Mycobacterium isolates obtained from PAH-contaminated and uncontaminated matrices were evaluated for their ability to degrade three-, four- and five-ring PAHs. PAH enrichment studies were prepared using pyrene and inocula obtained from manufacturing gas plant (MGP) soil, uncontaminated agricultural soil, and faeces from Macropus fuliginosus (Western Grey Kangaroo). Three pyrene-degrading microorganisms isolated from the corresponding enrichment cultures had broad substrate ranges, however, isolates could be differentiated based on surfactant, phenol, hydrocarbon and PAH utilisation. 16S rRNA analysis identified all three isolates as Mycobacterium sp. The Mycobacterium spp. could rapidly degrade phenanthrene and pyrene, however, no strain had the capacity to utilise fluorene or benzo[a]pyrene. When pyrene mineralisation experiments were performed, 70–79% of added 14C was evolved as 14CO2 after 10 days. The present study demonstrates that PAH degrading microorganisms may be isolated from a diverse range of environmental matrices. The present study demonstrates that prior exposure to PAHs was not a prerequisite for PAH catabolic activity for two of these Mycobacterium isolates. 1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants that have been widely distributed as a result of anthropogenic activities including the combustion of fossil fuels and organic matter, coal liquefaction and gasification processes, oil seepage, and accidental spillage of hydrocarbons [1, 2]. Due to their acute toxicity and/or mutagenic, teratogenic, or carcinogenic properties [3–5], there is toxicological concern about the presence of PAHs in the environment. As a result, the need to develop inexpensive and practical remediation technologies for PAH-contaminated soil is evident. Over the past 20 years, bioremediation has been promoted as a potential economic strategy for the remediation of PAH-contaminated soil. A wealth of information has accumulated in the scientific literature on bacterial PAH degradation including rate and extent of degradation, metabolic pathways, molecular mechanisms of PAH degradation, and application of these organisms to bioremediation strategies. A number of papers have reviewed the catabolic diversity of bacterial, fungal, and algal degradation of PAHs [6–8], whilst the review of Juhasz and Naidu [2] focussed on the microbial degradation of benzo[a]pyrene. It is apparent from these reviews and earlier suggestions by Kastner et al. [9], that nocardioform bacteria, in particular Mycobacteria, may play a crucial
References
[1]
W. F. Guerin and G. E. Jones, “Two stage mineralization of phenanthrene by estuarine enrichment cultures,” Applied and Environmental Microbiology, vol. 54, no. 4, pp. 929–936, 1988.
[2]
A. L. Juhasz and R. Naidu, “Bioremediation of high molecular weight polycyclic aromatic hydrocarbons: A review of the microbial degradation of benzo(a)pyrene,” International Biodeterioration and Biodegradation, vol. 45, no. 1-2, pp. 57–88, 2000.
[3]
W. Lijinsky, “The formation and occurrence of polynuclear aromatic hydrocarbons associated with food,” Mutation Research, vol. 259, no. 3-4, pp. 251–261, 1991.
[4]
D. H. Phillips, “Fifty years of benzo[a]pyrene,” Nature, vol. 303, no. 5917, pp. 468–472, 1983.
[5]
R. C. Sims and M. R. Overcash, “Fate of polynuclear aromatic compounds (PNAs) in soil-plant systems,” Residue Reviews, vol. 88, pp. 1–68, 1983.
[6]
C. E. Cerniglia, “Biodegradation of polycyclic aromatic hydrocarbons,” Biodegradation, vol. 3, no. 2-3, pp. 351–368, 1992.
[7]
R. Kanaly and S. Harayama, “Biodegradation of high-molecular weight polycyclic aromatic hydrocarbons by bacteria,” Journal of Bacteriology, vol. 182, no. 8, pp. 2059–2067, 2000.
[8]
S. C. Wilson and K. Jones, “Bioremediation of soil contaminated with polynuclear aromatic hydrocarbons (PAHs): a review,” Environmental Pollution, vol. 81, no. 3, pp. 229–249, 1993.
[9]
M. Kastner, M. Breuer-Jammali, and B. Mahro, “Enumeration and characterization of the soil microflora from hydrocarbon-contaminated soil sites able to mineralize polycyclic aromatic hydrocarbons (PAH),” Applied Microbiology and Biotechnology, vol. 41, no. 2, pp. 267–273, 1994.
[10]
R. Margesin, D. Labbe, F. Schninner, C.W. Greer, and L.G. Whyte, “Characterisation of hydrocarbon-degrading microbial populations in contaminated and pristine contaminated soils,” Applied and Environmental Microbiology, vol. 69, pp. 3985–3092, 2003.
[11]
R. A. Kanaly, R. Bartha, K. Watanabe, and S. Harayama, “Rapid mineralization of benzo[a]pyrene by a microbial consortium growing on diesel fuel,” Applied and Environmental Microbiology, vol. 66, no. 10, pp. 4205–4211, 2000.
[12]
A. L. Juhasz and R. Naidu, “Enrichment and isolation of non-specific aromatic degraders from unique uncontaminated (plant and faecal material) sources and contaminated soils,” Journal of Applied Microbiology, vol. 89, no. 4, pp. 642–650, 2000.
[13]
A. L. Juhasz, M. L. Britz, and G. A. Stanley, “Degradation of fluoranthene, pyrene, benz[a]anthracene and dibenz[a,h]anthracene by Burkholderia cepacia,” Journal of Applied Microbiology, vol. 83, no. 2, pp. 189–198, 1997.
[14]
C. E. Dandie, S. M. Thomas, R. H. Bentham, and N. C. McClure, “Physiological characterization of Mycobacterium sp. strain 1B isolated from a bacterial culture able to degrade high-molecular-weight polycyclic aromatic hydrocarbons,” Journal of Applied Microbiology, vol. 97, no. 2, pp. 246–255, 2004.
[15]
W. G. Weisburg, S. M. Barns, D. A. Pelletier, and D. J. Lane, “16S ribosomal DNA amplification for phylogenetic study,” Journal of Bacteriology, vol. 173, no. 2, pp. 697–703, 1991.
[16]
W. G. COCHRAN, “Estimation of bacterial densities by means of the "most probable number",” Biometrics, vol. 6, no. 2, pp. 105–116, 1950.
[17]
USEPA, “Polynuclear aromatic hydrocarbons method 8100,” in Us Environmental Protection Agency Methods, pp. 8101–8110, 1996.
[18]
P. Cheung and B. Kinkle, “Mycobacterium diversity and pyrene mineralization in petroleum contaminated soils,” Applied and Environmental Microbiology, vol. 67, no. 5, pp. 2222–2229, 2001.
[19]
N. M. Leys, A. Ryngaert, L. Bastiaens et al., “Occurrence and community composition of fast-growing Mycobacterium in soils contaminated with polycyclic aromatic hydrocarbons,” FEMS Microbiology Ecology, vol. 51, no. 3, pp. 375–388, 2005.
[20]
D. Dean-Ross and C. E. Cerniglia, “Degradation of pyrene by Mycobacterium flavescens,” Applied Microbiology and Biotechnology, vol. 46, no. 3, pp. 307–312, 1996.
[21]
B. Boldrin, A. Tiehm, and E. Fritzsche, “Degradation of phenanthrene, fluorene, fluoranthene, and pyrene by amycobacterium sp,” Applied and Environmental Microbiology, vol. 59, no. 6, pp. 1927–1930, 1993.
[22]
T. Schr？der, G. Schuffenhauer, B. Sielaff, and J. R. Andreesen, “High morpholine degradation rates and formation of cytochrome P450 during growth on different cyclic amines by newly isolated Mycobacterium sp. strain HE5,” Microbiology, vol. 146, no. 5, pp. 1091–1098, 2000.
[23]
J. D. Band, J. I. Ward, D. W. Fraser, et al., “Peritonitis due aMycobacterium chelonei-like organism associated with intermittent chronic peritoneal dialysis,” Journal of Infectious Diseases, vol. 145, no. 1, pp. 9–17, 1982.
[24]
C. D. Miller, K. Hall, Y. N. Liang et al., “Isolation and characterization of polycyclic aromatic hydrocarbon-degrading mycobacterium isolates from soil,” Microbial Ecology, vol. 48, no. 2, pp. 230–238, 2004.
[25]
M. A. Heitkamp, W. Franklin, and C. E. Cerniglia, “Microbial metabolism of polycyclic aromatic hydrocarbons: isolation and characterization of a pyrene-degrading bacterium,” Applied and Environmental Microbiology, vol. 54, no. 10, pp. 2549–2555, 1988.
[26]
R. Van Herwijnen, B. Joffe, A. Ryngaert et al., “Effect of bioaugmentation and supplementary carbon sources on degradation of polycyclic aromatic hydrocarbons by a soil-derived culture,” FEMS Microbiology Ecology, vol. 55, no. 1, pp. 122–129, 2006.
[27]
J. E. Bauer and D. G. Capone, “Effects of co-occurring aromatic hydrocarbons on degradation of individual polycyclic aromatic hydrocarbons in marine sediment slurries,” Applied and Environmental Microbiology, vol. 54, no. 7, pp. 1649–1655, 1988.
[28]
R. J. Grosser, D. Warshawsky, and J. R. Vestal, “Indigenous and enhanced mineralization of pyrene, benzo[a]pyrene, and carbazole in soils,” Applied and Environmental Microbiology, vol. 57, no. 12, pp. 3462–3469, 1991.
[29]
M. A. Heitkamp and C. E. Cerniglia, “Mineralization of polycyclic aromatic hydrocarbons by a bacterium isolated from sediment below an oil field,” Applied and Environmental Microbiology, vol. 54, no. 6, pp. 1612–1614, 1988.
[30]
R. Y. Hamzah and B. S. Al-Baharna, “Catechol ring-cleavage in Pseudomonas cepacia: the simultaneous induction of ortho and meta pathways,” Applied Microbiology and Biotechnology, vol. 41, no. 2, pp. 250–256, 1994.
[31]
Y. H. Kim, J. D. Moody, J. P. Freeman, B. Brezna, K-H Engesser, and C. E. Cerniglia, “Evidence for the existence of PAH-quinone reductase and catechol-O-methyltransferase in Mycobacterium vanbaalenii PYR-1,” Journal of Industrial Microbiology and Biotechnology, vol. 31, no. 11, pp. 507–516, 2004.
[32]
M. A. Heitkamp, J. Freeman, D. Miller, and C. E. Cerniglia, “Pyrene degradation by a Mycobacterium sp: identification of ring oxidation and ring fission products,” Applied and Environmental Microbiology, vol. 54, no. 10, pp. 2556–2565, 1988.
[33]
I. Y. Jimenez and R. Bartha, “Solvent-augmented mineralization of pyrene by a Mycobacterium sp,” Applied and Environmental Microbiology, vol. 62, no. 7, pp. 2311–2316, 1996.
[34]
J. Schneider, R. Grosser, K. Jayasimhulu, H. Weiling, and D. Warshawsky, “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, vol. 62, no. 1, pp. 13–19, 1996.
[35]
J. L. Bolton, M. A. Michael, T. M. Penning, G. Dryhurst, and T. J. Monks, “Role of quinones in toxicology,” Chemical Research in Toxicology, vol. 13, no. 3, pp. 135–160, 2000.
