Non-random patterns of activated sludge floc assembly

AYARZA, JOAQUIN; GUERRERO, LEANDRO; ERIJMAN, LEONARDO

Aalborg, Dinamarca

Congreso; Microbial Population Dynamics in Biological Wastewater Treatment (ASPD 5); 2009

International Water Association

Efficient aggregation is essential to the activated sludge process, since the operational success depends largely on good settling sludge. The continuous recycle in conventional activated sludge, and the repetition of the settling process in sequential batch reactors, select for microorganisms living in flocs. Yet activated sludge systems with stable performance exhibit pronounced fluctuations in species composition, revealing that the systems are not at equilibrium. Research from several laboratories showed that lab-scale replicate reactors frequently diverge on a temporal scale, the degree of divergence depending upon the severity of perturbation to which they are exposed. However, even in the absence of external disturbances activated sludge communities are constantly changing. It has been recently shown that industrial wastewater imposes a selective pressure upon the bacterial communities within bioreactors, which can influence the taxa turnover of the taxa-time relationship. The question remains as whether the shifting patterns observed, especially when selective pressure is low (e.g. with domestic wastewater) arise from the recruitment of different populations of bacteria that are drawn stochastically from a pool of redundant species, or whether there are assembly rules, which albeit unknown, determine the observed patterns. To approach this question we investigated, using appropriate statistical tests, whether the changing patterns of community structure, observed during floc development in replicate reactors, were significantly different from what might be expected by stochastic assembly from the same source of species. In order to study the dynamics of flocculation it was necessary to disrupt the activated sludge flocs into smaller particles and to enrich the biomass in planktonic populations, while maintaining the richness of the original sample. Sludge was collected from the aeration basin of a full-scale domestic wastewater treatment plant (WWTP). After vigorous vortexing, samples were incubated at 20 ºC for 7 days, with daily addition of synthetic sewage (SS), without biomass wasting. The non-flocculent biomass suspension was then diluted four-fold with SS and used to seed laboratory-scale bioreactors. Reactors A (two replicates) were operated as chemostats, with the hydraulic retention time (HRT) equal to the solid retention time (SRT) of 2 days. Reactors B and C were operated in a sequential batch mode with biomass retention, which included settling and decanting phases. In these cases HRT were 2 days and SRT were set at 4 days (four replicates) and 6 days (two replicates). Microscopic imaging showed that the size of flocs in reactors with biomass retention increased during the first few days until a steady state size was reached. The diversity and community structure of the sludge in all reactors were analyzed during a period of up to 10 SRT using denaturing gradient gel electrophoresis (DGGE) of RT-PCR amplified, as well as PCR amplified rDNA 16S genes. Analysis of rRNA was selected in order to reduce the interference from nonactively growing bacteria in the community. High rates of change in DGGE profiles from consecutive sampling points (moving window analysis) suggested a high level of dynamics in all reactors. This conclusion was confirmed by the application of the Raup and Crick probability-based similarity index (Src) for the comparison of RNA based fingerprinting patterns, which indicated that bacterial communities within reactors were not significantly similar after 3 SRT (Src<0.7) and became significantly dissimilar after 5 SRT (Src<0.05). More importantly, significant similarity among replicate reactors were observed at all times analyzed (Src>0.95). Identical results were obtained for the analysis based on rDNA profiles. Therefore, in the absence of external perturbations, bacterial communities shift in the temporal scale, reflecting the “internal dynamics” of the populations, and the fact that the patterns between replicates were more reproducible than expected by chance under highly dynamic conditions allowed us to reject the null hypothesis that activated sludge floc communities have assembled neutrally. We suggest that communities progressively recruit from the available pool of bacterial species, each with particular ecological requirements that determine their time of emergence into the community. It is conceivable that reproducibility in closed laboratory systems may be constrained by the limited pool of potential invasive species. In that regard, it is important to note that our bioreactors are open, and located inside a temperature-controlled cabinet (a BOD chamber) and thus behave as neighbor islands where exchange of species is possible. Therefore, the observation of assembly rules in these lab-scale communities might depend upon the possibility of re-immigration (from neighbor reactors) of taxa, which may have eventually fluctuated to become locally extinct. This dependency on reimmigration may contribute to differences reported from similar experiments in other laboratories, and also may explain why the bacterial diversity of small closed biological treatment communities are often very unstable, as opposed to large biological treatment systems.