<Emphasis Type="Italic">Thermincola ferriacetica</Emphasis> sp. nov<Emphasis Type="Italic">.,</Emphasis> a new anaerobic,thermophilic, facultatively chemolithoautotrophic bacterium capable of dissimilatory Fe(III) reduction

A moderately thermophilic, sporeforming bacterium able to reduce amorphous Fe(III)-hydroxide was isolated from ferric deposits of a terrestrial hydrothermal spring, Kunashir Island (Kurils), and designated as strain Z-0001. Cells of strain Z-0001 were straight, Gram-positive rods, slowly motile. Strain Z-0001 was found to be an obligate anaerobe. It grew in the temperature range from 45 to 70°C with an optimum at 57–60°C, in a pH range from 5.9 to 8.0 with an optimum at 7.0–7.2, and in NaCl concentration range 0–3.5% with an optimum at 0%. Molecular hydrogen, acetate, peptone, yeast and beef extracts, glycogen, glycolate, pyruvate, betaine, choline, N-acetyl-d-glucosamine and casamino acids were used as energy substrates for growth in presence of Fe(III) as an electron acceptor. Sugars did not support growth. Magnetite, Mn(IV) and anthraquinone-2,6-disulfonate served as the alternative electron acceptors, supporting the growth of isolate Z-0001 with acetate as electron donor. Formation of magnetite was observed when amorphous Fe(III) hydroxide was used as electron acceptor. Yeast extract, if added, stimulated growth, but was not required. Isolate Z-0001 was able to grow chemolithoautotrophicaly with molecular hydrogen as the only energy substrate, Fe(III) as electron acceptor and CO₂ as the carbon source. Isolate Z-0001 was able to grow with 100% CO as the sole energy source, producing H₂ and CO₂, requiring the presence of 0.2 g l⁻¹ of acetate as the carbon source. The G+C content of strain Z-0001^T DNA G+C was 47.8 mol%. Based on 16S rRNA sequence analyses strain Z-0001 fell into the cluster of family Peptococcaceae, within the low G+C content Gram-Positive bacteria, clustering with Thermincola carboxydophila (98% similarity). DNA–DNA hybridization with T. carboxydophila was 27%. On the basis of physiological and phylogenetic data it is proposed that strain Z-0001^T (=DSMZ 14005, VKM B-2307) should be placed in the genus Thermincola as a new species Thermincola ferriacetica sp. nov. The GenBank accession number for the sequence reported in the paper is AY 631277.     

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.     

Geovibrio ferrireducens,a phylogenetically distinct dissimilatory Fe(III)-reducing bacterium

A new, phylogenetically distinct, dissimilatory, Fe(III)-reducing bacterium was isolated from surface sediment of a hydrocarbon-contaminated ditch. The isolate, designated strain PAL-1, was an obligately anaerobic, non-fermentative, motile, gram-negative vibrio. PAL-1 grew in a defined medium with acetate as electron donor and ferric pyrophosphate, ferric oxyhydroxide, ferric citrate, Co(III)-EDTA, or elemental sulfur as sole electron acceptor. PAL-1 also used proline, hydrogen, lactate, propionate, succinate, fumarate, pyruvate, or yeast extract as electron donors for Fe(III) reduction. It is the first bacterium known to couple the oxidation of an amino acid to Fe(III) reduction. PAl-1 did not reduce oxygen, Mn(IV), U(VI), Cr(VI), nitrate, sulfate, sulfite, or thiosulfate with acetate as the electron donor. Cell suspensions of PAL-1 exhibited dithionite-reduced minus air-oxidized difference spectra that were characteristic of c-type cytochromes. Analysis of the 16S rRNA gene sequence of PAL-1 showed that the strain is not related to any of the described metal-reducing bacteria in the Proteobacteria and, together with Flexistipes sinusarabici, forms a separate line of descent within the Bacteria. Phenotypically and phylogenetically, strain PAl-1 differs from all other described bacteria, and represents the type strain of a new genus and species, Geovibrio ferrireducens. Received: 26 September 1995 / Accepted: 28 February 1996     

Influence of Biogenic Fe(II) on Bacterial Crystalline Fe(III) Oxide Reduction

Bacterial crystalline Fe(III) oxide reduction has the potential to significantly influence the biogeochemistry of anaerobic sedimentary environments where crystalline Fe(III) oxides are abundant relative to poorly crystalline (amorphous) phases. A review of published data on solid-phase Fe(III) abundance and speciation indicates that crystalline Fe(III) oxides are frequently 2- to S 10-fold more abundant than amorphous Fe(III) oxides in shallow subsurface sediments not yet subjected to microbial Fe(III) oxide reduction activity. Incubation experiments with coastal plain aquifer sediments demonstrated that crystalline Fe(III) oxide reduction can contribute substantially to Fe(II) production in the presence of added electron donors and nutrients. Controls on crystalline Fe(III) oxide reduction are therefore an important consideration in relation to the biogeochemical impacts of bacterial Fe(III) oxide reduction in subsurface environments. In this paper, the influence of biogenic Fe(II) on bacterial reduction of crystalline Fe(III) oxides is reviewed and analyzed in light of new experiments conducted with the acetate-oxidizing, Fe(III)-reducing bacterium (FeRB) Geobacter metallireducens . Previous experiments with Shewanella algae strain BrY indicated that adsorption and/or surface precipitation of Fe(II) on Fe(III) oxide and FeRB cell surfaces is primarily responsible for cessation of goethite ( f -FeOOH) reduction activity after only a relatively small fraction (generally < 10%) of the oxide is reduced. Similar conclusions are drawn from analogous studies with G. metallireducens . Although accumulation of aqueous Fe(II) has the potential to impose thermodynamic constraints on the extent of crystalline Fe(III) oxide reduction, our data on bacterial goethite reduction suggest that this phenomenon cannot universally explain the low microbial reducibility of this mineral. Experiments examining the influence of exogenous Fe(II) (20 mM FeCl 2 ) on soluble Fe(III)-citrate reduction by G. metallireducens and S. algae showed that high concentrations of Fe(II) did not inhibit Fe(III)-citrate reduction by freshly grown cells, which indicates that surface-bound Fe(II) does not inhibit Fe(III) reduction through a classical end-product enzyme inhibition mechanism. However, prolonged exposure of G. metallireducens and S. algae cells to high concentrations of soluble Fe(II) did cause inhibition of soluble Fe(III) reduction. These findings, together with recent documentation of the formation of Fe(II) surface precipitates on FeRB in Fe(III)-citrate medium, provide further evidence for the impact of Fe(II) sorption by FeRB on enzymatic Fe(III) reduction. Two different, but not mutually exclusive, mechanisms whereby accumulation of Fe(II) coatings on Fe(III) oxide and FeRB surfaces may lead to inhibition of enzymatic Fe(III) oxide reduction activity (in the absence of soluble electron shuttles and/or Fe(III) chelators) are identified and discussed in relation to recent experimental work and theoretical considerations.     

Role of iron chelators in growth-promoting effect on mouse hybridoma cells in a chemically defined medium

Summary The role of various iron chelators on the multiplication of mouse hybridoma cells in an albumin-free, transferrin-deficient defined medium was investigated. Fe(III)-dihydroxyethylglycine, Fe(III)-glycylglycine, Fe(III)-ethylenediamine-N,N′-dipropionic acid, or Fe(III)-iminodiacetic acid supported the excellent growth of the cells. In addition, the growth of the iron-starved cells, which had been preincubated in a protein-, iron- and chelator-free defined medium, restored rapidly when the medium was supplemented with holotransfeerrin, ferric iron, and chelator compared to that when supplemented with holotransferin, but without iron and chelator. The results suggest that such chelators modulate a progression of transferrn cycle in the presence of transferin and ferric iron. An alternative explantation is that there is a decrease in generation of iron-catalyzed free radicals.     

Ferric iron reduction by Desulfovibrio vulgaris Hildenborough wild type and energy metabolism mutants

Desulfovibrio vulgaris Hildenborough wild type and its hyn1, hyd and hmc mutants, lacking genes for periplasmic [NiFe] hydrogenase-1, periplasmic [FeFe] hydrogenase or the transmembrane high molecular weight cytochrome (Hmc) complex, respectively, were able to reduce Fe(III) chelated with nitrilotriacetic acid (NTA), but not insoluble ferric oxide, with lactate as the electron donor. The rate and extent of Fe(III)-NTA reduction followed the order hyn = WT > hmc >> hyd, suggesting that reduction of soluble Fe(III) is a periplasmic process that requires the presence of periplasmic [FeFe] hydrogenase. Reduction of Fe(III)-NTA was not coupled to cell growth. In fact cell concentrations declined when D. vulgaris was incubated with Fe(III)-NTA as the only electron acceptor. Wild type and mutant cells reducing a limiting concentration of sulfate (2 mM), reduced Fe(III)-NTA with similar rates. However, these were similarly incapable of catalyzing subsequent lactate-dependent reduction of Fe(III)-NTA to completion. Periplasmic reduction of Fe(III)-NTA appeared to inhibit the productive, sulfate-reducing metabolism of D. vulgaris, possibly because it prevents the cycling of reducing equivalents needed to achieve a net bioenergetic benefit.     

The effect of albumin,ceruloplasmin, and other serum constitutents on Fe(II) oxidation

This study has analyzed the role of several serum constituents, that have been proposed to effect the following reactionin situ: {fx1-1} {fx1-2} These reactions were monitored by measuring the rate of Fe(II) oxidation in the presence of apo-transferrin (reaction A) and Fe(III)-transferrin formation (reaction B) at 465 nm. Reactions A and B were found to be kinetically equivalent. The results show that, singly or in combination, bicarbonate, orthophosphate, citrate, apo-transferrin, and/or albumin have less than one-tenth of the ability to enhance the oxidation of Fe(II) compared to the serum enzyme, ceruloplasmin. It was also found that the rate of Fe(II) oxidation by serum Fe-ligands was influenced by the efficiency of oxygen utilization. Whereas ceruloplasmin produces a 4∶1 ratio of Fe(II) oxidized to oxygen utilized, the non-enzymic components yield a 2∶1 or 3.09∶1 ratio. These data support the role of ceruloplasmin as an antioxidant that prevents the formation of the intermediate active oxygen species O ₂ ⁻ · and H₂O ₂ ^· through the Fe(II) auto-oxidation reaction. A hitherto unrecognized factor in the control of nonenzymic oxidation of Fe(II) was serum albumin. This protein, at >25 μM, was found to sharply dampen the rate of Fe(II) oxidation in the presence of a physiological concentration of bicarbonate, citrate, and transferrin Albumin did not appear to affect the ceruloplasmin catalyzed oxidation of Fe(II) at pH 7.0. The addition of ceruloplasmin effected up to a 44 × increase in the rate of Fe(II) oxidation and Fe(III)-transferrin formation even in the presence of 0.60 mM albumin.     

α-Amino acid behaves differently from β- or γ-amino acids as treated by trimetaphosphate

Summary. The condensation reactions of sodium trimetaphosphate with single amino acids, namely glycine, L-alanine, β-alanine and γ-aminobutyric acid or pairs of these amino acids were reinvestigated by electrospray ion-trap mass spectrometry and high performance liquid chromatography. It was found when mixtures were treated by sodium trimetaphosphate only in the presence of α-amino acid dipeptides were formed. Without addition of α-amino acids, the β-amino acid or γ-aminobutyric acid could not form peptide either by themselves or with their mixtures under the same conditions. From the data it is concluded that phosphate might select α-amino acids to produce the peptides being important precursors for the origin of life. Authors’ address: Dr. Pengxiang Xu, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemistry, Xiamen University, Xiamen 361005, China     

Colonization of biofilms by bacteria capable of biodegrading sodium dodecyl sulphate (SDS) at clean and polluted riverine sites

Fifty cyanobacterial strains (10 genera) were tested in batch culture for their ability to use organic phosphorus compounds (1 mg liter⁻¹ P) as their sole P source. Two monoesters, Na₂-β-glycerophosphate and π-nitrophenyl phosphate (πNPP), supported growth of all strains, and the diester bis-π-nitrophenyl phosphate (bis-π-NPP) and herring sperm DNA supported almost all strains. ATP was either a very favorable or poor P source and failed to support growth of nine strains, seven of which were Rivulariaceae with trichomes ending in a hair or long tapered region. Phytic acid was in general the least favorable P source. P-limited cultures grown initially with inorganic phosphate to conditions of P limitation were also tested for cell-bound and extracellular phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities at two pH values (7.6, 10.3) using πNPP and bis-πNPP as substrates. Cell-bound PMEase was inducible in all strains and cell-bound PDEase in most strains. Most showed extracellular PMEase, but not extracellular PDEase. The highest values (μM πNPP or bis-πNPP hydrolyzed mg dry weight⁻¹ hour⁻¹) all occurred in strains ofGloeotrichia as follows: cell-bound PMEase at pH 7.6, 2.7 μM in strain D602; cell-bound PMEase at pH 10.3, 5.2 μM in D602; extracellular PMEase at pH 7.6, 0.73 μM in D281; extracellular PMEase at pH 10.3, 6.6 μM in D281; cell-bound PDEase at 7.6, 0.40 μM in D613; cell-bound PDEase at pH 10.3, 1.0 μM in D613. The results were compared to see if they indicated possible relationships between phosphatase activity and taxonomic or ecological grouping. The following differences were significant (P<0.05). Rivulariaceae produced higher yields than filamentous non-Rivulariaceae with β-glycerophosphate, πNPP, and DNA. Rivulariaceae with the ability to form hairs in culture showed poorer growth in ATP than non-hair-forming Rivulariaceae, but were more effective at utilizing phytic acid. Strains from calcareous environments had higher PMEase activity at pH 10.3 than strains from noncalcareous environments (P<0.01).     

Iron limitation results in induction of ferricyanide reductase and ferric chelate reductase activities in Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii Dangeard CW-15 exhibited very low rates of plasma-membrane Fe(III) reductase activity when grown under Fe-sufficient conditions. After switching the medium to an Fe-free formulation, both ferricyanide reductase and ferric chelate reductase activities rapidly increased, reaching a maximum after 3 d under iron-free conditions. Both of the Fe(III) reductase activities increased in parallel over time, they exhibited similar K _m values (approximately 10 μM) with respect to Fe(III), displayed the same pH profile of activity, and both exhibited the same degree of light stimulation which could be inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). Furthermore, ferricyanide competitively inhibited ferric chelate reduction by iron-limited cells. These results indicate that both Fe(III) reductase activities were mediated by the same iron-limitation-induced plasma-membrane reductase. No evidence was found for the presence of Fe(III)-reducing substances in the culture medium, or for the involvement of active oxygen species in the process of Fe(III) reduction. Chlamydomonas reinhardtii appears to respond to iron limitation in a manner similar to Strategy I higher plants. Received: 24 June 1997 / Accepted: 2 August 1997     

The influence of chelating agents upon the dissimilatory reduction of Fe(III) by Shewanella putrefaciens

The ability of S. putrefaciens to reduce Fe(III) complexed by a variety of ligands has been investigated. All of the ligands tested caused the cation to be more susceptible to reduction by harvested whole cells than when uncomplexed, although some complexes were more readily reduced than others. Monitoring rates of reduction by a ferrozine assay for Fe(II) formation proved inadequate using Fe(III) ligands giving Fe(II) complexes of low kinetic lability (e.g. EDTA). A more suitable assay for Fe(III) reduction in the presence of such ligands proved to be the observation of associated cytochrome oxidation and re-reduction. Where possible, an assay for Fe(III) reduction based upon the disappearance of Fe(III) complex was also employed. Reduction of all Fe(III) complexes tested was totally inhibited by the presence of O₂, partially inhibited by HQNO and slower in the absence of a physiological electron donor. Upon cell fractionation, Fe(III) reductase activity was detected exclusively in the membranes. Using different physiological electron donors in assays on membranes, relative reduction rates of Fe(III) complexes complemented the data from whole cells. The differences in susceptibility to reduction of the various complexes are discussed, as is evidence for the respiratory nature of the reduction.     

Reduction of Fe(II)EDTA-NO by a newly isolated <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain DN-2 in NO<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript> scrubber solution

Biological reduction of nitric oxide (NO) chelated by ferrous ethylenediaminetetraacetate (Fe(II)EDTA) to N₂ is one of the core processes in a chemical absorption–biological reduction integrated technique for nitrogen oxide (NO _x ) removal from flue gases. A new isolate, identified as Pseudomonas sp. DN-2 by 16S rRNA sequence analysis, was able to reduce Fe(II)EDTA-NO. The specific reduction capacity as measured by NO was up to 4.17 mmol g DCW⁻¹ h⁻¹. Strain DN-2 can simultaneously use glucose and Fe(II)EDTA as electron donors for Fe(II)EDTA-NO reduction. Fe(III)EDTA, the oxidation of Fe(II)EDTA by oxygen, can also serve as electron acceptor by strain DN-2. The interdependency between various chemical species, e.g., Fe(II)EDTA-NO, Fe(II)EDTA, or Fe (III)EDTA, was investigated. Though each complex, e.g., Fe(II)EDTA-NO or Fe(III)EDTA, can be reduced by its own dedicated bacterial strain, strain DN-2 capable of reducing Fe(III)EDTA can enhance the regeneration of Fe(II)EDTA, hence can enlarge NO elimination capacity. Additionally, the inhibition of Fe(II)EDTA-NO on the Fe(III)EDTA reduction has been explored previously. Strain DN-2 is probably one of the major contributors for the continual removal of NO _x due to the high Fe(II)EDTA-NO reduction rate and the ability of Fe(III)EDTA reduction.     

Interactions of Cu (II) and Fe (III) with mangal humic substances studied by synchronous fluorescence spectroscopy and potentiometric titration

Humic (HA) and fulvic (FA) acids isolated from mangrove sediments of Sundarban, the largest delta on earth in the estuarine phase of the river Ganges, were studied and attempts were made to characterize their binding sites by quenching of Synchronous fluorescence (SyF) bands with Fe (III) and Cu (II). A modified Stern-Volmer relationship applicable for static quenching was applied for the determination of conditional stability constants and the data were compared with those determined by potentiometric titration. In the excited state HA and FA showed different acidity constant compared to the ground state. Values of the conditional stability constant (log K_c) for Fe (III) and Cu (II) indicated that binding sites were bidentate in nature. FA were better chelators than the HA fractions. High energy binding sites of both FA & HA were occupied by Fe(III) and the low energy binding sites, mainly responsible for mobilization and immobilization of metal, were occupied by Cu(II).     

异化铁还原细菌LQ25还原重金属Cr (VI)的特性研究

[背景] 异化铁还原细菌能够在还原Fe （III）的同时将毒性较大的Cr （VI）还原成毒性较小的Cr （III）,解决铬污染的问题。[目的] 基于丁酸梭菌（Clostridium butyricum） LQ25异化铁还原过程制备生物磁铁矿,开展异化铁还原细菌还原Cr （VI）的特性研究。[方法] 构建以氢氧化铁为电子受体和葡萄糖为电子供体的异化铁培养体系。菌株LQ25培养结束时制备生物磁铁矿。设置不同初始Cr （VI）浓度（5、10、15、25和30 mg/L）,分别测定菌株LQ25对Cr （VI）还原效率以及生物磁铁矿对Cr （VI）的还原效率。[结果] 菌株LQ25在设置的Cr （VI）浓度范围内都能良好生长。当Cr （VI）浓度为15 mg/L时,在异化铁培养条件下,菌株LQ25对Cr （VI）的还原率为63.45%±5.13%,生物磁铁矿对Cr （VI）的还原率为87.73%±9.12%,相比菌株还原Cr （VI）的效率提高38%。pH变化能影响生物磁铁矿对Cr （VI）的还原率,当pH 2.0时,生物磁铁矿对Cr （VI）的还原率最高,几乎达到100%。电子显微镜观察发现生物磁铁矿表面有许多孔隙,X-射线衍射图谱显示生物磁铁矿中Fe （II）的存在形式是Fe （OH）₂。[结论] 基于异化铁还原细菌制备生物磁铁矿可用于还原Cr （VI）,这是一种有效去除Cr （VI）的途径。     

Anaerobic benzene degradation

Although many studies have indicated that benzene persists under anaerobic conditions in petroleum-contaminated environments, it has recently been documented that benzene can be anaerobically oxidized with most commonlyconsidered electron acceptors for anaerobic respiration. These include: Fe(III),sulfate, nitrate, and possibly humic substances. Benzene can also be convertedto methane and carbon dioxide under methanogenic conditions. There is evidencethat benzene can be degraded under in situ conditions in petroleum-contaminatedaquifers in which either Fe(III) reduction or methane production is the predominant terminal electron-accepting process. Furthermore, evidence from laboratory studies suggests that benzene may be anaerobically degraded in petroleum-contaminated marine sediments under sulfate-reducing conditions. Laboratory studies have suggested that within the Fe(III) reduction zone of petroleum-contaminated aquifers, benzene degradation can be stimulated with the addition of synthetic chelators which make Fe(III) more available for microbial reduction. The addition of humic substances and other compounds that contain quinone moieties can also stimulate anaerobic benzene degradation in laboratory incubations of Fe(III)-reducing aquifer sediments by providing an electron shuttle between Fe(III)-reducing microorganisms and insoluble Fe(III) oxides. Anaerobic benzene degradation in aquifer sediments can be stimulated with the addition of sulfate, but in some instances an inoculum of benzene-oxidizing,sulfate-reducing microorganisms must also be added. In a field trial, sulfate addition to the methanogenic zone of a petroleum-contaminated aquifer stimulated the growth and activity of sulfate-reducing microorganisms and enhanced benzene removal. Molecular phylogenetic studies have provided indications of what microorganisms might be involved in anaerobic benzene degradation in aquifers. The major factor limiting further understanding of anaerobic benzene degradation is the lack of a pure culture of an organism capable of anaerobic benzene degradation.     

Secondary Mineral Formation During Ferrihydrite Reduction by Shewanella oneidensis MR-1 Depends on Incubation Vessel Orientation and Resulting Gradients of Cells,Fe2+ and Fe Minerals

In previous studies on microbial ferric iron (Fe(III)) reduction varying results regarding reduction rates and secondary mineral formation have been reported for almost identical conditions regarding temperature, pH, medium composition, Fe(III) mineral identity and bulk iron concentration. Here we show that in addition to physico-chemical parameters also geometric aspects, i.e., incubation orientation and dimension of cultivation vessels, influence the reduction rates and mineralogy. We incubated the Fe(III)-reducer Shewanella oneidensis MR-1 in test tubes at ferrihydrite (FH) concentrations of 1.3–50 mM either in vertical or horizontal orientation. Cells and minerals formed a pellet at the bottom of the tubes with different thicknesses at the same initial FH concentration depending on the incubation orientation. In vertically incubated tubes thick FH pellets were present at the bottom of the tubes and magnetite was formed in all setups with ≥2.5 mM initial FH. In tubes that were incubated horizontally no magnetite was formed in presence of <5 mM initial FH. Spatially resolved analysis of the supernatant and mineral sediment including voltammetric microelectrodes, Xray diffraction and Mössbauer spectroscopy revealed strong gradients of Fe²⁺ in both the aqueous supernatant and mineral pellets, whereas a heterogeneous distribution of cells and minerals in the sediment pellet was detected. The highest cell density and, consequently, the initiation of FH reduction was found at the mineral-supernatant interface. This study demonstrates that small changes in incubation conditions can significantly influence and even change the experimental results of geomicrobiological experiments.     

Fluorescent siderophore-based chemosensors: iron(III) quantitative determinations

 A highly sensitive and selective method is described for a rapid and easy determination of iron(III). This procedure is based on fluorimetric detection combined with the attractive properties of siderophores and biomimetic ligands, which are strong and selective ferric chelators. Azotobactin δ, a bacterial fluorescent siderophore, three fluorescent derivatives of desferriferrioxamine B with a linear structure (NBD-, MA-, NCP-desferriferrioxamine B) and one tripodal biomimetic ligand of desferriferrichrome carrying an anthracenyl fluorescent probe were examined. A very efficient static quenching mechanism by iron was observed for all the ligands considered in this work. Our results identify azotobactin δ as the most promising chemosensor of ferric traces in water, more sensitive than the NBD-desferriferrioxamine B fluorescent ligand. Under more lipophilic conditions, the anthryl-desferriferrichrome biomimetic analogue showed similar analytical potential and was found to be more sensitive than the lipophilic MA- and NCP-desferriferrioxamine B. Their detection limits were respectively 0.5 ng mL^–1 for azotobactin δ and 0.6 ng mL^–1 for the anthryl tripodal chelator. The calibration curves were linear over the range 0–95 ng mL^–1 and 0–180 ng mL^–1. Various foreign cations have been examined and only copper(II) and aluminium(III) were shown to interfere when present in similar concentrations as iron(III). The developed procedure using fluorescent siderophores or biomimetic ligands of iron(III) may be applied (1) to monitor iron(III)-dependent biological systems and (2) to determine iron(III) quantitatively in natural waters and in biological systems. Received: 12 September 1998 · Accepted: 19 January 1999     

Adhesion of Dissimilatory Fe(III)-Reducing Bacteria to Fe(III) Minerals

The metabolism of dissimilatory iron-reducing bacteria (DIRB) may provide a means of remediating contaminated subsurface soils. The factors controlling the rate and extent of bacterial F(III) mineral reduction are poorly understood. Recent research suggests that molecular-scale interactions between DIRB cells and Fe(III) mineral particles play an important role in this process. One of these interactions, cell adhesion to Fe(III) mineral particles, appears to be a complex process that is, at least in part, mediated by a variety of surface proteins. This study examined the hypothesis that the flagellum serves as an adhesin to different Fe(III) minerals that range in their surface area and degree of crystallinity. Deflagellated cells of the DIRB Shewanella algae BrY showed a reduced ability to adhere to hydrous ferric oxide (HFO) relative to flagellated cells. Flagellated cells were also more hydrophobic than deflagellated cells. This was significant because hydrophobic interactions have been previously shown to dominate S. algae cell adhesion to Fe(III) minerals. Pre-incubating HFO, goethite, or hematite with purified flagella inhibited the adhesion of S. algae BrY cells to these minerals. Transposon mutagenesis was used to generate a flagellum-deficient mutant designated S. algae strain NF. There was a significant difference in the rate and extent of S. algae NF adhesion to HFO, goethite, and hematite relative to that of S. algae BrY. Amiloride, a specific inhibitor of Na + -driven flagellar motors, inhibited S. algae BrY motility but did not affect the adhesion of S. algae BrY to HFO. S.algae NF reduced HFO at the same rate as S. algae BrY. Collectively, the results of this study support the hypothesis that the flagellum of S. algae functions as a specific Fe(III) mineral adhesin. However, these results suggest that flagellum-mediated adhesion is not requisite for Fe(III) mineral 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.