Hydrophobins from Aspergillus mediate fungal interactions with microplastics

Abstract

Microplastics present myriad ecological and human health risks including serving as a vector for pathogens in human and animal food chains. However, the specific mechanisms by which pathogenic fungi colonize these microplastics have yet to be explored. In this work, we examine the opportunistic fungal pathogen, Aspergillus fumigatus, and other common soil and marine Aspergilli, which we found bind microplastics tightly. Up to 3.85+/- 1.48 g microplastic plastic/g fungi were bound and flocculated for polypropylene (PP), polyethylene (PE), and polyethylene terephthalate (PET) powders and particles ranging in size from 0.05 - 5 mm. Gene knockouts revealed hydrophobins as a key biomolecule driving microplastic-fungi binding. Moreover, purified hydrophobins were still able to flocculate microplastics independent of the fungus. Our work elucidates a role for hydrophobins in fungal colonization of microplastics and highlights a potential target for mitigating the harm of microplastics through engineered fungal-microplastic interactions.

Keywords: flocculation; fungi; hydrophobin; microplastic.

Figure 1:. Aspergillus ubiquitously capture microplastics from solution.

(A) Polyethylene particles captured from solution via flocculation by fungal isolate. (B) Microplastics recovery of a variety of ‘pristine’ and post-consumer plastics shows ubiquitous recovery near 100%.

Figure 2:

(A) Phylogenetic tree of 45 Aspergillus strains built using complete ITS sequences, with strains used in this study boxed in red. A neighbor joining tree was constructed with 100 bootstrap iterations, using Metarhizium anisopliaei as the outgroup. Tree is rooted to the outgroup. (B) Species tree confirming taxonomic identification of AF UD1. The tree was built by FastTree based on orthofinder clustering.

Figure 3:

Biomass normalized flocculation of two plastic types by Aspergillus strains across the genome. Images above each bar are 5 mL liquid cultures of each strain with flocculated LDPE particles.

Figure 4:. Hydrophobins drive microplastics flocculation.

(A) Confocal microscopy image of Aspergillus fumigatus AF-UD1 (blue) stained with calcofluor white using hyphal interactions to grab fluorescent LDPE beads (red). (B) SEM image showing dense hyphal network of Aspergillus fumigatus AF-UD1 holding microplastic beads in a floc. (C) Images showing microplastics flocculation by AF-UD1 in the absence (left) and no flocculation in the presence (right) of beta-mercaptoethanol.

Figure 5:. Hydrophobins are responsible for microplastics flocculation by AF-UD1.

(A) Microplastics flocculation by Aspergillus strains with hydrophobin genes knocked out from the genome. Aspergillus fumigatus strains with each hydrophobin gene knocked out have reduced flocculation ability, indicating the importance of hydrophobins in microplastics recovery. (B) Recovery of green fluorescent LDPE beads by pure RodA (right) relative to a water control (left).