Metagenomics of prokaryotes

MetaHIT project
  Group members
  Collaborations
  Publications

Exploration of the microbial diversity in wastewater processing using activated sludge
  For many years the microbial world has been studied using methods based on the isolation and cultivation of bacteria. However, classical culture techniques have only succeeded in characterizing a small group of species. These cultivable bacteria, however, only represent a small portion of the bacteria present in all natural environments, and are the current basis of the majority of our knowledge about microbial diversity and microbiology. Progress in molecular biology has made it possible to demonstrate the enormous range of microbial diversity in all natural habitats. These studies, mainly based on the sequence of 16S ribosomal DNA (Pace, 1997), have revealed the existence of numerous unknown species (Bacteria and Archaea) which are considered to be non-cultivable because they have never been isolated from samples from natural environments. The sequence of 16S rDNA is also used to establish phylogenetic relationships between the species which have been identified in this way (Woese et al., 1990).
  This approach, which has the advantage of reconstituting the phylogenesis of the bacterial and archaeal world in an acceptable way, is insufficient for the exploration of microbial diversity.
 
  Cloaca maxima
 

Bassins de la station d’épuration d’Evry. Photo de P. Ginestet, P. Carmacho (Suez Environnement)

Biodiversity is not limited to rDNA. It is the reflection of the different ways in which micro-organisms obtain energy and interact with the environment. It is this great diversity in genes and metabolism that we wish to explore.
  In order to contribute to the inventory of prokaryotic genes, the Cloaca maxima project started in 2000. For fundamental and practical reasons we have chosen to examine the microbial diversity of the different basins used in the process of purification of domestic waste water.
  From a fundamental point of view, these environments are the seat of intense metabolic activity, the result of which is the conversion of a large portion of polluting organic substances to solid components and biogas. The stations in a water purification plant are a series of black boxes which degrade polymers, pollutants, organic matter, and eliminate phosphates. This is also the site of elimination of ammonia by nitrification-denitrification reactions and the anammox process.
 

Boues d’épuration de la station d’Evry. Photo de P. Ginestet, P.Carmacho (Suez Environnement)

The purification process is of great practical importance. The increase in the population and industrial activities generates new types of pollution and increasing need for treatment of wastes, especially domestic waste water. Current sewage treatment techniques are based on the use of active micro-organisms (which form what we call activated sludge) in the absorption and/or the degradation of dissolved substances or material in suspension (organic compounds, minerals, etc.) as well as in phenomena of decantation and aggregation. The identification of the microbial actors in sewage treatment is essential in order to understand, manage and prevent dysfunction in sewage treatment stations. The process of treatment of wastewater and its last step, anaerobic digestion, are key elements in sustainable development.
 
  We have focused our exploration on the basins of the Evry sewage treatment plant which treats mainly urban wastewater.
 
 

Objectives
 
  Diversity
  The first goal of Cloaca maxima is to establish an inventory of micro-organisms present at the different stages of sewage treatment which is as complete as possible. This inventory is based on cloning and analysis of 16S rDNA sequences from DNA from the environment after amplification by PCR. In order to produce a complete inventory of the microflora and to create a view of the true diversity, we have used both universal primers which target rDNA from both Archaeal and Bacterial domains (Woese & al., 1990), and primers which are specific for the divisions of the domain Bacteria which include few or no cultivable representatives, such as Planctomycetes, Verrucomicrobiales, Acidobacteriales or the candidate divisions WS6, BCR1 and SAR11. The PCR products are then separated by cloning and sequenced. We have analyzed more than 4000 sequences in this way, from samples from the various basins (aerobic, anoxic and mesophilic digester) in the sewage treatment plant.
 
  Exploration of the Metagenome
  The second objective of Cloaca maxima is the exploration of the metagenome, which means the ensemble of the microbial genomes (bacteria and archaea) which are present in a natural environment (Rondon & al, 2000). The main goals are to make a massive contribution to the inventory of prokaryotic genes, to initiate an inventory of enzymatic activities and metabolic processes.
  For this purpose two large metagenomic libraries (a total of 1.5 million clones) have been constructed from DNA from two different environments: one BAC library from the aerobic basin and a fosmid library from the mesophilic anaerobic digester. Systematic sequencing of the ends of the inserts has been undertaken, and for each of these reads, the ORFs (open reading frames) have been determined.
 

Results :
 
  A large diversity of micro-organisms have been cloned in the form of BACs and fosmids. In addition to representatives of the Archaea, genomic fragments of 23 bacterial divisions have been cloned, including 8 candidate divisions which do not yet have any cultivable representative. The screening method which we have developed has also made it possible to demonstrate the existence of bacteria which belong to a new candidate bacterial division: WWE3. Representatives of WWE3 have detected by PCR in 19/52 anaerobic digesters analyzed (Germany, Spain, France, Ireland, Italy, Mexico, Czech Republic and Switzerland).
 

Some BACs and fosmids identified by their 16S rDNA sequence and for which the phylogenetic position is especially interesting (a group with no representative which has been cultivated to date, for instance) have been chosen for sequencing. Thus the fosmids which are affiliated with several phylotypes from WWE1 (a bacterial candidate division which has been described recently, Chouari et al., 2005), have been completely sequenced in order to explore their level of similarity. Starting with a fosmid containing a 16S rDNA from WWE1, we have attempted to extend this region by a process of iterative assembly. This method has allowed us to reconstitute the complete genome of a representative of the WWE1 line: “Candidatus Cloacamonas acidaminovorans”.
  The ensemble of this information will make it possible to undertake an in-depth study of the differences between the aerobic basin and the anaerobic digester. We will pay special attention to the metabolism of anaerobiosis, a system which is not as well characterized as that of aerobic life.
 
 
  Bibliography :

• Pace NR.
    “A molecular view of microbial diversity and the biosphere”.
    Science. 1997 May 2;276(5313):734-40. Review.
• Rondon MR, August PR, Bettermann AD, Brady SF, Grossman TH, Liles MR, Loiacono KA, Lynch BA, MacNeil IA, Minor C, Tiong CL, Gilman M, Osburne MS, Clardy J, Handelsman J, Goodman RM.
    “Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms”.
    Appl Environ Microbiol. 2000 Jun;66(6): 2541-7.

• Woese CR, Kandler O, Wheelis ML.
    “Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya”.
    Proc Natl Acad Sci U S A. 1990 Jun;87(12):4576-9.