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

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 uncultured Rhodocyclus-related PAO, two morphotypes hybridizing with gene probes for the gram-positive Actinobacteria were also actively involved in uptake of orthophosphate (P i ). 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 Tetrasphaera within the family Intrasporangiaceae of the Actinobacteria (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 Bacteria hybridizing with general bacterial probes), while the cocci in tetrads (targeted by probe Actino-221) made up 7%. Both morphotypes took up P i 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 Rhodocyclus-related PAO, and its identity is still unknown. Growth and uptake of P i 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 Rhodocyclus-related PAO, thus playing a very important role in the EBPR process.In the wastewater treatment industry, enhanced biological phosphorus removal (EBPR) has been widely used to remove orthophosphate (P i ) from wastewater to protect the receiving water bodies against eutrophication. In EBPR processes, the principle is to enrich microorganisms that can accumulate excessive amounts of intracellular polyphosphate [poly(P)] by using sequential anaerobic-aerobic and/or anaerobic-denitrifying conditions (37). The understanding of the biochemical pathways of these poly(P)-accumulating organisms (PAO) is based mainly on analysis of chemical transformations in enriched laboratory-scale EBPR systems, because no pure cultures are available (25, 34, 37). The generally accepted hypothesis (25, 34) proposes that PAO take up organic matter (usually assumed to be acetate) during the anaerobic period by using poly(P) as an energy source and sequester the acetate taken up into polyhydroxyalkanoates (PHA). The reducing power to form PHA is provided by hydrolysis of an intracellular glycogen pool through the glycolytic pathway. The PHA are used as energy and carbon sources in the following aerobic or denitrifying...

A metabolic model for members of the genus Tetrasphaera involved in enhanced biological phosphorus removal

“…Both could take up phosphate aerobically and store it intracellularly as polyphosphate, and assimilate a range of substrates under anaerobic conditions. However, in situ staining has failed to show that Tetrasphaera store these in the form of intracellular PHA in EBPR systems, in contrast to the situation with Accumulibacter (Kong et al, 2005;Nguyen et al, 2011). Furthermore, Tetrasphaera appears able to ferment glucose (Kong et al, 2008;Nguyen et al, 2011), but like Accumulibacter, Tetrasphaera also assimilates phosphate into polyphosphate granules under aerobic conditions only if in a previous anaerobic phase organic substrates have been available to them.…”

“…Thus, the ecophysiology of Tetrasphaera seems to be more versatile than that of Accumulibacter. Tetrasphaera-related PAOs can be detected in high relative abundances in many full-scale EBPR plants where they account for up to 30% of the total bacterial biovolume (Kong et al, 2005;Nguyen et al, 2011). They are also phylogenetically diverse, comprising three distinct clades, where cells within each clade exhibit a range of morphologies (Nguyen et al, 2011).…”

“…Members of the Actinobacterial genus Tetrasphaera were suggested to be putative PAOs (Maszenan et al, 2000;Kong et al, 2005), but with a markedly different physiology to Accumulibacter. Both could take up phosphate aerobically and store it intracellularly as polyphosphate, and assimilate a range of substrates under anaerobic conditions.…”

The effects of carbon/phosphorus ratio on polyphosphate- and glycogen-accumulating organisms in aerobic granular sludge

“…However, the presence of Tetrasphaera in GSBR was confirmed with the broader probes designed by Nguyen et al 2011. The controversy regarding the ability of Tetrasphaera PAOs to utilize acetate (Kong et al 2005;Nguyen et al 2011) may be explained by the broad coverage of the latter probes (Nguyen et al 2011). …”

“…In full-scale systems, Tetrasphaerarelated PAOs are usually more abundant than Accumulibacter, but in laboratory-scale reactors, those bacteria were overlooked (Nguyen et al 2011). However, two actinobacterial probes Actino-221 and Actino-658, which had been widely used (Kong et al 2005), cover only a part of the three Tetrasphaera clades (Nguyen et al 2011). GAOs in the seeding sludge did not exceed 2 % of EUBmix, and they were represented only by Competibacter.…”