# Wastewater Treatment with Bacterial Representatives of the _Thiothrix_ Morphotype
## 3 Methods for Identification of _Thiothrix_ in Wastewater Treatment Plants
### Enhanced Biological Removal of Phosphorus
Phosphoribbacter' [95]), _Microluntus phovovorus_, _Dechromonas_, _Thiohtrix_ and others [96]. _'Ca_. Accumulibacter' and _'Ca_. Phosphoribbacter' are considered the main ones [97]. PAO metabolism by _'Ca_. Accumulibacter' is considered classical. These bacteria, under anaerobic conditions, (i) take up VFAs and store them in the form of the intracellular polymer PHA, (ii) use glycogen as a source of reducing equivalents (carbon is also included in PHA) and (iii) hydrolyze internal polyphosphate (poly-P) to form the essential ATP. Under aerobic conditions, PAOs (i) utilize the PHA reserves to grow and replenish the glycogen reserves and (ii) use the ATP generated by oxidative phosphorylation to take up and convert phosphate to poly-P [98] (Figure 5a). Unlike _'Ca_. Accumulibacter', representatives of _Tetrasphaera_ spp. do not possess the ability to store PHA. However, like representatives of _'Ca_. Accumulibacter', they synthesize poly-P inside cells aerobically and use it anaerobically. It is assumed that amino acids and glucose are substrates in the cycle of poly-P transformation in anaerobiosis. During the reaction, these substrates are presumably converted into free amino acids and glycogen, although the role of glycogen is unclear, since it is not found in the activated sludge of _Tetrasphaera_ spp. representatives [95] (Figure 5b). Figure 4: Possible pathways of phosphorus metabolism in bacterial cells. For the depicted names of the enzyme proteins, see the text. The figure was adapted from [PERSON] et al. [92] and [PERSON] et al. [93] under the terms of the Creative Commons Attribution 4.0 International License. _Thiohtrix_ enzymes are shown based on the published data [4; 11; 47; 49; 56; 84; 94]. Figure 5: PAO metabolism in (**a**) ‘_Ca._ Accumulibacter’ and (**b**) _Tetrasphaera_ (= ‘_Ca._ Phosphorbincter’ [96]) and (**c**) predicted functional potential members of the genus _Thiodtrix_. The schemes in panels (**a**,**b**) are based on [PERSON] et al. [97] under the terms of the Creative Commons Attribution 4.0 International License; panel (**c**) considers data from published articles [4; 5; 47; 56; 94]. In panel (**c**), the metabolic pathways outlined by the dashed line are putative. Internal, internal reserve substances in the cell; PHA, poly-\(\beta\)-hydroxy-alkanoateates; \(\mathrm{S}^{0}\), elemental sulfur; VFAs, volatile fatty acids; PolyP, polyphosphate; TCA, tricarboxylic acid cycle; ETC, electron transport chain. It was shown that members of the genera _Thiothrix_[47] and _Thiothine_[11] can play a key role in biological phosphorus removal. As shown under aerobic conditions in EBPR systems, in an environment with low organic content but enriched in reduced sulfur compounds, _T. calidfontis_ could behave as an efficient phosphate-removing PAO with mixotrophic metabolism. Phosphorus removal from the environment in the aerobic phase proceeds with the oxidation of the intracellular sulfur pool to provide PAO with an additional source of energy [47]. In the same EBPR system, under anaerobic conditions, this species can store carbon in the form of PHA and generate the required energy via the hydrolysis of poly-P stored in the aerobic phase. In the aerobic period, PHA was used as the source of carbon and energy for the growth and synthesis of poly-P (Figure 5c). On the other hand, _Thiothrix_ sp. that received glutamate as the only source of nitrogen and carbon was able to store poly-P, probably avoiding PHA synthesis [99]. It should be noted that the growth conditions in EBPR systems differ markedly from those of batch cultivation and often provide preferences for new and rare species. Several representatives of _Thiothrix_ were described resulting from the assembly of MAGs obtained from the activated sludge in EBPR systems [11; 56; 81]. One of the resulting MAGs was closely related to the species _Thiothrix disciformis_ (_= Thiolinea disciformis_) [10; 11] and two MAGs were described as new species with _Candidatus_ status, namely _'Ca_. Thiothrix moscovensis' and _'Ca_. Thiothrix singaporeensis' [56].