Mechanism of disinfection

Certain biochemical aspects of mechanism of disinfection are discussed in the paper. The total dehydrogenase activity of intact E. coli cells is affected by chlorine dose and this correlates with per cent survivals. The succinic dehydrogenase activity in the crude extract of E. coli is affected by chlorine within the bactericidal concentrations. The dose required for inhibition of above enzyme is less than that for intact cells. To deliniate the mechanism of inhibition of succinic dehydrogenase, the effect of chlorine on thiol groups present in crude extract is studied. After chlorine exposure there is a reduction in thiol groups indicating the thiol groups to be vulnerable to the oxidising influence of chlorine. The inhibition of succinic dehydrogenase induced due to lower doses of chlorine can significantly be reversed with reduced glutathione. The ATP-ase which is localised in cytoplasmic membrane of cell, like succinic dehydrogenase, is not affected to any degree by chlorine in bactericidal concentrations. The catalase activity of E. coli is not affected.     

Chlorine dioxide reactivity with proteins

Studies were conducted to evaluate chlorine dioxide reactivity with proteins and the role of these reactions in the inactivation of the bacterial virus f2 with chlorine dioxide. The effect of chlorine dioxide on the ability of f2 to specifically attach to its host Escherichia coli K13 was compared to the inactivation of virus during the initial seconds of contact time. At pH 7.2 and and 5°C, the virus was rapidly inactivated with 0.6 mg l⁻¹ of chlorine dioxide. Approximately 2 log units of inactivation were observed within 30 s. The loss of protein specific attachment function nearly paralleled intact virus inactivation with 1.2 log units of attachment inhibition occurring within 30 s. The inactivation of virus and the inhibition of specific attachment both increased with increasing pH and increasing disinfectant concentration.Inactivation of f2 was hypothesized to occur as the result of chlorine dioxide reacting with discrete chemical moieties in the viral protein. Cysteine, tyrosine and tryptophan reacted with chlorine dioxide within a time frame that could affect viral inactivation. In denatured f2 capsid protein monomers, these amino acids were almost totally degraded within 2 min by chlorine dioxide. Only tyrosine reacted with chlorine dioxide following treatment of the intact virion with disinfectant. Even though the degradation of tyrosine residues occurred at a much slower rate than the rate of virus inactivation, the protein component of f2 virus appeared to be the site of the lethal lesion produced by chlorine dioxide. These tyrosine reactions with chlorine dioxide appeared to alter the virus such that specific attachment was inhibited.     

纯培养条件下细菌需氯量和存活性的试验研究

以埃希氏大肠杆菌和粪肠球菌作为研究对象,在纯培养条件下考察了自由氯、氯胺和二氧化氯的投加量以及细菌初始浓度对需氯量的影响.结果表明:灭活率达到99.9％时,两种细菌的需氯量均随初始菌浓度和消毒剂投加量的提高而增大,粪肠球菌的需氯量大于埃希氏大肠杆菌;投加自由氯时两种细菌的需氯量差别最大,其次是二氧化氯,氯胺的差别最小.     

Inactivation of heterotrophic bacterial populations in finished drinking water by chlorine and chloramines

The die-off of heterotrophic bacteria from a finished drinking water reservoir, located in southern California, was evaluated using chloramines and free chlorine. Understanding the inactivation of hetertrophic populations is valuable because it reflects the response to disinfection of naturally occurring organisms in their native state and environments. Disinfection studies with the heterotrophic bacterial group were performed during summer and winter months using different chloramine application techniques at pH 6 and 8. In addition, bacteria surviving exposure to chloramines and free chlorine was influenced predominantly by the presence of highly chlorine tolerant, red-pigmented bacteria, identified as Flavobacterium spp. Inactivation by chloramines, though, was chiefly dependent upon the method of application and pH. Viable bacteria recovered from the indigenous population after 60 min of exposure to chloramines (1.0 mg l⁻¹, pH 8) included members of Pseudomonas, Acinetobacter and Flavobacterium. In general, with contact times of 1 h or more, free chlorine and chloramine solutions (1.0–1.6 mg l⁻¹, pH 8) reduced total count levels below 50 colony forming units ml⁻¹.     

二氧化氯、氯胺顺序投加联合消毒工艺的研究

以天津市某自来水厂的滤后水为研究对象,采用二氧化氯、氯胺顺序投加联合消毒工艺对其进行消毒处理。运用化学分析和生物学试验的方法,考察了经不同投量的组合消毒后消毒剂残余量的变化,以及联合工艺的消毒效果和持续消毒能力。试验结果表明,经不同投量组合的二氧化氯、氯胺顺序投加联合消毒后,出水中的细菌总数及总大肠菌群均达到《生活饮用水卫生标准》的要求。氯胺投量对总余氯和二氧化氯残余量的影响显著,而二氧化氯的投加量对其残余量的影响不大。二氧化氯、氯胺顺序投加联合消毒工艺在低投量组合下就能达到良好的消毒效果,且持续消毒能力强。     

Effect of chlorine on filamentous microorganisms present in activated sludge as evaluated by respirometry and INT-dehydrogenase activity

Activated sludge technology is more used than any other for biological treatment of wastewater. However, filamentous bulking is a very common problem in activated sludge plants, chlorine being the chemical agent normally used to control it. In this work the effect of chlorine on microorganisms present in activated sludge flocs was assessed by a respirometric technique (oxygen uptake rate, OUR) and by the INT-dehydrogenase activity test (DHA) measured by two techniques: spectrophotometry (DHA(a)) and image analysis (DHA(i)). Both DHA tests were optimized and correlated with the respirometric technique (OUR) using pure cultures of a filamentous microorganism (Sphaerotilus natans) under chlorine inhibition. Using these correlations the tested methods were applied to determine the action of chlorine on respiratory activity in activated sludge. The OUR and the DHA(a) quantifies the action of chlorine on the total respiratory activity (RA) of flocs (filamentous and floc-forming bacteria); in contrast, the DHA(i) test evaluates specific action of chlorine on the RA of filamentous microorganisms. In activated sludge flocs containing filamentous microorganisms, a chlorine dose of 4.75 mgCl(2) (gVSS)(-1) with a contact time of 20 min reduced about 80% of the RA of filamentous bacteria while affecting only 50-60% of the total RA of flocs. Besides, a chlorine dose of 7.9 mgCl(2) (gVSS)(-1) produced the total respiratory inactivation of filamentous microorganisms after 10 min contact, however, with this dose the total RA of activated sludge flocs was reduced only about 45-65%; controlling filamentous bulking without affecting too much floc-forming bacteria. At the tested chlorine concentrations the inhibition of filamentous microorganisms was higher than in the whole activated sludge. Although floc-forming microorganisms were demonstrated to be more susceptible to chlorine than filamentous in pure cultures, results obtained in the present work confirmed that it is the location of the filamentous microorganisms in the flocs and the presence of extracellular polymer substances which largely determines their higher susceptibility to chlorine; consequently this feature plays a critical role in bulking control.     

Mode d'action du bioxyde de chlore sur les composes organiques en milieu aqueux: Consommations de bioxyde de chlore et reactions sur les composes phenoliques

The aim of our work was to study the reaction between chlorine dioxide (ClO₂) and organic substances. In the first part of our survey, chlorine dioxide demands were measured in diluted aqueous solutions of various kinds of organic compounds (5 × 10⁻⁵ −10 M) at pH 7. In the second part, the study of the action of chlorine dioxide on phenols (phenol, di and triphenols) was undertaken by observing the change of the organic substance through global parameter controls (COT, u.v. spectrum) and by trying to identify a certain number of oxidation products by means of chromatographic analysis (HPLC and GC), of mass spectrometry and of Nuclear Magnetic Resonance spectrometry (NMR).     

二氧化氯对水中细菌的灭菌效果

通过对不同消毒剂投量、接触时间和ＰＨ值等条件研究，证明ＣｌＯ２来菌效果优于或相当于液氯。ＣｌＯ２可在广泛的ＰＨ值范围内具有较强的灭菌能力，而液氯仅在ＰＨ６．８－８．５范围内具有较强的灭菌能力。还初步探讨了ＣｌＯ２对细菌的杀灭机理。研究结果表明，ＣｌＯ２是一种优良的替代消毒剂。     

Modeling of chlorine effect on floc forming and filamentous micro-organisms of activated sludges

Chlorination is the most economical, non-specific method to control the excessive growth of filamentous micro-organisms causing bulking in activated sludge systems in the treatment of food industrial wastewaters; it was one of the first methods used to control filamentous bulking and is still widely employed. Considering that chlorination affects both floc-forming and filamentous micro-organisms and leaves undesirable disinfection by-products, it is necessary to define the adequate doses to control bulking, minimizing the effect on floc-forming bacteria.In the present work the effect of biomass concentration and type of micro-organism on chlorine decay kinetics was evaluated; the inactivation of either a filamentous (Sphaerotilus natans) or a floc-forming (Acinetobacter anitratus) micro-organism due to chlorination was also analyzed.For chlorine decay assays, the samples were treated in a batch system with sodium hypochlorite ranging between 9.8 and 56.6 mg Cl(2) (gVSS)(-1). Respirometric assays were used to evaluate the effect of chlorine on micro-organisms respiratory activity; in these cases, sodium hypochlorite doses ranged between 2.5 and 18 mgCl(2) (gVSS)(-1).A model that allowed to predict simultaneously chlorine consumption and respiratory activity decay for both micro-organisms as a function of time was proposed. The model includes three coupled differential equations corresponding to respiratory inhibition, readily organic matter oxidation by chlorine and chlorine decay. The rate of chlorine decay depended on both, type and concentration of the micro-organisms in the system. Chlorine consumption rate due to S. natans was 2-4 times faster than A. anitratus. Using the proposed model initial critical chlorine doses (the lowest initial dose that leads to a total inhibition of the respiratory activity) were calculated for both micro-organisms and values of 11.9 mgCl(2) (gVSS)(-1) for S. natans and 4.5 mgCl(2) (gVSS)(-1) for A. anitratus were obtained. These critical doses indicated that in non flocculated pure cultures, floc-former bacteria A. anitratus was more susceptible to chlorine action than S. natans.     

Avoidance response of rainbow trout (Salmo gairdneri) to single-dose chlorination in a power plant discharge canal

Rainbow trout (Salmo gairdneri) were exposed to 20 min single-dose chlorine additions designed to achieve maximum total residual chlorine (TRC) concentrations of 0.04, 0.2, 0.6, and 1.0 mg l⁻¹. First retreats from the chlorine front occurred at 0.05 mg l⁻¹ TRC. Approximately 95% of the fish had moved downstream when TRC reached 0.5 mg l⁻¹, well before cumulative time-dose exposure approached lethal limits. Percentage of fish remaining near the discharge decreased linearly as TRC concentration rose, suggesting that a rapid rise in receiving water chlorine level might be beneficial in reducing cumulative time-dose exposure. Rainbow trout demonstrated the initial sensitivity to avoid lethal chlorine exposure, but complete assessment of the utility of the avoidance response must also consider distribution throughout chlorination and the potential for repeated exposure.     

Comparative disinfection of treated sewage with chlorine and ozone: Effect of nitrification

Chlorine and ozone were compared in pilot plants (capacity about 3.2 m³ h⁻¹), which were fed with the same activated sludge treated and filtered water. Together with physico-chemical analysis the water was analysed for different types of microorganisms, including vegetative bacteria (total and thermotolerant coliforms, faecal streptococci and Pseudomonas aeruginosa), bacterial spores (spores of aerobic bacteria at 37°C and sulphite reducing clostridia) and bacterial viruses (somatic coliphages and F-specific bacteriophages).The average chlorine and ozone dose were, respectively, 3.65 and 15.3 mg l⁻¹ of water, while after a contact time for both of about 25 min the average residual concentrations were 1.79 and 0.35 mg l⁻¹ of water. These residuals were measured with the DPD-method. The ammonia-N concentration varied greatly (0.06–72.0 mg l⁻¹) and was used to group the data into four classes: (1) non-nitrified water, defined as water in which nitrate-N was smaller than ammonia-N; (2) moderately nitrified water, in which nitrate-N was larger than ammonia-N and the ammonia-N was higher than 2 mg l⁻¹; (3) well nitrified water, defined as water in which ammonia-N was lower than 2 mg l⁻¹; (4) very well nitrified water, in which ammonia-N was smaller than 0.5 mg l⁻¹.This classification indicated that the concentrations of most other impurities decreased with a better nitrification. Statistical analysis of the data showed also that ozone was a better disinfectant than chlorine in the case where the disinfection is based upon their residual content. The degree of nitrification had a greater effect on chlorine disinfection than on ozone disinfection.During chlorination the total residual chlorine decreased, with better nitrification; the chlorine demand increased; the composition of the residual chlorine changed very much and the inactivation of bacterial viruses improved. The vegetative bacteria showed a varying pattern; most were inactivated in moderately nitrified water, when the dichloramine concentration was highest and false positive FAC concentration was lowest of the four classes. Reduction of bacterial spores was not observed.During ozonization other effects were indicated. Reductions of most organims increased slightly with better nitrification; only reductions of F-specific bacteriophages decreased. There was also a small decrease of bacterial spores. The treated effluent had a high ozone consumption and the inactivation of the organisms was low in relation to ozone dose and residual ozone.The bromide concentration (0.3–2.9 mg l⁻¹) effected the chemistry of chlorine and ozone and had a positive effect on chlorine and ozone disinfection of total coliforms.For most types of micoorganisms the disinfection coefficients of the Selleck model and the germicidal efficiencies could be determined.     

Kinetics of membrane damage to high (HNA) and low (LNA) nucleic acid bacterial clusters in drinking water by ozone, chlorine, chlorine dioxide, monochloramine, ferrate(VI), and permanganate

Drinking water was treated with ozone, chlorine, chlorine dioxide, monochloramine, ferrate(VI), and permanganate to investigate the kinetics of membrane damage of native drinking water bacterial cells. Membrane damage was measured by flow cytometry using a combination of SYBR Green I and propidium iodide (SGI+PI) staining as indicator for cells with permeabilized membranes and SGI alone to measure total cell concentration. SGI+PI staining revealed that the cells were permeabilized upon relatively low oxidant exposures of all tested oxidants without a detectable lag phase. However, only ozonation resulted in a decrease of the total cell concentrations for the investigated reaction times. Rate constants for the membrane damage reaction varied over seven orders of magnitude in the following order: ozone > chlorine > chlorine dioxide ≈ ferrate > permanganate > chloramine. The rate constants were compared to literature data and were in general smaller than previously measured rate constants. This confirmed that membrane integrity is a conservative and therefore safe parameter for disinfection control. Interestingly, the cell membranes of high nucleic acid (HNA) content bacteria were damaged much faster than those of low nucleic acid (LNA) content bacteria during treatment with chlorine dioxide and permanganate. However, only small differences were observed during treatment with chlorine and chloramine, and no difference was observed for ferrate treatment. Based on the different reactivity of these oxidants it was suggested that HNA and LNA bacterial cell membranes have a different chemical constitution.     

Mutagenic activity in humic water and alum flocculated humic water treated with alternative disinfectants

Mutagenic activity in Salmonella typhimurium strains TA 100 , TA 98 and TA 97 has been determined for humic water and alum flocculated humic water, treated with the alternative disinfectants chlorine, ozone, chlorine dioxide, ozone/chlorine and chlorine/chlorine dioxide.The most pronounced activity was found for chlorine treated water tested on strain TA 100 without metabolic activation (S9 mix). Ozone treatment prior to chlorination did not alter the activity, while treatment with chlorine in combination with chlorine dioxide reduced the activity to a level somewhat over the background. No mutagenic response was detected in waters treated with ozone or chlorine dioxide alone. In presence of S9 mix all water extracts studied were non-mutagenic.     

Effect of oxidants on microalgal flocculation

The effects of chlorine, ozone and chlorine dioxide on Scenedesmus sp. cultures were studied. Algal cell viability and chlorophyll concentration decreased, and the concentration of dissolved organic substances increased with increasing applied oxidant concentration. Pretreatment with chlorine dioxide (1, 3 or 5 mg l⁻¹) or ozone (2.6, 4.6 or 8.1 mg l⁻¹) on algal cultures enhanced algal flocculation with alum, while prechlorination with 10 or 20 mg l⁻¹ increased the required dosage of alum by 15%. Scanning electron micrographs of oxidized cells revealed drastically adverse effects upon the cell surface architecture: in addition to the oxidation of noncellular organic materials, the oxidants damaged both cell surface structures and intracellular components. A model explaining the effects of the different oxidants on microalgal flocculation is suggested.     

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.     

In vitro toxicity of surface water disinfected by different sequential treatments

The in vitro toxicity of extracts of Hanjiang water disinfected by different sequential treatments was evaluated. Hanjiang water was disinfected using ozone, chloride dioxide or chlorine as the primary disinfectant followed by chlorine as the secondary disinfectant. HepG₂ cells were exposed to extracts corresponding to concentrations of 0.2, 1, 5, 25 and 125 mL water/mL medium. Compared with control, HepG₂ cells exposed to extracts of raw water and all disinfected water for 24 h increased oxidative stress level, DNA damage and micronuclei frequency, and decreased cell viability. Water disinfected by Cl₂ + Cl₂ had the highest DNA double-strand breaks. All disinfected water and raw water increased micronuclei frequency via clastogenic and aneugenic effects. Oxidative stress induced DNA strand breaks and micronuclei frequency and therefore reduced cell viability either in disinfected water or raw water. Compared with raw water, water after disinfection increased DNA strand breaks, decreased cell viability and changed oxidative stress potential. Compared with chlorination, sequential treatment using O₃ or ClO₂ as primary disinfectant followed by chlorine disinfection reduced chlorinated by-products, DNA double-strand breaks and cell viability, but did not decrease micronuclei frequency and other DNA damage such as DNA single-strand break, alkali liable sites and incomplete excision sites. Sequential treatments did not significantly reduce in vivo toxicity of disinfected Hanjiang water.     

The toxicity of chlorine to a common vascular aquatic plant

Myriophyllum spicatum was exposed to various chlorine concentrations on a continuous and intermittent basis in 96-h toxicity studies utilizing a proportional diluter. Continuous exposure to chlorine concentrations as low as 0.05 mg l⁻¹ total residual chlorine (TRC) depressed shoot and total plant dry weights approx. 30% relative to controls. Shoot length was depressed approx. 16% at this concentration. Chlorophyll a was depressed 25% at 0.1 mg l⁻¹ TRC. However, intermittent exposure of plants to chlorine for three 2-h periods daily for 96 h indicated an insensitivity to repeated short term chlorine exposure at all concentrations but 1.0 mg l⁻¹ TRC. These results indicate that high level chlorine discharges from waste water facilities and electric generating plants could be a contributing factor impacting nearby submerged aquatic vegetation.     

Differential effect of chlorine on the oxidative stress generation in dormant and active cells within colony biofilm

In an effort to better control bacterial biofilm, we examined the effects of various oxidative antimicrobial chemicals including silver, paraquat, hydrogen peroxide, and chlorine depending on the physiological status of cells in biofilm. The metabolically heterogeneous cells within colony biofilm were physically fractionated and the oxidative stress generated in each fraction was monitored by soxS and oxyS promoter reporter systems. Chlorine induced soxS to a greater degree in the dormant cells than active cells of biofilm. In addition, chlorine-dependent induction of soxS was more prominent in aerobically grown cells compared with anaerobically grown cells. On the contrary, the soxS induction by other chemicals such as paraquat and silver, and the oxyS induction by hydrogen peroxide were higher in active biofilm cells and aerobically grown cells. Our results suggest that chlorine might generate strong oxidative stress by direct modification of the 2Fe-2S cluster in an O₂-independent manner, which provides the molecular basis of our previous report showing that chlorine has a more efficient killing effect on dormant cells in biofilm and cells grown under unaerobic conditions. This study shows that chlorine may be particularly promising for the control of anaerobic bacteria and biofilm where dormant cells are hard to control.     

二氧化氯对水中典型菌种的试验研究

研究了二氧化氯（ＣｌＯ２）对水中某些典型细菌的灭菌效果，考察了在消毒剂投量、ｐＨ值及接触时间等不同试验条件下，二氧化氯的灭菌效果．与液氯比较，二氧化氯的灭菌效果优于或与液氯相当．二氧化氯可在广泛的ｐＨ范围内有效地杀灭细菌．