Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure

Abstract

Bacterial community structures in activated sludge samples from aeration tanks of a two-stage system with a high-load first stage and a low-load second stage were analyzed with oligonucleotide probes. The probes were complementary to conserved regions of the rRNA of the alpha, beta, and gamma subclasses of proteobacteria and of all bacteria. Group-specific cell counts were determined by in situ hybridization with fluorescent probe derivatives. Contributions of the proteobacterial subclasses to total bacterial rRNA were quantified by dot blot hybridization with digoxigenin-labeled oligonucleotides. The activated sludge samples were dominated by proteobacteria from the alpha, beta, or gamma subclass. These proteobacteria account for about 80% of all active bacteria found in the activated sludge. For both samples the community structures determined with molecular techniques were compared with the composition of the heterotrophic saprophyte flora isolated on nutrient-rich medium. Probes were used to rapidly classify the isolates and to directly monitor population shifts in nutrient-amended, activated sludge samples. The rich medium favored growth of gamma-subclass proteobacteria (e.g., enterobacteria) and selected against beta-subclass proteobacteria. The culture-dependent community structure analysis of activated sludge produced partial and heavily biased results. A more realistic view will be obtained by using in situ techniques.

Occurrence and diversity of bacterial communities in Tuber magnatum during truffle maturation

“…The comparison of the quantitative and qualitative information on bacterial diversity in T. magnatum, collected with the above-mentioned methods, showed that only a few 16S rDNA sequences of the isolates were comparable to those of environmental clones. This confirmed the importance of non-cultivation approaches in studying bacterial diversity in truffle (Barbieri et al, 2005a), notably because cultivation-based diversity surveys are rather unlikely to reflect the true microbial community structure present in situ because of inherent qualitative and quantitative biases (Wagner et al, 1993;Hugenholtz et al, 1998;Ellis et al, 2003). Nevertheless, the 16S rDNA sequences obtained from the bacterial isolates extended the taxonomic databases of Tuber-associated bacteria useful for comparative systematic studies.…”

Both widespread PEP‐CTERM proteins and exopolysaccharides are required for floc formation of Zoogloea resiniphila and other activated sludge bacteria

“…Zoogloea species have long been considered to be predominant floc-forming bacteria of activated sludge. Fluorescence in-situ hybridization analysis using the 16S rRNA-targeted oligonucleotide probes revealed that Zoogloea cells accounted for up to approximately 10% of the total cell numbers of activated sludge samples (Wagner et al, 1993;Rossellomora et al, 1995). High throughput 454 pyrosequencing of amplicons of 16S rDNAs also revealed that Zoogloea existed in high abundance (1.38-11.1%) in the activated sludge of municipal sewage treatment plants (Wang et al, 2012;Zhang et al, 2012).…”

In situ identification of polyphosphate‐ and polyhydroxyalkanoate‐accumulating traits for microbial populations in a biological phosphorus removal process

“…failed to perform the biochemical transformations that take place in the treatment process and constituted only a smaller fraction of total bacterial cells in the sludge. This bias arises mainly because of the low fraction of culturable organisms present in the sludge (Wagner et al, 1993;1994a).…”