INTRODUCTION

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Biodegradation of industrial organic pollutants, especially heterocyclic compounds such as morpholine, is of special environmental interest. Morpholine has great industrial importance and a wide range of applications; it is used as an anticorrosive agent in water boiling systems and as a chemical intermediate (catalyst, solvent, antioxidant, etc.) in the manufacture of rubber additives and in the textile industry. To date, only strains belonging to the genus Mycobacterium have been reported to be able to use morpholine as a sole source of carbon, nitrogen, and energy (1, 2, 4, 7, 8, 14-16, 20-22, 24). Moreover, the high water solubility of this compound and the potential for conversion to the potent mutagen and carcinogen N-nitrosomorpholine make it a xenobiotic compound of special interest from an environmental point of view (9, 11, 26).

The metabolic pathway involved in biodegradation of morpholine has been very difficult to establish, because this chemical does not possess any chromophore and is highly soluble in water, which does not allow easy extraction. Consequently, no tool for direct detection of intermediates or even morpholine has been available. Only indirect strategies have been developed previously; these strategies include chemical oxygen demand, optical density, and NH₃ measurements, growth on intermediates, and in vitro enzymatic assays. Recently, we developed a new approach, in which ¹H-nuclear magnetic resonance (¹H-NMR) spectroscopy is performed directly with culture supernatants without preliminary purification to identify some metabolic intermediates of morpholine and thiomorpholine degradation by two Mycobacterium strains, Mycobacterium aurum MO1 and Mycobacterium sp. strain RP1 (1, 7, 8, 21) (Fig. 1).

View larger version (8K): [in this window] [in a new window]   FIG. 1.   Intermediates of the morpholine and thiomorpholine biodegradation pathway in M. aurum and Mycobacterium sp. strain RP1, as shown by in situ 1H-NMR (1, 7, 8, 21). 1, 2-(2-aminoethoxy)acetic acid; 2, glycolic acid; 3, sulfoxide; 4, thiodiglycolic acid.

The result of the first step in morpholine degradation is either cleavage of the C---N bond, which leads to an amino acid, or oxidation of the sulfur atom to sulfoxide prior to the ring-opening step. By using a specific inhibitor, metyrapone, we showed previously that these reactions are initiated by a cytochrome P450 (8, 21). This finding was the first evidence of the presence of a cytochrome P450 in mycobacteria. Since then, Poupin et al. have shown that a soluble cytochrome P450 is induced during degradation of morpholine in nine bacterial strains isolated from three different environments (22). Also, cloning and characterization of the genes encoding a cytochrome P450 involved in piperidine and pyrrolidine utilization and its regulatory protein have been described recently for Mycobacterium smegmatis mc2155 (23). The complete genome sequence of Mycobacterium tuberculosis contains 20 genes that potentially code for cytochrome P450 proteins (6). All of these findings stress the key role played by cytochrome P450 in mycobacteria, especially in biodegradative processes.

In order to see if the biodegradation pathways for morpholine and thiomorpholine observed with M. aurum MO1 could be generalized to other saturated nitrogen heterocyclic compounds, we performed new experiments in which ¹H-NMR was used. A new technique, direct ionspray mass spectrometry of the incubation supernatants, was also used to confirm the structures of some metabolites during morpholine and thiomorpholine degradation. The results obtained with these two techniques suggested a general pathway for biodegradation of nitrogen heterocyclic compounds by M. aurum MO1.

	MATERIALS AND METHODS

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Chemicals. Morpholine, thiomorpholine, piperidine, dioxane, 2,6-dimethylmorpholine, diglycolic acid, glycolic acid, and glutaric acid were purchased from Aldrich, and tetradeuterated sodium trimethylsilylpropionate (TSPd₄) was purchased from EurisoTop (Saint Aubin, France).

Growth conditions. M. aurum MO1 was isolated by Cech et al. (4) and was grown in 100-ml portions of Trypticase soy broth (bioMérieux, Marcy l'Etoile, France) in 500-ml Erlenmeyer flasks incubated at 30°C with agitation at 200 rpm. The cells were harvested after 48 h of incubation.

Incubation with xenobiotic compounds. Cells were harvested by centrifugation at 9,000 × g for 15 min at 5°C, and the pellet was washed twice with Knapp buffer (containing [per liter of distilled water] 1 g of KH₂PO₄, 1 g of K₂HPO₄, 4 mg of FeCl₃, and 40 mg of MgSO₄ · 7H₂O; pH 6.6) and then resuspended in this buffer (5 g of wet cells in 50 ml of buffer). The cells were incubated with a xenobiotic compound at a concentration of 10 mM as the only source of energy in a 500-ml Erlenmeyer flask at 30°C with agitation (200 rpm). The negative controls consisted of preparations incubated under the same conditions without a substrate or cells. Samples (1 ml) were taken regularly and centrifuged at 12,000 × g for 5 min. The supernatants were isolated and immediately frozen until an NMR analysis was performed.

¹H-NMR spectroscopy. The methods used to prepare NMR samples and the methods used to obtain a spectrum with a Bruker model Avance 300 DSX spectrometer (Larmor frequency, 300.13 MHz) at 21°C with 5-mm diameter tubes have been described previously (7), as has the method used for quantification of the metabolites.

Ionspray mass spectrometry. (i) Preparation of samples. Supernatant (500 µl) was filtered (Analypore; pore size, 0.22 µm; Fischer Scientific), and the pH was measured.

(ii) Ionspray spectra. The ionspray mass spectrometry analysis was performed with a Perkin-Elmer model Sciex API 165 mass spectrometer. The instrument was operated with pressure ionization by utilizing a PE Sciex Turboionspray interface. An Apple Macintosh System 8.1 computer with the Mass Chrom v1.0 application was used for data acquisition and processing.

	RESULTS AND DISCUSSION

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Degradation of piperidine by M. aurum MO1 as determined by ¹H-NMR. The spectra for the supernatants collected after 2 and 6 h of incubation of M. aurum MO1 with 10 mM piperidine are shown in Fig. 2A and B, respectively. The spectra which we obtained were compared with the spectra for reaction mixtures without cells or substrates.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Piperidine degradation by M. aurum MO1. Resting cells (5 g [wet weight] of cells in 50 ml of Knapp buffer [1 g of KH2PO4 per liter, 1 g of K2HPO4 per liter, 4 mg of FeCl3 per liter, 40 mg of MgSO4 · 7H2O per liter; pH 6.6]) were incubated with 10 mM piperidine at 30°C with agitation (200 rpm). Samples (1 ml) were collected every hour for 12 h and from time to time until 30 h; after centrifugation, the supernatants of these samples were analyzed by 1H-NMR spectroscopy at 300.13 MHz. TSPd4 was used as a reference compound for chemical shifts and quantification. (A) 1H-NMR spectrum of a sample collected at 2 h (B) 1H-NMR spectrum of a sample collected at 6 h. (C) Time courses for the concentrations of piperidine () and glutarate (×). Compounds were quantified by integrating the signals in 1H-NMR spectra relative to the area for the reference compound TSPd4.

View larger version (14K): [in this window] [in a new window]   FIG. 3.   General metabolic pathway for degradation of nitrogen heterocyclic compounds by M. aurum MO1.

Identification of morpholine and thiomorpholine metabolites by ionspray spectrometry. Based on data collected in our previous and present studies, a general pathway for degradation of nitrogen heterocyclic compounds by M. aurum MO1 can be suggested (Fig. 3). Cleavage of the C---N bond leads to formation of an amino acid, which undergoes deamination and oxidation. In the case of thiomorpholine and piperidine, a dicarboxylic acid was produced under these conditions (thiodiglycolic acid and glutaric acid, respectively). In the case of morpholine, a singlet resonating at 3.95 ppm was assigned to glycolate (by coincidence after the commercial compound was added to the sample). We also showed that this intermediate was integrated in central metabolism (7). However this raises the following question: Is diglycolate an intermediate of degradation, as observed for the other heterocyclic compounds? Unfortunately, the ¹H chemical shift of diglycolate is the same (difference, less that 5 × 10³ ppm) as that of glycolate whatever the pH (so ¹H-NMR is not suitable for detecting this metabolite). In contrast, the two compounds have different ¹³C chemical shifts (64.19 and 72.37 ppm for the methylene groups of glycolate and diglycolate, respectively), but the concentration of metabolites was too low to detect ¹³C resonance either directly (one-dimensional spectra) or indirectly (two-dimensional HMQC experiments). Consequently, we used a new approach to detect the presence of diglycolic acid. Again, this technique had to be performed directly with the culture medium as no purification of the products was possible; it also had to be rather noninvasive (so the metabolites were not destroyed). Ionspray mass spectrometry was an ideal tool, except that the presence of nonvolatile ions, particularly phosphate ions (concentration in Knapp buffer, 15 mM), had to be avoided.

Incubation of M. aurum MO1 in the absence of inorganic phosphate. Our first attempts to eliminate phosphates, in which we used barium hydroxide precipitation followed by Dowex exchange, were unsuccessful. We decided to incubate Mycobacterium cells directly in distilled water. Under these new conditions, degradation of morpholine (10 mM) by M. aurum MO1 was monitored as previously described (7). Figure 4 shows a comparison of two spectra collected after 12 h of incubation in Knapp buffer (pH 7.31) (Fig. 4A) and after 13 h of incubation in distilled water (pH 7.35) (Fig. 4B). The kinetics of degradation and the observed metabolites were the same as those obtained previously with Knapp buffer. Similar results were obtained for degradation of thiomorpholine (data not shown). Mycobacterium cells are able to grow without inorganic phosphate. This is probably due to the presence of inorganic phosphate polymers present in various bacteria, particularly in members of the genus Mycobacterium (17, 18, 25). These polymers can be used by the cells under P_i-limited conditions.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Morpholine degradation by M. aurum MO1 cells. 1H-NMR spectra of samples were obtained after 12 h of incubation in Knapp buffer (spectrum A) and after 13 h of incubation in distilled water (spectrum B).

Degradation of morpholine. Mycobacterium cells (5 g [wet weight] of cells in 50 ml of distilled water) were incubated as previously described in the presence of 10 mM morpholine. Each sample was analyzed in parallel by ¹H-NMR and ionspray mass spectrometry. For the latter technique, after centrifugation of the samples, the supernatants were filtered through a 0.22-µm-pore-size membrane to eliminate the cell debris before injection and analysis with the mass spectrometer. Ionspray mass spectra were recorded under a positive mode and under a negative mode.

View larger version (34K): [in this window] [in a new window]   FIG. 5.   Ionspray mass spectra recorded under negative (A) and positive (B) ionization with an infusion pump for a sample collected after 13 h of incubation of M. aurum MO1 cells (100 g liter1) in distilled water supplemented with 10 mM morpholine.

Degradation of thiomorpholine. The ionspray mass spectrometry method used to study morpholine degradation was applied to thiomorpholine degradation. Our aims were (i) to validate this new approach as a general technique, (ii) to confirm the structure of the metabolites deduced by ¹H-NMR analyses, and (iii) to eventually identify new metabolites.

View larger version (39K): [in this window] [in a new window]   FIG. 6.   Results of an analysis performed after 14 h of incubation of M. aurum MO1 cells (100 g liter1) in distilled water in the presence of thiomorpholine (10 mM). (A) 1H-NMR spectrum. (B) Mass spectrometry spectra obtained under positive (upper spectrum) and negative (lower spectrum) ionization with an infusion pump. In the positive mode, the OR voltage applied was 20 V and the RNG voltage was 220 V. In the negative mode, the OR voltage was 60 V and the RNG voltage was 330 V.

Conclusions. In this paper we describe a new method in which we performed ionspray mass spectrometry directly with incubation medium without purification. Until now, only liquid chromatography or gas chromatography coupled with mass spectrometry has been used to analyze biological fluids containing natural metabolites or xenobiotic compounds (3, 5, 10, 13). In our study, heterocyclic compounds could not be purified by chromatography, so we had to develop a direct approach. Using this technique, we found a new metabolite, diglycolate, which is not detectable by ¹H-NMR. Identification of this dicarboxylate was essential for the proposal for a general pathway for heterocyclic compound degradation by Mycobacterium strains. Also, we could confirm unambiguously the identities of other morpholine and thiomorpholine metabolites.