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Keywords:

  • Polycyclic aromatic hydrocarbons;
  • Chrysene;
  • Sphingomonas;
  • Biofilm;
  • Biphasic culture medium;
  • Non-aqueous-phase liquid;
  • [14C]Chrysene mineralization

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

A bacterial strain able to grow in pure culture with chrysene as sole added carbon and energy source was isolated from PAH-contaminated soil after successive enrichment cultures in a biphasic growth medium. Initially, growth occurred in the form of a biofilm at the interface between the aqueous and non-aqueous liquid phases. However, after a certain time, a transition occurred in the enrichment cultures, with growth occurring in suspension and a concomitant increase in the rate of chrysene degradation. The strain responsible for chrysene degradation in these cultures, named Sphingomonas sp. CHY-1, was identified by 16S rDNA sequencing as a novel sphingomonad, the closest relative in the databases being Sphingomonas xenophaga BN6T (96% sequence identity). Both these strains clustered with members of the genera Sphingobium and Rhizomonas, but could not be categorically assigned to either genus. Sphingomonas sp. CHY-1 was characterized in terms of its growth on chrysene and other PAH, and the kinetics of chrysene degradation and 14C-chrysene mineralization were measured. At an initial chrysene concentration of 0.5 g l−1 silicone oil, and an organic/aqueous phase ratio of 1:4, chrysene was 50% degraded after 5 days incubation and 97.5% degraded after 35 days. The protein content of cultures reached a maximum value of 11.5 μg ml−1 aqueous phase, corresponding to 92 mg g−1 chrysene. 14C-labelled chrysene was 50% mineralized after 6–8 weeks incubation, 10.7% of the radioactivity was incorporated into cell biomass and 8.4% was found in the aqueous culture supernatant. Sphingomonas sp. CHY-1 also grew on naphthalene, phenanthrene and anthracene, and naphthalene was the preferred substrate, with a doubling time of 6.9 h.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants, many of which are mutagenic and/or carcinogenic or cytotoxic [1–3]. Although low-molecular-weight PAHs composed of 2 or 3 aromatic rings can be biodegraded under favourable conditions, PAHs of 4 rings or more are recalcitrant to bioremediation and may persist for long periods in the environment. Further knowledge of the microorganisms degrading high-molecular-weight PAHs and the metabolic pathways involved may therefore lead to improved strategies for bioremediation.

A large number of bacterial strains have been isolated which are able to degrade the 4-ring PAH, pyrene, and many of these are able to grow with this PAH as sole added carbon source [3,4]. Many intermediates in the pathways of pyrene degradation have been identified and hypothetical metabolic pathways have been proposed. Several intermediates in the biodegradation of benz(a)anthracene, another 4-ring PAH, have also been identified [5,6]. On the other hand, very little is known about the biodegradation of chrysene, a carcinogenic, 4-ring PAH, which was actually the first PAH compound to be detected in a non-industrial environment [7]. The isolation of bacterial strains able to degrade chrysene, either by co-metabolism [8,9] or by use as growth substrate [10–13] has been briefly reported, but no biochemical studies of chrysene degradation were carried out. However, studies with a mutant strain (B8/36) of Sphingobium yanoikuyae B1, which contains a broad-substrate-specificity biphenyl dioxygenase system, showed that chrysene was hydroxylated in the 3,4 positions to yield chrysene cis-3,4-dihydrodiol, which may correspond to the initial intermediate in the chrysene biodegradation pathway [14].

In order to carry out biochemical studies of chrysene degradation, we wished to isolate a bacterial strain that was able to grow in pure culture with chrysene as sole added carbon source, using a biphasic growth medium in which the PAH is dissolved in a non-aqueous-phase liquid (NAPL) such as silicone oil. The use of a NAPL allows an increase in the amount of substrate that can be provided in the culture for bacterial growth [15]. Here, we describe the isolation of such a strain, its taxonomic affiliation and its growth characteristics on chrysene and other aromatic and non-aromatic carbon substrates.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Isolation and growth of strain CHY-1

Soil samples heavily contaminated with PAHs (>4000 ppm, including 300 ppm chrysene) from a coal gasification site in the Rhône-Alpes region, France, were provided by R. Gourdon (INSA, Lyon, France). An aliquot of soil (5 g) was placed in a sterile 250-ml Erlenmeyer flask pre-treated with Aquasil (Pierce, PERBIO, France) to minimize adherence of chrysene to the inside of the flask, and mixed with 50 ml of a biphasic culture medium composed of 40 ml mineral salts medium (MSM; [10]) and 10 ml silicone oil (Rhodorsil 4V20; Sodipro, France) containing 0.5 g l−1 chrysene, which was dissolved by heating. The culture was incubated in a rotary shaker (Novotron TR-225, Infors) at 200 rpm and 25 °C. At weekly intervals, samples were withdrawn from the oil phase for measurement of chrysene concentration, and from the aqueous phase for bacterial growth measurements. After two months, when no further decrease in the concentration of chrysene in the oil phase was observed, the contents of the flask were allowed to settle and 4 ml of the aqueous phase was used to inoculate a culture flask prepared as above, but without soil. For isolation of bacterial strains, aqueous phase samples were diluted and plated on 20-fold diluted PTYG (PTYG20) agar medium [16], which was incubated at 30 °C for 10 days. Individual colonies were then picked and purified three times by serial streaking on PTYG20 agar plates. Purified strains were then tested for chrysene degradation, either by streaking on MSM agar plates, followed by UV observation, or by inoculation into biphasic culture medium containing chrysene.

2.2Measurement of chrysene concentration and detection of metabolites

The concentration of chrysene dissolved in silicone oil was measured by centrifuging samples at 12,000g for 5 min in a microcentrifuge and extracting 100 μl of the upper phase with 1 ml of acetonitrile. The absorbance of the diluted extract was then scanned between 210 and 350 nm, using an HP8452A diode array spectrophotometer, and the concentration of chrysene determined from the height of the characteristic absorbance peak at 268 nm, using an experimentally determined extinction coefficient (ε268) of 126 mM−1 cm−1.

The total amount of chrysene in culture flasks was assayed as follows. The contents of the flask was poured into a siliconized, sterile Corex glass centrifuge tube and centrifuged at 10,000g and 15 °C for 10 min. After removal of the majority of the aqueous and oil phases, the residual, interfacial material (2–3 ml) was extracted twice with 10 ml hexane, and the concentration of chrysene in each extract was measured spectrophotometrically. The empty culture flasks were then extracted three times each with 10 ml hexane, then once with methanol, and the chrysene concentrations determined as before. Finally, the bacterial cell pellets obtained after centrifugation of the culture medium were dried overnight in a 60 °C oven, then extracted with 1 ml hexane prior to cell dry weight and protein measurements.

GC-MS analysis of chrysene dissolved in acetonitrile was carried out as described previously [4]. Chrysene displayed a retention time of 22.4 min under the chromatographic conditions used. For analysis of polar metabolites, aqueous phase samples were acidified to pH 2 with 1 M H2SO4 and extracted three times with an equal volume of ethyl acetate. After drying with anhydrous Na2SO4 the extracts were evaporated under vacuum and the residue dissolved in a small volume of methanol. Aliquots were then dried, dissolved in 50 μl acetonitrile and derivatized with 100 μl Bis(trimethylsilyl)trifluoroacetamide (BSTFA)/Trimethylchlorosilane (TCMS) (99:1) (Supelco), prior to GC-MS analysis as above.

2.316S rDNA analysis

DNA was extracted from cells grown on PTYG20 agar plates using the EasyDNA Kit (Invitrogen). Amplification, sequencing and sequence analysis of 16S rDNA was carried out as described previously [17,18]. Cloning and RFLP analysis of PCR-amplified 16S rDNA were performed as described by Mouné et al. [18]. The partial 16S rDNA sequence of Sphingomonas sp. CHY-1 (1300 bp) has been deposited in the EMBL/GenBank/DDJB databases under the Accession No. AJ715526.

2.414C-Chrysene mineralization experiments

[5,6,11,12-14C]Chrysene (47.4 mCi mmol−1; 370 μCi ml−1) was purchased from ChemSyn Laboratories, Lenexa, KA, USA, and diluted to 0.74 μCi ml−1 in silicone oil containing 0.5 g l−1 of unlabelled chrysene. Cultures were grown as above, except that 20 ml of biphasic growth medium (4:1 ratio aqueous phase/non-aqueous liquid phase) was used, in a 125-ml Erlenmeyer flask sealed with a silicone rubber stopper (Saint Gobain Verneret, Charny, France), and inoculated either with cells grown on PTYG20 agar or with a preculture grown on unlabelled chrysene. At various times the culture flask was flushed with sterile air into a 1 N NaOH trap and the amount of 14CO2 in the gas phase was determined by scintillation counting. The total amount of non-mineralized radioactivity was determined by centrifugation and hexane extraction of the various culture fractions, as described above.

2.5Bacterial growth measurements

Bacterial growth was followed by measurement of the absorbance of cultures at 550 nm. In biphasic cultures direct measurement of the A550 was precluded by the presence in the aqueous phase of emulsified oil droplets. However, these could be removed by centrifugation and resuspension of the bacterial pellet in an equivalent volume of MSM medium. For measurement of cell dry weights bacterial pellets were washed once in MSM medium, by centrifugation in pre-weighed Eppendorf tubes and dried overnight in a 60 °C oven. The dried pellets were then extracted with 1 ml hexane to remove any residual silicone oil before weighing. For protein determinations cell pellets were dried as above, dissolved in 0.4% sodium deoxycholate/1 N NaOH and heated for 10 min at 100 °C in a boiling water bath. After cooling to room temperature the solution was neutralized with 1 N HCl and the protein content was assayed with bicinchoninic acid (BCA) reagent (Pierce, PERBIO, France) using bovine serum albumin as standard.

2.6Substrate utilization and growth tests

The utilization of individual PAHs as growth substrates was tested in biphasic cultures as decribed above, with PAHs being dissolved in silicone oil at the following concentrations: naphthalene, phenanthrene, fluoranthene, fluorene, acenaphthene (5 g l−1); anthracene, pyrene (1 g l−1); benz(a)anthracene, benzo(a)pyrene (0.5 g l−1). In experiments to determine PAH degradation, PAHs were provided at 50 μg ml−1 in silicone oil and degradation was followed spectrophotometrically. For growth experiments with mixtures of chrysene (0.5 g l−1), and either naphthalene or phenanthrene, the latter substrates were used at a concentration of 1 g l−1, and their degradation, which preceded that of chrysene, was followed by measuring the absorbance of acetonitrile extracts at 220 and 252 nm, respectively.

For growth tests on other carbon sources MSM medium was supplemented with aromatic acids (5 mM final concentration), glucose (10 mM), succinate (10 mM) or acetate (30 mM) and growth was followed by measurement of the A550. Assimilation of other sugars and organic acids as well as nitrate reduction and other enzymatic tests were carried out using the API 20 NE identification system (bioMérieux S.A., Lyon, France).

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

3.1Degradation of chrysene in biphasic enrichment cultures and formation of a biofilm

Biphasic enrichment cultures on chrysene were established as described in Section 2 and subcultured every 8–9 weeks over a period of two years. After the first two subcultures a stable mixed culture was established, as determined by serial dilution and plating. At least six visually distinct colony types were observed on PTYG20 plates, which remained in constant proportions throughout the experiment. However, none of the individual colony types was able to grow on or degrade chrysene in pure culture.

In the mixed cultures chrysene was degraded about 70% over a period of two months (Fig. 1, curve A). The solubility of chrysene in hot silicone oil was about 0.5 g l−1, but decreased to 50 μg ml−1 after addition to the growth medium, resulting in an opaque layer of precipitated chrysene at the oil–water interface. Monitoring of the concentration of chrysene in the oil phase was therefore not a good indicator of the total amount of chrysene remaining in the culture and the kinetics of chrysene degradation were followed by sacrificing replicate cultures at various time points. Separation and extraction of different culture fractions showed that, in 3 week-old cultures, significant amounts of chrysene were found in the oil phase and at the interface, whereas in 6 or 9 week-old cultures the amount of chrysene in these fractions was very low and most of the residual chrysene was present in a gelatinous slurry which adhered to the inner surfaces of the flask after pouring out the growth medium. The use of siliconized flasks decreased the prevalence of this form and led to an increase in the overall rate of chrysene degradation.

image

Figure 1. Kinetics of chrysene degradation in enrichment cultures before (A) and after (B) the evolutionary transition. Cultures were inoculated with 10% (v/v) of a previous enrichment culture into multiple 250-ml Erlenmeyer flasks containing 40 ml MSM medium supplemented with 10 ml silicone oil containing 0.5 g l−1 chrysene. At the time intervals shown, triplicate cultures were sacrificed and the total amount of residual chrysene in each flask was determined as described in Section 2. The mean ± SE of the three values is plotted and expressed as a percentage of the initial amount of chrysene present in the flask, i.e. 5 mg. The insert curve shows the initial rate of chrysene degradation in pure cultures of Sphingomonas sp. strain CHY-1 grown and incubated under the same conditions.

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When the growth medium from 6 week-old or 9-week old cultures was centrifuged in a siliconized glass tube to separate the two phases, then an opaque, white, membrane-like film was observed at the interface. After careful removal of the upper oil phase and the lower aqueous phase, the intact biofilm could be removed with a heat-sealed Pasteur pipette, rinsed in sterile medium and transferred to a clean tube. Upon extraction with hexane the film became translucid and the hexane extract showed the characteristic UV absorption spectrum of chrysene. Biofilms from 50-ml cultures contained 423 ± 19 μg chrysene (assayed spectrophotometrically) and their dry weight was 1.17 ± 0.27 mg after hexane extraction. Analysis of the extracted biofilms showed them to contain 175 ± 25 μg protein mg−1 dry weight, and 400 ± 50 ng DNA was extracted from each intact biofilm. When 3 week-old cultures were centrifuged, gelatinous material was observed at the interface, but no intact biofilm, suggesting that the biofilm was not fully formed at this time.

After 18 months of repetitive subculturing an abrupt transition in the enrichment cultures was observed, resulting in the loss of biofilm formation and a marked increase in both the rate and extent of chrysene degradation (Fig. 1, curve B). Chrysene was 70% degraded after only 7 days incubation and 97.5% degraded after 35 days. After 8–9 weeks the amount of residual chrysene in the cultures was <1% of the initial value, as determined by spectrophotometric analysis and GC-MS. Compared to previous cultures, a large increase in the number of slow-growing (8–10 days), whitish to pale-yellow colonies was observed after plating on PTYG20. Extensive purification of these colonies yielded a strain, designated CHY-1, that was able to grow in pure culture and degraded chrysene at similar rates to the mixed enrichment culture (Fig. 1, insert). Subculturing of the enrichment cultures was continued for a further 6 months, but no further increase in the rate of chrysene degradation was observed.

3.2Phylogeny of strain CHY-1

The partial sequence of 16S rDNA from strain CHY-1 was determined and the closest relative in the databases was found to be Sphingomonas xenophaga (96% sequence identity) (Fig. 2). S. xenophaga BN6T was isolated for its ability to degrade naphthalene sulphonates [19], but it has not been reported to degrade PAHs. The classification of Sphingomonas species was recently reassessed and three additional genera were proposed, namely Sphingobium, Novosphingobium and Sphingopyxis[20]. S. xenophaga and strain CHY-1 clustered with members of the genus Sphingobium on the basis of 16S rDNA sequences (Fig. 2) but were no more closely related to these species than to Rhizomonas suberifaciens. Signature 16S rRNA nucleotides were identified for the various genera [20]. However, both strain CHY-1 and S. xenophaga lack one of the signature nucleotides characteristic of the genus Sphingobium (Table 1). Further taxonomic work is needed to determine whether these two organisms represent a separate genus or if the definition of the Sphingobium genus should be extended to include them. We therefore retain the provisional name Sphingomonas sp. CHY-1 for the newly isolated strain.

image

Figure 2. Phylogenetic tree showing the affiliation of Sphingomonas sp. CHY-1 with the sphingomonad group based on 16S rRNA sequences. Sequences were aligned by the Clustal method and the tree, based on a comparison of 1302 nucleotide positions, was constructed using the MEGALIGN program of the DNASTAR software package (DNASTAR Inc., Madison, WI, USA). Bootstrap analysis was performed using the Seqboot, Dnadist, Neighbour and Consense programs of the PHYLIP software package (version 3.5c) [22]. Bootstrap values supporting each node are shown as a percentage of 200 replicates. Brevundimonas diminuta DSM 1635T was included as an outgroup to facilitate rooting of the tree and was specified as the outgroup for bootstrap analysis.

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Table 1.  16S rRNA nucleotide signatures distinguishing Sphingomonas sp. CHY-1 and Sphingomonas xenophaga BN6T from species of the four most closely related genera [20]
Genus/species/strainSignature at positiona
 52:359134593987:1218990:1215
  1. aEscherichia coli numbering system (Accession No. J01859).

S. xenophaga BN6T/Sphingomonas sp. CHY-1U:AGUG:CU:G
SphingobiumU:AGUA:UU:G
RhizomonasC:GAAG:CU:G
BlastomonasC:GGUG:CU:A
SphingomonasC:GGGG:CU:G

Bacterial 16S rDNA was amplified from DNA extracted from the biofilm described above and cloned. RFLP analysis of individual clones, using the Rsa1 and AluI restriction enzymes, showed that 6/15 clones gave identical restriction patterns to 16S rDNA from Sphingomonas sp. CHY-1, indicating that this strain was present in the biofilm. The other nine clones fell into six different RFLP groups, suggesting that the bacterial population in the biofilm was complex. Further work is needed to determine whether the evolutionary transition observed in the enrichment cultures arose from a mutation in strain CHY-1, or by lateral gene transfer from another species in the biofilm. The 16S rDNA sequences were determined for a further six isolates with the same colony morphotype and were identical to that of strain CHY-1. This result, taken together with the fact that the kinetics of chrysene degradation by the isolated strain were identical to those of the rapidly growing enrichment cultures, indicates that Sphingomonas sp. CHY-1 was the principal strain responsible for chrysene degradation following the transition from biofilm growth to growth in suspension.

3.3Growth characteristics of Sphingomonas sp. CHY-1

Strain CHY-1 grew in pure culture on chrysene and degraded chrysene at a rate of 13 μg day−1 ml−1 culture (Fig. 1, insert). When the cultures were inoculated with cells grown on PTYG20 agar medium then a variable lag period of 7–14 days was observed before chrysene degradation commenced. However, this lag period was abolished when a preculture grown on chrysene was used as inoculum. Bacterial growth, followed by measurements of absorbance and protein content, also began without a detectable lag period, reaching final values of 0.19 ± 0.04 (A550) and 11.5 μg ml−1 protein, respectively (no increase in A550 or protein content was observed in controls without chrysene). Kinetic measurements showed that both A550 and protein content (not shown) reached approximately 80% of their final values after 3 weeks incubation when the level of residual chrysene had fallen to < 5% of the initial value, then continued to increase slowly to a plateau value after 6 to 7 weeks incubation. The number of colony forming units per ml culture increased to a maximum value of between 3.6 ± 1.2 × 108 and then decreased to 9.8 ± 6.8 × 106 (i.e. 30- to 100-fold) at the onset of stationary phase. A rapid loss of the ability to form colonies in stationary phase was also reported for S. xenophaga BN6T[19]. Wet-mounted or DAPI-stained cells appeared as short rods or cocci that were estimated to be 0.5–1.0 μm in diameter.

Using the same biphasic culture medium as for chrysene, Sphingomonas sp. CHY-1 was able to grow with naphthalene, phenanthrene or anthracene, but not with the other PAHs tested (see Section 2). Other PAHs were also not degraded at significant rates when provided individually, although co-metabolism was not tested. Growth was fastest on naphthalene, with a doubling time of 6.9 h; on anthracene, it was relatively poor, and the growth medium became red due to the accumulation of 2,3-dihydroxynaphthalene, which was identified by GC/MS. Sphingomonas sp. CHY-1 also grew well on mixtures (2:1 mass ratio) of naphthalene plus chrysene or phenanthrene plus chrysene. In these cultures the yield of biomass from cultures was three times greater, and the rate of chrysene degradation 2- to 3-fold higher, than in cultures grown on chrysene alone. These substrate combinations could therefore be used to produce increased amounts of cell material for biochemical studies of chrysene degradation.

Sphingomonas sp. CHY-1 failed to grow on any of the monoaromatic substrates tested including benzoate, mandelate, salicylate, protocatechate, phthalate, gentisate and cinnamate, but grew on acetate, succinate and glucose, with doubling times of 10, 15 and 23 h, respectively. In the API 20 NE identification system, Sphingomonas sp. CHY-1 gave negative results for all tests, except for glucose and malate assimilation, which were weakly positive after 4 days incubation at 30 °C. This strain therefore does not reduce nitrate to nitrite or nitrogen, produce indole from tryptophan, ferment glucose or arginine, produce urease or β-galactosidase, hydrolyse esculin or gelatin, or assimilate arabinose, mannose, mannitol, N-acetylglucosamine, maltose, gluconate, caprate, adipate, citrate or phenylacetate.

3.414C-Chrysene mineralization experiments

The kinetics of mineralization of [5,6,11,12-14C]chrysene by Sphingomonas sp. CHY-1 growing in pure culture on chrysene are shown in Fig. 3 (curve A). As expected, the rate of mineralization was greater in cultures growing on a mixture of naphthalene and chrysene (Fig. 3, curve B). However, in this case, a lag period of two days was observed, corresponding to the time required for the consumption of naphthalene (Fig. 3, insert). At the end of each experiment the cultures were fractionated and the amount of radioactivity in each fraction determined (Table 2). Overall, only about 70% of the initial radioactivity was recovered from cultures grown on chrysene, and 85% from cultures grown on naphthalene plus chrysene. In uninoculated control flasks the recovery of chrysene was >95% after 8 weeks incubation, suggesting that at least some of the missing radioactivity may have been due to covalent or to strong, non-covalent binding of chrysene oxidation products to the glass wall of the culture flasks. In support of this, approximately 0.4% of the initial radioactivity (non-extractable by hexane, dichloromethane or toluene) was extracted by methanol, and the UV spectrum of the methanol extract was different from that of chrysene. Moreover, an additional 2% radioactivity was extracted by heating at 100 °C for 24 h with either 2 N HCl or 1% Triton X-100, but not with 2 N NaOH or 1% SDS. Control experiments suggested that leakage of 14CO2 from the culture flasks was negligible during the time-course of the experiments.

image

Figure 3. Kinetics of [14C]Chrysene mineralization by cultures of Sphingomonas sp. CHY-1 grown in the presence of [14C]-labelled chrysene (A) or [14C]-labelled chrysene plus naphthalene (B). Cultures were prepared and inoculated in triplicate, as decribed in Section 2, and at the time intervals shown, gas samples were withdrawn from each culture for measurement of 14CO2 content. The culture flasks were then flushed with sterile air before being reincubated. The cumulative amount of radioactivity evolved into the gas phase in each culture was then calculated and expressed as a percentage of the initial radioactivity, which corresponded to 249 × 103 cpm per flask. The insert curve shows the disappearance of naphthalene from the oil phase (open squares) and the concomitant increase in A550 due to bacterial growth (solid squares), measured in parallel cultures containing unlabelled chrysene. 100% residual napthalene was equivalent to 1 g l−1 silicone oil.

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Table 2.  Distribution of radioactivity in cultures of Sphingomonas sp. CHY-1 grown in the presence of [14C]-labelled chrysene or [14C]-labelled chrysene plus naphthalene
Culture fractionaRadioactivity (% total added)b
 [14C] Chrysene[14C] Chrysene + naphthalene
  1. aCultures were incubated for 56 days ([14C] Chrysene) or 35 days ([14C] Chrysene + naphthalene) in sealed 100-ml Erlenmeyer flasks, then fractionated and analysed as described in Section 2. The amount of radioactivity in the interface fractions and in the hexane extracts of cell pellets was negligible.

  2. b100% radioactivity was equivalent to 249 × 103 cpm. The values shown are the mean ± SE of determinations on separate cultures (number in brackets).

Gas phase (CO2)49.2 ± 1.8 (5)60.7 ± 0.1 (2)
Cell pellet (hexane-extracted)10.7 ± 1.9 (5)5.7 ± 0.2 (2)
Culture supernatant (aqueous phase)8.4 ± 1.6 (5)15.9 ± 5.1 (2)
Oil phase0.5 ± 0.2 (5)0.6 ± 0.1 (2)
Hexane wash of flask0.5 ± 0.3 (5)0.9 ± 0.2 (2)
Methanol wash of flask0.4 ± 0.3 (5)0.4 ± 0.2 (2)
   
Total69.7 ± 3.0 (5)84.2 ± 4.7 (2)

The radioactivity detected in the aqueous culture medium was presumably due to the release of hydrosoluble chrysene degradation products. However, in extracts prepared from 2 l of culture medium only one potential metabolite was detected after silylation, with a GC retention time of 22.45 min. Its structure could not be deduced from the mass spectrum, but the size of the heaviest mass peak observed (m/z= 521) suggests that it contained at least three reactive groups. Some of the soluble radioactivity may be due to very small, acidic compounds, such as acetate, which would not have been detected in our analyses.

Deduction of the metabolic pathways of PAH degradation by bacteria has relied heavily on the identification of metabolic intermediates that are excreted into the culture medium during growth and metabolism [3]. However, despite reports of bacterial strains that are able to co-metabolize chrysene, such as S. paucimobilis EPA 505 [8], Stenotrophomonas maltophilia VUN10,010 [9] and Pseudomonas fluorescens VUN10,011 [9], or to use it as sole carbon source for growth, i.e., Rhodococcus sp. UW1 [10], P. fluorescens P2a [11,13] or Alcaligenes odorans P20 [12], no metabolic intermediates have so far been identified. The elucidation of the pathway(s) of chrysene catabolism may therefore require the identification and characterization of the enyzmes catalysing each step. In this regard, it is interesting to note that an E. coli strain overexpressing a dioxygenase gene from Sphingomonas sp. CHY-1 is able to convert chrysene to a single product, identified as a cis-dihydrodiol, which presumably corresponds to the initial product of chrysene oxidation by this bacterium [21].

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

This work was supported by grants from the Rhône-Alpes region (XI Contrat de Plan Etat-Région), the Agence de l'Environnement et de la Maîtrise de l'Energie (ADEME), the CNRS and the CEA. I would like to thank Drs. Yves Jouanneau and Sandrine Demanèche for helpful discussions, Sandrine Bagnos for technical assistance, and Georgia Plouchart, Aude Coutin, Isabelle Ponton, and Nathalie Arsac for their enthusiastic participation at various stages of this work.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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