*2.4. Iron Quantification*

Due to the importance of ferrous and ferric ion concentrations on the corrosion characteristics of carbon steel, the total iron (Fe2+ and Fe3+) concentration in this study was determined using a colorimetric technique using 1, 10-phenanthroline according to the procedure outlined in reference [31].

#### *2.5. Microscopic and Biofilm Cross-Sectional Analysis*

2.5.1. Development of Biofilms on Carbon Steel for Microscopic Imaging and FIB-SEM Analysis

The carbon steel specimens, mounted in epoxy resin (as described in Section 2.3) were attached to a polypropylene fishing line, and immersed in 250 mL of sterilized test solutions in a 250 mL Duran bottle. Using aseptic techniques, 1 mL of bacterial culture

was taken from a 3-day old stock culture of IRB and inoculated into the Duran bottle as pure culture. The solution was not de-aerated. An abiotic control was also set up under the same conditions. The carbon steel specimen was exposed to the bacterial culture (or abiotic conditions) for a period of 72 h or 168 h at 30 ◦C. At least two samples were observed for each condition in order to examine reproducibility.

#### 2.5.2. Biofilm Fixation and Environmental Scanning Electron Microscopy (ESEM) Imaging

The morphology of the biofilm on the carbon steel specimen was observed using the 3D Quanta FEI in ESEM mode. Biofilms were fixed using 2% glutaraldehyde solution for 1 h and then subject to two washes in deionised water, each wash for 5 min. The use of relatively high pressures (~650 Pa, or 1 mbar) and water vapor in the specimen chamber in the ESEM mode allowed the samples to be viewed in a hydrated state, and without the need for metallic coating, as is often required in SEM sample preparation which introduces artifacts. Minimum of two samples were observed under ESEM for each condition.

#### 2.5.3. Focused Ion Beam—Scanning Electron Microscopy (FIB-SEM)

FIB-SEM technique was used to investigate sub-surface features of the biofilm. FIB milling was carried out using the FEI Quanta 3D FEG instrument. As this instrument also has SEM and EDS capabilities, the subsurface structure could be viewed, and elemental composition analysed following the milling. Due to the sample being placed in a high vacuum chamber (~10−<sup>6</sup> Pa), in addition to the sample preparation steps detailed in Section 2.5.2 for ESEM, the sample was also dehydrated and sputter coated with 3 nm of platinum coating in order to avoid charging. The surface of the specimen was first viewed under SEM to locate a site of interest in the biofilm for subsequent cross-sectional analysis. The site chosen for FIB milling was typical and representative for the condition under study. A platinum strip, 1 μm in width and 1 μm in thickness, was applied across the length of the area of interest in order to protect the biofilm from ion beam degradation during the milling process [32]. The sample was then tilted to 52◦ so that the milling could be performed using current gallium (Ga+) beam (in the range of 5 nA). Beam currents were lowered (in the pA range) for subsequent cleaning of cross-sections to remove material re-deposited on the area during the initial rough milling.

#### 2.5.4. Attenuated Total Reflectance—Fourier Transform Infrared (ATR-FTIR) Spectroscopy

The specimens exposed to biotic and abiotic conditions were removed after 72 h and any loosely attached bacterial cells were rinsed off with phosphate buffer solution. The specimens were left in a desiccator for at least 24 h to dehydrate. FTIR spectra were obtained using a Perkin Elmer Spectrum 100 series spectrometer. The spectral acquisition (128 coadded scans at 8 cm<sup>−</sup><sup>1</sup> of spectral resolution in the 4000 cm<sup>−</sup><sup>1</sup> to 600 cm<sup>−</sup><sup>1</sup> range).
