Phosphorus sequestration: contribution of magnetotactic bacteria in oxic–anoxic transition zones – PHOSTORE

1) We carried out a detailed physicochemical profile along the water column of Lac Pavin. After locating the oxic-anoxc transition zone (OATZ) and the MTB communities by optical microscopy, a sampling of the OATZ with a step of 20 cm (9 depths) was carried out and analyzed with regard to the concentration of the different morphotypes. of MTB (magnetic sorting and light microscopy approaches). The proportion of magnetotactic cocci containing or not containing PolyP inclusions was evaluated as a function of the depth in the water column using transmission electron microscopy ( STEM & HR-TEM) and Energy Dispersive X-ray Spectrometry (EDXS). The statistical relationships between the abundance of MTB phenotypes and morphotypes, and different physicochemical and geochemical parameters was assessed using multivariate statistics.

2) Water column samples were collected in order to identify the different populations of magnetotactic bacteria. A single-cell sorting approach was carried out by micromanipulation under an optical microscope. As different MTB populations are easily distinguished by optical microscopy due to different parameters (e.g., cell morphology, presence of inclusions, mobility) such an approach has thus made it possible to specifically target the MTB populations of interest. Once the cells were sorted, their genome was amplified in order to have sufficient DNA available for sequencing their 16S rRNA and then their genome.

- The convergence of projects related to the study of magnetotactic bacteria in the water column of Lake Pavin, led by various members of this consortium (ANR SIGMAG, ANR PHOSTORE, CNRS INSU-EC2CO Microbien 2020), resulted in the development of an efficient method of magnetic concentration coupled with high-resolution sampling by in-line pumping (Busigny et al. 2021, Env. Microbiol. doi: 10.1111 / 1462-2920.15458). This development now allows detailed studies of the structure and composition of MTB populations and their ecological niches, and makes it possible to sample large volumes of water.
- A new MTB morphotype was recently described by our consortium, in the oxic-anoxic transition zone located in the water column of Pavin Lake: rods forming amorphous calcium carbonate inclusions, belonging to an undescribed genus within the Rhodospirillaceae family of the Alphaproteobacteria class (Monteil et al. 2021, ISME J doi: 10.1038 / s41396-020-00747-3).
- We have demonstrated the stratification of 2 main populations of magnetotactic bacteria in the water column of Lake Pavin. These bacteria exhibited distinct sequestration capacities (polyphosphates of magnesium, calcium or potassium, and amorphous calcium carbonates, respectively). We identified biogeochemical niches of MTB populations, in particular cocci forming intracellular polyphosphates, and we proposed some key factors associated with their structuration in natural environments.

Metagenomics will be use in order to characterize the functional potential and molecular diversity of MTB communities in the Lake Pavin water column. The metabolisms linked to the environmental factors structuring the population of P-accumulating cocci will be explored through this gene centric approach. In addition, the contribution of these bacteria to the P cycle in Pavin Lake will be evaluated, by analyzing sediments and the export of phosphorus by MTB from the water column.

- Monteil CL, Benzerara K, Menguy N, Bidaud CC, Michot-Achdjian E, Bolzoni R, Mathon F, Coutaud M, Alonso B, Garau C, Jézéquel D, Viollier E, Ginet N, Floriani M, Swaraj S, Sachse M, Busigny V, Duprat E, Guyot, F, Lefèvre CT (2021) Intracellular amorphous Ca-carbonate and magnetite biomineralization by a magnetotactic bacterium affiliated to the Alphaproteobacteria. ISME J. 15:1-18 doi.org/10.1038/s41396-020-00747-3 hal-cea.archives-ouvertes.fr/cea-02961539

- Busigny V, Mathon FP, Jézéquel D, Bidaud CC, Viollier E, Bardoux G, Bourrand JJ, Benzerara K, Duprat E, Menguy N, Monteil CL, Lefèvre CT (2021) Mass collection of magnetotactic bacteria from the permanently stratified ferruginous Lake Pavin, France. Env. Microbiol. doi.org/10.1111/1462-2920.15458 hal.archives-ouvertes.fr/hal-03171041

- Bidaud CC, Monteil CL, Menguy N, Busigny V, Jézéquel D, Viollier E, Travert C, Skouri-Panet F, Benzerara K, Lefevre CT, Duprat E. Population structure of magnetotactic bacteria forming intracellular polyphosphates in the water column of Lake Pavin, a freshwater ferruginous environment, EGU General Assembly 2021, online, 19–30 Apr 2021, EGU21-11061 doi.org/10.5194/egusphere-egu21-11061

The role of microorganisms in the geochemical cycle of phosphorus (P) has received great interest in the context of bioremediation and the formation of sedimentary P resources (phosphorites). In particular, some microorganisms particularly prone in sequestering P have been observed in oxic-anoxic transition zones (OATZ). However, only few models are known yet, most of which have been identified at water-sediment interfaces.
We recently evidenced some magnetotactic bacteria (MTB) affiliated to the Magnetococcaceae family, strongly accumulating intracellular polyphosphates (PolyP) at the OATZ in the water column of the ferruginous Lake Pavin (Massif Central, France). We propose that these MTB are a new and valuable model to study the cycling of P by microorganisms in freshwater environment, the molecular mechanisms of P sequestration and how it is impacted by environmental parameters.
Here, we propose to combine in situ, in vitro and in silico multidisciplinary approaches in order to (i) further identify MTB strains particularly efficient in accumulating PolyP, (ii) measure the size and dynamics of this P-reservoir and therefore the impact of MTB on the P cycle in Lake Pavin, by their active storage of P in the biomass or by favoring the precipitation of phosphate mineral phases, and (iii) determine the genetic, metabolic and environmental determinants of these processes. This project, with an inherent questioning about the interactions between living systems and solid inorganic phases, will be carried out by a highly cohesive and complementary consortium combining recognized expertise in microbiology and ecology of MTB, (bio)mineralogy and geochemistry of solid and aqueous phases, (environmental) genomics, field- and laboratory-work.
Our work program will consist in (i) exploring the P cycling in Lake Pavin, in particular correlations between biological (MTB diversity, PolyP accumulation) and geochemical parameters (solution geochemistry). The MTB and other PolyP-bearing microbial species will be described using cell sorting strategies followed by taxonomic identification. The size, dynamics and fate of this biological reservoir of P will be determined, with a quantification of the contribution of MTB to P sequestration and export; (ii) in vitro cultivation of MTB isolated from Lake Pavin under varying controlled conditions with parallel monitoring of P speciation and accumulation kinetics, metabolic and mineralogical traits, and gene expression. The P sequestration capabilities of MTB isolated from Lake Pavin will be compared with those of phylogenetically close/distant model strains, isolated from other OATZ environments. Further insight on intracellular PolyP formation will be provided by their characterization at the micro- and nano-meter scale using advanced microscopies and spectroscopies; (iii) deciphering the functional potential of several MTB enriched from Lake Pavin as a function of their taxonomic identity, with a focus on the abundance of gene families involved in the bacterial metabolism of P. These genomic repertoires will be compared with those of model MTB strains and other already-known bacterial P-hyperaccumulators with available genome.
We will provide new microbial models for efficient P sequestration and produce fundamental knowledge on the dependence of this process on environmental chemical conditions. In the longer term, this project may enable the development of “green” depollution/bioremediation strategies for P removal and/or heavy metal sequestration using MTB, which offer the advantage of being easily sorted by a magnetic field. This could be also a strategy to recover efficiently P from wastewater and for further use as an alternative to the exploitation of sedimentary deposits. Microbial populations sequestering PolyP efficiently such as those found in Lake Pavin may therefore provide ecosystem services and/or inspire new ways to fix efficiently P in the form of biomass and minerals.