Imaginary admittance and charge transfer resistance correlate to the physiological status of Shewanella oneidensis cultures in real time.

Monitoring microbial activity is essential for industrial and environmental applications to proceed efficiently. To minimize time and labor-intensive monitoring, a new paradigm is required for in-situ, real time analysis. Since bioconversion of organics is accomplished by microorganisms through the oxidation of feedstocks linked to the reduction of electron acceptors, microorganisms can be viewed as electrochemical catalysts. In this respect, cell membranes have an electrical potential, which is analogous to a conventional capacitor and linked dynamically to cellular activity. Here we demonstrate the use of electrochemical impedance spectrometry (EIS) and cyclic voltammetry (CV) for monitoring microbial metabolic activity in real time, in-situ. The effect of organic electron donors as a function of concentration to the physiological status of strains of Shewanella oneidensis was determined. In this study, the pyomelanin overproducer (S. oneidensis ΔhmgA) and the pyomelanin deficient mutant (S. oneidensis ΔmelA) were chosen due to different surface electrochemical characteristics along with differences in oxygen utilization efficiency. CV, relative admittance, phase shift and permittivity changed with growth status and correlated with electron flow from organic carbon sources and terminal electron acceptor availability. This work offers a novel and inexpensive approach to real time monitoring with the advantage of abundant data.

The Impact of Storms on Legionella pneumophila in Cooling Tower Water, Implications for Human Health.

At the U.S. Department of Energy's Savannah River Site (SRS) in Aiken, SC, cooling tower water is routinely monitored for Legionella pneumophila concentrations using a direct fluorescent antibody (DFA) technique. Historically, 25-30 operating SRS cooling towers have varying concentrations of Legionella in all seasons of the year, with patterns that are unpredictable. Legionellosis, or Legionnaires' disease (LD), is a pneumonia caused by Legionella bacteria that thrive both in man-made water distribution systems and natural surface waters including lakes, streams, and wet soil. Legionnaires' disease is typically contracted by inhaling L. pneumophila, most often in aerosolized mists that contain the bacteria. At the SRS, L. pneumophila is typically found in cooling towers ranging from non-detectable up to 108 cells/L in cooling tower water systems. Extreme weather conditions contributed to elevations in L. pneumophila to 107-108 cells/L in SRS cooling tower water systems in July-August 2017. L. pneumophila concentrations in Cooling Tower 785-A/2A located in SRS A-Area, stayed in the 108 cells/L range despite biocide addition. During this time, other SRS cooling towers did not demonstrate this L. pneumophila increase. No significant difference was observed in the mean L. pneumophila mean concentrations for the towers (p < 0.05). There was a significant variance observed in the 285-2A/A Tower L. pneumophila results (p < 0.05). Looking to see if we could find "effects" led to model development by analyzing 13 months of water chemistry and microbial data for the main factors influencing the L. pneumophila concentrations in five cooling towers for this year. It indicated chlorine and dissolved oxygen had a significant impact (p < 0.0002) on cooling tower 785A/2A. Thus, while the variation in the log count data for the A-area tower is statistically greater than that of the other four towers, the average of the log count data for the A-Area tower was in line with that of the other towers. It was also observed that the location of 785A/2A and basin resulted in more debris entering the system during storm events. Our results suggest that future analyses should evaluate the impact of environmental conditions and cooling tower design on L. pneumophila water concentrations and human health.

Convenient non-invasive electrochemical techniques to monitor microbial processes: current state and perspectives.

Real-time electrochemical monitoring in bioprocesses is an improvement over existing systems because it is versatile and provides more information to the user than periodic measurements of cell density or metabolic activity. Real-time electrochemical monitoring provides the ability to monitor the physiological status of actively growing cells related to electron transfer activity and potential changes in the proton gradient of the cells. Voltammetric and amperometric techniques offer opportunities to monitor electron transfer reactions when electrogenic microbes are used in microbial fuel cells or bioelectrochemical synthesis. Impedance techniques provide the ability to monitor the physiological status of a wide range of microorganisms in conventional bioprocesses. Impedance techniques involve scanning a range of frequencies to define physiological activity in terms of equivalent electrical circuits, thereby enabling the use of computer modeling to evaluate specific growth parameters. Electrochemical monitoring of microbial activity has applications throughout the biotechnology industry for generating real-time data and offers the potential for automated process controls for specific bioprocesses.

In-situ electrochemical analysis of microbial activity.

Microbes have a wide range of metabolic capabilities available that makes them industrially useful organisms. Monitoring these metabolic processes is a crucial component in efficient industrial application. Unfortunately, monitoring these metabolic processes can often be invasive and time consuming and expensive, especially within an anaerobic environment. Electrochemical techniques, such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) offer a non-invasive approach to monitor microbial activity and growth. EIS and CV were used to monitor Clostridium phytofermentans, an anaerobic and endospore-forming bacterium. C. phytofermentans ferments a wide range of sugars into hydrogen, acetate, and ethanol as fermentation by-products. For this study, both traditional microbiological and electrochemical techniques were used to monitor the growth of C. phytofermentans and the formation of fermentation products. An irreversible reduction peak was observed using CV beginning at mid-logarithmic phase of growth. This peak was associated with C. phytofermentans and not the spent medium and was indicative of a decrease in carbon and energy sources to the cells. Additionally, EIS analysis during growth provided information related to increased charge transfer resistance of the culture also as a function of carbon and energy source depletion. Results demonstrate that CV and EIS are useful tools in the monitoring the physiological status of bioprocesses.

Review of concrete biodeterioration in relation to nuclear waste.

Storage of radioactive waste in concrete structures is a means of containing wastes and related radionuclides generated from nuclear operations in many countries. Previous efforts related to microbial impacts on concrete structures that are used to contain radioactive waste showed that microbial activity can play a significant role in the process of concrete degradation and ultimately structural deterioration. This literature review examines the research in this field and is focused on specific parameters that are applicable to modeling and prediction of the fate of concrete structures used to store or dispose of radioactive waste. Rates of concrete biodegradation vary with the environmental conditions, illustrating a need to understand the bioavailability of key compounds involved in microbial activity. Specific parameters require pH and osmotic pressure to be within a certain range to allow for microbial growth as well as the availability and abundance of energy sources such as components involved in sulfur, iron and nitrogen oxidation. Carbon flow and availability are also factors to consider in predicting concrete biodegradation. The microbial contribution to degradation of the concrete structures containing radioactive waste is a constant possibility. The rate and degree of concrete biodegradation is dependent on numerous physical, chemical and biological parameters. Parameters to focus on for modeling activities and possible options for mitigation that would minimize concrete biodegradation are discussed and include key conditions that drive microbial activity on concrete surfaces.

Gamma radiation interacts with melanin to alter its oxidation-reduction potential and results in electric current production.

The presence of melanin pigments in organisms is implicated in radioprotection and in some cases, enhanced growth in the presence of high levels of ionizing radiation. An understanding of this phenomenon will be useful in the design of radioprotective materials. However, the protective mechanism of microbial melanin in ionizing radiation fields has not yet been elucidated. Here we demonstrate through the electrochemical techniques of chronoamperometry, chronopotentiometry and cyclic voltammetry that microbial melanin is continuously oxidized in the presence of gamma radiation. Our findings establish that ionizing radiation interacts with melanin to alter its oxidation-reduction potential. Sustained oxidation resulted in electric current production and was most pronounced in the presence of a reductant, which extended the redox cycling capacity of melanin. This work is the first to establish that gamma radiation alters the oxidation-reduction behavior of melanin, resulting in electric current production. The significance of the work is that it provides the first step in understanding the initial interactions between melanin and ionizing radiation taking place and offers some insight for production of biomimetic radioprotective materials.

The role of 4-hydroxyphenylpyruvate dioxygenase in enhancement of solid-phase electron transfer by Shewanella oneidensis MR-1.

We hypothesized that Shewanella oneidensis MR-1, a model dissimilatory metal-reducing bacterium, could utilize environmentally relevant concentrations of tyrosine to produce pyomelanin for enhanced Fe(III) oxide reduction. Because homogentisate is an intermediate of the tyrosine degradation pathway, and a precursor of a redox-cycling metabolite, pyomelanin, we evaluated the process of homogentisate production by S. oneidensis MR-1, in order to identify the key steps involved in pyomelanin production. We determined that two enzymes involved in this pathway, 4-hydroxyphenylpyruvate dioxygenase and homogentisate 1,2-dioxygenase are responsible for homogentisate production and oxidation, respectively. We used genetic analysis and physiological characterization of MR-1 strains either deficient in or displaying substantially increased pyomelanin production. The relative significance imparted by pyomelanin on solid-phase electron transfer was also addressed using electrochemical techniques, which allowed us to extend the genetic and physiological findings to biogeochemical cycling of metals. Based on our findings, environmental production of pyomelanin from available organic precursors could contribute to the survival of S. oneidensis MR-1 when dissolved oxygen concentrations become low, by providing an increased capacity for solid-phase metal reduction. This study demonstrates the role of organic precursors and their concentrations in pyomelanin production, solid phase metal reduction and biogeochemical cycling of iron.

Pyomelanin is produced by Shewanella algae BrY and affected by exogenous iron.

Melanin production by Shewanella algae BrY occurred during late- and (or) post-exponential growth in lactate basal salts liquid medium supplemented with tyrosine or phenylalanine. The antioxidant ascorbate inhibited melanin production but not production of the melanin precursor homogentisic acid. In the absence of ascorbate, melanin production was inhibited by the 4-hydroxyphenylpyruvate dioxygenase inhibitor sulcotrione and by concentrations of Fe >or= 0.38 mmol L(-1). These data support the hypothesis that pigment production by S. algae BrY was a result of the conversion of tyrosine or phenylalanine to homogentisic acid, which was excreted, auto-oxidized, and self-polymerized to form pyomelanin. Pyomelanin production by S. algae BrY may play an important role in the biogeochemical cycling of Fe in the environment.

In situ uranium stabilization by microbial metabolites.

Microbial melanin production by autochthonous bacteria was explored in this study as a means to increase U immobilization in U contaminated soil. This article demonstrates the application of bacterial physiology and soil ecology for enhanced U immobilization in order to develop an in situ, U bio-immobilization technology. We have demonstrated microbial production of a metal chelating biopolymer, pyomelanin, in U contaminated soil from the Tims Branch area of the Department of Energy (DOE), Savannah River Site (SRS), South Carolina, as a result of tyrosine amendments. Bacterial densities of pyomelanin producers were >10(6) cells per g wet soil. Pyomelanin demonstrated U complexing and mineral binding capacities at pH 4 and 7. In laboratory studies, in the presence of goethite or illite, pyomelanin enhanced U sequestration by these minerals. Tyrosine amended soils in a field test demonstrated increased U sequestration capacity following pyomelanin production up to 13 months after tyrosine treatments.

Identification of marker proteins for Bacillus anthracis using MALDI-TOF MS and ion trap MS/MS after direct extraction or electrophoretic separation.

Direct extraction of bacterial vegetative cells or spores followed by matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI TOF MS) has become popular for bacterial identification, since it is simple to perform and mass spectra are readily interpreted. However, only high-abundance proteins that are of low mass and ionize readily are observed. In the case of B. anthracis spores, small acid-soluble spore proteins (SASPs) have been the most widely studied. Additional information can be obtained using tandem mass spectrometry (MS-MS) to confirm the identity of proteins by sequencing. This is most readily accomplished using ion trap (IT) MS-MS. However, enzymatic digestion of these proteins is needed to generate peptides that are within the mass range of the ion trap. The use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), or other forms of electrophoresis, allows one to focus on specific proteins of interest (e.g. the high mass exosporium glycoproteins BcIA and BcIB) that provide additional species- and strain-specific discrimination.

Electron transfer from Shewanella algae BrY to hydrous ferric oxide is mediated by cell-associated melanin.

Shewanella algae BrY uses insoluble mineral oxides as terminal electron acceptors, but the mechanism of electron transfer from cell surface to mineral surface is not well understood. We tested the hypothesis that cell-associated melanin produced by S. algae BrY serves as an electron conduit for bacterial-mineral reduction. Results from Fourier transform infrared spectroscopy and cell surface hydrophobicity assays indicated that extracellular melanin was associated with the cell surface. With H(2) as electron donor, washed cell suspensions of melanin-coated S. algae BrY reduced hydrous ferric oxide (HFO) 10 times faster than cells without melanin. The addition of melanin (20 microg ml(-1)) to these melanin-free cells increased their HFO reduction rate two-fold. These results suggest that cell-associated melanin acts as an electron conduit for iron mineral reduction by S. algae BrY.

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.