Amplified Ribosomal DNA Restriction Analysis ARDRA

The amplified 16S rDNA, full-length inserts 1500 bp), were selected for restriction enzyme digestion to define restriction fragment length polymorphism (RFLP) patterns using restriction endonuclease Cfol (Promega, USA). To determine the similarities of bacterial populations present in three clone libraries, comparisons of the RFLP types within and among each of clone libraries were performed using GelCompar II (version 1.5) software. The constituent populations in the aerobic granules were defined in terms of operational taxonomic units (OTUs). The relative abundance of individual clones within each OTU was also assessed.

There was considerable diversity in each clone library and there were variations in microbial diversity among the three different clone libraries. This suggests a shift in the composition of the microbial communities. Microorganisms associated with five restriction fragment length polymorphism (RFLP) types (A, B, C, D, and E) appeared to play an important role in the development of aerobic granules.

A total of 144 clones containing the full-length inserts (~1500bp), were digested with restriction enzyme Cfol, which has been shown to be particularly effective at defining operational taxonomic units (OTUs) (Moyer et al., 1996; Chandler et al., 1997). Cfol digestion of full-length inserts resulted in 2-6 easily resolved bands, which were used for RFLP cluster analysis (data not shown). The results of 16S rDNA clone library and ARDRA are summarized in Table 7.1.

A total of 56 different RFLP types (OTUs), were identified from the three clone libraries. 21 RFLP types were detected from 45 clones derived from young granules, 27 types were identified from 52 clones from mature granules, and 23 types were recognized from 47 clones from old granules.

Table 7.1. Summary of 16S rDNA clones and RFLP types recovered from aerobic granule samples

Table 7.1. Summary of 16S rDNA clones and RFLP types recovered from aerobic granule samples

Growth stages

Full-length clones

RFLP types

Unique clones

Coverage %



















^ <<<<<<<<^<<<<<<<<<<«<5<<<miDiDiii

E3 Young

25 20 15 10

25 20 15 10


^ <<<<<<<<^<<<<<<<<<<<<<<<<<mmmm

M Mature

<mo ow IL<3x — -J -J gzo occ toi-s> ax> N<m oo wIL &x-j T* jg zoocc to i-3>5 x>N <mo Q

S Old

Fig. 7.3. Distribution and relative abundance of 16S rDNA clones from different growth stages of aerobic granules.

The distribution of clones in each clone library, both in RFLP types and relative abundance is plotted in Fig. 7.3.

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