Use of 16S rRNA Gene Terminal Restriction Fragment Analysis To Assess the Impact of Solids Retention Time on the Bacterial Diversity of Activated Sludge

Abstract

ABSTRACT Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes was used to investigate the reproducibility and stability in the bacterial community structure of laboratory-scale sequencing batch bioreactors (SBR) and to assess the impact of solids retention time (SRT) on bacterial diversity. Two experiments were performed. In each experiment two sets of replicate SBRs were operated for a periods of three times the SRT. One set was operated at an SRT of 2 days and another set was operated at an SRT of 8 days. Samples for T-RFLP analysis were collected from the two sets of replicate reactors. HhaI, MspI, and RsaI T-RFLP profiles were analyzed using cluster analysis and diversity statistics. Cluster analysis with Ward's method using Jaccard distance and Hellinger distance showed that the bacterial community structure in both sets of reactors from both experimental runs was dynamic and that replicate reactors were clustered together and evolved similarly from startup. Richness ( S ), evenness ( E ), the Shannon-Weaver index ( H ), and the reciprocal of Simpson's index (1/ D ) were calculated, and the values were compared between the two sets of reactors. Evenness values were higher for reactors operated at an SRT of 2 days. Statistically significant differences in diversity ( H and D ) between the two sets of reactors were tested using a randomization procedure, and the results showed that reactors from both experimental runs that were operated at an SRT of 2 days had higher diversity ( H and D ) at the 5% level. T-RFLP analysis with diversity indices proved to be a powerful tool to analyze changes in the bacterial community diversity in response to changes in the operational parameters of activated-sludge systems.