Identity and Ecophysiology of Uncultured Actinobacterial Polyphosphate-Accumulating Organisms in Full-Scale Enhanced Biological Phosphorus Removal Plants

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Abstract
Microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was used to screen for potential polyphosphate-accumulating organisms (PAO) in a full-scale enhanced biological phosphorus removal (EBPR) plant. The results showed that, in addition to unculturedRhodocyclus-related PAO, two morphotypes hybridizing with gene probes for the gram-positiveActinobacteriawere also actively involved in uptake of orthophosphate (Pi). Clone library analysis and further investigations by MAR-FISH using two new oligonucleotide probes revealed that both morphotypes, cocci in clusters of tetrads and short rods in clumps, were relatively closely related to the genusTetrasphaerawithin the familyIntrasporangiaceaeof theActinobacteria(93 to 98% similarity in their 16S rRNA genes). FISH analysis of the community biomass in the treatment plant investigated showed that the short rods (targeted by probe Actino-658) were the most abundant (12% of allBacteriahybridizing with general bacterial probes), while the cocci in tetrads (targeted by probe Actino-221) made up 7%. Both morphotypes took up Piaerobically only if, in a previous anaerobic phase, they had taken up organic matter from wastewater or a mixture of amino acids. They could not take up short-chain fatty acids (e.g., acetate), glucose, or ethanol under anaerobic or aerobic conditions. The storage compound produced during the anaerobic period was not polyhydroxyalkanoates, as forRhodocyclus-related PAO, and its identity is still unknown. Growth and uptake of Pitook place in the presence of oxygen and nitrate but not nitrite, indicating a lack of denitrifying ability. A survey of the occurrence of these actinobacterial PAO in 10 full-scale EBPR plants revealed that both morphotypes were widely present, and in several plants more abundant than theRhodocyclus-related PAO, thus playing a very important role in the EBPR process.