异化铁还原细菌Clostridium sp.LQ25分离及其产氢与铁还原特性

[背景] 一些异化铁还原细菌兼具铁还原和发酵产氢能力,可作为发酵型异化铁还原细菌还原机制研究的对象。[目的] 筛选出一株发酵型异化铁还原细菌。在异化铁还原细菌培养体系中,设置不同电子供体并分析电子供体。[方法] 通过三层平板法从海洋沉积物中筛选纯菌株,基于16S rRNA基因序列进行菌株鉴定。通过测定细菌培养液Fe （II）浓度及发酵产氢量分析菌株异化铁还原和产氢性质。[结果] 菌株LQ25与Clostridium butyricum的16S rRNA基因序列相似性达到100%,结合电镜形态观察,菌株命名为Clostridium sp.LQ25。在氢氧化铁为电子受体培养条件下,菌株生长较对照组（未添加氢氧化铁）显著提高。菌株LQ25能够利用丙酮酸钠、葡萄糖和乳酸钠进行生长。丙酮酸钠为电子供体时,菌株LQ25细胞生长和异化铁还原效率最高,菌体蛋白质含量是（78.88±3.40） mg/L,累积产生Fe （II）浓度为（8.27±0.23） mg/L。以葡萄糖为电子供体时,菌株LQ25发酵产氢量最高,达（475.2±14.4） mL/L,相比对照组（未添加氢氧化铁）产氢量提高87.7%。[结论] 筛选到一株具有异化铁还原和发酵产氢能力的菌株Clostridium sp.LQ25,为探究发酵型异化铁还原细菌胞外电子传递机制提供了新的实验材料。     

Shewanella oneidensis MR‐1 mutants selected for their inability to produce soluble organic‐Fe(III) complexes are unable to respire Fe(III) as anaerobic electron acceptor

Summary Recent voltammetric analyses indicate that Shewanella putrefaciens strain 200 produces soluble organic‐Fe(III) complexes during anaerobic respiration of sparingly soluble Fe(III) oxides. Results of the present study expand the range of Shewanella species capable of producing soluble organic‐Fe(III) complexes to include Shewanella oneidensis MR‐1. Soluble organic‐Fe(III) was produced by S. oneidensis cultures incubated anaerobically with Fe(III) oxides, or with Fe(III) oxides and the alternate electron acceptor fumarate, but not in the presence of O₂, nitrate or trimethylamine‐N‐oxide. Chemical mutagenesis procedures were combined with a novel MicroElectrode Screening Array (MESA) to identify four (designated Sol) mutants with impaired ability to produce soluble organic‐Fe(III) during anaerobic respiration of Fe(III) oxides. Two of the Sol mutants were deficient in anaerobic growth on both soluble Fe(III)‐citrate and Fe(III) oxide, yet retained the ability to grow on a suite of seven alternate electron acceptors. The rates of soluble organic‐Fe(III) production were proportional to the rates of iron reduction by the S. oneidensis wild‐type and Sol mutant strains, and all four Sol mutants retained wild‐type siderophore production capability. Results of this study indicate that the production of soluble organic‐Fe(III) may be an important intermediate step in the anaerobic respiration of both soluble and sparingly soluble forms of Fe(III) by S. oneidensis.     

Microbial Fe(III) oxide reduction potential in Chocolate Pots hot spring,Yellowstone National Park

Chocolate Pots hot springs (CP) is a unique, circumneutral pH, iron‐rich, geothermal feature in Yellowstone National Park. Prior research at CP has focused on photosynthetically driven Fe(II) oxidation as a model for mineralization of microbial mats and deposition of Archean banded iron formations. However, geochemical and stable Fe isotopic data have suggested that dissimilatory microbial iron reduction (DIR) may be active within CP deposits. In this study, the potential for microbial reduction of native CP Fe(III) oxides was investigated, using a combination of cultivation dependent and independent approaches, to assess the potential involvement of DIR in Fe redox cycling and associated stable Fe isotope fractionation in the CP hot springs. Endogenous microbial communities were able to reduce native CP Fe(III) oxides, as documented by most probable number enumerations and enrichment culture studies. Enrichment cultures demonstrated sustained DIR driven by oxidation of acetate, lactate, and H₂. Inhibitor studies and molecular analyses indicate that sulfate reduction did not contribute to observed rates of DIR in the enrichment cultures through abiotic reaction pathways. Enrichment cultures produced isotopically light Fe(II) during DIR relative to the bulk solid‐phase Fe(III) oxides. Pyrosequencing of 16S rRNA genes from enrichment cultures showed dominant sequences closely affiliated with Geobacter metallireducens, a mesophilic Fe(III) oxide reducer. Shotgun metagenomic analysis of enrichment cultures confirmed the presence of a dominant G. metallireducens‐like population and other less dominant populations from the phylum Ignavibacteriae, which appear to be capable of DIR. Gene (protein) searches revealed the presence of heat‐shock proteins that may be involved in increased thermotolerance in the organisms present in the enrichments as well as porin–cytochrome complexes previously shown to be involved in extracellular electron transport. This analysis offers the first detailed insight into how DIR may impact the Fe geochemistry and isotope composition of a Fe‐rich, circumneutral pH geothermal environment.     

The potential significance of microbial Fe(III) reduction during deposition of Precambrian banded iron formations

During deposition of late Archean–early Palaeoproterozoic Precambrian banded iron formations (BIFs) the downward flux of ferric hydroxide (Fe(OH)₃) and phytoplankton biomass should have facilitated microbial Fe(III) reduction. However, quantifying the significance of such a metabolic pathway in the Precambrian is extremely difficult, considering the post‐depositional alteration of the rocks and the lack of ideal modern analogues. Consequently, we have very few constraints on the Fe cycle at that time, namely (i) the concentration of dissolved Fe(II) in the ocean waters; (ii) by what mechanisms Fe(II) was oxidized (chemical, photochemical or biological, the latter using either O₂ or light); (iii) where the ferric hydroxide was precipitated (over the shelf vs. open ocean); (iv) the amount of phytoplankton biomass, which relates to the nutrient status of the surface waters; (v) the relative importance of Fe(III) reduction vs. the other types of metabolic pathways utilized by sea floor microbial communities; and (vi) the proportion of primary vs. diagenetic Fe(II) in BIF. Furthermore, although estimates can be made regarding the quantity of reducing equivalents necessary to account for the diagenetic Fe(II) component in Fe‐rich BIF layers, those same estimates do not offer any insights into the magnitude of Fe(III) actually generated within the water column, and hence, the efficiency of Fe and C recycling prior to burial. Accordingly, in this study, we have attempted to model the ancient Fe cycle, based simply on conservative experimental rates of photosynthetic Fe(II) oxidation in the euphotic zone. We estimate here that under ideal growth conditions, as much as 70% of the biologically formed Fe(III) could have been recycled back into the water column via fermentation and organic carbon oxidation coupled to microbial Fe(III) reduction. By comparing the potential amount of biomass generated phototrophically with the reducing equivalents required for Fe(III) reduction and magnetite formation, we also hypothesize that another anaerobic metabolic pathway might have been utilized in the surface sediment to oxidize the fermentation by‐products. Based on the premise that the deep ocean waters were anoxic, this role could have been fulfilled by methanogens, and maybe even methanotrophs that employed Fe(III) reduction.     

Fe(III) Oxide Reduction by Anaerobic Biofilm Formation-DeficientS-Ribosylhomocysteine Lyase (LuxS) Mutant of Shewanella oneidensis

Shewanella oneidensis respires a variety of terminal electron acceptors, including solid phase Fe(III) oxides. S. oneidensis transfers electrons to Fe(III) oxides via direct (outer membrane- or nanowire-localized c-type cytochromes) and indirect (electron shuttling and Fe(III) solubilization) pathways. In the present study, the influence of anaerobic biofilm formation on Fe(III) oxide reduction by S. oneidensis was determined. The gene encoding the activated methyl cycle (AMC) enzyme S-ribosylhomocysteine lyase (LuxS) was deleted in-frame to generate the corresponding mutant ΔluxS. Conventional biofilm assays and visual inspection via confocal laser scanning microscopy indicated that the wild-type strain formed anaerobic biofilms on Fe(III) oxide-coated silica surfaces, while the ΔluxS mutant was severely impaired in anaerobic biofilm formation on such surfaces. Cell-hematite attachment isotherms demonstrated that the ΔluxS mutant was also severely impaired in attachment to hematite surfaces under anaerobic conditions. The S. oneidensis ΔluxS mutant, however, reduced Fe(III) at wild-type rates during anaerobic incubation with Fe(III) oxide-coated silica surfaces or in batch cultures with Fe(III) oxide or hematite as a terminal electron acceptor. Anaerobic biofilm formation by the ΔluxS mutant was restored to wild-type rates by providing a wild-type copy of luxS in trans or by the addition of AMC or transsulfurylation pathway metabolites involved in organic sulfur metabolism. LuxS is thus required for wild-type anaerobic biofilm formation on Fe(III) oxide surfaces, yet the inability to form wild-type anaerobic biofilms on Fe(III) oxide surfaces does not alter Fe(III) oxide reduction activity.     

Effect of Oxidation Rate and Fe(II) State on Microbial Nitrate-Dependent Fe(III) Mineral Formation

A nitrate-dependent Fe(II)-oxidizing bacterium was isolated and used to evaluate whether Fe(II) chemical form or oxidation rate had an effect on the mineralogy of biogenic Fe(III) (hydr)oxides resulting from nitrate-dependent Fe(II) oxidation. The isolate (designated FW33AN) had 99% 16S rRNA sequence similarity to Klebsiella oxytoca. FW33AN produced Fe(III) (hydr)oxides by oxidation of soluble Fe(II) [Fe(II)_sol] or FeS under nitrate-reducing conditions. Based on X-ray diffraction (XRD) analysis, Fe(III) (hydr)oxide produced by oxidation of FeS was shown to be amorphous, while oxidation of Fe(II)_sol yielded goethite. The rate of Fe(II) oxidation was then manipulated by incubating various cell concentrations of FW33AN with Fe(II)_sol and nitrate. Characterization of products revealed that as Fe(II) oxidation rates slowed, a stronger goethite signal was observed by XRD and a larger proportion of Fe(III) was in the crystalline fraction. Since the mineralogy of Fe(III) (hydr)oxides may control the extent of subsequent Fe(III) reduction, the variables we identify here may have an effect on the biogeochemical cycling of Fe in anoxic ecosystems.     

电子穿梭体对菌株Clostridium butyricum LQ25异化铁还原性质影响

【目的】在异化铁还原细菌培养体系中,通过外加电子穿梭体,分析电子穿梭体种类与浓度对细菌异化铁还原性质的影响。【方法】以一株发酵型异化铁还原细菌Clostridium butyricum LQ25为研究对象,设置水溶性介体蒽醌-2-磺酸钠和核黄素作为外加电子穿梭体。【结果】在氢氧化铁为电子受体、葡萄糖为电子供体培养条件下,不同浓度蒽醌-2-磺酸钠和核黄素对菌株LQ25异化铁还原效率影响具有显著性差异。外加蒽醌-2-磺酸钠浓度为0.5 mmol/L时,菌株累积产生Fe(Ⅱ)浓度最高,为12.95±0.08 mg/L,相比对照组提高88%。核黄素浓度为100mg/L时,菌株累积产生Fe(Ⅱ)浓度是11.06±0.04mg/L,相比对照组提高61%。外加电子穿梭体能够改变菌株LQ25发酵产物中丁酸和乙酸浓度,提高乙酸相对含量。【结论】蒽醌-2-磺酸钠和核黄素作为外加电子穿梭体能显著促进细菌异化铁还原效率,为揭示发酵型异化铁还原细菌胞外电子传递机制提供实验支持。     

Fe(III) reduction during pyruvate fermentation by Desulfotomaculum reducens strain MI‐1

Desulfotomaculum reducens MI‐1 is a Gram‐positive, sulfate‐reducing bacterium also capable of reducing several metals, among which is Fe(III). Very limited knowledge is available on the potential mechanism(s) of metal reduction among Gram‐positive bacteria, despite their preponderance in the microbial communities that inhabit some inhospitable environments (e.g., thermal or hyperthermal ecosystems, extreme pH or salinity environments, heavy metal or radionuclide contaminated sediments). Here, we show that in the presence of pyruvate, this micro‐organism is capable of reducing both soluble Fe(III)‐citrate and solid‐phase hydrous ferric oxide, although growth is sustained by pyruvate fermentation rather than Fe(III) respiration. Despite the fact that Fe(III) reduction does not support direct energy conservation, D. reducens uses it as a complementary means of discarding excess reducing equivalent after H₂ accumulation in the culture headspace renders proton reduction unfavorable. Thus, Fe(III) reduction permits the oxidation of greater amounts of pyruvate than fermentation alone. Fe(III) reduction by D. reducens is mediated by a soluble electron carrier, most likely riboflavin. Additionally, an intracellular electron storage molecule acts as a capacitor and accumulates electrons during pyruvate oxidation for slow release to Fe(III). The reductase responsible for the transfer of electrons from the capacitor to the soluble carrier has not been identified, but data presented here argue against the involvement of c‐type cytochromes.     

Regulation of Dissimilatory Fe(III) Reduction Activity in Shewanella putrefaciens

Under anaerobic conditions, Shewanella putrefaciens is capable of respiratory-chain-linked, high-rate dissimilatory iron reduction via both a constitutive and inducible Fe(III)-reducing system. In the presence of low levels of dissolved oxygen, however, iron reduction by this microorganism is extremely slow. Fe(II)-trapping experiments in which Fe(III) and O₂ were presented simultaneously to batch cultures of S. putrefaciens indicated that autoxidation of Fe(II) was not responsible for the absence of Fe(III) reduction. Inhibition of cytochrome oxidase with CN⁻ resulted in a high rate of Fe(III) reduction in the presence of dissolved O₂, which suggested that respiratory control mechanisms did not involve inhibition of Fe(III) reductase activities or Fe(III) transport by molecular oxygen. Decreasing the intracellular ATP concentrations by using an uncoupler, 2,4-dinitrophenol, did not increase Fe(III) reduction, indicating that the reduction rate was not controlled by the energy status of the cell. Control of electron transport at branch points could account for the observed pattern of respiration in the presence of the competing electron acceptors Fe(III) and O₂.     

Adhesion of the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY to crystalline Fe(III) oxides

Shewanella alga BrY adhesion to hydrous ferric oxide, goethite, and hematite was examined. Adhesion to each oxide followed the Langmuir adsorption model. No correlation between adhesion and Fe(III) oxide surface area or crystallinity was observed. Zeta potential measurements suggested that electrostatic interactions do not influence S. alga BrY adhesion to these minerals. Cell adhesion does not appear to explain the recalcitrance of crystalline Fe(III) oxides to bacterial reduction. Received: 12 May 2000 / Accepted: 19 June 2000     

Evidence for Microbial Fe(III) Reduction in Anoxic, Mining-Impacted Lake Sediments (Lake Coeur d'Alene, Idaho)

Mining-impacted sediments of Lake Coeur d'Alene, Idaho, contain more than 10% metals on a dry weight basis, approximately 80% of which is iron. Since iron (hydr)oxides adsorb toxic, ore-associated elements, such as arsenic, iron (hydr)oxide reduction may in part control the mobility and bioavailability of these elements. Geochemical and microbiological data were collected to examine the ecological role of dissimilatory Fe(III)-reducing bacteria in this habitat. The concentration of mild-acid-extractable Fe(II) increased with sediment depth up to 50 g kg⁻¹, suggesting that iron reduction has occurred recently. The maximum concentrations of dissolved Fe(II) in interstitial water (41 mg liter⁻¹) occurred 10 to 15 cm beneath the sediment-water interface, suggesting that sulfidogenesis may not be the predominant terminal electron-accepting process in this environment and that dissolved Fe(II) arises from biological reductive dissolution of iron (hydr)oxides. The concentration of sedimentary magnetite (Fe₃O₄), a common product of bacterial Fe(III) hydroxide reduction, was as much as 15.5 g kg⁻¹. Most-probable-number enrichment cultures revealed that the mean density of Fe(III)-reducing bacteria was 8.3 × 10⁵ cells g (dry weight) of sediment⁻¹. Two new strains of dissimilatory Fe(III)-reducing bacteria were isolated from surface sediments. Collectively, the results of this study support the hypothesis that dissimilatory reduction of iron has been and continues to be an important biogeochemical process in the environment examined.     

Hyperthermophilic Anaerobic Nitrate-Dependent Fe(II) Oxidization by Tibetan Hot Spring Microbiota and the Formation of Fe Minerals

Fe(II) in geothermal fluids was among the most important electron and energy sources for extremophiles and early life, and microbial oxidation of Fe(II) in turn contributed to the global Fe deposits such as banded iron formation (BIF). However, information was rare on Fe(II) bio-oxidation and consequent mineral formation in geothermal systems. In the present study, we investigated the anaerobic nitrate-depending Fe(II) oxidation (ANDFO) in the Tibetan hot springs with temperature ranging 52–86°C. ANDFO cultivation was established by inoculating sediments from the studied hot springs. Positive ANDFO reaction was observed in the cultures from three high-temperature hot springs (>80°C). Phylogenetic analysis showed that bacteria in the three obtained ANDFO cultures were mainly affiliated with phyla of Betaproteobacteria, Alphaproteobacteria, and Firmicutes. In the obtained ANDFO cultures, ferrous iron oxidation occurred with nitrate reduction, accompanied with the formation of magnetite and/or siderite, which could be finished within one week. The resulting euhedral magnetite was at the micrometer scale, which was larger in size and showed better crystallinity than its counterparts (usually <1?µm) formed by chemical reactions. Thus, it can be concluded that ANDFO bacteria and denitrifiers played important roles in the magnetite and siderite precipitation in the studied Tibetan hot springs. The coupling between Fe(II) oxidation and nitrate reduction mediated by thermophiles might provide a new mechanism for euhedral magnetite and siderite deposition in BIFs during the Precambrian period.     

Interactions Between Fe(III)-Oxides and Fe(III)-Phyllosilicates During Microbial Reduction 1: Synthetic Sediments

Fe(III)-oxides and Fe(III)-bearing phyllosilicates are the two major iron sources utilized as electron acceptors by dissimilatory iron-reducing bacteria (DIRB) in anoxic soils and sediments. Although there have been many studies on microbial Fe(III)-oxide and Fe(III)-phyllosilicate reduction with both natural and specimen materials, no controlled experimental information is available on the interaction between these two phases when both are available for microbial reduction. In this study, the model DIRB Geobacter sulfurreducens was used to examine the pathways of Fe(III) reduction in Fe(III)-oxide stripped subsurface sediment that was coated with different amounts of synthetic high surface area (HSA) goethite. Cryogenic (12K) ⁵⁷Fe Mössbauer spectroscopy was used to determine changes in the relative abundances of Fe(III)-oxide, Fe(III)-phyllosilicate, and phyllosilicate-associated Fe(II) [Fe(II)-phyllosilicate] in bioreduced samples. Analogous Mössbauer analyses were performed on samples from abiotic Fe(II) sorption experiments in which sediments were exposed to a quantity of exogenous soluble Fe(II) (FeCl₂?2H₂O) comparable to the amount of Fe(II) produced during microbial reduction. A Fe partitioning model was developed to analyze the fate of Fe(II) and assess the potential for abiotic Fe(II)-catalyzed reduction of Fe(III)-phyllosilicates. The microbial reduction experiments indicated that although reduction of Fe(III)-oxide accounted for virtually all of the observed bulk Fe(III) reduction activity, there was no significant abiotic electron transfer between oxide-derived Fe(II) and Fe(III)-phyllosilicatesilicates, with 26–87% of biogenic Fe(II) appearing as sorbed Fe(II) in the Fe(II)-phyllosilicate pool. In contrast, the abiotic Fe(II) sorption experiments showed that 41 and 24% of the added Fe(II) engaged in electron transfer to Fe(III)-phyllosilicate surfaces in synthetic goethite-coated and uncoated sediment. Differences in the rate of Fe(II) addition and system redox potential may account for the microbial and abiotic reaction systems. Our experiments provide new insight into pathways for Fe(III) reduction in mixed Fe(III)-oxide/Fe(III)-phyllosilicate assemblages, and provide key mechanistic insight for interpreting microbial reduction experiments and field data from complex natural soils and sediments.     

Dissimilatory Fe(III) reduction by Clostridium beijerinckii isolated from freshwater sediment using Fe(III) maltol enrichment

A microorganism which reduces Fe(III) during the fermentation of glucose was isolated from freshwater sediment. The Fe(III) was supplied to enrichment cultures as a soluble complex with the bidentate ligand maltol (3-hydroxy-2-methyl-4-pyrone). Advantages that were afforded by the use of Fe(III)(maltol)3 over previously published methods included negation of the requirement for assays of Fe(II) formation. Because Fe(III)(maltol)3 has a characteristic deep red colour, Fe(III) reduction could be quantified spectrophotometrically by monitoring the disappearance of the complex in liquid cultures. Furthermore, Fe(III) reduction on agar plates containing the complex was apparent by zones of decolourisation around the bacterial colonies. 16S rRNA gene sequencing indicated the isolate to be a strain of Clostridium beijerinckii. Growth experiments were performed on the isolate in batch cultures with varying concentrations of Fe(III) citrate and 50 mM glucose. Increasing the level of Fe(III) citrate present was found to alter the fermentation balance, with less acidic products being formed. The presence of Fe(III) led to increases in the growth rate and growth yield, which were both approximately doubled when the supply of the cation reached 25 mM. A NAD(P)H-dependent Fe(III) reductase activity was localised to the bacterial membrane and found not to be sensitive to respiratory inhibitors. Taken together, these data suggest that dissimilatory Fe(III) reduction by the isolate provides a means of utilising the cation as an electron sink, thus facilitating pyridine nucleotide to be recycled during fermentative metabolism.     

Direct and Fe(II)-Mediated Reduction of Technetium by Fe(III)-Reducing Bacteria

The dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens reduced and precipitated Tc(VII) by two mechanisms. Washed cell suspensions coupled the oxidation of hydrogen to enzymatic reduction of Tc(VII) to Tc(IV), leading to the precipitation of TcO₂ at the periphery of the cell. An indirect, Fe(II)-mediated mechanism was also identified. Acetate, although not utilized efficiently as an electron donor for direct cell-mediated reduction of technetium, supported the reduction of Fe(III), and the Fe(II) formed was able to transfer electrons abiotically to Tc(VII). Tc(VII) reduction was comparatively inefficient via this indirect mechanism when soluble Fe(III) citrate was supplied to the cultures but was enhanced in the presence of solid Fe(III) oxide. The rate of Tc(VII) reduction was optimal, however, when Fe(III) oxide reduction was stimulated by the addition of the humic analog and electron shuttle anthaquinone-2,6-disulfonate, leading to the rapid formation of the Fe(II)-bearing mineral magnetite. Under these conditions, Tc(VII) was reduced and precipitated abiotically on the nanocrystals of biogenic magnetite as TcO₂ and was removed from solution to concentrations below the limit of detection by scintillation counting. Cultures of Fe(III)-reducing bacteria enriched from radionuclide-contaminated sediment using Fe(III) oxide as an electron acceptor in the presence of 25 μM Tc(VII) contained a single Geobacter sp. detected by 16S ribosomal DNA analysis and were also able to reduce and precipitate the radionuclide via biogenic magnetite. Fe(III) reduction was stimulated in aquifer material, resulting in the formation of Fe(II)-containing minerals that were able to reduce and precipitate Tc(VII). These results suggest that Fe(III)-reducing bacteria may play an important role in immobilizing technetium in sediments via direct and indirect mechanisms.     

Reduction of Fe(III), Mn(III), and Cu(II) chelates by roots of pea (Pisum sativum L.) or soybean (Glycine max)

Neither the reduction of Fe(III) to Fe(II) by roots nor its induction by Fe-deficiency are unique characteristics of the reductive activities of roots. We show that chelated Mn(III) or chelated Cu(II), as well as chelated Fe(III), may be reduced by Fe-stressed roots of pea (Pisum sativum L.). Deficiency of Fe stimulated the reduction of Fe(III)EDTA about 20-fold, the reduction of Mn(III)CDTA about 11-fold, the reduction of Cu(II)(BPDS)₂ about 5-fold, and the reduction of Fe(III)(CN)₆ by only about 50%. Not only are metals other than Fe reduced as part of the Fe-stress response, but deficiencies of metals other than Fe stimulate the reductive activity of roots. We show that depriving peas or soybeans (Glycine max) of Cu or Zn stimulates the reduction of Fe(III).     

Fe(III) oxide reduction and carbon tetrachloride dechlorination by a newly isolated Klebsiella pneumoniae strain L17

Aims:  To isolate an iron-reducing bacterium and examine its ability of Fe(III) oxide reduction and dechlorination.
Methods and Results:  A fermentative facultative anaerobe, strain L17 isolated from subterranean sediment, can reduce Fe(III) oxides and carbon tetrachloride (CT). It was identified as Klebsiella pneumoniae by 16S rRNA sequence analysis. Strain L17 can metabolize fermentable substrates such as citrate, glycerol, glucose and sucrose coupled with the reduction of hydrous ferric oxide, goethite, lepidocrocite and hematite. Fe(III) reduction was influenced by crystal structure of Fe(III) oxide, type of fermentable substrate, metabolic status of the strain, and significantly enhanced by addition of anthraquinone-2,6-disulfonate (AQDS). Strain L17 could dechlorinate CT to chloroform, and the rate was accelerated in the presence of Fe(III) oxide and AQDS. Biotic dechlorination by strain L17 and abiotic dechlorination by sorbed Fe(II) were proposed as the two main mechanisms. AQDS might accelerate the dechlorination by transferring electrons from strain L17 to Fe(III) oxide and CT.
Conclusions:  K. pneumoniae L17 can reduce Fe(III) oxides and CT. The two reductions can occur simultaneously, and be significantly promoted by AQDS.
Significance and Impact of the Study:  This is the first report of a strain of K. pneumoniae capable of reducing Fe(III) oxides and CT. As a strain of environmental origin, strain L17 may have the potential for bioremediation of chlorinated compound-contaminated sites.     

Lack of Production of Electron-Shuttling Compounds or Solubilization of Fe(III) during Reduction of Insoluble Fe(III) Oxide by Geobacter metallireducens

Studies with the dissimilatory Fe(III)-reducing microorganism Geobacter metallireducens demonstrated that the common technique of separating Fe(III)-reducing microorganisms and Fe(III) oxides with semipermeable membranes in order to determine whether the Fe(III) reducers release electron-shuttling compounds and/or Fe(III) chelators is invalid. This raised doubts about the mechanisms for Fe(III) oxide reduction by this organism. However, several experimental approaches indicated that G. metallireducens does not release electron-shuttling compounds and does not significantly solubilize Fe(III) during Fe(III) oxide reduction. These results suggest that G. metallireducens directly reduces insoluble Fe(III) oxide.     

Mechanisms for Accessing Insoluble Fe(III) Oxide during Dissimilatory Fe(III) Reduction by Geothrix fermentans

Mechanisms for Fe(III) oxide reduction were investigated in Geothrix fermentans, a dissimilatory Fe(III)-reducing microorganism found within the Fe(III) reduction zone of subsurface environments. Culture filtrates of G. fermentans stimulated the reduction of poorly crystalline Fe(III) oxide by washed cell suspensions, suggesting that G. fermentans released one or more extracellular compounds that promoted Fe(III) oxide reduction. In order to determine if G. fermentans released electron-shuttling compounds, poorly crystalline Fe(III) oxide was incorporated into microporous alginate beads, which prevented contact between G. fermentans and the Fe(III) oxide. G. fermentans reduced the Fe(III) within the beads, suggesting that one of the compounds that G. fermentans releases is an electron-shuttling compound that can transfer electrons from the cell to Fe(III) oxide that is not in contact with the organism. Analysis of culture filtrates by thin-layer chromatography suggested that the electron shuttle has characteristics similar to those of a water-soluble quinone. Analysis of filtrates by ion chromatography demonstrated that there was as much as 250 μM dissolved Fe(III) in cultures of G. fermentans growing with Fe(III) oxide as the electron acceptor, suggesting that G. fermentans released one or more compounds capable of chelating and solubilizing Fe(III). Solubilizing Fe(III) is another strategy for alleviating the need for contact between cells and Fe(III) oxide for Fe(III) reduction. This is the first demonstration of a microorganism that, in defined medium without added electron shuttles or chelators, can reduce Fe(III) derived from Fe(III) oxide without directly contacting the Fe(III) oxide. These results are in marked contrast to those with Geobacter metallireducens, which does not produce electron shuttles or Fe(III) chelators. These results demonstrate that phylogenetically distinct Fe(III)-reducing microorganisms may use significantly different strategies for Fe(III) reduction. Thus, it is important to know which Fe(III)-reducing microorganisms predominate in a given environment in order to understand the mechanisms for Fe(III) reduction in the environment of interest.     

The influence of chelating agents upon the dissimilatory reduction of Fe(III) byShewanella putrefaciens. Part 2. Oxo-and hydroxo-bridged polynuclear Fe(III) complexes

The susceptibility to dissimilatory reduction of polynuclear oxo- and hydroxo-bridged Fe(III) complexes byShewanella putrefaciens intact cells and membranes has been investigated. These complexes were ligated by the potential tetradentates heidi (H₃heidi =N-(2-hydroxyethyl)iminodiacetic acid) or nta (H₃nta = nitrilotriacetic acid), or the potential tridentate ida (H₂ida = iminodiacetic acid). A number of defined small complexes with varied nuclearity and solubility properties were employed, as well as undefined species prepared by mixing different molar ratios of ida or heidi:Fe(III) in solution. The rates of Fe(III) reduction determined by an assay for Fe(II) formation with ferrozine were validated by monitoringc-type cytochrome oxidation and re-reduction associated with electron transport. For the undefined Fe(III) polymeric species, reduction rates in whole cells and membranes were considerably faster in the presence of heidi compared to ida. This is believed to result from generally smaller and more reactive clusters forming with heidi as a consequence of the alkoxo function of this ligand being able to bridge between Fe(III) nuclei, with access to an Fe(III) reductase located at the cytoplasmic membrane being of some importance. The increases in reduction rates of the undefined ida species with Fe(III) using membranes relative to whole cells reinforce such a view. Using soluble synthetic Fe(III) clusters, slow reduction was noted for an oxo-bridged dimer coordinatively saturated with ida and featuring unligated carboxylates. This suggests that sterically hindering the cation can influence enzyme action. A heidi dimer and a heidi multimer (17 or 19 Fe(III) nuclei), which are both of poor solubility, were found to be reduced by whole cells, but dissimilation rates increased markedly using membranes. These data suggest that Fe(III) reductase activity may be located at both the outer membrane and the cytoplasmic membrane ofS. putrefaciens. Slower reduction of the heidi multimer relative to the heidi dimer reflects the presence of a central hydroxo(oxo)-bridged core containing nine Fe(III) nuclei within the former cluster. This unit is a poor substrate for dissimilation, owing to the fact that the Fe(III) is not ligated by aminocarboxylate. The faster reduction noted for the heidi dimer in membranes than for a soluble ida monomer suggests that the presence of ligating water molecules may relieve steric hindrance to enzyme attack. Furthermore, reduction of an insoluble oxo-bridged nta dimer featuring ligating water molecules in intact cells was faster than that of a soluble monomer coordinatively saturated by nta and possessing an unligated carboxylate. This suggests that steric factors may override solubility considerations with respect to the susceptibility to reduction of certain Fe(III) complexes by the bacterium.Previous paper in this series: Dobbin PS, Powell AK, McEwan AG, Richardson DJ. 1995 The influence of chelating agents upon the dissimilatory reduction of Fe(III) byShewanella putefraciens.BioMetals 8, 163–173.