In the present study, the characteristics of the combined ultraviolet (UV) and ozone disinfection process were investigated from kinetic and mechanistic viewpoints employing Escherichia coli (E. coli) as an indicator microorganism. Compared to individual unit processes, the combined UV/O3 tests produced excess hydroxyl radicals (HO•) and yielded synergistic inactivation of E. coli in the initial phase of reaction. The presence of O3 during UV exposure caused the destruction of cell structure, and then repressed bacteria regrowth after treatment. Moreover, the formation of malondialdehyde (MDA) showed that the improved generation of intermediate HO• via ozone photolysis accelerated the decomposition of bacterial cell surfaces, which was further confirmed by the leakage of intracellular potassium ions (K+). The results suggested that the synergistic bactericidal effect of combined UV/O3 owed mainly to the enhanced destruction of bacterial cell structure.
During recent decades several technologies have been applied for water disinfection to prevent the spread of diseases caused by waterborne pathogens. Traditional biocidal compounds, such as chlorinated agents and ozone, are efficient for inactivation of a broad range of microorganisms, but can produce disinfection byproducts (DBPs) that have a negative impact on the aquatic environment (Lian et al. 2014; Lyon et al. 2014). Ultraviolet irradiation in the UV-C spectral region has been considered an excellent disinfection technology for not forming dangerous or malodorous halogenated compounds in low UV dose (Sisti et al. 2014). Nevertheless, some species of microorganisms have shown high resistance to UV-C irradiation, and many bacteria have regrowth potential after UV treatment (Guo et al. 2012).
Recently, combining UV radiation with ozone (UV/O3) has been proposed as a promising disinfection technology with high effectiveness while being environmentally friendly (Bustos et al. 2010). Several researchers have reported the synergistic bactericidal effect of the UV/O3 process (Tawabini et al. 2013), and evidence has also shown that the presence of UV radiation during ozone exposure can reduce the formation of toxic byproducts (Zhao et al. 2013). However, the disinfection efficiency of the UV/O3 process in the literature has been significantly influenced by the species of microorganisms (Liu et al. 2014), and some authors have stated that hastened decomposition of ozone under UV radiation could diminish its efficacy as a disinfectant (Venosa et al. 1984).
Compared to the large database on kinetics, the microbial inactivation mechanism of the UV/O3 process is relatively poorly established. Generally, direct impairment in intracellular functions is considered to be the primary reason for bacteria growth inhibition after UV radiation treatment (Beck et al. 2014), while cell death by ozone is primarily related to cell surface damage (Cho et al. 2010). Short wavelength UV light can directly attack DNA to form pyrimidine dimers, which can prohibit the replication of DNA and cause the inactivation of microorganisms (Poepping et al. 2014). In the case of UV/O3 disinfection, not only UV radiation, but also molecule ozone and intermediate reactive oxygen species (ROS) can attack bacterial cells. However, few systematic studies have examined the multiple bactericidal mechanism of the UV/O3 process.
The objective of this study was to evaluate the disinfection efficacy of the combined UV/O3 process, and to elucidate the bacteria inactivation mechanism. The roles of UV, ozone and HO• in the UV/O3 process were estimated, and the destruction of cell structure was examined.
MATERIALS AND METHODS
Preparation of bacteria culture
The Escherichia coli (strain DH5α) bacteria was pre-cultured aerobically in Luria-Bertani (LB) nutrient broth based on procedures reported elsewhere (Wu et al. 2011). Briefly, after incubation at 37°C for 20 h, the bacterial cells were harvested from culture by centrifugation, and were then washed three times using saline water (0.9%, w/w). The sediment was resuspended in ultrapure water to give a cell concentration of approximately 108 colony-forming units per millilitre (CFU/mL) for tests.
All experiments were performed at room temperature (20 ± 2°C) and pH 7.1. Tests under each experimental condition were conducted in triplicate with their mean and standard deviations being reported. At fixed time intervals, 10.0 mL bacterial suspension was harvested from the reactor and dosed with 0.2 mL 0.1 mol/L Na2S2O3 to stop the inactivation reaction.
A series of 10-fold dilutions was performed and 0.1 mL of each dilution was plated on LB agar plates. All plates were incubated at 37°C for 24 h, and the numbers of colonies on the plates were counted.
To determine the E. coli photoreactivation after treatment by various processes, samples were transferred into several 50 mL glass flasks, and then were exposed to white fluorescent light with light intensity of 1.1 × 104 lx. At each time interval, a 0.1 mL sample was transferred and used immediately for cell viability assays.
Atomic force microscopy
Bacterial cells after various treatments were characterized directly by atomic force microscopy (AFM). Briefly, a 0.2 mL specimen was dropped on a glass slide, followed by air-drying in a clean bench. Specimens were observed and photographed under an AFM system (BioScope, Veeco, Germany).
UV-C fluence rate was determined by a UV irradiance meter (Photoelectric Instrument Factory of Beijing Normal University, China). Ozone concentration was measured by the iodometric method (APHA 1998). The temperature and pH were measured with a Hach sensION378 meter (USA). The HO• concentration was assayed using the p-chlorobenzoic acid (pCBA) method (Cho et al. 2004). The quantity of malondialdehyde (MDA) was measured using the thiobarbituric acid (TBA) method (Esterbauer & Cheeseman 1990). The K+ concentration was determined by an inductively coupled plasma optical emission spectrometer (ICP-OES, Optima 5300DV, Perkin-Elmer, Inc., USA).
RESULTS AND DISCUSSION
Inactivation may be temporary or permanent, depending upon the type and dose of disinfectant used (Magbanua et al. 2006). As shown in Figure 2(b), photoreactivation of E. coli occurred in samples of the UV-only process, and the count of reactivation bacteria cells was positive with exposure time, especially in the initial phase. After exposure to white ﬂuorescent light for 1 h, bacteria counts were approximately ten times higher than that obtained right after UV radiation. Conversely, extended bacterial mortality was observed after reactions with the presence of ozone, suggesting that the UV/O3 process could overcome the limitation of bacteria regrowth in the UV-only process.
Destruction of cell structure checked by AFM
Cell structure damage was an important cause of bacteria inactivation. The above AFM investigation indicated that ozone played a critical role in bacteria cell disruption, and the presence of UV radiation during ozonation expedited the decomposition of cell structure. This may have been mainly due to the improved generation of HO• in the UV/O3 process. On the other hand, cell damage induced by UVC radiation during UV/O3 disinfection might depress cell antioxidant capacity (Santos et al. 2013), thereby enhancing the bactericidal effects of ozone and HO•.
Evidence of cell membrane damage
The cell membrane plays a vital role in providing a barrier of selective permeability for bacteria. The leakage of K+, which exists universally in bacteria and plays an important role in protein synthesis and regulation of polysome content (Ren et al. 2009), was utilized to reflect the permeability of the cell membrane. Similar to the results of MDA formation (Figure 5(a)), the curves described in Figure 5(b) suggest that UV radiation was ineffective for K+ leakage, while ozone caused the leaked K+ concentration to increase significantly. The K+ leakage rate in the UV/O3 process was faster than that in the ozone-only test, with approximately 1.05 mg/L and 0.84 mg/L K+ being detected after 10 s, respectively. The maximum K+ concentration in the ozone-only or UV/O3 tests was about 1.23 mg/L at 180 s treatment.
The above observations agree with the results of AFM (Figure 4), that the combination of ozone with UV radiation expedited the destruction of bacterial cell structure, and then accelerated the leakage of intracellular components. The gradual leakage of intracellular substances from impaired cells might also induce the inactivation of bacteria after O3 and UV/O3 exposure, which was probably a reason for the extended bacterial mortality that is depicted in Figure 2(b).
This work revealed that the application of ozone in conjunction with UV radiation could hasten and enhance the inactivation of E. coli cells. UV and ozone were mainly responsible for E. coli inactivation in the coupled process, and the improved disinfection efficiency could have partly resulted from the abundant production of HO•, which expedited the destruction of bacterial cell structures. Restated, not only can the combined treatment synergistically inactivate harmful cells in water, but also the limitations of using ozone and UV separately can be reduced. It is concluded that UV/O3 is an efficient technology for water disinfection, and the multiple forms of damage caused by ozone, UV and HO• result in enhanced inactivation efficiency.
We are grateful for grants from Natural Science Foundation of Jiangsu Province (No. BK20,130,835), China Postdoctoral Science Foundation (No. 2013M541,600), Fundamental Research Funds for the Central Universities of Hohai University (No. 2013B13,020,026), and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.