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Molecular diagnostics of polycyclic aromatic hydrocarbon biodegradation in manufactured gas plant soils

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Abstract

Traditional methods for quantifying specific catabolic bacterial populations underestimate the true population count due to the limitations of the necessary laboratory cultivation methods. Likewise,in situ activity is also difficult to assess in the laboratory without altering the sample environment. To circumvent these problems and achieve a truein situ bacterial population count and activity measurement, new methods based on molecular biological analysis of bacterial nucleic acids were applied to soils heavily contaminated with polycyclic aromatic hydrocarbons (PAH). In addition, a naphthalene-lux reporter system was used to determine bioavailability of naphthalene within these soils. DNA extracted from seven PAH-contaminated soils and hybridized with thenahA gene probe indicated that the naphthalene degradative genes were present in all samples in the range of 0.06 to 0.95 ng/100 µl DNA extract which was calculated to represent 3.2×106 to 1.1×1010 cells/g soil (assuming one copy of these genes per cell).14C-naphthalene mineralization was observed in all contaminated soils with14CO2 mineralization rates ranging from 3.2×10−5 to 304,920.0×10−5 µg g soil−1h−1. Phenanthrene, anthracene, and benzo(a)pyrene were mineralized also in several soils. Messenger RNA transcripts ofnahA were isolated and quantified from 4 soils. Only one soil tested, soil B, was inducible with salicylate above thein situ nahA gene transcript level. Two of the soils, C and G, were already fully inducedin situ. The naphthalene mineralization rate correlated positively with the amount ofnahA gene transcripts present (r=0.99). Naphthalene was bioavailable in soils A, D, E, G, and N as determined by a bioluminescent response from the naphthalene-lux reporter system. Taken together, these data provided information on what the naphthalene-degrading bacterial population was experiencingin situ and what approaches would be necessary to increase activity.

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References

  • Barkay T & Sayler GS (1988) Gene probes as a tool for the detection of specific genomes in the environment. In: Adams WJ, Chapman GA & Landis WG (Eds) Aquatic Toxicology and Hazard Assessment, Vol 10 (pp 29–36). American Society for Testing and Materials, Philadelphia

    Google Scholar 

  • Bauer, JE & Capone DG (1988) Effects of co-occurring aromatic hydrocarbons on the degradation of individual polycyclic aromatic hydrocarbons in marine sediment slurries. Appl. Environ. Microbiol. 54: 1649–1655

    Google Scholar 

  • Blackburn JW (1989) Is there an “uncertainty principle” in microbial waste treatment? Biotreatment of Agricultural Wastewater (pp 149–161). CRC Press, Boca Raton, FLA

    Google Scholar 

  • Burlage RS, Sayler GS & Larimer F (1990) Monitoring of naphthalene catabolism by bioluminescence withnah-lux transcriptional fusions. J. Bacteriol. 172: 4749–4757

    Google Scholar 

  • Cerniglia CE (1981) Aromatic hydrocarbons: metabolism by bacteria, fungi and algae. In: Hodgson E, Bend JR & Philpot RM (Eds) Reviews in Biochemical Toxicology, Vol 3 (pp 321–361). Elsevier Norh Holland, Inc

  • Cerniglia CE (1984) Microbial metabolism of polycyclic aromatic hydrocarbons. Adv. Appl. Microbiol. 30: 31–71

    Google Scholar 

  • Cernigla CE & Heitkamp MA (1989) Microbial degradation of polycyclic aromatic hydrocarbons in the aquatic environment. In: Varanasi U (Ed) Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment (pp 41–68). CRC Press, Boca Raton, FLA

    Google Scholar 

  • Church GM & Gilbert W (1984) Genomic sequencing. Proc. Natl. Acad. Sci. 81: 1991–1995

    Google Scholar 

  • Cushy MA & Morgan DJ (1990) Biological Treatment of Soils Containing Manufactured Gas Plant Residues. Topical Report. Gas Research Institute. Contract No. 5086-254-1334

  • Environmental Research & Technology (1984) Handbook of Manufactured Gas Plant Sites. Prepared for the Utility Solid Waste Activities Group, Superfund Committee, Washington, D.C

  • Fleming J, Sanseverino J & Sayler GS (1993) Quantitative relationship between naphthalene catabolic frequency and expression in predicting PAH degradation in soils at town gas manufacturing sites. Env. Sci. Technol. 27: 1068–1074

    Google Scholar 

  • Gibson DT (Ed) (1984) Microbial Degradation of Organic Compounds. Marcel Dekker, New York

    Google Scholar 

  • Heitkamp MA, Freeman JP & Cerniglia CE (1987) Naphthalene biodegradation in environmental microcosms: Estimates of degradation rates and characterization of metabolites. Appl. Environ. Microbiol 53: 129–136

    Google Scholar 

  • Heitzer A, Webb OF, Thonnard JE & Sayler GS (1992) Specific and quantitative assessment of naphthalene and salicylate bioavailability using a bioluminescent catabolic reporter bacterium. Appl. Environ. Microbiol 58: 1839–1846

    Google Scholar 

  • Herbes SE & Schwall LR (1978) Microbial transformation of polycyclic aromatic hydrocarbons in pristine and petroleum-contaminated sediments. Appl. Environ. Microbiol 35: 306–316

    Google Scholar 

  • Herbes SE (1981) Rates of microbial transformation of polycyclic aromatic hydrocarbons in water and sediments in the vicinity of a coal-coking wastewater discharge. Appl. Environ. Microbiol 41: 20–28

    Google Scholar 

  • Holben WE, Jansson JK, Chelm BK & Tiedje JM (1988) DNA probe method for the detection of specific microorganisms in the soil bacterial community. Appl. Environ. Microbiol. 54: 703–711

    Google Scholar 

  • International Agency for Research on Cancer (1985) Cancer Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man, Vol 35. Polynuclear Aromatic Compounds, Part 4, Bitumens, Coal-tars and Derived Products, Shale Oils and Soots. World Health Organization, Lyon, France

    Google Scholar 

  • Jacobsen CS & Rasmussen OF (1992) Development and application of a new method to extract bacterial DNA from soil based on separation of bacteria from soil with cation-exchange resin. Appl. Environ. Microbiol. 58: 2458–2462

    Google Scholar 

  • Jain RK, Burlage RS & Sayler GS (1988) Methods for detecting recombinant DNA in the environment. CRC Crit. Rev. Microbiol. 8: 33–84

    Google Scholar 

  • Jones KC, Stratford JA, Waterhouse KS, Furlong ET, Giger W, Hites RA, Schaffner C & Jonston AE (1989) Increase in the polynuclear aromatic hydrocarbon content on an agricultural soil over the last century. Environ. Sci. Technol. 23: 95–101

    Google Scholar 

  • Kanige K, Sory M, Delor I, Saegerman C, Limet JN & Cornelis GR (1992) Monitoring ofYersinia enterocolitica in murine and bovine feces on the basis of the chromosomally integratedlux-AB marker gene. Appl. Environ. Microbiol. 58: 1024–1026

    Google Scholar 

  • King JMH, DiGrazia PM, Applegate B, Burlage RB, Sanseverino J, Dunbar P, Larimer F & Sayler GS (1990) Rapid, sensitive bioluminescent reporter technology for naphthalene exposure and biodegradation. Science 249: 778–781

    Google Scholar 

  • Kiyohara H, Nagao K & Yano K (1982) Rapid screen for bacteria degrading water-insoluble, solid hydrocarbons on agar plates. Appl. Environ. Microbiol. 43: 454–457

    Google Scholar 

  • Kukor JJ & Olson RH (1990) Molecular cloning, characterization, and regulation of aPseudomonas pickettii PKO1 gene encoding phenol hydroxylase and expression of the gene inPseudomonas aeruginosa PAO1c. J. Bacteriol. 172: 4624–4630

    Google Scholar 

  • Lane WF & Loehr RC (1992) Estimating the equilibrium aqueous concentrations of polynuclear aromatic hydrocarbons in complex mixtures. Environ. Sci. Technol. 26: 983–990

    Google Scholar 

  • Lee K & Ruby EG (1992) Detection of the light organ symbiont,Vibrio fishceri, in Hawaiian seawater by usinglux gene probes. Appl. Environ. Microbiol. 58: 942–947

    Google Scholar 

  • Leisinger T, Cook AM, Hutter R & Nuesch J (1981) Microbial Degradation of Xenobiotics and Recalcitrant Compounds. Academic Press, London

    Google Scholar 

  • Leisinger T (1983) Microorganisms and xenobiotic compounds. Experentia 39: 1183–1191

    Google Scholar 

  • Menn F-M, Applegate BM & Sayler GS (1993) NAH plasmid-mediated catabolism of anthracene and phenanthrene to naphthoic acids. Appl. Environ. Microbiol. 59: 1938–1942

    Google Scholar 

  • Neilson J, Josephson KL, Pillai SD & Pepper I (1992) Polymerase chain reaction and gene probe detection of the 2,4-dichlorophenoxyacetic acid degradation plasmid, pJP4. Appl. Environ. Microbiol. 58: 1271–1275

    Google Scholar 

  • Oelmuller U, Kruger N, Steinbuchel A & Friedrich CG (1990) Isolation of prokaryotic RNA and detection of specific mRNA with biotinylated probes. J. Microbiol. Methods. 11: 73–84

    Google Scholar 

  • Ogram A, Sayler GS & Barkay T (1987) The extraction and purification of microbial DNA from sediments. J. Microbiol. Methods. 7: 57–66

    Google Scholar 

  • Ogram AV & Sayler GS (1988) The use of gene probes in the rapid analysis of natural microbial communities. J. Ind. Microbiol. 3: 281–292

    Google Scholar 

  • Ogunseitan OA, Delgado IL, Tsai YL & Olson BH (1991) Effect of 2-hydroxybenzoate on the maintenance of naphthalene-degrading pseudomonads in seeded and unseeded soil. Appl. Environ. Microbiol. 57: 2873–2879

    Google Scholar 

  • Ogunseitan OA, Sayler GS & Miller RV (1992) Application of DNA probes to analysis of bacteriophage distribution patterns in the environment. Appl. Environ. Microbiol. 58: 2046–2052

    Google Scholar 

  • Pettigrew CA & Sayler GS (1986) The use of DNA:DNA colony hybridization in the rapid isolation of 4-chlorobiphenyl degradative bacterial phenotypes. J. Microbiol. Methods. 5: 205–213

    Google Scholar 

  • Picard C, Ponsonnet C, Paget E, Nesme X & Simonet P (1992) Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Appl. Environ. Microbiol 58: 2717–2722

    Google Scholar 

  • Rochkind-Dubinsky ML, Sayler GS & Blackburn JW (1987) Microbiological Decomposition of Chlorinated Aromatic Compounds. Marcel Dekker, New York

    Google Scholar 

  • Sanseverino J, Applegate BM, King JMH & Sayler GS (1993) Plasmid-mediated mineralization of naphthalene, phenanthrene, and anthracene. Appl. Environ. Microbiol. 59: 1931–1937

    Google Scholar 

  • Sayler GS, Lund LC, Shiaris MP, Sherrill TW & Perkins RE (1979) Comparative effects of AroClor 1254 (polychlorinated biphenyls) and phenanthrene on glucose uptake by freshwater microbial populations. Appl. Environ. Microbiol. 37: 878–885.

    Google Scholar 

  • Sayler GS, Sherrill TW, Perkins RE, Mallory LM, Shiaris MP & Pederson D (1982) Impact of coal-coking effluent on sediment microbial communities: a multivariate approach. Appl. Environ. Microbiol. 44: 1118–1129

    Google Scholar 

  • Sayler GS, Perkins RE, Sherrill TW, Perkins BK, Reid MC, Shields MS, Kong HL & Davis JW (1983) Microcosm and experimental pond evaluation of microbial community response to synthetic oil contamination in freshwater sediments. Appl. Environ. Microbiol. 46: 211–219

    Google Scholar 

  • Sayler GS & Sherrill TW (1983) Polyaromatic hydrocarbon biodegradation and fate in aquatic sediments. In: Babolini G & Girbino G (Eds) Environment and Lung Disease (pp 184–216). La Grafica Editoriale, Messina, Italy

    Google Scholar 

  • Sayler GS, Shields MS, Tedford ET, Breen A, Hooper SW, Sirotkin KM & Davis JW (1985) Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples. Appl. Environ. Microbiol. 49: 1295–1303

    Google Scholar 

  • Sayler GS, Harris C, Pettigrew C, Pacia D, Breen A & Sirotkin KM. (1986) Evaluating the maintenance and effects of genetically engineered microorganisms. Dev. Ind. Microbiol. 27: 135–149

    Google Scholar 

  • Sayler GS, Fleming J, Applegate B, Werner C & Nikbakht K (1989) Microbial community analysis using environmental nucleic acid extracts. In: Hattori T, Ishida Y, Maruyama Y, Morita RY & Uchida A (Eds) Recent Advances in Microbial Ecology. Tokyo, Japan Scientific Publishers

    Google Scholar 

  • Sayler GS & Layton AC (1990) Environmental application of nucleic acid hybridization. Ann. Rev. Microbiol. 44: 625–648

    Google Scholar 

  • Sayler GS, Nikbakht K, Fleming J & Packard J (1991a) Application of molecular techniques to soil biochemistry. In: Stotzky G & Bollag J-M (Eds) Soil Biochemistry, Vol 7 (pp 131–172). Marcel Dekker, Inc. New York

    Google Scholar 

  • Sayler GS, Fleming JT, Applegate B & Werner C (1991b) In: Wellington EM & van Elsas JD (Eds) Genetic Interactions between Microorganisms in the Natural Environment (pp 237–255). Manchester Press, Manchester, UK

    Google Scholar 

  • Sayler GS & Fox RD (1991) Environmental biotechnology: Perceptions, reality, and applications. In: Sayler GS, Fox R & Blackburn JW (Eds) Environmental Biotechnology for Waste Treatment. Plenum Press, New York

    Google Scholar 

  • Selenska S & Klingmuller W (1992) Direct recovery and molcular analysis of DNA and RNA from soil. Microbial Releases. In press

  • Shaw JJ, Settles LG & Kado CI (1988) Transposon Tn4431 mutagenesis ofXanthomonas campestris pv.campestris: Characterization of a nonpathogenic mutant and cloning of a locus for pathogenicity. Mol. Plant-Microbe Interact. 1: 39–45

    Google Scholar 

  • Sherrill TW (1982) Evaluation of the role of environmental contamination in the microbial degradation of polyaromatic hydrocarbons. Dissertation presented for the Doctor of Philosophy Degree. University of Tennessee, Knoxville, Tennessee

    Google Scholar 

  • Shiaris MP (1989) Seasonal biotransformation of naphthalene, phenanthrene, and benzo(a)pyrene in surficial estuarine sediments. Appl. Environ. Microbiol. 55: 1391–1399

    Google Scholar 

  • Sims RC & Overcash MR (1983) Fate of polynuclear aromatic hydrocarbons (PNAs) in soil-plant systems. Residue Rev. 88: 1–68

    Google Scholar 

  • Smith GB & Tiedje JM (1992) Isolation and characterization of a nitrite reductase gene and its use as a probe for denitrifying bacteria. Appl. Environ. Microbiol. 58: 376–384

    Google Scholar 

  • Tsai YL, Park MJ & Olson BH (1991) Rapid method for direct extraction of mRNA from seeded soils. Appl. Environ. Microbiol. 57: 765–768

    Google Scholar 

  • Tsai YL & Olson BH (1991) Rapid method for direct extraction of DNA from soil and sediments. Appl. Environ. Microbiol. 57: 1070–1074

    Google Scholar 

  • Tsai YL & Olson BH (1992) Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction. Appl. Environ. Microbiol. 58: 2292–2295

    Google Scholar 

  • Tsien H & RS Hanson (1992) Soluble methane monooxygenase component B gene probe for identification of methanotrophs that rapidly degrade trichloroethylene. Appl. Environ. Microbiol. 58: 953–960

    Google Scholar 

  • U.S. EPA. (1985) Survey of Town Gas and By-product Production and Locations in the United States (1880–1950). EPA/600/7-85/004

  • Voordouw G, Voordouw JK, Karkhoff-Schweizer RR, Fedorak PM & Westlake DWS (1991) Reverse sample genome probing, a new technique for identification of bacteria in environmental samples by DNA hybridization and its application to the identification of sulfate-reducing bacteria in oil field samples. Appl. Environ. Microbiol. 57: 3070–3078

    Google Scholar 

  • Yen K-M & Serdar CM (1988) Genetics of naphthalene catabolism in pseudomonads. CRC Crit. Rev. Microbiol. 15: 247–268

    Google Scholar 

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Author notes

  1. J. M. H. King

    Present address: Unilever Research, Port Sunlight Laboratory, L63 3JW, Merseyside, England

Authors and Affiliations

  1. Center for Environmental Biotechnology and the Department of Microbiology, University of Tennessee, 37932, Knoxville, Tennessee, USA

    J. Sanseverino, C. Werner, J. Fleming, B. Applegate, J. M. H. King & G. S. Sayler

  2. Technology Development Division, IT Corporation, 37923, Knoxville, Tennessee, USA

    J. Sanseverino

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Sanseverino, J., Werner, C., Fleming, J. et al. Molecular diagnostics of polycyclic aromatic hydrocarbon biodegradation in manufactured gas plant soils. Biodegradation 4, 303–321 (1993). https://doi.org/10.1007/BF00695976

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