to determine the capability of Exiguobacterium sp. PMA to degrade 4-chloroindole in the soil, we performed microcosm studies using both sterile and non-sterile soil under optimized conditions. The optimized parameters were as follows: inoculum size 2 × 108 CFU g−1 soil, pH 7.2, temperature 30 ◦ C, and 100 ppm 4-chloroindole. In the microcosm with sterile soil, there was no degradation of 4-chloroindole at initial 3 days. The degradation was 50%, 70%, and 90% within 6, 8, and 9 days, respectively, and the complete degradation of 4-chloroindole was observed within 11 days (Fig. 4). In the microcosm with non-sterile soil, there was no degradation of 4-chloroindole at initial 4 days. The degradation was 40%, 73%, and 90% within 9, 11, and 13 days, respectively. The complete degradation of 4-chloroindole was observed within 14
Fig. 4. Microcosm studies. Degradation of 4-chloroindole in test microcosm with sterile soil (a), test microcosm with non-sterile soil (b), control microcosm with sterile soil (c), and control microcosm with non-sterile soil (d).
days. In control microcosm with sterile and non-sterile soil, there was no degradation of 4-chloroindole within 14 days. 4. Discussion Exiguobacterium sp. PMA previously isolated from a chemically contaminated site was screened for its ability to utilize indole derivatives including 4-chloroindole, indole-3-aceticacid, indole3-methyl, indole-3-carboxylic acid as its sole sources of carbon and energy. It was observed that Exiguobacterium sp. PMA utilized 4-chloroindole as its sole source of carbon and energy, and degraded it with release of stoichiometric amounts of chloride and ammonium ions. Exiguobacterium sp. PMA initially reductively dehalogenated 4-chloroindole to indole that was further degraded via isatin, anthranilic acid, and salicylic acid. Previous studies show that Exiguobacterium sp. PMA dehalogenated chlorinated aromatic compounds [19,24–26]. In the degradation pathway of 4-chloro-2-nitrophenol, strain PMA reductively dehalogenated 4-chloro-2-aminophenol to 2-aminophenol [19]. To the best of our knowledge, this is the first report of the complete degradation of 4-chloroindole. However, the catabolism of derivatives of chloroindole has been studied. Jensen et al. [16] studied catabolism of 4-chloroindole-3-acetic acid and 5-chloroindole3-acetic acid by Bradyrhizobium japonicum 61A24 and 110. 4-Chloroindole-3-acetic acid transformed to 4-chlorodioxindole via 4-chlorodioxindole-3-acetic acid, whereas 5-chloroindole-3acetic acid transformed 5-chloroanthranilic acid via 5-chloroisatin [16]. No chloride ions were released during the degradation of 4chloroindole-3-acetic acid and 5-chloroindole-3-acetic acid by B. japonicum [16]. However, the degradation of 4-chloroindole proceeded via release of stoichiometric amounts of chloride ions by Exiguobacterium sp. PMA. Perni et al. [27] studied biotransformation of 5-chloroindole (5-haloindole) into a pharmaceutical intermediate, 5-chlorotryptophan (5-halotryptophan) using Escherichia coli expressing a recombinant tryptophan synthase enzyme. In this study, we have not detected 4-chlorotryptophan as an intermediate product of degradation of 4-chloroindole. This data suggests that tryptophan synthase was not involved in the degradation of 4-chloroindole. In this study, we have detected indole as the first metabolite of degradation pathway of 4-chloroindole. Literature studies show that diverse biochemical mechanisms were involved in indole degradation under aerobic conditions [8,9]. Sakamoto et al. [8] reported that bacterial degradation of indole was occurred by an isatin pathway via the formation of indoxyl (3-hydroxyindole), 2,3dihydroxyindole, isatin, formylanthranilic acid, anthranilic acid, salicylic acid, and catechol. In this study, we have also observed isatin, anthranilic acid, salicylic acid as metabolites. However, we have not detected 2,3-dihydroxindole and formylanthranilic acid. Claus and Kutzner [9] identified another pathway of indole
P.K. Arora, H. Bae / Journal of Hazardous Materials 284 (2015) 261–268
267
5. Conclusion Exiguobacterium sp. PMA utilized 4-chloroindole as its sole source of carbon and energy, and degraded it via a novel pathway with the formation of indole, isatin, anthranilic acid, and salicylic acid as the metabolites. This strain was also able to degrade 4-chloroindole in soil; therefore, this strain may be used for bioremediation of 4-chloroindole-contaminated sites. This is the first report of the biodegradation of 4-chloroindole. Acknowledgment
Fig. 5. A proposed pathway of biodegradation of 4-chloroindole.
This work was carried out with the support of the NextGeneration Biogreen 21 Program (PJ00806302), Rural Development Administration, Republic of Korea. References
degradation in Alcaligenes sp. strain In3, in which indole was degraded via indoxyl, isatin, anthranilic acid, and gentisate. Indoxyl and gentisate were not detected as metabolites of degradation of 4chloroindole in strain PMA, therefore, the gentisate pathway should not be involved in degradation of 4-chloroindole in strain PMA. Another mechanism of indole degradation involves oxidation of indole into indoxyl, which oxidized spontaneously to indigo [28]. Indigo was not detected as degradation product in strain PMA. Fujioka and Wada [29] reported that indole was biodegraded to dihydroxyindole, which was then oxidized to anthranilic acid by a single enzyme step catalyzed by dihydroxyindole oxygenase and to catechol by the Gram-positive coccus. Catechol was not detected as a degradation product in strain PMA. On the basis of the above discussion, we have proposed a metabolic pathway of degradation of 4-chloroindole for strain PMA. 4-Chloroindole was initially dehalogenated to indole that was degraded via isatin, anthranilic acid, and salicylic acid (Fig. 5). We have also investigated the effects of different concentrations of 4-chloroindole on its degradation. It was observed that strain PMA was capable to degrade 4-chloroindole up to 0.5 mM concentrations. There was no degradation of 4-chloroindole when concentration was above 0.5 mM. These data suggest that high concentrations of 4-chloroindole are toxic to bacterial cells. Previously, several researchers have been studied effect of indole concentrations on degradation [30–36]. Katapodis et al. [30] studied indole toxicity on the thermophilic fungus Sporotrichum thermophile and determined the minimal inhibitory concentration (0.5 g L−1 ) of indole for the fungus. The minimal inhibitory concentration of indole for bacteria such as Pseudomonas sp. strain ST-200 [31] and Acinetobacter sp. ST-550 [32] was determined as 0.3 g L−1 . In this study, the minimal inhibitory concentration of 4-chloroindole for Exiguobacterium sp. PMA was 0.6 mM. It has been suggested that all studies for indole degradation should be carried out at low indole concentration to avoid toxic effects in microbes [33–36]. Chen et al. [37] reported that endophytic fungus Phomopsis liquidambari can degrade indole up to 100 g/L. Yin et al. [23] studied effects of the initial concentration of indole (0.3–3 mM) on the indole degradation by Pseudomonas aeruginosa Gs and reported the degradation of indole was the zero-order kinetic equation. We have also monitored the capacity of the cells of strain PMA to degrade 4-chloroindole in the soil microcosms using sterile and non-sterile soil. It was observed that strain PMA degraded 100 ppm 4-chloroindole within 11–14 days. The rate of the degradation of 4-chloroindole was faster in sterile soil than non-sterile soil. This data indicates that the biotic factors present in non-sterile soil were interrupting the degradation of 4-chloroindole in non-sterile soil microcosm. This is the first report of degradation of any indole derivative in soil.
[1] M. Novotny, J.W. Starand, S.L. Smith, D. Wiesler, F.J. Schwende, Compositional studies of coal tar by capillary gas chromatography/mass spectrometry, Fuel 60 (1981) 213–220. [2] W.W. Paudler, M. Cheplen, Nitrogen bases in solvent-refined coal, Fuel 58 (1979) 775–778. [3] F. Bak, F. Widdel, Anaerobic degradation of indolic compounds by sulfate-reducing enrichment cultures, and description of Desulfobacterium indolicum gen. nov., sp. nov, Arch. Microbiol. 146 (1986) 170–176. [4] J. Kim, H. Hong, A. Heo, W. Park, Indole toxicity involves the inhibition of adenosine triphosphate production and protein folding in Pseudomonas putida, FEMS Microbiol. Lett. 343 (2013) 89–99. [5] J. Kim, W. Park, Indole inhibits bacterial quorum sensing signal transmission by interfering with quorum sensing regulator folding, Microbiology 159 (2013) 2616–2625. [6] H. El-Shagi, U. Schulte, M.H. Zenk, Specific inhibition of anthraquinone formation by amino compounds in Morinda cellcultures, Naturwissenschaften 71 (1984) 267. [7] S. Lin, R.M. Carlson, Susceptibility of environmentally important heterocycles to chemical disinfection: reactions with aqueous chlorine dioxide, and chloramines, Environ. Sci. Technol. 18 (1984) 743–748. [8] Y. Sakamoto, M. Uchida, K. Ichibara, The bacterial decomposition of indole. I. Studies on its metabolic pathway by successive adaptation, Med. J. Osaka Univ. 3 (1953) 477–486. [9] G. Claus, H.J. Kutzner, Degradation of Indole by Alcaligenes spec, Syst. Appl. Microbiol. 4 (1983) 169–180. [10] Y.T. Wang, M.T. Suidan, J.T. Pfeffer, Anaerobic biodegradation of indole to methane, Appl. Environ. Microbiol. 48 (1984) 1058–1060. [11] D.F. Berry, E.L. Madsen, J.M. Bollag, Conversion of indole to oxindole under methanogenic conditions, Appl. Environ. Microbiol. 53 (1987) 180–182. [12] E.L. Madsen, A.J. Francis, J.M. Bollag, Environmental factors affecting indole metabolism under anaerobic conditions, Appl. Environ. Microbiol. 54 (1988) 74–78. [13] E.L. Madsen, J.M. Bollag, Pathway of indole metabolism by a denitrifying microbial community, Arch. Microbiol. 151 (1989) 71–76. [14] J.D. Gu, D.F. Berry, Degradation of substituted indoles by an indole-degrading methanogenic consortium, Appl. Environ. Microbiol. 57 (1991) 2622–2627. [15] X. Hong, X. Zhang, B. Liu, Y. Mao, Y. Liu, L. Zhao, Structural differentiation of bacterial communities in indole-degrading bioreactors under denitrifying and sulfate-reducing conditions, Res. Microbiol. 161 (2010) 687–693. [16] J.B. Jensen, H. Egsgaard, H. Van Onckelen, B.U. Jochimsen, Catabolism of indole-3-acetic acid and 4- and 5-chloroindole-3-acetic acid in Bradyrhizobium japonicum, J. Bacteriol. 177 (1995) 5762–5766. ˜ J.M. Peralta-Sánchez, L. Sánchez, S. Ananou, M. [17] M. Martín-Vivaldi, A. Pena, Ruiz-Rodríguez, J.J. Soler, Antimicrobial chemicals in hoopoe preen secretions are produced by symbiotic bacteria, Proc. Biol. Sci. 277 (2010) 123–130. [18] H.G. Tiedink, L.H. de Haan, W.M. Jongen, J.H. Koeman, In-vitro testing and the carcinogenic potential of several nitrosated indole compounds, Cell Biol. Toxicol. 7 (1991) 371–386. [19] P.K. Arora, A. Sharma, R. Mehta, B.D. Shenoy, A. Srivastava, V.P. Singh, Metabolism of 4-chloro-2-nitrophenol in a gram-positive bacterium, Exiguobacterium sp. PMA, Microb. Cell Fact. 11 (2012) 150. [20] P.K. Arora, R.K. Jain, Metabolism of 2-chloro-4-nitrophenol in a gram negative bacterium, Burkholderia sp. RKJ 800, PLoS ONE 7 (2012) e38676. [21] P.K. Arora, A. Srivastava, V.P. Singh, Degradation of 4-chloro-3-nitrophenol via a novel intermediate, 4-chlororesorcinol by Pseudomonas sp. JHN, Sci. Rep. 4 (2014) 4475. [22] P.K. Arora, A. Srivastava, V.P. Singh, Novel degradation pathway of 4-chloro-2-aminophenol via 4-chlorocatechol in Burkholderia sp. RKJ 800, Environ. Sci. Pollut. Res. 21 (2014) 2298–2304. [23] B. Yin, J.D. Gu, N. Wan, Degradation of indole by enrichment culture and Pseudomonas aeruginosa Gs isolated from mangrove sediment, Int. Biodeterior. Biodegrad. 56 (2005) 243–248.
268
P.K. Arora, H. Bae / Journal of Hazardous Materials 284 (2015) 261–268
[24] P.K. Arora, H. Bae, Bacterial degradation of chlorophenols and their derivatives, Microb. Cell Fact. 13 (2014) 31. [25] P.K. Arora, A. Srivastava, V.P. Singh, Bacterial degradation of nitrophenols and their derivatives, J. Hazard. Mater. 266 (2014) 42–59. [26] P.K. Arora, C. Sasikala, C.V. Ramana, Degradation of chlorinated nitroaromatic compounds, Appl. Microbiol. Biotechnol. 93 (2012) 2265–2277. [27] S. Perni, L. Hackett, R.J. Goss, M.J. Simmons, T.W. Overton, Optimisation of engineered Escherichia coli biofilms for enzymatic biosynthesis of l-halotryptophans, AMB Express 3 (2013) 66. [28] O.K. Sebek, H. Jager, Divergent pathways on indole metabolism in Chromobacterium violaceum, Nature 196 (1962) 793–795. [29] M. Fujioka, H. Wada, The bacterial oxidation of indole, Biochim. Biophys. Acta 158 (1968) 70–78. [30] P. Katapodis, M. Moukouli, P. Christakopoulos, Biodegradation of indole at high concentration by persolvent fermentation with the thermophilic fungus Sporotrichum thermophile, Inter. Biodeterior. Biodegrad. 60 (2007) 267–272. [31] N. Doukyu, R. Aono, Biodegradation of indole at high concentration by persolvent fermentation with Pseudomonas sp. ST-200, Extremophiles 1 (1997) 100–105.
[32] N. Doukyu, T. Nakano, Y. Okuyama, R. Aono, Isolation of an Acinetobacter sp. ST-550 which produces a high level of indigo in a water-organic solvent two-phase system containing high levels of indole, Appl. Microbiol. Biotechnol. 58 (2002) 543–546. [33] R.W. Eaton, P.J. Chapman, Formation of indigo and related compounds from indolecarboxylic acids by aromatic acid degrading bacteria: chromofenic reactions for cloning genes encoding dioxygenases that act on aromatic acids, J. Bacteriol. 177 (1995) 6983–6988. [34] N. Mermod, S. Harayama, K.N. Timmis, New route to bacterial production of indigo, Biotechnology 4 (1996) 321–324. [35] B. Yin, J.D. Gu, Aerobic degradation of 3-methylindole by Pseudomonas aeruginosa Gs isolated from mangrove sediment, Hum. Ecol. Risk Assess. 12 (2006) 248–258. [36] A.V. Kamath, C.S. Vaidyanathan, New pathway for the biodegradation of indole in Aspergillus niger, Appl. Environ. Microbiol. 56 (1990) 275–280. [37] Y. Chen, X.G. Xie, C.G. Ren, C.C. Dai, Degradation of N-heterocyclic indole by a novel endophytic fungus Phomopsis liquidambari, Bioresour. Technol. 129 (2013) 568–574.
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Biodegradation of 4-chloroindole by Exiguobacterium sp. PMA Pankaj Kumar Arora ∗ , Hanhong Bae ∗ School of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk 712-749, Republic of Korea
h i g h l i g h t s • • • •
Exiguobacterium sp. PMA utilized 4-chloroindole as its sole source of carbon and energy. Strain PMA degraded 4-chloroindole via formation of indole, isatin, anthranilic acid, and salicylic acid. Strain PMA also degraded 4-chloroindole in the soil. This is the first report of the bacterial degradation of 4-chloroindole.
a r t i c l e
i n f o
Article history: Received 11 August 2014 Received in revised form 5 November 2014 Accepted 14 November 2014 Available online 20 November 2014 Keywords: Biodegradation 4-Chloroindole Isatin Anthranilic acid Salicylic acid
a b s t r a c t Exiguobacterium sp. PMA utilized 4-chloroindole as its sole source of carbon and energy. The effect of initial concentrations of substrate on the 4-chloroindole degradation was studied and observed that strain PMA was capable of degrading 4-chloroindole up to concentration of 0.5 mM. The degradation pathway of 4-chloroindole was studied for Exiguobacterium sp. PMA based on metabolites identified by gas chromatography–mass spectrometry. 4-Chloroindole was initially dehalogenated to indole that was further degraded via isatin, anthranilic acid, and salicylic acid. The potential of strain PMA to degrade 4-chloroindole in soil was monitored using soil microcosms, and it was observed that the cells of strain PMA efficiently degraded 4-chloroindole in the soil. The results of microcosm studies show that strain PMA may be used for bioremediation of 4-chloroindole-contaminated sites. This is the first report of the bacterial degradation of 4-chloroindole. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Indole and its derivatives constitute a major group of Nheterocyclic compounds which are widely used for synthesis of pharmaceuticals, cosmetics, pesticides, disinfectants, agrochemicals, and dyestuffs [1–3]. These compounds have been considered as environmental pollutants due to their toxicity and worldwide occurrence. The high concentrations of indole and its derivatives are toxic to cells due to perturbations in membrane potential [4]. Kim et al. [4] showed that indole inhibits adenosine triphosphate production and protein folding in Pseudomonas putida. Indole inhibits bacterial quorum sensing signal transmission by interfering with quorum sensing regulator folding [5]. Indole may cause low pigmentation in plant tissues by inhibition of anthraquinone biosynthesis [6]. Furthermore, indole may produce toxic chlorinated aromatic compounds when dilute aqueous solution of
∗ Corresponding authors. Tel.: +82 538103031. E-mail addresses: [email protected] (P.K. Arora), [email protected] (H. Bae). http://dx.doi.org/10.1016/j.jhazmat.2014.11.021 0304-3894/© 2014 Elsevier B.V. All rights reserved.
chlorine or chlorine dioxide are used for the chemical disinfection of indole-containing wastewater [7]. Metabolism of indole has been extensively studied in microbes under aerobic as well as anaerobic conditions [8–11]. Several aerobic degradation pathways have been studied for metabolism of indole [8,9]. One of them is known as the isatin pathway in which indole was degraded via the formation of indoxyl, 2,3dihydroxyindole, isatin, formylanthranilic acid, anthranilic acid, salicylic acid, and catechol [8]. Another pathway is the gentisate pathway in which indole was degraded via indoxyl, isatin, anthranilic acid, and gentisate [9]. Under anaerobic conditions, indole degraded to methane and carbon dioxide [10]. Bak and Widdel [3] reported that a sulfate-reducing bacterium, Desulfobacterium indolicum utilized indole as an electron donor and its carbon source. Under anaerobic and denitrifying conditions, indole was degraded via oxindole and isatin by the bacterial consortia [11–13]. An indole-degrading methanogenic consortium enriched from sewage sludge degraded indole and 3-methylindole via oxindole and 3-methyloxindole [14]. Hong et al. [15] studied anaerobic bacterial communities in indole-degrading bioreactors under denitrifying and sulfate-reducing conditions. In the denitrifying
262
P.K. Arora, H. Bae / Journal of Hazardous Materials 284 (2015) 261–268
bioreactor, -proteobacteria including bacteria from genera Alicycliphilus, Acaligenes, and Thauera were dominant and responsible for indole degradation whereas in the sulfate-reducing bioreactor, Clostridia, and Actinobacteria were dominant bacteria [15]. Literature studies show that there are several reports of bacterial degradation of indole [8–15]. However, only few reports are available for degradation of chloro derivatives of indole [16], which are highly toxic to living beings. For example, 4-chloroindole causes eye and skin irritation, and may also cause irritation of the lungs and respiratory system. The antimicrobial activity of 4-chloroindole has also been investigated against several grampositive and gram-negative bacteria [17]. Tiedink et al. [18] studied the genotoxic effects of nitrosated 4-chloroindole and 4-chloro-6methoxyindole, and reported that both of the chloroindoles were highly mutagenic to Salmonella typhimurium TA100 as well as potent inducers of sister chromatid exchanges. Despite the fact that 4-chloroindole is a toxic compound, there is no report of its degradation. In this communication, we report the aerobic degradation of 4-chloroindole by a previously isolated bacterium, Exiguobacterium sp. PMA [19]. 2. Material and methods 2.1. Bacterial strain The bacterial strain used in this study was Exiguobacterium sp. PMA isolated previously from a chemically-contaminated site of India by enrichment method which utilized 4-chloro2-nitrophenol, 2-nitrophenol, 4-chloro-2-aminophenol, and 2aminophenol as its sole carbon and energy sources [17]. 2.2. Screening for degradation of indole derivatives Exiguobacterium sp. PMA was screened for its ability to degrade indole derivatives. For screening, Exiguobacterium sp. PMA was streaked on minimal agar plates containing 0.2 mM test compound as its sole carbon and energy source. Minimal agar plates were prepared as described previously [19]. 4-Chloroindole, indole-3-acetic acid, indole-3-methyl, indole-3-carboxylic acid were used as test compounds. The growth of Exiguobacterium sp. PMA on minimal agar plate containing an indole derivative was considered positive result. 2.3. Degradation and growth studies For degradation studies, strain PMA was grown on minimal medium containing different concentrations of 4-chloroindole (0.2–0.6 mM). Minimal medium was prepared by dissolving the following compounds in 100 ml of double distilled water: 0.4 g Na2 HPO4 , 0.2 g KH2 PO4 , 0.08 g NaNO3 , 0.08 g MgSO4 ·7H2 O, 0.1 ml trace element solution [19]. The composition of trace element solution was exactly same as described previously [19–22]. 4Chloroindole was added into medium at before the autoclave [19–22]. The media was autoclaved at 15 lbs for 20 min. The 2% inoculums of overnight grown cells of strain PMA (2 × 108 CFU/ml) were added into the medium and the flasks were incubated at 30 ◦ C under shaking conditions (200 rpm). The samples were collected at regular intervals and centrifuged. 4-Chloroindole was analyzed by spectrophotometrically or by high performance liquid chromatography using a method as described by Yin et al. [23]. For growth studies, strain PMA was grown on minimal media containing 0.5 mM 4-chloroindole, samples (2 ml) were collected at regular intervals (0 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, 48 h, 52 h, and 56 h) and the bacterial growth was measured by taking optical density at 600 nm using a spectrophotometer.
2.4. Detection of ammonia and chloride ions Chloride ions were analyzed using QuantiChromTM Chloride assay kit (DICL-250) from BioAssay Systems, Hayward, CA. Ammonia ions were detected with the ‘Ammonia Assay Kit’ from Sigma–Aldrich (GmbH, Germany) according to the manufacturer’s instructions [19–22].
2.5. Effect of different inoculum sizes on 4-chloroindole degradation Exiguobacterium sp. PMA was grown on 250 ml nutrient broth at 30 ◦ C under shaking condition. When the culture reached the late logarithmic phase of growth, the cells were harvested by centrifugation at 10 000 × g for 20 min at 4 ◦ C, washed with minimal medium [19–22]. The resultant pellets were re-suspended in double distilled water [19–22]. To study the effect of different inoculum sizes on degradation, different quantities of cells suspension were added to 200 ml minimal media containing 0.5 mM 4-chloroindole as its sole source of carbon and energy. At different time intervals, the 4-chloroindole degradation was monitored. The final concentrations of the inoculum used in this study were: 2.0 × 106 , 2 × 107 , and 2 × 108 CFU/ml which were confirmed at the start of the experiment by the plate count method.
2.6. Identification of metabolites Strain PMA was grown on minimal medium containing 0.5 mM 4-chloroindole. Samples were collected at regular intervals (0, 12, 24, 36, and 48 h), centrifuged and extracted with ethyl acetate. The extracted samples were dissolved into 100 l methanol, and analyzed by gas chromatography–mass spectrometry (GC–MS). Agilent gas chromatography system mconnected to model high throughput time-of-flight mass spectrometer (HT-TOFMS) was used with a HP-5 (30 m × 0.320 mm × 0.25 m) column for detection of the metabolites [21]. Separation was performed under the following temperature program on the low-polarity columns (HP5ms) with helium as a carrier gas at 1.5 ml/min; 50 ◦ C held for 1 min, and the temperature was increased at 25◦ /min to 280 ◦ C and held for 5 min [21]. The samples (1 l) were injected in splitless mode. The temperatures of the transfer line and ion source (electron ionisation mode, EI, 70 eV) were 225 ◦ C and 250 ◦ C, respectively [21].
2.7. Enzyme assay for 4-chloroindole dehalogenase The activity for 4-chloroindole dehalogenase was monitored by the detection of the total released chloride ions at room temperature in a reaction mixture containing 100 mM Tris-Acetate buffer (pH 7.5), 0.2 mM NADPH, 200 mg of cell-free lysate, and 500 M of 4-chloroindole. The final volume of the reaction mixture was 5 ml and the reaction mixture without crude extract was used as a control [19]. Samples were collected at regular intervals and assayed for chloride ions. Standard curve was prepared using NaCl as standard to quantify the chloride ions [19]. Samples were also analyzed by GC–MS to detect the product of the reaction [19]. The cell free lysate was prepared as described previously [20]. The overnight grown cells of strain PMA (1%, v/v) were inoculated into 500 ml minimal media containing 20 mM sodium succinate and 0.5 mM 4-chloroindole [20]. The bacterial cells were harvested upon the incubation of 24 h, washed with phosphate buffer (50 mM, pH 7.5) and re-dissolved in the same buffer. The cells were sonicated as described previously [20] and were centrifuged at 4 ◦ C for 15 min. The supernatant was used for enzyme assays.
P.K. Arora, H. Bae / Journal of Hazardous Materials 284 (2015) 261–268
2.8. Enzyme assay for anthranilic acid deaminase The activity for anthranilic acid deaminase was monitored by the detection of the total released ammonium ions at room temperature in a reaction mixture containing 100 mM Tris-Acetate buffer (pH 7.5), 0.2 mM NADPH, 100 mg of cell-free lysate, and 500 M of anthranilic acid. The final volume of the reaction mixture was 2 ml. After 10 min, the reaction mixture was centrifuged and supernatant was divided into two parts. One part (0.5 ml) was used to detect the ammonium ions and the second part (1.5 ml) was extracted with ethyl acetate and analyzed by the GC–MS for identification of the product. 2.9. Microcosm studies Soil used in microcosm studies was collected from outside the campus. Microcosm studies were performed as described previously [19–22]. The soil contained 54% sand, 30% silt, 16.0% clay, 2.4% organic matters, 1.8 ppm phosphorus, 107 ppm potassium, 80 ppm nitrogen, and had a pH 7.2. Microcosms were prepared using 250 ml glass beakers and each backer contained 50 g of soil
263
spiked with 100 ppm 4-chloroindole. Four types of microcosms were prepared (a) test microcosm with sterile soil, (b) test microcosms with non-sterile soil, (c) control microcosm with sterile soil, and (d) control microcosm with non-sterile soil [19–22]. Test microcosms with non-sterile and sterile soils were inoculated with pre-grown and 4-chloroindole induced cells of Exiguobacterium sp. PMA at ∼2 × 108 cells colony-forming units (CFUs) g−1 soil, whereas the control microcosms with sterile and non sterile soils were left non-bioaugmented. The bioaugmentation was performed by thorough mixing of the pre-grown cells of Exiguobacterium sp. PMA with the soil samples [19–22]. All the microcosms were covered with perforated aluminum foil and incubated at 30 ◦ C for 15 days. During the incubation period all the microcosms were sprinkled with distilled water at regular intervals to compensate the loss of water via evaporation [19–22]. Soil samples were removed at regular intervals, and extracted for analysis as described previously [19–22]. The various factors such as inoculum size, pH, temperature, and substrate concentration, affecting 4-chloroindole degradation in microcosm were optimized prior to the study as described previously [19–22]. All experiments were carried out in triplicate.
Fig. 1. Degradation and Growth Studies. (a) Degradation of various concentrations of 4-chloroindole by Exiguobacterium sp. PMA. (b) Growth of Exiguobacterium sp. PMA on the minimal media containing 4-chloroindole as its sole source of carbon and energy. (c) Analysis of chloride and ammonia releases from 4-chloroindole by Exiguobacterium sp. PMA. (d) Degradation of 4-chloroindole by different inoculum sizes.
264
P.K. Arora, H. Bae / Journal of Hazardous Materials 284 (2015) 261–268
Fig. 2. GC–MS profiles of various samples of degradation of 4-chloroindole by Exiguobacterium sp. PMA.
P.K. Arora, H. Bae / Journal of Hazardous Materials 284 (2015) 261–268
3. Results 3.1. Screening for indole degradation Screening results show that Exiguobacterium sp. PMA grow on minimal agar plates containing 0.2 mM 4-chloroindole as its sole source of carbon energy, however strain PMA did not grow on the minimal agar plates containing any other indole derivative including indole-3-aceticacid, indole-3-methyl, indole-3-carboxylic acid. This data suggests that strain PMA utilized only 4-chloroindole as its sole source of carbon and energy. 4-Chloroindole was selected for its degradation study by strain PMA.
265
degradation was observed when the concentration of 4chloroindole was above 0.5 mM. The optimum concentration for degradation of 4-chloroindole was determined as 0.5 mM. All experiments were performed using the optimum concentration. For growth study, strain PMA was grown on minimal media containing 0.5 mM 4-chloroindole. At the initial 12 h, no growth was observed. The exponential growth of strain PMA was observed between 16 and 36 h (Fig. 1(b)). After 36 h, bacterial growth became slow due to utilization of most of the 4-chloroindole (about 0.4 mM). After 48 h, no more growth was observed due to complete consumption of 4-chloroindole. 3.3. Detection of ammonia and chloride ions
3.2. Degradation and growth studies Degradation studies showed that strain PMA degraded 4chloroindole up to concentration of 0.5 mM (Fig. 1(a)). No
4-Chloroindole was degraded via the removal of the chloride and ammonium ions. At the initial 12 h, neither chloride nor ammonium ions were detected. The detection of chloride ions was
Fig. 3. Mass spectra of metabolites and authentic standards.
266
P.K. Arora, H. Bae / Journal of Hazardous Materials 284 (2015) 261–268
initiated from the sample of the 16 h whereas the detection of the ammonium ions was initiated from the sample of 20 h. The stoichiometric amounts of chloride and ammonium ions were detected at the sample of 44 h and 48 h, respectively (Fig. 1c). These data suggest that chloride release occurred before the ammonia release. 3.4. Effect of different inoculum sizes We have also monitored the effects of the various inoculum sizes on the 4-chloroindole degradation by Exiguobacterium sp. PMA. It was observed that inoculum sizes significantly affected the degradation of 4-chloroindole. The larger inoculum sizes degraded 4-chloroindole more rapidly as compared to lower inoculum sizes (Fig. 1(d)). The culture with highest cell densities (2 × 108 ) degraded 4-chloroindole completely within 44 h, whereas the cultures with the cell densities of 2 × 107 and 2 × 106 degraded 4-chloroindole within 48 and 52 h, respectively. 3.5. Identification of metabolites GC–MS analysis of the samples collected from regular intervals confirmed the complete degradation of 4-chloroindole (Fig. 2). In the 4-chloroindole degradation, no metabolite was detected in the samples of 0 h and 12 h. Only 4-chloroindole was detected in the samples of 0 h and 12 h. In the sample of 24 h, metabolites I and II were detected along with 4-chloroindole. The molecular ions of metabolites I and II were observed at m/z 117 and m/z 147, respectively (Fig. 3(a) and (b)). Apart from 4-chloroindole, metabolite III, and metabolite IV were detected in the sample of 36 h with molecular ions at m/z 137 and m/z 138, respectively (Fig. 3(c) and (d)). In the sample of 48 h, neither 4-chloroindole nor any metabolite was detected. The mass spectra of these four metabolites were exactly matched to the authentic standards of indole, isatin, anthranilic acid, and salicylic acid (Fig. 3(e–h). 3.6. 4-Chloroindole dehalogenase activity 4-Chloroindole dehalogenase catalyzed the conversion of 4-chloroindole to indole with removal of chloride ion. The stoichiometric amounts of chloride ions were detected during the enzyme assay and indole was detected as a product. In the control, neither chloride release was observed nor indole was detected. 3.7. Anthranilic acid deaminase activity This enzyme catalyzed the conversion of anthranilic acid to salicylic acid with release of ammonium ions. The stoichiometric amounts of ammonium ions were detected during the enzyme assay and salicylic acid was detected as a product. 3.8. Microcosm studies In order to determine the capability of Exiguobacterium sp. PMA to degrade 4-chloroindole in the soil, we performed microcosm studies using both sterile and non-sterile soil under optimized conditions. The optimized parameters were as follows: inoculum size 2 × 108 CFU g−1 soil, pH 7.2, temperature 30 ◦ C, and 100 ppm 4-chloroindole. In the microcosm with sterile soil, there was no degradation of 4-chloroindole at initial 3 days. The degradation was 50%, 70%, and 90% within 6, 8, and 9 days, respectively, and the complete degradation of 4-chloroindole was observed within 11 days (Fig. 4). In the microcosm with non-sterile soil, there was no degradation of 4-chloroindole at initial 4 days. The degradation was 40%, 73%, and 90% within 9, 11, and 13 days, respectively. The complete degradation of 4-chloroindole was observed within 14
Fig. 4. Microcosm studies. Degradation of 4-chloroindole in test microcosm with sterile soil (a), test microcosm with non-sterile soil (b), control microcosm with sterile soil (c), and control microcosm with non-sterile soil (d).
days. In control microcosm with sterile and non-sterile soil, there was no degradation of 4-chloroindole within 14 days. 4. Discussion Exiguobacterium sp. PMA previously isolated from a chemically contaminated site was screened for its ability to utilize indole derivatives including 4-chloroindole, indole-3-aceticacid, indole3-methyl, indole-3-carboxylic acid as its sole sources of carbon and energy. It was observed that Exiguobacterium sp. PMA utilized 4-chloroindole as its sole source of carbon and energy, and degraded it with release of stoichiometric amounts of chloride and ammonium ions. Exiguobacterium sp. PMA initially reductively dehalogenated 4-chloroindole to indole that was further degraded via isatin, anthranilic acid, and salicylic acid. Previous studies show that Exiguobacterium sp. PMA dehalogenated chlorinated aromatic compounds [19,24–26]. In the degradation pathway of 4-chloro-2-nitrophenol, strain PMA reductively dehalogenated 4-chloro-2-aminophenol to 2-aminophenol [19]. To the best of our knowledge, this is the first report of the complete degradation of 4-chloroindole. However, the catabolism of derivatives of chloroindole has been studied. Jensen et al. [16] studied catabolism of 4-chloroindole-3-acetic acid and 5-chloroindole3-acetic acid by Bradyrhizobium japonicum 61A24 and 110. 4-Chloroindole-3-acetic acid transformed to 4-chlorodioxindole via 4-chlorodioxindole-3-acetic acid, whereas 5-chloroindole-3acetic acid transformed 5-chloroanthranilic acid via 5-chloroisatin [16]. No chloride ions were released during the degradation of 4chloroindole-3-acetic acid and 5-chloroindole-3-acetic acid by B. japonicum [16]. However, the degradation of 4-chloroindole proceeded via release of stoichiometric amounts of chloride ions by Exiguobacterium sp. PMA. Perni et al. [27] studied biotransformation of 5-chloroindole (5-haloindole) into a pharmaceutical intermediate, 5-chlorotryptophan (5-halotryptophan) using Escherichia coli expressing a recombinant tryptophan synthase enzyme. In this study, we have not detected 4-chlorotryptophan as an intermediate product of degradation of 4-chloroindole. This data suggests that tryptophan synthase was not involved in the degradation of 4-chloroindole. In this study, we have detected indole as the first metabolite of degradation pathway of 4-chloroindole. Literature studies show that diverse biochemical mechanisms were involved in indole degradation under aerobic conditions [8,9]. Sakamoto et al. [8] reported that bacterial degradation of indole was occurred by an isatin pathway via the formation of indoxyl (3-hydroxyindole), 2,3dihydroxyindole, isatin, formylanthranilic acid, anthranilic acid, salicylic acid, and catechol. In this study, we have also observed isatin, anthranilic acid, salicylic acid as metabolites. However, we have not detected 2,3-dihydroxindole and formylanthranilic acid. Claus and Kutzner [9] identified another pathway of indole
P.K. Arora, H. Bae / Journal of Hazardous Materials 284 (2015) 261–268
267
5. Conclusion Exiguobacterium sp. PMA utilized 4-chloroindole as its sole source of carbon and energy, and degraded it via a novel pathway with the formation of indole, isatin, anthranilic acid, and salicylic acid as the metabolites. This strain was also able to degrade 4-chloroindole in soil; therefore, this strain may be used for bioremediation of 4-chloroindole-contaminated sites. This is the first report of the biodegradation of 4-chloroindole. Acknowledgment
Fig. 5. A proposed pathway of biodegradation of 4-chloroindole.
This work was carried out with the support of the NextGeneration Biogreen 21 Program (PJ00806302), Rural Development Administration, Republic of Korea. References
degradation in Alcaligenes sp. strain In3, in which indole was degraded via indoxyl, isatin, anthranilic acid, and gentisate. Indoxyl and gentisate were not detected as metabolites of degradation of 4chloroindole in strain PMA, therefore, the gentisate pathway should not be involved in degradation of 4-chloroindole in strain PMA. Another mechanism of indole degradation involves oxidation of indole into indoxyl, which oxidized spontaneously to indigo [28]. Indigo was not detected as degradation product in strain PMA. Fujioka and Wada [29] reported that indole was biodegraded to dihydroxyindole, which was then oxidized to anthranilic acid by a single enzyme step catalyzed by dihydroxyindole oxygenase and to catechol by the Gram-positive coccus. Catechol was not detected as a degradation product in strain PMA. On the basis of the above discussion, we have proposed a metabolic pathway of degradation of 4-chloroindole for strain PMA. 4-Chloroindole was initially dehalogenated to indole that was degraded via isatin, anthranilic acid, and salicylic acid (Fig. 5). We have also investigated the effects of different concentrations of 4-chloroindole on its degradation. It was observed that strain PMA was capable to degrade 4-chloroindole up to 0.5 mM concentrations. There was no degradation of 4-chloroindole when concentration was above 0.5 mM. These data suggest that high concentrations of 4-chloroindole are toxic to bacterial cells. Previously, several researchers have been studied effect of indole concentrations on degradation [30–36]. Katapodis et al. [30] studied indole toxicity on the thermophilic fungus Sporotrichum thermophile and determined the minimal inhibitory concentration (0.5 g L−1 ) of indole for the fungus. The minimal inhibitory concentration of indole for bacteria such as Pseudomonas sp. strain ST-200 [31] and Acinetobacter sp. ST-550 [32] was determined as 0.3 g L−1 . In this study, the minimal inhibitory concentration of 4-chloroindole for Exiguobacterium sp. PMA was 0.6 mM. It has been suggested that all studies for indole degradation should be carried out at low indole concentration to avoid toxic effects in microbes [33–36]. Chen et al. [37] reported that endophytic fungus Phomopsis liquidambari can degrade indole up to 100 g/L. Yin et al. [23] studied effects of the initial concentration of indole (0.3–3 mM) on the indole degradation by Pseudomonas aeruginosa Gs and reported the degradation of indole was the zero-order kinetic equation. We have also monitored the capacity of the cells of strain PMA to degrade 4-chloroindole in the soil microcosms using sterile and non-sterile soil. It was observed that strain PMA degraded 100 ppm 4-chloroindole within 11–14 days. The rate of the degradation of 4-chloroindole was faster in sterile soil than non-sterile soil. This data indicates that the biotic factors present in non-sterile soil were interrupting the degradation of 4-chloroindole in non-sterile soil microcosm. This is the first report of degradation of any indole derivative in soil.
[1] M. Novotny, J.W. Starand, S.L. Smith, D. Wiesler, F.J. Schwende, Compositional studies of coal tar by capillary gas chromatography/mass spectrometry, Fuel 60 (1981) 213–220. [2] W.W. Paudler, M. Cheplen, Nitrogen bases in solvent-refined coal, Fuel 58 (1979) 775–778. [3] F. Bak, F. Widdel, Anaerobic degradation of indolic compounds by sulfate-reducing enrichment cultures, and description of Desulfobacterium indolicum gen. nov., sp. nov, Arch. Microbiol. 146 (1986) 170–176. [4] J. Kim, H. Hong, A. Heo, W. Park, Indole toxicity involves the inhibition of adenosine triphosphate production and protein folding in Pseudomonas putida, FEMS Microbiol. Lett. 343 (2013) 89–99. [5] J. Kim, W. Park, Indole inhibits bacterial quorum sensing signal transmission by interfering with quorum sensing regulator folding, Microbiology 159 (2013) 2616–2625. [6] H. El-Shagi, U. Schulte, M.H. Zenk, Specific inhibition of anthraquinone formation by amino compounds in Morinda cellcultures, Naturwissenschaften 71 (1984) 267. [7] S. Lin, R.M. Carlson, Susceptibility of environmentally important heterocycles to chemical disinfection: reactions with aqueous chlorine dioxide, and chloramines, Environ. Sci. Technol. 18 (1984) 743–748. [8] Y. Sakamoto, M. Uchida, K. Ichibara, The bacterial decomposition of indole. I. Studies on its metabolic pathway by successive adaptation, Med. J. Osaka Univ. 3 (1953) 477–486. [9] G. Claus, H.J. Kutzner, Degradation of Indole by Alcaligenes spec, Syst. Appl. Microbiol. 4 (1983) 169–180. [10] Y.T. Wang, M.T. Suidan, J.T. Pfeffer, Anaerobic biodegradation of indole to methane, Appl. Environ. Microbiol. 48 (1984) 1058–1060. [11] D.F. Berry, E.L. Madsen, J.M. Bollag, Conversion of indole to oxindole under methanogenic conditions, Appl. Environ. Microbiol. 53 (1987) 180–182. [12] E.L. Madsen, A.J. Francis, J.M. Bollag, Environmental factors affecting indole metabolism under anaerobic conditions, Appl. Environ. Microbiol. 54 (1988) 74–78. [13] E.L. Madsen, J.M. Bollag, Pathway of indole metabolism by a denitrifying microbial community, Arch. Microbiol. 151 (1989) 71–76. [14] J.D. Gu, D.F. Berry, Degradation of substituted indoles by an indole-degrading methanogenic consortium, Appl. Environ. Microbiol. 57 (1991) 2622–2627. [15] X. Hong, X. Zhang, B. Liu, Y. Mao, Y. Liu, L. Zhao, Structural differentiation of bacterial communities in indole-degrading bioreactors under denitrifying and sulfate-reducing conditions, Res. Microbiol. 161 (2010) 687–693. [16] J.B. Jensen, H. Egsgaard, H. Van Onckelen, B.U. Jochimsen, Catabolism of indole-3-acetic acid and 4- and 5-chloroindole-3-acetic acid in Bradyrhizobium japonicum, J. Bacteriol. 177 (1995) 5762–5766. ˜ J.M. Peralta-Sánchez, L. Sánchez, S. Ananou, M. [17] M. Martín-Vivaldi, A. Pena, Ruiz-Rodríguez, J.J. Soler, Antimicrobial chemicals in hoopoe preen secretions are produced by symbiotic bacteria, Proc. Biol. Sci. 277 (2010) 123–130. [18] H.G. Tiedink, L.H. de Haan, W.M. Jongen, J.H. Koeman, In-vitro testing and the carcinogenic potential of several nitrosated indole compounds, Cell Biol. Toxicol. 7 (1991) 371–386. [19] P.K. Arora, A. Sharma, R. Mehta, B.D. Shenoy, A. Srivastava, V.P. Singh, Metabolism of 4-chloro-2-nitrophenol in a gram-positive bacterium, Exiguobacterium sp. PMA, Microb. Cell Fact. 11 (2012) 150. [20] P.K. Arora, R.K. Jain, Metabolism of 2-chloro-4-nitrophenol in a gram negative bacterium, Burkholderia sp. RKJ 800, PLoS ONE 7 (2012) e38676. [21] P.K. Arora, A. Srivastava, V.P. Singh, Degradation of 4-chloro-3-nitrophenol via a novel intermediate, 4-chlororesorcinol by Pseudomonas sp. JHN, Sci. Rep. 4 (2014) 4475. [22] P.K. Arora, A. Srivastava, V.P. Singh, Novel degradation pathway of 4-chloro-2-aminophenol via 4-chlorocatechol in Burkholderia sp. RKJ 800, Environ. Sci. Pollut. Res. 21 (2014) 2298–2304. [23] B. Yin, J.D. Gu, N. Wan, Degradation of indole by enrichment culture and Pseudomonas aeruginosa Gs isolated from mangrove sediment, Int. Biodeterior. Biodegrad. 56 (2005) 243–248.
268
P.K. Arora, H. Bae / Journal of Hazardous Materials 284 (2015) 261–268
[24] P.K. Arora, H. Bae, Bacterial degradation of chlorophenols and their derivatives, Microb. Cell Fact. 13 (2014) 31. [25] P.K. Arora, A. Srivastava, V.P. Singh, Bacterial degradation of nitrophenols and their derivatives, J. Hazard. Mater. 266 (2014) 42–59. [26] P.K. Arora, C. Sasikala, C.V. Ramana, Degradation of chlorinated nitroaromatic compounds, Appl. Microbiol. Biotechnol. 93 (2012) 2265–2277. [27] S. Perni, L. Hackett, R.J. Goss, M.J. Simmons, T.W. Overton, Optimisation of engineered Escherichia coli biofilms for enzymatic biosynthesis of l-halotryptophans, AMB Express 3 (2013) 66. [28] O.K. Sebek, H. Jager, Divergent pathways on indole metabolism in Chromobacterium violaceum, Nature 196 (1962) 793–795. [29] M. Fujioka, H. Wada, The bacterial oxidation of indole, Biochim. Biophys. Acta 158 (1968) 70–78. [30] P. Katapodis, M. Moukouli, P. Christakopoulos, Biodegradation of indole at high concentration by persolvent fermentation with the thermophilic fungus Sporotrichum thermophile, Inter. Biodeterior. Biodegrad. 60 (2007) 267–272. [31] N. Doukyu, R. Aono, Biodegradation of indole at high concentration by persolvent fermentation with Pseudomonas sp. ST-200, Extremophiles 1 (1997) 100–105.
[32] N. Doukyu, T. Nakano, Y. Okuyama, R. Aono, Isolation of an Acinetobacter sp. ST-550 which produces a high level of indigo in a water-organic solvent two-phase system containing high levels of indole, Appl. Microbiol. Biotechnol. 58 (2002) 543–546. [33] R.W. Eaton, P.J. Chapman, Formation of indigo and related compounds from indolecarboxylic acids by aromatic acid degrading bacteria: chromofenic reactions for cloning genes encoding dioxygenases that act on aromatic acids, J. Bacteriol. 177 (1995) 6983–6988. [34] N. Mermod, S. Harayama, K.N. Timmis, New route to bacterial production of indigo, Biotechnology 4 (1996) 321–324. [35] B. Yin, J.D. Gu, Aerobic degradation of 3-methylindole by Pseudomonas aeruginosa Gs isolated from mangrove sediment, Hum. Ecol. Risk Assess. 12 (2006) 248–258. [36] A.V. Kamath, C.S. Vaidyanathan, New pathway for the biodegradation of indole in Aspergillus niger, Appl. Environ. Microbiol. 56 (1990) 275–280. [37] Y. Chen, X.G. Xie, C.G. Ren, C.C. Dai, Degradation of N-heterocyclic indole by a novel endophytic fungus Phomopsis liquidambari, Bioresour. Technol. 129 (2013) 568–574.
