Disinfection of Water Containing Natural Organic Matter by Using Ozone-Initiated Radical Reactions

Ozone is widely used to disinfect drinking water and wastewater due to its strong biocidal oxidizing properties. Recently, it was reported that hydroxyl radicals (^·OH), resulting from ozone decomposition, play a significant role in microbial inactivation when Bacillus subtilis endospores were used as the test microorganisms in pH controlled distilled water. However, it is not yet known how natural organic matter (NOM), which is ubiquitous in sources of drinking water, affects this process of disinfection by ozone-initiated radical reactions. Two types of water matrix were considered for this study. One is water containing humic acid, which is commercially available. The other is water from the Han River. This study reported that hydroxyl radicals, initiated by the ozone chain reaction, were significantly effective at B. subtilis endospore inactivation in water containing NOM, as well as in pH-controlled distilled water. The type of NOM and the pH have a considerable effect on the percentage of disinfection by hydroxyl radicals, which ranged from 20 to 50%. In addition, the theoretical T value of hydroxyl radicals for 2-log B. subtilis removal was estimated to be about 2.4 × 10⁴ times smaller than that of ozone, assuming that there is no synergistic activity between ozone and hydroxyl radicals.     

Comparison of the inactivation of Bacillus subtilis spores and MS2 bacteriophage by MIOX, ClorTec and hypochlorite

AIMS: To compare the disinfection ability of two widely used electrolytic generation systems (ClorTec and MIOX) and the conventional chlorine disinfectant (sodium hypochlorite) using three strains of Bacillus subtilis spores and MS2 bacteriophage. METHODS AND RESULTS: Three B. subtilis aerobic spore strains (ATCC1A1, 35021 and 35946) and the bacteriophage MS2 (ATCC 15597-B1) were propagated and sporulated. Four indicator organisms were exposed to four disinfectant treatments for comparing the effectiveness of inactivation: hypochlorite, ClorTec, MIOX and MIOX-anode. The results indicated that the two electrolytic generation systems were as effective as the conventional chlorination for the inactivation of micro-organisms used. Some data points showed the variation using anova analysis, in which the inactivation of MIOX and ClorTec was higher than that of hypochlorite. CONCLUSIONS: The ClorTec and MIOX systems are quite similar to hypochlorite in the inactivation-effectiveness for aerobic spores and bacteriophage in drinking water. SIGNIFICANCE AND IMPACT OF THE STUDY: Laboratory-scale investigation proved that gaseous chlorine could be replaced by either ClorTec or MIOX systems for the drinking water treatment utilities, which still could maintain the same disinfection efficiency.     

Solar disinfection of drinking water contained in transparent plastic bottles : characterizing the bacterial inactivation process

A series of experiments is reported to identify and characterize the inactivation process in operation when drinking water, heavily contaminated with a Kenyan isolate of Escherichia coli , is stored in transparent plastic bottles that are then exposed to sunlight. The roles of optical and thermal inactivation mechanisms are studied in detail by simulating conditions of optical irradiance, water turbidity and temperature, which were recorded during a series of solar disinfection measurements carried out in the Kenyan Rift Valley. Optical inactivation effects are observed even in highly turbid water (200 ntu) and at low irradiances of only 10 mW cm⁻². Thermal inactivation is found to be important only at water temperatures above 45 °C, at which point strong synergy between optical and thermal inactivation processes is observed. The results confirm that, where strong sunshine is available, solar disinfection of drinking water is an effective, low cost method for improving water quality and may be of particular use to refugee camps in disaster areas. Strategies for improving bacterial inactivation are discussed.     

Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection

Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log(10) units) with LP-UV (20 mJ/cm(2)) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log(10) unit being inactivated. These results demonstrate the inability of MIOX to inactivate Cryptosporidium. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water.     

Comparative inactivation of poliovirus type 3 and MS2 coliphage in demand-free phosphate buffer by using ozone.

MS2 coliphage (ATCC 15597-B1) has been proposed by the U.S. Environmental Protection Agency as a surrogate for enteric viruses to determine the engineering requirements of chemical disinfection systems on the basis of previous experience with chlorine. The objective of this study was to determine whether MS2 coliphage was a suitable indicator for the inactivation of enteric viruses when ozone disinfection systems were used. Bench-scale experiments were conducted in 2-liter-batch shrinking reactors containing ozone demand-free 0.05 M phosphate buffer (pH 6.9) at 22 degrees C. Ozone was added as a side stream from a concentrated stock solution. It was found that an ozone residual of less than 40 micrograms/liter at the end of 20 s inactivated greater than 99.99% of MS2 coliphage in the demand-free buffer. When MS2 was compared directly with poliovirus type 3 in paired experiments, 1.6 log units more inactivation was observed with MS2 coliphage than with poliovirus type 3. It was concluded that the use of MS2 coliphage as a surrogate organism for studies of enteric virus with ozone disinfection systems overestimated the inactivation of enteric viruses. It is recommended that the regulatory agencies evaluate their recommendations for using MS2 coliphage as an indicator of enteric viruses.     

Comparative inactivation of poliovirus type 3 and MS2 coliphage in demand-free phosphate buffer by using ozone.

MS2 coliphage (ATCC 15597-B1) has been proposed by the U.S. Environmental Protection Agency as a surrogate for enteric viruses to determine the engineering requirements of chemical disinfection systems on the basis of previous experience with chlorine. The objective of this study was to determine whether MS2 coliphage was a suitable indicator for the inactivation of enteric viruses when ozone disinfection systems were used. Bench-scale experiments were conducted in 2-liter-batch shrinking reactors containing ozone demand-free 0.05 M phosphate buffer (pH 6.9) at 22 degrees C. Ozone was added as a side stream from a concentrated stock solution. It was found that an ozone residual of less than 40 micrograms/liter at the end of 20 s inactivated greater than 99.99% of MS2 coliphage in the demand-free buffer. When MS2 was compared directly with poliovirus type 3 in paired experiments, 1.6 log units more inactivation was observed with MS2 coliphage than with poliovirus type 3. It was concluded that the use of MS2 coliphage as a surrogate organism for studies of enteric virus with ozone disinfection systems overestimated the inactivation of enteric viruses. It is recommended that the regulatory agencies evaluate their recommendations for using MS2 coliphage as an indicator of enteric viruses.     

Bactericidal effect of solar water disinfection under real sunlight conditions

Batch solar disinfection (SODIS) inactivation kinetics are reported for suspensions in water of Campylobacter jejuni, Yersinia enterocolitica, enteropathogenic Escherichia coli, Staphylococcus epidermidis, and endospores of Bacillus subtilis, exposed to strong natural sunlight in Spain and Bolivia. The exposure time required for complete inactivation (at least 4-log-unit reduction and below the limit of detection, 17 CFU/ml) under conditions of strong natural sunlight (maximum global irradiance, approximately 1,050 W m(-2) +/- 10 W m(-2)) was as follows: C. jejuni, 20 min; S. epidermidis, 45 min; enteropathogenic E. coli, 90 min; Y. enterocolitica, 150 min. Following incomplete inactivation of B. subtilis endospores after the first day, reexposure of these samples on the following day found that 4% (standard error, 3%) of the endospores remained viable after a cumulative exposure time of 16 h of strong natural sunlight. SODIS is shown to be effective against the vegetative cells of a number of emerging waterborne pathogens; however, bacterial species which are spore forming may survive this intervention process.     

Vitamin C (ascorbic acid) induced hydroxyl radical formation in copper contaminated household drinking water: role of bicarbonate concentration

We have previously shown that Vitamin C (ascorbic acid) can trigger hydroxyl radical formation in copper contaminated household drinking water. We report here that the capacity of ascorbic acid to catalyze hydroxyl radical generation in the drinking water samples is strongly dependent on the bicarbonate concentration (buffer capacity and pH) of the samples. We found that at least 50 mg/l bicarbonate was required in the water samples to maintain the pH over 5.0 after ascorbic acid addition. At this pH, that is higher than the pKa

1

4.25 of ascorbic acid, a hydroxyl radical generating redox cycling reaction involving the mono-anion of vitamin C and copper could take place. The ascorbic acid induced hydroxyl radical generating reaction could easily be mimicked in Milli-Q water by supplementing the water with copper and bicarbonate. Our results demonstrate that ascorbic acid can induce a pH dependent hydroxyl radical generating reaction in copper contaminated household tap water that is buffered with bicarbonate. The impact of consuming ascorbic acid together with copper and bicarbonate containing drinking water on human health is discussed.     

Modelling the combined effects of pH,temperature and sodium chloride stresses on the thermal inactivation of Bacillus subtilis spores in a buffer system

AIMS: The inactivation of Bacillus subtilis 168 spores subjected to the combined stress of pH, temperature and sodium chloride in a buffer system was modelled. METHODS AND RESULTS: Bacillus subtilis 168 spore suspension in 50 mmol l-1 potassium phosphate buffer was heated in an open system using a block heater. A second order polynomial equation was used to describe the relationship between pH, temperature, sodium chloride concentration and the logarithm of the decimal reduction time (D-value) of the spores. Response surface graphs were constructed to predict the inactivation within the experimental domain. The data obtained were also compared with those reported for B. subtilis in different media and foods included in a large reference-based database of thermal inactivation (ThermoKill Database, TKDB R9100), which was constructed in the laboratory. CONCLUSIONS: All the variables studied seemed to have a significant effect on the inactivation of B. subtilis 168 spores in potassium phosphate buffer. The coefficient of determination, r2, and an analysis of the residuals from the model indicated the adequacy of the model to predict the inactivation of B. subtilis spores within the range of the experimental variables studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this study will enable a better understanding of the inactivation of B. subtilis spores under the influence of the studied environmental variables. The model can be used by food industries to assess and monitor the shelf life of food products in the event of a chance contamination by B. subtilis spores.     

Role of oxyradicals in the inactivation of catalase by ozone

The antioxidant enzymes, catalase and superoxide dismutase, are inactivated upon exposure to ozone. In this study, the mechanism of this inactivation was examined using catalase as a model system. The data show that the inactivation of catalase is dependent on ozone concentration, time of exposure, and pH. Loss of catalase activity is accompanied with loss of the heme spectra. Tiron, desferal-Mn, trolox-c, and pyruvate protect the enzyme against ozone inactivation. SOD is less effective due to its inactivation by ozone. On the other hand, alcohols do not provide significant protection. The data suggest the possible involvement of superoxide radicals in the inactivation of catalase by ozone.     

Cell Culture-Taqman PCR Assay for Evaluation of Cryptosporidium parvum Disinfection

Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log₁₀ units) with LP-UV (20 mJ/cm²) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log₁₀ unit being inactivated. These results demonstrate the inability of MIOX to inactivate Cryptosporidium. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water.     

Leukotriene B4, C4, D4 and E4 inactivation by hydroxyl radicals

Leukotriene B4 chemotactic activity and leukotriene C4, D4 and E4 slow reacting substance activity were rapidly decreased by hydroxyl radicals generated by two different iron-supplemented acetaldehyde-xanthine oxidase systems. At low Fe2+, leukotriene inactivation was inhibited by catalase, superoxide dismutase, mannitol and ethanol, suggesting involvement of hydroxyl radicals generated by the iron-catalyzed interaction of superoxide and H2O2 (Haber-Weiss reaction). Leukotriene inactivation increased at high Fe2+ concentrations, but was no longer inhibitable by superoxide dismutase, suggesting that inactivation resulted from a direct interaction between H2O2 and Fe2+ to form hydroxyl radicals (Fenton reaction). The inactivation of leukotrienes by hydroxyl radicals suggests that oxygen metabolites generated by phagocytes may play a role in modulating leukotriene activity.     

Inactivation of Escherichia coli by photochemical reaction of ferrioxalate at slightly acidic and near-neutral pHs

Fenton chemistry, which is known to play an effective role in degrading toxic chemicals, is difficult to apply to disinfection in water treatment, since its reaction is effective only at the acidic pH of 3. The presence of oxalate ions and UV-visible light, which is known as a photoferrioxalate system, allows the Fe(III) to be dissolved at slightly acidic and near-neutral pHs and maintains the catalytic reaction of iron. This study indicates that the main oxidizing species in the photoferrioxalate system responsible for microorganism inactivation is OH radical. Escherichia coli was used as an indicator microorganism. The CT value (OH radical concentration x contact time; used to indicate the effect of the combination of the concentration of the disinfectant and the contact time on inactivation) for a 2-log inactivation of E. coli was approximately 1.5 x 10(-5) mg/liter/min, which is approximately 2,700 times lower than that of ozone as estimated by the delayed Chick-Watson model. Since the light emitted by the black light blue lamp is similar to sunlight in the specific wavelength range of 300 to 420 nm, the photoferrioxalate system, which can have a dual function, treating water for both organic pollutants and microorganisms simultaneously, shows promise for the treatment of water or wastewater in remote or rural sites. However, the photoferrioxalate disinfection system is slower in inactivating microorganisms than conventional disinfectants are.     

Disinfection of model indicator organisms in a drinking water pilot plant by using PEROXONE

PEROXONE is an advanced oxidation process generated by combining ozone and hydrogen peroxide. This process stimulates the production of hydroxyl radicals, which have been shown to be superior to ozone for the destruction of some organic contaminants. In this study, pilot-scale experiments were conducted to evaluate the microbicidal effectiveness of PEROXONE and ozone against three model indicator groups. Escherichia coli and MS2 coliphage were seeded into the influent to the preozonation contactors of a pilot plant simulating conventional water treatment and were exposed to four ozone dosages (0.5, 1.0, 2.0, and 4.0 mg/liter), four hydrogen peroxide/ozone (H2O2/O3) weight ratios (0, 0.3, 0.5, and 0.8), and four contact times (4, 5, 12, and 16 min) in two source waters--Colorado River water and state project water--of different quality. The removal of heterotrophic plate count bacteria was also monitored. Results of the study indicated that the microbicidal activity of PEROXONE was greatly affected by the applied ozone dose, H2O2/O3 ratio, contact time, source water quality, and type of microorganism tested. At contact times of 5 min or less, ozone alone was a more potent bactericide than PEROXONE at all H2O2/O3 ratios tested. However, this decrease in the bactericidal potency of PEROXONE was dramatic only as the H2O2/O3 ratio was increased from 0.5 to 0.8. The fact that the bactericidal activity of PEROXONE generally decreased with increasing H2O2/O3 ratios was thought to be related to the lower ozone residuals produced. The viricidal activity of PEROXONE and ozone was comparable at all of the H2O2/O3 ratios. Heterotrophic plate count bacteria were the most resistant group of organisms. Greater inactivation of E. coli and MS2 was observed in Colorado River water than in state project water and appeared to result from differences in the turbidity and alkalinity of the two waters. Regardless of source water, greater than 4.5 log10 of E. coli and MS2 was inactivated at an applied ozone dosage of 2.0 mg/liter (and a 4-min contact time) when the H2O2/O3 ratio was less than or equal to 0.5. Comparative disinfection experiments indicated that free chlorine was the most potent bactericidal agent, followed (in descending order of effectiveness) by ozone, PEROXONE, and chloramines. These results indicate that the PEROXONE process must be optimized for each source water to achieve microbicidal effectiveness.     

Disinfection of model indicator organisms in a drinking water pilot plant by using PEROXONE.

PEROXONE is an advanced oxidation process generated by combining ozone and hydrogen peroxide. This process stimulates the production of hydroxyl radicals, which have been shown to be superior to ozone for the destruction of some organic contaminants. In this study, pilot-scale experiments were conducted to evaluate the microbicidal effectiveness of PEROXONE and ozone against three model indicator groups. Escherichia coli and MS2 coliphage were seeded into the influent to the preozonation contactors of a pilot plant simulating conventional water treatment and were exposed to four ozone dosages (0.5, 1.0, 2.0, and 4.0 mg/liter), four hydrogen peroxide/ozone (H2O2/O3) weight ratios (0, 0.3, 0.5, and 0.8), and four contact times (4, 5, 12, and 16 min) in two source waters--Colorado River water and state project water--of different quality. The removal of heterotrophic plate count bacteria was also monitored. Results of the study indicated that the microbicidal activity of PEROXONE was greatly affected by the applied ozone dose, H2O2/O3 ratio, contact time, source water quality, and type of microorganism tested. At contact times of 5 min or less, ozone alone was a more potent bactericide than PEROXONE at all H2O2/O3 ratios tested. However, this decrease in the bactericidal potency of PEROXONE was dramatic only as the H2O2/O3 ratio was increased from 0.5 to 0.8. The fact that the bactericidal activity of PEROXONE generally decreased with increasing H2O2/O3 ratios was thought to be related to the lower ozone residuals produced. The viricidal activity of PEROXONE and ozone was comparable at all of the H2O2/O3 ratios. Heterotrophic plate count bacteria were the most resistant group of organisms. Greater inactivation of E. coli and MS2 was observed in Colorado River water than in state project water and appeared to result from differences in the turbidity and alkalinity of the two waters. Regardless of source water, greater than 4.5 log10 of E. coli and MS2 was inactivated at an applied ozone dosage of 2.0 mg/liter (and a 4-min contact time) when the H2O2/O3 ratio was less than or equal to 0.5. Comparative disinfection experiments indicated that free chlorine was the most potent bactericidal agent, followed (in descending order of effectiveness) by ozone, PEROXONE, and chloramines. These results indicate that the PEROXONE process must be optimized for each source water to achieve microbicidal effectiveness.     

Sterilization effect of atmospheric plasma on Escherichia coli and Bacillus subtilis endospores

Aims:  Escherichia coli and Bacillus subtilis spores were treated with an atmospheric plasma mixture created by the ionization of helium and oxygen to investigate the inactivation efficiency of a low-temperature plasma below 70°C.
Methods and results:  An electrical discharge plasma was produced at a radio frequency (RF) of 13·56 MHz, connected to a perforated circular electrode with a discharge spacing of 1–15 mm. The discharge gas was helium with 0–2% oxygen. For the plasma treatment, a dried E. coli cell or B. subtilis endospore suspension on a cover-glass was exposed to oxygen downstream of the plasma from holes in an RF-powered electrode. The sterilization effect of the RF plasma was highest with 0·2% oxygen, corresponding to the maximum production of oxygen radicals.
Conclusions:  Oxygen radicals generated by RF plasma are effective for the destruction of bacterial cells and endospores.
Significance and Impact of the study:  Low-temperature atmospheric plasma can be used for the disinfection of diverse objects, especially for the inactivation of bacterial endospores.     

Comparison of ozone inactivation, in flowing water, of hepatitis A virus, poliovirus 1, and indicator organisms.

In steadily flowing water at 20 degrees C and pH 7, five organisms had the following order of resistance to ozone (at constant levels of ozone): poliovirus 1 (PV1) less than Escherichia coli less than hepatitis A virus (HAV) less than Legionella pneumophila serogroup 6 less than Bacillus subtilis spores. The tests were repeated at 10 degrees C with HAV, PV1, and E. coli. Ozone inactivation of HAV and E. coli was faster at 10 degrees C than at 20 degrees C. At 20 degrees C, 0.25 to 0.38 mg of O3 per liter was required for complete inactivation of HAV but only 0.13 mg of O3 per liter was required for complete inactivation of PV1.     

Comparison of ozone inactivation, in flowing water, of hepatitis A virus, poliovirus 1, and indicator organisms

In steadily flowing water at 20 degrees C and pH 7, five organisms had the following order of resistance to ozone (at constant levels of ozone): poliovirus 1 (PV1) less than Escherichia coli less than hepatitis A virus (HAV) less than Legionella pneumophila serogroup 6 less than Bacillus subtilis spores. The tests were repeated at 10 degrees C with HAV, PV1, and E. coli. Ozone inactivation of HAV and E. coli was faster at 10 degrees C than at 20 degrees C. At 20 degrees C, 0.25 to 0.38 mg of O3 per liter was required for complete inactivation of HAV but only 0.13 mg of O3 per liter was required for complete inactivation of PV1.     

Mechanism of site-specific DNA damage induced by ozone

Ozone has been shown to induce lung tumors in mice. The reactivity of ozone with DNA in an aqueous solution was investigated by a DNA sequencing technique using 32P-labeled DNA fragments. Ozone induced cleavages in the deoxyribose-phosphate backbone of double-stranded DNA, which were reduced by hydroxyl radical scavengers, suggesting the participation of hydroxyl radicals in the cleavages. The ozone-induced DNA cleavages were enhanced with piperidine treatment, which induces cleavages at sites of base modification, but the inhibitory effect of hydroxyl radical scavengers on the piperidine-induced cleavages was limited. Main piperidine-labile sites were guanine and thymine residues. Cleavages at some guanine and thymine residues after piperidine treatment became more predominant with denatured single-stranded DNA. Exposure of calf thymus DNA to ozone resulted in a dose-dependent increase of the 8-oxo-7,8-dihydro-2'-deoxyguanosine formation, which was partially inhibited by hydroxyl radical scavengers. ESR studies using 5,5-dimethylpyrroline-N-oxide (DMPO) showed that aqueous ozone produced the hydroxyl radical adduct of DMPO. In addition, the fluorescein-dependent chemiluminescence was detected during the decomposition of ozone in a buffer solution and the enhancing effect of D2O was observed, suggesting the formation of singlet oxygen. However, no or little enhancing effect of D2O on the ozone-induced DNA damage was observed. These results suggest that DNA backbone cleavages were caused by ozone via the production of hydroxyl radicals, while DNA base modifications were mainly caused by ozone itself and the participation of hydroxyl radicals and/or singlet oxygen in base modifications is small, if any. A possible link of ozone-induced DNA damage to inflammation-associated carcinogenesis as well as air pollution-related carcinogenesis is discussed.