LAi

11 i-iii

Fig. 9.4 T-RFLP profiles obtained with bacterial (16 S) amplicons from soil polluted with different doses of (heavy metal enriched) pyrite sludge. In the electropherograms, the x-axis marks the size of the fragment while the _y-axis marks the fluorescence intensity of each fragment. T-RF fragment size (OTUs) and abundance change as the heavy metal concentration in the soil increases, implying gradual changes in the soil bacterial community

Fig. 9.4 T-RFLP profiles obtained with bacterial (16 S) amplicons from soil polluted with different doses of (heavy metal enriched) pyrite sludge. In the electropherograms, the x-axis marks the size of the fragment while the _y-axis marks the fluorescence intensity of each fragment. T-RF fragment size (OTUs) and abundance change as the heavy metal concentration in the soil increases, implying gradual changes in the soil bacterial community

and has high running costs, both of which limit its routine use in ecological assessments. In addition, although the species corresponding to each profile can be inferred, it is not possible to directly clone the DNA bands of interest. Despite these limitations, T-RFLP is very useful tool for studying soil microbial diversity (Liu et al. 1997; Tiedje et al. 1999; Osborn et al. 2000). Recently, TRF patterns have been analyzed by matching the resulting profile with an existing database of restriction patterns. However, the association of sequenced clones with patterns in a database is problematic, since related organisms commonly produce TRFs of the same length, requiring several enzyme digests to distinguish among similar community members. In any case, the potential of the T-RLFP method to discriminate between soil microbial communities in polluted and unpolluted soils has been demonstrated. The T-RFLP approach has been applied to a number of marker genes and has been shown to be a powerful method for describing differences and changes in bacterial (Turpeinen et al. 2004, Hartmann et al. 2005; Lazaro et al. 2008) and arbuscular mycorrhizal fungi (Tonin et al. 2001) communities in heavy metal polluted soil.

Singh et al. (2006) developed and validated a rapid and sensitive method that could be used to simultaneously analyze communities of several microbial taxa with one PCR. They called this multiplex terminal restriction fragment length polymorphism (M-TRFLP). Recently, Macdonald et al. (2008) used M-TRFLP to assess the impact of heavy metal contamination on bacterial, fungal, and archaeal communities simultaneously, and T-RFLP to assess the impact of this contamination on actinobacterial populations. They showed that such contamination is responsible for population shifts, and that various genotypic markers (as represented by individual TRFs) within a community differ in their levels and directions of response to increasing metal contamination.

9.6.2.5 Ribosomal Intergenic Spacer Analysis (RISA)

RISA allows ribosomally based fingerprinting of the microbial community. In this method, the intergenic spacer (IGS) region that is located between the small (16 S) and large (23 S) rRNA subunit genes is amplified by PCR, denatured, and resolved by polyacrylamide gel electrophoresis under denaturing conditions. The IGS region varies in both sequence and length (50-1,500 bp) depending on the species, and this unique feature facilitates taxonomic identification of organisms (Spiegelman et al. 2005). In RISA, sequence polymorphisms are detected by silver staining, whereas the forward primer is fluorescently labeled and automatically detected using the automated RISA method (ARISA). Both methods provide highly reproducible bacterial community profiles, but ARISA increases the sensitivity of the method and reduces the time needed to perform it. RISA is a very rapid and simple rRNA fingerprinting method, but its applicability to microbial community analysis from contaminated sources is limited, partly due to the limited database for ribosomal IGS, which is not as large or as comprehensive as the 16 S sequence database (Spiegelman et al. 2005). As a result, community analysis using RISA could reduce its effectiveness at identifying unknown or nonculturable microbial species from contaminated sources. Furthermore, RISA sequence variability may be too great for environmental applications. Its level of taxonomic resolution is greater than 16 S rRNA, which can lead to very complex community profiles.

Ranjard et al. (2006) evaluated the short-term effects of single and combined additions of copper, cadmium, and mercury at different doses on soil bacterial community structure using the bacterial automated ribosomal intergenic spacer analysis (B-ARISA) fingerprinting technique. Their results suggested that there was no simple negative correlation between pollution stresses and genetic diversity in microbial communities. Thus, it is assumed that the increase in stress reduced the innate competitive exclusion of populations and induced the enrichment and predominance of other types of populations, leading to a potential increase in diversity that was followed, when the stress reached a critical level, by the progressive extinction of organisms, leading to a loss of diversity. Gleeson et al. (2005) also demonstrated that an elevated heavy metal concentration had a profound impact on bacterial community structure, and found strong relationships between certain ribo-types and particular chemical/heavy metal elements.

9.6.2.6 Fluorescent In Situ Hybridization

Fluorescent in situ hybridization (FISH) allows the direct identification and quantification of specific and/or general taxonomic groups of microorganisms within their natural microhabitats. In FISH, whole cells are fixed, their 16 S or 23 S rRNA is hybridized with fluorescently labeled taxon-specific oligonucleotide probes, and then the labeled cells are viewed by scanning confocal laser microscopy. This technique allows artifacts arising from biases in DNA extraction, PCR amplification, and cloning to be avoided. FISH can detect microorganisms across all phylogenetic levels, whereas immunofluorescence techniques are limited to the species and subspecies levels. In addition, scanning confocal laser microscopy surpasses epifluorescence microscopy in sensitivity and has the ability to view the distributions of several taxonomic groups simultaneously as a three-dimensional image. The community structures of soils contaminated with low and high levels of metal have been investigated by hybridization with group-specific phylogenetic probes (a-proteobacteria, b-proteobacteria, g-proteobacteria, 8-proteobacteria, Cytophaga-Flexibacter-Bacteroides, Gram-positive bacteria with a low mol %G + C, Gram-positive bacteria with a high mol %G+C) (Sandaa et al. 1999, 2001). The most abundant group of clones in the soil contaminated with a low level of metal was the Cytophaga-Flexibacter Bacteroides group. This group was twice as abundant in the low-level compared to the high-level contaminated soil. In the soil contaminated with a high level of metal, clones belonging to the a-proteobacteria were numerically dominant. With respect to the isolates, 30-37% of them belonged to Gram-positive bacteria with low mol %G+C. In the soil contaminated with a high level of metal, the abundances of isolates and clones belonging to the a-proteobacteria subclass differed markedly, as the percentage of clones was 38%

and that of isolates was only 14%. Analysis with FISH also has been extensively used in other types of studies, such as those aiming at the identification of acido-philes in bioleaching operations or AMD in metal mines (Espejo and Romero 1997; Norris et al. 1996).

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