Intracellular Accumulation of Glycine in Polyphosphate-Accumulating Organisms in Activated Sludge, a Novel Storage Mechanism under Dynamic Anaerobic-Aerobic Conditions

Abstract

Dynamic anaerobic-aerobic feast-famine conditions are applied to wastewater treatment plants to select polyphosphate-accumulating organisms to carry out enhanced biological phosphorus removal. Acetate is a well-known substrate to stimulate this process, and here we show that different amino acids also are suitable substrates, with glycine as the most promising.13 C-labeled glycine and nuclear magnetic resonance (NMR) were applied to investigate uptake and potential storage products when activated sludge was fed with glycine under anaerobic conditions. Glycine was consumed by the biomass, and the majority was stored intracellularly as free glycine and fermentation products. Subsequently, in the aerobic phase without addition of external substrate, the stored glycine was consumed. The uptake of glycine and oxidation of intracellular metabolites took place along with a release and uptake of orthophosphate, respectively. Fluorescence in situ hybridization combined with microautoradiography using 3 H-labeled glycine revealed uncultured actinobacterial Tetrasphaera as a dominant glycine consumer. Experiments with Tetrasphaera elongata as representative of uncultured Tetrasphaera showed that under anaerobic conditions it was able to take up labeled glycine and accumulate this and other labeled metabolites to an intracellular concentration of approximately 4 mM. All components were consumed under subsequent aerobic conditions. Intracellular accumulation of amino acids seems to be a novel storage strategy for polyphosphate-accumulating bacteria under dynamic anaerobic-aerobic feast-famine conditions. T he wastewater treatment process known as enhanced biological phosphorus removal (EBPR) is a widely employed process in wastewater treatment for removal of phosphorus (P). The principle is to create favorable conditions for the enrichment of microorganisms capable of excess uptake and storage of orthophosphate (P i ) as polyphosphate [poly(P)], based on alternating anaerobic and aerobic periods (1). Early metabolic models detailed that in the anaerobic phase, polyphosphateaccumulating organisms (PAOs) take up volatile fatty acids (e.g., acetate and propionate), using polyphosphate as an energy source. These substrates are reduced and stored as intracellular polyhydroxyalkanoates (PHA) with energy obtained from hydrolysis of intracellular polyphosphate and energy and reducing power from glycolysis of intracellular glycogen (2), the tricarboxylic acid (TCA) cycle (3), or both (4). In the subsequent aerobic or denitrifying phase, the PAOs use PHA for growth and for replenishing their polyphosphate and glycogen reserves (5). As the quantity of phosphate removed during the aerobic stage is greater than that released during the initial anaerobic stage, a net removal of phosphate from the wastewater is achieved.While PAOs share the metabolic ability for accumulation of polyphosphate as an energy storage compound, they are phylogenetically diverse (6-9). Rhodocyclus-related bacteria ("Candidatus Accumulibacter"), belonging to the B...

Resolving the individual contribution of key microbial populations to enhanced biological phosphorus removal with Raman-FISH

“…The strength of the current FISH-Raman method is that it does not rely on such inferences and that it can directly measure the polymer in question in its native state at a single-cell level. It has been shown that T. elongata can grow by fermentation 19 , and probe-defined Tetrasphaera in full-scale EBPR plants likely ferment glucose after several of days anaerobic conditions 54 , so fermentation is likely a key metabolic feature in tandem with poly-P formation/degradation in their successful competition to many other microbes in EBPR plants.…”

“…Annotation of representative genomes for the genus suggests that glucose could be stored as glycogen (but not PHAs) under anaerobic conditions, and some un-polymerised fermentation products and amino acids have been shown to accumulate in the cell. Under subsequent aerobic conditions, glycogen and accumulated intracellular substrates are suggested to be utilized for growth and the replenishment of poly-P reserves 18; 19 .…”

A Full-Scale Comparative Study of Conventional and Side-Stream Enhanced Biological Phosphorus Removal Processes

“…Three Tetrasphaera-related clades, including Clade 1, Clade 2A, and Clade 3 were dominant in the culture at similar relative abundances. Although Tetrasphaera Clade 1 and a portion of Clade 3 cannot take up acetate, they are all capable of fermentation, which leads to Tetrasphaera thriving in WRRFs that receive complex organic matter (Nguyen et al, 2011(Nguyen et al, , 2015. In the side-stream reactor of an S2EBPR system, it is likely that Tetrasphaera may play a crucial role that firstly fermenting complex carbon sources for energy generation, and Accumulibacter could then take up the fermentation products (e.g., acetate or propionate) generated by Tetrasphaera for PHA storage.…”

The proteome of Tetrasphaera elongata is adapted to changing conditions in wastewater treatment plants

“…The acquired data indicated that this is not the case for T. elongata and that it does indeed show a high level of metabolic robustness and readiness [39]. The theoretical advantage of being able to keep growing without major metabolic adaptations will be enhanced under in situ conditions as T. elongata can store excessive amounts of poly-P for rapid supplementation of energy or accumulation of carbon substrates under anaerobic conditions [16]. The proteomic and metabolomic data helps to complement the existing metabolic model [15] (Fig.…”

“…Lp2 and E. coli str. K-12 were cultivated in modified R2A (minimal) medium [16] to be comparable with previous studies. Inoculation was performed from liquid overnight cultures to an optical density (OD) at 600 nm of 0.01.…”