Molecular monitoring of inactivation efficiencies of bacteria during pulsed electric field treatment of clinical wastewater.

AIMS: The applicability of an alternative wastewater disinfection concept based on the pulsed electric field (PEF) treatment is tested with molecular biology techniques using clinical wastewaters. METHODS AND RESULTS: Hospital wastewater was treated with the PEF technology. The inactivation efficiencies of bacteria were successfully monitored with real-time polymerase chain reaction (PCR). As the differentiation between living and dead bacterial cells is important for the determination of the disinfection efficiency, propidium monoazide (PMA) was applied. PMA selectively penetrates cells with compromised membranes and intercalates into the DNA inhibiting a subsequent PCR amplification. The rates of reduction were examined for specific pathogens and wastewater populations using PCR-denaturing gradient gel electrophoresis. The results showed that the main part of the bacterial population could be inactivated efficiently with the PEF treatment. Moreover, it was demonstrated that naturally occurring nuclease activities were not affected by the PEF treatment in contrast to a thermal treatment. CONCLUSIONS: The results indicated that the PEF treatment is an appropriate alternative disinfection concept for the treatment of clinical wastewaters and surpass the disadvantages of other disinfection methods. SIGNIFICANCE AND IMPACT OF THE STUDY: With the use of propidium monoazide for live-dead distinction, a new concept could be developed for the evaluation of disinfection methods.

Effects of cetyltrimethylammonium bromide (CTAB) surfactant upon the dielectric properties of yeast cells.

The dielectric properties of yeast cells in the absence and presence of cetyltrimethylammonium bromide (CTAB) were investigated. The surfactant concentration range was between 0.0 and 1.0 mM. The experimental permittivity and conductivity spectra of frequency were analyzed by means of the two-shell electrical cell model (Irimajiri et al., Bull. Inst. Chem. Res., Kyoto Univ. 69 (1991) 421-438), and the electrical phase parameters of cells were subsequently evaluated. The cytoplasm conductivity and the conductivity of the vacuole interior decreased drastically by treating the cells with surfactant. The apparent capacitance of the plasma membrane increased systematically from 0.65 microF/cm2, for untreated cells, up to about 0.75 microF/cm2, at 0.3 mM CTAB. This growth was ascribed to the increase in the folding of the membrane surface associated with the surfactant-induced cell shrinkage. A further addition of the surfactant entailed a gradual decrease of the capacitance that was assigned to the membrane solubilization by the surfactant molecules. Within the accuracy of the data, the specific capacitance of the vacuole membrane was nearly constant (0.544+/-0.021 microF/cm2) over the whole surfactant concentration range. Also, the cytoplasm permittivity remained constant at 64.3+/-4.5.

Non-linearity of molecular transport through human skin due to electric stimulus.

The multilamellar bilayer system of the skin's stratum corneum (SC) provides the main barrier to transdermal transport of ions and charged molecules. Electrically driven transport of charged species at low trans-SC voltages (U(SC)<5 V) occurs predominantly via pre-existing aqueous pathways. In contrast, high voltage, (HV; U(SC)>50 V) has been hypothesized to involve electroporation within the SC's multilamellar bilayer membranes, creating new aqueous pathways that contribute to a rapid, large increase in transport. Thus, it might be expected that HV-pulses would always increase subsequent iontophoresis. Here we show, however, that for some charged molecules the opposite occurs, because the low skin resistance due to new aqueous pathways leads to an actual decrease in U(SC) for the same applied current, and the transport of some, highly charged molecules has a highly nonlinear dependence on U(SC).

Surface area involved in transdermal transport of charged species due to skin electroporation.

The electroporative effect on the stratum corneum (SC) is highly localized. However, the fractional area for the transport of small ions and larger ionic species differs considerably during and after high voltage (HV) application. Electroporation of SC creates new aqueous pathways, accessible for small ions, such as Cl(-) and Na(+) ions. The pores are distributed across the skin surface yielding a fractional area for current flow during electroporation of up to 0.1%. An increased permeability after high voltage application persists within a fractional area on the order of 10(-3)%. The permeabilization of SC for larger, charged molecules (M > 200 g/mol) involves Joule heating and a phase transition of the long chain sphingolipids within local transport regions (LTR). The transport area for these molecules (approximately 10(-3)%) changes only negligibly after high voltage application.

Perturbation of human skin due to application of high voltage.

Electroporation is believed to be the effect that greatly enhances the transport of water-soluble molecules across the stratum corneum (SC) by application of short high voltage pulses. However, electroporation was originally a phenomenon investigated at the level of cell and model membranes, which is only partially comparable to the complicated structure of the stratum corneum. Here, we show, that electroporation is accompanied by other effects, which may be primarily involved in creation of new pathways and altering existing pathways, respectively. Experimental evidence shows that the dramatic increase in skin permeability is due to synergistic effect of electric field and heating by high local current density. Heating starts at small spots, not related to a visible skin structure and results in a propagating heat front. The phase transition of the SC lipids plays a major role in skin permeability during the pulse. The permeability after a high voltage pulse correlates well with the surface area showing a permanent low electrical resistance after pulsing. The main transport of water-soluble molecules is facilitated by the electric field due to the electrophoretic driving force in conjunction with the high permeability due to the breakdown of the multilamellar system of the SC lipids.

Overcoming electrically induced artifacts in penetration studies with fluorescent tracers.

The use of model molecules in transdermal transport studies reveals transport behavior while providing an economical setup by detection of readily measured quantities such as fluorescence, radioactivity or absorbance of low-cost substances. Water soluble fluorescent tracers such as calcein have been repeatedly used as model molecules in transdermal transport studies. However, if electrically enhanced calcein transport across the human skin barrier is measured, artifacts due to interaction between calcein and electrode byproducts influence the result. Here, we describe an experimental setup which avoids known artifacts and makes calcein or other fluorescent tracers a suitable model for transdermal transport studies.