Carrel Maxence, Beltran Mario A, Morales Verónica L, Derlon Nicolas, Morgenroth Eberhard, Kaufmann Rolf, Holzner Markus
Institute of Environmental Engineering, ETH Zürich, Stefano Franscini-Platz 5, 8093 Zurich, Switzerland.
Swiss Federal Laboratories for Materials Science and Technology (EMPA), Dübendorf, Switzerland.
PLoS One. 2017 Jul 21;12(7):e0180374. doi: 10.1371/journal.pone.0180374. eCollection 2017.
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.
X射线断层扫描是一种强大的工具，可在中尺度上获取三维多孔介质中生物膜的形态。由于生物膜的含水量高，生物膜和水的衰减系数非常接近，在不使用造影剂的情况下难以区分生物膜和水。到目前为止，向液相中添加硫酸钡、镀银微粒或1-氯萘等造影剂可以对生物膜的三维形态进行成像。然而，这些造影剂并非被动的，注入样品时可能与生物膜发生相互作用。在此，我们使用一种天然无机化合物，即硫酸铁，作为造影剂，在生物膜生长过程中以稀释或胶体形式逐渐结合到胞外聚合物（EPS）基质中。通过在X射线实验室源上采用非常长的源到探测器距离，并在断层重建之前实施洛伦兹滤波器，我们显著提高了生物膜与周围液体之间的对比度，从而能够揭示生物膜的三维形态。将这种新方法与Davit等人（Davit等人，2011年）提出的使用硫酸钡作为造影剂标记液相的方法进行了比较。两种方法之间的定量评估显示，从两种方法获得的体积分数存在显著差异。具体而言，硫酸钡注入过程中发生的造影剂-生物膜相互作用（如生物膜脱落）导致生物膜体积分数降低超过50%，并使生物膜斑块在柱中的其他位置发生位移。新提出的方法的两个关键优点是，被动添加硫酸铁在成像前保持了生物膜的完整性，并且生物膜本身由造影剂标记，而不是像其他现有方法那样标记液相。所提出的硫酸铁方法可用于理解多孔材料中生物膜的形成和生物堵塞机制，所获得的生物膜形态可能是用于研究生物膜-流动耦合的水动力条件三维数值计算的理想基础。
