Nondestructive 3D Imaging and Quantification of Hydrated Biofilm-Sediment Aggregates Using X‑ray Microcomputed Tomography

Biofilm-sediment aggregate (BSA) contains a high water content, either within internal pores and channels or bound by extracellular polymeric substances (EPS) forming a highly hydrated biofilm matrix. Desiccation of BSAs alters the biofilm morphology and thus the physical characteristics of porous media, such as the binding matrix within BSA and internal pore geometry. Observing BSAs in their naturally hydrated form is essential but hampered due to the lack of techniques for imaging and discerning hydrated materials. Generally, imagery techniques (scanning electron microscopy (SEM), transmission electron microscopy (TEM), and focused ion beam nanotomography (FIB-nt)) involve the desiccation of BSAs (freeze-drying or acetone dehydration) or prevent differentiation between BSA components such as inorganic particles and pore water (confocal laser scanning microscopic (CLSM)). Here, we propose a novel methodology that simultaneously achieves the 3D visualization and quantification of BSAs and their components in their hydrated form at a submicron resolution using X-ray microcomputed tomography (μ-CT). It enables the high-resolution detection of comparable morphology of multiphase components within a hydrated aggregate: each single inorganic particle and the hydrated biofilm matrix. This allows the estimation of aggregate density and the illustration of biofilm-sediment binding matrix. This information provides valuable insights into investigations of the transport of BSAs and aggregate-associated sediment particles, contaminants (such as microplastics), organic carbon, and their impacts on aquatic biogeochemical cycling.