## 4 Discussion  
Manganese is often the most recalcitrant transition metal found in elevated concentrations in mine drainage waters in terms of how amenable it is to be removed by chemical means. Thermodynamically, Mn (II) should oxidize spontaneously to Mn (IV) in aerated, neutral pH waters, but the activation energy required is relatively high, which impedes the reaction [18]. This barrier can be overcome by Mn (II)-oxidizing microorganisms that catalyze the oxidation of divalent, soluble Mn (II) to insoluble Mn oxides. Other studies have reported biological manganese oxidation at low pH (5.5) in a manganese deposit influenced by metal-rich groundwater [6] and in low pH uranium-contaminated groundwater and soil material from a uranium mine area in Germany [5]. Manganese (II) can, however, also be removed from solution by adsorption onto solid phase Mn (IV) oxides [1] without being oxidized. Manganese oxides are known for their ability to absorb metals and for their cation exchange capacities [19]. The former derives from negative charges of Mn (IV) oxides due to the presence of some Mn (III) or vacant sites within the mineral lattice structure [3]. Consequently, cationic transition metal as well as alkali and alkaline earth metals can be adsorbed to neutralize this negative charge, though the polarity of the charge borne by manganese (IV) oxides is pH-dependent. [PERSON] et al. [11] determined the point of zero charge (pH\({}_{\text{PZC}}\)), i.e., the pH above which the Mn (IV) oxides is net negatively charged and below which it is net positively charged, of a similar material to that used in the present study, to be 5.5. Mineralogical analysis of the biofilms was not performed, though previous work using a similar system identified the Mn (IV) mineral phase as birnessite [11], which is the most common Mn (IV) bio-oxide in aquatic environments [3]. Solution pH would therefore have had an important influence on both adsorption and (microbial) oxidation on Mn. The oxidation of Mn (II) and formation of insoluble Mn (IV) is a proton-generating reaction:  
\[\text{Mn (II)}\rightarrow\text{Mn (III)}+\text{e}^{-} \tag{1}\]  
\[\text{Mn (III)}+2\text{H}_{2}\text{O}\rightarrow\text{MnO(OH)}+3\text{H}^{+} \tag{2}\]  
\[\text{MnO(OH)}\rightarrow\text{Mn}_{2}+\text{H}^{+}+\text{e}^{-} \tag{3}\]  
In theory, oxidation and precipitation of 150 uM Mn (II) should generate 300 uM proton acidity (equivalent to pH 3.5 if generated in pure water) and it is interesting that the pH of the influent liquor (5.0 or 6.5) had little impact on that of the effluent generated. The Mn (IV) biofilm-colonized pebbles used in the bioreactors removed soluble Mn from the partly processed synthetic mine water very effectively at the start of phase C1, but this was not sustained in the longer term. This indicated that the initial removal of Mn (II) was due to adsorption onto the Mn (IV) biofilms, and that once adsorption sites had been more or less saturated the rate-limiting factor was likely to be the subsequent oxidation of immobilized Mn which would have generated additional Mn (IV) (hydroxy)oxides, thereby allowing the process to perpetuate. Increasing the pH of the feed liquor from 5.0 to 6.5 in phase C2a (using a different MnOB) did not improve immobilization of Mn, though combining this with a larger HRT did (phase C2b). Increasing the HRT still further in phase C3 and using a two-stage bioreactor system (one of which was again newly commissioned) resulted in a marked improvement in performance, with 90-100% of Mn removed over the first 44 days of operation. Although this subsequently declined to ~60% Mn removal, a one-off addition of a small amount of yeast extract brought about a rapid (though short-lived) improvement in the performance of the coupled MnOBs, with 99% Mn being removed from solution. The latter result suggested that the rate-limiting factor in determining how effective the bioreactors were in the longer-term was biological (the oxidation of Mn (II) to Mn (IV)) rather than chemical (adsorption of Mn (II)).