## 2 Characteristics of Biofilm Growth in Porous Media
### X-ray Tomography Technology  
X-ray tomography, as a nondestructive and in situ method, has already been used to obtain 3D images and characterize the structure of porous media as well as the distribution of fluids at the pore scale for decades [64; 65; 66; 67]. However, this technology has not been used to image biofilms in porous media until 2010, due to the significant challenge also ailing conventional contrast agents in distinguishing the biofilm from the fluid phase [54]. In an application for imaging the structure of biofilms in porous media, X-ray beams perpendicularly penetrate the column. The transmitted X-rays are then converted to visible light and a photograph is taken to obtain a two-dimensional (2D) grayscale map [65]. A large number of these 2D images at different depths can be used to reconstruct a 3D structure of biofilms in pore space [65; 55]. Since materials with different atomic mass absorb different degrees of X-rays, the water and the porous media can be distinguished because of their different atomic masses [65]. However, since there are more than 90% of water molecules in biofilms [3], a contrast agent is needed for dividing these two kinds of materials. Within the last decade, to divide the biofilm from bulk fluid, researchers have made progress towards determine contrast agents suitable for biofilm imaging in porous me \begin{table}
\begin{tabular}{c c c} \hline \hline
**Method** & **Porous Media** & **Grain Size (\(\mu\)m)** \\ \hline \multirow{4}{*}{X-ray} & Glass beads [54] & \textgreater{}1000 \\  & Expanded polystyrene beads [55] & 500–1500 \\  & Clay beads [20] & \textgreater{}1000 \\  & Nafion pellets [56] & 2500 \\  & Transparent Nafion pellets [57] & 2500 \\ \hline \multirow{4}{*}{NMR} & Glass beads [58] & 1000–1500 \\  & Monodisperse beads [39] & 241 \\  & Monodisperse beads [59] & 241 \\  & Glass beads [60] & 100 \\  & Soil [61] & - \\ \hline \hline CLSM & Glass beads [62] & 425–600 \\ \hline CCD & Silica sand [63] & 210–297 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Methods and materials. dia [20, 54, 55, 56, 57]. [PERSON] et al. [54] first used hollow silver-coated microspheres as a contrast agent to generate realistic 3D representations of biofilm grown in a glass bead pack. They further tried other contrast agents--a mixture of micrometer-sized barium sulfate and potassium iodide--to visualize the biofilms in polyamide and expanded-polystyrene-beads-modeled porous media [55]. The images are restricted to the external structure of biofilm since the agent particles have full coverage over the biofilm surface. Nevertheless, the obtained 3D images of biofilm structure may provide new ideas and experimental bases for studying the heterogeneity of mass transport and pore space caused by biomass growth. 1-Chloronaphtalene was used as a contrast agent to prevent the drawbacks of classical agents, such as fast sedimentation and heterogeneous distribution [20]. Characteristics (for example, partial pore clogging), especially at the sites of nutrient entry, can be observed by imaging experiment. A quantitative analysis on the microstructure (volume fractions and specific surface area) of the biofilm developed in columns of clay beads is also proposed based on the 3D images obtained in that study. The methods mentioned above share another trait in that the agents are potentially destructive to the biofilm morphology. Accordingly, iron sulfate (FeSO4) was chosen as a nontoxic inorganic compound and added to the feed solution during biofilm growth [56]. In the contrasting experiment, barium sulfate was used, and more than 50% of biofilms were washed out due to abrasion and shear detachment, emphasizing the importance of nondestructive contrast agents (Figure 3). The oxidation of Fe (II) to Fe (III), which is less soluble and tends to form colloids, was observed quickly after the startup of the system, complicating the biofilm-filled system.