Storage Stability and Disinfection Performance on Escherichia Coli of Electrolyzed Seawater
Total Page：16
File Type：pdf, Size：1020Kb
water Article Storage Stability and Disinfection Performance on Escherichia coli of Electrolyzed Seawater Regina G. Damalerio 1, Aileen H. Orbecido 1, Marigold O. Uba 2, Patricio Elvin L. Cantiller 2 and Arnel B. Beltran 1,* 1 Chemical Engineering Department, De La Salle University, Malate, Manila 1004, Philippines; [email protected] (R.G.D.); [email protected] (A.H.O.) 2 Biology Department, De La Salle University, Malate, Manila 1004, Philippines; [email protected] (M.O.U.); [email protected] (P.E.L.C.) * Correspondence: [email protected]; Tel.: +632-524-4611 (local 218) Received: 22 March 2019; Accepted: 24 April 2019; Published: 10 May 2019 Abstract: The study investigated the effect of storage conditions on the stability of electrolyzed seawater (ESW)’s physicochemical properties (pH, oxidation-reduction potential (ORP), and free chlorine (FC) concentration), and bactericidal efficiency on the fecal coliform Escherichia coli for 30 days. Preliminary experiments were conducted to determine the optimal current and electrolysis time. Two batches of 2750 mL filtered seawater were electrolyzed using 50 mm 192 mm platinum–titanium × mesh electrodes at a current of 1.5 A for 20 min. One hundred milliliters of electrolyzed solution was transferred into each amber glass and high-density polyethylene (HDPE) bottles. The bottles were stored in a dark area at ambient temperature. The results showed an increase in pH and a decrease in ORP and FC concentration through time. Hypochlorous acid remained as the dominant component since the pH levels of the solutions remained below 7.5. FC decay was investigated using Chick’s Law. It was determined that the decay in HDPE bottles (k = 0.066 day 1) was faster compared to amber − − glass bottles (k = 0.046 day 1). Nonetheless, HDPE bottles could still be used as an alternative − − container for 30 days only due to observed instability beyond 30 days. ESW remained effective since no surviving population of E. coli was observed throughout the experimentation. Keywords: disinfection performance; Escherichia coli; electrolysis; kinetics; storage stability; seawater 1. Introduction Electrochemical disinfection was introduced as an alternative due to its advantages over utilization of chlorine reagents generated from chlorine gas [1–5]. It is performed by passing current through chloride-containing water, which can be made synthetically (brine solution) or collected from a natural source (seawater) [2,6]. The presence of sodium chloride (NaCl) in the solution is important for the formation of oxidizing agents, such as free chlorine (FC), sodium hypochlorite (NaClO), or hypochlorous acid (HOCl) [5,7]. These compounds are responsible for inactivating pathogenic micro-organisms by oxidizing or attacking both the inner and outer membrane of the cell [8–10]. Other advantages of electrochemical disinfection include on-site production, raw material availability, an environment-friendly process, a less-hazardous reaction, low cytotoxicity, and no reported microbial resistance [10]. Furthermore, it has been utilized in several sectors, such as agricultural and food industries, since the disinfectant can be produced over wide pH ranges [11–15]. Xie et al. [12] investigated the effect of acidic electrolyzed water stored at different temperatures on contaminated raw shrimp, while Kasai et al. [16] investigated the effect of electrolyzed seawater (ESW) in naturally contaminated oysters. Moreover, the disinfectant can be applied in sanitizing materials and equipment. Water 2019, 11, 980; doi:10.3390/w11050980 www.mdpi.com/journal/water Water 2019, 11, 980 2 of 11 Osafune et al. [10] concluded that electrolyzed acidic water is effective as it destroyed the cellular structures of the three bacterial species cultivated from the kendo equipment. Seawater has 3.5% salinity, which predominantly consists of sodium and chloride ions. Furthermore, it also contains bromine, which is converted into an oxidant through electrolysis. These oxidizing compounds are detrimental towards pathogenic micro-organisms [4,14,17,18]. Thus, ESW has been employed and studied as a disinfectant [9,14,16–19]. However, the main problem lies in its storage stability through time [1,2]. The disinfectant is considered stable when it is still effective for inactivating pathogenic microorganisms. Residual oxidants or FC remains present and dominant, despite the changes in the physicochemical properties and storage conditions. No studies related to storage stability of ESW can be found in the literature since it is more often viewed as an alternative to water disinfection and as a disinfectant to be utilized immediately. Therefore, there is a possibility that the production and utilization of the disinfectant will be maximized by investigating its storage stability through time. The study could benefit people living near coastal areas, where raw material is abundant and accessible. Furthermore, this could serve as temporary alternative solution when sanitation is inaccessible (e.g., calamities). The storage stability of ESW is determined by investigating the effects of the storage conditions and changes in the physicochemical properties, such as pH, oxidation-reduction potential (ORP), and FC concentration through time, to its bactericidal efficiency on the fecal coliform Escherichia coli. The rate of FC decay in amber glass and high-density polyethylene (HDPE) bottles is investigated using Chick’s Law to determine the possibility of using HDPE bottles as an alternative storage container to amber glass bottles. 2. Materials and Methods 2.1. Materials Glassware, platinum–titanium mesh electrodes, and reagents, such as N,N-dimethyl-p- phenylenediamine (DPD) powder, phosphate-buffered saline, nutrient agar, and broth powder, were purchased from several local suppliers. Purified stock culture of E. coli was acquired from the microbiology laboratory. The seawater sample utilized in the experiment was collected five meters away from the beach shore of Oriental Mindoro, Philippines. It was filtered using a PM10 microfilter before being stored at ambient temperature. 2.2. Electrolysis Setup Fifty by 192 mm titanium–platinum mesh electrodes (mesh size: 10 5 mm; electrode surface × area = 23 cm2) were sonicated for 30 min to remove unwanted particles on the surface [18,19], and submerged vertically with a 0.5 cm gap in a 4 L Pyrex beaker. An alternating current (AC) power supply was utilized as an electrical source (Figure1). Twenty each of 120 mL amber glass bottles and 250 mL HDPE bottles were cleaned and labeled prior to the day of the electrolysis experiment. Water 20192019,, 1111,, 980x FOR PEER REVIEW 33 of of 11 Figure 1. Electrolysis Setup. 2.3. Electrolysis of Seawater Figure 1. Electrolysis Setup 2.3. ElectrolysisTwo batches of Seawater of 2750 mL seawater were electrolyzed at a current of 1.5 A for 20 min. The applied current and electrolysis time were determined based on preliminary electrolysis experiments. It wasTwo carried batches out of by2750 investigating mL seawater the were eff ectselectrolyzed of varying at a current current andof 1.5 electrolysis A for 20 minutes. duration The on theapplied physicochemical current and electrolysis properties oftime ESW. were determined based on preliminary electrolysis experiments. It was carried out by investigating the effects of varying current and electrolysis duration on the 2.4.physicochemical Storage Experiment properties of ESW. About 100 mL of ESW was transferred into each amber glass and HDPE bottle. Bottles were stored 2.4. Storage Experiment in a dark place at ambient temperature. The physicochemical properties and bactericidal efficiency of ESWAbout were evaluated 100 mL of for ESW 30 days. was transferred into each amber glass and HDPE bottle. Bottles were stored in a dark place at ambient temperature. The physicochemical properties and bactericidal 2.5.efficiency Preparation of ESW of thewere Working evaluated Culture for of30 E. days. coli A purified stock culture of E. coli was cultivated every two weeks with nutrient agar and maintained 2.5. Preparation of the Working Culture of E. coli at 4–10 ◦C for the duration of the study. A working bacterial suspension, prepared from the stock culture,A purified was standardized stock culture prior of to E. every coli disinfectionwas cultivated experiment. every two This weeks was performed with nutrient by inoculating agar and freshmaintained colonies at of4–10E. coli°C forin sterilethe duration distilled of waterthe study. until A the working turbidity bacterial was adjusted suspension, to equal prepared McFarland from Standardthe stock culture, #0.5, which was approximatesstandardized 1.5prior 10to 8everycolony disinfection forming units experiment. (CFU) per This mL was [20 performed]. A standard by × plateinoculating count wasfresh also colonies performed of E. coli to confirm in sterile the distilled microbial water density until ofthe the turb standardizedidity was adjusted turbidity to[ 1equal,2]. McFarland Standard #0.5, which approximates 1.5 × 108 colony forming units (CFU) per mL [20]. A 2.6.standard Disinfection plate Experimentscount was also performed to confirm the microbial density of the standardized turbidityTo determine [1,2]. the effect of ESW on E. coli, 1 mL of the standardized E. coli suspension was mixed with and exposed to 9 mL ESW for one minute. Then, 1 mL of the latter mixture was added to 9 mL of Phosphate-Bu2.6. Disinfectionffered Expe Salineriments for one minute to render the ESW ineffective.