Task 3 – Validation of the transfer properties of the nano-tracers

Responsible:  IFSTTAR & LEM –Task Leaders P.E. Peyneau & V. Rodriguez Nava
Participants: IFSTTAR & LEM with the support of ILM, Nano-H, Monash U., and IGE


This task aims at the selection and validation of the engineered nano-tracers. Determination of their properties (size and charge) to mimic real nano-pollutants and bacteria. Characterization of the transfer properties of the engineered nano-tracers under various dynamic conditions using laboratory columns: model porous media, homogeneous flow (homogeneous porous media) and strongly heterogeneous flow (macropored porous media). Test on the field with comparison between the engineered nano-tracers and real pollutants and bacteria in stormwater.

Description of work

The first step consists in the characterization of emerging pollutants (anthropic nanoparticles) and bacteria in stormwatrer and runoff water. Water and solid aliquots will be sampled in Django Reinhardt infiltration basin. The population of nanoparticles contained in the fraction < 5 µm of suspended solids will be studied and characterized in terms of their Zeta potential and size distribution using NDLS method and SEM (Scanning Electron Microscope) analyses. The composition of stormwater and runoff water in terms of Nocardia and Pseudomonas cells will be conducted by analysis of hsp65 and tpm DNA sequences. We anticipate P. aeruginosa to be a significant part of the percolating species but that only a limited number of genotypes of this species will be concerned. Such cells can be differentiated and tracked by several DNA markers such as tpm [30]. The tpm gene is found among major bacterial pathogens belonging to the gamma-proteobacteria such as Pseudomonas aeruginosa, Legionella pneumophila, Bordetella pertussis, Vibrio cholerea and Aeromonas caviae. Another genetic marker will be used in these investigations to complete our view of the bacterial taxa, i.e. the hsp65 marker will be used since it allows a differentiation of actinobacteria [76]. Range of sizes observed among each genotype will be investigated by flow sight cytometry or using a laser granulometer [12]. Zeta potential analyses of these distinct lineages will be performed to quantify charges that should be harbored by the nano-tracers. Similarly, from the hsp65 dataset, N. cyriacigeorgica genotypes will be identified and analyzed as indicated above (size, charge). These works will give a priority list of anthropic nano-pollutants and bacteria species to study.

The second step consists of transfer studies of the transfer properties of the engineered nano-tracers in model porous media, under controlled flow conditions (lab column). Experiments will be conducted with both homogeneous porous media made of glass beads and the similar medium with an inserted macropore [45, 50] and also materials encountered on the field. These synthetic systems will be submitted to different flow conditions (unsaturated, saturated, several flowrates) and fed with the solution of engineered nano-tracers on one hand and with real stormwater containing emerging pollutants and bacteria (containing tpm and hsp65 harboring species) on the other hand. Macropored columns will allow investigating the incidence of the size of macropores and flow conditions (unsaturated, saturated, variable flow rates) on nanoparticle and bacterial transfer. Elution and deposition patterns will be compared between the engineered nano-tracers and the targeted emerging pollutants & bacteria.

The columns will be inspected at the end of leaching experiments and during the experiments using MRI for the engineered nano-tracers (AguIX nanoparticles). Bacterial concentrations will be either estimated from fluorescence emissions using gfp-labelled bacterial cells (equipment to be bought) or by quantitative PCR. Physico-chemical parameters such as pH, conductivity, UV spectrometry will be continuously registered. The breakthrough curves of solutes and nano-pollutants will be monitored by fluorescence spectroscopy. All these experiments (at column scale) will be combined with a modelling approach at several scales (with DEEP & IFSTTAR, LEHNA, Monash University, IGE).

The third step consists in field observations and tests. Field observations previously obtained for Django Reinhardt infiltration basin over the last 10 years [87] and mostly for heavy metals (e.g. Cu & Zn) will be completed for the studied emerging pollutants and bacterial taxa by LEM and LEHNA. A set of water-solute-nanoparticle infiltration experiments will be performed in the field to compare the transfer of the engineered nano-tracers, the emerging pollutants and bacteria. As described for the in vitro experiments, hsp65 and tpm DNA trackings will be used to estimate the DjR filtration capacity of bacterial taxa. The fate of tpm and hsp65 sequences will be monitored, and correlations between the distributions of the observed OTU (operational taxonomic units) and nano-tracers will be investigated. Illumina miseq sequencings of tpm/hsp65 PCR products will be performed at Biofidal (20000 reads per sample). Reads will be analysed by the Mothur (www.mothur.org) suite and Vegan package (in R) in order to compare tpm/hsp65 genetic structures. Correlation analyses will be performed through the CoNet application implemented in the Cytoscape software [29].

Role of participants

The column experiments dedicated to the transfer of pollutants and bacteria in synthetic porous media will be part of a joint activity between IFSTTAR and LEM teams. IFSTTAR will design and provide the synthetic porous media. The injection of nano-tracer and anthropic nanoparticles will be performed in the synthetic porous media at IFSTTAR. All columns with injection of bacteria will be performed at LEM at SFR41 SEDAQUA platform. LEM will benefit from IFSTTAR for the synthetic porous media and the supervision of column experiments.

Modelling of nano-tracer transfers will be conducted by IFSTTAR and LEHNA with the help of ILM (for nano-tracer reactivity).

Modelling of bacterial transfers will be conducted by LEM with Monash and IGE for modelling bacteria reactivity and growth.

Risks and contingency plan

This task is crucial since it will be active all along the project, from the determination of properties nano-pollutant and bacteria in stormwater before engineering the nano-tracers to the validation of engineered nano-tracers. The validation will help to optimize the engineered nano-tracers throughout the whole project and through a back and forth procedure between tasks. However, a final deadline will be fixed to save enough time to complete the injection and infiltration experiments for the final version of engineered nano-tracers. The consortium has relevant skills and devices required for the realization of the task. Methods and protocols for the realization of synthetic porous media containing macropores are already available and column leaching experiments are routinely conducted at IFSTTAR. LEHNA and IFSTTAR already have the experimental devices and methodology for both injection and detection of tracer, reactive solutes and nanoparticles. The participation of partners skilled in the use and characterization of both nanoparticles and bacteria properties will minimize the risks associated with such complex experiments. Special attention during the preparation of the experimental protocols will be paid to the stability of background chemical conditions in columns to avoid impacts on the nanoparticle suspensions and physiological status of the bacteria.

Deliverables / milestones

  • D3.1 Report on the characterization of emerging nano-pollutants and bacteria in stormwater and runoff water
  • D3.2 Report on the validation of engineered nano-tracers
  • M3.1 Characterization of emerging pollutants in stormwater
  • M3.2 Characterization of bacteria in stormwater
  • M3.3 Tests of designed nano-tracers in synthetic porous media using columns
  • M3.4 Tests of designed nano-tracers on the field