Effect of water compounds on photo-disinfection efficacy of TiO2 NP-embedded cellulose acetate film in natural water

Photocatalysis disinfection has great potential for irrigation water disinfection to improve fresh produce safety. Titanium dioxide (TiO2) nanoparticle (NP)-embedded cellulose acetate (CA) film has shown effectiveness against Escherichia coli (E. coli) O157:H7 in water. The current study evaluated the effect of natural water compounds on the photo-disinfection efficacy of TiO2 NP-embedded CA film. Humic acid, calcium carbonate (CaCO3), and kaolin clay solutions were prepared at four concentrations, respectively. When concentration increased from 0 to 20 ml/L, inactivation of E. coli O157:H7 in humic acid, CaCO3, and kaolin clay solutions decreased from 6 log to 5, 4, and 2 log CFU/ ml, respectively after 3 h treatment. Turbidity, UVT-254, water hardness, total suspended solids (TSS), and total organic carbon (TOC) of the solutions were measured. UVT-254 and turbidity had the highest correlation with the inhibition effect of water compounds on photo-disinfection efficacy. A prediction equation was developed with UVT-254 and water hardness as independent variables to predict photo-disinfection efficacy in natural water. E. coli O157:H7 decreased by 1 and 2.5 log CFU/ ml in unfiltered and filtered natural creek water samples after treatment. The results from this study showed promise in the use of TiO2 NP-embedded CA film to inactivate pathogens in natural water.


INTRODUCTION
Agriculture consumes 70% of the freshwater used worldwide, and the number increases to 95% in developing countries (FAO ). As a crucial part of agricultural water, irrigation water is one of the main pathways by which pathogenic microorganisms can reach fresh produce (FDA ). Irrigating produce to be eaten raw with contaminated water may increase the risk of foodborne illness (Chalmers et al. ; Gu et al. ; Rodrigues et al. ). Evidence of produce contamination through contaminated irrigation water has been found in epidemiological investigations (Steele & Odumeru ). Irrigation water has been implicated in several E. coli O157:H7 outbreaks (Ackers et  Chlorination is the most commonly used disinfection method, however, disinfection byproducts (DBPs) that are potentially carcinogenic may be produced when chlorine reacts with natural organic matter (Marugán et al. ).
Treatment methods such as UV and ozone are effective and have been demonstrated for drinking water treatment (Solomon et al. ; Loeb et al. ). However, these methods are costly and may not be suitable for irrigation water disinfection. The development and implementation of alternative disinfection methods are therefore needed.
Photocatalysis disinfection inactivates microorganisms by reactive oxygen species (ROS) generated when a semiconductor photocatalyst is activated by light in the presence of water and oxygen. Photocatalysis disinfection is an economic and environmentally friendly process and might be an alternative water disinfection method for irrigation water (Dasgupta et al. ). Titanium dioxide (TiO 2 ) has been considered the most suitable photocatalyst for water disinfection due to its strong oxidizing abilities, chemical stability, low cost, and low toxicity (Chong et al. ). TiO 2 NP-embedded cellulose acetate (CA) film has shown effectiveness in the inactivation of bacteria in water (Xie & Hung ). This film might be combined with different types of solar photocatalytic reactors for water treatment. However, when applyied in natural water, various organic and inorganic compounds may detrimentally affect photocatalysis disinfection of water. The effect of these water compounds on photocatalysis disinfection needs to be understood before this technology can be practically applied. Humic substances constitute the major fraction of natural organic matter (NOM) in natural water and 90% of total dissolved organic carbon (DOC) in surface water may come from humic acid (Alkan et al. ). It has been reported that humic acid can inhibit the photocatalysis process by acting as an ·OH scavenger (Garbin et al. ; Cheng et al. ). Calcium carbonate (CaCO 3 ) occurs in rocks, soil, and natural water world-wide.
The presence of CaCO 3 affects water hardness and alkalinity, and it is the predominant component of scales deposited from natural water (Wang et al. ). The effect of CaCO 3 on photocatalysis inactivation is rarely reported. Kaolin clay is one of the common inorganic particles in natural water, and it contributes to turbidity in water and might be the worst particle for shielding (Liu & Zhang ). The effect of kaolin clay on photocatalysis disinfection in water has not been reported.
Also, there is a lack of information on the comparison of the inhibition effect of different water compounds on photocatalysis disinfection.
The overall purpose of this study was to determine the effect of natural water compounds on the photo-disinfection efficacy of TiO 2 NP-embedded CA films. Specific objectives include: (i) to determine the photo-disinfection efficacy of TiO 2 NPembedded CA film in water containing humic acid, CaCO 3 , and kaolin clay; (ii) to evaluate the correlation between water quality parameters and the photo-disinfection efficacy of TiO 2 NP-embedded CA film in water; (iii) to develop a prediction equation for predicting the disinfection efficacy of TiO 2 NP-embedded CA film in natural water.

MATERIALS AND METHODS
Preparation of TiO 2 NPs-embedded film Aeroxide ® P25 TiO 2 nanoparticles (anatase-rutile), acetone, cellulose acetate (average Mn ∼30,000 by GPC), and triethyl citrate (TEC) (>99%) were purchased from Sigma-Aldrich (St Louis, MO, USA). TiO 2 NPs-embedded CA film was prepared using a solution casting method as described in Xie & Hung (). An optimum TiO 2 NPs concentration at 0.82 mg/cm 2 on the film to achieve the highest bactericidal effect had been determined in a previous study (Xie & Hung  submitted). To fabricate the film, 4 g of CA and 0.4 g of TiO 2 were dissolved and suspended in 20 ml of acetone at room temperature (24 C), separately. TEC (1.2 g) was added to TiO 2 NPs suspension as plasticizer. An ultra-sonication bath (Model FS60, Fisher Scientific, Waltham, MA, USA) was used to assist the suspension of TiO 2 NPs. After sonication, TiO 2 -solvent suspension was added into the CA solution gradually using a pipette with stirring, and the solution was continuously stirred for 2 h. Five millilitres of the mixed solution was added into each glass Petri dish (88 mm in diameter, Corning ® , Sigma-Aldrich (St Louis, MO, USA)) and allowed to dry with Petri dish lid covering in a fume hood at 24 C overnight, and then stored in a vacuum desiccator for 24 h. The thickness of the prepared film was 53.5-54.7 μm.
The strains were stored at À70 C in tryptic soy broth One isolated colony of each strain was then transferred into 10 ml of tryptic soy broth supplemented with 50 μg/ml nalidixic acid (TSBNA) using a sterile inoculation loop and incubated at 37 C overnight. Then one loopful (10 μL) of bacterial culture was transferred again into 10 ml of TSBNA and incubated overnight. After incubation, bacterial suspension was centrifuged at 4,000 × g for 12 min, and the supernatant was decanted and the cell pellet was resuspended in 9 ml of sterilized phosphate-buffered saline (Acros Organics, NJ, USA; PBS, pH 7.2). The inoculum was prepared by mixing 9 ml of each strain to obtain a five-strain cocktail with a concentration of about 9 log CFU/ml. The inoculum was further adjusted to a concentration of about 8 log CFU/ml by making ten-fold dilution.

Sampling and bacterial enumeration
Water samples were taken hourly for three hours and 1 ml of sample was taken each time. Serial dilutions were made in PBS and appropriate dilutions were plated on SMACNA agar and incubated at 37 C for 24 h. Colonies were counted and recorded as log CFU/ml.

Water quality parameter measurement
Water quality parameters including turbidity, UV transmittance at 254 nm (UVT-254), total organic carbon (TOC), total suspended solids (TSS), and total hardness of all the lab-prepared water samples and natural water samples were measured. Turbidity, TOC, and TSS were measured following Hach methods 8237, 10129, and 8006, respectively, using a DR/90 colorimeter (HACH, Loveland, CO, USA). UVT-254, which measures the percentage of light that passes through a water sample at 254 nm, was measured using a UV-vis spectrophotometer (Orion™ AquaMate 8000, Thermo Fisher Scientific, Waltham, MA, USA). Total hardness was determined following USEPA method 8226.

Statistical analysis
Experiments were replicated at least twice. Duplicate measurements were made on each sample. Pearson correlation analysis and partial correlation analysis on the water quality parameters and bacterial reduction were conducted using JMP 14 (SAS Institute, Cary, NC, USA).
The regression equation was developed by least squares regression also using JMP 14. All the tests were performed at a significance level of 0.05.

RESULTS AND DISCUSSION
Effect of water compound on photo-disinfection efficacy of TiO 2 embedded CA film where C/C 0 is the reduction in bacterial concentration, k is the disinfection rate constant, and t is the treatment time.
The delayed Chick-Watson model (Cho et al. ) was developed to accommodate any initial lag time (t 0 ): The modified Chick-Watson model (Cho et al. ) was to fit either an initial shoulder or a tail at the end of the reaction: The Homs model (Hom ) was reported for predicting a curvilinear or non-linear function: where h is the second parameter.
The data generated in the current study were fitted using these empirical models and RMSE (root mean square error) was calculated using the following equation: where N is the number of observations, y n is the observed value, and yn is the predicted value. The results in Table 1 show that the delayed Chick-Watson model provided a better fit with the smallest RMSE for all water compound solutions tested. The three water compounds inhibited the photo-disinfection of CA film following the order of: humic acid > CaCO 3 > kaolin clay.

Effect of kaolin clay on water property and disinfection efficacy
The effects of kaolin clay on water quality parameters and bacterial reduction are shown in Table 2. Kaolin clay did not have a strong inhibition effect on photo-disinfection in the current study compared with humic acid and CaCO 3 .
Results of water property analysis showed that increasing kaolin clay concentration in water did not significantly change TOC and hardness. UVT-254 slightly decreased when kaolin clay concentration increased. However, kaolin clay concentration significantly affected the total suspended solids (TSS) and turbidity. It has been reported that kaolin clay might be the worst particle for shielding light (Liu & Zhang ) and hence may affect photo-disinfection efficacy. However, the current study showed that when the turbidity and TSS of kaolin clay solution were increased to 24 FAU and 24 mg/L, respectively, more than 4.2 log bacterial reductions were achieved. This indicates that turbidity and TSS might not be significant parameters affecting photo-disinfection efficacy.

Effect of humic acid on water properties and disinfection efficacy
Results in Table 2 show that increasing humic acid concentration significantly increased TOC, turbidity, and TSS in water, whereas the UVT-254 reading decreased significantly. as other compounds such as humic acid.

Prediction equation development
As discussed above, various water compounds such as humic acid and CaCO 3 have strong inhibition effects on the TiO 2 NP-embedded CA film photo-disinfection effect.
However, in a practical situation, it is unrealistic to monitor the level of all compounds in water. Identifying common water quality parameters that can be used as water quality indicators for photo-disinfection is a more efficient solution.
To select the proper water quality parameters that can be used for predicting TiO 2 NP-embedded CA film disinfection efficacy, the Pearson correlation of all the water quality parameters reported in Table 2 was first performed and the results are presented in Table 3. It shows that several variables are strongly correlated with another variable. To determine which variable should be excluded from model development, a partial correlation between the predictor variables and the response variable was performed and the results are shown in Table 4. Pearson correlations reported in Table 3   A linear prediction equation is then developed using the least squares method and listed in Equation (7) Table 5. Table 6 shows the observed and predicted bacterial reductions using TiO 2 NP-embedded CA film in these simulated water samples.  Table 7 shows that in natural creek water, bacteria can be reduced by 1 log CFU/ml after 3 h treatment using TiO 2 NP-embedded CA film. After filtration using a 0.45 μm filter, 1.4 log reductions were achieved. After filtration using a 0.2 μm filter, bacterial reductions increased to 2.5 log CFU/ml. As shown in Table 7, filtration also improved water quality. The hardness, turbidity, TSS, and TOC all decreased after filtration, and UVT-254 increased with filtration. Filtration can remove insoluble particles that cause the increase of these parameters and can therefore improve water quality (O'Melia ). Bacterial reductions were also calculated using Equation (7). The predicted bacterial reduction was À0.9 CFU/ml reduction for the water sample without filtration, and 0.9 and 1.1 CFU/ml reductions for samples filtered with 0.2 and 0.45 μm filters, respectively. The total hardness of the unfiltered water sample was over the ranges of the hardness of water samples used for the development of the prediction equation, which may affect the prediction accuracy. More types of natural water and more water property information are needed to further improve the prediction equation.
Nevertheless, the current study has demonstrated that using TiO 2 NP-embedded CA film has the potential to inactivate pathogens in natural water. Filtration has also been found as a simple and effective method to remove water compounds that may affect bacterial inactivation during the photo-disinfection process.

CONCLUSIONS
The results of this study showed that natural water compounds such as humic acid, CaCO 3 , and kaolin clay can all affect the photo-disinfection efficacy of TiO 2 NPembedded CA film. Humic acid had the highest inhibition effect on photo-disinfection, followed by CaCO 3 and kaolin clay. The effect of different water quality parameters on TiO 2 NP-embedded CA film indicates that different water compounds affect photo-disinfection efficacy through different mechanisms. It was found that UVT-254 and turbidity can be used as indicators for predicting the effect of natural water compounds on photo-disinfection efficacy. A predictive equation was developed using UVT-254 and turbidity as independent variables. Photo-disinfection using TiO 2 NP-embedded CA film reduced E. coli O157:H7 by 1 log CFU/ml in the inoculated natural creek water sample.
After filtration using a 0.2 μm filter, about 2.5 log CFU/ml reductions were achieved with the same treatment. Hence, filtration can be used as an effective pre-treatment for photo-disinfection by TiO 2 NP-embedded CA film.