INTRODUCTION
General Introduction
One of the most important sources of water pollution, especially in urban areas, is sewage, which is made up of many diverse groups of microorganisms. Due to a continuous increase in the urban population, the threat of water pollution from sewage is alarming. Photosensitization appears to be promising under the current scenario, using an active substance, a photosensitizer that enhances the disinfection action of sunlight (Ergaieg and Seux, 2009).
In Type II mechanism, singlet oxygen is generated via an energy transfer process during a collision of the excited sensitizer with triplet oxygen (DeRosa and Crutchley, 2002; Kuznetsova et al., 2007). Besides singlet oxygen species, hydroxyl radicals and superoxide ions are also produced for disinfection. Hydroxyl is known to be very reactive, possibly more reactive than singlet oxygen especially in lipids, where singlet oxygen exhibits a long lifetime and is thought to be less reactive (Chen et al., 2011).
In order for photosensitive compounds to be used efficiently for the inactivation of microorganisms in water, in addition to understanding their mechanism of action, the effect of the nature and concentration of these compounds, pH, type of microorganisms on the disinfection efficiency should be examined. in detail.
Aim and objectives
The statistical interpretation of the results is in the form of analysis of variance (ANOVA), student's t-test, p-value, F-value, etc. The experimental design and the range of the parameters used were as defined in Table 5. Experiments were designed using concentrations of the components as 0 and +1. The components and their concentrations used are summarized in Table 7.
Since the increase in the pH of the cell suspension also showed a significant effect on photo-inactivation, but it depended on the levels of the combination of two other factors, the initial concentration of MB and the initial number of viable cells. The accuracy and precision of the models, in the form of coefficient of determination (R2), adjusted R2, standard deviation (SD) and predicted residual error sum of squares (PRESS) are presented in Table 4.1. The accuracy and precision of the models, in the form of coefficient of determination (R2), adjusted R2, standard deviation (SD) and predicted residual error sum of squares (PRESS), suggest that the models were very efficient in predicting photo- experimental inactivation. results (Mahanty et al., 2010).
Statistical analysis of the results showed that MB has a significant effect in the case of Gram-positive E.
Organization of thesis
LITERATURE REVIEW
Water pollution
- Sources of pollution
ANOVA (Table 4.5) of the photoinactivation results obtained at a 30-minute incubation period in the dark with a high Fischer 'F' value and a low probability 'P' value of the regression model indicates its validity in explaining the variation in the results. To better understand the combined effect of PS on the inactivation of these two bacteria, we performed statistical analysis of the results in the form of analysis of variance (ANOVA) and student's 't' test.
Microorganism in water
- Classes of microorganism
- Indicator microorganism
- Tests for viability
- Direct methods
- Indirect methods
Water disinfection
- Physical disinfection
- Chemical disinfection
- Disinfection by-products and other emerging contaminants of
In general, no harmful byproducts were created during the process. 2012) studied the efficiency of UV radiation for the inactivation of Aspergillus sp. 2013) studied UV disinfection on both total heterotrophic bacteria and antibiotic-resistant bacteria present in secondary effluent samples from a municipal wastewater treatment plant. 2014) studied the inactivation of bacteria and yeasts at different growth stages under controlled temperature by high-frequency ultrasound.
The chlorine compounds commonly used for wastewater disinfection are chlorine, sodium hypochlorite, calcium hypochlorite and chlorine dioxide (Winward et al., 2008). They reported sequential disinfection, which showed that synergistic effect was most effective for E. coli inactivation due to the presence of low levels of chlorine dioxide. 2014) investigated chlorine dioxide effect on cell integrity, toxin degradation and disinfection by product formation of Microcystis aeruginosa.
2014) investigated the effectiveness of ozonation on the removal of organic residues and pathogenic microorganisms from municipal secondary wastewater.
Photosensitization
- Types of photosensitizers
- Organic dyes and aromatic hydrocarbons
- Porphyrins, pthalocyanines and related tetrapyrroles
- Transition metal complexes
- Lasers
- Role of Oxygen
- Properties of Singlet Oxygen
- Mechanism of Microbial Inactivation
Several groups of molecules that absorb in the UV-vis range have shown the ability to generate singlet oxygen. Methylene blue belonging to the phenothiazinium group has a strong absorption in the range 550-700 nm with a quantum yield of 0.52, while the absorption bands of xanthene dyes such as rose bengal and eosin lie in the green region of the visible spectrum (480-550 pm) (De Rosa and Crutchley, 2002). Lee and Rodgers (2008) studied the laser flash photolysis for the production of triplet state of the xanthene dye and rose bengal and their reaction with oxygen to form singlet oxygen and superoxide anion radical.
This difference in the organism's behavior was explained on the basis of charge and the structure of the bacterial cell wall. At low pH, triplet methylene blue (3MB+) is expected to be in the protonated. 3MBH2+ is formed before it reacts with oxygen to generate singlet oxygen. The obtained results showed decrease in the survival of the bacteria with the increase in RB concentration and illumination time.
The properties of porphyrins depend on the substituents of the macrocycle, metal ions coordinated at its center and ligands attached to the axial positions of the metal ion. Therefore, they concluded that the rate of bacteriophage photoinactivation and the efficiency of the photosensitizer vary with the charge and the substituents in the meso positions of the porphyrin macrocycle. 1992) investigated the photosensitizing efficacy of deuteroporphyrin (DP) in the presence of the polycationic agent polymyxin nonapeptide (PMNP) on Escherichia coli and Pseudomonas aeruginosa. Most of the studies involve organic molecules for the production of singlet oxygen in photosensitization, but some inorganic complexes have been reported to be effective photosensitizers.
The temperature range depends on the type of filament (strip, spiral or double spiral) and the strength of the passing current. The light emitted by these lamps must be filtered to narrow the spectral band in the absorption maximum of the photosensitizer. When using these lamps, it is necessary to use filters to narrow the emission in the visible range, because most photosensitizers have a maximum absorption in this range, and it is also good that UV-B causes DNA destruction in bacterial cells.
Photosensitization starts with the absorption of a photon of light by a sensitizer, which results in the excitation of the molecules from the ground state (S0) to the extremely unstable excited singlet state (S1) with a half-life in the range s. These states differ from each other only by the structure of the π-antibonding orbitals in the electronic configurations.
Design of Experiments
- Factorial design of Experiments
- Plackett Burman design of Experiment
- Analysis of Variance
- Student ‘t’ Test
The design of the experiment and the range of parameters used were defined in Table 3.3 and Table 3.4, respectively. In Table 4.1, which presents the ANOVA of the photoinactivation results obtained at a 30-minute incubation period in the dark, the high Fischer 'F' value and low probability 'P' value of the regression model indicate its validity in explaining the variation in the results. Lipid peroxidation and protein carbonyl index results (Table 4.8), which showed that changes occur in membrane lipids and proteins that cause cell damage.
MATERIALS AND METHODS
Chemicals and reagents
Photosensitive compounds used in this study, methylene blue (MB) and sodium anthraquinone-2-sulfonate (SAQS), were purchased from Sigma Aldrich, Germany. Phosphate buffer salt components (NaCl, KCl, Na2HPO4 and KHPO4) and agar were purchased from Merck, India. Analytical reagents propidium iodide, dihydrochlorofluorescin diacetate (DCFDA), tricarboxylic acid (TCA), guanidine hydrochloride, and 2,4-dinitrophenylhydrazine (DNPH) were purchased from Sigma Aldrich, Germany.
Microorganisms and its maintenance
Seed culture preparation
Photoinactivation of E. coli and E. hirae in aqueous solution using
- Effect of concentration of photosensitizer, pH of solution and
- Effect of dark incubation on photo inactivation efficiency
- Studies on cell death using Flow cytometry
- Mechanism of photo inactivation of bacteria
- Reactive Oxygen Species (ROS) determination
- Lipid peroxidation assay
- Estimation of Protein carbonyl
Viable cells in the culture plates were counted using the colony counting method (Vilela et al. 2012), which involves counting distinct viable colonies using a colony counter. The mean results of percent inactivation of microorganisms from each duplicate run in the study were calculated according to the following equation. Photo-inactivation experiments were performed to first study the combined effect of methylene blue concentration, cell suspension pH and initial number of viable cells (dilution) using the statistically valid full factorial design of experiment.
The serially diluted suspension (10 μl) was then spread on agar plates and incubated for 24 h at 37°C. Photoinactivation of microorganisms requires dark incubation with constant shaking to obtain a homogeneous mixture of bacterial suspension and the light-sensitive dye (Ergaieg and Seux, 2009). Therefore, to investigate the effect of the dark incubation period on photoinactivation using methylene blue, three different periods of dark incubation (5, 15 and 30 minutes) with constant shaking on a gel shaker were investigated.
For each set of experiments, the concentration of Methylene Blue was in the range of 0.73 μmol/l to 1.25 μmol/l, the dilution was up to thousands of times, and the pH was in the range of 7.5 to 9.00. To confirm the microbial inactivation results obtained from the photoinactivation experiments using the colony counting method, the flow cytometry method was used. To gain further insight into the mechanism of photoinactivation analysis, reactive oxygen species (ROS), lipid peroxidation and protein carbonyl index were performed.
Sample from each photoinactivation experimental run as presented in Table 2 was added with 20μl of 20μM dihydrochlorofluorescein diacetate (DCFDA) and incubated for 30 minutes at 37°C. The suspension was then added with MB and kept on a gel tumbler in the dark for 30 minutes. The suspension was checked for 2,7 dichlorofluorescin (DCF) fluorescence by excitation at 488nm and emission spectra were analyzed in the range 510-540 nm using Fluoromax 4.
Bacterial cell suspension was prepared and inactivation experiments were performed as described in section 3.4. The treated bacterial cells were obtained in the form of pellets and the lipid peroxidation products of the cell lysate were determined as reactive substances to thiobarbituric acid (Trivedi, 2005).
Photoinactivation of E. coli and E. hirae in aqueous solution using
- Effect of Concentration of Photosensitizer, pH of Solution,
- Mechanism of photo inactivation
- Statistical analysis
In Table 4.11, which presents ANOVA of photoinactivation results obtained for combined effect of PS, the high Fischer's 'F' value (9.86) and a low probability 'P' value (0.004) of the regression model indicate its validity in explaining the variations in the results. This is reported in the form of shift in the fluorescence peak of the propidium iodide with increasing concentration of the PS. Statistical analysis of the results revealed that besides the significant individual effect due to concentration of PS, pH of bacterial.
SUMMARY AND CONCLUSIONS
