Investigation of the biological activity of solutions activated electrochemically in a membrane electrolyzer

The biological activity of the catholyte and anolyte of double distilled water was studied in experiments on the germination of wheat grains in the period from March to May. The activity of the solutions, which was characterized by a growth index, was high early in this period, then decreased almost to zero in the middle of the period, and then increased to about the initial value by the end of the period. Throughout, the efficiency of the anolyte of double distilled water generally exceeded the efficiency of the catholyte. Early and late in the period, the stimulatory effect of the anolyte exceeded that of the catholyte by a factor of 5–5.5. The changes in the biological activity of the catholyte and anolyte of double distilled water were also compared with the changes in the biological activity of the catholyte of nutrient medium M9. The stimulatory effect of the catholyte of the nutrient medium was evaluated from the change in the growth of E. coli cells. Early in the period at a cultivation temperature of 20°C, the stimulatory effect determined from the increase in the optical density of the cell suspension in the experiment with respect to a reference value was 55–60%. Next, the stimulatory effect decreased almost to zero in the middle of the period and increased to approximately initial value by the end of the period. It was assumed that the physicochemical mechanisms of action of the catholyte and anolyte of double distilled water on the wheat seed germination and of the catholyte of the nutrient medium on E. coli cell growth are of different nature.     

Biological activity of electrochemically activated solutions obtained in a diaphragm electrolyser

The biological activity of the catholyte and anolyte of bidistilled water in experiments with the germination of wheat grains in the period from March to May has been studied. The activity of solutions, which was characterized by the grain germination index, was high at the beginning of the period, then it gradually decreased and was equal to zero at the middle of the period; at the end of the period it gradually increased almost to initial values. It has been established that the effectiveness of bidistilled water anolyte was as a rule higher than that of catholyte throughout the observation period. At the beginning and end, the stimulating effect of anolyte was 5-5.5 times greater than that of catholyte. The seasonal changes in the biological activity of M 9 medium catholyte were compared with those of bidistilled water anolyte and catholyte. The stimulating effect of M 9 catholyte was estimated by changes in the growth of E. coli cells. The stimulating effect, which was estimated from an increase in the optical density of cell suspension in the initial period at a cultivation temperature of 20 degrees C was 55-60% relative to control (untreated medium). Then it decreased almost to zero in the middle of the period to increase again approximately to the initial values. The assumption has been made that the physicochemical causes of the influence of catholyte and anolyte of bidistilled water on wheat grains and of the culture medium catholyte on E. coli cells are of different nature.     

The causes of the biological action of electrochemically activated solutions by changes in the growth of Escherichia coli cells

To study the causes of the biological effect of electrochemically activated solutions, nutrient growth media M 9 were prepared using catholyte and anolyte solutions containing separate components of the nutrient medium, such as distilled water, phosphate buffer, phosphate buffer with chlorides (NaCl, NH4Cl), and chlorides. The biological activity of different nutrient media was assessed by a comparison with the stimulation or inhibition of the growth of Escherichia coli cells in the catholyte and anolyte of the complete nutrient medium M 9. It was shown that medium M 9 prepared on the catholytes of different initial solutions acquired the stimulating properties only if the initial solution contained salts containing chlorine. The stimulating effect of the initial solution was 18-24%. Electrochemical treatment of solutions containing no chlorides (distilled water, phosphate buffer) and subsequent addition of the components of nutrient medium to exposed solutions had neither a stimulating nor the inhibiting effect on cell growth. The cultivation of cells in a nutrient medium based on the catholyte of preliminarily treated hydrochloric acid showed that it is the presence of chlorine ions in solution during electrolysis that causes the stimulating effect of the nutrient medium based on the catholyte. The formation of oxidizers and the inhibitory effect of the anolyte described previously was also observed if the solution contained chlorine ions during electrolysis. Possible mechanisms of the biological effect of catholytes containing chlorides during electrolysis were discussed.     

Nonequilibrium state of electrochemically activated water and its biological activity]

Changes in the physicochemical parameters (pH, redox potential and electroconductivity) of catholyte and anolyte produced by membrane electrolysis of distilled water and dilute (c < 10(-3) M) sodium chloride solutions were studied. The relaxation of these parameters after electrolysis and the influence of catholyte and anolyte on the growth of roots of Tradescantia viridis grafts, the development of duckweed, and the motive activity of infusoria Spirostomum ambiguum were investigated. It was found that the anolyte of distilled water stimulated development of these biological objects. The direction of shift of physicochemical parameters of catholyte and anolyte from equilibrium values and the type of their biological activity (stimulation or inhibition) depend on salt concentration in initial solution. Barbotage of initial distilled water with argon or nitrogen leads to a greater decrease in the redox potential of catholyte during electrolysis. The physicochemical parameters relax to equilibrium values, and the biological activity of catholite and anolyte decreases with time and practically disappears by the end of the day. It was found that the oxidation of reducing agent by atmospheric oxygen is not the sole cause of the relaxation of catalyte redox potential. The increase in the ionic strength of catholite and anolyte by the addition of concentrated sodium chloride after electrolysis decreases the rate of redox potential relaxation several times. The redox potential can be maintained for long periods by freezing.     

On the fluorescence of bidistilled water after electrochemical treatment

The short-wave fluorescence of bidistilled water treated in the cathode and anode chambers of two types of electrolysers made from different materials was investigated by fluorescence spectroscopy. It was shown that the electrochemical treatment of water did not induce intrinsic short-wave flyorescence of catholyte and anolyte in the case of quartz glass electrolyser. The increase observed in the intensity of intrinsic short-wavelength fluorescence of bidistilled water in the catholyte and anolyte of the electrolyser made from technical organic glass was caused by microcontaminations released from electrolyser material in the samples.     

Causes of fluorescence in bidistilled water after electrochemical treatment

Fluorescence spectroscopy was used to study the shortwave fluorescence of bidistilled water treated in the cathode and anode chambers of two types of electrolyzers made of different materials. Electrochemical treatment in the quartz glass electrolyzer did not induce intrinsic fluorescence of the anolyte or catholyte. An increase in the shortwave fluorescence of the anolyte and catholyte was observed in the electrolyzer made of Plexiglass, which was probably due to the release of microcontaminants from components of the electrolyzer.     

Level of hydrogen peroxide in electrochemically activated solutions and study of its effect on growth of Escherichia coli

The formation of hydrogen peroxide in catholytes and anolytes of electrochemically activated solutions: bidistilled water and solutions of sodium chloride and nutrition medium M9 was studied. The concentration of hydrogen peroxide was determined by the method of enhanced chemiluminescence in a system peroxidase-luminol-p-iodophenol. It was shown that the concentration of hydrogen peroxide depends on the ionic content of the solution and varies from a few fractions of a micromole in catholytes of bidistilled water and sodium chloride solutions (10(-5) divided by 10(-2) M) to 20-25 microM in catholytes of medium M9. The concentration of H2O2 in anolytes of various solutions was 15-20 times lower than in the corresponding catholytes and was equal to a few nanomoles in bidistilled water and a few micromoles in medium M9. The biological activity of the catholyte of medium M9 was determined from changes in the growth of E. coli cells. It was found that this catholyte stimulates the cell growth. The stimulating effect was 20-25% and did not change after the decomposition of hydrogen peroxide in the catholyte by catalase. The addition of H2O2 at the corresponding concentration to the inactivated nutrient medium produced no stimulating effect. These data suggest that hydrogen peroxide formed in the catholyte of nutrient medium M9 does not affect its biological activity.     

Surfactant production by the Rhodococcus erythropolis sH-5 bacterium grown on various carbon sources

It has been shown that the Rhodococcus erythropolis sH-5 strain can produce surfactants associated and not associated with the cell wall. Their content depends on medium composition, the nature of the carbon source, and oxygen supply. The highest biosurfactant (bioSF) yield is achieved by growing R. erythropolis sH-5 in medium with 2% kerosene at neutral pH. It has been found that the bioSF yield and emulsification index for various hydrocarbons depend on the kind of the nitrogen source used by the bacterium, increasing with replacement of KNO3 by NaNO3. The yields of biomass and bioSF in R. erythropolis depend on growth temperatures (max at 30 degrees C) but not on water quality (bidistillate, catholyte, or anolyte). It has been found that sH-5 produces more cell-associated bioSF than extracellular species.     

Stabilization of pH gradients in buffer electrofocusing on polyacrylamide gel.

pH gradients in buffer electrofocusing on polyacrylamide gel (BEF) have been stabilized for at least 100 hr, 25°C, by replacing the strongly acidic and basic anolyte and catholyte with isoelectric buffers identical to the terminal constituents of the pH gradient and gel. Such stabilization leads to a constant pI position of an electrofocused protein. No stabilization of pH gradients is achieved under otherwise identical conditions when strongly acidic and basic anolyte and catholyte are used.     

Surfactant production by the Rhodococcus erythropolis sH-5 bacterium grown on various carbon sources

It has been shown that the Rhodococcus erythropolis sH-5 strain can produce surfactants associated and not associated with the cell wall. Their content depends on medium composition, the nature of the carbon source, and oxygen supply. The highest biosurfactant (bioSF) yield is achieved by growing R. erythropolis sH-5 in medium with 2% kerosene at neutral pH. It has been found that the bioSF yield and emulsification index for various hydrocarbons depend on the kind of the nitrogen source used by the bacterium, increasing with replacement of KNO₃ by NaNO₃. The yields of biomass and bioSF in R. erythropolis depend on growth temperatures (max at 30°C) but not on water quality (bidistillate, catholyte, or anolyte). It has been found that sH-5 produces more cell-associated bioSF than extracellular species.     

Ammonium as a sustainable proton shuttle in bioelectrochemical systems

This work examines a pH control method using ammonium (NH(4)(+)) as a sustainable proton shuttle in a CEM-equipped BES. Current generation was sustained by adding NH(3) or ammonium hydroxide (NH(4)OH) to the anolyte, controlling its pH at 7. Ammonium ion migration maintained the catholyte pH at approximately 9.25. Such NH(4)(+)/NH(3) migration accounted for 90±10% of the ionic flux in the BES. Reintroducing the volatilized NH(3) from the cathode into the anolyte maintained a suitable anolyte pH for sustained microbial-driven current generation. Hence, NH(4)(+)/NH(3) acted as a proton shuttle that is not consumed in the process.     

Insights in the anode chamber influences on cathodic bioelectromethanogenesis – systematic comparison of anode materials and anolytes

Cathode and catholyte are usually optimized to improve microbial electrosynthesis process, whereas the anodic counter reaction was not systematically investigated and optimized for these applications yet. Nevertheless, the anolyte and especially the anode material can limit the cathodic bioelectrochemical process. This paper compares for the first time the performance of different anode materials as counter electrodes for a cathodic bioelectrochemical process, the bioelectromethanogenesis. It was observed that depending on the anode material the cathodic methane production varies from 0.96 µmol/d with a carbon fabric anode to 25.44 µmol/d with a carbon felt anode of the same geometrical surface area. The used anolyte also affected the methane production rate at the cathode. Especially, the pH of the anolyte showed an impact on the system; an anolyte with pH 5 produced up to 2.0 times more methane compared to one with pH 8.5. The proton availability is discussed as one reason for this effect. Although some of the measured effects cannot be explained completely so far this study advises researchers to strongly consider the anode impact during process development and optimization of a cathodic bioelectrochemical synthesis process.     

Gel electrophoresis of protein-dodecyl sulfate complexes in a pH gradient and improved resolution of poliovirus polypeptides.

Electrophoresis of poliovirus capsid polypeptides and of nonviral test proteins was carried out in 12.5% acrylamide gels in the presence of sodium dodecyl sulfate. The gels were prepared at pH 7.2. The electrode buffers were (i) both at pH 7.2 (normal conditions), (ii) both at pH 9, 10, or 11, or (iii) the catholyte was at pH 11 and the anolyte was at pH 6.5. The VP1 = 3 group of poliovirus polypeptides yielded the classical three bands under the first (i) set of conditions, except that VP2 and VP3 each yielded two bands in protracted runs; up to six bands were obtained under the second (ii) and third (iii) sets of conditions. When the catholyte was pH 11, there was a molecular weight-dependent, progressive deceleration of the migration of all proteins. In addition, a pH gradient was formed in the gels, and these expanded markedly. The improved resolution of the poliovirus polypeptides is discussed in the light of these observations.     

A biofilm enhanced miniature microbial fuel cell using Shewanella oneidensis DSP10 and oxygen reduction cathodes

A miniature-microbial fuel cell (mini-MFC, chamber volume: 1.2 mL) was used to monitor biofilm development from a pure culture of Shewanella oneidensis DSP10 on graphite felt (GF) under minimal nutrient conditions. ESEM evidence of biofilm formation on GF is supported by substantial power density (per device cross-section) from the mini-MFC when using an acellular minimal media anolyte (1500 mW/m2). These experiments demonstrate that power density per volume for a biofilm flow reactor MFC should be calculated using the anode chamber volume alone (250W/m3), rather than with the full anolyte volume. Two oxygen reduction cathodes (uncoated GF or a Pt/vulcanized carbon coating on GF) were also compared to a cathode using uncoated GF and a 50mM ferricyanide catholyte solution. The Pt/C-GF (2-4% Pt by mass) electrodes with liquid cultures of DSP10 produced one order of magnitude larger power density (150W/m3) than bare graphite felt (12W/m3) in this design. These advances are some of the required modifications to enable the mini-MFC to be used in real-time, long-term environmental power generating situations.     

Analysis of single erythrocytes by injection-based capillary isoelectric focusing with laser-induced native fluorescence detection

A modified version of capillary isoelectric focusing (cIEF) was developed to separate hemoglobin variants contained within single human erythrocytes. Laser-induced native fluorescence with 275 nm excitation was used to detect the separated hemoglobins. In this method, baseline fluctuations were minimized and detection sensitivity was improved by using dilute solutions of anolyte, catholyte, and carrier ampholytes (with methylcellulose). Since electrosmotic flow was used for mobilization of the focused bands, separation and detection were integrated into a single step. The capillary was first filled with only ampholyte solution, and the cell (or standard) was injected as in capillary zone electrophoresis. The ∼90 fl injection volume for individual cells is 7×10⁴ times lower than those previously reported. Adult (normal and elevated A₁), sickle (heterozygous), and fetal erythrocytes were analyzed, with the amounts of hemoglobins A₀, A_1c, S and F determined. The pH gradient for cIEF was linear (r² = 0.9984), which allowed tentative identification of Hb F_ac. Variants differing by as little as 0.025 pI units were resolved.     

Capillary isoelectric focusing of proteins and microorganisms in dynamically modified fused silica with UV detection

We suggest a method for the reproducible and efficient capillary isoelectric focusing of proteins and microorganisms in the pH gradient 3-10. The method involves the segmental injection of the simple ampholytes, the solution of the selected electrolytes, and the sample mixture of bioanalytes and carrier ampholytes to the fused silica capillaries dynamically modified by poly(ethylene glycol), PEG 4000, which is added to the catholyte, the anolyte and injected solutions. In order to receive the reproducible results, the capillaries were rinsed by the mixture of acetone/ethanol between analyses. For the tracing of the pH gradients the low-molecular-mass pI markers were used. The simple proteins and the mixed cultures of microorganisms, Saccharomyces cerevisiae CCM 8191, Escherichia coli CCM 3954, Candida albicans CCM 8180, Candida parapsilosis, Candida krusei, Staphylococcus aureus, Streptococcus agalactiae CCM 6187, Enterococcus faecalis CCM 4224, Staphylococcus epidermidis CCM 4418 and Stenotrophomonas maltophilia, were focused and separated by the method suggested. The minimum detectable number of microbial cells was 5x10(2) to 1x10(3) with on-column UV detection at 280 nm.     

Lack of evidence of chalone activity in used medium and extract of JB-1 tumor cells in vitro. A flow cytometry study.

In cell-free mouse ascites fluid from the JB-1 ascites tumor in the plateau phase of growth low-molecular chalone substances have been found which reversibly and specifically arrest JB-1 cells in the G1 and G2 phase of the cell cycle. The aim of this study was to investigate whether chalones were involved in the regulation of in vitro growth of JB-1 tumor cells. Used medium and cell extract from confluent, stationary JB-1 cell cultures were investigated for proliferation-inhibitory properties. JB-1 cells from stationary cultures were explanted in test cultures and the traverse of cells through the S phase was investigated by means of flow cytometry (FCM). Inhibition--expressed as a delay of the traverse of cells through the S phase--was not observed when a surplus of used medium, concentrated and fractionated used medium or concentrated and fractionated cell extract from JB-1 cells in vitro was added to test cultures. On the contrary, used medium and concentrated and fractionated used medium stimulated growth. Thus, no involvement of chalones in the growth regulation of JB-1 tumor cells in vitro was detected.     

Effect of magnesium on the growth and cell cycle of transformed and non-transformed epithelial rat liver cells in vitro

The effects of magnesium (Mg) restriction on cell growth and the cell cycle were determined in transformed (TRL-8) and non-transformed (TRL-12-15) epithelial-like rat liver cells. Cells were cultured in RPMI 1640 medium in which the Mg concentration was reduced to 0.5, 0.1, and 0 × the concentration in the regular RPMI 1640 media (100mg/l). Cell growth in the transformed cells was not influenced by the Mg restriction as greatly as in the non-transformed cell line. Transit through the cell cycle also exhibited an independence of the Mg in the medium in the transformed cells. When transformed cells were grown for two generations in Mg-limited medium, the growth rate slowed to a rate similar to that demonstrated by the non-transformed cells. Analysis by flow cytometry showed that transit through the cell cycle was minimally slowed in Mg deficient transformed cells; however, transit through the G₁ and S phases in the non-transformed cells was slowed. The TRL-8 cells in Mg-limited medium resulted in fewer nuclei in G₁ with subsequent increases in the percentages of S-phase nuclei. The TRL 12-15 cells reacted oppositely with the number of G₁ nuclei increased and the number of S-phase nuclei decreased. In respect to growth, these results show that epithelial cells respond in a similar manner to Mg-limitation as do fibroblast cells. The transformed cells exhibited a level of independence from Mg in respect to growth, reproduction, and cell-cycle kinetics.     

Utilization of glucose and amino acids in insect cell cultures: Quantifying the metabolic flows within the primary pathways and medium development

The current understanding of insect cell metabolism is very limited. In order to gain some insight into the growth and metabolism of insect cells Spodoptera frugiperda (Sf9), a comprehensive characterization of culture conditions for cells grown in the IPL-41 medium was made by measuring the amino acid composition of the growth medium and the cell extract, the macromolecular composition of the cells (DNA, RNA, and protein), medium concentrations of various metabolites and sugars, and the evolved CO(2). Since in the IPL-41-based serum-free medium all of the amino acids except cysteine are in great excess of what is needed by the cells for energy and protein production, a medium formulation with an osmolarity similar to the IPL-41 but with a lower amino acid content than IPL-41 was also developed. The new medium also lacks maltose and sucrose (contains only glucose), supported cell growth to a high cell density of 8 x 10(6) cells/mL. The cellular and energetic yields indicated that a tight coupling between the biosynthetic and energetic reactions was attained for cells grown in the new medium. Moreover, it was found that the intermittent feeding of glucose may not be required as the cell yield and growth rate were comparable whether the same total amount of glucose was provided intermittently or was included initially in the medium. The eventual cessation of growth in the new medium is believed to be due to the amino acid limitation because concentrations of both glutamine and glutamate were very low at the end of the growth phase. Thus, further optimization, which may include higher initial glutamine in the medium or its intermittent feeding, could lead to a further increase in the cell density. Finally, a stoichiometrically based analysis of metabolic reactions confirmed the operation of the key pathways and was used to quantify the distribution of metabolites among primary metabolic reactions. The quantitative flow values were used to highlight some key aspects of insect cell metabolism. (c) 1993 John Wiley & Sons, Inc.     

Serum-free growth of normal and transformed fibroblasts in milk: differential requirements for fibronectin

Bovine milk may be used as a supplement for the serum-free growth of certain fibroblastic cells in culture. The growth properties of three representative cell types in milk-supplemented medium were examined; fibroblastic cell strains, fibroblastic cell lines, and transformed fibroblasts. Transformed fibroblasts, which included RNA and DNA tumor virus-transformed cells and carcinogen-transformed cells, grew in milk. Instead of growing attached to the culture dishes, as they normally do in serum, transformed fibroblasts grew in milk as large clusters in suspension. In contrast, nontransformed fibroblastic cell strains and cell lines did not grow in milk-supplemented medium. Fibroblasts transformed by a temperature-sensitive transformation mutant of Rous sarcoma virus were temperature-sensitive for growth in milk. The failure of cells to adhere to the substratum in milk-supplemented medium suggested that milk might be deficient in attachment factors for fibroblasts. When the attachment of fibroblastic cells in milk- supplemented medium was facilitated by pretreating culture dishes with fibronectin, (a) transformed cells grew attached rather than in suspension, (b) normal cell lines attached and grew to confluence, and (c) normal cell strains adhered and survived but did not exhibit appreciable cell proliferation.