## 2 Materials and Methods  
### Commissioning and Operation of Mn(II)-Oxidizing bioreactors (MnOBs)  
Small pebbles (1.5-5.0 cm in diameter) covered with thin black coatings (Supplementary Figure S1) were collected from a catchment stream (pH 6.5, containing \(\sim\)30 uM soluble manganese) located in the Snowodonia National Park, North Wales, UK (GPS coordinates: 53\({}^{\circ}\)11'51.5112\" N, 4\({}^{\circ}\)7\({}^{\prime}\)39.7596\" W), and stored in plastic bottles at 4 \({}^{\circ}\)C until use. [PERSON] et al. [11] have previously shown that the coatings on similar pebbles contained Mn (IV) by positive reaction with hydrogen peroxide. The pebbles were packed into 32.0 by 6.8 cm Perspex(r) columns to a depth of 24.5 cm (Figure 1), and partly processed synthetic mine water (\(\sim\)600 mL) was added, to 3.5 cm above the pebbles, giving the column bioreactor a working volume of 1.02 L. The partly processed synthetic mine water (pH 5.0) was that draining the sulfidogenic bioreactor in the integrated system (Supplementary Figure S2; [10]) and was adjusted, in some cases, to pH 6.5 by adding sodium hydroxide. Nonserile atmospheric air was bubbled (at \(\sim\)1 L min\({}^{-1}\)) into the packed bed bioreactors through an air stone, influent liquor was pumped into its base through an L-shaped Perspex(r) tube and drained via a fixed tube attached to the top of the columns, which maintained the reactors at a fixed volume. The systems were operated in continuous flow mode at flow rates ranging between 23 and 92 mL h\({}^{-1}\) (equivalent to hydraulic retention times (HRTs) of \(\sim\)45 and \(\sim\)11 h, respectively) at 30 \({}^{\circ}\)C. A sampling port was connected to the outlet of each bioreactor.  
Variations in Operational and Set Up Conditions of MnOBs  
The packed bed bioreactors were tested in four experimental phases over 264 days in order to determine optimum conditions for removal of manganese from the partly processed synthetic mine water. Phases C1, C2a and C2b used a single-column system (Figure 1) and evaluated how this was impacted by variations in feed liquor pH and HRT. In phase C1, the bioreactor was packed with biofilm-colonized pebbles and the partly processed synthetic mine water (pH 5.0) that drained the sulfidogenic bioreactor was used directly as the influent liquor. A new column was commissioned in phase C2, and the pH of the feed liquor was adjusted to 6.5 with NaOH (the pH of the stream from which the pebbles were collected). This column was prepared as before but had a thin layer (\(\sim\)6 cm) of pale-colored inert gravel (average diameter: 1.0 cm) placed between two layers of biofilm-coated pebbles in order to visualize to what extent, if any, fresh rock surfaces became coated with dark-colored biofilms over the course of the experiment (Supplementary Figure S3). Phase C3 used an in-line two-column system (Supplementary Figure S4), where effluent liquor from the first packed bed bioreactor was pumped into the base of a second column. Sampling ports were attached to the drain points of the column to analyze the chemistry of water draining each bioreactor. In phase C3, the first column was that used in phase C2, while the second was a third newly commissioned backed bed bioreactor. Table 1 lists the different operational parameters used in each of the experimental phases.  
Figure 1: Schematic representation of the aerobic Mn (II)-oxidizing bioreactor. Mn-containing partly processed synthetic mine water was pumped into the bottom of the column, drained at the top and collected into an effluent bottle.