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

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  • Yunhong Kong
    Department of Life Sciences, Section of Environmental Engineering, Aalborg University, DK-9000 Aalborg, Denmark
  • Jeppe Lund Nielsen
    Department of Life Sciences, Section of Environmental Engineering, Aalborg University, DK-9000 Aalborg, Denmark
  • Per Halkjær Nielsen
    Department of Life Sciences, Section of Environmental Engineering, Aalborg University, DK-9000 Aalborg, Denmark

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<jats:title>ABSTRACT</jats:title> <jats:p> 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 uncultured <jats:italic>Rhodocyclus</jats:italic> -related PAO, two morphotypes hybridizing with gene probes for the gram-positive <jats:italic>Actinobacteria</jats:italic> were also actively involved in uptake of orthophosphate (P <jats:sub>i</jats:sub> ). 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 genus <jats:italic>Tetrasphaera</jats:italic> within the family <jats:italic>Intrasporangiaceae</jats:italic> of the <jats:italic>Actinobacteria</jats:italic> (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 all <jats:italic>Bacteria</jats:italic> hybridizing with general bacterial probes), while the cocci in tetrads (targeted by probe Actino-221) made up 7%. Both morphotypes took up P <jats:sub>i</jats:sub> aerobically 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 for <jats:italic>Rhodocyclus</jats:italic> -related PAO, and its identity is still unknown. Growth and uptake of P <jats:sub>i</jats:sub> took 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 the <jats:italic>Rhodocyclus</jats:italic> -related PAO, thus playing a very important role in the EBPR process. </jats:p>

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