Design and optimization of field protocols suitable for an operational context.
Design and testing of pollution scenarii considering chemical characteristics of each HNS group
A complete and versatile bioremediation test bench (BTB) has been designed to mimic as much as possible ecosystems impacted by HNS (land, salt marsh, mudflat and sea). This BTB will be optimized to use both biostimulation and bioaugmentation strategies. Depending on the prioritized fungi selected during WP1, part or entirety of the proposed BTB would be realized for WP4. In order to be able to monitor HNS biodegradation, chemical analysis protocols will be optimized upstream according to the sampling matrix. If necessary, this bench could recreate an ecotone between land and sea thanks to facilities available from the different partners in controlled conditions (light, temperature, agitation, oxygenation, humidity):
- Incubators from LUBEM and BIODIMAR will be used with microcosm (250 mL) to simulate surface oceanic environment with reconstituted seawater.
- The tidal bench of Cedre can simulate the tide in mesocosm. This tidal bench can mimic the tide and the surf on a beach, vegetated salt marsh or mudflat with constantly refreshed seawater.
- Greenhouses and phytotrons from LUBEM and BIODIMAR will be used for shore and land simulation.
- Biodegradation activity hit on the stable isomer of picric acid registered during WP1 will be studied on hyperbaric microcosm to test the ability of fungal enzymes or fungi to degrade this HNS under moderate hydrostatic pressure (100-200 bars) in seawater by Leo Viridis. The final aim would be to assess the degradation potential of Picric acid from immersed leaking ammo directly on the oceanic floor.
To assess the potential synergy between fungus and plant in bioremediation, two phytosystems traditionally involved in bioremediation will be used to mimic shore and land polluted ecosystems.
After validation and optimization of procedures, contaminated and control soils and sediments will be surveyed during a long term exposure experiment. Samples from bulk soil and rhizosphere will be collected in mesocosms during 3 months. The diversity and dynamics of bacterial (target: 16S) and fungal (target: ITS and 18S) communities will be determined using high-throughput sequencing, as already implemented by LUBEM partner. Microbial co-occurrence networks, in addition to measuring microbiota diversity per se, may be important for detecting changes in the ecosystem functioning in response to bioremediation. In addition to this in-depth characterization of plant and soil-associated microbiota, the disease index for each pathosystem will be evaluated after inoculation of the specific pathogen (i.e. Claviceps purpurea and Colletotrichum salicis).
Design and testing of remediation - biocontrol scenarii according to candidates selected during WP1-2
Biostimulation by using fungal metabolites to directly stimulate HNS biodegradation (e.g. surfactants) or by inducing systemic resistance to plant pathogens.
Bioaugmentation by using living fungi directly inoculated in water, soils and sediments.
Bioremediation scenarios will be carried out according to results from the WP1 using the BTB and chemical analysis protocols. To fulfill this objective, an experimental matrix will be designed in function of the targeted HNS and their main environments impacted. In the case of hits would be recorded on the 4 HNS during WP1, the experimental matrix would be simplified to the more impacted environment based on literature data for the sake of feasibility.
For the 2 strategies of bioremediation, fresh enzymes extracts and fungal biomass will be produced at the LUBEM according to protocols and culture parameters optimized in WP1. Feedstock of mycosurfactants produced during WP3 will be used for WP4.
The aim will be to monitor the degradation rate of HNS along with the resilience of water, soils and sediments microbial communities using a metabarcoding approach. In order to highlight putative stringent modifications of microbial communities due to the bio- augmentation step and/or the HNS degradation, snapshots of microbial diversity will be generated after different incubation times to follow the dynamic of microbial communities using iTag sequencing, coupled to qPCR analyses specifically directed towards the bio- augmented fungal isolate to have a better idea of the resilience of each fungal isolate used here for bioremediation processes. At the same time, quantification of HNS will be processed to look for correlation between HNS degradation and evolution of microbial communities.
If the degradation of HNS is studied on land or salt marsh ecosystems, the possible beneficial effect of biostimulation or bioaugmentation on the fitness of bioremediation plants confronted to their reference parasites will be assessed (Spartina maritima vs Claviceps purpurea and Salix purpurea vs Colletotrichum salicis).
Definition of the best way of formulation and propagation of the tested solution considering the operational context.
The objective will be the optimization of the α protocol suitable with a more challenging operational context: β protocol (TRL8).
Bioremediation strategies will be optimized in the most pragmatic way suitable with operational context and cost limitations. By benefiting from the cumulated feedback from previous tasks, efficiency of bioremediation protocols will be improved testing immobilization approach (β protocol). The effect of hydrodynamism, solubility and resilience of both bioremediation strategies will be analyzed and conclusions will drive to the best way to enhance processes performances using already existing options on shelf. Based on literature, several resilient (e.g. non-soluble) immobilization matrices will be tested if requested for biostimulation as well as bioaugmentation. They will be designed considering the targeted environment of operation. They must be biodegradable in the long term. For example, in the case of submarine pollution (e.g. picric acid from submerged ammunition), the immobilization matrix should be able to sink but also to present a shape compatible with potentially important in situ hydrodynamism in order to avoid a too important dispersion during inoculation operation (e.g. pebble shaped of different sizes). Activated charcoal, biopolymeric porous matrix (e.g. xanthan, chitosan), natural fibres (cellulose based material), inorganic matrix (e.g. soft sol-gel process of silica and citric acid) are most probable and affordable candidates. Such immobilization matrices have been already employed in the past for bioremediation (bioaugmentation and biostimulation) and showed good results.
D4.1.1: A versatile, validated and complete bioremediation test bench (BTB) to adapt to WP1 results
D4.1.2: Optimized chemical analysis protocols for HNS biodegradation monitoring
D4.1.3: Characterization of the microbial communities dynamic (bioaugmentation) and network interactions during HNS bioremediation
D4.2.1: Validated bioremediation activities thanks to complete monitoring approaches (e.g. fungal iTag profiles, chemistry analysis, biometry)
D4.2.2: Preliminary efficient fungal bioremediation strategies on the 4 targeted HNS (bioaugmentation and/or biostimulation)
D4.3: A validated set of immobilization solutions compatible with fungal bioremediation