*2.7. Bacteria Fixing and Biocementation*

To achieve biocementation, a two-stage process was applied involving (i) the injection of a bacterial suspension, followed by (ii) injections of cementation medium (treatments), as per Table 4. The first treatment, using CM1, is injected immediately after the bacterial suspension and has the effect of fixing the bacteria within the columns in addition to initiating MICP. Harkes et al. [29] found that when a cementation solution consisting of 1 M equimolar urea and calcium chloride was injected into columns immediately after a bacterial suspension this resulted in 100% retention of bacteria, as determined by optical density measurement of effluent samples. Divalent cationic ions such as Ca2+ may enhance the attachment of bacteria to surfaces by reducing electrostatic repulsion [31]. The bacterial suspension was injected upwards into the base of all columns simultaneously using a peristaltic pump and constant 1.5 mL/min pumping rate, followed immediately by one and a half pore volumes of CM1. The outlet tubing from columns was then drained and reconnected. Tubing was closed off with clamps and disconnected from the pump tubing following each treatment. On each of the following four days, one and a half pore volumes of CM2 were pumped through the columns, as per the schedule detailed in Table 4.


**Table 4.** Columns treatment schedule.

The timing between treatments had been determined by preliminary studies undertaken as part of this research, and based upon the time taken to deplete the calcium source in columns containing jute, in which this occurred fastest. While injecting CM2 the e ffluent was collected in a series of 5 mL quantities in 15 mL polypropylene tubes.

### *2.8. Measurement of Electrical Conductivity, pH and Evaluation of Bacterial Fixing*

The measurement of pH and electrical conductivity can provide an indication of the extent of substrate conversion, and thus bacterial or urease activity within columns. The e ffluent displaced from columns during the injection of new CM was collected in a series of centrifuge tubes in 5 mL quantities. For each 5 mL column e ffluent collected, the conductivity and pH of the e ffluent was measured using a Consort multi parameter analyser C3010, pH probe and conductivity probe with temperature compensation. Following the first CM treatment, 1 mL of e ffluent from the 5–10 mL sample of e ffluent from each tube was taken to measure optical density, as an indication of biomass concentration, to determine e ffectiveness of bacteria fixing. Optical density was measured using a spectrophotometer (Hach DR 6000, Loveland, CO, USA) at a wavelength of 600 nm. The first 5 mL was not used since this may include some CM that had been retained in the column outlet after treatment.
