GSF National Research Center for Environment and Health, Institute of Ecological Chemistry, Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany1
Technical University Munich, Chair of Ecological Chemistry and Environmental Analytics, D-85350 Freising, Germany2
Author for correspondence: Klaus Rehmann. Tel: +49 89 31872514. Fax: +49 89 31873372. e-mail: rehmann{at}gsf.de
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ABSTRACT |
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Keywords: biodegradation, degradation products, degradation pathway, polycyclic aromatic hydrocarbons, ring cleavage
Abbreviations: COSY, correlated spectroscopy; HMBC, heteronuclear multiple bond correlation; HMQC, heteronuclear multiple quantum correlation; LC, liquid chromatography; MSTFA, N-methyl-N-(trimethylsilyl) trifluoroacetamide; NOESY, nuclear Overhauser and exchange spectroscopy; PAH, polycyclic aromatic hydrocarbon; TMS, trimethylsilyl; UV-Vis, UVvisible
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INTRODUCTION |
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During the last 15 years it has become evident that PAHs with more than three rings, despite their low water solubility, may serve as growth substrates for a number of soil bacteria. However, our knowledge of microbial transformation capabilities for PAHs with more than three rings is still limited (Kanaly & Harayama, 2000 ; Sutherland et al., 1995
). The utilization of fluoranthene as a sole source of carbon and energy by a pure bacterial strain was first described by Weißenfels et al. (1990)
and Mueller et al. (1990)
. Despite the description of several other fluoranthene-utilizing strains (Boldrin et al., 1993
; Bouchez et al., 1995
; Bryniok, 1994
; Dagher et al., 1997
; Ho et al., 2000
; Juhasz et al., 1997
; Kästner et al., 1994
; Kleespies et al., 1996
; Lloyd-Jones & Hunter, 1997
; Mueller et al., 1997
; Sepic et al., 1998
; Thibault et al., 1996
; Walter et al., 1991
; Willumsen et al., 1998
), data on initial metabolites are relatively scarce.
In this study we investigated the fluoranthene-degrading capability of Mycobacterium sp. strain KR20 (Rehmann et al., 1999 ), presenting information on the time course of fluoranthene degradation as well as detailed structural data of several metabolites. We discuss the implication of our results with respect to our current understanding of the bacterial degradation of fluoranthene, focussing on the degradation pathways known to date.
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METHODS |
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Isolation and characterization of the organism.
A fluoranthene-degrading mixed culture was enriched from a PAH-contaminated soil (former gasworks site, Kassel, Germany) using a mineral salts medium (MSM) (Weißenfels et al., 1990 ) with fluoranthene added as the sole source of carbon and energy (see below). A suspected fluoranthene-utilizing member of this bacterial community was identified by its ability to produce clear zones on fluoranthene-coated MSM agar plates (Kiyohara et al., 1982
) and isolated by conventional microbial techniques. It was preliminarily characterized by Gram and acid-fast staining. The mycolic acid pattern of the isolate was determined using the TLC method of Minnikin et al. (1975)
. Utilization of PAHs other than fluoranthene and of Tween 80 was studied in liquid media containing 0·5 mg ml-1 of the respective substrate and inoculated with MSM-washed KR20 cells pre-grown in R2A medium (Reasoner & Geldreich, 1985
). R2A agar plates (20 ml) used in colony-forming units (c.f.u.) plate-counting experiments were incubated at 25 °C.
Cultivation of Mycobacterium sp. strain KR20.
Standard cultivation conditions were as described by Rehmann et al. (1998) in the presence of a nominal concentration of 0·5 mg fluoranthene ml-1. All cultures were incubated on a rotary shaker at 20 °C and 100 r.p.m. The optical density of 10 ml cultures was determined at 578 nm using a UVICAM 5675 spectrophotometer by means of tube-connected cuvettes (diameter 10 mm), which were mounted onto the screw caps of the culture vessels. Starter cultures (10 ml) were inoculated from R2A agar slants. After reaching an OD578 of 0·30·5 these cultures served as an inoculant (10%, v/v) for new 10 ml or 100 ml cultures. Medium-term maintenance of the strain (up to 3 months) was performed on R2A agar slants which, after confluent growth had appeared, were stored at 4 °C, whereas -80 °C deep-frozen suspensions of R2A-agar grown cells in 50% (w/v) glycerol were used for long-term storage.
Chromatographic analysis, preparation and identification of fluoranthene metabolites.
Sample preparation for quantitative HPLC analysis was performed as described by Rehmann et al. (1998) . Ethyl acetate extracts prepared from acidified (pH 1·5, 1 M HCl) 10 ml whole cultures were dried over anhydrous Na2SO4. The solvent was removed under reduced pressure and the residues dissolved in a suitable volume of methanol. Samples were analysed by HPLC (HP1090, Hewlett Packard) applying UV-Vis diode array detection and using a Baker Widebore RP18 column (J. T. Baker Inc.) at 40 °C. Fluoranthene determination was performed isocratically using a methanol/8 mM phosphoric acid 85:15 (v/v) mixture at a flow of 0·8 ml min-1, applying a detection wavelength of 235 nm. Metabolite separation was achieved in a linear 8 mM phosphoric acid/methanol gradient: 0% to 100% methanol within 30 min at a flow of 0·8 ml min-1. Metabolites were detected at 235 nm and 254 nm. UV-Vis spectra were recorded at the peak maxima and were corrected for solvent background. The recovery of fluoranthene under the extraction conditions described was >85% down to a concentration of 5 µg ml-1.
For the purification of metabolites, eight samples of 5- to 8-d-old 100 ml cultures were filtered through glass wool to remove residual fluoranthene crystals. Combined filtrates were extracted three times with 250 ml ethyl acetate to separate excess fluoranthene and non-dissociating metabolites. The filtrates (aqueous phases) were subsequently acidified (pH 1·5, 1 M HCl) and subjected to a second threefold ethyl acetate extraction. Corresponding extracts were pooled, dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure at 35 °C. Extraction residues dissolved in a suitable volume of methanol were fractionated by liquid chromatography (LC) (Merck-Hitachi: pump L6200, auto-sampler AS2000A, UV-Vis detector L4000, Merck) at room temperature on an RP18 column (250x10 mm, Merck Lichroprep, Merck). Selected mixtures (composition depending on the respective metabolite) of methanol and acetic acid (4%, v/v), followed by pure methanol, served to separate the constituents of the acidic extract at a flow of 2 ml min-1. For the purification of the constituents of the neutral extract, acetic acid was replaced by water. Fractions were collected according to the UV-absorbance profile of the eluates at 235 nm or 254 nm. The solvent was removed (see above) and the metabolites were redissolved in methanol. Metabolite I was further purified by TLC on silica gel plates (60F254, 200x200 mm, thickness 0·25 mm, Merck) in an ethyl acetate/methanol 8:2 solvent system. The purity of the metabolites was routinely checked by HPLC and was finally 90%.
1H- and 2D-NMR spectra were recorded with a Bruker DMX 500 spectrometer using Bruker standard software (Bruker Analytik; proton frequency 500·13 MHz) employing 2·0 mm capillaries and an inverse-geometry TXI 2·5 mm probehead (90°: 9·4 µs 1H; 10·0 µs 13C) in methanol-d4, acetone-d6 and chloroform-d1, respectively, at 30 °C (1H/13C: 3·30/49·00, 2·04/29·00 and 7·24/77·00 p.p.m.). 1H,13C-HMBC (heteronuclear multiple bond correlation) spectra to determine the position of carbons without directly bonded protons were recorded in the absolute-value mode F1: 6800 Hz, using coupling constants of 10 Hz (metabolites I, IV), and 5, 7·5, 10 and 15 Hz (metabolite III). 1H,13C-HMQC (heteronuclear multiple quantum correlation) spectra to assign carbons bearing a proton were acquired by use of BIRD (bilinear rotation decoupling) pulses and 13C-GARP (globally optimized alternating-phase rectangular pulses) decoupling [BIRD: 500 ms, GARP: 70 µs, aq: 95320 ms, sw (F2): 5400 Hz, d1: 1·52·5 s, 1J(CH): 145 Hz (metabolite II), 160 Hz (metabolites I, III, IV), number of increments in F1: 60256]. The 13C-NMR spectra were recorded with a 2·5 mm dual probehead (90°: 9·0 µs) with broad-band decoupling and an acquisition time of 1·01·9 s (relaxation delay d1: 3·55 s).
Absolute-value DQ-1H,1H-COSY (double-quantum 1H,1H-correlated spectroscopy) spectra (aq: 200 ms) were acquired on a Bruker AC 400 NMR spectrometer (Bruker Analytik, proton frequency: 400·13 MHz) using an inverse-geometry 5 mm probehead (90°: 8·0 µs). Phase-sensitive TPPI (time-proportional phase increment) 1H,1H-NOESY (nuclear Overhauser and exchange spectroscopy) spectra were recorded on the same instrument with mixing times of 800 ms (metabolites III, V) and 650 ms (metabolite IV). COSY and NOESY spectra served to establish the succession of protons in metabolites II, III, IV and V.
For GC-MS (capillary column gas chromatography-mass spectrometry) analysis, trimethylsilyl (TMS) derivatives of the metabolites were prepared according to Zink & Lorber (1995) . The analysis was performed in the EI mode (70 eV) on an HP 5890 series II chromatograph (Hewlett Packard) equipped with a DB5 capillary column, 0·25 mm inside diameter by 60 m, coating 0·1 µm, coupled to a Finnigan Mat SSQ 7000 quadrupole spectrometer (Finnigan MAT GmbH). Samples were injected into the GC at 90 °C, held isothermally for 1 min, programmed to 270 °C at 15 °C min-1, and held isothermally for another 20 min. A similar method was successfully applied for the analysis of bacterial fluoranthene metabolites by Sepic et al. (1998)
and Sepic & Leskovsek (1999)
.
Direct-infusion LC-MS was performed on a Perkin Elmer Sciex API 300 LC-MS/MS system (Perkin Elmer Überlingen). Samples were dissolved in water/methanol (1:1, v/v) and injected into the mass spectrometer via a syringe pump (Harvard Apparatus) at a flow of 5 µl min-1. Ionization was achieved in the negative mode with the ion spray interface set at -3·5 kV. Nitrogen was used as nebulizer gas (1·5 l min-1) and curtain gas (1·2 l min-1). Lens and quadrupole parameters were set as follows: orifice -30 V, focusing ring -200 V, Q0 10 V. All other parameters were optimized with regard to signal intensity. LC2 Tune 1.2 and Multiview 1.2 software (Perkin Elmer Sciex) were used for data acquisition and evaluation.
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RESULTS |
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Although metabolites were produced from several other PAHs (Table 1) none of these compounds was able to support the growth of strain KR20, i.e. they failed to increase the number of c.f.u. over a 14 d incubation period.
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Time course of fluoranthene degradation
Fluoranthene metabolism of Mycobacterium sp. strain KR20 was monitored over 624 h (Fig. 3) using 10 ml batch cultures which were inoculated to an OD578 of 0·03 with cells pre-grown on fluoranthene for 6 d. Fluoranthene degradation started without an apparent lag and cell counts began to rise after 48 h. Maximum c.f.u. numbers (about 6·2x108 cells ml-1), a 30-fold increase compared to the inoculum, were obtained between day 10 and 14 of the cultivation period, when
60% of the initial fluoranthene content was metabolized. The c.f.u. numbers decreased considerably afterwards. The fluoranthene degradation eventually reached >96%, whereas sterile controls run in parallel showed no loss of fluoranthene after 624 h.
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The situation when non-fluoranthene-grown inocula were used was slightly different (data not shown). As indicated by the turbidity increase of these cultures, cells started to grow only after a lag phase of about 72 h. Depending on the respective experiment a temporary accumulation of cis-2,3-fluoranthene dihydrodiol during the first 120 h up to 192 h of incubation could be observed.
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DISCUSSION |
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The five fluoranthene metabolites identified from cultures of Mycobacterium sp. strain KR20 supported a degradation route (Fig. 3, right column) previously outlined by Rehmann et al. (1999)
. Fluoranthene metabolism by this isolate obviously seems to start with a dioxygenation of the fluoranthene molecule in the 2,3-position, yielding cis-2,3-fluoranthene dihydrodiol (metabolite V). The follow-up product expected, 2,3-dihydroxyfluoranthene, could not, however, be detected. But this also holds for the analogous ortho-dihydroxy PAH derivatives in the degradation pathways of anthracene (Fernley et al., 1964
) and pyrene (Dean-Ross & Cerniglia, 1996
; Heitkamp et al., 1988
; Rehmann et al., 1998
; Walter et al., 1991
), probably due to a strong association of the dihydroxy metabolites with the enzymes involved in their turnover. The ortho ring cleavage of 2,3-dihydroxyfluoranthene leads to the formation of metabolite IV, which subsequently probably loses a C2-unit, hypothetically producing 1-carboxyfluorene-9-one. This compound, in contrast to its successor, metabolite II, could not be detected in cultures of strain KR20.
The consequence of this hypothesis is that strain KR20, Mycobacterium sp. strain PYR 1 (Kelley et al., 1993 ) and Pasteurella sp. IFA (Sepic et al., 1998
) might use homologous entry reactions for fluoranthene degradation, implying that the initial dioxygenation by the latter strains also takes place in the 2,3-position. However, the final aromatic metabolites identified by Kelley et al. (1993)
and Sepic et al. (1998)
suggest a divergence of the metabolic routes in a later stage of degradation, i.e. the breakdown of the fluorene scaffold.
Metabolite II in turn represents the substrate for the second ring-opening reaction, which might proceed by two alternative mechanisms: either by a dehydrogenation of the secondary hydroxy group at C-1 resulting in the formation of a spontaneously hydrolysing 1,3-diketone whose hydrolysis product under acidic conditions (as prevailing during metabolite clean-up) should easily dehydrate to yield the corresponding -lactone (Grifoll et al., 1994
), or by a biological BaeyerVilliger reaction as suggested for the fluoranthene degradation pathway proposed by Weißenfels et al. (1990)
. However, the resulting product, metabolite III, after hydrolysis is most likely metabolized analogously to biphenyl (Higson, 1992
), since the formation of metabolite I (hemimellitic acid), which represented the last detectable aromatic intermediate, requires the opening of the hydroxylated ring of metabolite III in a biphenyl-like manner. The fate of metabolite I is unclear at the moment and warrants further investigation.
This fluoranthene degradation pathway seems also to be operative in Mycobacterium hodleri inasmuch as Kleespies et al. (1996) reported cis-2,3-fluoranthene dihydrodiol as the only identifiable metabolite (without any structural proof) and the UV-Vis spectra of two other fluoranthene metabolites of this isolate were identical to the UV-Vis spectra of Mycobacterium sp. KR20 metabolites III and IV, respectively (H. Kneifel, personal communication).
The degradative route outlined closely corresponds to a fluorene degradation pathway discovered in a Pseudomonas sp. by Grifoll et al. (1994) . Starting with the second dihydrodiol metabolite (metabolite II) of the fluoranthene degradation pathway of strain KR20, characterized intermediates of both pathways are homologues: metabolite II versus cis-1,9a-dihydroxy-1-hydrofluorene-9-one, metabolite III versus 4-hydroxy-benzo[c]chromene-6-one, and metabolite I versus phthalic acid. The fluoranthene descendants differ from their respective fluorene counterparts each by one additional carboxy group in one of the aromatic rings. This homology might point to an evolutionary relation between the two sets of degradative enzymes, but, as already mentioned, strain KR20 was not able to utilize fluorene as a growth substrate. The reason for the latter observation might be based on the requirement of the dioxygenase involved in the formation of the angular dihydrodiol of an activated carbon atom on position 9 as given when attacking fluoren-9-one (Bressler & Fedorak, 2000
). However, this hypothesis still has to be investigated.
The fact that isolate KR20 did not produce any fluoranthene metabolite attributable to the acenaphthenone route of fluoranthene degradation present in the strains investigated by Kelley et al. (1993) , Sepic et al. (1998)
, Weißenfels et al. (1990)
and J. G. Mueller, S. E. Lantz & C. E. Cerniglia (unpublished results) in our opinion might be related to the inability of Mycobacterium sp. strain KR20 to use any other PAH as growth substrate, but this question also requires more detailed investigations.
Time course of fluoranthene degradation
Strain KR20 pre-grown on fluoranthene degraded the same compound in batch cultures without any apparent lag phase, thereby increasing its cell number about 30-fold within 1014 d and excreting limited amounts of the metabolites reported above. As mentioned, the situation with non-fluoranthene-grown inocula was different. The temporary accumulation of cis-2,3-fluoranthene dihydrodiol observed in this case might be caused by an induction event, i.e. the dihydrodiol accumulated only until the degradation pathway enzymes were fully induced. A comparable transient accumulation of primary dihydrodiol metabolites was reported for the degradation of phenanthrene and pyrene by Mycobacterium sp. strain KR2 (Rehmann et al., 1996 , 1998
).
Since under any conditions, none of the metabolites accumulated in a 1:1 stoichiometric ratio to the amount of fluoranthene added, it may be assumed that no real dead-end metabolites (in the sense of being not substrates for any degradative enzyme of the strain) were produced. Rather, the amount of degradation products found might be interpreted as spillover of the cells due to the large amount of growth substrate available or compounds which cannot be reassimilated, e.g. due to their ionic character.
Fluoranthene-degrading bacteria
Mycobacterium sp. strain KR20 represents one of about 50 bacterial fluoranthene degraders described during recent years (Table 7). Currently the number of Gram-negative isolates, especially Sphingomonas sp., matches or even outnumbers the number of Gram-positive fluoranthene-utilizing bacteria (Mueller et al., 1997
; Ho et al., 2000
). This finding is in contrast to the situation with pyrene-degrading bacteria, where nocardioforms (Gram-positives) constitute the majority of species described so far (Ho et al., 2000
; Kästner et al., 1994
; Rehmann et al., 1998
), a fact that was ascribed to the facilitated interaction of hydrophobic PAH with the outer cell surface of Gram-positive bacteria, which is also hydrophobic in nature (Rehmann et al., 1998
). However, it is still open to discussion (Mueller et al., 1997
) to what extent the enrichment techniques used bias the ratio depicted in Table 7
. For example, Bastiaens et al. (2000)
demonstrated that the type of fluoranthene degrader isolated from the same soil sample was clearly dependent on the kind of enrichment technique applied, whereas Mueller et al. (1997)
, in several cases, obtained similar spectra of fluoranthene-degrading organisms despite applying different kinds of enrichment procedures to the same soil sample.
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ACKNOWLEDGEMENTS |
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Received 2 March 2001;
revised 30 May 2001;
accepted 8 June 2001.