Degradation of 2,4,6-trinitrotoluene by P. aeruginosa and characterization of some metabolites.

Degradation of 2,4,6-trinitrotoluene (TNT), a nitroaromatic explosive found in the soil and ground water, was investigated using Pseudomonas aeruginosa in in vitro experiments . Biodegradable abilitiy of this bacteria was performed with 50 and 75 mg L (-1) TNT concentrations in a defined liquid medium for 96 h time period. Treatment of TNT in supernatant samples taken at 0, 6, 12, 24, 48, 72 and 96 h from agitated vessels was followed by reverse-phase high-performance liquid chromatography (HPLC). In cultures supplemented with 50 and 75 mgL (-1) TNT, after 96 h of incubation 46% and 59% reduction were detected respectively. Two metabolites as degradation intermediates with nitrite release into the medium, 2,4-dinitrotoluene (2,4-DNT) and 4-aminodinitrotoluene (4-ADNT), were elucidated by thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS). These findings clearly indicate that Pseudomonas aeruginosa can be used in bioremediation of TNT contaminated sites.

Introduction

Oligonitroaromatic compounds are well-known environmental pollutants which are used in many industrial processes, including the production of pesticides, dyes, polymers, plastics, herbicides, explosives, textile, paper and pharmaceuticals ( Ju and Parales, 2010 ). These pollutants over several decades have leaked to soils, groundwaters, surfacewaters and wastewaters around manufacturing sites as a result of their release in industrial effluents ( Robertson ve Jjemba, 2005 ; Kalderis ; Lotufo ).

2,4,6-trinitrotoluene is a nitroaromatic explosive which has been extensively used for military purposes due to its low melting point, chemical and thermal stability, low sensitivity to impact and friction ( Park ; Robertson ve Jjemba, 2005 ; Kalderis ; Lin ). It was reported that its production reached a maximum during World War II ( Zheng ; Ayoub ; Zhang ). Toxic and mutagenic effects of TNT to various organisms ranging from microorganisms and humans have been described even at low concentration (its lethal dose 0.8–13 g/kg) ( Seth-Smith 2002 ; Dodard ; Ayoub ). TNT as possible human carcinogenic (class C) has been classified by United States Environmental Protection Agency ( Gandia-Herrero ) and also refered to cause diseases such as rashes, toxic hepatitis, dermatitis, cyanosis, sneezing, cough, peripheral neuritis, cataract, muscular pain, aplastic anemia and kidney damage when exposure to it ( Kalderis ; Yang ).

Owing to these impacts, improving the effective technology for remediation of TNT-polluted sites is immediately needed ( Fleischmann ). Until today, several methods for treating of TNT contamined site have been developed, including incineration, compositing, chemical oxidation and adsorption ( Baretto-Rodrigues ; Wang ) Limitations containing the retention of untreated and undefinied compounds, costliness, the requirement of additional nutrients and incomplete degradation of TNT, recent researches have caused to focus on biotreatment processes which are an efficient, cost effective and environmental friendly ( Fu ).

This hazardous chemical which is persistent to degradation in environmental, is sensitivity to microbial attack. Evidences of TNT transformation by aerobic, anaerobic and combined processes are available in the literature ( Robertson and Jjemba, 2005 ; Moshe ; Montgomery ). The present paper aims to investigate the transformation capacity of TNT to non-toxic byproduct by Pseudomonas aeruginosa which was isolated from soil.

Materials and Methods

Isolation of Pseudomonas

For isolation of microorganism, soil sample was collected from the NATO military industry region, Izmir, Turkey. 1 g of soil sample in 99 mL of Glutamate Starch Phenol Red (GSP) medium ( Coral and Karagoz 2005 ) containing the following (g L −1 distilled water) sodium L(+) glutamate (10), soluble starch (3.5), KH 2 PO 4 (2), MgSO 4 .7H 2 O (0.5), phenol red (0.36), was incubated on an orbital shaker (180 rpm as aeration speed) at 30 °C for 72 h. And then 100 μL of growth culture was transferred to an GSP agar plate for preliminary isolation. The plate was incubated at 30 °C for 72 h under aerobically conditions. After incubation the selected colonies which were 2–3 mm in diameter, red and red-zone around theirs on plate, were maintained on an Nutrient agar slant. Isolates tested by Gram staining and biochemical tests were defined using VITEK bacterial identification system.

Evaluation of tolerance

Tolerance of the isolated strains to TNT was analysed by growing in an aqueous medium contained different concentrations of TNT (5, 10, 15, 30, 50, 75 and 100 mg L −1 ). This degradation medium which consisted of (g L −1 ) K 2 HPO 4 , 6.18; KH 2 PO 4 , 1.93; yeast extract, 1; pH adjusted to 7.2 using 1 N NaOH, was prepared by adding from the stock solution of TNT prepared in acetone (1000 mg L −1 ) was filtered through (0.22 μm) sterilized ( Tope ).

Bioremediation

The capability of the selected isolate to remove TNT was investigated by two processes: (i) Preproduction: The isolate was grown in liquid medium including (g L −1 ) meat extract, 10; meat peptone, 10; NaCl, 5; K 2 HPO 4 , 2; KH 2 PO 4 , 1; MgSO 4 .7H 2 O, 0.2; pH 7.2, ( Tope ) at 30 °C with shaking (180 rpm) for 24 h. After incubation, separated cells by centrifugated (9000 × g for 10 min +4 °C) were washed twice with 50 mM potasium phosphate buffer (pH 7.3). A final optical density (OD 600 ) of acquired bacterial suspension was adjusted about 4.8 and 50 mL of this biomass was evaluated as inoculum culture for second process. (ii) Incubation of inoculums in different TNT concentaration (50 and 75 mg L −1 ) containing degradation medium and monitoring of bacterial growth: In shaking flask, the biomass was grown at 30 °C (180 rpm) in the dark for 96 h. Throughout the experiment, the growth was followed by recording the absorbance at 600 nm and counting viable colonies on plate count agar (PCA). Uninoculated flasks were utilized as controls, as well as flasks containing the bacteria without TNT.

Following TNT transformation

Extraction

To identify the major intermediates of TNT by Pseudomonas aeruginosa, 50 mL of bacterial cultures and control groups were sacrificed at each time point (0, 6, 12, 24, 48, 72 and 96 h), centrifuged at 9000 × g for 1 h at 4 °C. The acidified culture supernatants (20 mL) with 1 N HCl 160 μL for per 1 mL sample, were extracted twice with equal volume of ethyl acetate. After acidification, H 2 O was removed from the extracts over anhydrous Na 2 SO 4 . And then the extracts were concentrated by removing excess solvent with rotary evaporation at 35 °C. The reconstituted residues in 1 mL of acetonitrile, were preserved at +4 °C in 1.5 mL teflon coated brownglass vial for HPLC, GC MS and TLC analysis ( Schackmann and Müller 1991 ).

Analysis of TNT

Analysis of TNT transformation was carried out using a Schimadzu, Japan HPLC equipped with a UV detector at 254 nm. Chromatographic separation was performed by injected a sample volume of 20 μL into a C18 reverse phase column at 30 °C. The mobile phase comprised of 21% (v/v) acetonitrile; 35% methanol and 44% deionized water. This phase mixture was isocratically delivered to column at a flow rate of 0.7 mL min −1 . TNT concentration in samples was determined by comprasion a peak height that yielded with reference standarts ( Mercimek ).

Identification of degradation products by TLC and GC MS analyses

Final confirmation of target products made whereby GC/MS and TLC. In GC/MS analyses, TNT intermadiates were identified by comparison of retentions times and mass spectra between samples and standart chemicals. The samples were chromatographed with a gradient temprature programme using TR5-MS column (60 mx0.25 mm, 0.25 μm film thickness) and ionization detector. Initial oven temperature 80 °C for 1 min was ramped to 300 °C with a rise 10 °C min −1 and held for 12 min final hold. The injector temperature was 240 °C. Helium flow rate was adjusted at 1 mL min −1 and the injection volume was 1 μL ( Oh and Kim, 1998 ; Mercimek ).

In TLC analyses, the metabolic products were separated by the ascending method with a solvent system consisting of ethyl acetate/hexan (40:60 v/v) on pre-coated plates of silica gel F254 purchased from Merck ( Nam 1997 ). For identifiaction of spots indicating TNT metabolits, TLC sheets were visualized under UV illumination. Rf values of the spots were determined by comparing those of the authentic standards.

Other analyses

During the degradation, demineralization of TNT that is responsible for nitrite ion release was analyzed by spectrophotometrically ( Nyanhongo ). From 20 mL of the cultures withdrawn, was centrifuged at 9000 x g for 1 h. The acquired supernatant (200 μL) was transfered to test tubes including 50 μL sulphanilamide solution which was prepared by dissolving 5 g sulphanilamide in 26 mL HCl and adjusting total volume of solution to 500 mL in distilled water. After incubation at room temperature for 5 min, 50 μL N -(1-naphthly) ethylenediamine dihydrochloride solution (0.5 g N -(1-naphthyl) ethylenediamine dihydrochloride in 500 mL deoinized water) as the coloring reagent was added to reaction mixture and incubated for 20 min at room temperature. Total volume of reaction mixture was adjusted at 1 mL with deoinized water and nitrite concentration was determined the OD at 540 nm. The oxidation of released nitrite to nitrate was followed by mixing 5 mL of supernatant with 1 mL 1 N HCl and then examining at 220 nm ( Al-Absi 2008 ).

Results

Isolation and identification of the Pseudomonas sp

In NATO soil sample, bacterial population that is capability of growth in the presence of TNT, was screened on GSP agar. This medium contains glutamate and strach as its sole nutrients. While many microorganisms can not metabolize these compounds, Aeromonas was able to hydrolyze with acid production the starch, causing phenol red to change yellow but not by Pseudomonas . These bacteria formed a red precipitation zone surronding colonies, 2–3 mm in diameter, on GSP agar plates. All of the selected strains were revealed belonging to the Pseudomonas genus, with possible similarities such as Gram negative, bacilli-shaped, oxidase and citrate positive. It was finally defined that isolated ten strains were Pseudomonas sp. by VITEK bacterial identification system.

Selection of Pseudomonas sp that used in TNT transformation

It was observed that only one of the ten isolates was capable to grow in an aqueous medium contained 50 and 75 mg L −1 TNT concentrations. So, this isolate, Pseudomonas aeruginosa , was selected to be used for further degradation studies.

Growth of P. aeuroginosa and biodegradation of TNT as sole carbon source

P. aeuroginosa was inoculated in liquid mediums contaning 50 and 75 mg L −1 TNT, to evaluate its capability to utilization of TNT as carbon source by aerobic pathway. It was observed that color of culture mediums that were treated by P. aeuroginosa , turned to red color from yellow during degradation. Within 4 days of culturing, degradation efficiency for different concentrations (50 and 75 mg L −1 ) of TNT, was recorded as 46% and 59% respectively ( Figure 1 ).

Figure 1

Degradation of TNT by cell suspensions of P. aeruginosa .

The role of abiotic processes on biotransformation of TNT, was also examined in the uninoculated controls. In these control cultures, at initial concentrations of TNT were determined a decline with time by HPLC ( Figure 1 ). The certain microbial degradation of TNT by P. aeuroginosa was calculated by expulsion values of chemical hydrolysis.

During incubation, correlation between decreasing TNT concentrations and increasing density of cells in cultures was followed to prove that P. aeuroginosa used TNT as nutritional source. Time-dependent changes in microbial growth (turbidity and colony-froming units (CFU)) were reported in Figure 2 . Bacterial growth based on the initial inoculum that was about 102.5 × 10 5 cfu mL −1 , arose following degradation and reached maximum levels at 12 h (178 × 10 5 cfu mL −1 ) and 24 h (208.5 × 10 5 cfu mL −1 ) of incubation in liquid cultures including respectively 50 and 75 mg L −1 TNT ( Figure 2a ). From that period to by the end of incubation, it was determined that bacterial growth in both concentrations decreased gradually ( Figure 2a–b ). But biotransformation was shown to proceed with the residual viable cells. In control cultures without TNT, maximum growth was recorded as 131 × 10 5 cfu mL −1 at 48 h ( Figure 2a ).

Figure 2

Changes in bacterial growth following incubation period; (a) viable bacterial counts (cfu mL −1 ) and (b) turbidity of culture mediums with and without TNT inoculated P. aeruginosa .

Estimation of nitrite and nitrate from TNT

During degradation time, nitrite analysis was continued to determine the accumulation of nitrite from TNT, as a positive evidence of demineralization and degradation. The findings of nitrite analyses were graphically demonstrated in Figure 3 . In bacterial cultures containing 50 mg L −1 TNT, the increase in measurable nitrite concentrations after incubating for 6 h, continued up to 48 h, reached a maximum of 0.28 mg L −1 , at 72 h. This value showed the negligible decrease (0.01 mg L −1 ) and thereafter it remained constant level at 0.27 mg L −1 . During the biotransformation, the nitrite amounts in biotic cultures with 75 mg L −1 TNT showed a gradual rise and the highest nitrite concentration released from TNT was defined 0.47 mg L −1 at 72 h.

Figure 3

Release of nitrite during TNT removal by P. aeruginosa .

In uninoculated media including TNT, nitrite accumulation was observed depending upon the chemical degradation. At the end of 96 h when was arrived maximum biotransformation, amount of nitrite that measured for both of abiotic cultures was 0.15 mg L −1 . A significant result with regard to forming nitrate was not achieved. The results of nitrate assays are presented in Figure 4 .

Figure 4

Detection of nitrate by forming via nitrite reduction.

The conversion of TNT to reduction products

In all of the experiments conducted, removal of TNT was associated to an accumulation the possible degradation products. These products in biotic and abiotic cultures supernatans were decomposed by GC-MS and preparative TLC plates. The assignment of TNT and its metabolits were based on the match with standart mass spectrum in the GC-MS library database. The retention times of TNT, 2,4-DNT and 4-ADNT standarts based on mass/charge were detected as 17.54, 15.65 and 20.14 min, respectively ( Figure 5a ). From degraded samples of TNT, retention times of peaks at 20.13 and 20.69 min were identified as 4-ADNT by comprasion of standarts ( Figure 5b–c ). The metabolite containing amino group (aminodinitrotoluenes) could only be determined in the biotic culture supernatants by GC-MS.

Figure 5

GC-MS spectra of peaks belonging to analytical standarts (a) and samples obtained from cultures contaning respectively 50 (b) and 75 (c) mg L −1 TNT.

TLC analyses were performed to monitor the removal of TNT and formation of its intermediates. TNT, 2,4-DNT and 4-ADNT standarts migrated with Rf values 0.83, 0.67 and 0.5 ( Figure 6a ). At the end of degradation, it was revealed the presence two spots on TLC plates, corresponding to the spots belonging to the standarts ( Figure 6b–c ). These spots at Rf values 0.8 and 0.63 were accepted as TNT and 2,4-DNT. 2,4-DNT couldn’t be encountered in the un-inoculated media.

Figure 6

TLC monitoring of intermediates formed during transformation by P. aeruginosa. (a) represents the reference standarts on TLC plates. (b) and (c) present residual TNT and its metabolits that accumulated in biotic and abiotic cultures.

Discussion

In our experience, it was researched the possibility that TNT degradation by Pseudomonas aeruginosa strain, carried out in liquid medium. In the presence of P. aeruginosa , maximum specific removal rates for cultures including 50 and 75 mg L −1 TNT were calculated as 46% and 59% within 96 h, respectively. These differences among experiments in the degradation efficiency were mainly related to differences in initial TNT concentrations. Similarly, some workers were published that P. aeruginosa and other Pseudomonas sp were ability to grow in the presence of TNT and transform it ( Alvarez ; Tope and Jamil, 2000 ; Park ; Zaripov ; Maeda ; Kubota ; Nyanhongo ). Accordingly our results, chemical hydrolysis of TNT based on agitating in the present studies, was also reported ( Oh ; Claus ).

Generally, we observed that TNT metabolism was correlated with the increasing in microbial growth of P. aeruginosa accompanied by decrasing of TNT amounts in the cultures.

In time-dependent TNT biotransformation, bacteria-mediated reduction was detected to elevate with a high cell density of P. aeruginosa in the log phase. In control cultures without TNT, low levels of bacterial population indicated that P. aeruginosa can be used TNT as nutrient in building their cells. Also, in this present work was documented that the rate of TNT removal by P. aeruginosa declined as it entered stationary phase, which may be due to nutrient depletion and a reduction in the microbial population based on toxicity of the accumulating transformation products. These datas are in agreement with those acquired by prior researches on TNT-biodegrading ability of P. aeruginosa ( Tope and Jamil, 2000 ; Oh ; Maeda ). Which is contrast to our investigations, in a number of earlier experiments was noted that efficient of TNT removal rised with decreasing in the bacterial growth ( Tope ).

The findings with regard to nitrite accumulation corresponded to distribution of TNT from culture medium throughout incubation. A rise in the level of recorded nitrite ions was substantially revealed by the decrease in TNT concentrations. In the light of the above findings, it was clearly a evidence that liberation nitrite by utilization TNT as a growth substrate under enrichment cultures with yeast extract. The occurence of nitrite ions manifests a release of nitrogen from the aromatic ring during degradation reaction. Besides, nitrite ions as single transformation product in abiotic cultures were identified. So, estimation of nitrite in cultures without bacteria, was verified chemical degradation correlated with decrease of TNT concentrations. Previous researchers denoted supportively our results on nitrite release by reduction during microbial processes ( Oh ; Litake ; Maeda ; Mercimek ).

The further reduction of the released nitrite to nitrate was associated with decreasing of nitrite in all cultures during incubation time. But a distinct formation of nitrate as a result of direct oxidation of nitrite to nitrate by bacteria and chemical reactions, was not encountered. In contast to our datas, some searchers indicated the findings of nitrate in the cultures of Clavibacterium , Sphingomonas and their mixture during destruction of TNT ( Rahal and Moussa, 2011 ).

During removal process of TNT by P. aeruginosa , all of the cultures except abiotic were appeared color changes. It was observed that the color of the culture mediums returned to a pinksih red to brown around 10–12 h of incubation, gradually darkened and changed a deep red color for a prolonged period. In previous reports of TNT biotransformation, the hydride-Meisenheimer complex of TNT formed by aerobic microbial degradation that occured via the elimination of nitro groups, were detected to be responsible for these color changes ( Kim and Song, 2000 ; Pak ; Litake ; Nyanhongo ).

Intermadiates of TNT from red-coloured supernatant were analyzed to clarify the mechanisms by which metabolic pathway of TNT removal. The incorporated bacteria converted TNT to 2,4-DNT and 4-ADNT as well as nitrite, was determined by TLC and GC-MS analyses. The presence of two degradation products in P. aeruginosa cultures with nitrite, and in abiotic controls demonstrated that absence of 2,4-DNT and 4-ADNT, indicated that transformation TNT was biologically mediated by P. aeruginosa . The presence of impurities in originating from TNT was also shown by this analytical methods. This results supported to HPLC datas that indicated TNT was not completely used by P. aeruginosa . Formation of 2,4-DNT and 4-ADNT from TNT proves that reduction by two different biochemical routes: 1. the elimination nitro groups to nitrite/nitrate; 2. the reduction of nitro groups to amines.

As earlier mentioned, TNT is a symmetrical nitroaromatic compound with nitro groups present on a carbon ring at the 2, 4 and 6 positions and a methyl group localate in the 1 position ( Gallagher 2010 ). These arrangement that procured stability of aromatic ring and the electron-withdrawing properties of nitro groups surronding its aromatic ring created steric hindrance ( Claus ; Kubota ). Due to the presence of multiple nitro groups on the aromatic ring, TNT has electron deficient in π-orbitals. The potentiality of electrophilic attack that leading to ring fission declines as the number of nitro groups on aromatic ring increases ( Han 2008 ). So, microbial activites on TNT be limited to elimination of nitro groups from aromatic ring and metabolism of the remaining carbon skeleton ( Spain 1995 ).

Aerobic reduction of TNT was summarized in three categories: 1. ring oxygenation that leads to accumulation of nitrite ions, 2. nucleophilic attack by the addition of a hidride ion to form a hydride-Meisenheimer complex that reduce the aromatic ring to di-nitro derivate ( Kulkarni and Chaudhari, 2007 ) 3. partial reduction of the nitro group to a hydroxylamine derivate ( Spain 1995 ). In case of anaerobic processes, TNT was degraded either by respiration of the NO 2− groups or by removal of the NO 2− groups to be used as a terminal electron acceptor or as a nitrogen source ( Gallagher 2010 ). TNT was catalyzed to 2,4,6-triaminotoluene (TAT) as final product in anaerobic conditions ( Hawari ; Esteve-Núñez ).

Upon review of the above information, in this study appearance of red color during degradation and the accumulation of 2,4-DNT, 4-ADNT and nitrite as biotransformation products, demonstrated that P. aeruginosa can reduce the aromatic ring to dinitro-compounds by forming hydride-Meisenheimer complex, which subsequently caused the release of nitrite. And it was described that the further reduction of the nitro groups to amino groups occured by using the nitro-group as electron acceptor in the second biochemical reeaction.