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.

Results of mathematical modeling and whole cell 16S ribosomal RNA-targeted fluorescence in situ hybridizations challenge the widely held perception that microbial populations in “steady-state” activated sludge systems share a common net growth rate that is proportional to the inverse of the mean cell residence time. Our results are significant because they encourage bioprocess engineers to appreciate the differences in growth physiology among individual microbial populations in complex mixed microbial communities such as suspended growth activated sludge bioreactor systems.

Conventional activated sludge systems require bacteria to grow to avoid washout through decay and routine solids wasting. Recently we developed a procedure targeting precursor 16S ribosomal RNA to measure the in situ growth activity of phylogenetically defined microbial populations, and this procedure was used to study the growth of bacteria in activated sludge systems. The current study significantly expands this previous work by quantifying levels of precursor 16S ribosomal RNA within individual cells of pure cultures of bacteria exposed to various culture conditions. Initially, three ranges (Type I, Type II, and Type III) of precursor 16S ribosomal RNA levels were defined by whole cell fluorescence in situ hybridization of a pure culture of Acinetobacter calcoaceticusT prepared in three culture conditions. Low levels of precursor 16S ribosomal RNA (Type I) corresponded to a stationary phase culture prepared overnight in Luria-Bertani medium. Intermediate levels of precursor 16S ribosomal RNA (Type II) corresponded to a culture transferred into fresh Luria-Bertani medium, and high levels of precursor 16S ribosomal RNA (Type III) corresponded to a culture treated with the growth inhibiting antibiotic chloramphenicol. Subsequently, the abundance of individual cells of each Type were measured in four different pure cultures after exposure to 0.45-microm filtered primary effluent collected from four different conventional activated sludge treatment plants in Cincinnati, OH, USA. Individual cells of each Type were observed in the culture of A. calcoaceticusT exposed to each of the four primary effluents. Only Type I cells were observed in cultures of A. johnsoniiT, A. johnsonii strain 210a, and Escherichia coliT exposed to each of the four primary effluents. These results suggest that the growth of A. calcoaceticusT was inhibited by an unidentified component of filtered primary effluent present in each of the four wastewaters; whereas the growth of A. johnsoniiT, A. johnsonii strain 210a, and E. coliT were not inhibited. These results have significance for understanding the growth of phylogenetically defined microbial populations within activated sludge treatment systems. If the pattern of elevated p16S rRNA levels observed in A. calcoaceticusT is prevalent in many microbial populations in activated sludge systems, this may have implications for preventing washout of critical microbial populations that may be experiencing growth inhibition.

IMPACT OF INFLUENT MICROORGANISMS UPON POOR SOLIDS SEPARATION IN THE QUIESCENT ZONE OF AN INDUSTRIAL WASTEWATER TREATMENT SYSTEMConventional and molecular biology tools were employed to diagnose the biological origin of poor sludge settling in the secondary clarifier of an industrial activated sludge system treating a waste stream from a dairy food processing plant. Gram and acridine orange staining indicated that viable, Gram-positive microorganisms were present in samples removed from the influent waste stream and...Author(s)D.B. OertherP.G. StrootR. ButlerS. GelmanI. BeydilliS. DudleyJ.M. SimpsonSourceProceedings of the Water Environment FederationSubjectSession 41 - Research Symposium: Chemical/Physical ProcessesDocument typeConference PaperPublisherWater Environment FederationPrint publication date Jan, 2002ISSN1938-6478SICI1938-6478(20020101)2002:13L.104;1-DOI10.2175/193864702784162840Volume / Issue2002 / 13Content sourceWEFTECFirst / last page(s)104 - 115Copyright2002Word count269