Antimicrobial Properties of Chitosan-Alumina / f-MWCNT Nanocomposites

1Department of Applied Chemistry and the DST/Mintek Nanotechnology Innovation Centre-Water Research Node, University of Johannesburg, P.O. Box 17011, Doornfontein, Johannesburg 2028, South Africa 2Nanotechnology and Water Sustainability Research Unit, College of Science, Engineering and Technology, University of South Africa, Florida, Johannesburg 1709, South Africa 3Water and Health Research Centre, University of Johannesburg, P.O. Box 17011, Doornfontein, Johannesburg 2028, South Africa


Introduction
Safe drinking water is still a dream for many South African rural settlements.Polluted water threatens political, economic, and social life for humanity; it is also an affront to human self-esteem [1].It is well known that the health of the communities is significantly affected by the quality of drinking water [2].Some water sources in South Africa rural areas are polluted by microbial pathogens derived from human or livestock wastes.Poor sanitation and poor maintenance of water supply systems also contribute a major role in the degradation of water sources by the microbes [3].The impact of waterborne diseases is substantial in South Africa, with an estimated 43000 deaths and over a million incidents of illness reported annually [4,5].The diseases mainly affect people with unfledged or compromised immune systems and in particular young and elderly people and those living with HIV/AIDS [2].
Many conventional methods used in water treatment such as disinfection using chlorine have been used.However, these are known to produce disinfection by products (DBPs) which are of environmental concern [6].Chitosan, which is a linear polysaccharide composed of randomly distributed -(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine (Figure 1), has a potential use in drinking water disinfection, as an antimicrobial agent in membranes, as an adsorbent (beads, sponges), and as a coating for water storage tanks [7][8][9].Furthermore, it has advantages over other disinfection materials since it is has high antimicrobial activities with lower toxicity to humans and animals.Chitosan is also  a bioactive and biocompatible material which is rendered useful in many applications including wound dressing, pharmaceutical and cosmetic products, and water treatment [10,11].
The antimicrobial activities of chitosan are due to its polycationic nature.These result from the electrostatic interaction between the positively charged amine groups of chitosan and the negatively charged cell membrane of the microbes [12].This electrostatic interaction results in a twofold interference: (i) by promoting changes in the properties of membrane wall permeability, thus provoking internal osmotic imbalances, and (ii) consequently inhibiting the growth of microorganisms [13,14].Secondly, the hydrolysis of the peptidoglycans in the microorganism wall leads to the leakage of intracellular electrolytes such as potassium ions and other low molecular weight proteinaceous constituents (e.g., proteins, nucleic acids, glucose, and lactate dehydrogenase) [15][16][17].This suggests that the higher the number of cationized amines, the higher the antimicrobial activity [18].The third mechanism of antimicrobial activities is the binding bacterial DNA and inhabitation of mRNA synthesis which occur via penetration of chitosan into the nuclei of the microorganisms thereby interfering with the synthesis of mRNA and proteins resulting in the death of these organisms [19,20].Finally, the chelation of metals, suppression of spore elements, and binding to essential nutrients to microbial growth lead to cell death [21].
Although chitosan has been proven to have antimicrobial properties, its applications are limited due to factors such as water solubility and working pH [19,22].Cross-linkers such as epichlorohydrin and glutaraldehyde and inorganic materials such as alumina, silica, and titania have been used to improve its chemical stability [23].The well-known characteristics of alumina as adsorbents are due to its high surface area (∼200 m 2 /g) and its amphoteric character of hydrous aluminum hydroxide.Its acid-based dissociation leads to the positive (OH 2+ ) at lower pH [24,25].Multiwalled carbon nanotubes (MWCNTs) have also become ideal materials to use as inorganic fillers for reinforcing or toughening polymeric materials [26].Given its favorable attributes as discussed above, this study investigated chitosan and its nanostructured composites in the removal of microbes from water.The synthesis of chitosan/alumina composites embedded with functionalized MWCNTs (f-MWCNTs) has not been reported by others.The materials demonstrated antimicrobial activity on twelve strains of bacteria.

Preparation of Chitosan/Alumina
Composites.Chitosan/ alumina gel (4 : 1 ratio of chitosan : alumina) was prepared by chelating alumina and chitosan using oxalic acid using a modified procedure reported by Li et al. [29] (Figure 2).Alumina was oxidized with 10% oxalic acid as a surface coating and then was washed with deionized water to pH = 7.The product was dried in an oven at 110 ∘ C overnight.The oxidized alumina powder was added to 2% chitosan gel prepared by a modified method reported by Ngah and Fatinathan [30] and Chatterjee and Woo [31].The mixture was stirred for 12 h and then degassed.

Preparation of Chitosan-Alumina/f-MWCNT Nanocomposites.
Chitosan-alumina/f-MWCNT nanocomposites (3.75 : 1 : 0.25) were prepared by adding a calculated amount of f-MWCNTs into a solution of chitosan-alumina prepared as described in Section 2.2 [29].The mixture was stirred until being homogeneous and then degassed.The nanocomposite gel was then precipitated and then dried at 60 ∘ C in an oven.The illustration of the formation of chitosan-alumina/f-MWCNT nanocomposites is shown in Figure 2. Oxalic acid acts as a cross-linking agent between chitosan, alumina, and f-MWCNTs, thus forming a hydrogen bonding. 1 were used to evaluate the antimicrobial properties of the chitosan and chitosan-alumina/f-MWCNT nanocomposites.The strains were plated and maintained on a Muller-Hinton agar (Oxoid, Cat.number CM0337) during the experiment.The plates were incubated at 37 ∘ C overnight and stored at 4 ∘ C. The bacterial strains were grown in liquid culture by inoculating Muller-Hinton broth (HI Media, Cat.number M391-500G) with a colony from the grown plates.All the liquid media were grown at 37 ∘ C with mild agitation (100 fpm) until an optimal density at 600 nm (OD 600 ) of 0.6 was reached.These cell suspensions, in media, were used in subsequent experiments.

Antimicrobial Activity of the Nanocomposites. The bacterial strains shown in Table
All tests for antimicrobial activity of the nanocomposites were performed in a sterile 96-well micro titre plates with lids (NUNC microwell 96F, Cat.number 167008) and a single plate was used to test chitosan and chitosan-alumina/f-MWCNT nanocomposites against specific selected bacterial strains [32].Sterile distilled water (50 L) was added to each of the wells after which 50 L of 2 g/L test solution (chitosan and chitosan-based nanocomposites in water) was added to the first well, mixed, and transferred to the next well.The    After addition of the alumina and f-MWCNTs, a surface interaction between chitosan and Al occurred and resulted in new peaks.The bands in the range of 1000-500 cm −1 were characteristics of aluminum oxide [33].The peaks at 775 and 679 cm −1 were assigned to the Al-O stretching mode and bending mode of O-Al-O, respectively.In addition to the characteristic peaks of Al, there were new sharp peaks at 1637, 1462, and 1320 cm −1 , which represented the O-C=O bending vibration of oxalic acid, C-O and C-C stretching vibration, and the C=O asymmetric stretching vibration, respectively.The peaks present in the chitosan-alumina spectrum were still present in the spectrum of chitosanalumina/f-MWCNTs.The only notable peak in spectrum of chitosan-alumina/f-MWCNTs was at 2912 cm −1 which was due to -C-H stretching vibrations.This suggested that there was a chemical interaction between chitosan, alumina, MWCNTs, and oxalic acid [29].

Thermal Analysis of Chitosan and Chitosan-Based Nanocomposites.
The thermograms and derivative weight plots of chitosan and chitosan-alumina/f-MWCNTs are presented in Figure 6.It was observed that chitosan lost its mass in two steps.The first step mass loss (i) in Figure 6(a) occurred in the range of 50-130 ∘ C.This first mass loss step was ascribed to the loss of water molecules since the polysaccharides normally have strong affinity for water and thus are easily hydrated.The second step mass loss started at 280 ∘ C and reached the maximum at 620 ∘ C corresponding to the thermal and oxidative decomposition of chitosan [34,35].Specifically, the degradation involved decomposition of amine groups (ii) and polymer chain degradation (iii) in Figure 6(a).
The chitosan-alumina thermogram demonstrated an additional mass loss (iv) observed at 710 ∘ C (Figure 6(b)).This mass loss was due to the dehydroxylation of Al 2 O 3 [36].For chitosan-alumina/f-MWCNT nanocomposite, there was also an additional mass loss (v) in Figure 6(c) observed at 890 ∘ C, which was attributed to thermal degradation of MWCNTs.Generally, the addition of Al and f-MWCNTs to chitosan polymer matrix increased the thermal stability of the polymer.Basically, chitosan completely decomposed in air while chitosan-alumina and chitosan-alumina/f-MWCNTs decomposed to about 75%.The remaining mass corresponds to the amount of the alumina and f-MWCNTs added to the polymer matrix.

Solubility of Chitosan and Chitosan-Based Nanocomposites.
The solubility of chitosan and chitosan-based nanocomposites is shown in Table 2.It was observed that chitosan composites were soluble in 0.1 M HAc (pH 3) solution but were insoluble in distilled water (pH = 7) and 0.1 M NaOH (pH = 9) solutions.Chitosan dissolved in acidic medium (pH 3) because the primary amino groups are protonated and this results in the degradation of the chitosan polymer chain.However, modification of chitosan with alumina and f-MWCNTs reduced their solubility.On the other hand, chitosan-alumina and chitosan-alumina/f-MWCNTs were found to be insoluble in all three media (Table 2).This was because there were fewer primary amino groups which can be protonated and cause dissolution by the cross-linker [37].
3.5.Antimicrobial Properties of the Nanocomposites.Figure 5 shows the antimicrobial effect of chitosan and chitosan-based  nanocomposites.All the nanocomposites (initial concentration = 2 mg/mL, i.e., 2 mg of beads in 1 mL of water) showed some growth inhibition to all bacterial strains.Typically, all the beads demonstrated complete (100%) inhibition at all concentrations (from high, i.e., 1 mg/mL = 2x, dilution in Figures 7(a 3).The antimicrobial activity of the nanocomposites was attributed to the positively charged active sites (-NH 2 and -OH groups in acidic media) of the materials, which interact with the negatively charged cell wall of the bacterial strains, thus causing the death of the bacteria.

Conclusions
Chitosan-alumina/f-MWCNT nanocomposites with antimicrobial properties were prepared by a simple phase inversion using oxalic acid as a cross-linker.The incorporation of alumina and f-MWCNTs on chitosan improved the thermal stability and reduced the solubility and swelling behaviour of chitosan.The chitosan-alumina/f-MWCNT nanocomposites showed similar antimicrobial activity as pristine chitosan; however, their physicochemical properties such as thermal stability and lower solubility were improved properties.

Figure 1 :
Figure 1: Structure of a chitosan polymer.

Figure 3 :
Figure 3: Example of a typical 96-well micro titre plate layout used for testing the nanocomposites.The clear wells show inhibition of bacterial growth and the purple wells indicate active cells reducing the INT.

Figure 4
shows SEM images and EDS spectra of chitosan, chitosan-alumina, and chitosan-alumina/f-MWCNT nanocomposites.A change in the internal morphology of chitosan after the incorporation of alumina and alumina/f-MWCNTs was observed.EDS confirmed that alumina and f-MWCNTs were included into the chitosan polymer matrix.This was evident by the presence of aluminum (Al), carbon (C), nitrogen (N), and oxygen (O) peaks detected on the EDS spectra of chitosan-alumina and chitosan-alumina/f-MWCNTs as shown in Figures4(b) and 4(c).

Figure 7 :
Figure 7: The antimicrobial effect of chitosan (a), chitosan-alumina (b), and chitosan-alumina/f-MWCNTs (c) on various strains of bacteria.Clear wells indicated that there was no growth/inhibition of the bacteria.Purple wells indicate growth (no inhibition) of the bacteria.
), 7(b), and 7(c) to low, that is, 31.25 g/mL = 64x, dilution in Figures 7(a), 7(b), and 7(c)) for E. faecalis bacterial strains.However, further dilution needed to be done in order to determine the minimum inhibition concentration (MIC) for E. faecalis.The MIC of the nanocomposites for other bacterial strains other than E. faecalis was in the range of 62.5 g/mL-0.25mg/L (Table

Table 1 :
Figure 2: A schematic representation of the formation of chitosan-alumina/f-MWCNT nanocomposites.Summary of bacterial strains used for the testing of the nanocomposites.

Table 2 :
Solubility effect of chitosan and chitosan-based nanocomposites.

Table 3 :
Summary of the highest dilutions (MICs) of the beads that inhibited the bacterial growth as determined by the INT assay.