Response surface modeling for the inactivation of Bacillus subtilis subsp. niger spores by chlorine dioxide gas in an enclosed space

ABSTRACT

Bacillus subtilis subsp. niger spores are a commonly used biological indicator to evaluate the disinfection of an enclosed space. In the present study, chlorine dioxide (ClO₂) gas was applied to inactivate B. subtilis subsp. niger spores in an enclosed space. The effects of the ClO₂ gas concentration (1-3 mg/l), relative humidity (RH, 30-70%) and exposure time (30-90 min) were investigated using a response surface methodology (RSM). A three-factor Box-Behnken experimental design was used. The obtained data were adequately fitted to a second-order polynomial model with an R_2adj of 0.992. The ClO₂ gas concentration, RH and exposure time all significantly (P<0.05) and positively correlated with the inactivation of B. subtilis subsp. niger spores. The interaction between the ClO₂ gas concentration and RH as well as that between the exposure time and RH indicated significant and synergistic effects (P<0.05). The predictive model was validated by additional eight experiments and proven to be with good accuracy. Overall, this model established by the RSM could show the trend of the inactivation of spores, indicate the interactions between important factors, and provide a reference to determine effective conditions for the disinfection in different enclosed spaces by ClO₂ gas.

Implications: The inactivation of indoor biological contaminants plays an important role in preventing the transmission of pathogens and ensuring human safety. The predictive model using response surface methodology indicates the influence and interaction of the main factors on the inactivation of Bacillus subtilis subsp. niger spores by ClO₂ gas, and can predict a ClO₂ gas treatment condition to achieve an effective sterilization of enclosed spaces. The results in this paper will provide a reference for the application of ClO₂ gas treatments for indoor disinfection.

Introduction

Infectious disease epidemics, such as those of the Ebola virus, Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome (SARS), have attracted increasing attention worldwide (Althaus et al., 2015; Petersen et al., 2015; Smith, 2006). The inactivation of indoor biological contaminants has been shown to play an important role in preventing the transmission of pathogens and ensuring human safety (Aygun et al., 2002). Therefore, a high-efficiency sterilization technique is required to effectively decontaminate indoor environments. Compared with liquid sterilization chemicals, fumigating decontaminants feature several advantages such as a high efficiency, large disinfection volume, good penetration ability, and simple operation and have been widely employed to sterilize enclosed spaces (Rogers et al., 2007; Goyal et al., 2014; Hudson et al., 2007).

Chlorine dioxide (ClO₂) is a strong oxidizing decontaminant with a broad and high biocidal effectiveness (Aieta and Berg, 1986). It has been approved as a disinfectant for agricultural, commercial, industrial, medical, and residential use (U.S. Environmental Protection Agency [EPA], 2006). As a promising fumigating decontaminant, ClO₂ gas is more effective, less toxic, and less time-consuming than traditional fumigants, such as ethylene oxide and formaldehyde, and it more effectively inactivates microorganisms than aqueous ClO₂ (Han et al., 2001; Vaid et al., 2010). Several studies have shown that ClO₂ gas can inactivate all bacteria and spores to completely decontaminate enclosed spaces such as hospital rooms (Lowe et al., 2013; Luftman et al., 2006), ambulances (Lowe et al., 2013), Biosafety Level 3 laboratories and associated ductwork (Lowe et al., 2012), cafeterias (Hsu et al., 2014), exposure chambers (Pottage et al., 2012), and entire buildings (Wood and Martin, 2009).

The ClO₂ gas concentration, relative humidity (RH), and exposure time have been shown to affect the sterilization effect of ClO₂ gas. Lowe et al. (2013) reported that the decontamination of an ambulance could achieve a complete inactivation of B. anthracis and B. atrophaeus spores by ClO₂ gas, and decrease in gas concentration, exposure time, and RH would reduce log reductions of spores. Morino et al. (2009) showed that the log reduction of feline calicivirus in the dry state was less than 1.0 in 45–55% RH but 6.1 in 75–85% RH when the organism was treated with 0.26 ppm ClO₂ gas for 24 hr. These previous studies mainly focused on the evaluation to the inactivation of different microorganisms by ClO₂ gas and demonstrated the significant effect of these parameters. However, the effect tendency and interactions of gas concentration, RH, and exposure time are little researched using multifactor experimental design and mathematical modeling. Additional studies are needed to elucidate the mechanisms underlying this effect as well as the interaction between these parameters.

Biological indicators are typically used to evaluate the sterilizing effect of different environments. Bacillus subtilis subsp. niger is one of the most common spore-forming strains and has the strongest resistance to the sanitizer among different types of pathogens. This strain has been used as a biological indicator in several evaluations of disinfection efficiency (Li et al., 2012; Jeng and Woodworth, 1990; Pottage et al., 2012). Furthermore, B. subtilis subsp. niger spores also can be used as an avirulent surrogate for B. anthracis spores because of the similar resistance to decontaminants. (Rogers et al., 2005, 2007).

Response surface methodology (RSM) is a collection of statistical and methodology techniques useful for developing, improving, and optimizing processes in which a response of interest is influenced by several variables (Baş and Boyacı, 2007). A predictive model for the inactivation of E. coli O157:H7 on green peppers by ClO₂ gas using RSM was built in a previous study (Han et al., 2001), but this model was primarily applied to foodborne pathogens. Furthermore, the inactivation of spores in an enclosed space has not yet been described by a mathematical model.

Therefore, the objectives of this study were to investigate the individual effects and interactions of the ClO₂ gas concentration (1–3 mg/L), RH (30–70%), and exposure time (30–90 min) on the inactivation of B. subtilis subsp. niger spores in an enclosed space and to develop a predictive model using RSM for determining appropriate treatment conditions to inactivate spores. The predictive model could illustrate the effect tendency of important factors in the ClO₂ gas treatment and provide a reference to apply ClO₂ gas to decontaminate environments.

Materials and methods

Spore preparation and coupon inoculation

Stock cultures of B. subtilis subsp. niger (ATCC 9372) were obtained from the China General Microbiological Culture Collection Center (Beijing, China) for this study. Spores were prepared according to the slightly modified standard method (Aouadhi et al., 2013). Cultures were grown in tryptic soy broth on a rotary shaker (HNY-2102C; Honour, Tianjin, China) at 37 °C for 18–24 hr to obtain a working culture. A aliquot (1 mL) of working culture was spread on sporulation agar plates (18 g/L nutrient broth, 15 g/L agar, 8 mg/L MnSO₄·H₂O, and 1 g/L CaCl₂·H₂O) and incubated at 37 °C for 7 days. Spores were gently dislodged from the plate using a sterile L-shape spreader and collected in a sterile centrifuge tube. The spores were washed three times with sterile distilled water and treated at 65 °C for 30 min to kill the vegetative cells. A spore population of >90% was verified via a spore stain and microscopic analysis. The spore stock was diluted to a final concentration of approximately 1 × 10⁹/mL and was stored at 4 °C.

As required, the samples of spore stock (2 mL) were diluted in a measured volume of sterile distilled water to obtain a spore suspension with a concentration of 5 × 10⁸/mL. Each sterile filter paper coupon (1 cm × 1 cm; type 103; Jiaojie, Shenyang, China) was inoculated with 10 μL of this suspension. After inoculation, the coupons were dried in a laminar flow biosafety hood for 1 hr at 23 °C. The prepared coupons were used within 24 hr of inoculation.

Test chamber

The inactivation experiments were conducted in a chamber (length × width × height, 1.25 m × 0.8 m × 1 m) equipped with a door, gloves, injection ports, and connection for electricity. A pass box was connected to the chamber. Four electric fans (HSL-2; Emicorcom, Leqing, China) were installed in the corner of the chamber to mix the fumigant. To maintain the desired RH during the exposure time, water vapor was injected into the chamber via an ultrasonic humidifier (PW103; Povos, Shanghai, China). A sensor (CHT3W1TLD; Honeywell, Morris Plains, NJ, USA) was placed inside the chamber to measure the temperature and RH. The structure of the test chamber is shown in Figure 1.

Figure 1. Diagram of the test chamber used for the inactivation experiment.

ClO₂ gas generation and measurement

Because ClO₂ gas is difficult to store and transport, it should be generated and used on site. In this study, ClO₂ gas was generated and injected into the test chamber using a ClO₂ gas generating system (Lab-01; National Bio-Protection Engineering Center, Tianjin, China) to obtain the desired gas concentration. A diaphragm pump (N86KTE; KNF Inc., Freiburg, Germany) was used to circulate the ClO₂ gas and air mixture inside the chamber, and a concentration sensor of ClO₂ gas (AF26F; Optex Inc., Tokyo, Japan) in the recirculation line continuously monitored the concentration of ClO₂ gas.

ClO₂ gas treatment

All experiments were repeated three times using three samples per experiment for a total of nine data points per inactivation treatment. In each treatment, three coupons were individual placed into different sealed and sterile centrifugal tubes (50 mL; Corning Inc., Corning, NY, USA). Another coupon was retained as positive control outside the chamber. The sealed centrifugal tubes and three sterile plates were placed inside the chamber within reach of the glove ports. The B. subtilis subsp. niger spores was then inactivated at varying gas concentrations (1–3 mg/L), RH (30–70%), and exposure times (30–90 min). The temperature in the test chamber was constant at 22–24 °C.

To achieve the expected experimental conditions, the humidifier and generator were used to transfer water vapor and ClO₂ gas into the chamber. When the desirable RH, ClO₂ gas concentration and temperature were stabilized as designed, the centrifugal tubes were opened, exposing the coupons and starting the exposure period. Because there was a slow decay of the ClO₂ gas during the exposure period, the ClO₂ gas was intermittently added to ensure that the gas concentration kept constant in the treatment. At the predetermined time point, the coupons were placed into sterile plates using sterile forceps, and the plates were then removed via a pass box to a biological safety cabinet in order to extract the spores. The chamber was aerated with air after all of the coupons had been removed.

Enumeration of viable spores

The number of viable spores in the untreated and ClO₂ gas–treated coupons was determined using the pour plate method. The test coupons and the untreated control coupons were aseptically placed into sterile tubes containing 10 mL of phosphate-buffered saline (PBS), 0.1% Tween 80, and a magnetic stir bar. The tubes were stirred (3 min) on a magnetic stirrer (RH basic; IKA Inc., Staufen, Germany) until the filter paper had disintegrated into pulp to aid the removal of spores from the coupons. The tubes containing the coupons were then vortexed (1 min) at a speed of 1000 r/min (MS1 Minishaker; IKA).

Serial dilutions (1 in 10) to 10⁻⁴ were prepared using sterile water. Aliquots (1 mL) of the appropriate dilution were pipetted and spread on nutrient broth agar plates in duplicate. These plates were incubated for 24 hr at 37 °C, and the colonies were then enumerated. Inactivation was expressed as a logarithmic viability reduction, log(N₀/N), where N and N₀ are the colony counts after sterilization and in the untreated coupons, respectively. The average number of colony-forming units (CFU) from positive controls was measured to be 5.13 × 10⁶ per coupon.

Design of experiments

The results of previous experiments showed that the ClO₂ gas concentration, RH, and exposure time were the most significant factors to influence the inactivation of microorganisms by ClO₂ gas (Han et al., 2001, 1999). In this study, single-factor experiments were designed to express the individual effects of the ClO₂ gas concentration (1–3 mg/L), RH (30–70%), and exposure time (30–90 min) on the log reduction of B. subtilis subsp. niger spores, respectively. Each single-factor experiment consisted of five levels of the experimental factor, and other nonexperimental factors were held at the middle value of the range studied. Based on the single-factor experiments, a Box-Behnken experimental design (Ferreira et al., 2007) with three independent factors was used to study the inactivation of B. subtilis subsp. niger spores by ClO₂ gas. Specifically, the factors investigated were the ClO₂ gas concentration, RH, and exposure time, which were designated as X₁, X₂, and X₃, respectively. Each factor was examined at three coded levels, which are indicated by −1, 0, and +1, as shown in Table 1. The real values of the independent variables were coded according to eq 1:

1

Table 1. Codes and levels of variables selected for the trials.

Factor	Symbols		Level^a
	Coded	Uncoded	−1	0	1
ClO2 gas concentration (mg/L)	x1	X1	1	2	3
Relative humidity (%)	x2	X2	30	50	70
Exposure time (min)	x3	X3	30	60	90

Notes: ^ax₁ = (X₁−2)/1; x₂ = (X₂−50)/20; x₃ = (X₃−60)/30.

where X_i is the real value of the independent variable; x_i is the coded value of the variable X_i; X₀ is the value of X_i at the center point, and ΔX is the step change. X and x values are shown in Table 1.

In the next step, the inactivation behavior of spores is explained by the second-order polynomial equation as follows:

2

where Y is the predicted response, which is the log reduction of B. subtilis subsp. niger spores (log(N₀/N)); B₀, B_i, B_ij, and B_ii are constant coefficients of intercept, linear, interaction, and quadratic terms, respectively; x_i and x_j are coded independent variables.

The software Design Expert (version 8.0.6; Stat-ease Inc., Minneapolis, MN, USA) was used for the analysis of variance (ANOVA) and estimation of the response surface with a multiple linear regression. The fit of the predictive model was assessed based on the adjusted coefficient of determination (R²_adj), and its statistical significance was checked by the Fisher’s F test in the same program. The three-dimensional graphical representation of the system behavior, called the response surface, was used to describe the interaction effects of the variables on the log reduction of spores.

Results

Single-factor experiments

The susceptibility of B. subtilis subsp. niger spores in the test chamber was tested by varying the ClO₂ gas concentration, RH, and exposure time according to the design of single-factor experiments. The spore viability decreased (log reduction increased) as the ClO₂ gas concentration increased from 1 to 3 mg/L at a constant RH of 50% and exposure time of 60 min, as shown in Figure 2. This finding suggested a positive correlation between the degree of inactivation and the gas concentration. Specifically, the highest log reduction of 5.06 was observed at 3 mg/L.

Figure 2. Individual effect of ClO₂ gas concentration on the inactivation of B. subtilis subsp. niger spores using ClO₂ gas. RH = 50%; exposure time = 60 min.

As shown in Figure 3, the inactivation of B. subtilis subsp. niger spores increased as the RH was increased from 30% to 70% at a ClO₂ gas concentration of 2 mg/L and an exposure time of 60 min. The maximum log reduction of 4.86 was achieved at 70% RH.

Figure 3. Individual effect of RH on the inactivation of B. subtilis subsp. niger spores using ClO₂ gas. Gas concentration = 2 mg/L; exposure time = 60 min.

Figure 4 shows that the exposure time of the spores to the ClO₂ gas affected the spore viability. In this study, exposure times of 30–90 min were examined, and the highest log reduction value of 4.42 of B. subtilis subsp. niger spores was obtained for an exposure time of 90 min at a gas concentration of 2 mg/L and RH of 50%.

Figure 4. Individual effect of exposure time on the inactivation of B. subtilis subsp. niger spores using ClO₂ gas. Gas concentration = 2 mg/L; RH = 50%.

Predictive model

Table 2 presents the results of varying the ClO₂ gas concentration, RH, and exposure time on the inactivation of B. subtilis subsp. niger spores in enclosed space by ClO₂ gas, using RSM. The response (Y) was the log reduction of this organism.

Table 2. Box-Behnken experimental design and data for inactivation of B. subtilis subsp. niger spores by ClO₂ gas using RSM.

Trial No.	x1	x2	x3	Y = log(N0/N)
				Observed	Predicted
1	−1	0	−1	0.55	0.53
2	1	0	−1	3.68	3.59
3	−1	0	1	3.01	3.10
4	1	0	1	6.71	6.73
5	−1	−1	0	0.65	0.49
6	1	−1	0	3.40	3.32
7	−1	1	0	2.91	3.00
8	1	1	0	6.71	6.86
9	0	−1	−1	0.49	0.66
10	0	−1	1	2.01	2.08
11	0	1	−1	2.32	2.25
12	0	1	1	6.71	6.54
13	0	0	0	2.61	2.60
14	0	0	0	2.51	2.60
15	0	0	0	2.44	2.60
16	0	0	0	2.75	2.60
17	0	0	0	2.67	2.60

Using a multiple regression analysis of the experimental data (Table 2), the response variables and test variables were related by the following second-order polynomial equation:

3

where Y is the predicted log reduction of spores, x₁, x₂, and x₃ are coded terms for three independent variables (viz., the ClO₂ gas concentration, RH, and exposure time).

The regression coefficients in eq 3 were calculated and tested for their significance using Design Expert, and are shown in Table 3. The positive signs of regression coefficients indicate the positive effect on the predicted response Y. The P value at the confidence level of α = 0.05 is used as a standard to assess the significance of each coefficient for being different from zero. The linear coefficients (B₁, B₂, and B₃), quadratic coefficients (B₁₁), and interaction coefficients (B₁₂ and B₂₃) were significant, as indicated by small P values (P < 0.05). The other term coefficients (B₁₃, B₂₂, and B₃₃) were not significant.

Table 3. Regression coefficients of the predictive model.

Model Term	Coefficient	Value	Standard Error	P Value
Intercept	B0	2.60	0.077	<0.001
x1	B1	1.67	0.061	<0.001
x2	B2	1.51	0.061	<0.001
x3	B3	1.42	0.061	<0.001
x1x2	B12	0.26	0.086	0.0187
x1x3	B13	0.14	0.086	0.1424
x2x3	B23	0.72	0.086	<0.001
	B11	0.71	0.084	<0.001
	B22	0.11	0.084	0.2387
	B33	0.18	0.084	0.0716

Table 4 presents a summary of the ANOVA for the predictive model. The results show that the predictive model was highly significant, as was evident from the Fisher’s F test (F = 230.84) with a very low probability value (P < 0.0001). The goodness of fit of the model was checked by the adjusted determination coefficient (R²_adj). The value was 0.992, which was close to 1, indicating a satisfactory adjustment of the model to the experimental data. The large P value for lack of fit (>0.05) shows that the the F test was insignificant, implying significant model correlation between the variables and responses.

Table 4. Analysis of variance (ANOVA) for the predictive model.

Source	Degree of Freedom	Sum of Squares	Mean Square	F Value	P Value
Model	9	61.78	6.86	230.84	<0.0001
Residual error	7	2.21	0.03
Lack of fit	3	0.15	0.049	3.21	0.1448
Pure error	4	0.061	0.015
Total	16	61.99

Response surfaces

The graphical representations of the regression eq 3 were obtained using the Design Expert. In Figures 5–7, the response surfaces and contour plots show the effect of the ClO₂ gas concentration, RH, and exposure time on the inactivation of B. subtilis subsp. niger spores in an enclosed space, and the contour lines in contour plots expressed the values of log reduction. In each figure, one factor remains constant at its optimal value determined by the design of experiments. A 6-log reduction of spores, which could reach the recommended sterility assurance level, was regarded as a criterion of the effective sterilization in this study (Bozkurt et al., 2014).

Figure 5. Interaction effects of ClO₂ gas concentration and RH on the inactivation of B. subtilis subsp. niger spores using ClO₂ gas while the exposure time was 60 min. (a) Contour plots of log reduction; (b) response surface.

Figure 6. Interaction effects of ClO₂ gas concentration and exposure time on the inactivation of B. subtilis subsp. niger spores using ClO₂ gas while the RH was 50%. (a) Contour plots of log reduction; (b) response surface.

Figure 7. Interaction effects of exposure time and RH on the inactivation of B. subtilis subsp. niger spores using ClO₂ gas while the ClO₂ gas concentration was 2 mg/L. (a) Contour plots of log reduction; (b) response surface.

Figure 5 shows the effect of the ClO₂ gas concentration and RH for a median exposure time (60 min, 0 level). Specifically, the log reduction of B. subtilis subsp. niger spores directly correlated with the ClO₂ gas concentration and RH. The model predicted a 6-log reduction when the gas concentration was in the range of 2.7–3 mg/L, and the RH was in the range of 60–70%.

The interaction of the ClO₂ gas concentration with the exposure time at a constant RH (50%, 0 level) is shown in Figure 6. The contour plot indicates that the reduction of B. subtilis subsp. niger spores increases with the increase in ClO₂ gas concentration and time. Meanwhile, the treatment at concentration of 2.75–3 mg/L and exposure time of 75–90 min was estimated to reach a 6-log reduction based on the model.

Figure 7 shows the log reduction of spores for varying exposure times and RH values at fixed ClO₂ gas concentration (2 mg/L, 0 level). Specifically, exposure times of 82–90 min and RH of 65–70% were predicted to result in a 6-log reduction of spores.

Validation of the model

To validate the accuracy of the predictive model, eight additional experimental parameters were tested in the range of the Box-Behnken design. The conditions and results of the experiments are shown in Table 5. The experimental values were found to be significantly in agreement with the predicted values (average absolute relative deviation = 5.43%). Therefore, the model reliably predicted the inactivation of B. subtilis subsp. niger spores by ClO₂ gas in an enclosed space for gas concentrations of 1–3 mg/L, RH of 30–70%, and exposure times of 30–90 min.

Table 5. Verified results of the predictive model.

Trial No.	Factors			Y = log(N0/N)
	x1 (mg/L)	x2 (%)	x3 (min)	Observed	Predicted
1	2	50	30	1.24	1.35
2	2	50	90	4.42	4.21
3	2	30	60	1.16	1.21
4	2	70	60	4.86	4.22
5	1	50	60	1.53	1.64
6	3	50	60	5.06	4.98
7	1	70	30	1.20	1.18
8	3	30	90	4.50	4.34

Discussion

In this study, the inactivation of B. subtilis subsp. niger spores by ClO₂ gas in an enclosed spaces was examined as a function of controlled ClO₂ gas concentration, RH value, and exposure time, and a predictive model using RSM was developed based on these experimental data.

An analysis of the individual effect of the ClO₂ gas concentration on the inactivation of spores (Figure 2) demonstrated that the log reduction of spores increased as the concentration increased from 1 to 3 mg/L. This finding is consistent with the previous work (Jeng and Woodworth, 1990), in which the log reduction of B. subtilis subsp. niger spores were 6.2, 5.9, and 4.0 when exposed to ClO₂ gas concentrations of 30, 15, and 7 mg/L for 30 min, respectively. The indoor disinfection by ClO₂ gas requires an appropriate range of concentration, considering the safety, operability, and efficiency. Low concentration leads to a slow inactivation rate and long treatment time, whereas excessive concentration brings strong causticity and high risk of operation. The range of concentration in this study is moderate and in accordance with the practical application in various enclosed spaces (Pottage et al., 2012; Lowe et al., 2013).

RH plays an important role in the inactivation of microorganisms by ClO₂ gas. The inactivation tendency of ClO₂ gas according to different levels of RH is shown in Figure 3. High level of RH leads to a high log reduction of B. subtilis subsp. niger spores. Several studies also obtained an efficient inactivation under conditions of high RH (>70%) (Wood and Martin, 2009; Luftman et al., 2006; Bhagat et al., 2010), whereas the disinfection effect of ClO₂ gas under low RH (<50%) is less mentioned. In the present study, different levels of RH were used to reflect the influence on the spore inactivation, and there was a nonlinear increase of the log reduction as the RH values increased from 30% to 70%. The rate of killing under high RH was faster than under low RH. Therefore, humidifying the environment to a high level of RH will accelerate the disinfection process by ClO₂ gas.

Exposure time is a common factor that can influence the degree of inactivation. In Figure 4, the increase of exposure time also reduced the number of B. subtilis subsp. niger spores. The disinfection by ClO₂ gas is a complex process that is affected by several factors. The single-factor experiments in which the ClO₂ gas concentration, RH, and exposure time were varied did not demonstrate interactions between different factors and predict a treatment condition that results in complete inactivation of B. subtilis subsp. niger spores. Therefore, RSM, an empirical modeling technique used to describe the individual effects of variables and the mutual interactions between variables, was employed in this study.

The RSM analysis (eq 3; Tables 3–5 and Figures 5–7) demonstrated the influence and interaction of the three controlled parameters on the inactivation of B. subtilis subsp. niger spores using ClO₂ gas. All the linear coefficients, B₁, B₂, and B₃, of the predictive model were significant (P < 0.001) and greater than zero in Table 3, confirming that these parameters all significantly and positively correlated with the inactivation of spores. Because of the descending order of the linear coefficients: B₁ (1.67) > B₂ (1.51) > B₃ (1.42), the ClO₂ gas concentration most significantly affected the inactivation, followed by the RH and exposure time in the respective ranges studied.

The interaction between the ClO₂ gas concentration and RH indicated a synergistic effect on the inactivation of spores due to the significance (P < 0.05) and positive sign of the interaction coefficient B₁₂ in Table 3. This phonemonon means that the combination of the gas concentration and RH has an additional and positive effect on the log reduction of spores except the individual effects of experimental factors. This considerable interaction could also verified by the nonlinear nature of the response surface (Figure 4b) and the respective contour plots (Figure 4a) (Yetilmezsoy et al., 2009). Several studies have also indicated that the combination of the ClO₂ gas concentration and RH represents a synergistic effect (Park and Kang, 2015; Han et al., 2001). This synergistic effect may be attributed to the following: Water vapor may condense onto the surface to surround the spores under high RH conditions (Pottage et al., 2012). Due to the high solubility of ClO₂ gas in water, the condensed water likely acts as the carrier of ClO₂ gas. Thus, the combination of high gas concentration and high RH will allow more ClO₂ act on the spores and lead to a rapid improvement in the inactivation efficiency.

The analysis of the model also shows that the RH and exposure time exert a synergistic effect on the inactivation of spores. The interaction coefficient B₂₃ (0.72) in Table 3 was significantly different from zero (P < 0.05) and greater than the coefficient B₁₂ (0.26), indicating a strong interaction between the RH and exposure time. When both the RH and exposure time increased, the huge increase in the log reduction in the response surface (Figure 7b) and the decrease in the spacing of the contour lines (Figure 7a) also verified the synergistic effect. The reason for this result may be as follows: Bacterial spores are highly resistant to disinfectants because of thick and firm spore coats, which protect the contents of spores from adverse environmental conditions (McKenney and Eichenberger, 2012). Increases in RH and time leads to spores swell and the expansion of the channels used by gases to access the spores (Westphal et al., 2003). Therefore, the combination of high RH and long exposure time will provide more effective exposure of ClO₂ gas to spores and exert a positive effect on the inactivation of B. subtilis subsp. niger spores.

The predictive model based on the RSM in this study can be used to illustrate the effect tendency and interactions of these experimental factors as well as predict a condition of ClO₂ gas treatment to achieve a 6-log reduction of B. subtilis subsp. niger spores according to the response surfaces and contour plots in Figures 5–7. Filter paper strip is one of the most common carrier types and is widely used for the validation of space decontamination (Jeng and Woodworth, 1990; Jia et al., 2013). However, materials of the spore carrier greatly affected the inactivation efficiency (Li et al., 2012; Rogers et al., 2007). It is important to note that the predicted results are based on a certain carrier of spores and that use of different indoor surface materials and/or treatment conditions may result in altered inactivation characteristics. Therefore, the predictive model only provided a reference for the ClO₂ gas treatment and further validation of calculated treatment conditions must be carried out before actually applying a treatment to the indoor disinfection.

Conclusions

An RSM involving experimental design and regression analysis was used to reveal the influence and interaction of the ClO₂ gas concentration, RH, and exposure time on the inactivation of B. subtilis subsp. niger spores in an enclosed space by ClO₂ gas. A predictive model was established and verified by the experiment data. The model could be used to describe the individual effect and interaction of important factors, and to predict a ClO₂ gas treatment condition to achieve a 6-log reduction of spores, which may aid the application of ClO₂ gas treatments for indoor disinfection.

Funding

This work was supported by the National Science and Technology Major Project of China (No. 2012ZX10004801) and the National High Technology Research and Development Program of China (No. 2014AA021405).

Additional information

Funding

This work was supported by the National Science and Technology Major Project of China (No. 2012ZX10004801) and the National High Technology Research and Development Program of China (No. 2014AA021405).

Notes on contributors

Jiancheng Qi

Tao Wang is a doctoral candidate, Jiancheng Qi is a research professor, Jinhui Wu is an associate professor, Limei Hao, Ying Yi, Song Lin, and Zongxing Zhang are research assistants at the Institute of Medical Equipment, Academy of Military Medical Sciences, and the National Bio-protection Engineering Center in Tianjin, People’s Republic of China.