Abstract

The problem of biological invasions attributed to ballast water release is an ongoing problem that threatens ecosystems and human health. Ultraviolet (UV) radiation has been increasingly used for ballast water treatment mainly due to the advantages of short contact time and minimized harmful disinfection by products. In this study, the impact of salinity on the inactivation of Vibrio cholerae (NCTC 7253) was examined, and comparison of inactivation level and disinfection kinetics after medium-pressure (MP) (1 kW) and low-pressure (LP) (10 W) UV irradiation was made. MP UV exposure resulted in higher inactivation efficacy against V. cholerae than LP UV exposure especially at lower UV doses (≤3 mJ cm−2) and salinity had a negative impact on both MP and LP UV disinfection, especially at higher UV doses (≥3 mJ cm−2 for MP and ≥4 mJ cm−2 for LP). To understand the mechanisms of salinity effect on V. cholerae, the enzyme-linked immunosorbent assay (ELISA) was employed to determine the number of cyclobutane pyrimidine dimers (CPDs), one major type of DNA damage. No significant effects of salinity were found at the CPDs level except for 3% artificial seawater after LP UV exposure case. It is imperative that site-specific conditions of salinity be taken into account in the design of UV reactors to treat V. cholerae and other species.

INTRODUCTION

Ballast water is water with its suspended matter carried by ships to ensure stability, trim and structural integrity (IMO 2004). Undesirable non-native organisms including epidemic cholera are introduced into ports throughout the world by the release of ballast water, which appears to be the world's largest invasion vector (Ruiz et al. 1997). Vibrio cholerae, a Gram-negative bacterium and the causative agent of cholera, has caused great concern owing to its toxigenicity and epidemic nature and its ability to adapt and grow in a new environment (Fykse et al. 2012). According to Regulation D2 (ballast water performance standard) set by the International Maritime Organization (IMO) in February 2004, ships are to discharge <1 colony forming unit (CFU) per 100 mL ballast water containing toxigenic V. cholerae. Only effective treatment of ballast water can bring down the species to innocuous levels. Different technologies exist to treat ballast water in order to reduce the spread of invasive species (Tsolaki & Diamadopoulos 2010), among which ultraviolet (UV) disinfection has been increasingly applied to microbial inactivation in ballast water (Sutherland et al. 2003), mainly due to the advantages of non-toxic by products and low maintenance costs.

Low-pressure (LP) and medium-pressure (MP) mercury lamps are the two UV sources predominantly used in water treatment. LP UV lamps emit monochromatic UV radiation at 254 nm, which is close to the optimum germicidal wavelength of 260 nm (Harm 1980). Compared to LP UV lamps, MP UV lamps emit a wider range of UV wavelengths (from 200 to 400 nm; Masschelein 2002), allowing them to affect biological molecules other than DNA. MP UV lamps have been gaining popularity and are used in a wide range of disinfection applications, mainly due to higher UV radiation intensity and savings in space and capital costs, despite the higher energy consumption and the shorter lifetime of about 4,000 h (Masschelein 2002). Studies on UV inactivation of V. cholerae focused mostly on LP UV lamps (Das et al. 1981; Wennberg et al. 2013), whereas little was known about the efficiency of MP UV disinfection and the comparison of the inactivation characteristics of LP and MP UV lamp.

UV light with the wavelengths from 100 to 400 nm can cause damage to DNA, cell membranes and cytoplasmic proteins (Schwarz 1998). The main types of photoproducts in irradiated DNA are cis-syn cyclobutane pyrimidine dimers (CPDs), pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), spore photoproduct, pyrimidine hydrates and DNA-protein crosslinks (Patrick & Rahn 1976), among which CPDs are the most abundant photoproduct accounting for ∼75% of DNA damage (Thoma 1999). CPDs can inactivate microbes through interference with either replication of DNA for the purpose of cellular reproduction or transcription of DNA into RNA for the purpose of making proteins (Harm 1980). Therefore, the relationship between CPDs formation (absorbance at 492 nm) and cell numbers of V. cholerae after UV irradiation was evaluated in the present study.

To our knowledge, the effects of different salt concentrations on UV disinfection are not well documented yet, although salinity is one important factor influencing UV performance, as is the case for Escherichia coli (Rubio et al. 2013) and fecal coliforms (Shang et al. 2009). Hence, the aim of the present work was to assess the effects of salinity on UV inactivation of V. cholerae as a ballast water treatment indicator microorganism after MP and LP UV disinfection. Both the cellular study and the enzyme-linked immunosorbent assay (ELISA)-based assay were used to investigate the inactivation efficiency.

MATERIALS AND METHODS

Bacteria

V. cholerae NCTC 7253 was purchased from the United Kingdom National Collection of Type Cultures (NCTC). One colony of the V. cholerae culture from an agar plate was inoculated into 30 mL of nutrient broth and shaken overnight at 37 °C to prepare an overnight phase. 1 mL of such overnight culture was added to 30 mL of fresh nutrient broth and incubated in a shaker for 4 h at 37 °C to obtain V. cholerae at exponential phase. The V. cholerae cells were harvested by centrifuging at 4,600 g for 10 min, washed twice with sterile distilled water, and resuspended in 30 mL of sterile distilled water. The suspension was further diluted in sterile distilled water to achieve an initial concentration of approximately 106 CFU mL−1 for the UV irradiation study.

UV irradiation experiments

UV irradiation was carried out using the Rayox® bench-scale collimated beam apparatus (Model PS1-1-220, Calgon Carbon Corporation) equipped with an interchangeable MP (1 kW) and LP (10 W) UV lamps. 10 mL of the diluted V. cholerae suspension (approximately 106 CFU mL−1) was dispensed into a 6 cm diameter sterile plastic Petri dish and exposed to either MP or LP UV radiation. The UV doses investigated ranged from 1 to 5 mJ cm−2, and the average germicidal irradiance values for LP and MP were approximately 0.037 mW cm−2 and 0.652 mW cm−2, respectively, calculated taking into account the various factors that can affect the measured UV intensity such as Petri-, water-, sensor- factor, etc., referring to previous studies (Zimmer & Slawson 2002; Guo et al. 2010; Zuo et al. 2015). Calculations of UV doses were performed as follows: UV dose (mJ cm−2) = irradiance (mW cm−2) × exposure time (s). All bacterial suspensions were stirred throughout the irradiation process. 0.1 mL samples were taken before and after irradiation for enumeration to confirm the expected log reduction.

Salinity experiments

V. cholerae were resuspended in two types of water (artificial seawater (ASW) and natural seawater (NSW)). ASW was prepared as described by Lleò et al. (2005), two levels of salinity of which (1% and 3%) were achieved using an Agilent 3200M Multi-Parameter Analyzer (Agilent Technologies Inc., USA) and represent a hyperosmotic environment of natural seawater down to a hypo-osmotic environment of brackish water (Lin et al. 2003). Natural seawater was taken from the western coast of Singapore and passed through a 0.45 μm sterile filter (Millipore, Co., USA). Some physicochemical characteristics of these waters are shown in Table 1.

Table 1

Some physicochemical characteristics of the types of water used in the salinity experiments

Suspension solution Salinity (%) Conductivity (mS cm−1TOC (mg L−1pH T (°C) 
ASW 16.5 – 8.09 22.9 
44.1 – 7.98 22.9 
NSW 43.7 3.47 7.88 22.7 
Suspension solution Salinity (%) Conductivity (mS cm−1TOC (mg L−1pH T (°C) 
ASW 16.5 – 8.09 22.9 
44.1 – 7.98 22.9 
NSW 43.7 3.47 7.88 22.7 

ASW, artificial seawater; NSW, natural seawater.

Cultivation assay

From appropriate dilutions of the microorganisms, the total number of V. cholerae was examined by spread plate on nutrient agar consisting of nutrient broth 13 g, agar 15 g obtained from Sigma-Aldrich Company (Singapore) per litre. Colonies were counted after 24 h incubation at 37 °C and recorded as CFU mL−1.

Enzyme-linked immunosorbent assay

The ELISA assay was used to determine the accumulation of CPDs. Briefly, DNA was extracted from 10 ml of lysed V. cholerae cells according to the protocol of DNeasy® Blood & Tissue kit (Qiagen, Germany). DNA concentration was determined with an ND-1000 spectrophotometer (NanoDrop, Fisher Thermo, Wilmington, DE, USA) to measure the absorbance at 260 nm, and the DNA samples were diluted in PBS to a final concentration of 0.2 μg mL−1 for ELISA. The CPDs content was measured according to the protocol of a commercial ELISA kit (Clone TDM-2, Cosmo Bio, Tokyo), and qualified with a Sunrise TECAN spectrophotometer (TECAN, Austria GmbH) at 492 nm. Samples were analyzed after UV irradiation at 5 mJ cm−2.

Data analysis

The Log reduction of the test microorganisms was calculated as:  
formula
(1)
where Ni is the initial concentration of V. cholerae before UV disinfection (CFU ml−1), and N0 is the concentration of V. cholerae immediately after UV disinfection (CFU mL−1).
A double first order kinetic model as suggested by Vélez-Colmenares et al. (2011) was applied to describe the kinetics of UV disinfection as follows:  
formula
(2)
where σ is fraction of microorganisms sensitive to UV radiation, 1-σ is fraction of microorganisms resistant to UV radiation, k1 is the inactivation rate for sensitive fraction of microorganisms associated with the slope of the first phase of the curves and with the process of inactivation, k2 is the inactivation rate for resistant fraction of microorganisms related to the slope of the second phase of the curves (tailing), and t is exposure time to UV light (s).

All experiments were conducted three times to ensure reproducibility of the experimental data. Data are presented in mean ± standard deviation. A t-test by SPSS 16.0 was used to identify differences in Log reduction and absorbance at 492 nm at the significance level of 0.05.

RESULTS AND DISCUSSION

UV inactivation of V. cholerae

Figure 1 shows the inactivation of V. cholerae following MP and LP UV disinfection in sterile distilled water (the controls). It can be seen that the UV doses between 1 and 5 mJ cm−2 resulted in 0.42 to 4.01 (for MP) and 0.52 to 4.16 (for LP) log reduction in distilled water (the controls).Such inactivation behavior was also reported by Das et al. (1981) who claimed that the survival curves for all three V. cholerae strains (569B, NIH 41 and 154) exhibited no tailing with the k-values ranging from 0.18–0.24 m2 J−1. It was also found that when the UV dose was lower than 4 mJ cm−2, higher log reduction values were achieved when MP UV radiation was employed, indicating that MP UV disinfection was more efficient than LP UV disinfection. This has been reported previously on E. coli (Hu et al. 2005), and is likely due to broader wavelength spectrum emitted by MP UV lamps that may cause damage to intercellular biomolecules other than DNA (Kalisvaart 2004). At higher doses (≥4 mJ cm−2), reduction in cell numbers were similar for LP and MP UV exposure, indicating that both types of UV lamps had similar effects on the inactivation of V. cholerae. Likely, Guo et al. (2009) also found that when the UV dose was 5–10 mJ cm−2, LP UV exposure resulted in higher inactivation level (3 log) against E. coli than MP UV exposure, whereas there was no difference in inactivation between LP and MP lamps at UV doses >10 mJ cm−2 (3–5 log). Additionally, k1 was 1.91 (0.188) and 1.88 (0.210) for MP and LP lamps, respectively (Table 2), which showed that inactivation of MP UV disinfection was faster than LP UV disinfection.

Table 2

Kinetic parameters of the double first order kinetic model applied to salinity experiments for V. cholerae

UV lamp Suspension solution Salinity (%) k1 (s−1k2 (s−1σ r2 
LP DI water – 1.88 1.88 1.00 0.999 
LP ASW 0.0707 0.00342 0.995 0.974 
0.0813 4.50E-16 0.996 0.971 
LP NSW 0.0793 0.001 0.995 0.99 
MP DI water – 1.91 1.91 1.00 0.983 
MP ASW 1.21 0.100 0.993 0.908 
1.61 0.253 0.993 0.995 
MP NSW 1.33 0.573 0.900 0.988 
UV lamp Suspension solution Salinity (%) k1 (s−1k2 (s−1σ r2 
LP DI water – 1.88 1.88 1.00 0.999 
LP ASW 0.0707 0.00342 0.995 0.974 
0.0813 4.50E-16 0.996 0.971 
LP NSW 0.0793 0.001 0.995 0.99 
MP DI water – 1.91 1.91 1.00 0.983 
MP ASW 1.21 0.100 0.993 0.908 
1.61 0.253 0.993 0.995 
MP NSW 1.33 0.573 0.900 0.988 

DI water, distilled water; LP, low-pressure; MP, medium-pressure; ASW, artificial seawater; NSW, natural seawater.

Figure 1

UV inactivation of V. cholerae by medium-pressure (MP) and low-pressure (LP) UV disinfection with UV dose ranging from 1 to 5 mJ cm−2 in sterilized distilled water. Error bars represent standard deviations of three experiments.

Figure 1

UV inactivation of V. cholerae by medium-pressure (MP) and low-pressure (LP) UV disinfection with UV dose ranging from 1 to 5 mJ cm−2 in sterilized distilled water. Error bars represent standard deviations of three experiments.

Previous studies have shown the sensitivity of E. coli to range from 10 to 15 mJ cm−2 (MP) (Quek et al. 2006) and 5 to 11 mJ cm−2 (LP) (Butler et al. 1987) for a 4 Log10 inactivation, while for V. cholerae, a UV dose of 5 mJ cm−2 was needed to result in the same level of reduction, revealing that V. cholerae is more sensitive to UV light than E. coli, which was in good agreement with previous studies (Hijnen et al. 2006; Coohill & Sagripanti 2008). The relative sensitivity of V. cholerae was 0.5 to 1.0 for E. coli (Coohill & Sagripanti 2008). The k-values varied from 0.506–0.642 cm2 mJ−1 for E. coli to 1.341 cm2 mJ−1 for V. cholerae (Hijnen et al. 2006), demonstrating V. cholerae was relatively higher UV sensitive.

Effect of salinity on UV inactivation

The effect of salinity on log removal of V. cholerae by two types of UV lamps is shown in Figure 2. It was observed that at 1% or higher, salinity had a negative impact on both MP and LP UV disinfection especially at higher UV doses (≥3 mJ cm−2 for MP and ≥4 mJ cm−2 for LP). After MP or LP exposure, UV inactivation was significantly suppressed in artificial seawater (ASW) or natural seawater than in sterile distilled water with a lower log reduction and inactivation rate (k1) shown in Figure 2 and Table 2.

Figure 2

The effect of salinity on UV inactivation of V. cholerae by (a) medium-pressure (MP) and (b) low-pressure (LP) UV disinfection. Error bars represent standard deviations of three experiments. ASW, artificial seawater; NSW, natural seawater. The * denote values that are significantly different (*P < 0.05) from the control value.

Figure 2

The effect of salinity on UV inactivation of V. cholerae by (a) medium-pressure (MP) and (b) low-pressure (LP) UV disinfection. Error bars represent standard deviations of three experiments. ASW, artificial seawater; NSW, natural seawater. The * denote values that are significantly different (*P < 0.05) from the control value.

The effect of salinity on UV disinfection is currently under debate. Rubio et al. (2013) evaluated the disinfection efficiency of E. coli by UV radiation and found that the UV inactivation rate decreased when increasing the salt concentration, whereas Shang et al. (2009) observed that higher salinity resulted in higher level of inactivation of fecal coliform bacteria at a UV-C dose of 12 mJ cm−2. The formation of radicals that interact with bacteria such as dissolved molecular oxygen (Legrini et al. 1993; Buschmann et al. 2005) may contribute to the overall effect of salinity on UV inactivation. Another reason is clustering or adsorption of the micro-organisms to surfaces due to declined repulsive forces at high ionic strength values. Results indicated that salinity had a detrimental effect on inactivation of V. cholerae at high UV doses, probably due to the reason that the negative effect on clustering and other interface processes (adsorption) is probably higher. Therefore, at high salinity, UV disinfection performance can be improved by increasing UV doses in actual operation.

DNA damage using ELISA assay

As shown in Figure 3, after LP UV exposure, in 3% ASW significantly less CPDs were formed, which was in accordance with the decreasing net log reduction in cell numbers. In other cases, salinity of 1% or 3% led to a trend of decreased CPDs content, but the decreases were not statistically significant. The cell number reduction may be associated with DNA damage, as is the case for E. coli (Eischeid & Linden 2007), M. terrae (Bohrerova & Linden 2006) and A. variabilis (Sakai et al. 2007). Further support is provided by the study of Cairns & MacDougall (1995) showing that the presence of the CPDs could prevent the accurate reading of the genetic code in the microorganisms for important cellular processes such as protein synthesis during growth or nucleic acid replication during cell division, and such mutations ultimately lead to cell death. However, for V. cholerae, no significant effects of salinity were found at the CPDs level, which did not agree with those observed in the cellular study. Further studies should be conducted on the quantification of UV-induced pyrimidine dimers in genomic DNA to provide fundamental insight into the relationship between formation of CPDs and log inactivation.

Figure 3

Effect of salinity on formation of cyclobutane pyrimidine dimers (CPDs) after (a) medium-pressure (MP) and (b) low-pressure (LP) exposure at 5 mJ cm−2. Absorbance at 492 nm was used as a measure of induced CPDs. Error bars represent standard deviations of three experiments. ASW, artificial seawater; NSW, natural seawater.

Figure 3

Effect of salinity on formation of cyclobutane pyrimidine dimers (CPDs) after (a) medium-pressure (MP) and (b) low-pressure (LP) exposure at 5 mJ cm−2. Absorbance at 492 nm was used as a measure of induced CPDs. Error bars represent standard deviations of three experiments. ASW, artificial seawater; NSW, natural seawater.

CONCLUSION

In summary, V. cholerae was inactivated by either MP or LP UV irradiation, and the use of the MP lamp leads to a higher disinfection efficiency than LP. In general, increased salinity levels hindered MP or LP UV disinfection when UV doses were more than 3 or 4 mJ cm−2, respectively. DNA damage seems not likely to contribute to cell number reduction as to V. cholerae. Further research on salinity effects on UV inactivation at high doses (60–120 mJ cm−2; Oemcke et al. 2004) is needed to better simulate a ballast water treatment under realistic conditions by increasing the initial microorganism concentration.

ACKNOWLEDGEMENT

This study was supported by the Economic Development Board (SPORE, COY-15-EWI-RCFSA/N197-1) and the Maritime and Port Authority of Singapore (MPA).

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