Micromanipulation and further identification of FISH‐labelled microcolonies of a dominant denitrifying bacterium in activated sludge

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Abstract
The activated sludge process relies on the formation of strong microbial flocs. The knowledge about dominant floc‐forming bacteria is at present very limited, especially from a phylogenetic perspective. In this study, numerous microcolonies in the activated sludge flocs were found to be targeted by a Betaproteobacteria‐group‐specific oligonucleotide probe using fluorescence in situ hybridization (FISH). Some of these were micromanipulated and further identified by reverse transcriptase polymerase chain reaction (RT‐PCR) and sequencing to belong to the Aquaspirillum genus in the Neisseriaceae family. A specific oligonucleotide probe, Aqs997, was designed to target the identified bacteria. A survey in nine different wastewater treatment plants with nutrient removal (WWTP) showed a high abundance of bacteria hybridizing to the oligonucleotide probe developed. Microautoradiography (MAR) combined with FISH on activated sludge incubated with radiolabelled substrate showed uptake of substrate with oxygen, nitrate and nitrite as electron acceptor demonstrating a denitrifying potential of the bacteria investigated. The Aquaspirillum‐related bacteria seemed to be abundant denitrifiers in WWTPs with nitrogen removal and they were particularly numerous in plants mainly receiving domestic wastewater, where they constituted up to 30% of all bacteria.