Review articleThe use of molecular techniques to characterize the microbial communities in contaminated soil and water
Introduction
Traditionally, characterization of microbial community composition in contaminated soil and water has been limited to the ability to culture microorganisms from environmental samples. Unfortunately, only a fraction of the microorganisms involved in the biodegradation of contaminants in soil and water can currently be cultured in the laboratory. It has been estimated that the microbial community in one gram of soil may contain over one thousand different bacterial species (Rossello-Mora and Amann, 2001), but less than 1% of these may be culturable. It has been observed that fast growing organisms or strains best adapted to particular culture conditions grow preferentially than those which are not, and therefore do not accurately represent the actual microbial community composition of contaminated environments (Rappe and Giovannoni, 2003, Gilbride et al., 2006). Hence culture-dependent characterization of microorganisms at contaminated sites may limit the scope of microbial biodiversity and the ecological importance of unculturable organisms at contaminated sites may go undetected (Brockman, 1995, Van Hamme et al., 2003, Widada et al., 2002b).
Modern molecular techniques provide an exciting opportunity to overcome the requirement for culturing and have therefore greatly increased our understanding of microbial diversity and functionality in the environment. These methods rely on the characterization of cellular constituents such as nucleic acids, proteins, fatty acids and other taxa-specific compounds (Rossello-Mora and Amann, 2001). Such molecules can be extracted directly from environmental samples without the need for culturing and analysis of the molecular composition can be used to elucidate the composition of the microbial community (Amann et al., 1995, Greene and Voordouw, 2003). Another advantage of culture-independent molecular characterization includes the capacity to preserve in situ metabolic function and the microbial community composition by immediately preserving samples (Moller et al., 1998, Wilson et al., 1999a) or direct extraction of molecules of interest from environmental samples (Tsai and Olson, 1991).
This review is aimed at presenting and evaluating current molecular applications in the assessment of microbial community composition from contaminated soil and water environments in a bid to identify the dominant microbial communities or catabolic genes.
Section snippets
Phospholipids fatty acids (PLFA)
Phospholipids are important components of living cells membranes and constitute a significant proportion of organism biomass under natural conditions (Kozdroj and van Elsas, 2001). Microorganisms have the ability to change the lipid composition of their membranes in response to environmental conditions such as chemical stress (Frostegard et al., 1993) and temperatures fluctuations (Bartlett, 1999). PLFA rapidly degrade upon cell death thus making it good indicator of living organisms (Drenovsky
Polymerase chain reaction (PCR)
The polymerase chain reaction has the ability to produce millions of copies of a portion of a desired gene, entire gene or gene clusters with high fidelity within 3 to 4 h. It is the most widely used method for the amplification of 16S rRNA, or its gene, prior to fingerprinting studies. PCR-based methods have also used in the detection and quantification of microorganisms found in soil and water (Wilson et al., 1999b).
The technique can also be applied for the analysis of catabolic genes
Fluorescent in situ hybridization (FISH)
FISH is a method used to quantify the presence and relative abundance of microbial populations in a community sample. Microbial cells are treated with fixative, hybridized with specific probes (usually 15–25 bp oligonucleotide-fluorescently labelled probes) on a glass slide then visualised with either epiflourescence or confocal laser microscopy (Sanz and Kochling, 2007).
Hybridization with rRNA-targeted probes enhances the characterization of uncultured microorganisms and also facilitates the
Stable isotope probing (SIP)
Stable isotope probing (SIP) enables the characterization or identification of microbial populations actively involved in specific metabolic processes in the environment with the aim of linking the microbial phylogeny with function (Radajewski et al., 2000). SIP involves the incorporation of stable isotope-labelled substrates into cellular biomarkers that can be used to identify organisms assimilating the substrate (Boschker and Middelburg, 2002). Labelled biomarkers such as PLFA (Bull et al.,
Microarray technologies
DNA microarray technology is a very powerful taxonomic and functional tool that is widely used to study biological processes, including mixed microbial communities. This technique is similar to FISH, but provides a means for simultaneous analysis of many genes (Cho and Tiedje, 2002). DNA microarray is a miniaturized array of complementary DNA probes (∼ 500–5000 nucleotides in length) or oligonucleotides (15–70 bp) attached directly to a solid support, which permits simultaneous hybridization of
Conclusion
Culture independent molecular tools applied for the analyses of mixed microbial communities from contaminated soil and water have undoubtedly advanced our knowledge and understanding about microbial diversity and biochemistry of contaminants catabolism. Molecular techniques have contributed significantly to the detection and identification of microorganisms and catabolic genes especially in non-culturable organisms as well as the quantification or enumeration of the relative abundance of
References (169)
- et al.
Phylogenetic diversity of culturable bacteria from alpine permafrost in the Tianshan Mountains, northwestern China
Res Microbiol
(2006) - et al.
Stable isotopes and biomarkers in microbial ecology
FEMS Microbiol Ecol
(2002) - et al.
Phylogenetic analysis of aerobic freshwater and marine enrichment cultures efficient in hydrocarbon degradation: effect of profiling method
J Microbiol Methods
(2000) - et al.
Comparison of phospholipids fatty acid (PLFA) and total soil fatty acid methyl esters (TSFAME) for characterizing soil microbial communities
Soil Biol Biochem
(2004) - et al.
Real-time reverse transcription PCR analysis of expression of atrazine catabolism genes in two bacterial strains isolated from soil
J Microbiol Methods
(2004) - et al.
Exploration of soil bacterial communities for their potential as bioresource
Bioresour Technol
(2006) - et al.
T-RFLP analysis of bacterial communities in cyclodextrin-amended bioreactors developed for biodegradation of polychlorinated biphenyls
Res Microbiol
(2005) - et al.
Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis
Cell
(1979) - et al.
Molecular microbial ecology: land of the one-eyed king
Curr Opin Microbiol
(2004) - et al.
Microbiological changes during bioremediation of explosives-contaminated soils in laboratory and pilot-scale bioslurry reactors
Bioresour Technol
(2004)
DsrB gene-based DGGE for community and diversity surveys of sulfate-reducing bacteria
J Microbiol Methods
Molecular techniques in wastewater: understanding microbial communities, detecting pathogens, and real-time process control
J Microbiol Methods
Analysis of environmental microbial communities by reverse sample genome probing
J Microbiol Methods
Relative importance of nutrients and mortality factors on prokaryotic community composition in two lakes of different trophic status: microcosm experiments
FEMS Microbiol Ecol
Microbial activity and phospholipid fatty acid pattern in long-term tannery waste-contaminated soil
Ecotoxicol Environ Saf
Methods of studying soil microbial diversity
J Microbiol Methods
Comparison of phylogenetic relationships based on phospholipid fatty acid profiles and ribosomal RNA sequence similarities among dissimilatory sulfate-reducing bacteria
FEMS Microbiol Lett
Structural diversity of microorganisms in chemically perturbed soil assessed by molecular and cytochemical approaches
J Microbiol Methods
Evaluation of amplified ribosomal DNA restriction analysis (ARDRA) and species-specific PCR for identification of Bifidobacterium species
Syst Appl Microbiol
Terminal restriction fragment length polymorphism (T-RFLP): an emerging method for characterizing diversity among homologous populations of amplification products
Curr Opin Microbiol
Detection of catabolic genes in indigenous microbial consortia isolated from a diesel-contaminated soil
Bioresour Technol
Taxonomic and functional diversity of pseudomonads isolated from the roots of field-grown canola
FEMS Microbiol Ecol
DGGE/TGGE a method for identifying genes from natural ecosystems
Curr Opin Microbiol
The isotope array, a new tool that employs substrate-mediated labeling of rRNA for determination of microbial community structure and function
Appl Environ Microbiol
Mass spectrometry-based proteomics
Nature
Phylogenetic identification and in situ detection of individual microbial cells without cultivation
Microbiol Rev
Detection and enumeration of aromatic oxygenase genes by multiplex and real-time PCR
Appl Environ Microbiol
Microbial adaptations to the psychrosphere/piezosphere
J Mol Microbiol Biotechnol
A real-time polymerase chain reaction method for monitoring anaerobic, hydrocarbon-degrading bacteria based on a catabolic gene
Environ Sci Technol
Population diversity of Staphylococcus intermedius isolates from various host species: typing by 16S–23S intergenic ribosomal DNA spacer polymorphism analysis
J Clin Microbiol
Comparative analysis of the 16S to 23S Ribosomal intergenic spacer sequences of Bacillus thuringiensis strains and subspecies and of closely related species
Appl Environ Microbiol
Nucleic-acid-based methods for monitoring the performance of in situ bioremediation
Mol Ecol
Detection and classification of atmospheric methane oxidizing bacteria in soil
Nature
Development of a universal microarray based on the ligation detection reaction and 16S rrna gene polymorphism to target diversity of cyanobacteria
Appl Environ Microbiol
Suspension array analysis of 16S rRNA from Fe− and SO(4)2− reducing bacteria in uranium-contaminated sediments undergoing bioremediation
Appl Environ Microbiol
Diversity and characterization of sulfate-reducing bacteria in groundwater at a uranium mill tailings site
Appl Environ Microbiol
Quantitative detection of microbial genes by using DNA microarrays
Appl Environ Microbiol
Analysis of 16S–23S rRNA intergenic spacer of Vibrio cholerae and Vibrio mimicus for detection of these species
Methods Mol Biol
Accessing the black box of microbial diversity and ecophysiology: recent advances through polyphasic experiments
J Environ Sci Health A Tox Hazard Subst Environ Eng
Diversity of Geobacteraceae species inhabiting metal-polluted freshwater lake sediments ascertained by 16S rDNA analyses
Microb Ecol
Nature of polymorphisms in 16S–23S rRNA gene intergenic transcribed spacer fingerprinting of Bacillus and related genera
Appl Environ Microbiol
Assessing terminal restriction fragment length polymorphism suitability for the description of bacterial community structure and dynamics in hydrocarbon-polluted marine environments
Environ Microbiol
Validation of a more sensitive method for using spotted oligonucleotide DNA microarrays for functional genomics studies on bacterial communities
Environ Microbiol
Exploring the metabolic and genetic control of gene expression on a genomic scale
Science
Identification of a complete methane monooxygenase operon from soil by combining stable isotope probing and metagenomic analysis
Environ Microbiol
Assessment of microbial diversity in four southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis
Appl Environ Microbiol
Community analysis of nitrifying bacteria in an advanced and compact Gappei-Johkasou by FISH and PCR-DGGE
Water Sci Technol
Estimation of bacterial cell numbers in humic acid-rich salt marsh sediments with probes directed to 16S ribosomal DNA
Appl Environ Microbiol
Direct profiling of environmental microbial populations by thermal dissociation analysis of native rRNAs hybridized to oligonucleotide microarrays
Appl Environ Microbiol
Application of PCR-DGGE to analyse the yeast population dynamics in slurry reactors during degradation of polycyclic aromatic hydrocarbons in weathered oil
Yeast
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