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

Abstract

Members of the genus Tetrasphaera are considered to be putative polyphosphate accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) from wastewater. Although abundant in Danish full-scale wastewater EBPR plants, how similar their ecophysiology is to 'Candidatus Accumulibacter phosphatis' is unclear, although they may occupy different ecological niches in EBPR communities. The genomes of four Tetrasphaera isolates (T. australiensis, T. japonica, T. elongata and T. jenkinsii) were sequenced and annotated, and the data used to construct metabolic models. These models incorporate central aspects of carbon and phosphorus metabolism critical to understanding their behavior under the alternating anaerobic/aerobic conditions encountered in EBPR systems. Key features of these metabolic pathways were investigated in pure cultures, although poor growth limited their analyses to T. japonica and T. elongata. Based on the models, we propose that under anaerobic conditions the Tetrasphaera-related PAOs take up glucose and ferment this to succinate and other components. They also synthesize glycogen as a storage polymer, using energy generated from the degradation of stored polyphosphate and substrate fermentation. During the aerobic phase, the stored glycogen is catabolized to provide energy for growth and to replenish the intracellular polyphosphate reserves needed for subsequent anaerobic metabolism. They are also able to denitrify. This physiology is markedly different to that displayed by 'Candidatus Accumulibacter phosphatis', and reveals Tetrasphaera populations to be unusual and physiologically versatile PAOs carrying out denitrification, fermentation and polyphosphate accumulation.

Figure 1

Phylogenetic tree of the Tetrasphaera genus showing the grouping of Tetrasphaera isolates in three clades described by Nguyen et al. (2011). Names of isolates used to construct the Tetrasphaera genomes are indicated with boldface font. The outgroup used to construct this tree was nine strains of Nitrosomonas halophila.

Figure 2

Venn diagram of unique and conserved genes for four Tetrasphaera isolates (T. australiensis, T. elongata, T. japonica and T. jenkinsii). T. japonica has 2924 unique genes and T. elongata has 1183 unique genes. T. australiensis and T. jenkinsii, both belonging to clade 2, have 1974 and 1469 unique genes, respectively, and share 582 genes. The conserved genes constitute 1283 genes of each genome.

Figure 3

The ability of T. elongata to release Pi and consume glucose in the anaerobic phase (0–3 h) and take up Pi in the aerobic phase (3.5–6.5 h), and to produce and consume glycogen in the anaerobic and aerobic phases, respectively. The average concentration and standard deviation from duplicate experiments are shown for the Pi concentration of the media (diamonds) and glycogen content of the biomass (triangles). Media glucose concentration is also shown (squares).

Figure 4

Metabolic model for T. elongata. The key metabolic pathways enabling Tetrasphaera to compete in full-scale EBPR plants are shown. (a) In the anaerobic phase, glucose is taken up and either stored as glycogen or fermented to acetate, lactate, succinate and alanine (highlighted in blue circles). The energy required for glycogen synthesis is supplied by fermentation and polyphosphate degradation to orthophosphate (Pi). (b) In the aerobic phase, the stored glycogen is degraded, supplying energy for growth and replenishing the polyphosphate stores.