Activity and viability of polycyclic aromatic hydrocarbon‐degrading Sphingomonas sp. LB126 in a DC‐electrical field typical for electrobioremediation measures

There has been growing interest in employing electro‐bioremediation, a hybrid technology of bioremediation and electrokinetics for the treatment of contaminated soil. Knowledge however on the effect of weak electrokinetic conditions on the activity and viability of pollutant‐degrading microorganisms is scarce. Here we present data about the influence of direct current (DC) on the membrane integrity, adenosine triphosphate (ATP) pools, physico‐chemical cell surface properties, degradation kinetics and culturability of fluorene‐degrading Sphingomonas sp. LB126. Flow cytometry was applied to quantify the uptake of propidium iodide (PI) and the membrane potential‐related fluorescence intensities (MPRFI) of individual cells within a population. Adenosine tri‐phosphate contents and fluorene biodegradation rates of bulk cultures were determined and expressed on a per cell basis. The cells' surface hydrophobicity and electric charge were assessed by contact angle and zeta potential measurements respectively. Relative to the control, DC‐exposed cells exhibited up to 60% elevated intracellular ATP levels and yet remained unaffected on all other levels of cellular integrity and functionality tested. Our data suggest that direct current (X = 1 V cm^?1; J = 10.2 mA cm^?2) as typically used for electrobioremediation measures has no negative effect on the activity of the polycyclic aromatic hydrocarbon (PAH)‐degrading soil microorganism, thereby filling a serious gap of the current knowledge of the electrobioremediation methodology.     

Isolation and characterization of heavy polycyclic aromatic hydrocarbon-degrading bacteria adapted to electrokinetic conditions

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria capable of growing under electrokinetic conditions were isolated using an adjusted acclimation and enrichment procedure based on soil contaminated with heavy PAHs in the presence of an electric field. Their ability to degrade heavy PAHs under an electric field was individually investigated in artificially contaminated soils. The results showed that strains PB4 (Pseudomonas fluorescens) and FB6 (Kocuria sp.) were the most efficient heavy PAH degraders under electrokinetic conditions. They were re-inoculated into a polluted soil from an industrial site with a PAH concentration of 184.95 mg kg^?1. Compared to the experiments without an electric field, the degradation capability of Pseudomonas fluorescens and Kocuria sp. was enhanced in the industrially polluted soil under electrokinetic conditions. The degradation extents of total PAHs were increased by 15.4 and 14.0 % in the electrokinetic PB4 and FB6 experiments (PB4 + EK and FB6 + EK) relative to the PB4 and FB6 experiments without electrokinetic conditions (PB4 and FB6), respectively. These results indicated that P. fluorescens and Kocuria sp. could efficiently degrade heavy PAHs under electrokinetic conditions and have the potential to be used for the electro-bioremediation of PAH-contaminated soil, especially if the soil is contaminated with heavy PAHs.     

Influence of Growth Phase on Adhesion Kinetics of Escherichia coli D21g

The influence of bacterial growth stage and the evolution of surface macromolecules on cell adhesion have been examined by using a mutant of Escherichia coli K-12. To better understand the adhesion kinetics of bacteria in the mid-exponential and stationary growth phases under flow conditions, deposition experiments were conducted in a well-controlled radial stagnation point flow (RSPF) system. Complementary cell characterization techniques were conducted in combination with the RSPF experiments to evaluate the hydrophobicity, electrophoretic mobility, size, and titratable surface charge of the cells in the two growth phases considered. It was observed that cells in stationary phase were notably more adhesive than those in mid-exponential phase. This behavior is attributed to the high degree of local charge heterogeneity on the outer membranes of stationary-phase cells, which results in decreased electrostatic repulsion between the cells and a quartz surface. The mid-exponential-phase cells, on the other hand, have a more uniform charge distribution on the outer membrane, resulting in greater electrostatic repulsion and, subsequently, less adhesion. Our results suggest that the macromolecules responsible for this phenomenon are outer membrane-bound proteins and lipopolysaccharide-associated functional groups.     

Irreversible loss of negative surface charge and loss of colony-forming ability in Burkitt lymphoma cells after x-irradiation

The electrokinetic behavior of Burkitt lymphoma P 3H-R1 cells were studied following X-irradiation. The surface charge of irradiated cells decreased progressively with time after X-ray treatment, as compared with a mean mobility value (−0.677 ± 0.045 μsec⁻¹ V⁻¹ cm) of non-irradiated cells. The frequency distribution of cells treated with 3 000 R 4 h previously showed quite a different peak of lower mobility than that of non-irradiated cells with the boundary of −0.550 μsec⁻¹ V⁻¹ cm. Percentages of cells which showed the higher mobility than the boundary value 4 h after irradiation with 30, 50, 65, 100, 200 or 3 000 R were in accord with the percentage of cells which showed colony-forming ability in soft agar, respectively. Following cell proliferation the population of electrokinetically intact cells increased with time after irradiation; these values also coincided with the population of colony-forming cells. Thus, the present results indicate that there is a close relation between loss of negative surface charge and loss of colony forming ability in irradiated cells, suggesting that loss of colony forming ability of the cells may be determined as early as 4 h after irradiation.     

Influence of growth phase on adhesion kinetics of Escherichia coli D21g

The influence of bacterial growth stage and the evolution of surface macromolecules on cell adhesion have been examined by using a mutant of Escherichia coli K-12. To better understand the adhesion kinetics of bacteria in the mid-exponential and stationary growth phases under flow conditions, deposition experiments were conducted in a well-controlled radial stagnation point flow (RSPF) system. Complementary cell characterization techniques were conducted in combination with the RSPF experiments to evaluate the hydrophobicity, electrophoretic mobility, size, and titratable surface charge of the cells in the two growth phases considered. It was observed that cells in stationary phase were notably more adhesive than those in mid-exponential phase. This behavior is attributed to the high degree of local charge heterogeneity on the outer membranes of stationary-phase cells, which results in decreased electrostatic repulsion between the cells and a quartz surface. The mid-exponential-phase cells, on the other hand, have a more uniform charge distribution on the outer membrane, resulting in greater electrostatic repulsion and, subsequently, less adhesion. Our results suggest that the macromolecules responsible for this phenomenon are outer membrane-bound proteins and lipopolysaccharide-associated functional groups.     

Cell surface alterations induced by methylene blue and direct electric current in Escherichia coli.

Cell surface properties, including hydrophobicity, zeta potential, carbohydrate and fatty acid components, were altered on treatment of E. coli K12 with methylene blue (MB) and direct electric current (DC). The treatment of fimbriated E. coli cells with MB greatly increased the agglutination of yeast cells when compared to untreated bacteria. However, this increased agglutination was markedly reduced when the bacteria were treated with MB plus DC. These results suggest that MB modifies cell surface components in the absence of light and these alterations are more pronounced when cells are treated simultaneously with MB and DC.     

The effect of polycations on cell membrane stability and transport processes

Summary The interaction of poly-l-lysines of different molecular weights (PL) with Ehrlich ascites tumor cells was studied experimentally with respect to cell surface binding, cell electrophoresis, cytotoxicity and membrane permeability. Although they decrease the net negative charge of Ehrlich ascites cells similarly at low PL concentrations, low molecular weight PL was less cytotoxic and less damaging to the potassium transport mechanism than was high molecular weight PL. At certain PL concentrations, membrane damage was reversible on reincubation in PL-free media. The amount of bound polylysine as determined with fluorescent labeled polylysine was compared by electrophoresis to the amount of polylysine expressed on the electrokinetic surface. The results indicated that only a small fraction of polylysine bound to Ehrlich ascites tumor cells was electrokinetically detectable. The adsorption of polylysine to Ehrlich ascites tumor cells was not describable by the usual adsorption isotherms. It is suggested that the same number of monomeric lysine units of high and low molecular weight PL are adsorbed at the cell electrokinetic surface, but cytotoxicity is dependent on molecular weight. Although the negative charge of human red blood cells could be reversed at low PL concentrations, no such effect could be observed for ELD (a subline of Ehrlich ascites carcinoma) cells even at high PL concentrations. The relationship of PL binding to the stimulation of macromolecular uptake is discussed.     

Laminin and Fibronectin Treatment Leads to Generation of Dendritic Cells with Superior Endocytic Capacity

Background

Sampling the microenvironment at sites of microbial exposure by dendritic cells (DC) and their subsequent interaction with T cells in the paracortical area of lymph nodes are key events for initiating immune responses. Most of our knowledge of such events in human is based on in vitro studies performed in the absence of extracellular matrix (ECM) proteins. ECM in basement membranes and interstitial spaces of different tissues, including lymphoid organs, plays an important role in controlling specific cellular functions such as migration, intracellular signalling and differentiation. The aim of this study was, therefore, to investigate the impact of two abundant ECM components, fibronectin and laminin, on the phenotypical and functional properties of DC and how that might influence DC induced T-cell differentiation.

Methodology/Principal Findings

Human monocyte derived DC were treated with laminin and fibronectin for up to 48 hours and their morphology and phenotype was analyzed using scanning electron microscopy, flow cytometry and real time PCR. The endocytic ability of DC was determined using flow cytometry. Furthermore, co-culture of DC and T cells were established and T cell proliferation and cytokine profile was measured using H³-thymidine incorporation and ELISA respectively. Finally, we assessed formation of DC-T cell conjugates using different cell trackers and flow cytometry. Our data show that in the presence of ECM, DC maintain a ‘more immature’ phenotype and express higher levels of key endocytic receptors, and as a result become significantly better endocytic cells, but still fully able to mature in response to stimulation as evidenced by their superior ability to induce antigen-specific T cell differentiation.

Conclusion

These studies underline the importance of including ECM components in in vitro studies investigating DC biology and DC-T cell interaction. Within the context of antigen specific DC induced T cell proliferation, inclusion of ECM proteins could lead to development of more sensitive assays.     

Studies of Saccharomyces cerevisiae during fermentation--an in vivo electrokinetic investigation

An extensive in vivo study of the electrokinetic properties of six strains of the brewing yeast S. cerevisiae has been carried out. The yeasts were cultured under laboratory conditions. They were electrokinetically characterized by the electro-osmotic dipped cell technique, and data are presented as zeta-potentials. The effects of pH, fermentation time, successive fermentation cycles, and initial wort density have been established. The electrokinetic properties of an ale yeast which did not function correctly during commercial fermentation have also been examined. The results are discussed in the context of two controversial topics concerning the mechanism of yeast flocculation, the relative importance of yeast cell wall carboxyl and phosphate groups and the influence of wort components.     

The influence of phytohormones on zeta potential and electrokinetic charges of winter wheat cells

The zeta potential measurements of protoplasts obtained from winter wheat cell culture and phospholipid liposomes were performed to determine the electrokinetic charge in a medium containing various phytohormones (kinetin, 2,4-D and zearalenone) in absence and in presence of 2 x 10(-5) MCa2+. Calli were induced from immature inflorescences (inf) and embryos (emb) and cultured to obtain non-embryogenic (NE) and embryogenic (E) cell tissues. All investigated phytohormones indicate ability to adsorb to the negatively charged surfaces (latex, L88 - model negative adsorption site) both in water solutions and at the presence of mannitol and buffer (MES). In biological systems (protoplasts and liposomes - prepared from phospholipids of protoplasts) the electrokinetic charges were dependent on the phospholipid and protein composition of cells. The influence of protein groups on electrokinetic charge was calculated from charge values of protoplasts and liposomes, assuming additivity of surface charges. The comparison of calculated charges for protoplasts and liposomes indicate that 2,4-D is better adsorbed to the phospholipid and proteins of NE cells whereas kinetin is bound to the phospholipid and protein sites of E calli. This effect may be connected with embryogenesis process, where non-embryogenic culture of wheat requires 2,4-D in the medium, and embryogenic culture requires cytokinin rather. Zearalenone binding is especially dependent on the kind of explant.     

Comprehensive Analysis of Human Cells Motion under an Irrotational AC Electric Field in an Electro-Microfluidic Chip

AC electrokinetics is a versatile tool for contact-less manipulation or characterization of cells and has been widely used for separation based on genotype translation to electrical phenotypes. Cells responses to an AC electric field result in a complex combination of electrokinetic phenomena, mainly dielectrophoresis and electrohydrodynamic forces. Human cells behaviors to AC electrokinetics remain unclear over a large frequency spectrum as illustrated by the self-rotation effect observed recently. We here report and analyze human cells behaviors in different conditions of medium conductivity, electric field frequency and magnitude. We also observe the self-rotation of human cells, in the absence of a rotational electric field. Based on an analytical competitive model of electrokinetic forces, we propose an explanation of the cell self-rotation. These experimental results, coupled with our model, lead to the exploitation of the cell behaviors to measure the intrinsic dielectric properties of JURKAT, HEK and PC3 human cell lines.     

Motility of cultured fish epidermal cells in the presence and absence of direct current electric fields

The motile behavior and cytoskeletal structures of fish epidermal cells (keratocytes) in the presence and absence of direct current (DC) electric fields were examined. These cells spontaneously show highly directional locomotion in culture, migrating at rates of up to 1 micron/s. When DC electric fields between 0.5 and 15 V/cm are applied, single epidermal cells as well as cell clusters and cell sheets migrate towards the cathode. Cell clusters and sheets break apart into single migratory cells in the upper range of these field strengths. Cell shape and morphology are unaltered when the keratocytes are guided by an electric field. Neither the spontaneous locomotion nor the electrically guided motility were found to be microtubule dependent. 1 mM La3+, 10 mM Co2+, 50 microM verapamil, and 50 microM nitrendipine (calcium channel antagonists) reversibly inhibited lamellipod formation and cell locomotion in both spontaneously migrating and electrically guided cells. Ciba-Geigy Product 28392, which stimulates the opening of calcium channels, and is a competitive inhibitor of nitrendipine, has no effect on the locomotion of keratocytes. Cell motility was also unaffected by hyperpolarizing and depolarizing (low and high K+) media. It is argued that while a tissue cell may accommodate changes in resting membrane potential without becoming more or less motile, the cell may not be able to counterbalance the effects of depolarization and hyperpolarization simultaneously. In this context, a gradient of membrane potential, which is induced by an external DC electric field, will serve as a persistent stimulus for cell locomotion.     

Dielectrophoretic separation of cells: Continuous separation

Dielectrophoresis is the movement of particles in non-uniform alternating and direct current (AC, DC) electric fields. When nonuniform electric fields are created between microelectrodes, cells will redistribute themselves around the electrodes, the force holding the cells in place dependig on the local electric field and on the electrical properties of the cells themselves and the suspending medium. Steric drag forces produced by a gentle fluid flow in the chamber can be used to separate cells by selectively lifting cells from potential energy wells produced by the electric field. The technique is demonstrated in the batch separation of bacteria, yeast cells, and plant cells. Continuous separation and extraction of two cell types can be achieved by repeated reversing of the fluid flow direction in phase with the switching on and off of the applied voltage, and the efficacy of the technique is demonstrated for viable and nonviable (heat-treated) yeast cells. (c) 1995 John Wiley & Sons, Inc.     

Comparison of Flow Cytometry,Fluorescence Microscopy and Spectrofluorometry for Analysis of Gene Electrotransfer Efficiency

In this study, we compared three different methods used for quantification of gene electrotransfer efficiency: fluorescence microscopy, flow cytometry and spectrofluorometry. We used CHO and B16 cells in a suspension and plasmid coding for GFP. The aim of this study was to compare and analyse the results obtained by fluorescence microscopy, flow cytometry and spectrofluorometry and in addition to analyse the applicability of spectrofluorometry for quantifying gene electrotransfer on cells in a suspension. Our results show that all the three methods detected similar critical electric field strength, around 0.55 kV/cm for both cell lines. Moreover, results obtained on CHO cells showed that the total fluorescence intensity and percentage of transfection exhibit similar increase in response to increase electric field strength for all the three methods. For B16 cells, there was a good correlation at low electric field strengths, but at high field strengths, flow cytometer results deviated from results obtained by fluorescence microscope and spectrofluorometer. Our study showed that all the three methods detected similar critical electric field strengths and high correlations of results were obtained except for B16 cells at high electric field strengths. The results also demonstrated that flow cytometry measures higher values of percentage transfection compared to microscopy. Furthermore, we have demonstrated that spectrofluorometry can be used as a simple and consistent method to determine gene electrotransfer efficiency on cells in a suspension.     

A system for storage and retrieval of individual cells following flow cytometry.

A system has been developed to deposit cells in indexed locations on a gelatin-coated film following flow cytometry, allowing the measurements made of individual cells to be correlated with observed morphology or with subsequent microspectrophotometric measurements. Samples are deposited in a continuous track on the film by a deposition nib attached to the flow system below the observation point; laminar flow is preserved by adjusting the tape speed and the flow velocity. Locations of individual cells are indicated by etching the film with a spark triggered by the detection of a cell in the flow cytometer. After deposition, the film is dried by forced warm air. Cells on gelatin may be washed and restained with Papanicolaou and other stains with reasonable preservation of morphology. The system may be used for validation of automated cytodiagnostic procedures based on flow cytometry and for biomedical research.     

Quantitative study of electroporation-mediated molecular uptake and cell viability

Electroporation's use for laboratory transfection and clinical chemotherapy is limited by an incomplete understanding of the effects of electroporation parameters on molecular uptake and cell viability. To address this need, uptake of calcein and viability of DU 145 prostate cancer cells were quantified using flow cytometry for more than 200 different combinations of experimental conditions. The experimental parameters included field strength (0.1-3.3 kV/cm), pulse length (0.05-20 ms), number of pulses (1-10), calcein concentration (10-100 microM), and cell concentration (0.6-23% by volume). These data indicate that neither electrical charge nor energy was a good predictor of electroporation's effects. Instead, both uptake and viability showed a complex dependence on field strength, pulse length, and number of pulses. The effect of cell concentration was explained quantitatively by electric field perturbations caused by neighboring cells. Uptake was shown to vary linearly with external calcein concentration. This large quantitative data set may be used to optimize electroporation protocols, test theoretical models, and guide mechanistic interpretations.     

Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain.

A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a > 500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent. SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically > 10-fold brighter than that from intact organisms. Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli. Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility.     

Electrokinetic movement and biodegradation of 2,4-dichlorophenoxyacetic acid in silt soil

The coupling of electrokinetic movement of an organic contaminant, 2,4-dichlorophenoxyacetic acid (2,4-D), through soil and its biodegradation in situ has been demonstrated. In a first experiment, the direction and rate of movement of 2,4-D were determined using homogeneously contaminated soil (864 mg 2,4-D/kg dry weight soil) compacted into six individual compartments, 6 cm long, 3 cm wide, and 4 cm deep. Each compartment was bordered by a carbon felt anode and a stainless steel cathode. The application of a current density of 3.72 A/m(2) led to migration of 2,4-D towards the anode at a rate of approximately 4 cm/day. In a second experiment, electrokinetic movement and biodegradation were combined in situ. Sterilized silt soil contaminated with ring-labeled 14C-2,4-D (811 mg 2,4-D/kg dry weight soil) was compacted into a single soil compartment, 22 cm long, 7 cm wide, and 4 cm deep, in a 4.5 cm region adjacent to the cathode. The remainder of the compartment was filled with sterilized soil (to a total weight of 1,015 g). Burkholderia spp. RASC c2 (1.88 x 10(11) cells), a tetracycline-resistant bacterium with chromosomally encoded degradative genes for 2,4-D, was inoculated into the soil at a position 14-16 cm from the cathode. The reactor was placed within a sealed perspex box, with a constant air flow connected to sodium hydroxide traps. Under an applied current density of 0.89 A/m(2), the pollutant moved towards the bacteria. As it reached the inoculated region, its concentration decreased in the soil and 14CO2 was recovered in the traps. At the end of the experiment, 87.1% of radiolabel had been removed from the soil, 5.8% of which was recovered as 14CO2. A third, control, experiment showed a significant contrast in the absence of an electric current, where a slow rate of diffusion controlled the movement of both 2,4-D and bacteria in the soil and biodegradation occurred at the interface between the diffusing fronts.     

Modification of electrokinetic properties of nuclei in human buccal epithelial cells by electric fields

The influence of an alternating (50 Hz) electric field (5--110 V/cm) on the state of human buccal epithelium cells was studied by the methods of intracellular microelectrophoresis, heterochromatin staining with orcein, and indigo carmine staining for viability and membrane integrity evaluations. Electric field exposure induced an increase in electrophoretic mobility of cell nuclei, decreased numbers of heterochromatin granules near the inner membrane of cell nucleus, and induced cell membrane damage; but cell viability was conserved. Nuclear and cell membrane properties varied with electric field strength and age of the donors. The data obtained are interpreted as evidence of electric field induced activation of the functional state of nuclei.     

Electric Field-Assisted Delivery of Photofrin to Human Breast Carcinoma Cells

The influence of electroporation on the Photofrin uptake and distribution was evaluated in the breast adenocarcinoma cells (MCF-7) and normal Chinese hamster ovary cells (CHO) lacking voltage-dependent channels in vitro. Photofrin was used at a concentration of 5 and 25 μM. The uptake of Photofrin was assessed using flow cytometry and fluorescence microscopy methods. Cells viability was evaluated with crystal violet assay. Our results indicated that electropermeabilization of cells, in the presence of Photofrin, increased the uptake of the photosensitizer. Even at the lowest electric field intensity (700 V/cm) Photofrin transport was enhanced. Flow cytometry results for MCF-7 cells revealed ~1.7 times stronger fluorescence emission intensity for cells exposed to Photofrin and electric field of 700 V/cm than cells treated with Photofrin alone. Photofrin was effective only when irradiated with blue light. Our studies on combination of photodynamic reaction with electroporation suggested improved effectiveness of the treatment and showed intracellular distribution of Photofrin. This approach may be attractive for cancer treatment as enhanced cellular uptake of Photofrin in MCF-7 cells can help to reduce effective dose of the photosensitizer and exposure time in this type of cancer, diminishing side effects of the therapy.