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PEGASUS (Phylogeny of Eukaryotic Genomes in Activated Sludge and Untreated Sewage)

​​​​Investigating for and characterizing microorganisms that cannot be cultured in urban waste water treatment plants

Published on 24 October 2016

In the living world of eukaryotes, protists belong to numerous and diverse phyla and their biological diversity very probably exceeds that of land plants and animals combined. While model protists are now used to elucidate several essential aspects of biology such as cell structure, the development, ecology, evolution and diversity of protists has only been very partially characterized. Moreover, their participation in, and impact on, the planet's biogeochemical cycles are little known to date.

Activated mud from biological treatment plants constitutes a complex ecosystem. Within the mud, heterotrophic bacterial populations oxidize the dissolved and particulate organic materials in the waste water and transform it into CO2 and biomass. Their presence alone generally results in mud that is difficult to sediment and purified water of poor quality because of the burden of free bacteria and suspended materials. The presence of various protists such as flagellates, ameba and ciliates is necessary for a good ecological equilibrium of the mud. Their predatory activity contributes to clarifying interstitial water which is restituted to the receiving environment. Metazoa of large size contribute to re-oxygenation of the cores of the flocs by bioturbation. Protists and metazoa have slow growth rates and are very sensitive to toxic agents, oxygen deficiency and nutrient imbalances. In consequence, such organisms have long been used as bio-indicators of the correct operation of water-treatment plants. Their population is about 50,000 organisms per mL of mud. Their identification remains largely based on their morphology, necessitating long observer experience. In addition, the few tens of species usually identified in mud mask a great diversity of organisms that have not yet been described. Some of those organisms, capable of cyst formation, are pathogenic.

The molecular studies of the biodiversity of eukaryotes are much less complete than those of prokaryotes. The relative contributions of protists and metazoa in the purification process remains poorly defined.

We propose a multiphase approach to the study of protist diversity, which is intended to :

  1. identify, using molecular approaches (sequencing of the genes coding for rRNA 18S), the various eukaryote groups in the domestic and industrial waste water-treatment systems,
  2. study their ecology (development of specific nucleic probes enabling study of their morphology, abundance and in situ locations) and activity (metatranscriptomics of eukaryote RNA),
  3. isolating the microorganisms by micromanipulation and sequencing their genomes by single-cell DNA genome sequencing.

The initial analyses conducted on 1001 contigs derived from 12 different samples enabled evidencing 186 OTU. The phylogenetic analyses enabled us to identify, in our samples, certain ciliate protozoan and ameba phylotypes. More particularly, we evidenced and identified a great diversity of protists, of which unexpected majority groups such as mycelial eukaryotes (Fig. 1). The microorganisms are reported to be endowed with strong proteolytic potential and their contribution to the degradation of organic matter remains very little documented. Among the mycelial microorganisms, an imperfectly described group, the Cryptomycetes  appears predominant in some of our specimens.

The microorganisms have not been cultured (or have not yet been cultured!). We subsequently developed specific probes at various taxonomic levels and more particularly for the latter group. 

The probes were tested on various samples. Certain yielded positive signs of extracellular hybridization (Photo A) in the form of inclusions in the cytoplasm of certain ciliates (Photo B), rotifers (Photo C) and tardigrades (Photo D). The hybridization of the specific probes for Cryptomycota were co-localized with hybridization of the general probes directed against eukaryotes. The high stringency of the hybridizations tends to prove their specificity. The same microorganisms were described as predominant in certain activated mud purification plants strengthening our findings. If the endoparasites are dominant to that extent, their role in the equilibrium of the ecosystem is doubtless important and has yet to be explicitly determined. We are continuing to move forward in the analysis of the samples, which will enable us to describe the morphology and spatial interactions of the protist group by FISH methods.


Jones MD, Forn I, Gadelha C, Egan MJ, Bass D, Massana R, Richards TA. (2011) Discovery of novel intermediate forms redefines the fungal tree of life. Nature. 474(7350):200-3

Matsunaga, K., Kubota, K., Harada, H. (2014). Molecular diversity of eukaryotes in municipal wastewater treatment processes as revealed by 18S rRNA gene analysis. Microbes and Environments, 29(4), 401-407

Photo A : Valenton plant, 2 anoxia tanks, hybridization, 50% formamide, probe:  Crypto2-cy3 Euk-cy5 Syto9. Extracellular forms in the floc, cells organized in a concentric manner (arrows), (a):  phase-contrast microscopy;  (b) FISH observation

Photo B : Sample, Valenton treatment plant, 2 aerobic tanks, hybridization, formamide:  70%, probes, Euk-cy5 (a), Crypto1-cy3 (b), Syto9 (c), (d) Superimposition of (b) and (c), (multi-lobed elongated form inside the protozoa) © Photo credit:  PEGASUS PROJECT

Photo C : Sample, Valenton treatment plant, 2 aerobic tanks, hybridization, formamide:  70%, probes, (a) Euk-cy5 ; (b) Crypto2-cy3 ; (c) Syto9 (d) superimposition of (b) and (c) Cryptomycota in an amoeba-like microorganism © Photo credit:  PEGASUS PROJECT

Photo D : Photo D:  Sample from the Valenton plant 1B, aerated basin, probe, Crypto1-cy3BHQ, formamide: 35% A Tardigrade, water bear, colonized by Cryptomycota-like microorganisms © Photo credit:  PEGASUS PROJECT

Projet en cours de réalisation en collaboration avec :

- L'Institut de Biologie Cellulaire Intégrative :

Laboratoire de Génomique et Biodiversité microbienne des Biofilms, Institut de Biologie Cellulaire Intégrative, UMR 9198, CEA-CNRS-UPSud, Orsay, France

Contact : Loïc Morin, loic.morin ..At..

- L'Irstea, ex-CEMAGREF :

Unité de Recherches Hydrosystèmes et Bioprocédés, Irstea, Antony, France

Contact : Jean-Jacques Pernelle, Jean-Jacque​s.Pernelle ..At..

Financement : Conseil général de l'Essonne, PRES UniverSudParis (2013)​

​Lab​oratory involved​ : Laboratory of metagenomics of prokaryotes