Microbial Community Analysis

Fifty-six RFLP types (OTUs) were identified based on the results of cluster analysis. Diversity indices revealed that bacterial communities of mature and old granules were more diverse than in young granules. Shifts in microbial populations were also confirmed by ARDRA. Such changes were attributed to physiological adaptation of bacteria during aerobic granulation process. Microorganisms associated with 5 OTUs (A, B, C, D, and E) appeared in all three clone libraries at different growth stages, which suggests that these bacteria may have an important role in the development of aerobic granules.

Shifts in bacterial community were observed in three clone libraries (Fig. 7.3). For example, 46 RFLP types detected were unique to each clone library (13 in young granules, 17 in mature granules, and 16 in old granules). Different RFLP types were also observed to dominate different clone libraries. In young granules, RFLP types A, B, C, T, and U were the most frequently occurring, but were not uniformly abundant throughout the different growth stages. In mature granules, the most commonly occurring RFLP types were A, B, C, D, T, and V. RFLP types B, AN, AO, AP, AQ, and AR dominated the old granules.

Several RFLP types were also found in more than one clone library. Eight RFLP types (A, B, C, D, E, S, T, and U) from the young granules were also found in the mature granules. Seven types (A, B, C, D, E, AM, and AN) from the mature granules were also found in the old granules. Five RFLP types, A, B, C, D, and E, appeared in all three libraries, which suggest that these 5 RFLP types may have important roles in the development of aerobic granules. The relative abundance of RFLP type A decreased from young to mature to old granules. On the other hand, the relative abundance of RFLP types B, C, and D increased slightly from young to mature granules and decreased significantly in old granules. The relative abundance of RFLP type E did not change from young to mature to old granules. This finding is important since the changes in relative abundance may reflect the onset of granule lysis.

As a consequence, the clone libraries should not be viewed as quantitative representations of microbial abundance in the original community. Nevertheless, some researchers (Farrelly et al., 1995; Suzuki et al., 1996) have demonstrated that changes on the composition of clone libraries can signal temporal (or spatial) variations within identical environmental matrices, and therefore should represent qualitative changes in the microbial community. Results obtained in this study showed significant differences in clone abundance at different growth stages of aerobic granules, which probably reflected relative changes in abundance of that gene or organism in the original community.

In this study, ARDRA was chosen as an initial measure of genetic diversity within each clone library and the response of the microbial community during granule development. Community changes in bacterial composition and the relative abundance would be due to interactions among different groups of bacteria and the microniches in which they reside. Community changes are therefore a consequence of the natural phenomenon of physiological adaptation by bacteria to the surrounding environment and their mutual interactions.

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