Date Published: July 21, 2017
Publisher: Public Library of Science
Author(s): Maxence Carrel, Mario A. Beltran, Verónica L. Morales, Nicolas Derlon, Eberhard Morgenroth, Rolf Kaufmann, Markus Holzner, Meghan Byrne.
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.
Biofilms are ubiquitous sessile microorganisms embedded in a self-produced matrix consisting of extracellular polymeric substances (EPS) . The EPS matrix protects biofilms from their environment, so that they persistently develop and survive in industrial, natural or biomedical settings . In water saturated soils, most microorganisms develop sessile lifestyles . Biofilms are of high interest in this context, because of their natural contribution to the bioremediation of aquifers  or to reactive barriers , to microbial enhanced oil recovery  or to the sequestration of carbon dioxide [7, 8]. However, the growth of biofilms in porous media and the consequent bioclogging of the pore spaces  can also be detrimental as it can lead to the clogging of groundwater recharge wells  or deep geothermal systems . It can also lead to an enhanced non-Fickian spreading of solute contaminants in groundwater , substantially complexifying the modelling and upscaling of mass transport in these systems [13–18].
Fig 4(A) and 4(B) show slices of the LFeSO4 and BaSO4 datasets obtained after filtering with a 3D curvature-driven filter. On both images, the biofilm is visible (light gray zones in) A) and the darkest ones in (B) but it seems that there is substantially more biofilm on image (A). The biofilm gray values in (A) exhibit an important heterogeneity. On this image, it is possible to identify darker zones belonging to the liquid phase and lighter ones to the biofilm. Fig 4(D) and 4(E) show the corresponding segmented slices where the segmented biofilm phases obtained with both contrast agents roughly overlap. However, on both the pre-processed (Lorentzian when used and 3D curvature-driven filtering) and segmented images, some biofilm regions locally do not match. Fig 5. shows 3D renderings of the solid phase (Nafion grains) and the segmented datasets which reveal that the volume fraction of the biofilm (green) is much smaller for the BaSO4 dataset. Qualitatively, the biofilm appears more patchy for the BaSO4 than for the LFeSO4 dataset. For the latter, the biofilm exhibits complex corrugated shapes that are more interconnected. It appears that greater detail of the biofilm morphology is resolved when LFeSO4 is used as a contrast enhancing agent.
In this study, the combination of a non-destructive contrast agent, a long STD and Lorentzian filtering revealed intact 3D biofilm morphologies in porous media. This particular combination of contrast agent and imaging tools borrowed from phase contrast imaging allowed to substantially reduce noise and improve the contrast of the sample containing materials with small attenuation coefficient differences.
In this paper, we presented an innovative method to image biofilms in porous media combining iron sulfate as a contrast agent, a long STD and a Lorentzian filter. The non-toxic and non-destructive contrast agent was continuously added to the biofilm during the biofilm growth. The combination of using a large STD together with application of a Lorentzian Fourier filter allowed to exploit refraction effects. The reconstructed data showed a substantial reduction in noise and an increase in the contrast between materials exhibiting low attenuation coefficients differences, revealing the biofilm morphology. We found that in the porous medium and for the present growth conditions, the biofilm exhibits complex corrugated structures. We compared this method with an existing method using BaSO4 as as a contrast agent for the exact same sample and observed some differences in the biofilm morphology obtained due to interactions between the biofilm and the BaSO4. Namely, due to abrasion and shear detachment, more than 50% of the biofilm was washed out by the contrast agent emphasizing the need for non-destructive contrast agents for biofilm imaging in porous media. The method presented in this study delivers 3D biofilm morphologies in porous media non-destructively on a X-ray lab source. Possible applications are studies addressing the interplay between biofilms, their morphology and local hydrodynamic and mass transport processes in realistic porous media models.