Int.J.Curr.Microbiol.App.Sci (2013) 2(4): 20-30 ISSN: 2319-7706 Volume 2 Number 4 (2013) pp. 20-30 http://www.ijcmas.com Original Research Article The Antibacterial Activity of Activated Carbon, Silver, Silver Impregnated Activated Carbon and Silica Sand Nanoparticles against Pathogenic E. coli BL21 M.Karnib1, H.Holail1, Z.Olama1, A.Kabbani2 and M.Hines3 1Department of Biological and Environmental Sciences, Faculty of Science, Beirut Arab University, Lebanon. 2School of Arts and Sciences, Department of Chemistry, Lebanese American University, Lebanon. 3College of Sciences, Department of Biological Sciences, University of Massachusetts Lowell, U.S.A. *Corresponding author e-mail: [email protected] A B S T R A C T K e y w o r d s The ability of the activated carbon, silver impregnated activated carbon, and silica Antibacterial sand to eliminate and destroy water borne E. coli BL21 were tested under plate activity; assay and shake flask technique. Silver nanoparticles showed the highest activated antibacterial effect against E. coli BL21 with inhibition zone diameter 18 mm, on carbon; using the shake flask technique it was proved that bacterial count started to be silver reduced after one hour of incubation, while no bacterial growth was detected after nanoparticles; 2,3 and 24 hours using the activated carbon. Bacterial growth was completely silica sand; inhibited on using silver impregnated activated carbon at all the tested E. coli BL21. concentrations after one hour of incubation. Introduction to activated carbon particles through One of the most widely used nanoparticles strong Lifshitz van derWaals forces for water purification is activated carbon (Jucker, et al., 1996). Potable water due to its large surface area and high systems are considered low in ionic adsorption capacity (Ortiz-Ibarra, et al., strength so electrostatic interactions can 2007). Activated carbon has proven to offer the possibly of enhancing the remove bacteria like Pseudomonas efficacy of activated carbon to remove aeruginosa and Escherichia coli from microorganisms from water by positive fresh and potable water systems (Percival charge modification of the carbon and Walker, 1999; Quinlivan, et al., 2005). surfaces. Once there is a charge reversal, Despite electrostatic repulsion between the electrostatic attraction between negatively charged microorganisms and negatively charged microbial cell surfaces carbon surfaces, microorganisms attach and positively modified carbon particles 20 Int.J.Curr.Microbiol.App.Sci (2013) 2(4): 20-30 will be strong (Bos, et al., 1999; Shi et al., substrate resulting in enhanced 2007). Moreover, modification in the biodegradation (Pedrazzani, et al., 2004) activated carbon particles by coating with a quaternary ammonium compound gives the activated carbon particles bactericidal The main objective of the present study is properties (Shi et al., 2007) and decreases to evaluate the antibacterial efficacy of the the possibility of biofilm growth. In activated carbon nanoparticles, silver addition to the microorganisms charge, the impregnated activated carbon, and silica hydrophobicity of the surfaces that come sand against waterborne pathogenic E.coli in contact with microbes is important in strain, where the combination of the adhesion (Bos, et al., 1999). activated carbon and silver would take an important antibacterial advantage, due to Nanoparticles have attracted great interest the strength of these two nanoparticles and in their development as potential these materials used widely for application antibacterial drugs (Hsiao et al., 2006), in water purification. which has also been reported that many biophysical interactions occur between silver nanoparticles and bacteria including Materials and Methods biosorption, nanoparticles decomposition and cellular uptake, with effects bacterial Microorganism cell membrane damage and toxicity (Priester, et al., 2009; Brayner, et al., A standard Escherichia coli BL21 was 2006). kindly provided by MIRCENC (Microbiological Resource Center, Ain Shams University-Egypt), maintained on In Europe, silver has been used as a water Nutrient Agar slants and stored at 4°C disinfectant and has shown effectiveness with regular transfers at monthly intervals. against planktonic bacteria (Silvestry- For long preservation, the slants were Rodriguez, et al., 2007). Silver has gained folded with 25% glycerol. this efficacy through its binding to disulfide or sulfhydryl groups present in the cell wall proteins (Feng et al., 2000). Raw Materials Silver has been shown to bind DNA in the nucleus thus causing cell death. Activated carbon was washed with deionized water and modified by heat One draw back for the use of activated treatment (Yin 2007), Silver Nitrate, carbon is the lack of reversibility AgNO3 (99%, Sigma Aldrich), sodium (Sheintuch and Matatov-Meytal, 1999), borohydride (NaBH4, Sigma Aldrich), The uncertainty when determining whether M-Endo agar medium and Macconkey capacity has been reached. Silica based agar medium (Sigma Aldrich) Pure Sand nanoparticles used as immobilizers have (Sigma Aldrich), deionized water of high shown to enhance the non-selective purity (Beirut Arab University) was used capture of organic contaminants from in all experiments. Silica sand was wastewaters, thus increasing the time of supplied by Soliver glass production contact between microorganisms and company (Chweifat, Lebanon). 21 Int.J.Curr.Microbiol.App.Sci (2013) 2(4): 20-30 Preparation of seed culture Activated carbon impregnation with silver nanoparticles Transfers from single slant cultures (48 hours old) were taken into 50 ml aliquots Activated carbon (1g) was added to 20 ml of the seed medium containing (g/l): beef of different AgNO3 concentrations (a) 0.1; extract, 1; yeast extract, 2; peptone, 5; b) 1 and c) 1.5 mol.L-1) one at a time. sodium chloride, 5 and 1 liter of distilled After 24 hour of impregnation in the dark, water. Dispensed in 250 ml Erlenmeyer the powder samples were washed with flasks to initiate growth (OD<1). Standard water to remove loosely adsorbed AgNO3, inoculam of 2% (v/v) were taken from the until no AgNO3 was observed in the latter liquid culture after growth for 18 filtrate. The powder samples collected hours at 30°C ± 2 on a reciprocal shaker to after decantation was air-dried until the start growth in the fermentation flask next day. By adding 10 ml of 0.2 mol L-1 8 which is equivalent to 3× 10 colony NaBH4, impregnated AgNO3 was forming unit (CFU/ml) according to chemically reduced (over 24h) to form Ag McFarland scale 0.5. particles, then it was washed with water to remove the excess NaBH4 followed by drying (Bandyopadhyaya, et al., 2008). Preparation of silica sand Antibacterial test Silica sand (50g) was rinsed using de ionized water and then treated with Impregnated activated carbon, silver sodium acetate, sodium dithionate and nanoparticles and silica sand were tested sodium citrate to remove iron ions and for their antibacterial effect against hydrogen peroxide to remove organic waterborne pathogenic E.coli BL21 under matters. The silica sand was then saturated + test. If this organism is killed, as a with Na using 1M phosphate-buffered standard, all other borne-disease-causing saline (pH 7.0), sterilized and stabilized by organisms are assumed killed. extensive washing with sterilized de- ionized water (Chen and Zhu, 2005). (a) Plate Assay Method (qualitative test) Melted M-Endo Agar medium was Preparation of silver nanoparticles fortified with 3x108 CFU/ml medium of E .coli BL 21equivalent to 0.5 Mcfarland. Silver nanoparticles were prepared About 20 ml of the previously prepared according to the chemical reduction seeded agar was then dispensed in method adapted by Fang, et al., (2005). 50 petridishes, solidified by refrigerating for ml of 1x10-3 M silver nitrate was prepared, 4 to 6 hours. Seven mm diameter holes and then heated till boiling and 5 ml of 1% were made in the seeded agar using a tri-sodium citrate added drop by drop. The sterilized cork borer. 25 mg of different solution was mixed vigorously and heated nanoparticles under test were added one at until the colour changed to pale brown a time in these holes, using one to two followed by stirring until cooled to room drops of sterilized water. They were left at temperature. The aqueous solution was air 4°C for 1 hr then incubated at 37 C for dried up to 4 days so as to obtain a 24 hours and the antibacterial effect was powdered form of sliver nanoparticles. 22 Int.J.Curr.Microbiol.App.Sci (2013) 2(4): 20-30 measured referring to the inhibition zone lowest antibacterial effect with an diameter. inhibition zone of 8mm. Waterborne E. coli BL21 adhere only weakly to different (b)Shake flask test in saline activated carbon particles, and the main (Quantitative test) difference between different types of activated carbons is the number of For the shake flask test, 50 ml of sterile attractive sites revealed upon traversing of saline (0.9% NaCl) was inoculated with 1 a carbon particle through the outer ml bacterial suspension (3x108 CFU/ml) bacterial surface layer (Busscher et al., equivalent to 0.5 Mc Farland. 50 mg of 2008), while silica sand showed smaller different nanoparticles were added to the inhibition zone which could be due to the flasks, one at a time and the contents were texture and size of the sand granules since stirred on a rotary shaker at ambient finer sand fractions were more efficient in temperature. The samples were drawn bacterial removal than the coarse sand periodically (0,