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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 , , Silver Impregnated Activated Carbon and Silica Sand 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 and Sciences, Department of , 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 is activated carbon (Jucker, et al., 1996). Potable water due to its large surface area and high systems are considered low in ionic capacity (Ortiz-Ibarra, et al., strength so electrostatic interactions can 2007). Activated carbon has proven to offer the possibly of enhancing the remove 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 (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 (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 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 (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), (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 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 dithionate and nanoparticles and silica sand were tested sodium citrate to remove and for their antibacterial effect against peroxide to remove organic waterborne pathogenic E.coli BL21 under matters. The silica sand was then saturated + test. If this is killed, as a with Na using 1M phosphate-buffered standard, all other borne-disease-causing saline (pH 7.0), sterilized and stabilized by 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.

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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, 1, 3 and 24 hours) from used in this test (Gargiulo et al., 2007). the flask and tested for the number of surviving E.coli by plate count method on M-Endo agar, using standard procedures. Table.1 Inhibitory effect of the The percentage reduction of E. coli counts, nanoparticles under test against E.coli were obtained after treatment with several BL21, using plate assay method. nanoparticles according to this formula:

% Reduction of bacterial count= Inhibitory zone ((Viable count at time0 - Viable count at used diameter (mm) time x)/ Viable count at time0)x100 Activated carbon 10.00 Silver 18.00

Results and Discussion AC-Ag a 11.00

Antibacterial test AC-Ag b 14.00

AC-Ag c 16.00 (a) Plate Assay Method Silica sand 8.00 Results in Table 1 showed the inhibitory effect of activated carbon, silver, silver impregnated activated carbon and silica In a trial to test the antibacterial effect of sand. it was revealed that all the tested the tested nanoparticles against E.coli nanoparticles had an antibacterial effect BL21 under shaken conditions, it was exhibited by the diameters of the revealed that the number of non-adsorbed inhibition zones. Silver nanoparticles viable bacterial cells incubated with proved to be the most effective different nanoparticles reduced slightly antibacterial agent against E. coli BL21 within 1 hour after treatments with with an inhibition zone of 18 mm followed activated carbon, and silver impregnated by silver impregnated activated carbon activated carbon at different (AC-Ag a) then AC-Ag b, AC-Ag c and concentrations, and silica sand. However, activated carbon. Silica sand showed the after shaken for 3 hours, the total viable

23 Int.J.Curr.Microbiol.App.Sci (2013) 2(4): 20-30

E.coli count reached to zero with activated nanoparticles inhibited E. coli and Bacillus carbon and silica sand but with sand still sp. growth at low concentrations (0.1 have some bacterial colonies. µg/ml), as for: silver targets multiple Continuously, after contacting for 24 components in the bacterial cell, and the hours, the nanoparticles killed all bacterial mechanism behind its antibacterial activity cells and markedly proved to have is by weakening DNA replication and bactericidal effect against pathogenic inactivating proteins, as a result, resistance E.coli under test. Furthermore, As shown to bacteria cannot easily develop (Zhou et in Figure 2, The results showed slight al., 2012). difference in the percent reduction in E. coli count after treatment with the Moreover, other researchers showed that different nanoparticles under test, where activated carbon-Silver composite have a 100% reduction in E.coli count was superior antibacterial activity towards E. observed after 24 hours of contact with coli due to the synergistic effect of activated carbon and silica sand, as activated carbon and silver nanoparticles reported previously by Liu et al., 2012. (Sreeprasad and Pradeep, 2012). However, silica sand showed 100% reduction in bacterial count after 24 hours In general, Most of the bacterial reduction of contact (Fig 2) as reported by Chen and and inactivation took place during the first Zhu (2005). For both materials, a large three hours of incubation, and the fraction of cell death occured in the first mortality rate increases continuously with three hours of incubation. the increase of nanomaterial concentration and their antibacterial activities are time The antibacterial effect of Silver and concentration dependent (Liu et al., impregnated activated carbon at different 2012). Thus, Silver nanoparticles showed concentrations (a,b and c) against E. coli efficient antibacterial activity against BL21 were tested. Results in table 2 pathogenic E. coli that was similar to that showed no growth of bacteria starting one found by Rastogi et al., 2011. hour of incubation where the percentage reduction of the total viable count of E.coli The mechanism of inhibitory action was 100 % as shown in figure 2, while caused by silver nanoparticles on E.coli after 3 hours of incubation with activated BL21 is partially known, where some carbon, 100% reduction in the bacterial researchers reported that silver count was reported. Therefore, the change nanoparticles inhibit bacterial growth after 1 hour of incubation with the through binding to the leading nanoparticles under test is due to the fact to bacterial inactivation (Yan et al., 2012). that the loss of cell viability has Moreover, the Gram negative bacteria approached the complete inactivation (Liu have a layer of lipopolysaccharides at the et al., 2012). It was clear that the amount exterior that are composed of covalently of silver particles on the activated carbon linked lipids and polysaccharides; they considered to be the main factor for the lack strength and rigidity. Negative complete reduction in the total viable charges on the lipopolysaccharides are count of E.coli and responsible for the attracted towards the positive charges effective antimicrobial activity. The results available on silver nanoparticles (Santoro are in agreement with that obtained by et al., 2007). The opposite charges attract Zhou et al. (2012), where silver each other due to electrostatic forces. So

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Int.J.Curr.Microbiol.App.Sci (2013) 2(4): 20-30 once the nanoparticle comes in contact after three hours of incubation and the with the bacterial cell, it either inhibit the reduction rate was greatly influenced by cell wall synthesis, damage the the increase of silver concentrations cytoplasmic membrane, inhibit nucleic and contact time. The present study and synthesis or inhibit demonstrates the potential of activated specific enzyme systems which result in carbon composites for use in water the complete bacterial inhibition (Sadeghi purification and that silver impregnation is et al., 2010).The mechanism by which the very effective in producing potable nanoparticles are able to penetrate the drinking water. bacteria is not understood completely, but previous studies suggested that when Table. 2 Total viable count as affected by E.coli treated with silver, changes took the exposure time to different place in its membrane morphology that nanoparticles, using shake flask test. produced a significant increase in its permeability affecting proper transport Contact CFU/ml Nanoparticles through the plasma membrane, leaving the time (hr) x104 bacterial cells incapable of properly 0.0 97.0 regulating transport through the plasma Activated 1.0 50.0 membrane, resulting in cell death (Raffi et Carbon 3.0 23.0 al., 2008). Moreover, these studies showed 24.0 8.0 that bacterial inhibition caused once silver 0.0 15.0 nanoparticles penetrated inside the 1.0 4.0 Silver bacteria and caused damage by interacting 3.0 0.0 with phosphorus and containing 24.0 0.0 compounds such as DNA (Sadeghi et al., 0.0 0.0 2010). 1.0 0.0 AC-Ag a 3.0 0.0 The antibacterial effect of activated 24.0 0.0 carbon, silver nanoparticles, silver 0.0 0.0 impregnated activated carbon and silica 1.0 0.0 sand against waterborne pathogenic E.coli AC-Ag b BL21 was obtained and compared. Plate 3.0 0.0 assays and shake flask methods showed 24.0 0.0 that silica sand had the lowest antibacterial 0.0 0.0 1.0 0.0 activities compared to the other prepared AC-Ag c nanoparticles. However, impregnated 3.0 0.0 activated carbon with silver at the highest 24.0 0.0 concentration showed the maximum Silica sand 0.0 56.0 inhibitory effect against E. coli BL21. 1.0 30.0 Therefore, higher concentrations of silver 3.0 15.0 ions cause greater bactericidal effect and 24.0 0.0 antibacterial activities are time and concentration dependant. Most of the bacterial cell number reduction occurred

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Int.J.Curr.Microbiol.App.Sci (2013) 2(4): 20-30

Figure.1 Plate assay method for the detection of the Inhibitory effect of nanoparticles under test against E.coli BL2. a: activated carbon, b: AC-Aga, c: AC-Agb, d: AC-Agc and e: silica sand.

(a) (b)

(c)

(d) (e)

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Int.J.Curr.Microbiol.App.Sci (2013) 2(4): 20-30

Figure.2 Reduction percentage of the total viable count of E.coli BL21 as affected by the exposure time to (a): activated carbon, (b): silver, (c): Ac-Ag a , (d): Ac-Ag b, (e): Ac-Ag c and (f): silica sand.

(a (b)

(c ) (d)

(e) (f)

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Int.J.Curr.Microbiol.App.Sci (2013) 2(4): 20-30

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