Environ. Eng. Res. Vol. 12, No. 5, pp. 238～243, 2007Korean Society of Environmental Engineers

Photo or Solar Ferrioxalate Disinfection Technology without External Hydrogen Peroxide Supply

Min Cho1,2, Joonseon Jeong1, Jaeeun Kim1, and Jeyong Yoon1,†

1School of Chemical and Biological Engineering, College of Engineering, Seoul National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul, 151-742, Korea2Korea Interfacial Science and Engineering Institute, Yangjae-dong, Seocho-gu, Seoul, 137-899, Korea

Received October 2007, accepted December 2007

Abstract

The Fenton reaction, which refers to the reaction between ferrous ions and hydrogen peroxide to produce the OH radical, has not been widely applied to the disinfection of microorganisms despite being economic and environmentally friendly. Cho et al. have previously proposed the neutral photo ferrioxalate system as a solution to the problems posed by the Fenton reaction in acidic conditions,1) but this system still requires an external hydrogen peroxide supply. In the present study, we developed a simple disinfection technology using the photo or solar ferrioxalate reaction with-out the need for an external hydrogen peroxide supply. E. coli was employed as the indicating microorganism. The study results demonstrated the effectiveness of the photo ferrioxalate system in inactivating E. coli without any external hydrogen peroxide supply, as long as dissolved oxygen is supplied. Furthermore, the solar ferrioxalate system achieved faster inactivation of E. coli than an artificial light source at similar irradiance.

Keywords: E. coli, Inactivation, Ferrioxalate, Photo or solar ferrioxalate, OH radical

1. Introduction1

The Fenton reaction (Fe2+/H2O2) has been widely applied to the purification of non-biodegradable wastewater in the field of industrial wastewater treatment.2-4) Despite its dual advantages of being economic and environmentally friendly, the Fenton reaction, which refers to the reaction between ferrous ions and hydrogen peroxide to produce the OH radical,5-7) has not been widely used in microbial disinfection, due to the requirement of an acidic condition (pH of 3) and the formation of large amounts of iron sludge.

Fe(II) + H2O2 → ･OH equation (I)

Cho et al. recently reported that a modified version of the Fenton reaction, called the photo ferrioxalate system, can be used to inactivate microorganisms at near neutral pH, even under non biocidal light irradiation, as long as an appropriate amount of oxalate is present.1) However, one limitation of this technology is the requirement for an external hydrogen pero-xide supply, which is the key reagent of the Fenton reaction,

†Corresponding authorE-mail: jeyong@snu.ac.krTel: +82-2-880-8927, Fax: +82-2-876-8911

rendering the application of this technology difficult and com-plicated in terms of its operation and maintenance.4,8)

One method for producing in situ hydrogen peroxide is the complete utilization of the photochemical reaction of ferrioxa-late ([FeIII(C2O4)3]3-).9) This approach requires the use of dis-solved oxygen (O2), supplied by bubbling with oxygen, which becomes the precursor for hydrogen peroxide through a series of reactions.4)

CO2-･ + O2 → CO2 + O2

-･ equation (II)

Fe(II) + O2-･ + 2H+ → Fe(III) + H2O2 equation (III)

Accordingly, we examined the applicability of the photo fer-rioxalate system as disinfection technology by generating in situ hydrogen peroxide, and of the solar ferrioxalate system us-ing solar irradiation instead of artificial light (300-420 nm).10,11) Furthermore, we elucidated the role of reactive species formed during the photochemical ferrioxalate reaction for microbial inactivation.

Escherichia coli (E. coli) was selected as the indicator micro-organism in this study, due to its similar characteristics to water-borne bacterial pathogens such as Salmonella and Shigella and its use in many previous disinfection studies.12,13)

Photo or Solar Ferrioxalate Disinfection Technology without External Hydrogen Peroxide Supply 239

2. Materials and Methods

2.1. Preparation and Analysis of Chemicals

All solutions were prepared using de-ionized water (Millipore Co., USA), and analytical reagent grade chemicals (Aldrich Co. and Sigma Co., USA). All glassware used was washed with dis-tilled water and then sterilized by autoclaving at 121°C for 15 min. Stock solutions of Fe3+ (5 mM), oxalate (C2O4

2-, 300 mM) and hydrogen peroxide (H2O2, 10 mM) were prepared using the same procedure as that described in detail in the studies.9,14)

The concentrations of Fe3+ and hydrogen peroxide were mea-sured using the phenanthroline15) and titanium sulfate16) methods, respectively. The oxalic acid and para-chlorobenzoic acid (pCBA; 1.92 µM) concentrations were analyzed by high performance liquid chromatography (HPLC, Waters Co., USA) with a UV detector, employing carbohydrate (SUPELCOGEL C-610H, 300 mm × 7.8 mm, 9 μm) and C18 reverse-phase (XTerra Rp-18 reverse-phase column, 150 mm × 2.1 mm, 5 μm) columns, respectively.

2.2. Preparation and Analysis of E. coli

E. coli (ATCC strain 8739) stock was prepared following the procedure described in our previous work.12) In brief, E. coli was cultured by growing in tryptic soy broth for 18 hr at 37°C. The number of viable E. coli was measured by counting the E. coli colonies, which was formed using the spread plate method on nutrient agar (Difco Co., USA) after incubation at 37°C for 24 hr. This non-selective nutrient agar medium was chosen for the E. coli count in order to give a more conservative population estimate. During the disinfection experiments, the initial popu-lations of E. coli were adjusted within the range of 1.5 × 105 to 3.2 × 105 cfu/mL and 1 mL of solution was withdrawn at each sampling time and diluted 1/10 and 1/100. Three replicate 0.1 mL samples of the diluted and undiluted solutions were spread for the cell count and good reproducibility was achieved with 10% standard deviation.

2.3. Transmission Electron Microscopic (TEM) Assay

The structural change of E. coli induced by the disinfection step was examined with TEM.17) E. coli suspensions (108 cfu/mL), treated by the photo ferrioxalate system to achieve 1 log inacti-vation, were fixed in 2% para-formaldehyde for 2 - 4 hr at 4°C. Samples were centrifuged (5000 rpm, 10 min, 4°C) and rinsed three times in 0.05 M sodium cacodylate buffer (pH 7.2). The pellet was suspended in 0.05 M of sodium cacodylate buffer containing 1% of osmium tetraoxide and kept at 4°C for 2 hr. After the post fixation step, E. coli was rinsed twice using dis-tilled water and stained in 0.5% uranyl acetate at 4°C overnight. Samples, gradually dehydrated using ethanol, were placed in propylene oxide (100%) for 15 min, infiltrated using propylene oxide/Spurr’s resin (Low viscosity embedding kit, Electron Microscopy sciences Co., USA) for 30 min, and embedded in 100% Spurr’s resin and polymerized for 24 hr at 70°C. E. coli

was cut on an Ultra-microtome (MT-X, RMC, Tucson Co., USA) and sectioned samples were stained in 2% uranyl acetate for 7 min, followed by Reynold’s lead citrate for 7 min. Finally, grids were assayed using TEM (JEM-1010, JEOL Co., Japan).

2.4. Experimental Procedures

The procedures for the disinfection experiment with the photo ferrioxalate system were similar to those described in our pre-vious study.1) Experiments with artificial light were performed using 50 mL of the suspension in a 60 mL Pyrex reactor. The reactor was closed to the atmosphere with a cap. The suspension was irradiated from a distance of 2 cm and stirred vigorously at a temperature of 21 ± 1°C (maintained by air cooling). The in-cident photon flow of the four Black Light Blue (BLB) lamps (18 W, Philips Co., Netherlands), which emit in the wavelength range of 300-420 nm, was estimated by ferrioxalate actinometry18) to be 7.9 × 10-6 Einstein/L.sec while the irradiance was measured by a radiometer (UVX radiometer, UVP Co., USA) at 360 nm was 0.8 mW/cm2. The initial pH was adjusted to 5.8 with phos-phate buffer solution (PBS; KH2PO4 + NaOH; 5 mM). Aerobic conditions were obtained by bubbling high purity O2 or air gas through the solution for 20 min before starting the experiments and anaerobic conditions were obtained using N2 bubbling.

The solution used for the photolysis experiment was prepared by adding appropriate amounts of Fe3+ (0.04-0.20 mM), oxalate (5 mM) and PBS (5 mM), followed by spiking the diluted E. coli stock solution. The ferrioxalate solutions were protected from light by means of aluminium foil, prior to photolysis, in order to prevent any photochemical reactions from occurring. The photo ferrioxalate experiments were started by removing the aluminium foil. During all experiments, because the oxalate was continuously decomposed, the appropriate amount of stock oxa-late solution was injected into the reaction solution at each time interval by an automatic injection system, so that it was main-tained at an average of 75% of its initial concentration. Tert- butanol (t-BuOH; 20 mM) and pCBA were used as the OH rad-ical scavenger and OH radical probe compounds, respectively.12,19)

In general, 5-8 samples were taken during the disinfection experiments (20-240 min) to measure the microorganism popu-lations. The samples were quickly quenched with the appropriate amount of sodium sulfite (Na2SO3, 1 mM), which consumed any ferrioxalate-initiated oxidants and residual hydrogen pero-xide. The quenching reagent itself did not affect the viability of E. coli.

The ferrioxalate experiments with solar irradiation, termed the solar ferrioxalate system, were performed with a 50 mL quartz reactor to allow the UV-C (< 300 nm) component of sunlight to penetrate into the sample solution. The oxygen condition was maintained by bubbling air. The final concentration of Fe3+ and oxalate was controlled to 0.1 mM and 5.0 mM, respectively, and hydrogen peroxide (0.2 mM) was added in one initial pulse mode to achieve the rapid inactivation of the solar ferrioxalate system.

The disinfection experiments in the solar ferrioxalate system were conducted on a sunny summer day on the roof of our labo-

Min Cho, Joonseon Jeong, Jaeeun Kim, and Jeyong Yoon240

ratory building in Seoul (126°56´W, 37°27´N), Korea, in order to have a similar irradiance to that used for the photo ferrioxa-late system. During the experiments, the irradiance and temper-ature varied within the range of 0.92 ± 0.22 mW/cm2 and 21-32°C, respectively. The intensity of solar irradiation was measured with a radiometer at 360 nm. Because of the increased temper-ature with the solar irradiation itself, additional control experi-ments were conducted in PBS (30 mM) at 30°C.20-22)

During the control experiments of the photo ferrioxalate sys-tem, no E. coli inactivation was observed without BLB radiation or oxalate addition. Experiments were repeated three times and their averaged values with statistical deviations were used for the data analysis. The statistical analysis in this study was con-ducted using the Microsoft Excel program (version 2003, Micro-soft Co., USA).

3. Results and Discussion

3.1. Reaction Pathway of the Photo and Solar Ferrioxalate Systems

The reaction pathway for the photochemical reaction of ferri-oxalate is schematically presented in Fig. 1. When ferrioxalate ([FeIII(C2O4)3]3-) absorbs light, it produces Fe(II) and the oxalyl radical anion (C2O4

-･) through the ligand to metal charge

transfer.

[FeIII(C2O4)3]3- + hv → Fe(II) + 2C2O42- + C2O4

-･ equation (IV)

This reaction occurs even in the case of irradiation using a solar or artificial lamp within the wavelength range of 300-420 nm. The rate of this initiation reaction is linearly proportional to its primary quantum yield (less than unity), because of its back reaction and the rate of light absorption.5) The C2O4

-･ produced

as a result of photo-reduction is not significantly involved in any other reactions, due to its rapid decarboxylation dissocia-tion to the carbon dioxide radical anion (CO2

-･) and carbon

dioxide.2)

C2O4-･ + CO2

-･ + CO2 (reaction rate : 2×106 s-1) equation (V)

When dissolved oxygen is present, the photo-generated CO2-･

faces two major comparative pathways, namely reactions (b) and (c) in Fig. 1, depending on the concentrations of ferrioxa-late and oxygen (O2).

CO2-･ + [FeIII(C2O4)3]3- → Fe(II) + CO2 +

3C2O42-･(Reaction (b)) equation (VI)

CO2-･ + O2 →→ CO2 + H2O2 (Reaction (c)) equation (VII)

In an oxygen saturated condition, CO2-･ is mainly involved

in a reaction with oxygen, i.e. that is reactions (c) and (d), to produce hydrogen peroxide, after which the OH radical is pro-duced from the reaction between ferrous ion and hydrogen peroxide (reaction (a) in Fig. 1).

Fig. 1. Reaction pathway in the photo or solar ferrioxalate system.

To verify the formation of hydrogen peroxide and OH radical, demonstrated in Fig. 1, several experiments were conducted to analyze the concentration of hydrogen peroxide and the degrada-tion of an OH radical probe compound (pCBA), and the results are described in Fig. 2. Fig. 2(a) shows the generation of hydro-gen peroxide in the photo ferrioxalate system with oxygen bub-bling. In the presence of saturated oxygen, hydrogen peroxide was gradually generated, reaching at a concentration of 0.18 mM at 140 min (Fig 2(a)), supporting the reactions (c) & (d) in Fig. 1. Fig. 2(b) shows the degradation profiles of pCBA in the photo ferrioxalate system. pCBA was added three times sepa-rately during the hydrogen peroxide generation. Since pCBA is an OH radical probe compound,12) the degradation of pCBA in Fig. 2(b) apparently confirms the presence of OH radical indu-ced by reaction (a) in Fig. 1. In addition, the different rate of pCBA degradation in Fig. 2(b) could be explained by the diffe-rent concentrations of hydrogen peroxide (Fig. 2(a)) and OH radical (reaction (a)) during the experimental time.

3.2. E. coli Inactivation in the Photo Ferrioxalate System

The effect of oxygen on the E. coli inactivation by the photo ferrioxalate system was investigated according to the presence of a hydrogen peroxide supply (Fig. 3). Three important obser-vations can be made from the results of Fig. 3. First, E. coli was apparently inactivated in oxygenated conditions (-○-), even in the absence of an external hydrogen peroxide supply, as 96 min was required for 2 log inactivation. As shown in reaction (a) of Fig. 1, this observation is important since the external supply of hydrogen peroxide is the key reagent to generate the OH radical responsible for microbial inactivation in the photo ferrioxalate system. Therefore, a photo ferrioxalate system that does not require an external hydrogen peroxide supply will possess a major advantage in field applications. Second, no significant difference in the E. coli inactivation was apparent according to the type of oxygen supply, pure oxygen (O2 (-○-)) or air bubb-ling (-●-). This observation provides another advantage in the practical application of the photo ferrioxalate system. Third, a large lag phase of approximately 60 min (-○- in Fig. 3) was observed in the photo ferrioxalate disinfection practice, possi-bly due to the time required for (1) destroying significant sur-face components of the microorganism12,23) and (2) attaining a certain quantity of in situ hydrogen peroxide generation. The shortened lag phase of E. coli inactivation (-■- in Fig. 3.) resu-lting from the pulse input of hydrogen peroxide (0.2 mM) at the

Photo or Solar Ferrioxalate Disinfection Technology without External Hydrogen Peroxide Supply 241

(a) (b)

Fig. 2. Hydrogen peroxide formation (a) and pCBA degradation (b) supporting the presence of OH radicals in the photo ferrioxalate system (oxy-gen bubbling, [Fe3+] = 0.1 mM, [oxalate]0 = 5.0 mM, incident photon flow = 7.9 × 10-6 Einstein/L.sec, [oxalate]/[Fe3+] ≥ 25, pH = 5.8-6.5, tempe-rature = 21 ± 1°C, [pCBA]0 = 1.92 µM).

Fig. 3. Inactivation of E. coli in the photo ferrioxalate system without any external H2O2 supply ([Fe3+] = 0.1 mM, [oxalate]0 = 5.0 mM, inci-dent photon flow = 7.9 × 10-6 Einstein/L.sec, irradiance = 0.8 mW/cm2, [oxalate]/[Fe3+] ≥ 25, pH = 5.8-6.5, temperature = 21 ± 1°C)

beginning supports the last explanation about the required time for hydrogen peroxide generation. When hydrogen peroxide was injected once, the efficacy of microbial inactivation for 2 log E. coli inactivation was elevated by 53%, which is consis-tent with a relatively small lag phase in the hydrogen peroxide supplying system.1)

3.3. Effect of Ferric Ion

The effect of the ferric ion concentration on E. coli inactivation was examined in the photo ferrioxalate system without an exter-nal hydrogen peroxide supply, with a ferric ion concentration ranging from 0.04 to 0.20 mM (Fig. 4). E. coli inactivation was increased with increasing ferric ion concentration. As the initial ferric ion concentration increased from 0.04 to 0.20 mM, the time required for 2 log E. coli inactivation decreased by 57% (from

Fig. 4. Effect of ferric ion dose on E. coli inactivation in the photo ferrioxalate system without any external hydrogen peroxide supply (H2O2) ([Fe3+] = 0.04 - 0.2 mM, [oxalate]0 = 5.0 mM, incident photon flow = 7.9 × 10-6 Einstein/L.sec, irradiance = 0.8 mW/cm2, [oxalate]/ [Fe3+] ≥ 25, pH = 5.8-6.5, temperature = 21 ± 1°C)

180 to 78 min). The effect of the ferric ion concentration on E. coli inactivation was found to be similar regardless of the exter-nal hydrogen peroxide supply in the photo ferrioxalate system.1)

3.4. E. coli Inactivation in the Solar Ferrioxalate System

The solar ferrioxalate system is defined as the ferrioxalate system in which the artificial light is replaced with solar irradia-tion. Fig. 5 shows the results of E. coli inactivation in the solar ferrioxalate system. Two log E. coli inactivation was achieved four times faster with the ferrioxalate system in the presence of solar irradiation (BLB irradiance of 0.9 mW/cm2, temperature of 21-32°C, -■- in Fig. 5) than with the photo ferrioxalate sys-tem at similar experimental conditions (BLB irradiance of 0.8 mW/cm2, temperature of 21 ± 1°C, -■- in Fig. 3). The reaction

Min Cho, Joonseon Jeong, Jaeeun Kim, and Jeyong Yoon242

(a) (b)Fig. 6. Transmission electron microscopy images of E. coli inactivated with the photo ferrioxalate system: (a) before inactivation and (b) after one log inactivation.

Fig. 5. Inactivation of E. coli in the solar ferrioxalate system (one initial pulse input of 0.2 mM H2O2, air bubbling, ([Fe3+] = 0.1 mM, [oxalate]0 = 5.0 mM, irradiance = 0.92 ± 0.22 mW/cm2 at 360 nm, temperature = 21-32°C).

temperature in the solar ferrioxalate system was gradually incr-eased from 20°C to 32°C, possibly due to radiant heat. Since no significant E. coli inactivation was observed (data not shown), and as E. coli was left at 30°C for 60 min in PBS, the increased temperature alone cannot explain the significantly enhanced inac-tivation. However, it is well known that microbial inactivation becomes faster at higher temperature, when chemical treatments such as OH radical, ozone, chlorine are applied.12) Whereas, as shown in Fig. 5 (-□-), E. coli was only slightly inactivated by solar irradiation alone (1 log inactivation after 52 min) without ferrioxalate. This level of inactivation is expected by solar irra-diation itself.21) Therefore, the combined effect of the increased biocidal sensitivity at high temperature and the solar biocidal irradiation explains the superior performance of the solar ferrio-xalate system over the photo ferrioxalate system shown in Fig. 5.

3.5. Role of Reactive Species in the Photo Ferrioxalate System

In the photochemical ferrioxalate system, several reactive species (OH radical, H2O2, O2

-･ and CO2

-･) have the potential

to cause E. coli inactivation (Fig. 1). Several additional experi-ments were therefore conducted to elaborate the role of reactive species for E. coli inactivation formed in the photo ferrioxalate system.

In the absence of oxygen by nitrogen gas (N2) bubbling, it is clear that only CO2

-･ could exist among the reactive species of

the photo ferrioxalate system, due to the inhibition of reactions (a) and (c) in Fig. 1. However, no E. coli inactivation was ob-served in nitrogen condition (data not shown), indicating that CO2

-･ does not cause any significant E. coli inactivation under the given experimental conditions.

When photo ferrioxalate experiments were conducted in the presence of an excess amount of the OH radical scavenger (20 mM of t-BuOH), no E. coli inactivation was detected (data not shown). Because other reactive species (H2O2, O2

-･ and CO2

-･),

except the OH radical, exist in the OH radical scavenging condi-tion, this result confirms that the OH radical, which was produ-ced by the Fenton reaction (reaction (a) in Fig. 1),24,25) is the main species responsible for E. coli inactivation in the photo ferrioxalate system.

Fig. 6(b), showing the TEM image of E. coli when 1 log inac-tivation was attained in the photo ferrioxalate system, clearly illustrates the apparent disruption of various surface components, such as cell wall and membrane, and inner components of E. coli by OH radicals formed by the photo ferrioxalate reaction. Consi-dering the high reactivity of the OH radical (oxidation potential: 2.7 V) toward most E. coli components, it is plausible to assume that it must undergo rapid reaction with the various cell compo-nents.26,27)

4. Conclusion

This study has confirmed the effective utilization of a photo ferrioxalate system with artificial light (300-420 nm) for inac-tivating E. coli without an external hydrogen peroxide supply, as long as a level of dissolved oxygen is maintained. Even fas-

Photo or Solar Ferrioxalate Disinfection Technology without External Hydrogen Peroxide Supply 243

ter inactivation was observed in the solar ferrioxalate system. These results support the potential of the photo or solar ferrio-xalate system for disinfecting contaminated surface water or groundwater in remote rural places where sunlight is abundant.

Acknowledgements

This work was supported by the Brain Korea 21 Program in the Ministry of Education in Korea and the Ministry of Environ-ment as “The Eco-technopia 21 project”.

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