**Ecophysiology and Global Diversity of *Candidatus Microthrix* in Wastewater Treatment Plants: Insights into Species Distribution, Genomics, an...
Published on by Hossein Ataei Far, Deputy Manager of the Research, Technology Development, and Industry Relations Center at NWWEC
### Abstract 📜
*Candidatus* Microthrix, particularly *Ca. M. parvicella*, is a common filamentous microorganism in activated sludge wastewater treatment plants (WWTPs) worldwide. 🌐 Despite its prevalence, the global diversity and ecology of this genus are not well understood. This study leveraged the MiDAS 16S rRNA gene database and amplicon sequencing to explore the diversity, distribution, and environmental factors influencing *Ca. Microthrix* species on a global scale. The study identified two predominant species: *Ca. M. parvicella* and a novel species tentatively named *Ca. M. subdominans*. Both species were primarily found in European municipal WWTPs operating at low-to-moderate temperatures. ❄️🌡️ Genomic analysis suggested that *Ca. M. subdominans* may possess a more versatile metabolism than *Ca. M. parvicella*. 🔬 Novel fluorescence in situ hybridization (FISH) probes and Raman microspectroscopy were used to study the morphology and physiology of these species. These findings enhance our understanding of *Ca. Microthrix* ecology, potentially contributing to the development of improved control strategies in WWTPs. 🚰🔧
### Introduction 📚
The activated sludge process is the most widely adopted method for wastewater treatment globally, continuously evolving to meet the growing demands of urban populations and the shift towards sustainability and resource recovery. 🌆♻️ However, the overgrowth of filamentous bacteria, such as *Candidatus* Microthrix, often leads to sludge bulking and foaming, which compromise the stability and efficiency of WWTPs. Previous research has identified *Ca. Microthrix* species, particularly *Ca. M. parvicella* and *Ca. M. calida*, as significant contributors to these operational issues. Despite extensive studies, the ecophysiology of *Ca. Microthrix* in activated sludge remains poorly understood, necessitating further investigation to address these operational challenges. 🧪⚠️
### Results Summary 📊
- **Species Diversity and Distribution:** The study revealed low diversity within the *Ca. Microthrix* genus, identifying two abundant species: *Ca. M. parvicella* and the newly proposed *Ca. M. subdominans*. 🔍
- **Environmental Preferences:** Both species were predominantly found in European WWTPs, thriving at low-to-moderate temperatures and in plants with advanced process designs. 🏭🌍
- **Genomic Insights:** Metagenome-assembled genomes (MAGs) indicated high genetic similarity between *Ca. M. parvicella* and *Ca. M. subdominans*, with the latter displaying a potentially more versatile metabolism, including additional sugar transporters and a higher oxygen tolerance. 🍬🧬
- **Morphology and Physiology:** The use of novel FISH probes and Raman microspectroscopy confirmed similar filamentous morphologies in both species and provided insights into their intracellular polyphosphate (poly-P) storage capabilities. 🧫🔬
- **Seasonal Dynamics:** A 13-year survey of Danish WWTPs suggested that *Ca. M. parvicella* and *Ca. M. subdominans* may occupy slightly different ecological niches, as indicated by distinct seasonal patterns. 📅🌱
### Conclusion ✅
This study advances the understanding of *Ca. Microthrix* ecology in WWTPs, highlighting the low genus diversity and potential ecological differences between *Ca. M. parvicella* and the newly identified *Ca. M. subdominans*. The findings provide valuable genomic and ecological data that could inform more effective strategies for managing filamentous bacterial growth in wastewater treatment processes. 🌐🛠️
### Results and Discussion 🗣️
#### Diversity and Distribution of *Ca. Microthrix* Species in Full-Scale WWTPs by Amplicon Sequencing
The diversity and prevalence of *Ca. Microthrix* species in the activated sludge ecosystem were examined using data from long-term surveys of Danish WWTPs (Nierychlo et al., 2020a) and a global WWTP survey comprising 438 samples (Dueholm et al., 2021). 🌍🔬 The analysis revealed two widespread species: *Ca. M. parvicella* and a novel species, provisionally named midas_s_2 in the MiDAS taxonomy, here proposed as *Candidatus Microthrix subdominans* (see section Etymology). Other species within the genus were observed only at low abundances and were not prevalent in any WWTPs.
*Ca. M. parvicella* was dominant in most Danish and global plants, with average read abundances of 1.7-2.0% and 0.6-1.2%, respectively. In some instances, its abundance exceeded 14%, likely contributing to severe sludge separation issues and impaired plant performance. ⚠️ *Ca. M. subdominans* was generally observed at lower abundances, with average and maximum read abundances in Denmark of 1.1-1.3% and 13%, respectively, and globally of 0.2-0.5% and 4%, respectively. Both species co-existed in most plants, indicating similar ecological niches, with the highest global abundances recorded in several European countries. 🇪🇺
In Danish nutrient removal plants, *Ca. M. parvicella* was the second most abundant genus, underscoring its importance for wastewater treatment processes and floc properties. A long-term survey of Danish plants provided insights into the abundance levels and seasonal dynamics of the two species. In some plants, *Ca. Microthrix* was scarcely present, while in others, it was consistently found over many years. 🧑🔬
**Reference:**
[1] Nierychlo, M., et al. (2021). Low global diversity of *Candidatus* Microthrix, a troublesome filamentous organism in full-scale WWTPs. *Frontiers in Microbiology, 12*, 690251.
**Figure 1** | Composite FISH micrographs of *Ca. Microthrix* species in full-scale activated sludge. (A) *Ca. M. parvicella* visualized with species-specific probes Mpa177 (Cy3, red), *Ca. M. subdominans* visualized with probe Msu181 (6-FAM, green), other bacteria shown with EUBmix (Cy5, blue); (B) *Ca. M. parvicella* visualized with species-specific probe MPA60 (Cy3, red), *Ca. M. subdominans* visualized with probe Msu181 (6-FAM, green), other bacteria shown with EUBmix (Cy5, blue); (C) *Ca. M. parvicella* visualized with species-specific probes Mpa177 (Cy3, red) appear magenta, *Ca. M. subdominans* visualized with probe Msu181 (6-FAM, cyan) appear white, *Ca. Microthrix* genus visualized with probe MCX840 (Cy5, blue). Activated sludge was sampled from Randers WWTP. 🧫