Using Branching Fungus to Bioremediate Hard-to-reach Contaminants in Fractured Porous Media

Non-aqueous phase liquid (NAPL) trapped in stagnant or low permeability regions, such as dead-end fracture or rock matrices, are hard to remediate because they are mostly inaccessible by groundwater flow. Recent studies showed the potential of bioremediation technologies that utilize the chemotactic motility of bacteria [1-2]. However, bacteria cannot cross oil-water boundaries. Hence, such methods rely on diffusion and dissolution of contaminants from NAPL to aqueous phase. This process is slow and limited by the interfacial area. On the other hand, hyphae of fungi are known to generate a tremendous turgor pressure on their tips [3] and produce surfactants [4] that allow them to navigate through small pores and air pockets in porous media. However, to the best of our knowledge, there has been no direct visualization of fungal hyphal penetration into oil-water interfaces, and its implication on the bioremediation of NAPL has been unclear.

This study reports the active removal of NAPL by fungi using microfluidic experiments. We isolated naphthalene-degrading colonies from a local coal-tar contaminated site, and through the microbiome analysis, we identified and selected the fungal colony which constituted the major fungal populations in biofilms sampled from the site. The fungi were suspended in a minimal salt medium, and the solution was injected into a PDMS microfluidic chip with a flow channel surrounded by NAPL-saturated low porosity regions (Figure A). Vegetable oil with 1 g/L of naphthalene was used as the model NAPL. The fungal growth and the change of oil-water interfaces were recorded through a scientific CMOS camera at the pore scale. Our results showed the active removal of NAPL by fungi over 65 hours. We observed that clogging of preferential flow path by fungi induced flow instability which led to the viscous fingering-like displacement of trapped NAPL (Figure B). Moreover, fungal hyphae effectively penetrated water-oil interfaces and significantly enhanced the oil removal from low porosity regions (Figure C). In this contribution, we will further discuss the mechanisms behind the effective removal of NAPL by fungi.

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[2] Marx & Aitken (2000). ES&T, 34(16), 3379-3383

[3] Money (1995). CJB, 73(S1), 96-102

[4] Wösten et. al. (1999). Curr. Biol., 9(2), 85-88