Research on the mechanism of synergetic effect in UV enhanced chlorination disinfection First Author：Hongzhang Lin Order of Authors：Hongzhang Lin； Wei Liu； Jun He
Abstract：In this article Escherichia coli was selected as object of study to explore the inactivation effect in UV disinfection, chlorination and UV enhanced chlorination disinfection under different conditions. The analysis mean of ICP-AES was applied to study the internal reasons of the synergetic effect further.
The experimental results showed that there has synergetic effect in the disinfection of UV irradiation and chlorination, and it is closely related to the iron balance of cellular. When UV disinfection or chlorination alone, the iron content of E. coli did not vary obviously. However, the iron content of E. coli increased significantly when UV enhanced chlorination disinfection which at that time the synergetic effect is obvious. The same situation also occurred in the UV disinfection when added additional solution of ferrous.
After UV disinfection, the iron balance of E. coli was broken. With excessive absorption of E. coli from additional ferrous solution after UV irradiation, the synergetic effect in subsequent chlorination was enhanced furthermore, which indicated the sensitivity to chlorine caused by the intracellular iron overload in E. coli by UV irradiation was a reason of synergetic effect in UV enhanced chlorination disinfection.
Keywords：UV disinfection; chlorination; UV enhanced chlorination; synergetic effect; E. coli iron content
1.Introdution
With the development of the society, water pollution problems are increasingly serious. Drinking water disinfection is the most important measure to ensure drinking water safety[1]. Chlorine disinfection as a economic and effective drinking water disinfection method, has been used for over 100 years.
Since the beginning of the 20th century, chlorine disinfection is widely used in water disinfection process. At present, chlorine disinfection is still the most widely used chemical disinfection methods. It’s main features are: When handling quantity is bigger, the unit water treatment cost is low. After chlorine disinfection, water can keep a certain amount of residual chlorine over a long period of time, thereby it have continuous sterilization ability.
However, researchers give great attention to chlorine disinfection and disinfection by-products, as chlorine disinfection by-products trihalomethanes (THMs) were found in 1974 and it’s carcinogenicity has been reported. So far, about 300 species of chlorine disinfection by-products have been found. The international organization and some countries have issued regulations to limit the content of disinfection by-products in drinking water.
UV disinfection, refers to that the microorganism occurs photochemical reaction under the ultraviolet irradiation, and then microbes nucleic acids will be damaged. After that, microorganism will lose its activity. The cell's DNA and RNA absorb the high energy of short-wave UV whose wavelengths is in the range of 200 ~ 275 nm. It will lead adjacent nucleotides to form new combination. Then, it will create the dual structure or dimer in nucleic acids. The thymine basing in the DNA will be damaged photochemically. The neighbouring pyrimidines will happen dimer effect, and it will lead the cells to death.
Main advantages of UV disinfection: It can control UV additive amount automatically and effectively. It has small risk, for it won't produce by-products such as trihalomethanes(THMs) which will cause cancer. But there are also some disadvantages. UV disinfection cannot provide the continuous sterilization ability. When the water leave the reactor, Bacteria could reborn. There will be high cost of equipment and operating cost. The disinfection effect is not good when the water is poorer and has many suspended solid.
This article would analysis the Changes of the E. coli iron content at the time that synergetic effect was occurring in UV enhanced chlorination disinfection, and confirmed the suppositional mechanism of synergetic effect in UV enhanced chlorination disinfection.
2.Methods
2.1Experimental methods
2.1.1 Determination of UV Dose
UV dose is affected by two factors, UV intensity and irradiation time. UV dose can be calculated by measuring these two indicators. Here is the calculation formula:
UV dose（mJ/cm2）=Irradiation time(s)*UVC intensity （mW/cm2）
2.1.2 Number of Coliform Group Bacteria
Use Filter Membrane Method to detect coliform group bacteria.
2.1.3 The Determination of E.Coli Iron Content
2.1.3.1 Sterilization of Filter Membrane and the Filter
Put 100 pieces of membranes into the beaker, add 100ml 5% HNO3 and boil them up for 5-10 minutes. After that, put the beaker there for an hour, and then wash the beaker for 3-5 times with distilled water.
2.1.3.2 Drawing of the Standard Curve
Take iron 1000ppm standard solution to prepare a series of standard solution whose concentrations are respectively 0, 0.25, 0.5, 0.75, 1.0, 2.0, 4.0, 6.0 and 8.0ppm. Then sequentially determinate the absorbance of each standard sample using ICP-AES.
2.1.3.3 Determination of concentrations of sample iron
Take 100ml E.coli broth from different contact time during the disinfection process, add 100μl of 15% sodium thiosulfate solution, and then mix them to terminate the inactivation. Filter the sample through a 0.45μm microporous membrane in the glass filter, so that the E. Coli will be retained on the filter. After that, in order to wash away the iron of the extracellular and on the membrane, use 50ml of 0.05mol/L solution of disodium edentate to wash the filter membrane for 10 times. Then drain the membrane and move it into a 10ml beaker, add 2ml of concentrated HNO3, and 0.2ml of perchloric acid. Heat the sample on a electric hot plate until it turns colorless and transparent, and keep heating to discompose perchloric acid until the residue is almost dry, then cool down the beaker, use 5% HNO3 solution to dissolve the residue, and set the volume to 10ml. Determinate the absorbance of the sample by ICP-AES and calculate the concentration of iron in the water sample according to the standard curve. At the same time, determinate the blank sample, then divided by the number of E.Coli after the disinfection is terminated, then the E.Coli Iron Content can be found out.
2.2Experimental procedures
2.2.1 Take 1ml E. coli broth with pH=7, use the ten-fold dilution method to determinate its number of E. Coli, and refer the number to N0
2.2.2 Use UVC UV light intensity tester to determinate the UV intensity at the mouth of UV irradiation device collimator optical. Then take 20ml water sample into a petri dish of 5.5cm diameter, put it on the magnetic stirrer, with the petri dish positively facing the mouth of collimator optical, the liquid level should stay the same with the probe height when determinating UV intensity. Turn on the magnetic stirrer as well as the UV light. Use a stopwatch to record the radiation time when the plastic shading plate is quickly taken off. When the required radiation time is over, then quickly cover the shading-plate, then turn off the stirrer and UV light. Take 1ml water sample after radiation, then use the ten-fold dilution method to determinate its number of E. Coli, and refer the number to N1。
2.2.3 Take 3 beakers of 100ml water samples radiated by the UV light with their pH respectively being 6, 7, and 8.Add chlorine disinfectant into the sample water, with the dose respectively being 0.5mg/L, 0.75mg/L, and 1.0mg/L. Assimilate 1ml sample water each time when it’s about 4, 8, 12, 16 and 20 minutes (subject to the actual determination time), and terminate the reaction with an excess of sodium thiosulfate solution, then use the ten-fold dilution method to determinate its number of E. Coli, refer them respectively to Nn（n=2,3,4,5,6）. Chlorine disinfection contact time as the abscissa, LG (N0 / N) as the ordinate,draw the inactivation curve.
2.2.4 Take 100ml of sample water radiated by UV light, name it as sample A, and determinate its concentration of iron. Add solution of ferrous with concentration respectively being 0.3ppm and 0.6ppm, repeat steps 2.2 to determinate the concentration of iron.
2.2.5 Take 3 beakers of 100ml water samples radiated by the UV light with different concentration and their pH respectively being 6, 7, and 8. leach the sample through a 0.45μm filter membrane and remake ??it into bacterial suspension, add 0.5mg/L chlorine disinfectant, then assimilate 1ml sample water each time when the contact time is about 4, 8, 12, 16 and 20 minutes, then terminate the reaction with an excess of sodium thiosulfate solution, then use the ten-fold dilution method to determinate its number of E. Coli, refer them respectively to Nn（n=2,3,4,5,6）. Chlorine disinfection contact time as the abscissa, LG (N0 / N) as the ordinate, draw the inactivation curve.
3.Results and Discussion
3.1. Synergy Effect between UV Enhanced Chlorine Disinfection and E. Coli Inactivation
Under the same pH, chlorine dosage and reaction time, the inactivation rate of UV enhanced chlorine disinfection is significantly higher than the total rate of UV disinfection alone and Chlorine disinfection alone, that is to say, synergy effect happens. Take the condition where pH=7, 0.5 mg/L chlorine dosage as an example, the logarithmic inactivation rate is as shown in the fig.1.
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In the formula, Nx is the number of bacterial survival of chlorine disinfection alone, Ny is the number of bacterial survival of UV disinfection alone. N0 is the total number of bacteria in raw water. N is the number of bacterial survival when the two disinfection methods combine together. When M=0, these two disinfectants have no interaction; when M<0, they have antagonistic effect; when M>0, they have synergy.
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As is shown in the table, M is always greater than 0, so it is proved that there is synergy effect between UV enhanced chlorine disinfection and the E. coli inactivation.
3.2 Analysis to the Changes of E. Coli Iron Content during the UV Enhanced Chlorine Disinfection Process
3.2.1 Iron Content in E. Coli after Separate UV Irradiation
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Each E. coli has in itself about 1.2 × 106 iron atoms and the concentration of E. coli water samples prepared in this experiment is 2.73×108CFU/ml. Therefore, it can be calculated that the concentration of iron in the water sample is 0.311ppm and in the E. coli broth of this concentration, the content of iron is 1.139ppm/109. When 100ml water samples of different pH are exposed to different doses of UV irradiation, the iron concentration and the content of iron in the E. coli broth remain unchanged. As Min Cho [3] have found that UV irradiation will not affect E. coli’s cell wall so as to change the permeability of it, will not damage intracellular enzyme, and will not change Escherichia coli morphologically. Therefore, even if the UV irradiation causes DNA damage in cells and E. coli has lost its activity, it remains an intact cell structure and the iron within the cell body does not run out of the cell. That explains why the concentration and the content of iron in the E. coli broth remain unchanged after UV irradiation.
3.2.2 E. Coli iron content during Chlorine Disinfection Alone Process
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When 100ml water samples of different pH are exposed to Chlorine disinfection, the content of iron in the E. Coli broth remain basically unchanged as 1.139ppm/109 ,but the iron concentration in the broth shows a downward trend with the reaction time. As min Cho [2] found that, because of their relatively weak oxidizing, chlorine cannot react with the integral part of the cell wall of E. coli to change the permeability of the cell wall or the external morphology of the bacteria, but will penetrate into the interior of the cell directly and do harm to intracellular enzymes or other components. Therefore, chlorine disinfectants will not damage the E. coli iron balance.
3.2.3 Changes of E. Coli Iron Content during UV Enhanced Chlorine Disinfection Process
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After UV irradiation, the concentration of iron in the E. coli broth continuously reduce in subsequent chlorine disinfection process, while the E. coli iron content increased significantly in the subsequent chlorination process, the iron content can be up to 3.23 times the E. coli iron content before disinfection.
3.2.4 The Relationship between Synergy Effects of UV Enhanced Chlorine Disinfection and Changes of the E. Coli Iron Content
E. coli iron content significantly changes exactly when synergistic effect of UV enhanced chlorine disinfection happens. Therefore, we believe that the mechanism of synergy of UV enhanced chlorine disinfection is: UV irradiation leads to the disruption of cellular iron balance, so in the subsequent process of chlorine disinfection, when the inactivated E.coli releases iron, the E. coli with disrupted cellular iron balance will absorb the released iron, resulting in overload of iron content in the body.
Just as what people like Tatsuo Nunoshiba[3] found, to some extent, the inactivation of chlorine disinfectants on E. coli can produce damage to the DNA of the E. coli by generating hydroxyl radicals via the following form Harber-Weiss/Fenton reaction:
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Fur genes of E. coli are responsible for controlling vivo iron balance in the cell, and when fur gene of E. coli is damaged or missing, the iron balance will get out of control and cause iron overload in vivo because of excessive iron absorption into cell. Then, through the Fenton reaction, Iron overload will exacerbate the generation of free radicals and other substances that can damage DNA, and therefore, when the E. coli with damaged or missing fur gene is exposed to HOCl, the sensitivity to HOCl is higher than that in normal E. coli and the lethal effects also enhance, which means that the E. coli iron overload will increase Fenton reaction generated by HOCl.
3.2.5 Verification: Analysis on the Absorption of Additional Iron into E. Coli after UV Irradiation
Additionally add 0.3ppm ferrous solution to a E. coli broth whose pH=7, and after it has accepted 0mJ·cm-2, 10mJ·cm-2, 11mJ·cm-2 dose of UV irradiation, the iron concentration in the E. coli broth are separately 0.311, 0.473 and 0.528ppm, while E. coli iron content, are separately 1.139, 1.732 and 1.934ppm/109.
After additionally adding ferrous solution to the broth, under the condition without UV irradiation, E. coli iron content remains unchanged; but after the UV irradiation, the E. coli iron content is significantly improved, indicating that E. coli iron balance has been disrupted during UV irradiation, resulting in excess absorption of an additional iron into E. coli, then causing iron load in vivo exceeds the normal level, which will lead to increased sensitivity of E. coli to chlorine disinfectants and aggravate the Fenton reaction of HOCl, and thus synergistic effects are generated.
4.Conclusions
The experimental results show that, the inactivated rate of E. coli in UV enchanced chlorination was Greater than the sum of UV disinfection and chlorination. There has synergetic effect in the disinfection of UV irradiation and chlorination
According to these findings，this paper speculated that the mechanism of synergetic effect is that: the fur gene of E. coli would be damaged after UV irradiation，thus to break the Iron balance. In the subsequent chlorination, E. coli bacteria absorbed iron overload, and strengthened the Fenton reaction of HOCl. Therefore the inactivated effect of chlorine disinfectant would greater than chlorine disinfectant separately，thus to form synergy effect. (fig.2)
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References
[1]E Xue-li，WANG Li，XING Fang-xiao, Research Advance of Disinfection By-products and Standard Limits in Drinking Water , J Environ Health, January 2010, Vol．27, No.1.
[2]Cho M, Kim J, Kim JY, Yoon J, Kim JH. Mechanisms of Escherichia coli inactivation by several disinfectants. Water Res, 2010, 44(11): 3410-3418.
[3]Touati D, M. Jacques, B. Tardat, L. Bouchard, and S. Despied. 1995. Lethal oxidative damage and mutagenesis are generated by iron in Dfur mutants of Escherichia coli: protective role of superoxide dismutase. J Bacteriol, 1995, 177: 2305–2314.