K. S. Malsha Udayakanthaa,
Rohini M. de Silva*a,
K. M. Nalin de Silvaab and
Chamari Hettiarachchia
aDepartment of, Chemistry, University of Colombo, Colombo 03, Sri Lanka. E-mail: rohini@chem.cmb.ac.lk
bSri Lanka Institute of Nanotechnology, Nanotechnology and Science Park, Pitipana, Homagama, Sri Lanka
First published on 23rd July 2015
Activated carbon has been used for water purification since ancient times due to its well-known sorption properties. However it is not capable of disinfecting water borne pathogens such as bacteria. The main objective of this study was to incorporate antibacterial properties while maintaining the existing properties of Granular Activated Carbon (GAC). This was achieved by a biocompatible double coating on to GAC which consists of hydroxyapatite (HAP) nanoparticles and on top of those curcumin molecules. Coating of GAC with HAP was carried out using in situ precipitation of HAP under basic conditions. A layer of curcumin molecules was then attached on top of the HAP coating in order to obtain HAP-curcumin bi-coated GAC (HAP/C/GAC). Synthesized HAP/GAC and HAP/C/GAC were characterized using FT-IR spectroscopy, scanning electron microscopy (SEM), powder X-ray diffractometry (PXRD) and thermogravimetry (TGA). Characterization revealed that needle shaped HAP nanoparticles (50–100 nm in width and approximately 200–500 nm in length) can be anchored and immobilized successfully on GAC which in turn enhances the adhesion of curcumin on it. Antibacterial properties of pure GAC, HAP/GAC and HAP/C/GAC were then investigated using both Gram negative (Escherichia coli) and Gram positive (Staphylococcus aureus) bacteria. The results show that the antibacterial properties of HAP/C/GAC are remarkably higher than that of HAP/GAC and the antibacterial activity of pure GAC is negligible.
As for nanomaterials, silver nanoparticles,12,14 metal oxide nanoparticles such as TiO212 and ZnO,12 fullerenes12 and carbon nanotubes12 are reported in water purification as non-DBP producers.12 However silver nanoparticles are recognized to cause intoxication in humans leading to various health issues.15 High water solubility of ZnO brings down the opportunities for it in water treatment.12 Furthermore concentrations >5 mM of ZnO nanoparticles have reported to reduce human T cell viability.16 The susceptibility of coagulation of fullerenes by the salts present in water, and the toxicity to mammalian cells12 limits its audience in water treatment grounds.17
HAP is a calcium phosphate ceramic material with the molecular formula Ca10(PO4)6(OH)2 and nano HAP is a well-known material for removing heavy metal ions from water. HAP nanoparticles have become imperative tools in the medical industry due to its high biocompatibility with human physiology and has made it one of the best choices in drug and gene delivery.18–20 Also it has been reported in antibacterial applications, as a surface for adhesion of silver.21 Apart from that, it's evident that HAP/GAC nanocomposite has high heavy metal sorption capacity.22
On the other hand curcumin [1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] is a hydrophobic polyphenol, a natural compound extracted from the rhizome of plant Curcuma longa (turmeric). It has been in use for more than decades in ayurvedic medicine and Asian cuisine23 due to its antioxidant, anti-inflammatory, antimicrobial and anti-carcinogenic properties.24 Curcumin has well proven antibacterial properties as it inhibits the bacterial endotoxin induced cytokines secretion and related processes thereby directly suppressing the bacterial cell growth.25,26
The main objective of this study is to develop a multifunctional biocompatible filtering matrix with enhanced anti-bacterial properties. In our investigation, GAC was selected as the matrix material. By keeping the inherent properties of GAC intact, well known antibacterial materials such as nano HAP and curcumin were introduced to the GAC to improve the lacking antibacterial property.
Coating of HAP nanoparticles on to GAC was carried out by modifying our previously reported procedure.22 This HAP layer was further functionalized with curcumin molecules to develop HAP-curcumin bi-coated GAC. These two compounds were then tested for their antibacterial activity against E. coli and S. aureus as Gram negative and Gram positive bacteria respectively.
All the microbiological work was performed at the Laminar hood (BioBase). Bacterial growth media were autoclaved in Autoclave machine (ALP Co. Ltd. Model KT – 305D −230 V, 50/60 Hz) before use and all the glassware including Petri plates were incubated at 180 °C for 2 h in oven (Memmert Beschickung loading model 100-800) before use. Bacteria inoculated plates were incubated at 37 °C in incubator (Memmert Beschickung loading model 100-800) overnight for growth. For the sonication purposes Sonorox super RK 1028 CH, BANDELIN Electronics, Berlin was used. For the bacterial cell counting, haemocytometer (Neubauer, Germany) was used together with the optical microscope (OLYMPUS CK X41).
Filtered water was collected into autoclaved Pyrex tubes and was kept in an ice bath to inhibit bacterial multiplication. Bacterial contents of these water samples were analysed qualitatively using impregnation methods: by seeding 50.0 ml of autoclaved LB Agar media with 200.0 μl of the filtered water samples. The capacity of prolonged antibacterial efficacy of nano composites were analysed by draining bacterial suspension over 5 minutes and the resultant filtered water samples were analysed for the bacterial content using the same procedure. Quantitative analysis was done using the haemocytometer and spread plate technique.
As seen in the Fig. 1, FT-IR spectrum of HAP/GAC and FT-IR spectrum of pure HAP are very much similar to each other. In both HAP/GAC and pure HAP, the broad peak that appears at 3000–3500 cm−1 region accounts for the stretching vibration mode of H bonded OH groups present in HAP.27 Peak at 1092 cm−1 corresponds to the stretching mode of PO43− groups and the peaks at 603, 831 cm−1 are the bending modes of PO43− groups.27 The absence of broad band, at 2600–3000 cm−1 region and, peaks at 1600 cm−1 and 875–880 cm−1 which are characteristics of amorphous calcium phosphate, carbonate apatites and Ca deficient HAP respectively, indicates that these contaminant compounds are not formed during the synthesis of HAP nanoparticles.28 Therefore this confirms the presence of pure HAP nanoparticles on GAC. With compared to pure GAC spectrum, the HAP/GAC spectrum shows a broad band at 3000–3500 cm−1 indicating the increased amount of OH groups on GAC as a result of coating it with HAP.
Fig. 2 SEM images of (a) pure nano HAP (b) HAP/GAC low magnification (c) HAP/GAC high magnification. |
Fig. 2(a) shows the needle shaped HAP nanoparticles with the aspect ratio of approximately 50–100 nm in width and around 200–500 nm in length.
The SEM images obtained for HAP/GAC (Fig. 2(b) and (c)) show that the coating of HAP on GAC is not even as previously observed22 and instead, the needle shaped particles are scattered on the GAC surface permitting most of the GAC surface to be exposed. This observed change in coating may be attributed to the addition of ammonia solution at once, during the synthesis of HAP on GAC thereby allowing fast nucleation and quick separation between nucleation and nanoparticle growth leading to a monodispersed nanoparticle formation as suggested by LaMer model.29 According to Fig. 2(b) and (c), almost all the micropores of GAC are uncovered with HAP and this can be considered as an advantage since most of the adsorption occurs mainly at the pore sites.30 The image also reveals that HAP nanoparticles are held well in the porous matrix of GAC allowing a successful coat. It can also be suggested that the HAP nanoparticles are held strongly by the carbon matrix as calcium leaching was not observed for the leaching test carried out.
HAP-curcumin bi-coated GAC was analyzed using FT-IR spectroscopy and the spectra obtained for three samples namely, HAP/C/GAC, pure curcumin and HAP/GAC are compared in the Fig. 5.
Fig. 5 Comparison of FT-IR spectra of (a) HAP coated GAC (b) HAP-curcumin bi-coated GAC (c) curcumin. |
The spectrum of curcumin in Fig. 5 contains the broad band around 3500 cm−1 which is an indication of its H bonded phenolic OH group. The peak at 1628 cm−1 is due to the aryl substituted CC bond.34 The shoulder appearing on to its right at 1599 cm−1 accounts for the conjugated CC bonds of the aromatic ring. The peak at 1150 cm−1 stands for the C–O bond of the ether group (OCH3).35 These values are in accordance with the FT-IR peaks of curcumin reported previously.36 When comparing the three spectra the spectrum of HAP/C/GAC has similarities to both HAP/GAC as well as to pure curcumin. The broad peak presents in the region of 3000 cm−1 to 3500 cm−1 indicates the presence of both HAP coating as well as curcumin. However, the characteristic band of curcumin's carbonyl group seen at 1628 cm−1 present in HAP/C/GAC is absent in HAP/GAC. This confirms the successful coating of curcumin on top of the HAP coating. It was also confirmed that curcumin coats specifically onto HAP layer and not onto the carbon matrix. For this we carried out an experiment to coat curcumin onto pure GAC and pure HAP using the same coating procedure under HAP/C/GAC. The FT-IR spectra obtained for these experiments, (given in the ESI†) support the absence of curcumin on pure GAC. This selective adhesion of curcumin onto HAP can be attributed to the metal ion chelating ability of curcumin which ensures its binding to HAP via HAP's calcium ions.
Fig. 6 Comparison of TGA curves obtained for (a) curcumin (b) GAC (c) nano HAP (d) HAP/curcumin/GAC (e) HAP/GAC. |
As seen in the Fig. 6, it is evident that two of the main precursors present in synthesized nano-composites are not thermo-stable up to 1000 °C. GAC and curcumin has burnt under air leaving no residue during 600–800 °C. On contrary, HAP has shown a significant stability towards temperature. The first endothermic region of HAP's TGA curve corresponds to the removal of physically adsorbed water from the crystal lattice.32 Furthermore, absence of any other endothermic regions assure the absence of other contaminant compounds like CaHPO4, Ca(OH)2 thereby confirming the purity of HAP.37 Also it's visible that both the nanocomposites maintain a non-zero mass when 1000 °C is achieved. This is a clear evidence that these two nano composites contain HAP nanoparticles in them which withstand the higher temperatures.
Fig. 7 Inhibition zones. (A) Positive control (1.2 cm); (B) negative control (0.0 cm); (C and D) nano HAP (1.2 cm, 1.1 cm); (E and F) HAP coated with curcumin (1.5 cm, 1.3 cm). |
Compared to the positive control gentamycin (10 μg ml−1), HAP and HAP/C (100 μg ml−1) showed similar level of antibacterial activity against E. coli. These results clearly show that nano composites containing HAP and HAP/C have an effective antibacterial activity with the highest in HAP/C.
The ability of synthesized HAP/GAC and HAP/C/GAC nano-composites in removing bacteria in water, has been experimented using a known amount (5.2 × 109 CFU ml−1) of E. coli and S. aureus bacterial suspensions. This bacterial suspension was passed through columns prepared with HAP/GAC and HAP/C/GAC. The filtrates were analyzed using impregnation method. The results obtained are given in the Fig. 8.
Fig. 8 (a) Bacterial suspension used for filtration (b) filtrate of GAC filter (c) filtrate of HAP filter (d) filtrate of HAP/GAC filter. |
According to the results highest number of bacterial colonies is present in the filtrate of uncoated GAC filter. This is in accordance with the reported data where it mentions the formation of a biofilm on GAC which can facilitate the multiplication of coliforms rather than reducing their number.1 In general both the nanoparticle containing filters seemed to have reduced the bacterial contamination in water. However no bacterial colonies can be found in water samples that were passed through the columns containing pure HAP. Antibacterial activity observed with HAP/GAC nanocomposite is in between pure GAC and pure HAP. And it is evident that the antibacterial properties of synthesized HAP are retained even if they are immobilized by coating on to GAC.
The extended experimental studies on antibacterial activity of synthesized filter materials namely GAC, HAP/GAC and HAP/C/GAC were carried out in order to compare the antibacterial properties of HAP/C/GAC to pure GAC. The results obtained are shown in the Fig. 9.
In this experiment antibacterial activity was demonstrated against number of colony forming units (CFU). Compared to the number of colonies present in the original sample, the number of colonies found in HAP/GAC filtrate and HAP/C/GAC filtrate are extremely low for the case of S. aureus. A close observation shows that absolutely no colonies are found in the 1st ml for HAP/C/GAC as well as in HAP/GAC column. In HAP/GAC, a similar number of colonies are observed in the 2nd and 3rd ml followed by a slight increment in the 4th and 5th ml. In HAP/C/GAC, similar number of colonies are there in 2nd and 3rd ml and slight increment is observed up to 5th ml. However, compared to HAP/GAC the number of colonies are significantly low and this can be attributed to the presence of curcumin.
A spread plate technique was carried out for filter systems containing pure GAC, HAP/GAC and HAP/C/GAC in order to compare their activity against S. aureus. The results are given in the Fig. 10. As seen in the figure, the amount of growth of bacteria in the filtrate of pure GAC has given rise to a lawn of bacteria in the spread plate and the thickness of the lawn has increased when going from the 3rd towards 5th. The reason can be the same that was mentioned before, facilitation of the growth of bacteria by the filter bed rather than a reduction.
Nevertheless in both nanocomposite containing filters, the viable cell count in the filtrate has tremendously decreased compared to the pure GAC filter. The filtrate obtained from HAP/C/GAC shows absolutely no growth for the first fraction. Further, it is clear that the filtrate obtained from HAP/C/GAC for 2nd and 3rd fractions are having only a very few and similar number of colonies with the slight increment going from 4th to 5th. However with compared to the HAP/C/GAC the amount of colonies at the HAP/GAC has a significant increment in number. On the other hand, the gradual reduction of the antibacterial activity observed in both filters may be due to the saturation of the activity sites with bacteria due to the repeated use of filter. Therefore further studies should be carried out to determine the capacity of the filters towards bacterial disinfection.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra11518c |
This journal is © The Royal Society of Chemistry 2015 |