A compartmentalized microsystem helps understanding the uptake of benzo[a]pyrene by fungi during soil bioremediation processes.

Hydrophobic organic soil contaminants such as polycyclic aromatic hydrocarbons (PAH) are poorly mobile in the aqueous phase and tend to sorb to the soil matrix, resulting in low bioavailability. Some filamentous fungi are efficient in degrading this kind of pollutants. However, the mechanism of mobilization of hydrophobic compounds by non-motile microorganisms such as filamentous fungi needs investigations to improve pollutant bioavailability and bioremediation efficiency. Usual homogeneous media for microbial growth in the lab are poorly suited to model the soil, which is a compartmentalized and heterogeneous habitat. A microfluidic device was designed to implement a compartmentalization of the fungal inoculum and the source of the pollutant benzo[a]pyrene (BaP) as a deposit of solid crystals in order to gain a further insight into the mechanisms involved in the access to the contaminant and its uptake in soils. Thus in this device, two chambers are connected by an array of parallel microchannels that are wide enough to allow individual hyphae to grow through them. Macro-cultures of Talaromyces helicus in direct contact with BaP have shown its uptake and intracellular storage in lipid bodies despite the low propensity of BaP to cross aqueous phases as shown by simulation. Observations of T. helicus in the microfluidic device through laser scanning confocal microscopy indicate preferential uptake of BaP at a close range and through contact with the cell wall. However faint staining of some hyphae before contact with the deposit also suggests an extracellular transport phenomenon. Macro-culture filtrates analyses have shown that T. helicus releases extracellular non-lipidic surface-active compounds able to lower the surface tension of culture filtrates to 49.4 mN/m. Thus, these results highlight the significance of active mechanisms to reach hydrophobic contaminants before their uptake by filamentous fungi in compartmentalized micro-environments and the potential to improve them through biostimulation approaches for soil mycoremediation.

Microfluidic monitoring of the growth of individual hyphae in confined environments.

Soil fungi have the ability to form large mycelial networks. They rely on the resources available in the soil to produce biomass and are able to degrade complex biomolecules. Some of them can even degrade recalcitrant organic pollutants and are considered as promising candidates for soil bioremediation strategies. However, the success of this approach depends on the ability of fungi to colonize the soil matrix, where they encounter spatial and temporal variations of confinement, humidity and nutrient concentration. In this paper, we present a study of fungal growth at the scale of single hyphae in a microfluidic device, allowing fine control of nutrient and water supply. Time-lapse microscopy allowed simultaneous monitoring of the growth of dozens of hyphae of Talaromyces helicus, a soil isolate, and of the model fungus Neurospora crassa through parallel microchannels. The distributions of growth velocity obtained for each strain were compared with measurements obtained in macroscopic solid culture. For the two strains used in the study, confinement caused the growth velocity to drop in comparison with unconfined experiments. In addition, N. crassa was also limited in its growth by the nutrient supply, while the microfluidic culture conditions seemed better suited for T. helicus. Qualitative observations of fungi growing in microfluidic chambers without lateral confinement also revealed that side walls influence the branching behaviour of hyphae. This study is one of the first to consider the confinement degree within soil microporosities as a key factor of fungal growth, and to address its effect, along with physicochemical parameters, on soil colonization, notably for bioremediation purposes.