Biofilm imaging in porous media by laboratory X-Ray tomography: Combining a non-destructive contrast agent with propagation-based phase-contrast imaging tools

Abstract

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. A comparison of this new method with the method proposed by Davit et al (Davit et al., 2011), which uses barium sulfate as a contrast agent to mark the liquid phase was performed. Quantitative evaluations between the methods revealed substantial differences for the volumetric fractions obtained from both methods. Namely, contrast agent—biofilm interactions (e.g. biofilm detachment) occurring during barium sulfate injection caused a reduction of the biofilm volumetric fraction of more than 50% and displacement of biofilm patches elsewhere in the column. 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.

Quantitative 3D comparison of biofilm imaged by X‐ray micro‐tomography and two‐photon laser scanning microscopy

“…The Darcy velocity in Carrel et al . () was 1.06 mm/s, whereas the velocity for our flow cell was 52 mm/s. This increase in Darcy velocity could have generated more resistant biofilm structures that can sustain greater shear stress.…”

“…1-chloronaphtalene is an oily solvent which may induce complex two-phase flows in a water-saturated porous medium with a reduced penetration in small pores where capillary pressure is too high. Further, as noted by Carrel et al (2017), it is a powerful biocide that may significantly modify the biofilm structure upon contact with the matrix. BaSO 4 has the advantages of being nontoxic and to provide a very good contrast on X-ray laboratory machines (Carrel et al, 2017).…”

“…(), it is a powerful biocide that may significantly modify the biofilm structure upon contact with the matrix. BaSO 4 has the advantages of being nontoxic and to provide a very good contrast on X‐ray laboratory machines (Carrel et al ., ). Its main drawback is the high viscosity of the suspension, which can damage the biofilm structure upon introduction.…”

“…However, X-ray absorption coefficients for the biofilm and the surrounding aqueous phase are very similar and therefore contrast agents must be used to differentiate between phases. Contrast agents such as silvercoated micro-spheres , 1-chloronaphtalene (Rolland du Roscoat et al, 2014), iron sulphate (Carrel et al, 2017) and barium sulphate (BaSO4) (Davit et al, 2011) have been tested, but currently each of these has drawbacks restricting their application to specific experimental conditions. Silver-coated micro-spheres map the biofilm surface only, making it cumbersome and inaccurate to perform volume segmentation.…”

Emergent Properties of Microbial Activity in Heterogeneous Soil Microenvironments: Different Research Approaches Are Slowly Converging, Yet Major Challenges Remain

“…In many ways, the same comment pertains to the use of nuclear resonance imaging to detect “biofilms” in systems composed of polystyrene beads (Vogt et al, 2013). Sanderlin et al (2013) pioneered the use of a very promising low-field magnetic resonance system to visualize the 3D distribution of biofilms in glass beads and sand particles, whereas a number of other authors used X-ray tomography to assess the distribution of biofilms in systems of glass beads (Davit et al, 2011; Iltis et al, 2011; Peszynska et al, 2016) or 2.5 mm-diameter Nafion pellets (Carrel et al, 2017). In all these cases, the properties and geometry of the systems investigated are drastically different from those of actual soils, which are generally characterized by a spatially dispersed- rather than concentrated biomass, and it is not clear at all at this stage how the transition from artificial media to actual soils will be made.…”