Combination of microautoradiography and fluorescence in situ hybridization for identification of microorganisms degrading xenobiotic contaminants

One of the central topics in environmental bioremediation research is to identify microorganisms that are capable of degrading the contaminants of interest. Here we report application of combined microautoradiography (MAR) and fluorescence in situ hybridization (FISH). The method has previously been used in a number of systems; however, here we demonstrate its feasibility in studying the degradation of xenobiotic compounds. With a model system (coculture of Pseudomonas putida B2 and Sphingomonas stygia incubated with [14C] o‐nitrophenol), combination of MAR and FISH was shown to be able to successfully identify the microorganisms degrading o‐nitrophenol. Compared with the conventional techniques, MAR‐FISH allows fast and accurate identification of the microorganisms involved in environmental contaminant degradation.

[1]  R. Meulenberg,et al.  Degradation of 3-nitrophenol by Pseudomonas putida B2 occurs via 1,2,4-benzenetriol , 1996, Biodegradation.

[2]  G. Muyzer,et al.  Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology , 2004, Antonie van Leeuwenhoek.

[3]  J. Nielsen,et al.  Phylogenetic Identification and Substrate Uptake Patterns of Sulfate-Reducing Bacteria Inhabiting an Oxic-Anoxic Sewer Biofilm Determined by Combining Microautoradiography and Fluorescent In Situ Hybridization , 2002, Applied and Environmental Microbiology.

[4]  A. Hiraishi,et al.  Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses. , 2001, International journal of systematic and evolutionary microbiology.

[5]  M. Wagner,et al.  Nitrification in sequencing biofilm batch reactors: lessons from molecular approaches. , 2001, Water science and technology : a journal of the International Association on Water Pollution Research.

[6]  R Amann,et al.  Ribosomal RNA-targeted nucleic acid probes for studies in microbial ecology. , 2000, FEMS microbiology reviews.

[7]  J. Fuhrman,et al.  Marine Planktonic Archaea Take Up Amino Acids , 2000, Applied and Environmental Microbiology.

[8]  I. Head,et al.  Use of Combined Microautoradiography and Fluorescence In Situ Hybridization To Determine Carbon Metabolism in Mixed Natural Communities of Uncultured Bacteria from the GenusAchromatium , 2000, Applied and Environmental Microbiology.

[9]  J. Nielsen,et al.  Studies on the in situ physiology of Thiothrix spp. present in activated sludge. , 2000, Environmental microbiology.

[10]  M. Cottrell,et al.  Natural Assemblages of Marine Proteobacteria and Members of the Cytophaga-Flavobacter Cluster Consuming Low- and High-Molecular-Weight Dissolved Organic Matter , 2000, Applied and Environmental Microbiology.

[11]  Barbara A. Bekins,et al.  Microbial populations in contaminant plumes , 2000 .

[12]  E. Madsen Nucleic-acid characterization of the identity and activity of subsurface microorganisms , 2000 .

[13]  I. Head,et al.  Substrate Uptake by Uncultured Bacteria from the Genus Achromatium Determined by Microautoradiography , 1999, Applied and Environmental Microbiology.

[14]  J. Fuhrman,et al.  Microbial Desulfurization of a Crude Oil Middle-Distillate Fraction: Analysis of the Extent of Sulfur Removal and the Effect of Removal on Remaining Sulfur , 1999, Applied and Environmental Microbiology.

[15]  T. Marsh Terminal restriction fragment length polymorphism (T-RFLP): an emerging method for characterizing diversity among homologous populations of amplification products. , 1999, Current opinion in microbiology.

[16]  MacGregor Molecular approaches to the study of aquatic microbial communities , 1999, Current opinion in biotechnology.

[17]  G. Muyzer DGGE/TGGE a method for identifying genes from natural ecosystems. , 1999, Current opinion in microbiology.

[18]  K. Schleifer,et al.  Combination of Fluorescent In Situ Hybridization and Microautoradiography—a New Tool for Structure-Function Analyses in Microbial Ecology , 1999, Applied and Environmental Microbiology.

[19]  M. Wagner,et al.  Use of microautoradiography and fluorescent in situ hybridization for characterization of microbial activity in activated sludge , 1999 .

[20]  P. Nielsen,et al.  In situ characterization of substrate uptake by Microthrix parvicella using microautoradiography , 1998 .

[21]  Robert J. Seviour,et al.  Variability of type 021N in activated sludge as determined by in situ substrate uptake pattern and in situ hybridization with fluorescent rRNA targeted probes , 1998 .

[22]  D. Hahn,et al.  Analysis of bacterial community structure in bulk soil by in situ hybridization , 1997, Archives of Microbiology.

[23]  P. Nielsen,et al.  Application of microautoradiography to the study of substrate uptake by filamentous microorganisms in activated sludge , 1997, Applied and environmental microbiology.

[24]  D. Balkwill,et al.  Taxonomic study of aromatic-degrading bacteria from deep-terrestrial-subsurface sediments and description of Sphingomonas aromaticivorans sp. nov., Sphingomonas subterranea sp. nov., and Sphingomonas stygia sp. nov. , 1997, International journal of systematic bacteriology.

[25]  D. Balkwill,et al.  Aromatic-degrading Sphingomonas isolates from the deep subsurface , 1995, Applied and environmental microbiology.

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

[27]  K. Schleifer,et al.  Phylogenetic Oligodeoxynucleotide Probes for the Major Subclasses of Proteobacteria: Problems and Solutions , 1992 .

[28]  D A Stahl,et al.  Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology , 1990, Journal of bacteriology.

[29]  J. Zeyer,et al.  Purification and characterization of a bacterial nitrophenol oxygenase which converts ortho-nitrophenol to catechol and nitrite , 1988, Journal of bacteriology.

[30]  K. Timmis,et al.  Influence of para-substituents on the oxidative metabolism of o-nitrophenols by Pseudomonas putida B2 , 1986, Applied and environmental microbiology.

[31]  P. C. Kearney,et al.  Degradation of o-nitrophenol and m-nitrophenol by a Pseudomonas putida , 1984 .