Different Inactivation Behaviors of MS-2 Phage and Escherichia coli in TiO2 Photocatalytic Disinfection

Despite a wealth of experimental evidence concerning the efficacy of the biocidal action associated with the TiO₂ photocatalytic reaction, our understanding of the photochemical mechanism of this particular biocidal action remains largely unclear. It is generally accepted that the hydroxyl radical (·OH), which is generated on the surface of UV-illuminated TiO₂, plays the main role. However, our understanding of the exact mode of action of the hydroxyl radical in killing microorganisms is far from complete, and some studies report that other reactive oxygen species (ROS) (H₂O₂ and O₂·⁻, etc.) also play significant roles. In particular, whether hydroxyl radicals remain bound to the surface or diffuse into the solution bulk is under active debate. In order to examine the exact mode of action of ROS in inactivating the microorganism, we tested and compared the levels of photocatalytic inactivation of MS-2 phage and Escherichia coli as representative species of viruses and bacteria, respectively. To compare photocatalytic microbial inactivation with the photocatalytic chemical degradation reaction, para-chlorobenzoic acid, which rapidly reacts with a hydroxyl radical with a diffusion-limited rate, was used as a probe compound. Two different hydroxyl radical scavengers, tert-butanol and methanol, and an activator of the bulk phase hydroxyl radical generation, Fe²⁺, were used to investigate their effects on the photocatalytic mode of action of the hydroxyl radical in inactivating the microorganism. The results show that the biocidal modes of action of ROS are very different depending on the specific microorganism involved, although the reason for this is not clear. It seems that MS-2 phage is inactivated mainly by the free hydroxyl radical in the solution bulk but that E. coli is inactivated by both the free and the surface-bound hydroxyl radicals. E. coli might also be inactivated by other ROS, such as O₂·⁻ and H₂O₂, according to the present results.     

Enhancement of solar inactivation of Escherichia coli by titanium dioxide photocatalytic oxidation

AIMS: To improve solar water disinfection using a photocatalysing semi-conductor and to study the mechanisms involved in this process. METHODS AND RESULTS: Cells of Escherichia coli were used as the microbiological indicator to study the possibility of improving the efficiency of solar water disinfection using titanium dioxide (TiO2) as a photooxidizing semi-conductor. TiO2 was used either as a suspended powder or in an immobilized form. Both applications improved the efficiency of solar disinfection. TiO2 in suspension was more effective than the immobilized form, producing enhancement factors of 1.62 and 1.34, respectively. The concentration of TiO2 greatly affected efficiency, with a maximum effect at 1 mg ml(-1). Higher TiO2 concentrations reduced the efficiency. Dimethyl sulphoxide (DMSO) and cysteamine (Cys), hydroxyl radical (OH.) scavengers, were used to elucidate the mechanisms involved in the presence of TiO2. Both DMSO and Cys totally abolished the enhancing effect produced by the presence of TiO2. CONCLUSIONS: Sunlight has a potential water disinfecting capacity. The use of TiO2 greatly improved this efficiency. The effect of TiO2 was mainly concentration-dependent, giving maximum efficiency at 1 mg ml(-1). The presence of DMSO and Cys removed the TiO2-induced enhancement, indicating that OH. may be involved in the process of cell killing. SIGNIFICANCE AND IMPACT OF THE STUDY: The efficiency of solar disinfection is limited and time-consuming and needs to be improved. The use of a semi-conductor is promising as it reduces the time of exposure and therefore increases the efficiency of solar disinfection. This would allow for the availability of good quality water, and hence would improve the quality of life.     

Inactivation of MS-2 phage and poliovirus in groundwater

Since temperature affects the inactivation rate of viruses in natural water systems, the aim of this study was to determine if a temperature shift could influence the structural integrity of model viruses. When crude lysates of MS-2 phage were seeded into groundwater microcosms and incubated at 27 degrees C, complete virus inactivation took place in eight days. The temperature was then shifted to 4 degrees C. Three days after the temperature shift, a two-log increase in virus titer (reactivation) occurred. However, when purified MS-2 lysates were added to groundwater microcosms, no reactivation was obtained. No reactivation of poliovirus took place when similar microcosm experiments were done. The sedimentation coefficients of MS-2 shifted from 80S to 58S, 48S, 37S, 32S, and 18S as inactivation proceeded in groundwater and distilled water controls. Similarly, the sedimentation coefficients of polioviruses changed from 156S to 142S, 135S, 117S, 105S, 95S, and 80 S as inactivation took place. There was no correlation between % virus inactivation and % decrease in virions with intact sedimentation coefficients, as reported earlier for poliovirus inactivated by chlorine. The results presented support our hypothesis that virus inactivation proceeds gradually, involving the rearrangement and (or) loss of capsomere components that may eventually lead to the ejection of nucleic acids. The intermediate particles generated as inactivation proceeds may be in a reversibly inactivated state, and may revert back to a fully infectious state when chemical components stabilize the virus particle.     

Escherichia coli inactivation mechanism by pressurized CO2

The effects of pressurized CO2 on the survival of Escherichia coli and the mechanism of cell inactivation were studied. Bacterial cultures were inoculated in nutrient broth and incubated at 30 degrees C for 18 h. Exposure of the cells to CO2 under pressures ranging from 2.5 to 25 MPa and at temperatures between 8 and 40 degrees C was performed in a double-walled reactor with a 1 L capacity. The effect of the treatment on the cells was evaluated by plating and by transmission and scanning electron microscopy observation. Vapour CO2 generated a bacteriostatic effect. In liquid or supercritical state, CO2 provided a bactericidal effect. The bactericidal effect increased with pressure and temperature. The mechanism of cell inactivation by liquid CO2 involved two stages. First, cell stress caused by the CO2 penetration provoked cell wall collapse and cellular content precipitation. Second, the cell death caused by supercritical extraction of intracellular substances and cell envelope perforation resulted in leaking of intracellular constituents. In supercritical conditions, the cell inactivation process had one single phase: cellular death.     

TiO2 Photocatalysis Damages Lipids and Proteins in Escherichia coli

This study investigates the mechanisms of UV-A (315 to 400 nm) photocatalysis with titanium dioxide (TiO₂) applied to the degradation of Escherichia coli and their effects on two key cellular components: lipids and proteins. The impact of TiO₂ photocatalysis on E. coli survival was monitored by counting on agar plate and by assessing lipid peroxidation and performing proteomic analysis. We observed through malondialdehyde quantification that lipid peroxidation occurred during the photocatalytic process, and the addition of superoxide dismutase, which acts as a scavenger of the superoxide anion radical (O₂·⁻), inhibited this effect by half, showing us that O₂·⁻ radicals participate in the photocatalytic antimicrobial effect. Qualitative analysis using two-dimensional electrophoresis allowed selection of proteins for which spot modifications were observed during the applied treatments. Two-dimensional electrophoresis highlighted that among the selected protein spots, 7 and 19 spots had already disappeared in the dark in the presence of 0.1 g/liter and 0.4 g/liter TiO₂, respectively, which is accounted for by the cytotoxic effect of TiO₂. Exposure to 30 min of UV-A radiation in the presence of 0.1 g/liter and 0.4 g/liter TiO₂ increased the numbers of missing spots to 14 and 22, respectively. The proteins affected by photocatalytic oxidation were strongly heterogeneous in terms of location and functional category. We identified several porins, proteins implicated in stress response, in transport, and in bacterial metabolism. This study reveals the simultaneous effects of O₂·⁻ on lipid peroxidation and on the proteome during photocatalytic treatment and therefore contributes to a better understanding of molecular mechanisms in antibacterial photocatalytic treatment.     

Role of superinfecting phage in lysis inhibition with phage T4 in Escherichia coli

Rutberg, Blanka (Karolinska Institutet, Stockholm, Sweden), and Lars Rutberg. Role of superinfecting phage in lysis inhibition with phage T4 in Escherichia coli. J. Bacteriol. 90:891-894. 1965.-The ability of bacteriophage T4 to induce lysis inhibition upon superinfection was investigated after various treatments of the phage. This ability was found not to be a property of the external protein part of the phage, nor was it dependent on the functional and possibly structural integrity of the phage genetic material.     

Stepwise inactivation of Escherichia coli aspartokinase-homoserine dehydrogenase I

In the range of guanidine hydrochloride concentrations from 0.2 to 1.2 M, aspartokinase-homoserine dehydrogenase I loses its enzymatic properties, both kinase and dehydrogenase activities and their allosteric inhibition by L-threonine. Ligands which stabilize the tetrameric native structure protect the enzyme against inactivation. Under some conditions, all the functional properties do not disappear at the same rate: an intermediate species possessing only the kinase activity can be detected. Several arguments suggest that this partly active intermediate has a monomeric structure. These results show that deactivation of aspartokinase-homoserine dehydrogenase I is a stepwise process, compatible with the reverse of the previously described reactivation [Garel, J.-R., & Dautry-Varsat, A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3379-3383]. The same measurements performed with a monofunctional fragment carrying the dehydrogenase activity show that the loss of dehydrogenase activity is the same whether or not the polypeptide chain is intact or lacks the kinase region; this finding suggests that the protein is composed of independent regions. The influence of protein aggregation in studying unfolding-refolding of oligomeric enzymes is also discussed.