Preparation of a colored conductive paint electrode for electrochemical inactivation of bacteria

In this study we describe the preparation of a colored conductive paint electrode containing In(2)O(3), SnO(2), or TiO(2) for the electrochemical inactivation of marine bacteria. When each colored conductive paint electrode was immersed in seawater containing 10(6) cells/mL for 90 min, marine microbe attachment to the TiO(2)/SnO(2)/Sb electrode surface was minimal. Preparation of electrodes coated with 40% particles is shown to be more cost-effective, and because of their more translucent coatings they can be painted over with bright colors. When a potential of 1.0 V was applied for 30 min to the colored conductive paint electrode (40 wt% TiO(2)/SnO(2)/Sb) in sterile seawater, the survival ratio decreased to 55%. When 1.5 V vs. saturated calomel electrode (SCE) was applied, all attached cells were inactivated. Chlorine was not detected below an applied potential of 1.5 V. A change in pH was not observed in the range of 0 to 1.5 V. This method might be effective for preventing bacterial cell accumulation and the formation of biofilms.     

Electrochemical Prevention of Marine Biofouling with a Carbon-Chloroprene Sheet

A carbon-chloroprene sheet (CCS) electrode was used for the electrochemical disinfection of the marine gram-negative bacterium Vibrio alginolyticus. When the electrode was incubated in seawater containing 10⁵ cells per ml for 90 min, the amount of adsorbed cells was 4.5 × 10³ cells per cm². When a potential of 1.2 V versus a saturated calomel electrode was applied to the CCS for 20 min, 67% of adsorbed cells were killed. This disinfection was due to the direct electrochemical oxidation of cells and not to a change in pH or to the generation of toxic substances, such as chlorine. In a 1-year field experiment, marine biofouling of a CCS-coated cooling pipe caused by attachment of bacteria and invertebrates was considerably reduced by application of a potential of 1.2 V versus a saturated calomel electrode. Since this method requires low potential electrical energy, use of a CCS coating appears to be a suitable method for the clean prevention of marine biofouling.     

Electrochemical prevention of marine biofouling on a novel titanium-nitride-coated plate formed by radio-frequency arc spraying

We have developed a new method for forming titanium-nitride(TiN)-coated plates using radio-frequency arc spraying (RFAS). A TiN coating formed by RFAS has been used for electrochemical prevention of marine biofouling. X-ray diffraction and X-ray photoelectron spectroscopy indicate that a TiN composite film containing Ti was formed on a polyethylene terephthalate plate surface when Ti was sprayed by RFAS under atmospheric pressure. A cyclic voltammogram (scan rate 20 mV/s) of the TiN formed by RFAS revealed no oxidative and reductive peak currents in the range −0.6 V to 1.2 V against a saturated silver/silver chloride (Ag/AgCl) electrode. When a potential of 1.0 V against Ag/AgCl was applied to the electrode in seawater, no dissolved Ti was detected. Changes in pH and the chlorine concentration were not observed in this range. In all, only 4.5% of the Vibrio alginolyticus cells attached to the electrode survived when a potential of 0.8 V against Ag/AgCl was applied in seawater for 30 min. In field experiments, attachment of the organisms to the TiN electrode was inhibited by applying an alternating potential of 1.0 V and −0.6 V against Ag/AgCl. The TiN film can be formed by RFAS on large and intricately shaped surfaces, and it is a practical electrode for the electrochemical prevention of fouling of various marine structures. Received: 17 April 1998 / Received revision: 5 June 1998 / Accepted: 19 June 1998     

Electrochemical disinfection of bacteria in drinking water using activated carbon fibers

A novel electrochemical reactor employing activated carbon fiber (ACF) electrodes was constructed for disinfecting bacteria in drinking water. Escherichia coli adsorbed preferentially onto ACF rather than to carbon-cloth or granular-activated carbon. E. coli cells, which adsorbed onto the ACF, were killed electrochemically when a potential of 0.8 V vs. a saturated calomel electrode (SCE) was applied. Drinking water was passed through the reactor in stop-flow mode: 2mL/min for 12 h, o L/min for 24 h, and 1 mL/min for 6 h. At an applied potential of 0.8 V vs, SCE, viable cell concentration reamined below 30 cells/mL. In the absence of an applied potential, bacteria grew to a maximum concentration of 9.5 x 10(3) cells/mL. After continuous operation at 0.8 V vs. SCE, cells adsorbed onto the ACF could not be observed by scanning electron microscopy. In addition, chlorine in drinking water was completely removed by the reactor. Therefore, clean and efficient inactivation of bacteria in drinking water was successfully performed. (c) 1994 John Wiley & Sons, Inc.     

Electrochemical classification of gram-negative and gram-positive bacteria

Intestinal bacteria were classified as gram-positive or gram-negative by an electrode system with a basal plane pyrolytic graphite electrode and a porous nitrocellulose membrane filter to trap bacteria. When the potential of the graphite electrode was run in the range of 0 to 1.0 V versus the saturated calomel electrode (SCE), gram-positive bacteria gave peak currents at 0.65 to 0.69 V versus the SCE. The peak potentials of gram-negative bacteria were 0.70 to 0.74 V versus the SCE. Gram-negative bacteria and gram-positive bacteria were also classified based on the ratio of the second peak current to the first peak current when the potential cycle was repeated twice. The numbers of cells on the membrane filter were determined from the peak currents. It was found that the peak currents result from the electrochemical oxidation of coenzyme A in the cells of Escherichia coli and Lactobacillus acidophilus.     

Risk factors for fouling biomass: evidence from small vessels in Australia

Abstract

Invasive non-indigenous species (NIS) are a threat to marine biodiversity and marine reliant industries. Recreational vessels are recognised as an important vector of NIS translocation, particularly domestically. This paper reports on a novel application of multilevel modelling and multiple imputation in order to quantify the relationship between biofouling biomass (wet weight) and the vessel-level characteristics of recreational and fishing vessels. It was found that the number of days since the vessel was last cleaned strongly related to the biofouling biomass, yet differed dependent on vessel type. Similarly, the median number of trips undertaken was related to the biofouling biomass, and varied according to the type of antifouling paint (AF) used. No relationship was found between vessel size and biofouling biomass per sample unit. To reduce the spread of NIS, vessel owners should use an AF paint suitable to their vessel’s operational profile, and follow a maintenance schedule according to the paint manufacturer’s specifications.     

Mass spectrometry in demonstrating the site-specific nitration of hen egg white lysozyme by an improved electrochemical method

In producing a method for selective protein nitration, we previously demonstrated the electrochemical nitration of hen egg white lysozyme to be at Tyr23 initially, followed by bisnitration at Tyr20, but with no trisnitration at Tyr53. The nitration site was determined by sequencing a tryptic peptide that included Tyr23 and Tyr20, but possible effects on other regions of the protein were not determined. Moreover, the electrooxidation conditions were harsh, involving an oxidation potential of +1.2V (vs. saturated calomel electrode [SCE]), no added nitrogen source except the lysozyme itself, and long reaction periods with copper flag electrodes. Here we report a gentler procedure using much shorter reaction times with nitrite as the nitration source, a lower potential (+0.85V vs. SCE), and a platinum basket electrode. Intact protein analysis by electrospray Fourier transform ion cyclotron resonance mass spectrometry identified mono- and bisnitration products with mass increases of +45 and +90 Da, respectively, consistent with the substitution of NO(2) for H. In addition, the results revealed that no other covalent change in the protein occurred following electrooxidation. Nozzle skimmer dissociation of the intact mononitrated species localized the modification site to Tyr20 or Tyr23. Matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization time-of-flight analysis of the tryptic peptides of mononitrated lysozyme identified the site of nitration as Tyr23.     

Detection of human leucocytes by cyclic voltammetry and its application to monitoring of allergic reaction

Cyclic voltammetry was applied to the detection of human leucocytes and the monitoring of allergic reactions. A basal plane pyrolytic graphite electrode with attached leucocytes on a porous nitrocellulose membrane filter was employed as a working electrode. An anodic peak current appeared at 0.33 V versus the saturated calomel electrode (SCE) when the potential of the working electrode was scanned in the range of 0-1.0 V versus SCE. This peak current was attributed to the electrochemical oxidation of serotonin. When egg white was added to leucocytes obtained from patients who were allergic to egg, the peak current decreased owing to degranulation of leucocytes leading to serotonin release. The peak current decreased with increasing allergen concentration in the range of 5-50 micrograms ml-1. Leucocytes did not respond to other allergens such as soybean, milk and dinitrophenylated bovine serum albumin (DNP-BSA).     

Interactions of surface-confined DNA with electroreduced mitomycin C comparison with acid-activated mitomycin C

The anticancer activity of the antineoplastic drug mitomycin C (MC) was investigated using transfer stripping cyclic voltammetry (TSCV) with single-stranded DNA-modified hanging mercury drop electrode (HMDE). Reductive activation of MC is necessary for drug covalent binding to DNA, and we have found that some potential-controlled interactions of MC with DNA occur at the electrode, i.e. MC can be activated by electroreduction. Acid and electroreductive MC activations were compared and different adducts were subsequently generated, suggesting that the drug can bind to DNA in more than one way. Under conditions of acid activated MC, a monofunctional adduct between C-1 of MC and N-7 of guanine was formed on the electrode surface, reduced at - 0.44 V (vs. SCE). However, when the DNA-modified electrode was immersed in a MC solution and potentials corresponding to the quinone moiety reduction (- 0.3 V or more negative vs. SCE) were applied, an intrastrand bifunctional adduct between C-1 and C-10 of MC and two N-7 of a pair of adjacent guanines in ssDNA were formed at the electrode, reduced at - 0.49 V, i.e. 50 mV more negative than the monoadduct. The results presented in this paper show for the first time electrochemical detection of DNA-MC adducts at the hanging mercury drop electrode.     

Electrically controlled proliferation of human carcinoma cells cultured on the surface of an electrode.

Human carcinoma cells, MKN45, were cultured on the surface of a metal-coated plastic plate electrode the potential of which was controlled. The proliferation rate and cell morphology were altered depending on the applied potential. Cell proliferation was halted in the potential range above 0.4 V vs. Ag/AgCl, although cells started to proliferate again when the applied potential was shifted from 0.4 V to 0.1 V vs. Ag/AgCl. Fluorescence probe studies indicated that the fluidity of plasma membrane decreased in association with halting of cell proliferation. These results suggest that electrical stimulation causes cells to temporarily halt proliferation, and that cell proliferation was reversibly controlled by electrode potential. The mechanism is interpreted in relation to the change of plasma membrane structure represented by membrane fluidity.     

Effect of galvanically induced surface potentials on marine fouling

This paper reports the effect of a galvanically produced negative surface potential on the accumulation of marine biofouling. The potential was created by connecting pieces of copper or stainless steel to a layer of Indium/Tin Oxide semiconductor deposited on glass. It was shown that the negative potential significantly reduced the accumulation of biofouling. As the conductive layer is transparent, the technique was used to protect the windows of commercial optical instruments. This technology could provide an inexpensive way of extending deployment times for marine instruments.     

A paint incorporating silver to control mixed biofilms containingLegionella pneumophila

A three-stage chemostat containing a mixed consortium of microorganisms, includingLegionella pneumophila, was used to determine the suitability of a silver-containing paint to control biofouling in water systems. The paint was efficient in controlling total surface colonisation by heterotrophic microorganisms and growth of the pathogen over a 2-week period. Biodiversity was limited in the presence of the silver paint and this was thought to help controlL. pneumophila numbers. Glass control tiles suspended alongside the silver painted tiles also had reduced colonisation for the 2-week period, suggesting that low levels of silver leached from the paint surface. This loss of silver was confirmed since the inhibition of biofouling and inclusion of the pathogen was not maintained after the 2-week period. Although this paint was unsuitable for controlling biofouling over extended time periods, the data suggest that a reformulated paint or electrochemical method of introducing silver ions may be successful.     

A synthetic analogue for the active site of plant-type ferredoxin: two different coordination isomers by a four-cys-containing [20]-peptide.

The (Fe2S2)2+ complex of an artificial 20-peptide ligand, Ac-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys-Ala-Gly-Pro-Leu-Leu-T hr-Cys- Val-NH2, containing an invariant Cys-A-B-C-D-Cys-X-Y-Cys (A, B, C, D, X, Y = amino acid residues) fragment of plant-type ferredoxins was synthesized by a ligand exchange method with [Fe2S2(S-t-Bu)4]2-. 1H-nmr spectroscopic and electrochemical data of the complex indicate the presence of two coordination isomers. One of them having a Cys-X-Y-Cys bridging coordination to the two Fe(III) ions, has the (Fe2S2)2+ core environment similar to those of the denatured plant-type ferredoxins and exhibits a positive shifted redox potential at -0.64 V vs saturated colonel electrode (SCE) in N,N-dimethylformamide (DMF). Another isomer with the Cys-A-B-C-D-Cys bridging coordination shows a negative redox potential at -0.96 V vs SCE in DMF.     

Fluorometric observation of viable and dead adhering diatoms using TO-PRO-1 iodide and its application to the estimation of electrochemical treatment

A rapid method for the direct measurement of viable and dead adhering diatoms was developed using a fluorescent dye, TO-PRO-1 iodide. By staining the marine diatom, Nitzchia closterium, with TO-PRO-1 iodide, viable and dead cells were identified as red and yellow cells respectively, under an epifluorescence microscope employing blue excitation. Only dead cells were stained with TO-PRO-1 iodide. Viable cells were observed as red because of autofluorescence arising from intracellular chlorophyll, whereas dead cells were observed as yellow because of the fluorescence of TO-PRO-1 iodide. The percentage of TO-PRO-1-iodide-stained was correlated with the percentage of dead cells in N. closterium cells exposed to heat (60 °C, 15 min). Other microalgae containing intracellular chlorophyll could be also distinguished as viable or dead cells by this fluorometric staining method. This method was applied for the assessment of N. closterium cells killed by the electrochemical treatment and used to monitor biofouling populations and their viability directly on the electrode surface. When 1.0 V was applied against a saturated calomel electrode, 99% of the cells attached to graphite electrode were killed in 1 h. Received: 7 August 1998 / Received revision: 16 October 1998 / Accepted: 7 November 1998     

Electrical effects on the proliferation of living HeLa cells cultured on optically transparent electrode surface.

Low d.c. potential application induced changes of cellular morphology and growth of living cells on a potential-controlled electrode. At a potential range higher than +0.7 V (vs. Ag/AgCl), serious electric effects on cell viability, membrane permeability, and cytoskeletal morphology of HeLa cells were observed. On the other hand, at lower than +0.5 V no effect was observed. At the boundary potential range between +0.5 V to +0.7 V, where HeLa cells were cultured on the potential-controlled optically transparent In2O3 electrode (OTE) surface, intriguing effects on HeLa cells appeared. At this potential range, where HeLa cells cultured on a potential-applied OTE, all the cells were alive accompanying morphological change. The morphology of HeLa cells returned to their normal spindle shape, when potential application to the electrode was cut off. At a potential of +0.65 V, cell proliferation ratio of cultured cell on an electrode was about one-fifth of that on a non-controlled electrode. These results suggest that low d.c. electrical effects induce significant change in cellular morphology and function.     

Abundance of the Multiheme c-Type Cytochrome OmcB Increases in Outer Biofilm Layers of Electrode-Grown Geobacter sulfurreducens

When Geobacter sulfurreducens utilizes an electrode as its electron acceptor, cells embed themselves in a conductive biofilm tens of microns thick. While environmental conditions such as pH or redox potential have been shown to change close to the electrode, less is known about the response of G. sulfurreducens to growth in this biofilm environment. To investigate whether respiratory protein abundance varies with distance from the electrode, antibodies against an outer membrane multiheme cytochrome (OmcB) and cytoplasmic acetate kinase (AckA) were used to determine protein localization in slices spanning ∼25 µm-thick G. sulfurreducens biofilms growing on polished electrodes poised at +0.24 V (vs. Standard Hydrogen Electrode). Slices were immunogold labeled post-fixing, imaged via transmission electron microscopy, and digitally reassembled to create continuous images allowing subcellular location and abundance per cell to be quantified across an entire biofilm. OmcB was predominantly localized on cell membranes, and 3.6-fold more OmcB was detected on cells 10–20 µm distant from the electrode surface compared to inner layers (0–10 µm). In contrast, acetate kinase remained constant throughout the biofilm, and was always associated with the cell interior. This method for detecting proteins in intact conductive biofilms supports a model where the utilization of redox proteins changes with depth.     

Side-chain oxidative damage to cysteine on a glassy carbon electrode

In this paper, oxidative damage to the cysteine (CySH) side-chain on a glassy carbon electrode (GCE) was investigated. Voltammetric studies show that there are three anodic peaks for the oxidation of CySH, which arise from (1) the oxidation of the –SH side-chain, forming cystine (0.71 V, vs. SCE) and (2) CySO _x H, x = 2, 3 (0.98 V vs. SCE), and (3) the oxidation of the amino acid carboxyl group (around 1.51 V vs. SCE). The influence of dissolved oxygen, pH, scan rate, scan time, temperature and CySH concentration were investigated and the oxidative mechanism proposed. The peaks near 0.71 and 0.98 V are the promising candidates for measuring the oxidation of CySH on the GCE. This paper provides a new strategy for researching oxidative damage of amino acids, sulfur-containing peptides and proteins.     

Electrically promoted protein production by mammalian cells cultured on the electrode surface

Protein production of mammalian cells has been promoted by applying a small constant potential to the surface of an electrode on which cells are cultured. Human carcinoma line of MKN45 cells were cultured on the surface of a platinum-coated plastic plate electrode. Low d.c. voltage of constant potential was applied to the electrode during 4-day culture to modulate the production of carcinoembryonic antigen (CEA). The amounts of both secreted and membrane-bound CEA were dependent on the applied potential during culture. Secreted CEA was more than twice in amount in the potential range from 0.2 V to 0.6 V vs. Ag/Agcl as compared with that of normal culture. In the potential range, CEA was also increased in membrane-bound form. The potential-controlled cell culture may have an enhanced effect on protein production.     

Direct electron transfer of glucose oxidase promoted by carbon nanotubes

A stable suspension of carbon nanotubes (CNT) was obtained by dispersing the CNT in a solution of surfactant, such as cetyltrimethylammonium bromide (CTAB, a cationic surfactant). CNT (dispersed in the solution of 0.1% CTAB) has promotion effects on the direct electron transfer of glucose oxidase (GOx), which was immobilized onto the surface of CNT. The direct electron transfer rate of GOx was greatly enhanced after it was immobilized onto the surface of CNT. Cyclic voltammetric results showed a pair of well-defined redox peaks, which corresponded to the direct electron transfer of GOx, with a midpoint potential of about -0.466 V (vs SCE (saturated calomel electrode)) in the phosphate buffer solution (PBS, pH 6.9). The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (ks) and the value of midpoint potential (E1/2) were estimated. The dependence of E1/2 on solution pH indicated that the direct electron transfer reaction of GOx is a two-electron-transfer coupled with a two-proton-transfer reaction process. The experimental results also demonstrated that the immobilized GOx retained its bioelectrocatalytic activity for the oxidation of glucose, suggesting that the electrode may find use in biosensors (for example, it may be used as a bioanode in biofuel cells). The method presented here can be easily extended to immobilize and obtain the direct electrochemistry of other redox enzymes or proteins.     

Electricity production by Geobacter sulfurreducens attached to electrodes

Previous studies have suggested that members of the Geobacteraceae can use electrodes as electron acceptors for anaerobic respiration. In order to better understand this electron transfer process for energy production, Geobacter sulfurreducens was inoculated into chambers in which a graphite electrode served as the sole electron acceptor and acetate or hydrogen was the electron donor. The electron-accepting electrodes were maintained at oxidizing potentials by connecting them to similar electrodes in oxygenated medium (fuel cells) or to potentiostats that poised electrodes at +0.2 V versus an Ag/AgCl reference electrode (poised potential). When a small inoculum of G. sulfurreducens was introduced into electrode-containing chambers, electrical current production was dependent upon oxidation of acetate to carbon dioxide and increased exponentially, indicating for the first time that electrode reduction supported the growth of this organism. When the medium was replaced with an anaerobic buffer lacking nutrients required for growth, acetate-dependent electrical current production was unaffected and cells attached to these electrodes continued to generate electrical current for weeks. This represents the first report of microbial electricity production solely by cells attached to an electrode. Electrode-attached cells completely oxidized acetate to levels below detection (<10 micro M), and hydrogen was metabolized to a threshold of 3 Pa. The rates of electron transfer to electrodes (0.21 to 1.2 micro mol of electrons/mg of protein/min) were similar to those observed for respiration with Fe(III) citrate as the electron acceptor (E(o)' =+0.37 V). The production of current in microbial fuel cell (65 mA/m(2) of electrode surface) or poised-potential (163 to 1,143 mA/m(2)) mode was greater than what has been reported for other microbial systems, even those that employed higher cell densities and electron-shuttling compounds. Since acetate was completely oxidized, the efficiency of conversion of organic electron donor to electricity was significantly higher than in previously described microbial fuel cells. These results suggest that the effectiveness of microbial fuel cells can be increased with organisms such as G. sulfurreducens that can attach to electrodes and remain viable for long periods of time while completely oxidizing organic substrates with quantitative transfer of electrons to an electrode.