LI Shao-feng (李紹峰) ，RAN Zhi-lin (冉治霖)

1 Shenzhen Key Laboratory of Industrial Water Saving ＆Municipal Sewage Reclamation Technology，Shenzhen Polytechnic Institute，Shenzhen 518055，China

2 School of Transportation and the Environment，Shenzhen Institute of Information Technology，Shenzhen 518172，China

Introduction

Giardia intestinali(G.intestinali)is a diplomonadida that parasitizes the small intestine of virtually all vertebrates by direct attachment to the mucosal surface.It led to a high prevalence of long-lasting fatigue and abdominal symptom［1-3］.In particular，it also can cause a syndrome to the immuno-compromised hosts(such as infants，the elderly，and AIDS patients)，and even threaten their lives［4］.On one hand，in most developed countries，the poor catchment management and system failures often result in outbreaks of waterborne infection［5-7］.On the other hand，G.intestinali has a strong resistance to chlorine disinfectant and can survive for a few months in water［8-9］.Besides，it can survive for up to 1 year in artificial seawater［10］.

Many researchers have studied the susceptibility of G.intestinali to a range of disinfectants including UV，ozone，chlorine，and chloramine［11-13］.However，such studies had difficulties in achieving the required target for microbial inactivation.Ozonation had been demonstrated to be an effective oxidation technology in treating wastewater and drinking water.The application of O3in combination with hydrogen peroxide (H2O2/O3)brings about enhanced oxidative degradation of pollutants by the generation of highly reactive hydroxyl radicals.Previously，Wolfe's group［14-15］reported that the effect of H2O2/O3was superior to ozone for inactivating Giardia E.Coli，and MS-2 coli-phage without quantitative evaluation.A previous study mentioned that O3could effectively inactivate Giardia.In a similar way，the mechanism of Cryptosporidium cell damaged by O3was revealed using fluorescent in vivo staining and scanning electron microscopy(SEM)［16］.This study attempted not only to find out the optimal conditions of H2O2/O3reaction with G.intestinali(which included the optimum O3dosage，molar ratio of H2O2/O3)，but also to discuss the role of ·OH radicals in this system.Thereafter，with SEM，proteins released and damages of genomic DNA in this work to investigate the mechanism of G.intestinal cell that damaged by H2O2/O3.

1 Materials and Methods

1.1 Preparation of cysts

Giardia cysts were obtained from previous studies purified by Sheather's gradient centrifugation［17］or cesium chloride gradient centrifugation［18］.

1.2 Operating conditions and materials

Different doses of TBA were added interdmittently as an ·OH radical scavenger to examine the effect of ·OH radical in the H2O2/O3system.TBA (30 m mol/L)did not cause any inactivation to the microorganisms.The temperature during the experiments was controlled at 20℃ by immersing the batch reactor in a water bath.The pH was adjusted by adding H2SO4or NaOH.Dissolved ozone presented in the sample was quenched by mixing with indigo trisulfonate solution immediately upon collection［19］.

1.3 O3 and H2O2/O3 experiments

O3gas productrion procedure was achieved from the previous study［17］.The inactivation of G.intestinali with hydrogen peroxide was conducted using a 30% solution of H2O2which diluted into the required doses.UV-vis spectrophotometer(Hitachi U-3010， Japan) was used to determine the concentration of hydrogen peroxide in the samples stopped by sulfuric acid and titanium potassium oxalate.The selected wavelength is 385 nm［20］.The indigo stock solution was prepared by adding distilled water (500 mL)，phosphoric acid(85%，1 mL)and potassium indigo-trisulfonate (0.770 g)to a volumetric flask and diluted to 1L with distilled water.A 1∶100 dilution exhibits an absorbance of (0.20 ±0.010)cm-1at 600 nm and this stock solution is stable for about 4 months when stored in the dark.The ozone sample was introduced immersing the pipette to the bottom of the flask and gently shaking so that no ozone degassing occurred.The difference absorption of light at 600 nm between the blank and sample as measured using a UV-vis spectrophotometer.

1.4 Analytical methods

Cyst viability was determined using the fluorogenic vital dyes assay described by Campbell et al.［21］based on the morphology and the inclusion or exclusion of two vital dyes，4'，6-diamidino-2-phenylindole (DAPI)(Sigma，USA)and propidium iodide (PI)(Sigma，USA)as described by Ran et al［17］.The cysts which reacted with DAPI and PI analyzed by epifluorescence (Olympus BX51，Japan)equipped with a UV filter block for DAPI and a green filter block (500 nm excitation，630 nm emission)for PI.With 200 cysts in each subsample，the proportions of following categories of cysts to the total were determined:(1)viable cysts that included DAPI but exclude PI (DAPI + /PI -);(2)nonviable cysts that included DAPI and PI (DAPI + /PI +);(3)potentially infectious cysts that did not include either DAPI or PI but had“viable type”contents，as shown by differential interference contrast (DIC)microscopy (DAPI- /PI-，viable after further trigger);(4)empty cysts without recognizable contents，as shown by DIC microscopy，and that include neither DAPI nor PI (DAPI - /PI -，nonviable).Three replicate counts were performed for each subsample.Cysts with contents which were either DAPI+ /PI - or DAPI - /PI - were considered viable，thus，their total number represented the number of viable cysts.

1.5 Inactivation mechanisms

1.5.1 SEM

The method of SEM to observe the surface changes on G.intestinali was described in a previous study［17］.

1.5.2 Protein assay

The concentration of protein was determined using a Lowry assay［22］，which was a method to estimate the amount of protein in biological samples.Briefly，G.intestinali cells (2 × 106cell/mL)obtained at different levels of inactivation with O3and H2O2/O3were separated by centrifugation at 5 000 r/min for 5 min，4℃.The standard calibration was performed by using bovine serum albumin (Sigma，USA).The concentration of protein was calculated by measuring the absorbance at 700 nm with a spectrophotometer (Hitachi U-3010，Japan).

1.5.3 DNA gel analysis

Genomic DNA of G.intestinali treated with and without O3or H2O2/O3inactivation was extracted as described by Nishiguchi et al［23］.Briefly，the samples were collected by centrifugation at 8 000 r/min for 10 min then resuspended in Tris + EDTA buffer solution (TE) buffer.After centrifugation，50 μL of 10% sodium dodecyl sulfate (SDS)buffer was added and incubated at 65℃ for 20 min until clarification.After a series of treatments，approximately 15 μL of genomic DNA per lane was electrophoresed in 0.5% agarose gel with 5 μg/mL ethidium bromide，and the voltage and current were 150 V and 100 mA，respectively.The DNA in the gel was visualized under UV light and photographed［24］.

2 Results and Discussion

2.1 Inacitivation effect of G.intestinali by O3 and H2O2，respectively

Experiments were conducted to test the effect of O3alone on the inactivation of G.intestinali.Figure 1 illustrated the inactivation of G.intestinali by O3in water based on time.The results showed that when the dosages of O3were above 1.5 mg·L-1and the contact time was up to 10.0 min，the extinct rate of G.intestinali could achieve about 99.6% inactivation.Then，effect of H2O2alone on the inactivation of the G.intestinali was tested(Fig.1).Results showed no inactivation effects even when H2O2dosages up to 5.0 mg·L-1with the contact time increasing.These results are in excellent agreement with previously published data［25］.However，Susan et al's results showed inactivation of C.parvum at higher H2O2concentrations，and longer contact time than it used in this study［25］.

Fig.1 Effect of O3 and H2O2 alone on inactivation of G.intestinali

2.2 Optimum molar ratio of H2 O2/O3 for inactivation of G.intestinali

With initial concentration of O3at 1.0 mg·L-1，the molar ratio of H2O2/O3ranging from 0.1 to 1.2，the extinction rate of G.Intestinalis altered with the contact time changing，which was shown in Fig.2.

Fig.2 G.intestinali inactivation with different H2O2/O3 molar ratio

The H2O2/O3system had a faster effect on inactivating G.intestinali than O3.When the initial dosage of O3was 1.5 mg·L-1with contact time above 10.0 min，the extinction rate of G.intestinali could achieve 99.6%.After adding H2O2，the same inactivation effect could be achieved by H2O2/O3of 1.0 mg·L-1O3(［H2O2］/［O3］= 0.8)and contact time 7 min.The effect of inactivation reduced if the molar ratio of H2O2/O3was higher or lower than 0.8，which suggested the optimum molar ratio of H2O2/O3was 0.8.The results showed that not only the effect of G.intestinali inactivation was further strengthened，but also the O3dosage could be reduced significantly in the H2O2/O3synergistic process.The results are in excellent agreement with previously published data［26-27］.

2.3 Role of ·OH

For further research on the impact of H2O2on hydroxyl radicals，the changes of dosages of H2O2in the system with time were observed and showed in Fig.3.When H2O2(0.5 mg·L-1)was added into the ozone solution (initial ozone concentration 1.0 mg·L-1)，the consumption rate of H2O2was low (40%).However，after G.intestinali was added into the system，the consumption rate of H2O2increased one and a half times than the former with contact time 3 min.These results revealed that H2O2itself would react with O3and produce hydroxyl radicals，while consuming some amount of H2O2.If the reaction system included G.intestinali，the concentration of H2O2declined obviously，which implied that H2O2could be induced to produce more hydroxyl radicals from O3.The experiment above confirmed that the inactivation efficiency of peroxone was much better than O3alone.

Fig.4 Effect of TBA on inactivation of G.intestinali by H2O2/O3 system

To identify the existence of free radicals in the reaction system，free radical inhibitors such as TBA were added into the system.These free radical inhibitors consumed vast dosages of·OH，interrupting the free radical chain reaction，and thus affected the oxidation capacity.Figure 4 showed inhibition by different concentrations of TBA on inactivation of G.intestinali by H2O2/O3(molar ratio 0.8，reaction time 7 min，20℃).As TBA dosage increased，the G.intestinali inactivation rate reduced.When the concentration of TBA was 60 mg/L，the extinction rate of G.intestinali fell to 54.7%.From what has been mentioned above，it could be concluded that OH radicals acted as the main factor of H2O2/O3inactivating G.intestinali.

2.4 Mechanisms of G.intestinali inactivated by H2O2/O3

At present，there is limited information on the mechanisms of inactivation of G.intestinali with disinfection，and also few reports on the mechanisms of inactivation of G.intestinali by H2O2/O3.Inactivation of infectivity of G.intestinali is due to the damaging of the genome or destruction of the viral protein.If this is clear，mechanisms of inactivation of G.intestinali by disinfectants would be clarified，and more importantly，could give valuable guidance in the development of novel disinfectants and inactivation methods.

2.4.1 SEM examination of G.intestinali morphology variation

Surface structure of G.intestinali cells were investigated by SEM (Fig.5).It revealed that while exposed to O3and H2O2/O3，the morphological alterations of cysts were induced，and the extent of these morphological changes appeared to be dependent on the exposure time.

Fig.5 SEM photos of G.intestinali inactivated by O3 and H2O2/O3

Figure 5 (a)showed no exposure under O3and H2O2/O3in 5000 magnification，and the surface of the cell membrane was intact and smooth.In Fig.5 (b)，cysts were exposed to O3for 420 s，and the cell turned into folded.In Fig.5 (c)，cysts were exposed to H2O2/O3for 420 s，the cells were nearly destroyed，and the cell membrane had shrunk.After being treated by O3，the changes and damage of dermal cell wall indicated that the main effect on G.intestinali was a contact action.Through damage to the cell of G.intestinali，O3impacted the physiological activities of cysts.As it is known that the cell structure is the main function unit of a living body，the damage of cell structure will lead to the impairment of the function unit，which will irreversibly speed up the damage of cell structure.After adding H2O2into the cysts and O3system，during the same contact time，the damage of cell surface was enhanced and the cytoplasm leakage started.

2.4.2 Protein mass of G.intestinali treated with O3and H2O2/O3

Figure 6 compared the quantity of total proteins released to the solution during G.intestinali inactivation by O3and H2O2/O3.Proteins constitute of 15% of total E.coli mass which was estimated to 6.65 × 10-13g/cell.The G.intestinali cell almost 100 times more than E.coli cell (E.coli cell size is about 0.5 ×2 μm，the G.intestinali cell is about 12 ×9 μm).Therefore，approximately 20.0 mg/L of proteins would be extracted from 2 × 106cell/mL if all the proteins were released.

Fig.6 Protein mass of G.intestinali treated with O3 and H2O2/O3

At the beginning of reaction，just little protein had been released.As G.intestinali reacted with O3and H2O2/O3，the amount of proteins released changed gradually.For example，in the initial 3 min，the sum of released protein increased to 14.08 or 18.30 mg/L which were treated with O3or H2O2/O3respectively.After then，the protein content value started to reduce.And when reaction time reached 9 min，the sum of protein declined to 8.66 or 1.79 mg/L.This meant approximately 80% of released protein was degraded by H2O2/O3，comparatively，it was significantly higher than the cells disinfected by O3(30%).It demonstrated that H2O2/O3not only had a greater ability to damage cell wall of G.intestinali，but also degraded the released protein.Although the reagent protein may not accurately represent the protein in G.intestinal cells，the results provided a rough estimate on how much protein would be degraded after released from the cells due to reaction with disinfectant.

2.4.3 Genomic DNA of G.intestinali treated with H2O2，O3，and H2O2/O3

Experiments were performed to examine the damage degree of genomic DNA when G.intestinal cells were treated with H2O2，O3and H2O2/O3respectively.The dosages of O3and H2O2， and molar ratio of H2O2/O3were arranged as foredescribed.

Fig.7 Agarose gel electrophotogram of G.intestinali genomic DNA treated with H2O2，O3，and H2O2/O3

It showed that，no matter how long the contact time was，there was almost no genomic DNA damage after G.intestinal cells were treated with H2O2or O3，(Fig.7)respectively.It could be found that there was some trailing in agarose gel electrophotogram when the contact time with H2O2/O3system was up to 3 min.After 5 min reaction，the trailing of DNA increased.The above results indicated that H2O2/O3had a stronger damage effect on genomic DNA of G.intestinali than H2O2or O3respectively.

3 Conclusions

This paper examined the inactivation efficiency of H2O2/O3on G.intestinal using bench-scale experiments.The conclusions can be drawn as follows.

(1)The reaction with ·OH dominates during the G.intestinal inactivation by H2O2/O3system， the more consumption of H2O2after G.intestinal added suggested H2O2could be induced to produce more hydroxyl radicals from O3.

(2)O3alone process，dosage up to 1.5 mg·L-1with contact time above 10.0 min led to the extinct rate of G.intestinal achieving 99.6%.The inactivation on G.intestinal by H2O2/O3system required 1.0 mg·L-1O3(［H2O2］/［O3］= 0.8)with 7.0 min.The extinct rate was strengthened besides the O3dosage being reduced significantly in H2O2/O3synergistic process.

(3)In H2O2/O3synergistic process，the damage of G.intestinali cell surface was enhanced，and cells started the cytoplasm leakage.The effect of H2O2/O3could not only destroy the cell surface，but also degrade the protein and damage the genomic DNA of G.intestinali.

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