## 4 Discussion
It also highlighted that: (i) manganese oxidation appeared to be catalyzed by heterotrophic rather than autotrophic microorganisms; (ii) organic materials in the influent liquor were either present in insufficient concentrations or not readily metabolized by Mn-oxidizing populations in the MnOBs. Considering the latter, the average DOC concentration in the influent liquor was 9.10 mg L\({}^{-1}\), and [PERSON] and [PERSON] [10] previously identified that some of this DOC was glycerol and acetate, present in equivalent concentrations of 3.25 and 2.40 mg C L\({}^{-1}\), respectively. Assuming that, as is the case for glucose, yeast extract contains ~40% carbon, the addition of 100 mg of yeast extract to each liter of influent liquor would have increased its DOC content by ~500% (to ~49 mg L\({}^{-1}\)). This lends support to the hypothesis that concentrations of metabolizable organic materials in nonamended influent liquors were insufficient to support effective microbial growth and Mn (II) oxidation. Hydrodynamically, the biofilm formation and adhesion on the Mn (IV)-coated pebbles may possibly be improved by increasing fluid shear rate on the support surface as demonstrated by [PERSON] et al. [20]. The low superficial liquid velocity, due to relatively slow flow rates, might possibly also explain why there was limited, if any, formation of Mn (IV) biofilms on the gravel placed into one of the MnOBs, after 138 days. Two microorganisms present in the biofilm community were identified as Mn (II)-oxidizers. One was the bacterium _L. discophora_, which was identified both in clone libraries and by T-RFLP analysis (by calculating theoretical T-RF length). This bacterium is a strictly aerobic Gram-negative heterotroph, reported to grow on substrates such as peptone, yeast extract, glucose, pyruvate, malate but not on lactate, citrate and acetate, and to oxidize Mn (II) between pH 6 and pH 8 [21]. The other was a Mn (II)-oxidizing fungus, which was isolated from the surface of colonized pebbles from the MnOB. T-RFLP analysis of 18S rRNA genes (Haell III digests) amplified from the black biofilms on the fresh pebbles and in those coating the MnOBs throughout the experiment, showed a high relative abundance of a 280 nt T-RF, most notably in phase C3, which corresponded to the theoretical HaeIII digest of the 18S rRNA gene of the isolate, though some other eukaryotic clones obtained from the biofilm also had the same T-RF length as the fungal strain. Isolate MnI1 belongs to the order _Pleosporales_ (_Ascomycota_ phylum) and appears to be a novel species. Several Mn (II)-oxidizers have previously been described within this order [11, 22, 23, 24]. Isolate MnI1 catalyzed the removal of soluble manganese from liquid media, and accumulated brown/black-colored precipitates (presumably of Mn (IV)) on its mycelia when grown on Mn-containing plates. The centers of the fungal colonies (though not the filamentous outgrowths) on Mn-free plates also had a dark brown pigmentation, which was possibly due to the smaller amount of Mn (II) (18 uM) present in the trace elements solution used in the basic medium. Physiological tests carried out in Mn-containing liquid media showed that isolate MnI1 grew over a relatively wide range of pH (4.0-8.0), though the final culture pH values were always significantly lower than at the start, and typically ~pH 4. Manganese oxidation was not measured in the variable pH experiment with this isolate, though other experiments confirmed that this isolate oxidized Mn (II) and precipitated Mn (IV). Abiotic Mn oxidation requires pH values above 8, and therefore chemical oxidation was considered unlikely in the variable pH experiment. Isolate MnI1 also grew on a wide range of organic compounds, including glycerol and acetate, substrates identified in the influent liquors. This suggests that amendment with a more cost-effective substrate than yeast extract might be suitable to promote and sustain microbially-catalyzed Mn removal in the bioreactors.