Adaptation to copper stress influences biofilm formation in Alteromonas macleodii.

An Alteromonas macleodii strain was isolated from copper-containing coupons incubated in surface seawater (Key West, FL, USA). In addition to the original isolate, a copper-adapted mutant was created and maintained with 0.78 mM Cu2+. Biofilm formation was compared between the two strains under copper-amended and low-nutrient conditions. Biofilm formation was significantly increased in the original isolate under copper amendment, while biofilm formation was significantly higher in the mutant under low-nutrient conditions. Biofilm expression profiles of diguanylate cyclase (DGC) genes, as well as genes involved in secretion, differed between the strains. Comparative genomic analysis demonstrated that both strains possessed a large number of gene attachment harboring cyclic di-GMP synthesis and/or degradation domains. One of the DGC genes, induced at very high levels in the mutant, possessed a degradation domain in the original isolate that was lacking in the mutant. The genetic and transcriptional mechanisms contributing to biofilm formation are discussed.

Draft Genome Sequences of Four Alteromonas macleodii Strains Isolated from Copper Coupons and Grown Long-Term at Elevated Copper Levels.

Alteromonas macleodii is a marine bacterium involved in the early stages of biofouling on ship hulls treated with copper as an antifouling agent. We report here the draft genome sequences of an A. macleodii strain isolated from copper coupons and three laboratory mutants grown long-term at elevated copper levels.

Manganese deposition in drinking water distribution systems.

This study provides a physicochemical assessment of manganese deposits on brass and lead components from two fully operational drinking water distributions systems. One of the systems was maintained with chlorine; the other, with secondary chloramine disinfection. Synchrotron-based in-situ micro X-ray adsorption near edge structure was used to assess the mineralogy. In-situ micro X-ray fluorescence mapping was used to demonstrate the spatial relationships between manganese and potentially toxic adsorbed metal ions. The Mn deposits ranged in thickness from 0.01 to 400 µm. They were composed primarily of Mn oxides/oxhydroxides, birnessite (Mn(3+) and Mn(4+)) and hollandite (Mn(2+) and Mn(4+)), and a Mn silicate, braunite (Mn(2+) and Mn(4+)), in varying proportions. Iron, chromium, and strontium, in addition to the alloying elements lead and copper, were co-located within manganese deposits. With the exception of iron, all are related to specific health issues and are of concern to the U.S. Environmental Protection Agency (U.S. EPA). The specific properties of Mn deposits, i.e., adsorption of metals ions, oxidation of metal ions and resuspension are discussed with respect to their influence on drinking water quality.

Differences in Physical and Biochemical Properties of Thermus scotoductus SA-01 Cultured with Dielectric or Convection Heating.

A thermophile, Thermus scotoductus SA-01, was cultured within a constant-temperature (65°C) microwave (MW) digester to determine if MW-specific effects influenced the growth and physiology of the organism. As a control, T. scotoductus cells were also cultured using convection heating at the same temperature as the MW studies. Cell growth was analyzed by optical density (OD) measurements, and cell morphologies were characterized using electron microscopy imaging (scanning electron microscopy [SEM] and transmission electron microscopy [TEM]), dynamic light scattering (DLS), and atomic force microscopy (AFM). Biophysical properties (i.e., turgor pressure) were also calculated with AFM, and biochemical compositions (i.e., proteins, nucleic acids, fatty acids) were analyzed by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the fatty acid methyl esters extracted from cell membranes. Here we report successful cultivation of a thermophile with only dielectric heating. Under the MW conditions for growth, cell walls remained intact and there were no indications of membrane damage or cell leakage. Results from these studies also demonstrated that T. scotoductus cells grown with MW heating exhibited accelerated growth rates in addition to altered cell morphologies and biochemical compositions compared with oven-grown cells.

Mini-review: the morphology, mineralogy and microbiology of accumulated iron corrosion products.

Despite obvious differences in morphology, substratum chemistry and the electrolyte in which they form, accumulations of iron corrosion products have the following characteristics in common: stratification of iron oxides/hydroxides with a preponderance of α-FeOOH (goethite) and accumulation of metals. Bacteria, particularly iron-oxidizing and sulfate-reducing bacteria have been identified in some accumulations. Both biotic and abiotic mechanisms have been used to rationalize observations for particular sets of environmental data. This review is the first to compare observations and interpretations.

Issues for storing plant-based alternative fuels in marine environments.

Two coastal seawaters (Key West, FL, USA and the Persian Gulf, Bahrain, representing oligotrophic and eutrophic environments, respectively) were used to evaluate potential biodegradation and corrosion problems during exposure to alternative and conventional fuels. Uncoated carbon steel was exposed at the fuel/seawater interface and polarization resistance was monitored. Under typical marine storage conditions, dioxygen in natural seawater exposed to fuel and carbon steel was reduced to <0.1parts-per-million within 2d due to consumption by corrosion reactions and aerobic microbial respiration. Sulfides, produced by anaerobic sulfate-reducing bacteria, and chlorides were co-located in corrosion products. Transient dioxygen influenced both metabolic degradation pathways and resulting metabolites. Catechols, indicative of aerobic biodegradation, persisted after 90d exposures. Detection of catechols suggested that initial exposure to dioxygen resulted in the formation of aerobic metabolites that exacerbated subsequent corrosion processes.

Iron cycling at corroding carbon steel surfaces.

Surfaces of carbon steel (CS) exposed to mixed cultures of iron-oxidizing bacteria (FeOB) and dissimilatory iron-reducing bacteria (FeRB) in seawater media under aerobic conditions were rougher than surfaces of CS exposed to pure cultures of either type of microorganism. The roughened surface, demonstrated by profilometry, is an indication of loss of metal from the surface. In the presence of CS, aerobically grown FeOB produced tight, twisted helical stalks encrusted with iron oxides. When CS was exposed anaerobically in the presence of FeRB, some surface oxides were removed. However, when the same FeOB and FeRB were grown together in an aerobic medium, FeOB stalks were less encrusted with iron oxides and appeared less tightly coiled. These observations suggest that iron oxides on the stalks were reduced and solubilized by the FeRB. Roughened surfaces of CS and denuded stalks were replicated with culture combinations of different species of FeOB and FeRB under three experimental conditions. Measurements of electrochemical polarization resistance established different rates of corrosion of CS in aerobic and anaerobic media, but could not differentiate rate differences between sterile controls and inoculated exposures for a given bulk concentration of dissolved oxygen. Similarly, total iron in the electrolyte could not be used to differentiate treatments. The experiments demonstrate the potential for iron cycling (oxidation and reduction) on corroding CS in aerobic seawater media.

Strontium concentrations in corrosion products from residential drinking water distribution systems.

The United States Environmental Protection Agency (US EPA) will require some U.S. drinking water distribution systems (DWDS) to monitor nonradioactive strontium (Sr(2+)) in drinking water in 2013. Iron corrosion products from four DWDS were examined to assess the potential for Sr(2+) binding and release. Average Sr(2+) concentrations in the outermost layer of the corrosion products ranged from 3 to 54 mg kg(-1) and the Sr(2+) drinking water concentrations were all ≤0.3 mg L(-1). Micro-X-ray adsorption near edge structure spectroscopy and linear combination fitting determined that Sr(2+) was principally associated with CaCO3. Sr(2+) was also detected as a surface complex associated with α-FeOOH. Iron particulates deposited on a filter inside a home had an average Sr(2+) concentration of 40.3 mg kg(-1) and the associated drinking water at a tap was 210 µg L(-1). The data suggest that elevated Sr(2+) concentrations may be associated with iron corrosion products that, if disturbed, could increase Sr(2+) concentrations above the 0.3 µg L(-1) US EPA reporting threshold. Disassociation of very small particulates could result in drinking water Sr(2+) concentrations that exceed the US EPA health reference limit (4.20 mg kg(-1) body weight).

Shewanella frigidimarina microbial fuel cells and the influence of divalent cations on current output.

The genes involved in the proposed pathway for Shewanella extracellular electron transfer (EET) are highly conserved. While extensive studies involving EET from a fresh water Shewanella microbe (S. oneidensis MR-1) to soluble and insoluble electron acceptors have been published, only a few reports have examined EET from marine strains of Shewanella. Thus, Shewanella frigidimarina (an isolate from Antarctic Sea ice) was used within miniature microbial fuel cells (mini-MFC) to evaluate potential power output. During the course of this study several distinct differences were observed between S. oneidensis MR-1 and S. frigidimarina under comparable conditions. The maximum power density with S. frigidimarina was observed when the anolyte was half-strength marine broth (1/2 MB) (0.28 µW/cm(2)) compared to Luria-Bertani (LB) (0.07 µW/cm(2)) or a defined growth minimal medium (MM) (0.02 µW/cm(2)). The systematic modification of S. frigidimarina cultured in 1/2 MB and LB with divalent cations shows that a maximum current output can be generated independent of internal ionic ohmic losses and the presence of external mediators.

Sulphide production and corrosion in seawaters during exposure to FAME diesel.

Experiments were designed to evaluate the corrosion-related consequences of storing/transporting fatty acid methyl ester (FAME) alternative diesel fuel in contact with natural seawater. Coastal Key West, FL (KW), and Persian Gulf (PG) seawaters, representing an oligotrophic and a more organic- and inorganic mineral-rich environment, respectively, were used in 60 day incubations with unprotected carbon steel. The original microflora of the two seawaters were similar with respect to major taxonomic groups but with markedly different species. After exposure to FAME diesel, the microflora of the waters changed substantially, with Clostridiales (Firmicutes) becoming dominant in both. Despite low numbers of sulphate-reducing bacteria in the original waters and after FAME diesel exposure, sulphide levels and corrosion increased markedly due to microbial sulphide production. Corrosion morphology was in the form of isolated pits surrounded by an intact, passive surface with the deepest pits associated with the fuel/seawater interface in the KW exposure. In the presence of FAME diesel, the highest corrosion rates measured by linear polarization occurred in the KW exposure correlating with significantly higher concentrations of sulphur and chlorine (presumed sulphide and chloride, respectively) in the corrosion products.

Neutrophilic iron-oxidizing "zetaproteobacteria" and mild steel corrosion in nearshore marine environments.

Microbiologically influenced corrosion (MIC) of mild steel in seawater is an expensive and enduring problem. Little attention has been paid to the role of neutrophilic, lithotrophic, iron-oxidizing bacteria (FeOB) in MIC. The goal of this study was to determine if marine FeOB related to Mariprofundus are involved in this process. To examine this, field incubations and laboratory microcosm experiments were conducted. Mild steel samples incubated in nearshore environments were colonized by marine FeOB, as evidenced by the presence of helical iron-encrusted stalks diagnostic of the FeOB Mariprofundus ferrooxydans, a member of the candidate class "Zetaproteobacteria." Furthermore, Mariprofundus-like cells were enriched from MIC biofilms. The presence of Zetaproteobacteria was confirmed using a Zetaproteobacteria-specific small-subunit (SSU) rRNA gene primer set to amplify sequences related to M. ferrooxydans from both enrichments and in situ samples of MIC biofilms. Temporal in situ incubation studies showed a qualitative increase in stalk distribution on mild steel, suggesting progressive colonization by stalk-forming FeOB. We also isolated a novel FeOB, designated Mariprofundus sp. strain GSB2, from an iron oxide mat in a salt marsh. Strain GSB2 enhanced uniform corrosion from mild steel in laboratory microcosm experiments conducted over 4 days. Iron concentrations (including precipitates) in the medium were used as a measure of corrosion. The corrosion in biotic samples (7.4 ± 0.1 mM) was significantly higher than that in abiotic controls (5.0 ± 0.1 mM). These results have important implications for the role of FeOB in corrosion of steel in nearshore and estuarine environments. In addition, this work shows that the global distribution of Zetaproteobacteria is far greater than previously thought.

The utility of Shewanella japonica for microbial fuel cells.

Shewanella-containing microbial fuel cells (MFCs) typically use the fresh water wild-type strain Shewanella oneidensis MR-1 due to its metabolic diversity and facultative oxidant tolerance. However, S. oneidensis MR-1 is not capable of metabolizing polysaccharides for extracellular electron transfer. The applicability of Shewanella japonica (an agar-lytic Shewanella strain) for power applications was analyzed using a diverse array of carbon sources for current generation from MFCs, cellular physiological responses at an electrode surface, biofilm formation, and the presence of soluble extracellular mediators for electron transfer to carbon electrodes. Critically, air-exposed S. japonica utilizes biosynthesized extracellular mediators for electron transfer to carbon electrodes with sucrose as the sole carbon source.

An assessment of alternative diesel fuels: microbiological contamination and corrosion under storage conditions.

Experiments were designed to evaluate the nature and extent of microbial contamination and the potential for microbiologically influenced corrosion of alloys exposed in a conventional high sulfur diesel (L100) and alternative fuels, including 100% biodiesel (B100), ultra-low sulfur diesel (ULSD) and blends of ULSD and B100 (B5 and B20). In experiments with additions of distilled water, all fuels supported biofilm formation. Changes in the water pH did not correlate with observations related to corrosion. In all exposures, aluminum 5052 was susceptible to pitting while stainless steel 304L exhibited passive behavior. Carbon steel exhibited uniform corrosion in ULSD and L100, and passive behavior in B5, B20, and B100.

Simultaneous analysis of physiological and electrical output changes in an operating microbial fuel cell with Shewanella oneidensis.

Changes in metabolism and cellular physiology of facultative anaerobes during oxygen exposure can be substantial, but little is known about how these changes connect with electrical current output from an operating microbial fuel cell (MFC). A high-throughput voltage based screening assay (VBSA) was used to correlate current output from a MFC containing Shewanella oneidensis MR-1 to carbon source (glucose or lactate) utilization, culture conditions, and biofilm coverage over 250 h. Lactate induced an immediate current response from S. oneidensis MR-1, with both air-exposed and anaerobic anodes throughout the duration of the experiments. Glucose was initially utilized for current output by MR-1 when cultured and maintained in the presence of air. However, after repeated additions of glucose, the current output from the MFC decreased substantially while viable planktonic cell counts and biofilm coverage remained constant suggesting that extracellular electron transfer pathways were being inhibited. Shewanella maintained under an anaerobic atmosphere did not utilize glucose consistent with literature precedents. Operation of the VBSA permitted data collection from nine simultaneous S. oneidensis MR-1 MFC experiments in which each experiment was able to demonstrate organic carbon source utilization and oxygen dependent biofilm formation on a carbon electrode. These data provide the first direct evidence of complex cellular responses to electron donor and oxygen tension by Shewanella in an operating MFC at select time points.

An evaluation of carbon steel corrosion under stagnant seawater conditions.

Corrosion of 1020 carbon steel coupons in natural seawater over a 1-year period was more aggressive under strictly anaerobic stagnant conditions than under aerobic stagnant conditions as measured by weight loss and instantaneous corrosion rate (polarization resistance). Under oxygenated conditions, a two-tiered oxide layer of lepidocrocite/goethite formed. The inner layer was extremely tenacious and resistant to acid cleaning. Under anaerobic conditions, the corrosion product was initially a non-tenacious sulphur-rich corrosion product, mackinawite, with enmeshed bacteria. As more sulphide was produced the mackinawite was transformed to pyrrhotite. In both aerobic and anaerobic exposures, corrosion was more aggressive on horizontally oriented coupons compared to vertically oriented samples.

In situ measurement of Fe(III) reduction activity of Geobacter pelophilus by simultaneous in situ RT-PCR and XPS analysis.

Geobacter pelophilus is capable of dissimilatory Fe(III)-reduction on solid phase Fe(III)-oxides by means of surface attachment and direct electron transport to Fe(III), in part mediated by outer membrane c-type cytochromes. A study was undertaken to characterize surface colonization patterns, gene expression, and mineral transformation by this organism. The gene ferA (Geobacter sulfurreducens outer membrane Fe(III) reductase cytochrome c) was used as a target for PCR based molecular detection methods for visualizing G. pelophilus surface colonization. Protein extracts were prepared from solid-phase cultures, and cytochrome c content assessed. Mineral transformations were followed by X-ray photoelectron spectroscopy (XPS). Results of in situ (IS) RT-PCR experiments demonstrate that G. pelophilus attaches and grows at ferrihydrite mineral surfaces. Fluorescently-labeled cells were observed after IS-RT-PCR experiments, suggesting that G. pelophilus contains a cytochrome c sequence similar to ferA in G. sulfurreducens which is expressed in the presence of ferrihydrite. Protein extracts possessed high mass c-type cytochromes of similar size to those found in G. sulfurreducens. In addition, unique high-mass c-type cytochromes were also detected. XPS analysis demonstrated mineral transformation to occur, mediated by the surface associated population. This study demonstrates that G. pelophilus attaches to Fe(III)-oxide surfaces, reduces the Fe(III) oxides at the surface, produces c-type cytochromes under these growth conditions, and expresses cytochrome c-encoding genes as measured by in situ molecular detection techniques.

An evaluation of microbial growth and corrosion of 316L SS in glycol/seawater mixtures.

Glycol/seawater mixtures containing > 50% glycol inhibit corrosion of 316L stainless steel and do not support bacterial growth. The results indicate bacteria are able to use low concentrations of glycol (10%) as a growth medium, but bacterial growth decreased with increasing glycol concentration. Pitting potential, determined by anodic polarization, was used to evaluate susceptibility of 316L SS to corrosion in seawater-contaminated glycol. Mixture containing a minimum concentration of 50% propylene glycol-based coolant inhibited pitting corrosion. A slightly higher minimum concentration (55%) was needed for corrosion protection in ethylene glycol mixtures.

Electron energy loss spectroscopy techniques for the study of microbial chromium(VI) reduction.

Electron energy loss spectroscopy (EELS) techniques were used to determine oxidation state, at high spatial resolution, of chromium associated with the metal-reducing bacteria, Shewanella oneidensis, in anaerobic cultures containing Cr(VI)O4(2-). These techniques were applied to fixed cells examined in thin section by conventional transmission electron microscopy (TEM) as well as unfixed, hydrated bacteria examined by environmental cell (EC)-TEM. Two distinct populations of bacteria were observed by TEM: bacteria exhibiting low image contrast and bacteria exhibiting high contrast in their cell membrane (or boundary) structure which was often encrusted with high-contrast precipitates. Measurements by EELS demonstrated that cell boundaries became saturated with low concentrations of Cr and the precipitates encrusting bacterial cells contained a reduced form of Cr in oxidation state + 3 or lower.

In Situ Environmental Cell-Transmission Electron Microscopy Study of Microbial Reduction of Chromium(VI) Using Electron Energy Loss Spectroscopy.

Reduction of Cr(VI) by the bacterium, Shewanella oneidensis (previously classified Shewanella putrefaciens strain MR-1), was studied by absorption spectrophotometry and in situ, environmental cell-transmission electron microscopy (EC-TEM) coupled with electron energy loss spectroscopy (EELS). Bacteria from rinsed cultures were placed directly in the environmental cell of the transmission electron microscope and examined under 100 Torr pressure. Bright field EC-TEM images show two distinct populations of S. oneidensis in incubated cultures containing Cr(VI)O2- 4: those that exhibit low image contrast and heavily precipitate-encrusted cells exhibiting high image contrast. Several EELS techniques were applied to determine the oxidation state of Cr associated with encrusted cells. The encrusted cells are shown to contain a reduced form of Cr in oxidation state +3 or lower. These results demonstrate the capability to determine the chemistry and valence state of reduction products associated with unfixed, hydrated bacteria in an environmental cell transmission electron microscope.