SEARCH

SEARCH BY CITATION

Keywords:

  • bioremediation;
  • mycobacterium;
  • polycyclic aromatic hydrocarbons;
  • soil remediation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Chemicals
  6. Isolation, identification, and characterization
  7. Liquid-culture studies
  8. Pyrene mineralization in soil
  9. Results
  10. Identification and morphology
  11. Hydrophobicity
  12. Degradation of PAH and alkanes
  13. Discussion
  14. Acknowledgements
  15. References

Aims: Our goal was to characterize a newly isolated strain of Mycobacterium austroafricanum, obtained from manufactured gas plant (MGP) site soil and designated GTI-23, with respect to its ability to degrade polycyclic aromatic hydrocarbons (PAHs).

Methods and Results: GTI-23 is capable of growth on phenanthrene, fluoranthene, or pyrene as a sole source of carbon and energy; it also extensively mineralizes the latter two in liquid culture and is capable of extensive degradation of fluorene and benzo[a]pyrene, although this does not lead in either of these cases to mineralization. Supplementation of benzo[a]pyrene-containing cultures with phenanthrene had no significant effect on benzo[a]pyrene degradation; however, this process was substantially inhibited by the addition of pyrene. Extensive and rapid mineralization of pyrene by GTI-23 was also observed in pyrene-amended soil.

Conclusions: Strain GTI-23 shows considerable ability to mineralize a range of polycyclic aromatic hydrocarbons, both in liquid and soil environments. In this regard, GTI-23 differs markedly from the type strain of Myco. austroafricanum (ATCC 33464); the latter isolate displayed no (or very limited) mineralization of any tested PAH (phenanthrene, fluoranthene or pyrene). When grown in liquid culture, GTI-23 was also found to be capable of growing on and mineralizing two aliphatic hydrocarbons (dodecane and hexadecane).

Significance and Impact of the Study: These findings indicate that this isolate of Myco. austroafricanum may be useful for bioremediation of soils contaminated with complex mixtures of aromatic and aliphatic hydrocarbons.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Chemicals
  6. Isolation, identification, and characterization
  7. Liquid-culture studies
  8. Pyrene mineralization in soil
  9. Results
  10. Identification and morphology
  11. Hydrophobicity
  12. Degradation of PAH and alkanes
  13. Discussion
  14. Acknowledgements
  15. References

Bioremediation has long been proposed and applied as a treatment technology for the decontamination of hydrocarbon-contaminated soils. Many different bacteria are known to be capable of degrading, and in many cases, completely mineralizing, various individual xenobiotic compounds. For example, in the case of polycyclic aromatic hydrocarbons (PAHs), numerous bacteria are known that are capable of catabolizing various PAHs as sole sources of carbon and energy, making them good candidate species for site-remediation applications. The ability to degrade low-molecular-weight PAH compounds, such as naphthalene and phenanthrene, is widespread, and numerous researchers have identified bacteria capable of utilizing these compounds for growth (for reviews, see Cerniglia 1992; Sutherland et al. 1995). Growth on PAH containing four fused aromatic rings (e.g. chrysene, fluoranthene, pyrene, benz[a]anthracene) is somewhat more rare, although organisms are known that can utilize each of these as growth substrates (Heitkamp et al. 1988a,b; Mueller et al. 1990; Walter et al. 1991; Weissenfels et al. 1991; Boldrin et al. 1993; Caldini et al. 1995; Schneider et al. 1996; Bouchez et al. 1997; Juhasz et al. 1997; Churchill et al. 1999; Bastiaens et al. 2000, Vila et al. 2001). Currently, only a few bacterial isolates have been reported to degrade five-ring PAHs (e.g. benzo[a]pyrene); furthermore, this generally occurs through co-metabolism, during growth on simpler substrates (Schneider et al. 1996; Chen and Aitken 1999).

Many contaminated sites are characterized by the presence of complex mixtures of pollutants. For example, creosotes and the coal tars from which they are derived are typically comprised of a wide range of aromatic hydrocarbons, aliphatics, heterocyclic (N-, S- and O-containing) compounds, phenols and amines (Rhodes 1951; Nestler 1974; Nishioka et al. 1986; Mueller et al. 1989). Crude and refined oils present a similar situation; for example, a typical fuel oil (#2) consists of 45% cycloalkanes, 30% linear (straight chain and branched) aliphatics, and 25% aromatics (Arvin et al. 1988). It is, therefore, clear that the success of bioremediation approaches to soil treatment will hinge in part on the ability of bacteria (either versatile single strains or consortia in which multiple members can be simultaneously maintained) to degrade all of the components of complex hydrocarbon mixtures.

Members of the genus Mycobacterium may be particularly well suited to this role. Mycobacteria, for example, are known to possess extremely lipophilic cell surfaces (Rehmann et al. 1988), which may make them better suited to the direct uptake (Bouchez-Naïtali et al. 1999) of highly hydrophobic hydrocarbons, including high-molecular-weight PAHs (Kelley and Cerniglia 1995; Schneider et al. 1996) and highly branched aliphatic hydrocarbons (Berekaa and Steinbüchel 2000; Solano-Serena et al. 2000). This paper presents an initial characterization of a newly isolated strain (GTI-23) of Mycobacterium austroafricanum, which is capable of growth on, and/or degradation of, various PAHs (with up to five fused rings), as well as straight-chain aliphatic hydrocarbons (decane, dodecane, and hexadecane).

Chemicals

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Chemicals
  6. Isolation, identification, and characterization
  7. Liquid-culture studies
  8. Pyrene mineralization in soil
  9. Results
  10. Identification and morphology
  11. Hydrophobicity
  12. Degradation of PAH and alkanes
  13. Discussion
  14. Acknowledgements
  15. References

Fluorene (98%), phenanthrene (98%), and pyrene were obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI, USA). Fluoranthene (Practical grade) was obtained from Eastman Organic Chemicals (Rochester, NY, USA). Benzo[a]pyrene (98%), n-decane (99%+), n-dodecane (99%), n-hexadecane (99%+), and gelrite gellan gum were from Sigma Chemical Co. (St Louis, MO, USA), as were the following 14C-radiochemicals: 7-14C-benzo[a]pyrene (26·6 mCi  mmol−1), 9-14C-fluorene (14·2 mCi  mmol−1), 4,5,9,10-14C-pyrene (58·7 mCi  mmol−1), 1-14C-dodecane (4·1 mCi  mmol−1), and 1-14C-hexadecane (2·2 mCi mmol−1). 3-14C-Fluoranthene (45 mCi mmol−1) was from Moravek Biochemicals (Brea, CA, USA).

Isolation, identification, and characterization

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Chemicals
  6. Isolation, identification, and characterization
  7. Liquid-culture studies
  8. Pyrene mineralization in soil
  9. Results
  10. Identification and morphology
  11. Hydrophobicity
  12. Degradation of PAH and alkanes
  13. Discussion
  14. Acknowledgements
  15. References

A small amount of homogenized soil (∼1 g), obtained from a former manufactured gas plant site in Iowa, was suspended in 25 ml of sterile deionized water and serial 10-fold dilutions were made; appropriate dilutions (from 10−5 to 10−8) were spread onto gel plates [1·0 g KNO3, 0·38 g K2 HPO4, 0·2 g MgSO4·7H2O, 0·05 g FeCl3·6H2O, 17·0 g gelrite gellan gum (Sigma Chemical Co., St. Louis, MO, USA) per liter of distilled water, pH 7·0]. Pyrene (or other PAH of interest) was then sublimated onto the plates (Alley and Brown 2000), which were then incubated at 30°C. A clearing zone was observed around a smooth, yellowish colony within approximately 8–10 days.

This colony was further subcultured until cellular and colonial morphologies were uniform. Both pyrene- and phenanthrene-sublimated plates were employed during this procedure; the latter was occasionally used due to the fact that growth of the isolate was more rapid on these plates (generally requiring 2–3 days or less). Culture purity was assessed microscopically, and by streaking onto R2A agar (Difco Products, Detroit, MI, USA). Once an axenic culture was obtained, it was identified by sequencing of 16s rRNA gene (MIDI Labs, Newark, DE, USA). The resultant sequence was matched using the GenBank database.

Hydrophobicity of the isolate was determined according to the BATH (bacterial adhesion to hydrocarbons) method, as described by Bouchez-Naïtali et al. (1999). Briefly, cultures were grown in medium [YPS (see below) for Mycobacterium strains, mineral salt medium (MSM) for other isolates], supplemented with crystalline phenanthrene. When cultures became visibly turbid, cells were collected by centrifugation, washed once, and resuspended in phosphate buffer (0·15 M, pH 7·1). Cell suspensions were adjusted to a uniform A600 value (0·1–0·2), and 100 μl of hexadecane was added to 2-ml aliquots of cell suspension in 4-ml vials with Teflon-lined caps. These were extensively mixed (vortexed for 2 min) and the hexadecane layer was given 60 min to partition away from the aqueous phase. A600 measurements of the remaining aqueous layer were taken; hydrophobicity is expressed as the percent decrease of this value from the initial A600 of the cell suspension.

Liquid-culture studies

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Chemicals
  6. Isolation, identification, and characterization
  7. Liquid-culture studies
  8. Pyrene mineralization in soil
  9. Results
  10. Identification and morphology
  11. Hydrophobicity
  12. Degradation of PAH and alkanes
  13. Discussion
  14. Acknowledgements
  15. References

Two primary growth media were employed in this work. The media used in studies assessing the ability of GTI-23 to grow on hydrocarbons as sole sources of carbon and energy was the previously described (Bogan et al. 2001) MSM. The second medium (YPS medium), used for studies of co-metabolism and mineralization studies, was the Mycobacterium mineral salts medium, supplemented with yeast extract, peptone, and soluble starch (each at 250 mg l−1), originally described by Kelley and Cerniglia (1995), which is frequently used in studies of mycobacterial PAH degradation.

Studies examining growth of GTI-23 on alkanes as sole carbon and energy sources were conducted in 96-well microplates, which contained 150 μl of MSM and 5 μl of liquid hydrocarbon. Growth was monitored (A600) in an automated microplate reader (Dynex Technologies, Chantilly, VA, USA).

Liquid-culture experiments on mineralization and/or degradation of individual PAHs and alkanes were conducted in YPS medium (50 ml) in sealed serum bottles as previously described (Bogan et al. 2001). In the cases of PAHs, 15–20 mg were added to each bottle as previously described (Mueller et al. 1990); for alkanes, 10 μl of liquid hydrocarbon was added to each bottle. 14C-PAH or alkanes (30 000–80 000 dpm per culture) were added as methanolic stocks (≈5 μl). Experiments were typically run for 3–4 weeks, during which culture bottles were agitated constantly (150 rpm) on a shaking platform at room temperature (ca. 25°C); photodegradation of PAH (Miller et al. 1988) was avoided by wrapping culture bottles in aluminum foil during incubation.

Mineralization was assessed through the use of 1-ml NaOH traps, suspended in culture bottles as previously described (Bogan et al. 2001). Analyses of degradation of 14C-fluorene- and 14C-benzo[a]pyrene-supplemented cultures were conducted as follows. Cultures were acidified (pH ∼ 0·5) with concentrated H2SO4, and disrupted in a sonicator bath for ca. 5 min. Cultures were extracted twice with ethyl acetate (50 ml), which was then pooled. Aliquots of the remaining aqueous phase and the organic-extractable fractions were subjected to scintillation counting to determine the distribution of radioactivity between the two phases. To further characterize the organic-soluble fraction, the pooled ethyl acetate extracts were evaporated to dryness under a stream of N2. Following dissolution in acetonitrile (1 ml), the organic-soluble material was subjected to reverse-phase HPLC analysis as previously described (Bogan et al. 2001), with fractions (1-min intervals) of the HPLC eluent collected for quantitation of 14C.

Pyrene mineralization in soil

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Chemicals
  6. Isolation, identification, and characterization
  7. Liquid-culture studies
  8. Pyrene mineralization in soil
  9. Results
  10. Identification and morphology
  11. Hydrophobicity
  12. Degradation of PAH and alkanes
  13. Discussion
  14. Acknowledgements
  15. References

A commercial potting soil was screened (3·35-mm screen, #6 mesh), and allowed to air-dry. This soil had a pH of 5·3; hydrometer testing for particle-size distribution showed the soil to be 67% sand, 21% silt, and 12% clay, resulting in a classification, as per the USDA soil classification chart, of sandy loam. The soil was weighed into flasks (60 g per flask), and spiked with pyrene and 14C-pyrene (dissolved in 20 ml methylene chloride), such that the final soil pyrene concentration was 100 ppm, with a total of 150 000 dpm of 14C-pyrene per flask. After evaporation of solvent in a fume hood (approximately 48 h), these were then adjusted to a moisture content of ca. 25% (weight basis) and inoculated with an MSM suspension of Myco. austroafricanum GTI-23 (1 ml per flask of a suspension with A600 ˜ 2·5). Control cultures were established with MSM, which did not include bacterial inoculum; these, therefore, contained only the indigenous microbes in the potting soil. Both sets of flasks were attached to a constant airflow (ca. 25 ml  min−1); air exiting the microcosms was flushed through CO2 traps containing 20 ml of 0·5 M NaOH. Every 2–4 days, NaOH in the traps was replaced, and 5 ml was added to 15 ml of scintillation cocktail and subjected to liquid scintillation counting. Soil flasks were maintained at room temperature; although no effort was made to restrict light from the flasks, control mineralization results (see below) indicate that losses to photolysis were essentially zero. Water was periodically (every 1–2 days) added to microcosms to replace evaporative losses.

Identification and morphology

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Chemicals
  6. Isolation, identification, and characterization
  7. Liquid-culture studies
  8. Pyrene mineralization in soil
  9. Results
  10. Identification and morphology
  11. Hydrophobicity
  12. Degradation of PAH and alkanes
  13. Discussion
  14. Acknowledgements
  15. References

Searches of the GenBank database using the sequence data determined for the 16s rRNA gene of the GTI-23 isolate resulted in a 100% match to Myco. austroafricanum. GTI-23 was found to be a Gram-positive rod, typically in the range of 0·6 μm × 1·5 μm, which is in good agreement with the dimensions (0·5 μm × 2–6 μm) recognized for Myco. austroafricanum (Sneath et al. 1986).

Hydrophobicity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Chemicals
  6. Isolation, identification, and characterization
  7. Liquid-culture studies
  8. Pyrene mineralization in soil
  9. Results
  10. Identification and morphology
  11. Hydrophobicity
  12. Degradation of PAH and alkanes
  13. Discussion
  14. Acknowledgements
  15. References

Strain GTI-23 was considerably more hydrophobic than various strains of PAH-degrading soil bacteria (Acidovorax, Burkholderia, Pseudomonas, and Sphingomonas), which we have previously isolated; these data are presented in Table 1. Of the tested (non-mycobacterial) strains, only one (Sphingomonas GTI-8) was close to GTI-23 in net hydrophobicity; Mycobacterium sp. strain PYR-1 (obtained from Dr C. Cerniglia) had approximately the same hydrophobicity value as GTI-23 (52 vs 48%) when both were cultured on phenanthrene-supplemented YPS medium. Myco. austroafricanum ATCC 33464, the type strain of the species (Tsukamura et al. 1983), when cultured in YPS medium (phenanthrene omitted), was slightly less hydrophobic than either GTI-23 or PYR-1.

Table 1.  Hydrophobicity of Mycobacterium austroafricanum GTI-23, in comparison with various other PAH-degrading isolates (and the non-PAH-degrading Myco. austroafricanum type strain)
IsolateMediumCulture ageHydrophobicity (%)
Acidovorax temperans GTI-19MSM/phenanthrene1 day3
Burkholderia sp. GTI-3MSM/phenanthrene6 days15
Pseudomonas viridiflava GTI-5MSM/phenanthrene6 days4
Sphingomonas sp. GTI-7MSM/phenanthrene2 days15
Sphingomonas sp. GTI-8MSM/phenanthrene6 days26
Sphingomonas sp. GTI-10MSM/phenanthrene6 days0
Sphingomonas sp. GTI-11MSM/phenanthrene6 days18
Sphingomonas subarctica GTI-12MSM/phenanthrene2 days0
Myco. austroafricanum GTI-23YPS/phenanthrene6 days48
Myco. austroafricanum ATCC 33464YPS6 days41
Mycobacterium sp. PYR-1YPS/phenanthrene6 days52

Degradation of PAH and alkanes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Chemicals
  6. Isolation, identification, and characterization
  7. Liquid-culture studies
  8. Pyrene mineralization in soil
  9. Results
  10. Identification and morphology
  11. Hydrophobicity
  12. Degradation of PAH and alkanes
  13. Discussion
  14. Acknowledgements
  15. References

Myco. austroafricanum GTI-23 was originally recognized by, and chosen for, its ability to degrade pyrene, sublimated onto minimal agar (gelrite) medium. Clearing zones, indicative of PAH metabolism as a sole source of carbon and energy, were observed in the cases of pyrene (Fig. 1), as well as phenanthrene and fluoranthene. GTI-23 was also found, in YPS liquid medium, to be capable of mineralizing pyrene and fluoranthene, as shown in Fig. 2. Release of 14C from labeled pyrene was rapid, and totaled essentially 100%, implying (because of the distribution of the label across four positions within the molecule) near-complete mineralization, with little or no incorporation of pyrene-derived carbon into biomass. Mineralization of fluoranthene displayed a slight lag (5–7 days), and leveled off after approximately 60% of the input radioactivity had been released as 14CO2. Myco. austroafricanum ATCC 33464 (Tsukamura et al. 1983), displayed average mineralization totals of just 1·2, 1·0 and 0·6% for phenanthrene, fluoranthene, and pyrene, respectively, when grown in YPS medium for 21 days; it also failed to produce clearing zones on gel plates sublimated with these PAHs, even after a total exposure time of 3–4 weeks (data not shown).

image

Figure 1. Clearing zones formed by colonies of Mycobacterium austroafricanum strain GTI-23 on pyrene-sublimated gel plate (elapsed time=14 days)

Download figure to PowerPoint

image

Figure 2. Mineralization of fluoranthene (▪) and pyrene (♦) by Mycobacterium austroafricanum GTI-23 grown in YPS medium (initial PAH concentration=20 mg/50 ml). Data are means of duplicate cultures, with 95% confidence limits

Download figure to PowerPoint

GTI-23 evidenced no release of 14CO2 from either 14C-fluorene or 14C-benzo[a]pyrene at this same initial concentration (0·4 mg  ml−1). Essentially the entire input radioactivity was recovered at the conclusion of the experiment; for both compounds, the vast majority (>95%) remained in the organic-soluble fraction. However, analysis of these fractions by reverse-phase HPLC indicated that extensive transformation of fluorene had, in fact, occurred (Fig. 3). Although no attempt was made to characterize these products, they were clearly considerably more polar than fluorene, given their significantly earlier elution during reversed-phase HPLC. When benzo[a]pyrene was added at the same initial concentration, very little degradation occurred; HPLC analysis showed that less than 20% of recovered 14C was present in peaks other than BaP (data not shown). However, this was not true when benzo[a]pyrene was added at lower levels (0·5 vs 20 mg); Fig. 4 shows reverse-phase HPLC analysis of the ethyl acetate-extractable products of Myco. austroafricanum GTI-23 cultures that contained 14C-benzo[a]pyrene at this lower concentration. Approximately 83% of the starting material was converted to more-polar products; roughly half of this eluted in peaks that were near the void volume of the column, indicating a very high degree of polarity. Addition of phenanthrene (2 mg per culture) had no significant effect on BaP degradation; this is in contrast to Mycobacterium RJGII-135, in which the presence of phenanthrene stimulated BaP degradation (McClellan et al. 2002). Addition of pyrene (2 mg) had a significant inhibitory effect on transformation of benzo[a]pyrene (Fig. 4); this was also observed with strain RJGII-135 (McClellan et al. 2002). Under no circumstance was any significant 14CO2 evolution observed from benzo[a]pyrene.

image

Figure 3. Typical reverse-phase HPLC profile of ethyl acetate-soluble radioactive products derived from Mycobacterium austroafricanum cultures (YPS medium) supplemented with fluorene (20 mg per culture). Untransformed fluorene has a retention time of ca. 13 min, corresponding to the peak marked with an asterisk

Download figure to PowerPoint

image

Figure 4. Typical reverse-phase HPLC profiles of ethyl acetate-soluble radioactivity derived from Mycobacterium austroafricanum cultures (YPS medium) supplemented with 0·5 mg benzo[a]pyrene, either alone (top) or together with 2·0 mg of phenanthrene (center) or pyrene (bottom). Retention time of benzo[a]pyrene was ca. 26 min

Download figure to PowerPoint

Strain GTI-23 was found to be capable of growth on both decane and hexadecane (Fig. 5) as sole carbon sources (i.e. in MSM cultures), and of mineralization of hexadecane and dodecane when grown in YPS medium (Fig. 6). Growth on both of these compounds began to level off after approximately 20 days, a figure that coincided well with the timing of plateaus in 14CO2 liberation in mineralization experiments.

image

Figure 5. Growth of Mycobacterium austroafricanum GTI-23 in MSM supplemented with either decane (▪) or hexadecane (▴), as well as for unsupplemented, ‘blank’ cultures (♦). In all cases, data are means of triplicate cultures, with 95% confidence limits included

Download figure to PowerPoint

image

Figure 6. Mineralization (YPS media, n=2 cultures) of 14C-dodecane (▪) and hexadecane (♦) by GTI-23

Download figure to PowerPoint

When inoculated into pyrene-spiked soil, GTI-23 was capable of extensive 14C-pyrene mineralization, as shown in Fig. 7. As in liquid culture, the onset of mineralization was rapid, displaying no significant lag time, and persisted for several weeks of incubation time. Mock-inoculated (MSM, no GTI-23) microcosms released insignificant levels (<1000 dpm) of 14CO2 over this time frame (filled circles in Fig. 7), indicating (as expected) that no pyrene-mineralizing microorganisms were present in the commercial topsoil in the absence of inoculation.

image

Figure 7. Mineralization of 14C-pyrene (initial=100 ppm; 150 000 dpm) in non-sterile soil microcosms (n=3) inoculated with Mycobacterium austroafricanum GTI-23 (▪) or in mock-inoculated controls (•)

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Chemicals
  6. Isolation, identification, and characterization
  7. Liquid-culture studies
  8. Pyrene mineralization in soil
  9. Results
  10. Identification and morphology
  11. Hydrophobicity
  12. Degradation of PAH and alkanes
  13. Discussion
  14. Acknowledgements
  15. References

The ability to catabolize PAHs with more than three fused benzene rings is relatively rare among bacteria, particularly when compared to the ability to grow on lower-molecular-weight polycyclic aromatics. To date, growth on pyrene has been observed in some species of Rhodococcus (Walter et al. 1991; Bouchez et al. 1997) and strains of Burkholderia cepacia (Juhasz et al. 1997); additionally, cometabolism of pyrene has been observed in Pseudomonas saccharophila (Chen and Aitken 1999). However, the ability to grow on high-molecular-weight PAH substrates may be most widespread in the genus Mycobacterium, where this result has been reported for several different species and strains (Heitkamp et al. 1988b; Boldrin et al. 1993; Schneider et al. 1996; Churchill et al. 1999; Bastiaens et al. 2000; Vila et al. 2001). At least one of these, Mycobacterium PYR-1 (Rafii et al. 1992; Kelley and Cerniglia 1995), is, based on recently published evolutionary distance trees (Solano-Serena et al. 2000), very closely related to Myco. austroafricanum, although it has recently been classified as a separate new species, Myco. vanbaalenii (Khan et al. 2003).

Among the PAH-degrading Mycobacteria, some are also known to degrade aliphatic hydrocarbons. Solano-Serena et al. (2000) reported that Myco. austroafricanum strain IFP2173, originally isolated from gasoline-contaminated groundwater, was able to degrade some monoaromatics (e.g. toluene, m- and p-xylene), as well as a wide range of straight-chain and branched alkanes. This strain did not, however, degrade phenanthrene (Dr R. Marchal, personal communication). These findings indicate that the PAH-degrading capacity of this latter strain, at least under the particular growth conditions studied to date, is very limited. Several other strains that were identified as Mycobacteria were able to degrade and grow on alkanes (dodecane and hexadecane) as well as three- and four-ring PAHs (phenanthrene, fluoranthene, pyrene), although the authors (Lloyd-Jones and Hunter 1997) suggested that some of these strains might actually belong to a new genus, rather than to Mycobacterium. Growth substrates of Mycobacterium strain AP-1 included phenanthrene, fluoranthene, pyrene, and hexadecane (Vila et al. 2001). In addition to our findings with GTI-23, we have also observed mineralization (in YPS medium) of dodecane and hexadecane by Mycobacterium strain PYR-1 (data not shown), further extending within the genus Mycobacterium the ability to degrade both of these contaminant classes.

To our knowledge, GTI-23 is the first strain that has been reported to degrade both aliphatics and PAHs with five benzene rings (benzo[a]pyrene). Inasmuch as this latter compound is among the most hazardous individual PAHs (Smucker 2000), this finding has some significance. These findings are also of interest in light of the failure of the Myco. austroafricanum type strain to mineralize PAHs. Very little is known about the molecular biology of PAH degradation in Mycobacteria (Wang et al. 2000). Previous studies indicate that homology between mycobacterial genes for PAH-degrading enzymes and those known from other genera (e.g. the nah operon) is apparently very low (Churchill et al. 1999). Recent work has identified numerous cytochrome P450 genes in Myco. tuberculosis (Lamb et al. 2002); however, it is unclear what role enzymes of this type might play in PAH-degrading Mycobacteria. Clearly, the oxidative activity of P450 enzymes will not in itself result in mineralization of PAH, which would require a suite of enzymes analogous to those encoded by nah or other operons; P450 enzymes may, however, help to explain the levels of benzo[a]pyrene and fluorene oxidation (without mineralization) seen in this study. In either case, comparison of pairs of very closely related mycobacterial isolates, in which one strain degrades PAH and one does not, may provide insight into the molecular genetics underpinning this process in the genus; we are currently conducting studies to address this possibility.

Failure of inoculated bacteria to persist in soil is a frequent impediment to successful bioremediation (Van Dyke and Prosser 2000). In contrast to most of the studies of mycobacterial-contaminant degradation described above, the results reported herein provide clear evidence of GTI-23's ability to survive and degrade PAH in a non-sterile soil system. Pyrene mineralization continued for several weeks in soils inoculated with GTI-23, while no mineralization occurred in its absence. Our results thus concur with those of Cheung and Kinkle (2001), who reported that Mycobacterium strain RJGII-135 survived in soil systems for upwards of 80 days. Further examination of strain GTI-23 is underway, with a goal of better delineating its ability to survive, compete, and degrade contaminants in soil environments under various conditions.

The relative rarity of bacterial species capable of growth on PAHs containing four or more fused rings is most likely due to the difficulties inherent in uptake of highly hydrophobic compounds of this nature. This is generally accomplished through specialized adaptations, such as lipid-rich outer cell walls and/or production of biosurfactants (Bouchez-Naïtali et al. 1999; Bastiaens et al. 2000). Based on our results, Myco. austroafricanum GTI-23, as well as Myco. austroafricanum ATCC 33464, and the closely related Mycobacterium sp. PYR-1, are considerably more hydrophobic than any of the other PAH-degrading soil bacteria that we have isolated and tested. The precise nature of the adaptations that allow uptake of high-molecular-weight PAH compounds by Myco. austroafricanum GTI-23 is the subject of ongoing investigations in this laboratory.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Chemicals
  6. Isolation, identification, and characterization
  7. Liquid-culture studies
  8. Pyrene mineralization in soil
  9. Results
  10. Identification and morphology
  11. Hydrophobicity
  12. Degradation of PAH and alkanes
  13. Discussion
  14. Acknowledgements
  15. References

This work was supported by Research Contract #DE-AC26-99BC15223 from the United States Department of Energy, by Contracts #8054 and #8471 from the Gas Research Institute.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Chemicals
  6. Isolation, identification, and characterization
  7. Liquid-culture studies
  8. Pyrene mineralization in soil
  9. Results
  10. Identification and morphology
  11. Hydrophobicity
  12. Degradation of PAH and alkanes
  13. Discussion
  14. Acknowledgements
  15. References
  • Alley, J.F. and Brown, L.R. (2000) Use of sublimation to prepare solid microbial media with water-insoluble substrates. Applied and Environmental Microbiology 66, 439442.
  • Arvin, E., Jensen, B., Godsy, E.M. and Grbić-Galić, D. (1988) Microbial degradation of oil and creosote related aromatic compounds under aerobic and anaerobic conditions. In Physicochemical and Biological Detoxification of Hazardous Wastes. ed. Wu, Y.C. pp. 828847. Lancaster: Technomic Publishing.
  • Bastiaens, L., Springael, D., Wattiau, P., Harms, H. DeWachter, R., Verachtert, H. and Diels, L. (2000) Isolation of adherent polycyclic aromatic hydrocarbon (PAH)-degrading bacteria using PAH-sorbing carriers. Applied and Environmental Microbiology 66, 18341843.
  • Berekaa, M.M. and Steinbüchel, A. (2000) Microbial degradation of the multiply branched alkane 2,6,10,15,19,23-hexamethyltetracosane (squalane) by Mycobacterium fortuitum and Mycobacterium ratisbonense. Applied and Environmental Microbiology 66, 44624467.
  • Bogan, B.W., Lahner, L.M., Trbovic, V., Szajkovics, A.M. and Paterek, J.R. (2001) Effects of alkylphosphates and nitrous oxide on microbial degradation of polycyclic aromatic hydrocarbons. Applied and Environmental Microbiology 67, 21392144.
  • Boldrin, B., Tiehm, A. and Fritzsche, C. (1993) Degradation of phenanthrene, fluorene, fluoranthene, and pyrene by a Mycobacterium sp. Applied and Environmental Microbiology 59, 19271930.
  • Bouchez, M., Blanchet, D. and Vandecasteele, J.P. (1997) An interfacial uptake mechanism for the degradation of pyrene by a Rhodococcus strain. Microbiology 143, 10871093.
  • Bouchez-Naïtali, M., Rakatozafy, H., Marchal, R., Leveau, J.Y. and Vandecasteele, J.P. (1999) Diversity of bacterial strains degrading hexadecane in relation to the mode of substrate uptake. Journal of Applied Microbiology 86, 421428.
  • Caldini, G., Cenci, G., Maneti, R. and Morozzi, G. (1995) The ability of an environmental isolate of Pseudomonas fluorescens to utilize chrysene and other four-ring polynuclear aromatic hydrocarbons. Applied Microbiology and Biotechnology 44, 225229.
  • Cerniglia, C.E. (1992) Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation 3, 351368.
  • Chen, S.H. and Aitken, M.D. (1999) Salicylate stimulates the degradation of high-molecular weight polycyclic aromatic hydrocarbons by Pseudomonas saccharophila P15. Environmental Science and Technology 33, 435439.
  • Cheung, P.-Y. and Kinkle, B.K. (2001) Mycobacterium diversity and pyrene mineralization in petroleum-contaminated soils. Applied and Environmental Microbiology 67, 22222229.
  • Churchill, S.A., Harper, J.P. and Churchill, P.F. (1999) Isolation and characterization of a Mycobacterium species capable of degrading three- and four-ring aromatic and aliphatic hydrocarbons. Applied and Environmental Microbiology 65, 549552.
  • Heitkamp, M.A., Franklin, W. and Cerniglia, C.E. (1988a) Microbial metabolism of polycyclic aromatic hydrocarbons: isolation and characterization of a pyrene-degrading bacterium. Applied and Environmental Microbiology 54, 25492555.
  • Heitkamp, M.A., Freeman, J.P., Miller, D.W. and Cerniglia, C.E. (1988b) Pyrene degradation by a Mycobacterium sp.: identification of ring oxidation and ring fission products. Applied and Environmental Microbiology 54, 25562565.
  • Juhasz, A.L., Britz, M.L. and Stanley, G.A. (1997) Degradation of fluoranthene, pyrene, benz[a]anthracene and dibenz[a,h]anthracene by Burkholderia cepacia. Journal of Applied Microbiology 83, 189198.
  • Kelley, I. and Cerniglia, C.E. (1995) Degradation of a mixture of high-molecular-weight polycyclic aromatic hydrocarbons by a Mycobacterium strain PYR-1. Journal of Soil Contamination 4, 7791.
  • Khan, A.A., Kim, S.-J., Paine, D.D. and Cerniglia, C.E. (2002) Classification of a polycyclic aromatic hydrocarbon-metabolizing bacterium, Mycobacterium sp. strain PYR-1, as Mycobacterium vanbaalenii sp. nov. International Journal of Systematic and Evolutionary Microbiology. http://dx.doi.org/10.1099/IJS.0.02163-0.
  • Lamb, D.C., Skaug, T., Song, H.L., Jackson, C.J., Podust, L.M., Waterman, M.R., Kell, D.B., Kelly, D.E. and Kelly, S.L. (2002) The cytochrome P450 complement of (CYPome) of Streptomyces coelicolor A3(2). Journal of Biological Chemistry 277, 2400024005.
  • Lloyd-Jones, G. and Hunter, D.W.F. (1997) Characterization of fluoranthene- and pyrene-degrading Mycobacterium-like strains by RAPD and SSU sequencing. FEMS Microbiology Letters 153, 5156.
  • McClellan, S.L., Warshawsky, D. and Shann, J.R. (2002) The effect of polycyclic aromatic hydrocarbons on the degradation of benzo[a] pyrene by Mycobacterium sp. strain RJGII-135. Environmental Toxicology and Chemistry 21, 253259.
  • Miller, R.M., Singer, G.M., Rosen, J.D. and Bartha, R. (1988) Photolysis primes biodegradation of benzo[a]pyrene. Applied and Environmental Microbiology 54, 17241730.
  • Mueller, J.G., Chapman, P.J. and Pritchard, P.H. (1989) Creosote-contaminated sites: their potential for bioremediation. Environmental Science and Technology 23, 11971201.
  • Mueller, J.G., Chapman, P.J., Blattmann, B.O. and Pritchard, P.H. (1990) Isolation and characterization of a fluoranthene-utilizing strain of Pseudomonas paucimobilis. Applied and Environmental Microbiology 56, 10791086.
  • Nestler, F.H.M. (1974) The Characterization of Wood-preserving Creosote by Physical and Chemical Methods of Analysis. USDA Forest Service Research Paper FPL 195. Madison, WI: USDA Forest Service Forest Products Laboratory.
  • Nishioka, M., Chang, H.C. and Lee, M.L. (1986) Structural characteristics of polycyclic aromatic hydrocarbon isomers in coal tars and combustion products. Environmental Science and Technology 20, 10231027.
  • Rafii, F., Butler, W.R. and Cerniglia, C.E. (1992) Differentiation of a rapidly growing, scotochromogenic, polycyclic-aromatic-hydrocarbon-metabolizing strain of Mycobacterium sp. from other known Mycobacterium species. Archives of Microbiology 157, 512520.
  • Rehmann, K., Noll, H.P., Steinberg, C.E. and Kettrup, A.A. (1998) Pyrene degradation by Mycobacterium sp. strain KR2. Chemosphere 36, 29772992.
  • Rhodes, E.O. (1951) History of changes in chemical composition of creosote. Proceedings of the American Wood-Preservers’ Association 47, 4061.
  • Schneider, J., Grosser, R., Jayasimhulu, K., Xue, W. and Warshawsky, D. (1996) Degradation of pyrene, benz[a]anthracene, and benzo[a] pyrene by Mycobacterium sp. strain RJGII-135, isolated from a former coal gasification site. Applied and Environmental Microbiology 62, 1319.
  • Smucker, S.J. (2000) Region 9 Preliminary Remediation Goals – R9 PRG Tables (www.epa.gov/docs/region09/waste/sfund/prg/files/PRG2000.pdf). San Francisco: USEPA Region 9 Office.
  • Sneath, P.H.A., Mair, N.S., Sharpe, M.E. and Holt, J.G. (eds) (1986) Bergey's Manual of Systematic Bacteriology – Volume 2. Baltimore: Williams & Wilkins.
  • Solano-Serena, F., Marchal, R., Casarégola, S., Vasnier, C., Lebeault, J.M. and Vandecasteele, J.P. (2000) A Mycobacterium strain with extended capacities for degradation of gasoline hydrocarbons. Applied and Environmental Microbiology 66, 23922399.
  • Sutherland, J.B., Rafii, F., Khan, A.A. and Cerniglia, C.E. (1995) Mechanisms of polycyclic aromatic hydrocarbon degradation. In Microbial Transformation and Degradation of Toxic Organic Chemicals. ed. Young, L.Y. and Cerniglia, C.E. pp. 269306. New York: Wiley-Liss.
  • Tsukamura, M., van der Meulen, H.J. and Grabow, W.O.K. (1983) Numerical taxonomy of rapidly growing, scotochromogenic mycobacteria of the Mycobacterium parafortuitum complex: Mycobacterium austroafricanum sp. nov. and Mycobacterium diernhoferi sp. nov., nom. rev. International Journal of Systematic Bacteriology 33, 460469.
  • Van Dyke, M.I. and Prosser, J.I. (2000) Enhanced survival of Pseudomonas fluorescens in soil following establishment of inoculum in a sterile soil carrier. Soil Biology and Biochemistry 32, 13771382.
  • Vila, J., Lopez, Z., Sabate, J., Minguillon, C., Solanas, A.M. and Grifoll, M. (2001) Identification of a novel metabolite in the degradation of pyrene by Mycobacterium sp. strain AP1: Actions of the isolate on two- and three-ring polycyclic aromatic hydrocarbons. Applied and Environmental Microbiology 67, 54975505.
  • Walter, U., Beyer, M., Klein, J. and Rehm, H.J. (1991) Degradation of pyrene by Rhodococcus sp. UW1. Applied Microbiology and Biotechnology 34, 671676.
  • Wang, R.-F., Wennerstrom, D., Cao, W.-W., Khan, A.A. and Cerniglia, C.E. (2000) Cloning, expression, and characterization of the katG gene, encoding catalase-peroxidase, from the polycyclic aromatic hydrocarbon-degrading bacterium Mycobacterium sp. strain PYR-1. Applied and Environmental Microbiology 66, 43004304.
  • Weissenfels, W.D., Beyer, M., Klein, J. and Rehm, H.J. (1991) Microbial metabolism of fluoranthene: isolation and identification of ring-fission products. Applied Microbiology and Biotechnology 34, 528535.