16S rRNA in situ probing for the determination of the family level community structure implicated in enhanced biological nutrient removal

Knowledge of a discrete physiological group capable of excess biological phosphate removal (EBPR) remains unclear. Consequently, microbial community analysis of an enhanced continuous laboratory-scale activated sludge process displaying a strong EBPR mechanism was conducted. Unit design was configured upon the three-stage Phoredox process and characterization of the activated sludge bacterial community was carried out using fluorescent in situ hybridization (FISH) techniques. Fixed activated sludge samples were hybridized with fluorescently labeled oligonucleotide probes targeting the following bacterial phylogenetic divisions: a kingdom level probe specific for all bacteria (EUB338); family level probes specific for the alpha, beta and gamma subclasses of the class Proteobacteria; Gram positive bacteria with a high (G +C) DNA content (GPBHGC) or Actinobacteria; the Cytophaga-Flavabacterium (CF) subclass within the Cytophaga-Flavobacterium-Bacteriodes division; and genus level probes specific for Pseudomonas spp., Aeromonas spp., and Acinetobacter spp. Bacterial predominance between the anaerobic, anoxic and aerobic zones of the EBPR sludge were comparable and appeared as follows; beta (22%), alpha (19%), gamma (17%), GPBHGC (1 1 %) and CF (8%). The incidence of Acinetobacter spp. appeared to be generally low with counts amounting to <9% of the total bacterial count. A population shift in the alpha Proteobacteria subclass was evident between the non-nutrient removal seed sludge and the EBPR sludge, implicating this group in EBPR. The overall results indicate that the beta and alpha Proteobacteria can be metabolically functional in EBPR processes and reiterate the functional misconception of Acinetobacter spp. in EBPR systems.