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Bioleaching and biocorrosion are based on similar biochemical processes. Microbe-surface interaction, biofilm formation and concomitant extracellular polymeric substance (EPS) production gained increasing interest in the past decades. Nowadays it is generally accepted that biofilm formation and an accompanying formation of manganese oxides by manganese oxidizing bacteria such as Leptothrix spp. account for one type of pitting corrosion of stainless steel (SS). However, little is known about biofilm formation, EPS composition of manganese oxidizing microorganisms and their influence on microbiologically influenced corrosion. Consequently, we studied biofilm formation of Leptothrixdiscophora, the biooxidation of manganese in biofilms on floating filters as well as biofilm formation on stainless steel and the involved corrosion processes. Cells were visualized by epifluorescence (EFM) or confocal laser scanning –microscopy (CLSM). Additionally, the influence of biofilm formation and biooxidation of manganese by L. discophora on the open circuit potential (OCP) and pitting potential (Epit) of stainless steel was measured using a 3 electrode setup. L. discophora grew well in biofilms on floating filters and on SS coupons and incorporated in both conditions Mn2+ in the form of MnO2 from the bulk phase into the biofilm. OCP measurements of actively manganese-oxidizing biofilms on stainless steel showed a significant ennoblement of ≥200 mV.

Direct biotic and homogeneous abiotic Fe(II) oxidation rates as well as oxidation rates of Fe(II) with MnOX were determined in laboratory experiments and compared with biotic Mn(II) oxidation rates. In groundwaters and thermal installation waters, parameters for both Fe(II) oxidation steps were studied and products of biotic and abiotic Fe(II) and Mn(II) oxidation were analyzed. Direct Fe(II) oxidation of active Leptothrix cholodnii cultures can reach first-order rates of up to 1.17 ± 0.90 h−1. Second-order rates of Fe(II) oxidation with biogenic, Leptothrix-cholodnii- and Leptothrix-discophora-SS-1-originating MnOX lead to rates comparable with those as obtained with abiotic H+-birnessite.

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.

The growth characteristics of Sphaerotilus natans and Leptothrix discophora SS-1 (ATCC43182) were first used to fabricate the ferrite and manganese ferrite. The scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDS), X-ray diffraction (XRD), and vibrant sample magnetometer (VSM) were used to make clear their morphology microstructure, chemical composition, crystal structure, and magnetostatic characteristic. The result showed that the hollow ferrite fibers were prepared successfully, whose crystal structures were close to α-Fe2O3 and the average aspect ratio was over 50. Moreover, the soft magnetic manganese ferrite was fabricated with the saturation magnetization Ms at 25 emu/g and coercive force H c at 796 A/m.

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.

As biological manganese (Mn) removal becomes a more popular water treatment technology, there is still a large gap in understanding the key mechanisms and range of operational characteristics. This research aimed to expand on previous bench-scale experiments by directly comparing small filtration columns inoculated with indigenous biofilms from a Mn filtration plant and filtration columns inoculated with a liquid suspension of Leptothrix discophora SP-6. Batch tests found that in the absence of manganese oxidizing bacteria Mn was not removed by air alone, whereas a mixed population and Leptothrix strain achieved greater than 90% removal of Mn. The bench-scale biofiltration experiments found that biological filters can be inoculated with a pure culture of L. discophora SP-6 and achieve a similar removal of indigenous biofilm. While Mn oxidizing bacteria (MOB) seem to be necessary for the auto-catalytic nature of these biological filters, Mn removal is achieved with a combination of adsorption to Mn oxides and biological oxidation. Additionally, it was demonstrated that biological Mn removal is possible over a broader "field of activity" (e.g., Mn removal occurred at a pH level as low as 6.5) than has previously been reported. The ability of this treatment technology to work over a broader range of influent conditions allows for more communities to consider biological treatment as an option to remove Mn from their drinking water.

Manganese (II) and manganese-oxidizing bacteria were used as an efficient biological system for the degradation of the xenoestrogen 17 alpha-ethinylestradiol (EE2) at trace concentrations. Mn(2+)-derived higher oxidation states of Mn (Mn(3+), Mn(4+)) by Mn(2+)-oxidizing bacteria mediate the oxidative cleavage of the polycyclic target compound EE2. The presence of manganese (II) was found to be essential for the degradation of EE2 by Leptothrix discophora, Pseudomonas putida MB1, P. putida MB6 and P. putida MB29. Mn(2+)-dependent degradation of EE2 was found to be a slow process, which requires multi-fold excess of Mn(2+) and occurs in the late stationary phase of growth, implying a chemical process taking place. EE2-derived degradation products were shown to no longer exhibit undesirable estrogenic activity.

Leptothrix discophora SP-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 biocathode. 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 +500 mV(SCE) for 15 min was found to facilitate the ennoblement on carbon electrodes, and so did coating it 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 by L. discophora SP-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 forthe engineering applications.

UV Raman spectroscopic data measured at two distinct conditions, low and high laser power, establish that the manganese oxide (SP6-MnOx) produced by the freshwater bacterium (Leptothrix discophora SP-6) closely resembles the 3 × 3-tunnel todorokite among the MnO2 materials studied. Under the two conditions, the effect of hydration/cation and the phase transition of todorokite or SP6-MnOx to Mn3O4 or birnessite will be described. A higher concentration of Mg2+ incorporated in the framework of the SP6-MnOx than in todorokite is probably responsible for the formation of a new Raman band obtained at high laser powers, matching the most intense UV Raman band of synthetic birnessite. Also, we present the assignment of Raman bands for todorokite mineral and discuss the mutual exclusion principle that should hold for all the MnO2 materials studied. These experiments provide characterization of hydrous, poorly crystalline, or nanocrystalline metal oxides, which are frequently difficult to identify.

A gramicidin-S-producing Bacillus brevis 18-3 biofilm was shown to reduce corrosion rates of mild steel by inhibiting both the sulfate-reducing bacterium Desulfosporosinus orientis and the iron-oxidizing bacterium Leptothrix discophora SP-6. When L. discophora SP-6 was introduced along with D. orientis to a non-antimicrobial-producing biofilm control, Paenibacillus polymyxa ATCC 10401, a corrosive synergy was created and mild steel coupons underwent more severe corrosion than when only D. orientis was present, showing a 2.3-fold increase via electrochemical impedance spectroscopy (EIS) and a 1.8-fold difference via mass-loss measurements. However, when a gramicidin-S-producing, protective B. brevis 18-3 biofilm was established on mild steel, the metal coupons were protected against the simultaneous attack of D. orientis and L. discophora SP-6. EIS data showed that the protective B. brevis 18-3 biofilm decreased the corrosion rate about 20-fold compared with the non-gramicidin-producing P. polymyxa ATCC 10401 biofilm control. The mass loss for the protected mild steel coupons was also significantly lower than that for the unprotected ones (4-fold decrease). Scanning electron microscope images corroborated the corrosion inhibition by the gramicidin-S-producing B. brevis biofilm on mild steel by showing that the metal surface remained untarnished, i.e., the polishing grooves were still visible after exposure to the simultaneous attack of the sulfate-reducing bacterium and the iron-oxidizing bacterium.

Type 316L (UNS 31603) stainless steel (SS) was ennobled to an open-circuit potential (OCP) of 323 mV vs saturated calomel electrode (SCE) within a 5-day period due to the deposition of man...

In natural waters, manganese oxides (MnO x ) are important in mediating the bioavailability of trace metals such as Ni, Cu, Zn, Cd, and Pb, as these metals readily adsorb to the MnO x surface. Manganese from a variety of anthropogenic sources usually enters the aquatic environment in dissolved form as Mn 2+ . It is subsequently oxidized under oxic and neutral (pH = 6–7) conditions. Often this oxidation is catalyzed by bacteria, such as Leptothrix discophora , as part of their natural metabolic process. Mn K -edge X-ray Absorption Fine Structure Spectroscopy (XAFS) was used to investigate the local structure of manganese oxide on the sheath produced by the bacterium Leptothrix discophora SP-6. The features observed in the near edge region of the Mn K -edge spectrum indicate the presence of three oxidation states of manganese: Mn 2+ , Mn 3+ , and Mn 4+ . Fitting the experimental XAFS data identifies the bacterial MnO x as being composed of single-layer microcrystals with layers similar to those occurring in Na-birnessite. Some MnO 6 octahedra might lie outside the layer plane, sharing corners with those in the layer plane. X-ray diffraction results for the same samples are consistent with the single-layer structure.

Ennoblement of stainless steel (SS) by microbially deposited manganese oxides can lead to pitting corrosion at low chloride concentrations, causing unexpected material failures. We exposed 316L SS to manganese oxidizing bacteria Leptothrix discophora under well-defined laboratory conditions, and then placed the ennobled coupons in a 0.5 M sodium chloride solution until pitting developed. Using time-of-flight secondary ion mass spectroscopy we demonstrated that the pits and their immediate vicinity associated with microbial influenced corrosion had different chemical signatures than those associated with electrochemically induced pitting, suggesting a possibility that the microorganisms were directly involved in pit initiation. Based on the differences in the chemical signatures we were able to distinguish the microbially induced pits from those induced by anodic polarization.

It is a dubious but common practice to use growth parameters measured in suspended cultures to predict substrate concentration pro)les in bio)lms. To obtain bio)lm biokinetic parameters that apply to bio)lms, a reliable method is needed that allows the computation of biokinetic parameters from substrate concentration pro)les measured directly in bio)lms. We have developed such a method and demonstrated its utility by evaluating biokinetic parameters from oxygen concentration pro)les measured in bio)lms of Leptothrix discophora SP-6 grown on a membrane, which was placed on top of an agar plate by )tting the data to Monod or Tessier growth kinetics, including maintenance substrate consumptions. We found that the Monod model represented the growth of L. discophora SP-6 bio)lms marginally better than the Tessier model. The Monod half saturation coe8cient was 0 :333 ± 0:077 mg=l. ? 2003 Elsevier Ltd. All rights reserved.

The growth parameters of Leptothrix discophora SP-6 were quantified on the basis of the steady-state concentrations and utilization rates of pyruvate, dissolved oxygen, and concentration of microorganisms in a chemostat operated at 25 degrees C, pH 7.2, and an agitation rate of 350 rpm. The results showed that the microbial growth was limited by both pyruvate and dissolved oxygen. A combined growth kinetics model using Monod growth kinetics for pyruvate and Tessier growth kinetics for oxygen showed the best correlation with the experimental data when analyzed using an interactive multiple substrate model. The growth kinetics parameters and the respective confidence limits, estimated using the Monte Carlo simulation, were mu(max) = 0.576 +/- 0.021 h(-1), K(sMp) = 38.81 +/- 4.24 mg L(-1), K(sTo) = 0.39 +/- 0.04 mg L(-1), Y(X/p) = 0.150 (mg microorganism mg(-1) pyruvate), Y(X/o) = 1.24 (mg microorganism mg(-1) oxygen), the maintenance factors for pyruvate and oxygen were m(p) = 0.129 (mg pyruvate consumed mg(-1) microorganism h(-1)) and m(o) = 0.076 (mg oxygen consumed mg(-1) microorganism h(-1)), respectively.

The noble shift in corrosion potential to values between +300 and +400 mVSCE and the accompanying increase in cathodic current density and polarization slope at mild cathodic potentials that develop during microbial colonization of passive metals, are collectively known as ennoblement. This phenomenon is of concern as the noble shift in the corrosion potential may lead to pitting corrosion. We have demonstrated, by growing pure cultures of manganese oxidizing bacteria (MOB) Leptothrix discophora SP-6 under well defined conditions, that microbial deposition of manganese oxides causes ennoblement of 316L stainless steel (SS). Exposing 316L corrosion coupons in lakes and streams supported this conclusion; the rate and extent of ennoblement were positively correlated with the rates of deposition and the amounts of biomineralized manganese oxides deposited on the surfaces of the SS corrosion coupons. X-ray photoelectron spectroscopy (XPS) analyses of the deposits from the ennobled coupons revealed a mixture of manganese oxides, as expected. Many natural waters can support growth of MOB. When manganese-oxidizing biofilms accumulate on surfaces of passive metals there is a potential for manganese redox cycling on the metal surface. This process is initiated by depositing minute amounts of manganese oxides on the metal surface. These microbially deposited manganese oxides are then reduced by the electrons derived from anodic dissolution of the metal; the metal is corroding and the manganese oxides are reduced to divalent manganese ions. However, since the manganese ions are liberated within the manganese-oxidizing biofilm, the manganese ions are immediately reoxidized, and the cycle continues.

Pitting corrosion of 316L stainless steel ennobled in the presence of manganese-oxidizing bacteria, Leptothrix discophora, was studied in a low-concentration sodium chloride solution. Corrosion coupons were first exposed to the microorganisms in a batch reactor until ennoblement occurred, then sodium chloride was added, which initiated pitting. The pits had aspect ratios (length divided by width) and shapes closely resembling the aspect ratio and the shape of the bacteria, which suggested that the microorganisms were involved in pit initiation.

We have applied time-of-flight secondary ion mass spectroscopy (ToF-SIMS) to study microbially induced ennoblement of 316L stainless steel and Ti–6Al–4V surfaces exposed to manganese-oxidizing bacteria Leptothrix discophora SP-6. Our observations indicate that manganese biomineralization occurs in two steps: first, the divalent manganese (Mn 2+ ) is oxidized to manganese oxyhydroxide, MnOOH; then the MnOOH is further oxidized to manganese dioxide, MnO 2 . Both reactions contribute to an increase in the open circuit potential and hence to the ennoblement of the material. Manganese cycling at a surface of passive metals produces renewable cathodic reactants, manganese oxyhydroxide and manganese dioxide, and endangers the material integrity.

A poly(3-hydroxybutyrate) (PHB)-degrading, gram-negative, aerobic bacterium, strain HS(T), was isolated from a hot spring and chemotaxonomically and phylogenetically characterized. The oxidase-positive, weakly catalase-positive, non-pigmented cells (0.6 x 2.6 microm) exhibited a single polar flagellum and accumulated PHB granules. Strain HS(T) was capable of manganese oxidation. Highest growth rate was attained at 50 degrees C. The optimum pH for growth was 7-8. The major respiratory quinone was ubiquinone-8 and major cellular fatty acids were C16:0, C16:1 and C18:1. The G+C content of the DNA was 66.2 mol%. Comparative 16S rDNA analysis indicated that strain HS(T) is related to the Rubrivivax subgroup and the family Comamonadaceae. The nearest phylogenetic relatives were Ideonella dechloratans (92.1% similarity), Leptothrix discophora (93.6%), Roseateles depolymerans (92.4%) and Rubrivivax gelatinosus (92.2%). On the basis of its phylogenetic and phenotypic properties, it is proposed that this isolate be designated Caldimonas manganoxidans gen. nov., sp. nov.; the type strain is HS(T) (= JCM 10698T = IFO 16448T = ATCC BAA-369T).

The kinetics of Mn(II) oxidation by the bacterium Leptothrix discophora SS1 was investigated in this research. Cells were grown in a minimal mineral salts medium in which chemical speciation was well defined. Mn(II) oxidation was observed in a bioreactor under controlled conditions with pH, O2, and temperature regulation. Mn(II) oxidation experiments were performed at cell concentrations between 24 mg/L and 35 mg/L, over a pH range from 6 to 8.5, between temperatures of 10°C and 40°C, over a dissolved oxygen range of 0 to 8.05 mg/L, and with L. discophora SS1 cells that were grown in the presence of Cu concentrations ranging from zero to 0.1 μM. Mn(II) oxidation rates were determined when the cultures grew to stationary phase and were found to be directly proportional to O2 and cell concentrations over the ranges investigated. The optimum pH for Mn(II) oxidation was approximately 7.5, and the optimum temperature was 30°C. A Cu level as low as 0.02 μM was found to inhibit the growth rate and yield of L. discophora SS1 observed in shake flasks, while Cu levels between 0.02 and 0.1 μM stimulated the Mn(II) oxidation rate observed in bioreactors. An overall rate law for Mn(II) oxidation by L. discophora as a function of pH, temperature, dissolved oxygen concentration (D.O.), and Cu concentration is proposed. At circumneutral pH, the rate of biologically mediated Mn(II) oxidation is likely to exceed homogeneous abiotic Mn(II) oxidation at relatively low (≈μg/L) concentrations of Mn oxidizing bacteria.

The effects of iron and manganese (hydr)oxide formation processes on the trace metal adsorption properties of these metal (hydr)oxides and their mixtures was investigated by measuring lead adsorption by iron and manganese (hydr)oxides prepared by a variety of methods. Amorphous iron (hydr)oxide formed by fast precipitation at pH 7.5 exhibited greater Pb adsorption (gamma(max) = 50 mmol of Pb/mol of Fe at pH 6.0) than iron (hydr)oxide formed by slow, diffusion-controlled oxidation of Fe(II) at pH 4.5-7.0 or goethite. Biogenic manganese(III/IV) (hydr)oxide prepared by enzymatic oxidation of Mn(II) by the bacterium Leptothrix discophora SS-1 adsorbed five times more Pb (per mole of Mn) than an abiotic manganese (hydr)oxide prepared by oxidation of Mn(II) with permanganate, and 500-5000 times more Pb than pyrolusite oxides (betaMnO2). X-ray crystallography indicated that biogenic manganese (hydr)oxide and iron (hydr)oxide were predominantly amorphous or poorly crystalline and their X-ray diffraction patterns were not significantly affected by the presence of the other (hydr)oxide during formation. When iron and manganese (hydr)oxides were mixed after formation, or for Mn biologically oxidized with iron(III) (hydr)oxide present, observed Pb adsorption was similar to that expected for the mixture based on Langmuir parameters for the individual (hydr)oxides. These results indicate that interactions in iron/manganese (hydr)oxide mixtures related to the formation process and sequence of formation such as site masking, alterations in specific surface area, or changes in crystalline structure either did not occur or had a negligible effect on Pb adsorption by the mixtures.

To apply manganese-oxidizing bacteria to waste water treatment, basic performance of the manganese-oxidizing bacterium, Leptothrix discophora, distributed by ATCC was examined. In addition, the effect of carbon fiber on the oxidation by the bacterium was also investigated, since carbon fiber was reported to accelerate the growth of activated sludge during sewage treatment. The bacterium was found to be active in the medium containing 24 ppm Mn(II) ions, the concentration being about 8 times higher than the recommended one and practically useful level. The oxidation rate was higher with the static culture than the shaking culture. This was considered to be due to physical damage to the sheath structure of bacteria which is reported to be important to oxidize Mn(II) ions. The carbon fiber did not accelerate the microbial oxidation of Mn(II) ions. This is partly attributed to the lack of contact between the bacterial cell, which floated as thin membranes, and the carbon fiber sunk in the bottom of vessels. However, oxidized Mn species precipitated on the carbon fiber, which resulted in an improvement of the transparency of water. This effect is different from the one caused by activated carbons, since the carbon fiber has very low specific surface area and no pore structure. The effect was more remarkable in the shaking culture than the static culture, indicating that organic substances originated from the bacterium play an important role in the adsorption of oxidized Mn species.

During the past few years, biomineralized manganese has been shown to cause ennoblement of various stainless steels to open circuit potentials of 300–400 mV/SCE. We have demonstrated that ennoblement, caused by biologically deposited manganese minerals, along with a relatively low stainless steel pitting potential, caused by the presence of chloride, is sufficient to initiate and drive active pitting corrosion. Stainless steel samples (type 304L), chemically or microbiologically ennobled with manganese dioxide, were exposed to a 0.35% w/v NaCl solution; an environment otherwise not corrosive against the 304L stainless steel. In the first case, steel samples were ennobled by electroplating the sample with a thin film of manganese dioxide, except for a small anodic area. In the latter case, the manganese dioxide was deposited on the steel within biofilms of the manganese oxidizing bacterium Leptothrix discophora SP-6. After 24 h exposure to the chloride solution the samples were investigated by atomic force microscopy (AFM). Both types of ennobled samples were found severely pitted, whereas reference samples (w/o manganese minerals) had remained intact. Einfluss von biomineralischem Mangan auf die Lochkorrosion von nichtrostendem Stahl des Typ 304L In den letzten Jahren hat es sich gezeigt, dass biomineralisiertes Mangan die Veredelung von nichtrostendem Stahl zu freien Korrosionspotentialen von 300–400 mV/SCE verursachen kann. Wir haben gezeigt, dass die Veredelung von nichtrostendem Stahl durch mikrobiologisch gebildete Manganminerale kombiniert mit einem relativ niedrigen Lochkorrosionspotential des nichtrostenden Stahls in chloridhaltigem Wasser hinreichend ist, um aktive Lochkorrosion zu initiieren. Proben von nichtrostendem Stahl (Typ 304L), chemisch oder mikrobiologisch veredelt mit Manganoxid, wurden einer 0,35% w/v NaCl-Losung ausgesetzt; ein Milieu, das normalerweise bei 304L nichtrostendem Stahl keine Korrosion herbeifuhren kann. In dem ersten Experiment wurden die Stahlproben mit Ausnahme von einem kleinen anodischem Bereich mit einem dunnen Manganoxidfilm veredelt. In dem zweiten Experiment wurde das Manganoxid in Biofilmen, bestehend aus dem Mangan-oxidierenden Bakterium Leptothrix discophora SP-6, auf dem Stahl abgelagert. Nach 24 h in der Chloridlosung wurden die Proben mit Rasterkraftmikroskopie (AFM) untersucht. Bei beiden Typen von Veredelung wurden wesentliche Lochkorrosionsangriffe gefunden, gleichzeitig waren Kontrollproben (ohne Manganminerale) intakt.

The effect of Cu2+ on the Mn2+-oxidizing activity secreted by Leptothrix discophora strain SS-1 was studied. Cu2+ stimulated the activity of spent medium from stationary cultures when added during growth of the bacteria, although cell yield decreased with increasing Cu2+ concentrations. The stimulation was tentatively explained by involvement of the putative multicopper oxidase MofA in Mn2+ oxidation. Cu2+ did not enhance the activity when added directly to spent medium, possibly because the ions had to be incorporated during synthesis of the oxidizing factor. Sequence analysis of the region downstream from the mofA gene resulted in identification of mofB, which encodes a protein with a potential peptidyl-prolyl-cis-trans isomerase site, and mofC, which encodes a protein with a potential heme-binding site. The possibility that mofA, mofB, and mofC belong to one operon, and the possible functions of MofB and MofC, are discussed.

Biofilms of Leptothrix discophora SP-6, grown on 316L stainless steel (SS), ennobled the open circuit potential to 410 mV SCE . X-ray Photoelectron Spectroscopy (XPS) identified MnO 2 within these biofilms. Field exposed SS showed similar ennoblement after 30 days. XPS analysis of these samples revealed a mixture of different manganese minerals. Electrochemical reduction of MnO 2 was studied using electroplated SS. Plated MnO 2 was reduced amperometrically. The process was interrupted at different reduction stages. XPS analysis of remaining oxides showed that MnO 2 was reduced through MnOOH to Mn 2+ . We conclude that biomineralized MnO 2 may increase corrosion rates by serving as a cathodic reactant.

Pyrite oxidation in the underground mining environment of Iron Mountain, California, has created the most acidic pH values ever reported in aquatic systems. Sulfate values as high as 120 000 mg l−1 and iron as high as 27 600 mg l−1 have been measured in the mine water, which also carries abundant other dissolved metals including Al, Zn, Cu, Cd, Mn, Sb and Pb. Extreme acidity and high metal concentrations apparently do not preclude the presence of an underground acidophilic food web, which has developed with bacterial biomass at the base and heliozoans as top predators. Slimes, oil-like films, flexible and inflexible stalactites, sediments, water and precipitates were found to have distinctive communities. A variety of filamentous and non-filamentous bacteria grew in slimes in water having pH values

Fe and Mn oxides have long been recognized as important adsorbing phases governing the cycling of trace metals in aquatic environments (33, 35, 46, 62, 64). Fe oxides are often considered more important than other adsorbing phases because the high specific surface area of amorphous or colloidal Fe oxides is expected to result in high adsorption capacity, and thus trace metal adsorption to Fe oxides has been studied extensively (e.g., see reference 20). Less attention has been given to trace metal adsorption by Mn oxides, even though it is likely that fresh biologically oxidized Mn is poorly crystallized or amorphous (41, 58, 65) and could exhibit much greater trace metal adsorption than crystalline Mn oxides. The Mn oxidation states and mineral forms of biogenic Mn oxides have been characterized in several prior investigations (3, 23, 24, 41, 57); however, specific surface areas and trace metal binding characteristics of biogenic Mn oxides have not been reported previously. Moffett and Ho (44) reported that Co, Zn, Ce, and trivalent lanthanides could be incorporated into biogenic Mn oxides by being “coprocessed” via the same putative enzymatic pathways as those used for Mn oxidation, but adsorption of these elements to already formed Mn oxides was not described. He and Tebo (32) measured the surface area of Mn-oxidizing Bacillus sp. strain SG-1 spores and Cu adsorption to these spores, but Cu adsorption to Mn oxides formed by the spores was not reported. Tessier et al. (59) observed trace metal adsorption to freshwater sediment extracts containing Mn oxides, Fe oxides, and organic materials, but the specific role of Mn oxides was not elucidated because the extracted Mn oxides were contaminated with Fe oxides and residual organic material. Additional information on the metal adsorption capacity and specific surface area of biogenic Mn oxides under controlled laboratory conditions is needed to assess the relative importance of biogenic Mn oxides in controlling trace metal adsorption in natural aquatic environments. In the present work, Pb adsorption and specific surface area were measured for biogenic Mn oxides produced by the bacterium Leptothrix discophora SS-1 in a chemically defined medium. L. discophora is a well-characterized, model Mn-oxidizing bacterium whose structure, physiology, and phylogeny have been studied extensively (8, 26–28). In pure cultures of L. discophora, Mn oxidation has been shown to occur extracellularly (1–3, 8, 16, 17, 21, 22, 54, 58). The Mn oxides formed by L. discophora were previously shown to be mixed Mn(III, IV) oxides or oxyhydroxides with an average oxidation state of 3.6 (3). Pb adsorption by the biogenic Mn oxide (with cells and exopolymer) was compared to Pb adsorption by L. discophora cells and exopolymer alone (without Mn oxide). The adsorption properties of the biogenic Mn oxides were also compared to those of Mn oxides of abiotic origin. To unambiguously assess Pb adsorption to Mn oxides produced by L. discophora, it was necessary to grow the bacterium in a defined medium free of competing trace metals and undefined organic ligands or mineral precipitates that could interfere with Pb adsorption. Previous work with another strain, L. discophora SP-6, had shown that this organism could be grown in a defined mineral salts medium that contained 10 μM FeSO4 and a suite of vitamins (3). Therefore, to facilitate our experiments, it was first necessary to determine the minimal vitamin and Fe requirements of strain SS-1. These experiments revealed that vitamin B12 was required for SS-1 growth and also revealed that supplemental Fe enhanced Mn oxidation in the defined medium. Development of a chemically defined medium based on these determinations resulted in more accurate and meaningful measurement of Pb adsorption to the resulting biogenic Mn oxides.

A thermophilic bacterium that can grow on poly(3-hydroxybutyrate) (PHB) was isolated from a hot spring. The isolate, designated strain HS, was a gram-negative aerobic rod and grew optimally at 45–50°C. From its principal phenotypic characteristics, strain HS was revealed to be closely related to Leptothrix discophora. Sequence analysis of the 16S rRNA gene also supported this result. PHB depolymerase was purified to electrophoretic homogeneity from cell-free culture broth by hydrophobic interaction chromatography using Toyopearl HW-55F in combination with ammonium sulfate precipitation. The molecular weight of the enzyme was calculated to be 4.6 × 104 by electrophoresis and 4.3 × 104 by gel filtration. The enzyme was stable up to 65°C and the optimum reaction temperature was 70°C. A Km of 0.018 g/l was determined for PHB at 70°C. An isoelectric point of pH 8.6 was obtained by isoelectric focusing. The enzyme activity was inhibited by dithiothreitol but not by phenylmethylsulfonyl fluoride. The N-terminal amino acid sequence was unlike that of any other bacterial PHB depolymerase. Secretion of the enzyme was accelerated by the addition to the culture medium of 3-hydroxybutyrate which is the product of the enzyme.

The noble shift in open-circuit potential exhibited by microbially colonized stainless steel (ennoblement) was investigated by examining the relationship among surface colonization, manganese deposition, and open-circuit potential for stainless steel coupons exposed to batch cultures of the manganese-depositing bacterium Leptothrix discophora. Open-circuit potential shifted from -100 to +330 mV(infSCE) as a biofilm containing 75 nmol of MnO(infx) cm(sup-2) formed on the coupon surface but changed little further with continued MnO(infx) deposition up to 270 nmol cm(sup-2). Increased open-circuit potential corresponded to decreasing Mn(II) concentration in solution and to increased MnO(infx) accumulation and attached cell density on the coupon surfaces. MnO(infx) deposition was attributable to biological activity, and Mn(II) was observed to enhance cell attachment. The experimental results support a mechanism of ennoblement in which open-circuit potential is fixed near +350 mV(infSCE) by the cathodic activity of biomineralized MnO(infx).

A small amount of a manganese‐oxidizing protein was purified from spent culture medium of Leptothrix discophora strain SS‐1 and used to raise antibodies (αMOF). Expression libraries of L. discophora were constructed in λgtll and screened with αMOF. DNA inserts from the resulting αMOF‐positive λgt11 clones were used to isolate the corresponding gene (called mofA) from a genomic library. The mofA gene was mapped in the center of a NcoI‐EcoRI restriction fragment of 7262 bp. This fragment was cloned in a low‐copy broad‐host vector. The resulting plasmid (pGBM31) was used in an in vitro transcription‐translation experiment with an Escherichia coli S30 extract. Transcription of the mofA gene could be initiated on its own putative promoter region. Translation resulted in a protein of approximately 180 kD as determined by gel electrophoresis. DNA sequence analysis revealed an open reading frame of 4986 bp preceded by a potential promoter region and a ribosome binding site. The translation product, consisting of ...

The formation of layer silicates on capsuled bacterial cell walls was studied in freshwater microbial mats. The trends associated with Al, Si, Mn and Fe deposits with capsules are consistent with occurrence of layer silicates with 14, 10 and 7 Aa X-ray diffraction (XRD) patterns. Scanning electron microscope and transmission electron microscope (SEM and TEM) observations of the microbial mats revealed the presence of microcolonies of rod- and coccus-shaped bacteria with layer-silicate thin films. Field measurements of pH, temperature and Eh indicated that these conditions for bacterial crystallization of layer silicates and hydrated Fe/Mn oxides in freshwaters are as follows: pH 6.3 to 7.8, 12 to 20 degrees C and Eh -24 to +200 mV. Glass slides kept for 3 weeks in the beakers with natural freshwater and river sediments were coated with brown materials. These materials were identified as layer silicates and colonized bacteria formed under photosynthetic conditions. The well-developed holdfasts on Leptothrix discophora bacterial cells are mainly associated with poorly crystalline layer silicates and hydrated Fe-Mn oxides. Semiquantitative elemental analyses of holdfasts using energy-dispersive X-rays (EDX) indicated that layer-silicate crystallization covers the cell at an early stage. Iron and manganese crystallization develops at a later stage, where aluminum substitution occurs in crystal structures. Laboratory experimental results indicated that layer silicates grew from a biochemical origin, rather than from inorganic origins in freshwater. Layer-silicate formation is linked with bacteria in microbial mats.

Summary Strain Feox-1 was isolated from the sediment of a big freshwater lake (Chiemsee) in Upper Bavaria, Germany. The site of isolation was characterized by dark-brown ferromanganese deposits produced by sheated bacteria belonging to the genus Leptothirix . Cells of the newly isolated strain were gram-negative, rodshaped and highly motile by a single polar flagellum. Although sheaths were not formed strain Feox-1 could be affiliated to the genus Leptothrix by its ability to oxidize manganese at circumneutral pH values. A polyphasic approach was used to determine the taxonomic status of Feox-1 and of the other currently available Leptothrix strains which have been only partly described. Physiological tests, fatty acid analyses, DNA-DNA hybridization experiments and sequencing of 16S rRNA genes indicated that Feox-1 belongs to a new species, for which the name Leptothrix mobilis is proposed. Main characteristics of the newly proposed species are as follows: Good growth occurs in the range of 10 to 37°C and pH 6.5 to 8.5. Only very few compounds are utilized as sole carbon source. Cells contain as major fatty acids hexadecanoic acid, cis-9-hexadecenoic acid and octadecenoic acid; minor fatty acids are 3-hydroxydecanoic acid, dodecanoic acid, tetradecanoic acid and cis-9,10-methyleneoctadecanoic acid. The guanine-plus-cytosine content of the DNA is 68 mol%. Taxonomic studies of strains belonging to the non-validly described species “Leptothrix discophora” and to Leptottirix cbolodnii led to the description of Leptottirix discophora sp. nov. nom. rev. and to an emended description of Leptottirix cbolodnii. In addition, the two strains LMG 8142 and LMG 8143 originally assigned to "Leptottirix discophora" were transferred to Leptothrix cbolodnii .

Neutrophilic iron-depositing microorganisms include various groups of bacteria, algae, and protozoa. The most striking feature of these microorganisms is their ability to precipitate ferric iron around their cells and colonies in many different forms. Growth of these microorganisms has various practical implications, for example, formation of iron ore in many parts of the world, aging of water wells, and clogging of drinking water pipes. Morphological description of many genera and species of iron-depositing bacteria by microscopy dates back to the nineteenth century, but only very few pure cultures of bacteria such as Leptothrix discophora and Gallionella ferruginea have been obtained in the last decades. Therefore, little has been known on the physiology or phylogeny of these bacteria.

Summary Leptothrix discophora and Sphaerotilus natans are heterotrophic bacteria, characterized by the precipitation of iron oxides on their sheaths. In addition, L. discophora is known for its ability to precipitates manganese oxides. Here, the phylogenetic relationship of L. discophora and S. natans is inferred using 16S rDNA sequence analysis. Double-stranded PCR products obtained after the enzymatic amplification of the 16S rRNA gene were sequenced directly, and the sequences compared to sequences from other Proteobacteria. From this phylogenetic analysis it was found that both organisms belong to the β1-subdivision of the Proteobacteria.

Leptothrix discophora SP-6 was isolated from the outflow reservoir of an artificial iron seep. Its sheathforming phenotype was maintained by slow growth in a mineral salts-vitamin-pyruvate medium under minimal aeration at 20 to 25°C. A sheathless variant, SP-6(sl), was isolated from smooth colonies that appeared on spread plates after rapid growth of SP-6 in well-aerated cultures. SP-6 and SP-6(sl) are closely related but not identical to the previously studied sheathless strain SS-1 (ATCC 43182). Increasing Mn 2+ concentrations in the growth medium of SP-6 increased the phase density of the sheath, indicating increased Mn oxide deposition in the sheath. Electron microscopy of cultures grown without added Mn 2+ revealed that the sheath consisted of a well-defined inner layer, 30 to 100 nm thick, and a diffuse outer capsular layer of variable thickness. Mn oxides were identified in the sheath by their characteristic ultrastructure, electron density, and X-ray-dispersive energy spectra. In heavily encrusted sheaths, the Mn oxides were evenly distributed in both layers of the sheath. Sheathed cells retained more Mn-oxidizing activity than did sheathless cells after washing with distilled, deionized water; the sheath retained some of its activity after an EDTA-lysozyme-detergent treatment which removed the cells. An ultrafiltration-dialysis procedure significantly increased the recovery of activity from spent media of SP-6 over that reported previously for SS-1 (L.F. Adams and W.C. Ghiorse, J. Bacteriol. 169:1279-1285, 1987). A 108-kDa Mn-oxidizing protein was identified in concentrated spent media of SP-6 and SP-6(sl), and the activity of the concentrates showed stability in detergents comparable to that of SS-1 and patterns of heat inactivation and chemical inhibition similar to those of SS-1.

A new method for the quantification and characterization of manganese-oxidizing activity by spent culture medium of Leptothrix discophora SS-1 was developed. It is based on the formation of the dye Wurster blue from N,N,N′,N′ -Tetramethyl- p -phenylenediamine by oxidized manganese generated in the spent medium. The kinetic parameters thus obtained agreed well with data obtained with other methods. It was also possible to demonstrate iron oxidation by spent culture medium. The kinetics of the process and inhibition by enzyme poisons suggest that iron oxidation is enzymatically catalyzed. Probably two different factors are involved in manganese and iron oxidation.

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.

Extracellular electron transfer onto Fe(III) oxides in Geobacter sulfurreducens is considered to require proteins that must be exported to the outer surface of the cell. In order to investigate this, the putative gene for OxpG, the pseudopilin involved in a type II general secretion pathway of Gram-negative bacteria, was deleted. The mutant was unable to grow with insoluble Fe(III) oxide as the electron acceptor. Growth on soluble Fe(III) was not affected. An analysis of proteins that accumulated in the periplasm of the oxpG mutant, but not in the wild-type, led to the identification of a secreted protein, OmpB. OmpB is predicted to be a multicopper protein, with highest homology to the manganese oxidase, MofA, from Leptothrix discophora. OmpB contains a potential Fe(III)-binding site and a fibronectin type III domain, suggesting a possible role for this protein in accessing Fe(III) oxides. OmpB was localized to the membrane fraction of G. sulfurreducens and in the supernatant of growing cultures, consistent with the type II secretion system exporting OmpB. A mutant in which ompB was deleted had the same phenotype as the oxpG mutant, suggesting that the failure to export OmpB was responsible for the inability of the oxpG-deficient mutant to reduce Fe(III) oxide. This is the first report that proposes a role for a multicopper oxidase-like protein in an anaerobic organism. These results further emphasize the importance of outer-membrane proteins in Fe(III) oxide reduction and suggest that outer-membrane proteins other than c-type cytochromes are required for Fe(III) oxide reduction in Geobacter species.

We have operated a microbial fuel cell in which glucose was oxidized by Klebsiella pneumoniae in the anodic compartment, and biomineralized manganese oxides, deposited by Leptothrix discophora, were electrochemically reduced in the cathodic compartment. In the anodic compartment, to facilitate the electron transfer from glucose to the graphite electrode, we added a redox mediator, 2-hydroxy-1,4-naphthoquinone. We did not add any redox mediator to the cathodic compartment because the biomineralized manganese oxides were deposited on the surface of a graphite electrode and were reduced directly by electrons from the electrode. We have demonstrated that biomineralized manganese oxides are superiorto oxygen when used as cathodic reactants in microbial fuel cells. The current density delivered by using biomineralized manganese oxides as the cathodic reactant was almost 2 orders of magnitude higher than that delivered using oxygen. Several fuel cells were operated for 500 h, reaching anodic potentials of -441.5 +/- 31 mVscE and cathodic potentials of +384.5 +/- 64 mVscE. When the electrodes were connected by a 50 Ohms resistor, the fuel cell delivered the peak power density of 126.7 +/- 31.5 mW/m2.

The potential for filter-feeding bivalves to accumulate metals from a wide range of food sources is an important consideration when examining trophic transfer of metals up the food chain. The objective of this study was to determine the role of Leptothrix discophora in mediating metal uptake in the filter-feeding bivalve Mytilus trossulus. The bacterium L. discophora SP-6 was cultured in the absence or presence of Mn, allowing for a naturally formed Mn oxide sheath to develop. Secondary metals (Cd and Pb) were then added to the cultures, allowing for potential Cd and Pb adsorption to the Mn oxide sheath. Resulting bacterial aggregates of known diameter were then fed to the bivalve M. trossulus using a flow-through system. Initial concentrations of both Pb and Cd on the bacterium did not differ significantly in the presence or absence of the Mn oxide; conversely both Pb (F = 7.39, p0.0001) and Cd (F= 33.65, p0.0001) were found at lower concentrations in the mussel tissue when the Mn oxide was present. To determine whether these differences in metal uptake could be attributed to sorting by the mussel based on food quality, nutritional analysis was performed. Bacterial food matrixes containing Mn oxides were found to have significantly lower levels of carbon (F = 256, p0.0001). Particle clearance rates for the various food matrixes were positively correlated with organic content (R2 = 0.852, p0.008). The results of our study suggest that metal uptake in M. trossulus was significantly decreased for Cd with a similar trend for Pb when the SP-6 sheath contained Mn oxides. The mechanism mediating this differential uptake is best explained by food quality, in that a higher quality food source enhanced metal uptake due to an increased clearance rate of organic-rich particles by M. trossulus.