Design and Operation of Empirical Manganese-Removing Bioreactors and Integration into a Composite Modular System for Remediating and Recovering Metals from Acidic Mine Waters

Packed bed bioreactors were used to remove soluble manganese from a synthetic mine water as the final stage of an integrated bioremediation process. The synthetic mine water had undergone initial processing using a sulfidogenic bioreactor (pH 4.0–5.5) which removed all transition metals present in elevated concentrations (Cu, Ni, Zn and Co) apart from manganese. The aerobic bioreactors were packed with pebbles collected from a freshwater stream that were coated with black-colored, Mn(IV)-containing biofilms, and their capacity to remove soluble Mn (II) from the synthetic mine water was tested at varying hydraulic retention times (11–45 h) and influent liquor pH values (5.0 or 6.5). Over 99% of manganese was removed from the partly processed mine water when operated at pH 6.5 and a HRT of 45 h. Molecular techniques (clone libraries and T-RFLP analysis) were used to characterize the biofilms and identified two heterotrophic Mn-oxidizing microorganisms: the bacterium Leptothrix discophora and what appears to be a novel fungal species. The latter was isolated and characterized in vitro.

Development of a genetic system for a model manganese-oxidizing proteobacterium, Leptothrix discophora SS1

Understanding the molecular underpinnings of manganese oxidation in Leptothrix discophora SS1 has been hampered by the lack of a genetic system. In this report, we describe the development of a genetic system for L. discophora SS1. The antibiotic sensitivity was characterized, and a procedure for transformation with exogenous DNA via conjugation was developed and optimized, resulting in a maximum transfer frequency of 5.2×10−1 and a typical transfer frequency of the order of 1×10−3 transconjugants per donor. Genetic manipulation of L. discophora SS1 was demonstrated by disrupting pyrF via chromosomal integration with a plasmid containing a R6Kγ origin of replication through homologous recombination. This resulted in resistance to 5-fluoroorotidine, which was abolished by complementation with an ectopically expressed copy of pyrF cloned into pBBR1MCS. This system is expected to be amenable to a systematic genetic analysis of L. discophora SS1, including those genes responsible for manganese oxidation.

Iron Requirement for Mn(II) Oxidation by Leptothrix discophora SS-1

ABSTRACT A common form of biocatalysis of Mn(II) oxidation results in the formation of biogenic Mn(III, IV) oxides and is a key reaction in the geochemical cycling of Mn. In this study, we grew the model Mn(II)-oxidizing bacterium Leptothrix discophora SS-1 in media with limited iron (0.1 μM iron/5.8 mM pyruvate) and sufficient iron (0.2 μM iron/5.8 mM pyruvate). The influence of iron on the rate of extracellular Mn(II) oxidation was evaluated. Cultures in which cell growth was limited by iron exhibited reduced abilities to oxidize Mn(II) compared to cultures in medium with sufficient iron. While the extracellular Mn(II)-oxidizing factor (MOF) is thought to be a putative multicopper oxidase, Mn(II) oxidation in the presence of zero added Cu(II) was detected and the decrease in the observed Mn(II) oxidation rate in iron-limited cultures was not relieved when the medium was supplemented with Cu(II). The decline of Mn(II) oxidation under iron-limited conditions was not accompanied by siderophore production and is unlikely to be an artifact of siderophore complex formation with Mn(III). The temporal variations in mofA gene transcript levels under conditions of limited and abundant iron were similar, indicating that iron limitation did not interfere with the transcription of the mofA gene. Our quantitative PCR results provide a step forward in understanding the regulation of Mn(II) oxidation. The mechanistic role of iron in Mn(II) oxidation is uncertain; the data are consistent with a direct requirement for iron as a component of the MOF or an indirect effect of iron resulting from the limitation of one of many cellular functions requiring iron.

Carbon and Steel Surfaces Modified by Leptothrix Discophora SP-6: Characterization and Implications

Leptothrix discophoraSP-6, a type of manganese(Mn)-oxidizing bacteria, has been known to accumulate Mn oxides from the aqueous environment and thus play a key role in microbiologically influenced corrosion by increasing the electrochemical potential of steel and other metals. Similarly, this bacterium was found to modify the surface of glassy carbon in aqueous solution and increase its potential (i.e. ennoblement). In the latter case, biomineralized Mn oxides can be used as cathodic reactants for a new generation of microbial fuel cells featuring a bio-cathode. In this preliminary study, factors affecting the biofilm formation and biomineralization processes were examined. The inflow of air into the culture medium was found essential to sustain the ennoblement of substrate electrodes. The OCP and FESEM/EDS data indicated that a smoother initial substrate surface generally led to better ennoblement. Polarizing the carbon electrode at +500mVSCE for 15 minutes was found to facilitate the ennoblement on carbon electrodes, and so did the coating with a poly(L-lysine) layer. Independent of substrate material, initial surface roughness and pretreatment, there were three parameters in the EIS equivalent circuit that correlated well with the OCP indicating the level of ennoblement byL. discophoraSP-6, i.e., electrolyte resistance, double-layer capacitance, and low-frequencies capacitance. These fascinating findings merit further investigation as they may shed light on the fundamental bacteria/substrate interactions and help advance the knowledge base needed for the engineering applications.