Culture-dependent and -independent approaches establish the complexity of a PAH-degrading microbial consortium.

A microbial consortium (AM) obtained by sequential enrichment in liquid culture with a polycyclic aromatic hydrocarbon (PAH) mixture of three- and four-ringed PAHs as a sole source of carbon and energy was examined using a triple-approach method based on various cultivation strategies, denaturing gradient gel electrophoresis (DGGE), and the screening of 16S and 18S rRNA gene clone libraries. Eleven different sequences by culture-dependent techniques and seven by both DGGE and clone libraries were obtained. The comparison of three variable regions (V3-V5) of the 16S rRNA gene between the sequences obtained yielded 19 different microbial components. Proteobacteria were the dominant group, representing 83% of the total, while the Cytophaga-Flexibacter-Bacteroides group (CFB) was 11% and the Ascomycota fungi 6%. Beta-proteobacteria were predominant in the DGGE and clone library methods, whereas they were a minority in culturable strains. The highest diversity and number of noncoincident sequences were achieved by the cultivation method that showed members of the alpha-, beta-, and gamma-Proteobacteria; CFB bacterial group; and Ascomycota fungi. Only six of the 11 strains isolated showed PAH-degrading capability. The bacterial strain (AMS7) and the fungal strain (AMF1), which were similar to Sphingomonas sp. and Fusarium sp., respectively, achieved the greatest PAH depletion. The results indicate that polyphasic assessment is necessary for a proper understanding of the composition of a microbial consortium.

[1]  Zhongtang Yu,et al.  Comparisons of Different Hypervariable Regions of rrs Genes for Use in Fingerprinting of Microbial Communities by PCR-Denaturing Gradient Gel Electrophoresis , 2004, Applied and Environmental Microbiology.

[2]  James R. Cole,et al.  The RDP (Ribosomal Database Project) continues , 2000, Nucleic Acids Res..

[3]  A. Venosa,et al.  Selective enumeration of aromatic and aliphatic hydrocarbon degrading bacteria by a most-probable-number procedure. , 1996, Canadian journal of microbiology.

[4]  John Dunbar,et al.  Levels of Bacterial Community Diversity in Four Arid Soils Compared by Cultivation and 16S rRNA Gene Cloning , 1999, Applied and Environmental Microbiology.

[5]  R. Castenholz,et al.  Soil Microbial Community Structure across a Thermal Gradient following a Geothermal Heating Event , 2002, Applied and Environmental Microbiology.

[6]  H. Blöcker,et al.  Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. , 1989, Nucleic acids research.

[7]  J. Ellis-Evans,et al.  Bacterioplankton community diversity in a maritime Antarctic lake, determined by culture-dependent and culture-independent techniques. , 2003, FEMS microbiology ecology.

[8]  T. A. Hall,et al.  BIOEDIT: A USER-FRIENDLY BIOLOGICAL SEQUENCE ALIGNMENT EDITOR AND ANALYSIS PROGRAM FOR WINDOWS 95/98/ NT , 1999 .

[9]  D. Springael,et al.  Isolation of Adherent Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Bacteria Using PAH-Sorbing Carriers , 2000, Applied and Environmental Microbiology.

[10]  M. Bouchez,et al.  Efficiency of defined strains and of soil consortia in the biodegradation of polycyclic aromatic hydrocarbon (PAH) mixtures , 2004, Biodegradation.

[11]  O. Pantos,et al.  Characterization of the bacterial consortium associated with black band disease in coral using molecular microbiological techniques. , 2002, Environmental microbiology.

[12]  O. Ward,et al.  Community dynamics of a mixed-bacterial culture growing on petroleum hydrocarbons in batch culture. , 2000, Canadian journal of microbiology.

[13]  Richard J. Ellis,et al.  Cultivation-Dependent and -Independent Approaches for Determining Bacterial Diversity in Heavy-Metal-Contaminated Soil , 2003, Applied and Environmental Microbiology.

[14]  N. Urano,et al.  Sphingomonas cloacae sp. nov., a nonylphenol-degrading bacterium isolated from wastewater of a sewage-treatment plant in Tokyo. , 2001, International journal of systematic and evolutionary microbiology.

[15]  M. Bailey,et al.  Bacterial Community Structure and Physiological State within an Industrial Phenol Bioremediation System , 2000, Applied and Environmental Microbiology.

[16]  Margaret L. Britz,et al.  Degradation and Mineralization of High-Molecular-Weight Polycyclic Aromatic Hydrocarbons by Defined Fungal-Bacterial Cocultures , 2000, Applied and Environmental Microbiology.

[17]  R. Burns,et al.  Enrichment versus biofilm culture: a functional and phylogenetic comparison of polycyclic aromatic hydrocarbon-degrading microbial communities. , 2002, Environmental microbiology.

[18]  S. Macnaughton,et al.  Microbial Population Changes during Bioremediation of an Experimental Oil Spill , 1998, Applied and Environmental Microbiology.

[19]  L. Forney,et al.  Genetic Diversity through the Looking Glass: Effect of Enrichment Bias , 1997, Applied and environmental microbiology.

[20]  H. Kiyohara,et al.  Rapid Screen for Bacteria Degrading Water-Insoluble, Solid Hydrocarbons on Agar Plates , 1982, Applied and environmental microbiology.

[21]  B. Lal,et al.  Evaluation of Inoculum Addition To Stimulate In Situ Bioremediation of Oily-Sludge-Contaminated Soil , 2001, Applied and Environmental Microbiology.

[22]  A. M. Solanas,et al.  New metabolites in the degradation of fluorene by Arthrobacter sp. strain F101 , 1997, Applied and environmental microbiology.

[23]  M. Bouchez,et al.  Degradation of polycyclic aromatic hydrocarbons by pure strains and by defined strain associations: inhibition phenomena and cometabolism , 1995, Applied Microbiology and Biotechnology.

[24]  D. Fennell,et al.  Detection and Characterization of a Dehalogenating Microorganism by Terminal Restriction Fragment Length Polymorphism Fingerprinting of 16S rRNA in a Sulfidogenic, 2-Bromophenol-Utilizing Enrichment , 2004, Applied and Environmental Microbiology.

[25]  D. Lane.  16S/23S sequencing , 1991 .

[26]  L. Øvreås,et al.  Prokaryotic Diversity--Magnitude, Dynamics, and Controlling Factors , 2002, Science.

[27]  A. Solanas,et al.  Biodegradation of a crude oil by three microbial consortia of different origins and metabolic capabilities , 2002, Journal of Industrial Microbiology and Biotechnology.

[28]  A. Solanas,et al.  Isolation and characterization of a 9-fluorenone-degrading bacterial strain and its role in synergistic degradation of fluorene by a consortium , 1998 .

[29]  K. Schleifer,et al.  Phylogenetic identification and in situ detection of individual microbial cells without cultivation. , 1995, Microbiological reviews.

[30]  A. Solanas,et al.  Enhanced Biodegradation of Casablanca Crude Oil by A Microbial Consortium in Presence of a Rhamnolipid Produced by Pseudomonas Aeruginosa AT10 , 2004, Biodegradation.

[31]  N. Clipson,et al.  Microbial community changes during the bioremediation of creosote‐contaminated soil , 2007, Letters in applied microbiology.

[32]  A. M. Solanas,et al.  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 , 2001, Applied and Environmental Microbiology.

[33]  R. Prince,et al.  Petroleum spill bioremediation in marine environments. , 1993, Critical reviews in microbiology.

[34]  D. Wise.  Bioremediation of Contaminated Soils , 2000 .

[35]  D. T. Gilbson.  Microbial degradation of aromatic compounds. , 1968, Science.

[36]  M. Gealt,et al.  Biodegradation and Bioremediation. , 1996 .

[37]  R. Weller,et al.  Succession and convergence of biofilm communities in fixed-film reactors treating aromatic hydrocarbons in groundwater , 1997, Applied and environmental microbiology.

[38]  D. Bamford,et al.  Comparison of partial 16S rRNA gene sequences obtained from activated sludge bacteria , 1997, Applied Microbiology and Biotechnology.

[39]  P. de Vos,et al.  Synergistic Degradation of Linuron by a Bacterial Consortium and Isolation of a Single Linuron-Degrading Variovorax Strain , 2003, Applied and Environmental Microbiology.

[40]  P. Chapman,et al.  Actions of a versatile fluorene-degrading bacterial isolate on polycyclic aromatic compounds , 1995, Applied and environmental microbiology.

[41]  J. Sutherland,et al.  Detoxification of polycyclic aromatic hydrocarbons by fungi , 2005, Journal of Industrial Microbiology.

[42]  D. Crowley,et al.  Microbial phyllosphere populations are more complex than previously realized , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Kazuya Watanabe,et al.  Rhodanobacter sp. Strain BPC1 in a Benzo[a]pyrene-Mineralizing Bacterial Consortium , 2002, Applied and Environmental Microbiology.

[44]  L. Øvreås,et al.  Microbial Diversity and Community Structure in Two Different Agricultural Soil Communities , 1998, Microbial Ecology.

[45]  Shigeaki Harayama,et al.  Physicochemical Properties and Biodegradability of Crude Oil , 1997 .

[46]  Thomas J McDonald,et al.  Aerobic biodegradation of hopanes and other biomarkers by crude oil-degrading enrichment cultures. , 2002, Environmental science & technology.

[47]  C. Guasp,et al.  Molecular Methods for the Detection and Identification of Pseudomonas stutzeri in Pure Culture and Environmental Samples , 1998, Microbial Ecology.

[48]  A. M. Solanas,et al.  Laboratory-scale bioremediation experiments on hydrocarbon-contaminated soils , 2004 .

[49]  A. Juhasz,et al.  Microbial degradation and detoxification of high molecular weight polycyclic aromatic hydrocarbons by Stenotrophomonas maltophilia strain VUN 10,003 , 2000, Letters in applied microbiology.

[50]  E. Veignie,et al.  Bioremediation of an aged polycyclic aromatic hydrocarbons (PAHs)-contaminated soil by filamentous fungi isolated from the soil , 2004 .

[51]  J. V. van Elsas,et al.  Analysis of the dynamics of fungal communities in soil via fungal-specific PCR of soil DNA followed by denaturing gradient gel electrophoresis. , 2000, Journal of microbiological methods.

[52]  Owen P. Ward,et al.  Recent Advances in Petroleum Microbiology , 2003, Microbiology and Molecular Biology Reviews.

[53]  Gerrit Voordouw,et al.  Composition of Soil Microbial Communities Enriched on a Mixture of Aromatic Hydrocarbons , 2000, Applied and Environmental Microbiology.

[54]  Kazuya Watanabe,et al.  Rapid Mineralization of Benzo[a]pyrene by a Microbial Consortium Growing on Diesel Fuel , 2000, Applied and Environmental Microbiology.