One culture-independent method is the PCR-cloning method which includes the following steps: (1) isolating native DNA from the natural community; (2) PCR amplification of small subunit (SSU) rRNA gene sequences with universal primers; (3) screening of clones for genetic variability; and (4) using these detected variations to estimate genetic diversity and to select clones for subsequent sequencing to determine phylogenic affiliation. For screening SSU rDNA clone libraries, the amplified ribosomal DNA restriction analysis (ARDRA) of 16S rRNA genes has become an effective strategy to identify putative operational taxonomic units (OTUs) in various microbial communities such as those from hydrothermal vent system (Moyer et al., 1994); subsurface soil (Chandler et al., 1997); marine sediment (Urakawa et al., 1999); contaminated aquifer (Dojka et al., 1998); and also in activated sludge wastewater treatment plants (Gich et al., 2000).
In general, ARDRA is an effective strategy to provide a general view of how the clone libraries differ and to identify important OTUs for further analysis (Moyer et al., 1994; Chandler et al., 1997; Urakawa et al., 1999; Gich et al., 2000). Estimating community structure and diversity at the DNA level is an invaluable tool for microbial ecology, but this strategy also has its potential problems and limitations (von Wintzingerode et al., 1997; Head et al., 1998). Although the cloning-sequencing method requires considerable time and effort, it can provide new insights and a better understanding of the phylogenetic diversity of different microbial habitats.
Various diversity indices can be used to compare the bacterial communities associated with the three clone libraries. Species richness, which represents the total number of species or operational taxonomic units (OTUs), was calculated by rarefaction (Cho and Kim, 2000) with the online Rarefaction Calculator (http://gause.biology.ualberta.ca/jbrzusto/ rarefact.html). Bacterial diversity was calculated on the basis of RFLP types by using the Shannon-Weaver index (H), Pielou's evenness index (e), Simpson's dominance index (c), and equitability (J ). The estimated percentages of coverage (Dang and Lovell, 2000) for the different libraries were calculated as follows: [1-(n/N)] x 100, where n is the number of unique clones detected in a subsample (library) of size N.
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