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 ph osphate removal (EBPR) remains unclear. Consequently, microbial community a nalysis of an enhanced continuous laboratory-scale activated sludge process displaying a strong EBPR mechanism was conducted. Unit design was configur ed upon the three-stage Phoredox process and characterization of the activa ted sludge bacterial community was carried out using fluorescent in situ hy bridization (FISH) techniques. Fixed activated sludge samples were hybridiz ed with fluorescently labeled oligonucleotide probes targeting the followin g bacterial phylogenetic divisions: a kingdom level probe specific for all bacteria (EUB338); family level probes specific for the alpha, beta and gam ma subclasses of the class Proteobacteria; Gram positive bacteria with a hi gh (G +C) DNA content (GPBHGC) or Actinobacteria; the Cytophaga-Flavabacter ium (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 incid ence of Acinetobacter spp. appeared to be generally low with counts amounti ng to <9% of the total bacterial count. A population shift in the alpha Pro teobacteria subclass was evident between the non-nutrient removal seed slud ge and the EBPR sludge, implicating this group in EBPR. The overall results indicate that the beta and alpha Proteobacteria can be metabolically funct ional in EBPR processes and reiterate the functional misconception of Acine tobacter spp. in EBPR systems.