Inactivation of Bacillus subtilis spores with ozone and monochloramine

The inactivation kinetics of Bacillus subtilis spores with ozone and monochloramine was characterized by a lag phase followed by a pseudo-first-order rate of inactivation. The lag phase decreased and the post-lag phase rate constant increased with increasing temperature within the range investigated (1-30 degrees C for ozone, 1-20 degrees C for monochloramine). The corresponding activation energies were 46820 J/mol for ozone and 79640 J/mol for monochloramine. The CT concept was found to be valid within the concentration range investigated of 0.44-4.8 mg/l for ozone, and 3.8-7.7 mg/l as Cl(2) for monochloramine. The inactivation kinetics of B. subtilis spores with both ozone and monochloramine varied with pH within the range of pH 6-10 investigated. The fastest ozone and monochloramine inactivation rates were observed at pH 10 and 6, respectively. Different stocks of the same strain of B. subtilis spores had different resistance to ozone and monochloramine mainly because of discrepancies in the extent of the lag phase. B. subtilis spores might not be conservative surrogates for C. parvum oocysts for ozone disinfection at relatively low temperature mainly due to the spores having a lower activation energy compared to that for the oocysts. In contrast, the activation energy for monochloramine was comparable for both microorganisms but differences in the extent of the lag phase might result in the spores being overly conservative surrogates for the oocysts at relatively low temperature.     

Association and decontamination of Bacillus spores in a simulated drinking water system

The objective of this work was to elucidate the disinfectant susceptibility of Bacillus anthracis Sterne (BA) and a commercial preparation of Bacillus thuringiensis (BT) spores associated with a simulated drinking water system. Biofilms composed of indigenous water system bacteria were accumulated on copper and polyvinyl chloride (PVC) pipe material surfaces in a low-flow pipe loop and uniformly mixed tank reactor (CDC biofilm reactor). Application of a distributed shear during spore contact resulted in approximately a 1.0 and 1.6 log₁₀ increase in the number of spores associated with copper and PVC surfaces, respectively. Decontamination of spores in both free suspension and after association with biofilm-conditioned pipe materials was attempted using free chlorine and monochloramine. Associated spores required 5- to 10-fold higher disinfectant concentrations to observe the same reduction of viable spores as in suspension. High disinfectant concentrations (103 mg/L free chlorine and 49 mg/L monochloramine) yielded less than a 2-log₁₀ reduction in viable associated spores after 60 min. Spores associated with biofilms on copper surfaces consistently yielded higher Ct values than PVC.     

Investigating synergism during sequential inactivation of Bacillus subtilis spores with several disinfectants

The sequential application of ozone, chlorine dioxide, or UV followed by free chlorine was performed to investigate the synergistic inactivation of Bacillus subtilis spores. The greatest synergism was observed when chlorine dioxide was used as a primary disinfectant followed by secondary disinfection with free chlorine. A lesser synergistic effect was observed when ozone was used as the primary disinfectant, but no synergism was observed when UV was used as the primary disinfectant. When free chlorine was used as the primary disinfectant (i.e., sequential application in the reverse order), the synergistic effect was shown only when chlorine dioxide was applied as the secondary disinfectant. The synergistic effect observed could be related to damage to the spore coat during primary disinfection, suggested by the loss of proteins from spores during disinfectant treatment. The greatest synergism observed by the chlorine dioxide/free chlorine pair suggested that common reaction sites might exist for these disinfectants. The concept of percent synergistic effect was introduced to quantitatively compare the extent of synergistic effects in the sequential disinfection processes.     

Synergistic inactivation of Cryptosporidium parvum using ozone followed by monochloramine in two natural waters

The effect of sequential exposure to ozone followed by monochloramine on inactivation of Cryptosporidium parvum oocysts suspended in untreated natural surface water from two different sources was studied in bench-scale batch reactors. Animal infectivity using neonatal CD-1 mice was used to measure oocyst inactivation. A statistically significant synergistic effect on oocyst inactivation was measured in both natural water samples studied. The magnitude of the effect measured in the natural water with lower turbidity, colour, and organic carbon concentration was comparable to that previously reported for oocysts suspended in buffered de-ionized water but was reduced considerably in the natural water with higher turbidity, colour and organic carbon concentration. Synergy increased with initial pH and with the degree of ozone pre-treatment but was independent of temperature. For water treatment plants with adequate disinfectant contact times, ozone followed by monochloramine may be a practical means of achieving additional C. parvum inactivation, however, the influence of water quality characteristics should be considered.     

Investigating synergism during sequential inactivation of MS-2 phage and Bacillus subtilis spores with UV/H2O2 followed by free chlorine

A sequential application of UV as a primary disinfectant with and without H₂O₂ addition followed by free chlorine as secondary, residual disinfectant was performed to evaluate the synergistic inactivation of selected indicator microorganisms, MS-2 bacteriophage and Bacillus subtilis spores. No synergism was observed when the UV irradiation treatment was followed by free chlorine, i.e., the overall level of inactivation was the same as the sum of the inactivation levels achieved by each disinfection step. With the addition of H₂O₂ in the primary UV disinfection step, however, enhanced microbial inactivation was observed. The synergism was observed in two folds manners: (1) additional inactivation achieved by hydroxyl radicals generated from the photolysis of H₂O₂ in the primary UV disinfection step, and (2) damage to microorganisms in the primary step which facilitated the subsequent chlorine inactivation. Addition of H₂O₂ in the primary disinfection step was also found to be beneficial for the degradation of selected model organic pollutants including bisphenol-A (endocrine disruptor), geosmin (taste and odor causing compound) and 2,4-D (herbicide). The results suggest that the efficiency of UV/free chlorine sequential disinfection processes, which are widely employed in drinking water treatment, could be significantly enhanced by adding H₂O₂ in the primary step and hence converting the UV process to an advanced oxidation process.     

Source water quality effects on monochloramine inactivation of adenovirus, coxsackievirus, echovirus, and murine norovirus

There is a need for more information regarding monochloramine disinfection efficacy for viruses in water. In this study, monochloramine disinfection efficacy was investigated for coxsackievirus B5 (CVB5), echovirus 11 (E11), murine norovirus (MNV), and human adenovirus 2 (HAdV2) in one untreated ground water and two partially treated surface waters. Duplicate disinfection experiments were completed at pH 7 and 8 in source water at concentrations of 1 and 3 mg/L monochloramine at 5 and 15 °C. The Efficiency Factor Hom (EFH) model was used to calculate CT values (mg-min/L) required to achieve 2-, 3-, and 4-log₁₀ reductions in viral titers. In all water types, monochloramine disinfection was most effective for MNV, with 3-log₁₀ CT values at 5 °C ranging from 27 to 110. Monochloramine disinfection was least effective for HAdV2 and E11, depending on water type, with 3-log₁₀ CT values at 5 °C ranging from 1200 to 3300 and 810 to 2300, respectively. Overall, disinfection proceeded faster at 15 °C and pH 7 for all water types. Inactivation of the study viruses was significantly different between water types, but there was no indication that overall disinfection efficacy was enhanced or inhibited in any one water type. CT values for HAdV2 in two types of source water exceeded federal CT value recommendations in the US. The results of this study demonstrate that water quality impacts the inactivation of viruses and should be considered when developing chloramination plans.     

Factors influencing inactivation of Klebsiella pneumoniae by chlorine and chloramine

Inactivation of Klebsiella pneumoniae cultures by chlorine and chloramine was evaluated under different growth conditions by varying nutrient media dilution, concentrations of essential inorganic nutrients (FeCl3, MgSO4, phosphate, and ammonium salts), and temperature. All inactivation assays were performed at room temperature (22-23 degrees C) and near neutral pH (7.2-7.5). C*T(99.9) values for chlorine increased >20-fold and for chloramine increased 2.6-fold when cells were grown in 100-fold diluted nutrient broth (2NB) solutions (final TOC of 35-40 mg/L). Background levels of Mg: 6.75 x 10(-2) mM and Fe: 3.58 x 10(-5) mM or high levels of FeCl3 (0.01 mM) and MgSO4 (1 mM) during growth resulted in the highest resistances to chlorine with C*T(99.9) values of 13.06 (+/-0.91) and 13.78 (+/-1.97) mg-min/L, respectively. Addition of low levels of FeCl3 (0.001 mM) and MgSO4 (0.1 mM) to K. pneumoniae cultures during growth resulted in the lowest bacterial resistances to inactivation; C*T(99.9) values ranged from 0.28 (+/-0.06) to 1.88 (+/-0.53)mg-min/L in these cultures. Increase in growth temperature from 22.5 degrees C to 35 degrees C for unamended 2NB cultures resulted in a 42-fold decrease in C*T(99.9) values for chlorine. A similar change in temperature resulted in no significant change in C*T(99.9) values for chloramine. These results indicate that inactivation of K. pneumoniae cultures by chlorine was highly sensitive to changes in growth conditions unlike inactivation by chloramine.     

Adsorption of Bacillus subtilis on single-walled carbon nanotube aggregates, activated carbon and NanoCeram

Adsorption equilibrium and kinetics of Bacillus subtilis spores on single-walled carbon nanotube aggregates were investigated to explore the possibility of using single-walled carbon nanotubes for concentration, detection and removal of pathogens from contaminated water sources. Batch adsorption experiments were conducted to determine adsorption kinetics and adsorption equilibrium of B. subtilis spores on single-walled carbon nanotube aggregates, activated carbon and NanoCeram™. The adsorption kinetics data were analyzed with both the Lagergren pseudo first order and a pseudo second order models. The adsorption equilibrium data on three porous media were quantified by the Henry's law constant. It was observed that both the Lagergren first order rate model and the pseudo second order model correlate the adsorption kinetic data well although the calculated adsorption rate constants vary with adsorbate concentrations. The Henry's law adsorption equilibrium constant of B. subtilis spores on single-walled carbon nanotube aggregates is about 27-37 times higher than those on activated carbon and NanoCeram™. The high adsorption affinity of carbon nanotubes towards the B. subtilis spores is due to the mesoporous structure and unique surface properties of carbon nanotubes. These results suggest that single-walled carbon nanotube aggregates are good candidates as biosensors and adsorbent media for concentrating, detecting and removal of pathogens from contaminated water resources.     

Modeling Cryptosporidium parvum oocyst inactivation and bromate in a flow-through ozone contactor treating natural water

A reactive transport model was developed to simultaneously predict Cryptosporidium parvum oocyst inactivation and bromate formation during ozonation of natural water. A mechanistic model previously established to predict bromate formation in organic-free synthetic waters was coupled with an empirical ozone decay model and a one-dimensional axial dispersion reactor (ADR) model to represent the performance of a lab-scale flow-through ozone bubble-diffuser contactor. Dissolved ozone concentration, bromate concentration (in flow-through experiments only), hydroxyl radical exposure and C. parvum oocyst survival were measured in batch and flow-through experiments performed with filtered Ohio River water. The model successfully represented ozone concentration and C. parvum oocyst survival ratio in the flow-through reactor using parameters independently determined from batch and semi-batch experiments. Discrepancies between model prediction and experimental data for hydroxyl radical concentration and bromate formation were attributed to unaccounted for reactions, particularly those involving natural organic matter, hydrogen peroxide and carbonate radicals. Model simulations including some of these reactions resulted in closer agreement between predictions and experimental observations for bromate formation.     

Kinetic modeling of a mixed culture of Pseudomonas denitrificans and Bacillus subtilis under aerobic and anoxic operating conditions

The kinetics of biological denitrification have been studied and several models, with varying degree of complexity, to be used for design purposes have been presented in the recent years. However, most of these kinetic studies were performed with mixed (and not well defined) microbial systems, such as activated sludge. In the present work, kinetic experiments were carried out in order to study the dynamic characteristics of a defined mixed culture of the denitrifiers Pseudomonas denitrificans and Bacillus subtilis under anoxic and aerobic conditions in a defined synthetic medium involving a mixture of organic substrates, in the presence of nitrates and/or nitrites. Denitrification was assumed to occur by the consecutive reduction of nitrates to nitrites and then to nitrogen gas without accumulation of intermediate gaseous products. The behavior of these defined mixed cultures was predicted using a kinetic model based on the kinetic models that have already been developed for each bacterium separately and the predictions were compared with the results from mixed culture experiments. The overall mathematical model that was developed and validated in the present work is capable of describing the behavior of the mixed culture in the above conditions, i.e. the nitrates and nitrites reduction kinetics, the cell growth, and the organic carbon utilization rates.     

Synergistic inactivation of Cryptosporidium parvum using ozone followed by free chlorine in natural water

The synergistic effect of sequential exposure to ozone followed by free chlorine on inactivation of Cryptosporidium parvum oocysts suspended in natural waters was studied in bench-scale batch reactors. Animal infectivity using neonatal CD-1 mice was used to measure oocyst inactivation. The synergistic effect measured in two alkaline (pH 8.1) natural waters was statistically significant but was considerably smaller than previously reported in buffered de-ionized water at pH 6.0. Temperature, ozone primary treatment level, and water type did not have measurable impacts on the synergistic effect. Efforts to increase the synergistic effect by reducing the pH from 8 to 6 by acid addition were unsuccessful. In the two low alkalinity (pH 6.0) natural waters tested, the measured synergistic effect was greater than in the alkaline waters, but was still less than that measured previously in buffered de-ionized water. It was concluded that the synergistic effect reduction in the natural waters tested was due in part to alkalinity and in part to other unidentified water quality characteristics. Sequential treatment with ozone followed by free chlorine may only be a feasible strategy for achieving synergistic C. parvum inactivation credit for water treatment facilities with natural waters having a low pH (near 6.0).     

Protection against UV disinfection of E. coli bacteria and B. subtilis spores ingested by C. elegans nematodes

Nematodes, which occur abundantly in granular media filters of drinking water treatment plants and in distribution systems, can ingest and transport pathogenic bacteria and provide them protection against chemical disinfectants. However, protection against UV disinfection had not been investigated to date.In this study, Caenorhabditis elegans nematodes (wild-type strain N2) were allowed to feed on Escherichia coli OP50 and Bacillus subtilis spores before being exposed to 5 and 40 mJ/cm² UV fluences, using a collimated beam apparatus (LP, 254 nm). Sonication (15 W, 60 s) was used to extract bacteria from nematode guts following UV exposure in order to assess the amount of ingested bacteria that resisted the UV treatment using a standard culture method. Bacteria located inside the gut of C. elegans were shown to benefit from a significant protection against UV. Approximately 15% of the applied UV fluence of 40 mJ/cm² (as typically used in WTP) was found to reach the bacteria located inside nematode guts based on the inactivation of recovered bacteria (2.7 log reduction of E. coli bacteria and 0.7 log reduction of B. subtilis spores at 40 mJ/cm²). To our knowledge, this study is the first demonstration of the protection effect of bacterial internalization by higher organisms against UV treatment, using the specific case of E. coli and B. subtilis spores ingested by C. elegans.     

Mechanisms of Escherichia coli inactivation by several disinfectants

The objective of this study was to elucidate dominant mechanisms of inactivation, i.e. surface attack versus intracellular attack, during application of common water disinfectants such as ozone, chlorine dioxide, free chlorine and UV irradiation. Escherichia coli was used as a representative microorganism. During cell inactivation, protein release, lipid peroxidation, cell permeability change, damage in intracellular enzyme and morphological change were comparatively examined. For the same level of cell inactivation by chemical disinfectants, cell surface damage was more pronounced with strong oxidant such as ozone while damage in inner cell components was more apparent with weaker oxidant such as free chlorine. Chlorine dioxide showed the inactivation mechanism between these two disinfectants. The results suggest that the mechanism of cell inactivation is primarily related to the reactivity of chemical disinfectant. In contrast to chemical disinfectants, cell inactivation by UV occurred without any changes measurable with the methods employed. Understanding the differences in inactivation mechanisms presented herein is critical to identify rate-limiting steps involved in the inactivation process as well as to develop more effective disinfection strategies.     

Effect of turbulent gas-liquid contact in a static mixer on Cryptosporidium parvum oocyst inactivation by ozone

Static mixers may be used to dissolve gaseous ozone in water treatment facilities in order to provide protection against the waterborne parasite Cryptosporidium parvum. The objective of this study was to determine the effect of a brief exposure to turbulent gas-liquid mixing conditions in a static mixer on inactivation of C. parvum oocysts by ozone. Inactivation measured in an ozone contacting apparatus that employed a static mixer for ozone dissolution was compared to predictions based on a previously published kinetic model of C. parvum inactivation by dissolved ozone in gently stirred batch reactors. Although initial contact in the static mixer had no immediate effect on the oocysts, a 20% increase in the rate of inactivation during subsequent contact with dissolved ozone was observed. Increasing the degree of turbulence within the static mixer did not yield additional inactivation. Use of static mixers for dissolution of ozone in drinking water treatment systems may provide limited enhancement of C. parvum inactivation by dissolved ozone.     

Microbial toxicity of metal oxide nanoparticles (CuO, NiO, ZnO, and Sb2O3) to Escherichia coli, Bacillus subtilis, and Streptococcus aureus

In this study, the microbial toxicities of metal oxide nanoparticles were evaluated for Escherichia coli, Bacillus subtilis, and Streptococcus aureus in laboratory experiments. The nanoparticles tested were CuO, NiO, ZnO, and Sb₂O₃. The metal oxide nanoparticles were dispersed thoroughly in a culture medium, and the microorganisms were cultivated on Luria-Bertani agar plates containing different concentrations of metal oxide nanoparticles. The bacteria were counted in terms of colony forming units (CFU). The CFU was reduced in a culture medium containing metal oxide NP, and the dose-response relationship was characterized. CuO nanoparticles were found to be the most toxic among the tested nanoparticles, followed by ZnO (except S. aureus), NiO, and Sb₂O₃ nanoparticles. We determined that the intrinsic toxic properties of heavy metals are also associated with the toxicity of metal oxide nanoparticles. Ion toxicity was also evaluated to determine the effects of metal ions dissolved from metal oxide NPs, and the toxicity induced from the dissolved ions was determined to be negligible herein. To the best of our knowledge, this is the first study of the toxicity of NiO and Sb₂O₃ NPs on microorganisms. We also discuss the implications of our findings regarding the effects of the intrinsic toxic properties of heavy metals, and concluded that the apparent toxicities of metal oxide NPs can largely be understood as a matter of particle toxicity.     

Implications of sequential use of UV and ozone for drinking water quality

The formation of bromate levels exceeding the drinking water standard of 10 microg L-1 may impose the reduction of ozone doses used in the treatment of drinking water. This paper illustrates the procedure of evaluating the use of reduced ozone doses while implementing an additional UV disinfection step for an actual drinking water treatment plant. Ozonation was performed at low ozone doses in bench-scale experiments with a pretreated river water from the Paris area (France). At the low ozone dose of 0.5 mg L-1, bromate formation could be kept below 0.4 microg L-1, while inactivation of vegetative bacteria and UV-resistant viruses was calculated to exceed 5 log units, and a substantial decoloration (31% of the absorption at lambda=254 nm) was achieved. Based on the measured transient ozone and OH radical concentrations, the oxidation of micropollutants was calculated. Fast reacting micropollutants containing phenol, amine or double bond moieties, such as sulfamethoxazole, diclofenac and 17-alpha-ethinylestradiol, were completely oxidized. Slow-reacting synthetic micropollutants, e.g., atrazine, iopromide and methyl tertiary butyl ether (MTBE), were oxidized by only 20%, 20% and 10%, respectively, and the taste and odor compounds 2-methylisoborneol (MIB) and geosmin by 40% and 50%, respectively. The addition of an UV treatment step to the existing treatment train, which should guarantee disinfection of ozone-resistant pathogenic microorganisms, including Cryptosporidium parvum oocysts, has negligible effects on water matrix components but may induce significant transformation of micropollutants. Overall, the combination of ozonation at reduced doses and UV treatment leads to an improved water quality with regard to disinfection, oxidation of micropollutants and minimization of bromate.     

Effects of ozone and ozone/peroxide on trace organic contaminants and NDMA in drinking water and water reuse applications

An ozone and ozone/peroxide oxidation process was evaluated at pilot scale for trace organic contaminant (TOrC) mitigation and NDMA formation in both drinking water and water reuse applications. A reverse osmosis (RO) pilot was also evaluated as part of the water reuse treatment train. Ozone/peroxide showed lower electrical energy per order of removal (EEO) values for TOrCs in surface water treatment, but the addition of hydrogen peroxide increased EEO values during wastewater treatment. TOrC oxidation was correlated to changes in UV₂₅₄ absorbance and fluorescence offering a surrogate model for predicting contaminant removal. A decrease in N-nitrosodimethylamine (NDMA) formation potential (after chloramination) was observed after treatment with ozone and ozone/peroxide. However, during spiking experiments with surface water, ozone/peroxide achieved limited destruction of NDMA, while in wastewaters net direct formation of NDMA of 6-33 ng/L was observed after either ozone or ozone/peroxide treatment. Once formed during ozonation, NDMA passed through the subsequent RO membranes, which highlights the significance of the potential for direct NDMA formation during oxidation in reuse applications.     

A stochastic model of an ozonation reactor

Disinfection of some microorganisms is characterized by a lag-phase (a minimum required ozone exposure until disinfection occurs). This phenomenon is easy to model in laboratory batch reactors but not in continuous flow mixed reactors. This paper introduces a stochastic disinfection model where individual microorganisms are followed on their paths through full-scale reactors. Combining exponentially distributed transport processes with delayed exponential disinfection kinetics for large populations of microorganisms (up to 10,000 individuals) yields predictions which can be evaluated statistically. It could be shown that deterministic models work well for systems with good disinfection performance (more than 2 log units reduction of active microorganisms), for reactors with poor performance stochastic models have to be applied. It could be demonstrated for real reactors that Bacillus subtilis spores are poor surrogates for Cryptosporidium parvum oocysts. The differences between the two microorganisms are large for reactors that deviate significantly from plug-flow behaviour.     

Microbiological surveillance of private water supplies in England - The impact of environmental and climate factors on water quality

A passive surveillance system captured information on 34,904 microbiological samples from 11,233 private drinking water supplies within England as well as the associated constructional, climatic and environmental variables. Escherichia coli was detected in 6588 (18.87%) of samples and at least one positive sample was detected from 3638 (32.39%) of sites. However, this estimate of supplies failing to meet the European drinking water E. coli standard was probably an underestimate as the more samples taken per supply, the more likely the supply was to fail. A multivariable model of private water supplies data showed a strong seasonal impact, with samples between January and May being significantly less contaminated with E. coli than samples between June and December. Samples from springs (OR 2.5, CI 2.0-3.1) or surface waters (OR 2.4, CI 0.8-7.0) were more likely to fail than groundwater sources, as were supplies with no effective treatment (OR1.8, CI 1.5-2.3). Commercial supplies were less likely to fail than domestic supplies (OR 0.63, CI 0.48-0.83) and the probability of failure was linearly associated with the density of sheep in the area and rainfall on the previous day. A Monte Carlo modelling approach was used to estimate that, had sufficient samples been taken, 54% (95% confidence intervals 49-59%) of all private water supplies in England were likely to be unsatisfactory. These findings will be able to inform risk assessments of private water supplies prior to microbiological results being available.