*2.3. Bacteria Culture*

Non-pathogenic (ACDP Group 1) *Sporosarcina pasteurii*, commonly found in soil, was obtained from the American Type Culture Collection, Manassas, VA, USA, (ATCC 11859) as a freeze-dried culture and used to produce a stab culture for storage at 4 ◦C. Bacteria were transferred from the stab culture using a sterile inoculation loop onto plates of Luria–Bertani (LB) agar amended with 20 g/<sup>L</sup> syringe filtered urea. Growth medium for plates contained 5 g/<sup>L</sup> yeas<sup>t</sup> extract, 10 g/<sup>L</sup> tryptone, 10 g/<sup>L</sup> sodium chloride, 15 g/<sup>L</sup> agar and 20 g/<sup>L</sup> urea in deionised water. The inoculated plates, sealed with gas-permeable film, were incubated at 23 ◦C room temperature for 48 h. Single colonies from the plates were used to inoculate liquid growth medium. Triplicates of 50 mL liquid broth cultures were produced in 250 mL Erlenmeyer flasks, for use as an inoculant for further liquid broth cultures to be used in experiments. The liquid growth medium consisted of 13 g/<sup>L</sup> autoclave sterilised Oxoid CM0001 and 20 g/<sup>L</sup> syringe filtered urea in deionised water. Flasks were shaken at 23 ◦C, 150 rpm until the late-exponential phase of growth was reached after approximately 12 h and then stored at 4 ◦C. Liquid broth cultures were produced in 50 mL quantities using 250 mL Erlenmeyer flasks for the batch tests, followed by multiples of 150 mL in 500 mL flasks for the column studies for which greater volumes were required.

Bacterial cultures for use in experiments were inoculated using 100 μL liquid broth culture per 50 mL growth medium and aerobically grown at 23 ◦C, 150 rpm until an optical density at a wavelength of 600 nm (OD600) of 0.9–1.2 was obtained, which equates to approximately 7.5 × 107–1.1 × 10<sup>8</sup> cells/mL, according to the relationship reported by Ramachandran et al. [27]. For the column studies, freshly grown liquid broth cultures were transferred to 50 mL sterile polypropylene tubes, each containing 35 mL culture, and centrifuged at 5000 rpm for 20 min. The supernatant was then removed, and a sample taken from this to measure the optical density, to take into account any loss of bacteria in the supernatant. The bacteria were then resuspended in a small quantity of phosphate buffered saline (PBS), dispersed using a pipette and transferred to 15 mL centrifuge tubes from which bacteria would be injected in the columns. These bacterial suspensions were made up to 10 mL with additional PBS. Use of PBS ensured the bacteria would not undergo osmotic shock which would otherwise occur in water. Aseptic technique was followed throughout, and involves using lab practices which prevent contamination, to help ensure that the only bacteria present within the culturing flasks and columns was *Sporosarcina pasteurii*.

### *2.4. Preparation of Cemention Medium*

Two variations of the cementation medium (CM) were produced for column treatments, as per Table 2. The basic constituents of the CM as required for the process of MICP are urea and a calcium source. Calcium chloride dihydrate was selected for the calcium source. A slightly higher molarity of urea was used in comparison to calcium chloride dihydrate, since this helps ensure all calcium can be utilised. In addition, a source of nutrients, Oxoid CM0001, was added to the cementation medium to promote ongoing bacterial growth and therefore urease activity during treatment. CM1 consisted of 0.67 M urea and 0.50 M calcium chloride dihydrate, in addition to 3 g/<sup>L</sup> Oxoid CM0001 and was used as a fixation medium to fix the bacteria to the sand within the columns in addition to initiating MICP. CM2, based on the cementation medium used by Stocks-Fischer et al. [8] and Al Qabany and Soga [28], was as per CM1 with 6 g/<sup>L</sup> Oxoid CM0001, with 0.187 M ammonium chloride and 0.025 M sodium bicarbonate added. Sodium bicarbonate is added to stabilise the pH of the cementation medium before injections [28] and addition of ammonium chloride was found to help stimulate the MICP process beyond the initial CM injection.


**Table 2.** Cementation media composition and sterilisation methods.

The cementation media were prepared using tap water. Results from a batch test conducted as part of this study provided evidence of the beneficial effect of using tap water compared to deionised water. CM1 was produced by first autoclaving a solution containing calcium chloride dihydrate and Oxoid CM0001, into which a solution containing urea was syringe filtered. To prepare 2.0 L of CM2, firstly the ammonium chloride and Oxoid CM0001 were dissolved in 1.6 L tap water. This solution was adjusted to pH 6.0 using 2.0 M HCl prior to then adding the powdered calcium chloride dihydrate. The pH adjustment prevented the calcium precipitating out into the solution. This solution was autoclaved then made up to 2.0 L by adding a solution containing the urea and sodium bicarbonate using a 0.2 μm syringe filter.

### *2.5. Urease Activity and Batch Test*

Urease activity (mM urea hydrolysed/min) is calculated as per the relationship derived by Whiffin [7] below, based on a conductivity assay.

$$\text{LIRense Activity} = \text{Electrical conductivity} \left(\frac{\text{mS}}{\text{cm}} / \text{min}\right) \times 11.11 \left(R\_2 = 0.9988\right) \tag{3}$$

Electrical conductivity was measured over five minutes to obtain the average activity per minute, as per Harkes et al. [29]. This process was repeated 3 times for each sample tested and an average taken from the three results.

Specific urease activity (mM urea hydrolysed/min/OD600) is further defined by Whiffin [7] as the amount of urease activity per biomass, as per Equation (4).

$$Specific\text{ }U\text{-}measure\text{ }Activity = \frac{\text{ }U\text{-}case\text{ }Activity}{Biona\text{ }s\text{ }(OD\_{600})}\tag{4}$$

A batch test was conducted to determine effects on urease activity of (i) bacterial growth in tap water and deionised water, (ii) inoculation of medium with plate cultures or liquid broth culture, (iii) initial pH of growth medium. 50 mL liquid broth cultures were prepared using tap water or deionised water as described above and grown at 23 ◦C for 14–19 h, to achieve a stationary stage of growth. The nutrient medium was inoculated with either 100 μL of a liquid broth culture grown to 1.0 OD600 or with one colony from a plate culture. To test the effect of pH of the nutrient medium on urease activity the pH of the solution of Oxoid CM0001 nutrient broth in water was adjusted prior to autoclaving and adding urea, after which a 1 mL sample was taken to test the pH prior to inoculation.
