Biofilm Imaging in Porous Media by Laboratory X-ray Tomography: Combining a Non-Destructive Contrast Agent with Propagation-Based Phase-Contrast Imaging Tools

X-ray tomography is a powerful tool giving access to the morphology of biofilms, in 3D
porous media, at the mesoscale. Due to the high water content of biofilms, the
attenuation coefficient of biofilms and water are very close, hindering the distinction
between biofilms and water without the use of contrast agents. Until now, the use of
contrast agents such as barium sulfate, silver-coated micro-particles or
1-chloronaphtalene added to the liquid phase allowed imaging the biofilm 3D
morphology. However, these contrast agents are not passive and potentially interact
with the biofilm when injected into the sample. Here, we use a natural inorganic
compound, namely iron sulfate, as a contrast agent progressively bounded in dilute or
colloidal form into the EPS matrix during biofilm growth. By combining a very long
source-to-detector distance on a X-ray laboratory source with a Lorentzian filter
implemented prior to tomographic reconstruction, we substantially increase the contrast
between the biofilm and the surrounding liquid, which allows revealing the 3D biofilm
morphology. Two key advantages of the newly proposed method are that passive addition
of iron sulfate maintains the integrity of the biofilm prior to imaging, and that the biofilm itself
is marked by the contrast agent, rather than the liquid phase as in other available methods.
The iron sulfate method presented can be applied to understand biofilm development and
bioclogging mechanisms in porous materials and the obtained biofilm morphology could be an ideal
basis for 3D numerical calculations of hydrodynamic conditions to investigate biofilm-flow coupling.