Utilization of cathodic hydrogen by hydrogen-oxidizing bacteria

Summary Hydrogen is consumed by methanogenic, sulphate-reducing, and homoacetogenic bacteria and members of these bacterial groups are able to grow chemolithotrophically with hydrogen as sole energy source. Cathodic hydrogen consumption by sulphate-reducing bacteria has been proposed as one of the factors in the anaerobic corrosion of metals. Desulfovibrio spp. were able to utilize cathodic hydrogen from mild steel as the only source of energy for growth with sulphate or nitrate as terminal electron acceptor. Other hydrogen-oxidizing bacteria such as Methanospirillum hungatei, Acetobacterium woodii and Wolinella succinogenes were also able to utilize cathodic hydrogen from mild steel for energy generation and growth. Weight loss studies of mild steel coupons under different growth conditions of Desulfovibrio spp. indicated that hydrogen removal alone is not the cause of corrosion and the depolarization phenomenon probably plays a role only in the initiation of the anaerobic microbial corrosion process.     

Reduction of ferric green rust by Shewanella putrefaciens

AIMS: To reduce carbonated ferric green rust (GR*) using an iron respiring bacterium and obtain its reduced homologue, the mixed Fe(II)-Fe(III) carbonated green rust (GR). METHODS AND RESULTS: The GR* was chemically synthesized by oxidation of the GR and was incubated with Shewanella putrefaciens cells at a defined [Fe(III)]/[cell] ratio. Sodium methanoate served as the sole electron donor. The GR* was quickly transformed in GR (iron reducing rate = 8.7 mmol l(-1) h(-1)). CONCLUSIONS: Ferric green rust is available for S. putrefaciens respiration as an electron acceptor. The reversibility of the GR redox state can be driven by bacterial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This work suggests that GRs would act as an electronic balance in presence of bacteria. It provides also new perspectives for using iron reducing bacterial activity to regenerate the reactive form of GR during soil or water decontamination processes.     

Corroding iron as a hydrogen source for sulphate reduction in growing cultures of sulphate-reducing bacteria

Summary In anaerobic corrosion experiments, hydrogenase-positiveDesulfovibrio strains, grown with limiting lactate concentrations in the presence of steel wool, formed more sulphide than expected or observed with lactate alone. The additional sulphide obviously originated from sulphate reduction with cathodically formed hydrogen from the steel surface. The hydrogenasenegativeD. sapovorans did not produce additional sulphide. The observations agree with the theory of von Wolzogen Kühr and van der Vlugt (1934) that explains anaerobic corrosion as a cathodic depolarization of iron surfaces by hydrogen-consuming sulphate-reducing bacteria. The influence of the iron surface area, the salt concentration and the pH-value on the utilization of cathodically formed hydrogen was investigated. The significance of an additional organic electron donor for the corrosion of iron in aqueous environments is discussed.     

The role of biomineralization in microbiologically influenced corrosion

Synthetic iron oxides (goethite, -FeO·OH; hematite, Fe₂O₃; and ferrihydrite, Fe(OH)₃) were used as model compounds to simulate the mineralogy of surface films on carbon steel. Dissolution of these oxides exposed to pure cultures of the metal-reducing bacterium, Shewanella putrefaciens, was followed by direct atomic absorption spectroscopy measurement of ferrous iron coupled with microscopic analyses using confocal laser scanning and environmental scanning electron microscopies. During an 8-day exposure the organism colonized mineral surfaces and reduced solid ferric oxides to soluble ferrous ions. Elemental composition, as monitored by energy dispersive x-ray spectroscopy, indicated mineral replacement reactions with both ferrihydrite and goethite as iron reduction occurred. When carbon steel electrodes were exposed to S. putrefaciens, microbiologically influenced corrosion was demonstrated electrochemically and microscopically.     

Isolation of Acetogenic Bacteria That Induce Biocorrosion by Utilizing Metallic Iron as the Sole Electron Donor

Corrosion of iron occurring under anoxic conditions, which is termed microbiologically influenced corrosion (MIC) or biocorrosion, is mostly caused by microbial activities. Microbial activity that enhances corrosion via uptake of electrons from metallic iron [Fe(0)] has been regarded as one of the major causative factors. In addition to sulfate-reducing bacteria and methanogenic archaea in marine environments, acetogenic bacteria in freshwater environments have recently been suggested to cause MIC under anoxic conditions. However, no microorganisms that perform acetogenesis-dependent MIC have been isolated or had their MIC-inducing mechanisms characterized. Here, we enriched and isolated acetogenic bacteria that induce iron corrosion by utilizing Fe(0) as the sole electron donor under freshwater, sulfate-free, and anoxic conditions. The enriched communities produced significantly larger amounts of Fe(II) than the abiotic controls and produced acetate coupled with Fe(0) oxidation prior to CH₄ production. Microbial community analysis revealed that Sporomusa sp. and Desulfovibrio sp. dominated in the enrichments. Strain GT1, which is closely related to the acetogen Sporomusa sphaeroides, was eventually isolated from the enrichment. Strain GT1 grew acetogenetically with Fe(0) as the sole electron donor and enhanced iron corrosion, which is the first demonstration of MIC mediated by a pure culture of an acetogen. Other well-known acetogenic bacteria, including Sporomusa ovata and Acetobacterium spp., did not grow well on Fe(0). These results indicate that very few species of acetogens have specific mechanisms to efficiently utilize cathodic electrons derived from Fe(0) oxidation and induce iron corrosion.     

Isolation of anaerobic respiratory mutants of Shewannella putrefaciens and genetic analysis of mutants deficient in anaerobic growth on Fe3+.

A genetic approach was used to study (dissimilatory) ferric iron (Fe3+) reduction in Shewanella putrefaciens 200. Chemical mutagenesis procedures and two rapid plate assays were developed to facilitate the screening of Fe3+ reduction-deficient mutants. Sixty-two putative Fe3+ reduction-deficient mutants were identified, and each was subsequently tested for its ability to grow anaerobically on various compounds as sole terminal electron acceptors, including Fe3+, nitrate (NO3-), nitrite (NO2-), manganese oxide (Mn4+), sulfite (SO3(2-)), thiosulfate (S2O3(2-)), trimethylamine N-oxide, and fumarate. A broad spectrum of mutants deficient in anaerobic growth on one or more electron acceptors was identified. Nine of the 62 mutants (designated Fer mutants) were deficient only in anaerobic growth on Fe3+ and retained the ability to grow on all other electron acceptors. These results suggest that S. putrefaciens expresses at least one terminal Fe3+ reductase that is distinct from other terminal reductases coupled to anaerobic growth. The nine Fer mutants were conjugally mated with an S. putrefaciens genomic library harbored in Escherichia coli S17-1. Complemented S. putrefaciens transconjugants were identified by the acquired ability to grow anaerobically on Fe3+ as the sole terminal electron acceptor. All recombinant cosmids that conferred the Fer+ phenotype appeared to carry a common internal region.     

Melanin production and use as a soluble electron shuttle for Fe(III) oxide reduction and as a terminal electron acceptor by Shewanella algae BrY

Dissimilatory metal-reducing bacteria (DMRB) utilize numerous compounds as terminal electron acceptors, including insoluble iron oxides. The mechanism(s) of insoluble-mineral reduction by DMRB is not well understood. Here we report that extracellular melanin is produced by Shewanella algae BrY. The extracted melanin served as the sole terminal electron acceptor. Upon reduction the reduced, soluble melanin reduced insoluble hydrous ferric oxide in the absence of bacteria, thus demonstrating that melanin produced by S. algae BrY is a soluble Fe(III)-reducing compound. In the presence of bacteria, melanin acted as an electron conduit to Fe(III) minerals and increased Fe(III) mineral reduction rates. Growth of S. algae BrY occurred in anaerobic minimal medium supplemented with melanin extracted from previously grown aerobic cultures of S. algae BrY. Melanin produced by S. algae BrY imparts increased versatility to this organism as a soluble Fe(III) reductant, an electron conduit for iron mineral reduction, and a sole terminal electron acceptor that supports growth.     

Melanin Production and Use as a Soluble Electron Shuttle for Fe(III) Oxide Reduction and as a Terminal Electron Acceptor by Shewanella algae BrY

Dissimilatory metal-reducing bacteria (DMRB) utilize numerous compounds as terminal electron acceptors, including insoluble iron oxides. The mechanism(s) of insoluble-mineral reduction by DMRB is not well understood. Here we report that extracellular melanin is produced by Shewanella algae BrY. The extracted melanin served as the sole terminal electron acceptor. Upon reduction the reduced, soluble melanin reduced insoluble hydrous ferric oxide in the absence of bacteria, thus demonstrating that melanin produced by S. algae BrY is a soluble Fe(III)-reducing compound. In the presence of bacteria, melanin acted as an electron conduit to Fe(III) minerals and increased Fe(III) mineral reduction rates. Growth of S. algae BrY occurred in anaerobic minimal medium supplemented with melanin extracted from previously grown aerobic cultures of S. algae BrY. Melanin produced by S. algae BrY imparts increased versatility to this organism as a soluble Fe(III) reductant, an electron conduit for iron mineral reduction, and a sole terminal electron acceptor that supports growth.     

Organic matter mineralization with the reduction of ferric iron: A review

A review of the literature indicates that numerous microorganisms can reduce ferric iron during the metabolism of organic matter. In most cases, the reduction of ferric iron appears to be enzymatically catalyzed and, in some instances, may be coupled to an electron transport chain that could generate ATP. However, the physiology and biochemistry of ferric iron reduction are poorly understood. In pure culture, ferric iron‐reducing organisms metabolize fermentable substrates, such as glucose, primarily to typical fermentation products, and transfer only a minor portion of the electron equivalents in the fermentable substrates to ferric iron. However, fermentation products, especially hydrogen and acetate, may be important electron donors for ferric iron reduction in natural environments. The ability of some organisms to couple the oxidation of fermentation products to the reduction of ferric iron means that it is possible for a food chain of iron‐reducing organisms to completely mineralize nonrecalcitrant organic matter with ferric iron as the sole electron acceptor. The rate and extent of ferric iron reduction depend on the forms of ferric iron that are available. Most of the ferric iron in sediments is resistant to microbial reduction. Ferric iron‐reducing organisms can exclude sulfate reduction and methane production from the zone of ferric iron reduction in sediments by outcompeting sulfate‐reducing and methanogenic food chains for organic matter when ferric iron is available as amorphic ferric oxyhydroxide. There are few quantitative estimates of the rates of ferric iron reduction in natural environments, but there is evidence that ferric iron reduction can be an important pathway for organic matter decomposition in some environments. There is a strong need for further study on all aspects of microbial reduction of ferric iron.     

Iron Corrosion Induced by Nonhydrogenotrophic Nitrate-Reducing Prolixibacter sp. Strain MIC1-1

Microbiologically influenced corrosion (MIC) of metallic materials imposes a heavy economic burden. The mechanism of MIC of metallic iron (Fe⁰) under anaerobic conditions is usually explained as the consumption of cathodic hydrogen by hydrogenotrophic microorganisms that accelerates anodic Fe⁰ oxidation. In this study, we describe Fe⁰ corrosion induced by a nonhydrogenotrophic nitrate-reducing bacterium called MIC1-1, which was isolated from a crude-oil sample collected at an oil well in Akita, Japan. This strain requires specific electron donor-acceptor combinations and an organic carbon source to grow. For example, the strain grew anaerobically on nitrate as a sole electron acceptor with pyruvate as a carbon source and Fe⁰ as the sole electron donor. In addition, ferrous ion and l-cysteine served as electron donors, whereas molecular hydrogen did not. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MIC1-1 was a member of the genus Prolixibacter in the order Bacteroidales. Thus, Prolixibacter sp. strain MIC1-1 is the first Fe⁰-corroding representative belonging to the phylum Bacteroidetes. Under anaerobic conditions, Prolixibacter sp. MIC1-1 corroded Fe⁰ concomitantly with nitrate reduction, and the amount of iron dissolved by the strain was six times higher than that in an aseptic control. Scanning electron microscopy analyses revealed that microscopic crystals of FePO₄ developed on the surface of the Fe⁰ foils, and a layer of FeCO₃ covered the FePO₄ crystals. We propose that cells of Prolixibacter sp. MIC1-1 accept electrons directly from Fe⁰ to reduce nitrate.     

Shewanella putrefaciens adhesion and biofilm formation on food processing surfaces

Laboratory model systems were developed for studying Shewanella putrefaciens adhesion and biofilm formation under batch and flow conditions. S. putrefaciens plays a major role in food spoilage and may cause microbially induced corrosion on steel surfaces. S. putrefaciens bacteria suspended in buffer adhered readily to stainless steel surfaces. Maximum numbers of adherent bacteria per square centimeter were reached in 8 h at 25 degrees C and reflected the cell density in suspension. Numbers of adhering bacteria from a suspension containing 10(8) CFU/ml were much lower in a laminar flow system (modified Robbins device) (reaching 10(2) CFU/cm(2)) than in a batch system (reaching 10(7) CFU/cm(2)), and maximum numbers were reached after 24 h. When nutrients were supplied, S. putrefaciens grew in biofilms with layers of bacteria. The rate of biofilm formation and the thickness of the film were not dependent on the availability of carbohydrate (lactate or glucose) or on iron starvation. The number of S. putrefaciens bacteria on the surface was partly influenced by the presence of other bacteria (Pseudomonas fluorescens) which reduced the numbers of S. putrefaciens bacteria in the biofilm. Numbers of bacteria on the surface must be quantified to evaluate the influence of environmental factors on adhesion and biofilm formation. We used a combination of fluorescence microscopy (4',6'-diamidino-2-phenylindole staining and in situ hybridization, for mixed-culture studies), ultrasonic removal of bacteria from surfaces, and indirect conductometry and found this combination sufficient to quantify bacteria on surfaces.     

Anaerobic electron acceptor chemotaxis in Shewanella putrefaciens.

Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.     

Anaerobic electron acceptor chemotaxis in Shewanella putrefaciens

Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.     

Microbial Iron Respiration Can Protect Steel from Corrosion

Microbiologically influenced corrosion (MC) of steel has been attributed to the activity of biofilms that include anaerobic microorganisms such as iron-respiring bacteria, yet the mechanisms by which these organisms influence corrosion have been unclear. To study this process, we generated mutants of the iron-respiring bacterium Shewanella oneidensis strain MR-1 that were defective in biofilm formation and/or iron reduction. Electrochemical impedance spectroscopy was used to determine changes in the corrosion rate and corrosion potential as a function of time for these mutants in comparison to the wild type. Counter to prevailing theories of MC, our results indicate that biofilms comprising iron-respiring bacteria may reduce rather than accelerate the corrosion rate of steel. Corrosion inhibition appears to be due to reduction of ferric ions to ferrous ions and increased consumption of oxygen, both of which are direct consequences of microbial respiration.     

Microbial iron respiration can protect steel from corrosion

Microbiologically influenced corrosion (MC) of steel has been attributed to the activity of biofilms that include anaerobic microorganisms such as iron-respiring bacteria, yet the mechanisms by which these organisms influence corrosion have been unclear. To study this process, we generated mutants of the iron-respiring bacterium Shewanella oneidensis strain MR-1 that were defective in biofilm formation and/or iron reduction. Electrochemical impedance spectroscopy was used to determine changes in the corrosion rate and corrosion potential as a function of time for these mutants in comparison to the wild type. Counter to prevailing theories of MC, our results indicate that biofilms comprising iron-respiring bacteria may reduce rather than accelerate the corrosion rate of steel. Corrosion inhibition appears to be due to reduction of ferric ions to ferrous ions and increased consumption of oxygen, both of which are direct consequences of microbial respiration.     

Role of Thiobacillus ferrooxidans and sulphur (sulphide)-dependent ferric-ion-reducing activity in the oxidation of sulphide minerals

 Thiobacillus ferrooxidans oxidized the sulphide minerals e.g., pyrite, pyrrhotite and copper concentrate under anaerobic conditions in the presence of ferric ion as sole electron acceptor. Copper and iron were solubilized from sulphide ores by the sulphur (sulphide)-dependent ferric-ion oxidoreductase activity. Treatment of resting cells of T. ferrooxidans with 0.5% phenol for 30 min completely destroyed the iron- and copper-solubilizing activity. The above treatment destroyed the sulphur(sulphide)-dependent ferric-ion-reducing activity completely but did not affect the iron-oxidizing activity. The results suggest that sulphur(sulphide)-dependent ferric-ion-reducing activity actively participates in the oxidation of sulphide minerals under anaerobic conditions. The activity of sulphur(sulphide)-dependent ferric ion reduction in the solubilization of iron and copper from the sulphide ores were also observed under aerobic conditions in presence of sodium azide (0.1 μmol), which completely inhibits the iron-oxidizing activity. Received: 23 May 1995/Received revision: 10 October 1995/Accepted: 16 October 1995     

Structure and properties of novel inclusions in Shewanella putrefaciens

Cytoplasmic inclusions surrounded by a bilayer membrane were seen in thin sections. negatively stained and freeze-fractured preparations of Shewanella putrefaciens. Cells harvested from the late exponential and early stationary phase showed a higher number of these vesicles than bacteria isolated from early exponential or late stationary phase. Chemical dyes for polyphosphate or poly-beta-hydroxybutyrate did not stain the material enclosed within these vesicles. Elemental analysis of the material indicated that the content was organic in nature and might be a protein. HPLC analysis of the material showed that it was probably not a carbon source, nor an electron acceptor used by S. putrefaciens.     

Dissimilatory Fe(III) and Mn(IV) Reduction by Shewanella putrefaciens Requires ferE, a Homolog of the pulE (gspE) Type II Protein Secretion Gene

Shewanella putrefaciens strain 200 respires anaerobically on a wide range of compounds as the sole terminal electron acceptor, including ferric iron [Fe(III)] and manganese oxide [Mn(IV)]. Previous studies demonstrated that a 23.3-kb S. putrefaciens wild-type DNA fragment conferred metal reduction capability to a set of respiratory mutants with impaired Fe(III) and Mn(IV) reduction activities (T. DiChristina and E. DeLong, J. Bacteriol. 176:1468-1474, 1994). In the present study, the smallest complementing fragment was found to contain one open reading frame (ORF) (ferE) whose translated product displayed 87% sequence similarity to Aeromonas hydrophila ExeE, a member of the PulE (GspE) family of proteins found in type II protein secretion systems. Insertional mutants E726 and E912, constructed by targeted replacement of wild-type ferE with an insertionally inactivated ferE construct, were unable to respire anaerobically on Fe(III) or Mn(IV) yet retained the ability to grow on all other terminal electron acceptors. Nucleotide sequence analysis of regions flanking ferE revealed the presence of one partial and two complete ORFs whose translated products displayed 55 to 70% sequence similarity to the PulD, -F, and -G homologs of type II secretion systems. A contiguous cluster of 12 type II secretion genes (pulC to -N homologs) was found in the unannotated genome sequence of Shewanella oneidensis (formerly S. putrefaciens) MR-1. A 91-kDa heme-containing protein involved in Fe(III) reduction was present in the peripheral proteins loosely attached to the outside face of the outer membrane of the wild-type and complemented (Fer+) B31 transconjugates yet was missing from this location in Fer mutants E912 and B31 and in uncomplemented (Fer-) B31 transconjugates. Membrane fractionation studies with the wild-type strain supported this finding: the 91-kDa heme-containing protein was detected with the outer membrane fraction and not with the inner membrane or soluble fraction. These findings provide the first genetic evidence linking dissimilatory metal reduction to type II protein secretion and provide additional biochemical evidence supporting outer membrane localization of S. putrefaciens proteins involved in anaerobic respiration on Fe(III) and Mn(IV).     

Design and application of a rapid screening technique for isolation of selenite reduction-deficient mutants of Shewanella putrefaciens

A rapid screening technique for isolation of selenite (Se(IV)) reduction-deficient (Ser) mutants was developed and used to identify four Ser mutants of Shewanella putrefaciens. Two Ser mutants were unable to grow anaerobically on fumarate, nitrate or nitrite. Two other Ser mutants were unable to grow anaerobically on all compounds tested as sole terminal electron acceptor. Previously isolated Mn(IV) reduction-deficient mutants displayed Ser-positive phenotypes and reduced Se(IV) at wild-type rates, while two of nine Fe(III) reduction-deficient mutants displayed Ser-negative phenotypes and reduced Se(IV) at low rates. This study provides the first reported method for isolation of Ser mutants and demonstrates that Se(IV) reduction by S. putrefaciens is respiratory chain-linked.     

一种新的异育银鲫病原———腐败希瓦氏菌

【目的】江苏盐城一家养殖场的异育银鲫暴发疾病,通过对病原进行研究,旨在为该病的防治提供理论依据和参考。【方法】从病鱼体表病灶和内脏中分离出优势菌株,经人工感染试验证实为病原菌。采用传统的形态、生理生化表型鉴定与16S rDNA序列分析相结合的方法确定菌株的分类地位。运用K-B琼脂法对病原菌株进行药物敏感性测定。【结果】综合菌株形态、生理生化表型以及16S rDNA序列分析的结果,确定该分离株为腐败希瓦氏菌(Shewanella putrefaciens)。回接感染试验证实腐败希瓦氏菌即是导致此次异育银鲫发病死亡的致病原,其半数致死量(LD50)为2.1×103cfu/g。该株腐败希瓦氏菌对吡哌酸、萘啶酸、氟哌酸、氟啶酸、氟苯尼考、利福平、美满霉素、氟罗沙星、恩诺沙星、复达欣、菌必治、先锋Ⅳ、罗红霉素和左氟沙星等抗生素敏感。【结论】首次报道了异育银鲫一种新的病原,说明腐败希瓦氏菌作为一种潜在的新病原也可能会对异育银鲫的养殖造成威胁。