Effects of ethanol on the electrochemical removal of Bacillus subtilis spores from water
© Moghaddam Arjmand et al. 2015
Received: 18 January 2015
Accepted: 13 October 2015
Published: 18 November 2015
This study aimed to characterize the effects of ethanol on the monopolar electrochemical process to remove Bacillus subtilis spores from drinking water. In particular, spores’ destruction was tested by applying 20–100 mA current for 15–60 min to B. subtilis spores (102–104 CFU/mL density), with stainless steel electrodes. The experimental results showed electrochemical removal of spores in the presence of 0.4 M ethanol at 15, 45, and 60 min and 5 mA/cm2 current density. However, the use of ethanol or the electrochemical process alone did not eliminate B. subtilis spores at these time points. Overall, this study suggests that adding ethanol to the electrochemical process successfully removes B. subtilis spores from drinking water.
Fe3+ and OH− ions, generated at the electrodes surface, react to generate Fe(OH)3 compounds that can remove pollutants from aqueous solutions [9, 10]. Ethanol has also been used in water treatment processes, even though it has not been found to be an efficient sporicidal agent . According to previous studies, 875 mg/L ethanol is needed to reduce B. subtilis populations over 6 log10 . Ethanol is a membrane disrupter that induces rapid release of intracellular components and membrane disorganization, most likely due to the penetration of solvents into the hydrophobic region of the membrane bilayer. The aim of this study was to evaluate the efficiency of an electrochemical process in the presence of ethanol in water treatment. The effects of the operative parameters on Bacillus B. subtilis spore removal were also studied.
Materials and methods
Bacterial strain and culture conditions
The B. subtilis ATCC 6633 strain was obtained from the culture collection at the Tehran University (Iran). The strain was maintained on slant nutrient agar at 4 °C. Stocks were stored in aliquots containing 10 % glycerinated nutrient broth at −18 °C. 0.5 McFarland standards (corresponding to ~1.5 × 108 CFU/mL) spores were reactivated by incubation in 100 mL Erlenmeyer flasks containing 50 mL fresh trypticase soy broth (Merck) at 37 °C for 24 h, under aerobic conditions. Next, spore suspension was poured into sterile Erlenmeyer flasks and placed in a water-bath at 80 °C for 15 min to eliminate vegetative cells. Sporulation was confirmed by optical microscopy using the Gram staining technique, and spores were diluted into water. Total counts of bacterial spore suspensions were made using the pour plate method. Briefly, after incubation at 37 °C for 48 h, spore-forming bacteria were counted, and the results were expressed as the mean number of spores/mL. Culture media and equipment were sterilized by autoclaving at 121 °C for 15 min. The pH was adjusted at 0.1 M by adding NaOH or HCl.
Minimal inhibitory concentration (MIC)
Minimal inhibitory concentration (MIC) is defined as the lowest concentration of an antimicrobial agent that prevents the visible growth of a microorganism under certain in vitro condition. In this study, MICs were tested using the dilution broth method, according to the National Committee for Clinical Laboratory Standards . Briefly, 1 mL of the culture was transferred into a first sterile glass tube containing 10 mL TBS medium and 0.2 to 4 M ethanol. Next, after stirring, 1 mL was transferred from the first to the second tube and so on, to obtain a total of 12 dilutions. Tubes were incubated at optimal temperature for 24 and 48 h, prior to determine the MICs.
Experimental set up and operation
Analysis of the water quality
where I is the current through the solutions (A) and S is the area of the electrode (cm2). The pH level and conductivity of the solution were measured using a portable pH and EC meters (Eutech, Singapore). All the experiments were performed at a pH of 7.2. At the end of each experiment, the DC power source was switched off and the electrodes were removed from the water. During the experiments, samples were taken at 15 min interval and plated on TBS plates. All the experiments were repeated twice. The density of B. subtilis spore (102–104 m/l) removal was assessed in the electrochemical treatment using ethanol (0.2–0.4 M) at 20–100 mA current (1–5 mA/cm2).
These results demonstrated that the sporicidal efficiency is inversely proportional to the initial number of spore in solution. In addition, the sporicidal activity of the electrochemical treatment was directly proportional to ethanol concentrations, as shown in Figs. 4, 5 and 6. Overall, the results obtained in this study demonstrated that ethanol in combination with the electrochemical treatment improves the sporicidal efficiency of water disinfection, suggesting a synergistic effect between these two agents. The best result was obtained on 103 B. subtilis spores, using 0.4 M ethanol for 45 min at 5 mA/cm2 current density (Fig. 5).
Spores of B. subtilis are particularly resistant to conventional water disinfection treatments and, for this reason, they are used as surrogates for some waterborne pathogens such as Cryptosporidium spp, and as an indicator of hygienic quality of drinking water. Bacterial spores are more resistant to general sterilization and disinfection treatments such as heating, radiation, and the use of various chemicals than their vegetative cells . Several parameters participate in spore resistance, including impermeability, low water content, high levels of pyridine-2,6-dicarboxylic acid and divalent cations, and outer membrane thickness . In addition, spore DNA is protected against various types of damage . In recent years, increasing attention has been paid to electrochemical oxidation as an efficient technology for water disinfection. During this process, free chlorine is produced. This chemical damages the bacterial outer membrane, penetrates into the periplasm, destroys the inner membrane and degenerates cytoplasmic proteins. Also, the process can oxidize the microbes on the electrode surfaces . Usually, the oxidants of the electrochemical treatment are reactive oxygen species generated from the oxidation of water molecules . It was shown that the electrochemical treatment efficiently removes bacteria spores but not their vegetative cells . The aim of this study was to evaluate the efficiency of a combination of the electrochemical process and ethanol at low concentrations for disinfection of B. subtilis spores. Ethanol is a general bactericidal agent and has been widely applied for disinfection of human tissues and contaminated surfaces. According to the presented results, however, ethanol alone does not possess sporicidal activity . It has been reported that ethanol causes microbial membrane damage and denaturation of proteins thus interfering with cell metabolism and inducing cell lysis. According to the obtained results, maximum sporicidal effects were obtained by adding 0.4 M ethanol to104 B. subtilis spores at 100 mA current. Lower ethanol concentration (0.2 M) increased the reaction time to 90 min. Ethanol alone does not possess high sporicidal efficiency. Some studies have reported that the combination of ethanol with ferric chloride and ethylenediaminetetraacetic acid can act as sporicidal agent . Also, it has been shown that ethanol and anionic surfactants have sporicidal activity at low pH values. In this study, the sporicidal effects on B. subtilis spores was assessed by MIC. The results showed that ethanol alone has not sporicidal effect on B. subtilis spores. The electrochemical treatment exerted a low sporicidal effect on small numbers of B. subtilis spores, but it failed for large number of spores. These results indicated that the electrochemical treatment is an efficient method for water disinfection, but is not sufficient to remove disinfectant-resistant bacteria such as spore forming bacteria. This study showed that ethanol significantly increases the sporicidal efficiency of the electrochemical process.
The results obtained in this study show that B. subtilis spores were killed at 90 min by electrochemical water disinfection using ethanol. It was observed that increasing the operational time and adding ethanol to the electrochemical process improved the spore removal efficiency. Moreover, increasing the supporting electrolyte concentration in the solution reduces the specific electrical energy consumption.
The authors would like to thank the Tarbiat Modares University for funding and supporting this project (code: 1123803).
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