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Gum arabic as natural stabilizing agent in green synthesis of ZnO nanofluids for antibacterial application
Accepted Manuscript Title: Gum Arabic As Natural Stabilizing Agent In Green Synthesis Of ZnO Nanofluids For Antibacterial Application Authors: Norlin Pauzi, Norashikin Mat Zain, Nurul Amira Ahmad Yusof PII: DOI: Article Number:
S2213-3437(19)30454-3 https://doi.org/10.1016/j.jece.2019.103331 103331
Reference:
JECE 103331
To appear in: Received date: Revised date: Accepted date:
18 January 2019 27 July 2019 27 July 2019
Please cite this article as: Pauzi N, Zain NM, Yusof NAA, Gum Arabic As Natural Stabilizing Agent In Green Synthesis Of ZnO Nanofluids For Antibacterial Application, Journal of Environmental Chemical Engineering (2019), https://doi.org/10.1016/j.jece.2019.103331 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Gum Arabic As Natural Stabilizing Agent In Green Synthesis Of ZnO Nanofluids For Antibacterial Application. Norlin Pauzi1 , Norashikin Mat Zain1 and Nurul Amira Ahmad Yusof1 Faculty Of Chemical and Natural Resources Engineering. University Malaysia Pahang, Lebuhraya Tun Razak, 26300 Gambang, Kuantan, Pahang MALAYSIA
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Corresponding author: Tel: +6017-3281653, Fax: +609-549 3190, Email:[email protected]
Highlights
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Zinc oxide (ZnO) nanofluids were synthesized by a precipitating method assisted with microwave heating method. Agglomerated size of ZnO nanofluids was found to be around 200 to 350 nm compared to unstabilized ZnO nanofluids was 1020 nm. These ZnO nanofluids relatively stabilized for at least 6 months using gum arabic as a stabilizer. Stabilized ZnO nanofluids have better antibacterial properties compared to unstabilized ZnO nanofluids.
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Abstract. This work is part of a responsible green approach for the development of nanotechnology. Zinc oxide (ZnO) nanofluids are known as one of the versatile inorganic materials used in health-related applications with effective antibacterial activities. In this study, ZnO nanofluids were synthesized by a precipitating method assisted with microwave heating method. Zinc nitrate had been used as zinc salt, sodium hydroxide as reducing agent and gum arabic as a stabilizing agent. The objective of this study was to investigate the effect of gum arabic concentration on the size, optical properties, and stability of ZnO nanofluids. Nanosizer was used for the measurement of the particles size and the average size of ZnO nanofluids was found to be around 200 to 350 nm compared to unstabilized ZnO nanofluids was 1020 nm. The UV-vis absorption spectra were found in the range of 340 nm. The FTIR spectra showed peaks range from 424 to 475 cm-1 which indicated a standard peak of ZnO nanofluids. These ZnO nanofluids relatively stabilized for at least 6 months. The antibacterial activities of ZnO nanofluids were tested against Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Escherichia coli. Our results showed that stabilized ZnO nanofluids have better antibacterial properties compared to unstabilized ZnO nanofluids.
Keywords ZnO nanofluids; Gum arabic; Microwave heating; Antibacterial
1.
Introduction
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Since nanoparticles possess unique chemical, physical and biological properties, several kinds of nanofluids are being produced and commercialized. Hence, ZnO nanofluids have attracted marketable interest in various different areas of antibacterial applications such as paint industry [1], wastewater treatment [2], biomedical materials [3] and detergents [4]. ZnO nanofluids are less prone to bacterial resistance, thus possess greater effectiveness on resistant strains of bacteria [5]. The absorption 3.37 eV band gap by ZnO nanofluids can result in the generation of reactive oxygen species (ROS) [6]. ROS and Zn ions that are released from ZnO nanofluids will disrupt the integrity of the cell wall and destroy the bacteria [7].
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In general, ZnO nanofluids are not stable when dispersed in water [8] because of ZnO agglomeration in water due to high polarity of water which leads to the deposition. Thus, the industrialists use different surface modifying agents to improve the stability of ZnO nanofluids [1].
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Several methods are used to synthesize ZnO nanofluids, these include direct precipitation, homogeneous precipitation, solvothermal method, sonochemical method, reverse micelles, the sol-gel method, hydrothermal, and thermal decomposition [9]. Nevertheless, most of the identified methods utilized sophisticated equipment, toxic chemicals and generate environmentally risky by-products [10]. Frequently, the chemical synthesis can lead to the presence of some toxic substances adsorbed on the surface which is not suitable for clinical use [11]. Furthermore, the poisonous chemicals such as hydrazine, sodium citrate, amines, diethylenetriamine, sodium hypophosphite, and sodium borohydride are used as reducing and capping agents to create uniform suspensions [3]. These toxic substances are absorbed on the nanofluids' surfaces and contribute to higher toxicities are not safe for the environment and biomedical purposes.
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Thus, this drawback can be resolved using “green” synthesis methods to achieve more sustainable processes, focussing on reducing or preventing the use of toxic and risky substances. It is essential to reduce the potential risk associated with nanofluids during the design stage rather than downstream when manufacturing or at the end product [4, 5]. Recently, the stabilization of metallic nanofluids through natural polymers is widely studied. Stabilization is important in metallic nanofluids synthesis to ensure the shape control and increase the yield of monodisperse metallic nanoparticles. Nanofluids have the tendency of aggregating and agglomerating due to the high surface area and surface activity [12]. The agglomeration will not only cause settlement and clogging but decline the thermal conductivity of nanofluids [13]. Controlling the nanofluids by simplicities and costeffectiveness methods are the challenging drawbacks facing the preparation of stable nanoparticles. However, ZnO nanoparticles possess high surface energy which has a
tendency to agglomerate leading to the formation of bulk structures with lower reactivity [14]. This fastens the process that can affect the active surface area of the nanofluids [15]. On the other hand, a study had shown that the agglomeration of nanofluids was relevant to the drying methods [16]. Moreover, ZnO nanofluids are easily agglomerated by coalescence, this can be prevented by incorporation with a stable polymer [17]. Therefore, studying stability is an important issue influencing the properties of nanofluids applications.
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Moreover, the significant process needed to improve the stability of nanofluids using surfactants. In this study, gum arabic has been proposed as a stabilizing agent for green synthesis of ZnO nanofluids. Gum arabic is a complex polysaccharide, obtained as sticky exudates from the stems and branches of Acacia trees [18]. It is hydrophilic, non-toxic, phytochemical glycoprotein, biocompatible and biodegradable polymer. It is a weak polyelectrolyte that carries charged groups of carboxylate and amine groups. When adsorbed on a particle surface, it creates steric stabilization [19].
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A study had reported that gum arabic-mediated chemical synthesis was carried out to obtain ZnO nanoparticles (particle size of ZnO ≈ 40 nm) which were used for preparing ZnO–biopolymer nanocomposites used in the antibacterial application [19]. In addition, a study stated that the optimal amount of gum arabic to produce ZnO nanoparticles of 16 nm was 1.50 wt % [20]. It was used as a superb dispersant in the preparation of silver microspheres [18]. Highly stable gold nanoparticles have been successfully synthesized at room temperature to a little higher temperature by using gum arabic simultaneously as the reducing and stabilizing agents [21]. Furthermore, the hydroxyl groups in the gum arabic network hold the nanoparticles through hydrogen bonding, this helps them to stay separate and provide stability to the nanoparticles.
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Thus, the novelty in this study compared to previous studies is the use of natural gum arabic as a stabilizing agent through microwave-assisted synthesis method as a green approach for antibacterial application development in nanotechnology. In this study, Grampositive bacterium, S. aureus and Gram-negative bacterium E. coli were selected as model bacteria for most common pathogenic bacteria obtained in a clinical setting.
Materials and experimental methods
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Materials
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Zinc nitrate hexahydrate Zn(NO3)2.6H2O and sodium hydroxide (NaOH, 98.9%, Bendosen). of the analytical grade used without further chemical treatment and purification was used as the zinc cation and the hydroxide anion precursors, respectively. Gum arabic were functioning as stabilizing agents, were purchased from Sigma chemicals and used freshly after dissolving in distilled water to obtain reproducible results. The bacterial strain of S. aureus ATCC 6538 and E.coli ATCC 8739 were provided from Central Lab of University Malaysia Pahang.
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Synthesis of ZnO nanofluids
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The synthesis of ZnO nanofluids was carried out using precipitation method by using gum arabic as a stabilizer. 0.5, 1, 1.5, 2, 2.5 and 3 % of gum arabic was dissolved in 100 mL of distilled water and subjected to the microwave at 450W for 2 minutes to completely dissolve the gum arabic. 2.974 g of solid Zn(NO3)2.6H2O zinc nitrate was added into gum arabic solution and continue stirring. Then the obtained solution was heated in the microwave at 450W for 2 minutes. The initial pH of gum arabic-zinc nitrate solution was 4.5. NaOH solution was added drop-wise into the zinc nitrate and gum arabic solution under vigorous stirring until got pH value 10. The mixture solution was subjected to microwave heating for 5 minutes at 450 W. After 5 minutes, the mixture was left to cool at room temperature. The milky white solution of ZnO nanofluids was centrifuged for 2 minutes and the precipitated were washed two times with distilled water for removing excess zinc nitrate. These ZnO nanofluids were used for the characterization process.
Stability evaluation of ZnO nanofluids
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Sedimentation photograph of nanofluids was taken by a camera was used as a method for observing the stability of nanofluids. Zeta potential analysis is another method for evaluating the stability of nanofluids, 30 µL of ZnO solution was diluted in 2 mL of distilled water. The zeta potential in liquid suspension at pH 10, 25 °C was measured using Zeta potential (Malvern Instruments).
Characterization of ZnO nanofluids
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30 µL of ZnO nanofluids were diluted in 2 mL of distilled water. The nanoparticle sizes in liquid suspension at 25 °C were measured using Zetasizer Nano ZS (Malvern Instruments (ZEM5002, Malvern Instrument Ltd., Malvern, UK). Hydrodynamic diameter of ZnO nanofluids were measured. The optical properties of ZnO nanofluids were studied by diluting 0.1 mL of ZnO nanofluids in 1.9 mL of distilled water. The UV–vis absorption spectra of ZnO nanofluids were analyzed using the UV-vis spectrophotometer (U-1800 UV/VIS Spectrophotometer, Hitachi, Berkshire, United Kingdom) in the range of 250 nm to 800 nm. The chemical composition of the synthesized nanoparticles was studied using the FTIR spectrometer in the range of 400 cm-1- 4000 cm-1 using FTIR (iD7 ATR Nicolet iS5 Spectrometer, Thermo Fisher, USA). The size and the morphology of the ZnO nanofluids were examined using the field emission scanning electron microscope (FESEM)(JSM-7800F, JEOL, USA). 2.3.2. The ZnO nanoparticles were dispersed in distilled water and sonicated for 15 minutes. Next, the dispersed solutions of ZnO nanoparticles were dried on the carbon tape. The sample was coated with platinum in order to avoid charging during the observation.
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Antibacterial assays (disk diffusion method)
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The antibacterial activity of ZnO nanofluids synthesized with and without gum arabic as a stabilizing agent was evaluated using the disk diffusion method. The antibacterial test was achieved using Kirby-Bauer antibiotic testing, also called disc diffusion antibiotic sensitivity testing [22]. ZnO nanofluids were sterilized by autoclaving at 121 °C for 2 h. A single colony of E.coli and S.aureus was grown overnight in a sterile Tryptic Soy Broth (TSB) in incubator shaker at 37 ̊ C and 200 rpm until late log phase. 0.5 McFarland suspension (approximately cell density 1X108 CFU in sterile Phosphate buffer saline (PBS)) of E. coli and S. aureus were prepared in a tube and were vortexed to make sure the bacteria suspension were well mixed. A sterile cotton-tipped swab was dipped into the suspension and the excess liquid was removed from the swab by pressing it against the side of the tube. 50 μL of the bacteria samples were spread onto a solid Tryptic soy agar medium in Petri dishes using a sterile swab. 15 μL of the ZnO nanofluids were pipetted on the 1 cm of equal size autoclave-sterilized filter paper disk and left at room temperature to dry. The paper disks containing the ZnO nanoparticles samples were placed on the surface of the inoculated plates and incubated, lid down, at 37 °C for 24 hours. Triplicate experiments were carried out for each sample. The diameter of the inhibition zone will be measured after 24 hours of incubation.
Killing kinetics study
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The killing kinetics study was performed according to [23] with modifications. The inoculum culture of S. aureus and E. coli were prepared by transferring a single colony of E.coli and S.aureus into sterile Tryptic Soy Broth (TSB) at 37 ̊ C until late log phase. One mL of 0.5 McFarland of S. aureus and E. coli culture was transferred into the sterile Tryptic Soy Broth (TSB). The cultures were incubated in an incubator shaker at 37 ̊ C and 200 rpm. Optical density (OD) measurements from each flask were taken every 1 h to record the growth of the microbes. The growth rate of microbial cells interacting with the nanofluids was determined from a plot of the optical density versus time. ZnO nanofluids at 125 μg/mL were added into each flask at the mid-log phase of incubation for S. aureus and E. coli ( the mid-log phase time was decided before this experiment). Experiments were performed using flask without ZnO nanofluids as a control to track the normal growth of the microbial cells without nanofluids.
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Results and discussion
ZnO nanofluids were synthesized by a precipitating method assisted with microwave heating method. Reaction mechanism involved in ZnO nanofluids synthesis is given below. i) Zinc nitrate dissolves in gum arabic solution [Eq. (1)]. Zinc salt molecules are then formed in water and stabilized using the gum arabic solution [Eq. (2)].
Zn NO3 (aq) Zn2 (aq) 2 NO₃ˉ (aq) 2
(1)
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Zn2 (aq) GA(aq) Zn GA (aq)
(2)
ii) NaOH is added to the zinc salt (stabilized by gum arabic solution). Zinc nitrate reacts with NaOH to form zinc hydroxide, sodium nitrate and water molecules [Eq. (3 and 4)]. [Zn(GA)](2 ) (aq) 2 NaOH (aq) [Zn(OH )2 ]GA(s) 2 NaNO3 (aq) 2H 2O(aq) (3)
Zn OH GA (aq) 2NaOHˉ(aq) Zn OH GA(s) 2NaNO (aq) 2H ₂O(aq) 2
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2
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(4)
3
Zn OH ₄ ²ˉ GA(aq) ZnO GA (s) 2HOˉ(aq) H ₂O(aq)
(5)
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Nanoparticle, i.e. intermediate zinc nitrate hydroxide hydrate, is formed [24]. It is having a similar structure as that of structure Zn (OH) 2 [Eq. (6)]. 5Zn2 (aq) 8OHˉ(aq) 2 NO3ˉ(aq) 2 H 2O(aq) Zn5 NO3 OH .2 H ₂O( s) 2
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(6)
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Zinc nitrate hydroxide hydrate contains a positive ion denoted as [Zn₅ (OH)₈ ]²⁺ in the unit blocks of hydroxyl double salts. This positive layer is interrelated by water and nitrate anions to counter the positive charge [25]. Similar to Zn(OH)2, Zinc nitrate hydroxide hydrate nanofluids can easily decompose some surface nitrate ions and become positively charged. Therefore, it can then interact with the gum arabic molecules that are negatively charged [24]. Under the hydrothermal condition, Zinc nitrate hydroxide hydrate and Zn(OH)2 are unstable. Subsequently, they have transformed into ZnO nanoparticles [Eq. (7)] (Pholnak et al. 2014). Zn5 NO3 OH .2H 2O(aq) 5ZnO( s) 2 HNO3 (aq) 5H ₂O(aq) 8
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(7)
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Sedimentation method was used to evaluate the stability of ZnO nanofluids. Stability results in Fig. 1 show that the colloidal suspensions of ZnO nanofluids stabilized by gum arabic possessed greater stability upon storage in aqueous media (almost 6 months) and better dispersion of nanoparticles in water. The stability was obtained due to the protective role of gum arabic, as it slows down the growth and agglomeration of nanofluids by the steric effect. However with aging, the ZnO nanoparticle size increases and leads to the formation of the non-uniform and in the homogenous cluster, which may affect the stability of ZnO nanofluids after 6 months. From Fig. 1, it also can be seen that unstabilized ZnO nanofluids were easily coagulated within 30 minutes. The bare ZnO nanofluids stabilized themselves either by absorption of molecules from surroundings or by lowering the surface area through
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coagulation and agglomeration [27]. The settling behavior of dispersions may be influenced by the size and density of the dispersed particles [28]. Nanoparticles in the dispersion may stick together and form aggregates of the increasing size which may settle out due to gravity.
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Fig. 1. Stabilized ZnO nanofluids using gum arabic as stabilizing agent (left) compared to unstabilized ZnO nanofluids (right). (color should be used)
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The stability of colloidal suspensions can be assessed by measuring their zeta potential [29]. Colloids with high zeta potential (negative or positive) are electrically stabilized, while colloids with low zeta potentials tend to coagulate or flocculate. In general, a value of 25mV (positive or negative) can be taken as the arbitrary value that separates lowcharged surfaces from highly charged surfaces. The colloids with zeta potential from 40 to 60 mV are believed to be good stable, and those with more than 60 mV have excellent stability [30]. The magnitude of zeta potential determines the stability of the dispersion.
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To decrease the agglomeration among the smaller particles, gum arabic molecule containing many –OH functional groups was used to bind on the surface of nanofluids at the initial nucleation stage. Fig. 2, shows the zeta potential of unstabilized ZnO nanofluids and stabilized ZnO nanofluids at different gum arabic concentration. From this figure, it can be seen that zeta potential of the stabilized ZnO nanofluids was improved drastically with the increasing trend when gum arabic concentration increased. The zeta potential of the resultant dispersion was - 36 mV for stabilized ZnO nanofluids with 1.5 % gum arabic increased to 47 mV in 3.0 % gum arabic concentration indicated the stabilized condition of the nanofluids. It was far better than unstabilized ZnO nanoparticles which are – 8 mV. This might be due to the higher level of adsorption of gum arabic molecules on ZnO nanofluids surface.
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Fig. 2. Zeta potential of ZnO nanofluids at different gum arabic concentration.
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Agglomerated ZnO nanofluids of varying size are reported in Fig. 3. The results from DLS indicated that the ZnO nanofluids stabilized by gum arabic formed smaller agglomerates compared to the unstabilized ZnO nanofluids. The measurement of the average size of agglomerates ZnO nanofluids was found to be around 200 to 350 nm compared to 1020 nm of unstabilized ZnO nanofluids (not included in the graph).
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Fig. 3. ZnO nanofluids size at different gum arabic concentration based on DLS analysis.
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The rapid increase in the nanofluids size when the concentration of gum arabic is above 1.5 % proved that the mean size of nanofluids was sensitive to gum arabic concentration. The steric effect of gum arabic as a stabilizer is determined by the concentration of the stabilizer which will directly affect the size of nanoparticles. Selfassociation and aggregation properties of gum arabic over a large concentration range also can be the factor of the increasing size of agglomerated ZnO nanofluids above 1.5 %. However, at low concentration (0.5 %), agglomerated size of ZnO nanofluids increased. This is due to ZnO nanofluids are not enough to stabilize and the nanoparticles agglomerate more and influence the size of the nanofluids. Therefore, fewer gum arabic molecules are needed to covering the nanofluids surfaces sufficiently to provide a steric barrier against aggregation
[31]. In this study, 1.5 % of gum arabic concentration synthesized smallest ZnO nanofluids compare to other concentration. Therefore 1.5 % of gum arabic was selected to being investigated more for its characterization and its antibacterial study as it may serve as potential alternatives of antibacterial agent.
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Fig. 4 shows the DLS narrow size distribution of the best 1.5 % concentration of gum arabic is compared to unstabilized ZnO nanofluids. ZnO nanofluids stabilized by gum arabic 1.5 % displayed a narrow hydrodynamic particle size distribution averaging about 240 nm. The smaller size of the nanofluids produced using polymeric gum arabic indicates that gum arabic better passivate the nanoparticle against core growth. Nanofluids may grow by the aggregation of nanofluids of similar size, or by the addition of single ZnO atoms or small clusters to comparatively large particles. It may be accepted that polymeric gum arabic are better passivants against particle aggregation.
Fig. 4. Size distribution by the intensity of ZnO nanofluids a) synthesis at a concentration of gum arabic 1.5 % and b) synthesis without gum arabic stabilization.
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The size distribution in the intensity of the unstabilized ZnO nanofluids presents a multimodal distribution with several bumps in the range 100 nm –1 μm. Fig. 4b shows a few peaks at the different size of nanoparticles which are 200 nm, 1000 nm, and 8000 nm. The multimodal distributions might result from a process involving breakup of large particles, multiple sources of particles or variable growth mechanisms in the system [32]. Without stabilizer, the kinetics of nucleation and accretion increased rapidly, particle-particle interaction will be much higher, accounting the growth of large particles resulted in instability of the sample. The UV absorption spectra of ZnO nanofluids synthesized at different concentration of gum arabic were examined by UV- vis in Fig. 5. The absorbance increased from 0.351 to 0.478 with the increased in gum arabic concentration from 0.5 % to 1.5 %. However, when the concentration was higher than 1.5 %, the absorbance decreased. The maximum absorbance value for 2 %, 2.5 % and 3 % gum arabic concentration was 0.379, 0.344 and 0.299 respectively. A sharp peak was formed in the range of 340 nm – 350 nm, representing ZnO [33].
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Fig. 5. Uv-vis spectra of ZnO nanofluids synthesis at different concentration of gum arabic. GA= Gum arabic. (color should be used)
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FTIR measurement was conducted at room temperature in the acquired range of 4000400 cmˉ1. Fig. 6 shows the compositional analysis of the structure–bonding of 1.5 % gum arabic biopolymer stabilized ZnO nanofluids. The FTIR spectra of unstabilized ZnO nanofluids reveal main absorption bands at 419 and 412 cm-1 attributed to the Zn-O stretching vibration mode [34]. For the ZnO nanoparticles synthesized with gum arabic as a stabilizing agent, the absorption band has been split to a few bands. Gum arabic and ZnO nanofluids mainly interact through the surface hydroxyl groups of the gum arabic as biopolymer stabilizer. The broad band at 3447 cmˉ1 corresponded to the O-H mode of vibration within the hydroxyl groups [35]. The absorption band at 1352 cm-1 might be originated from the carboxylate group of the gum arabic [19,33]. The absorption bands near 1033-835 cm-1 are due to NO3 bonding on the ZnO surface which might be due to the absorption of the nitrate ion from the precursor. The absorption band in the region between 424 to 475 cm−1 is allotted to Zn–O stretching [37].
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Fig. 6. FTIR spectra of stabilized ZnO nanofluids synthesis with 1.5 % gum arabic concentration and unstabilized ZnO nanofluids. GA=Gum arabic
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The unstabilized ZnO nanofluids are branched star-shaped commonly referred to as nano-stars or nano-urchins with dimensions fluctuating in the range of 1000–1500 nm. Without capping agent or stabilizing agent, the nucleation and growth are continuously ongoing developing cluster aggregation and secondary formation of ZnO leading to a chemically unstable condition. Though stars are formed, the length of the spines is not uniform at some places. They are not well separated from each other. Some of the fully grown stars like structures with well-separated spines are observed in Fig. 7a. The formation of ZnO star-like particles is due to fourlings in ZnO. ZnO has different morphologies due to its hexagonal structure and its special atomic arrangement.
(a) (b) Fig. 7. FESEM image of the a) unstabilized ZnO nanofluids and b) Stabilized ZnO nanofluids with 1.5 % gum arabic concentration
Fig. 7b shows the morphology of uniformly dispersed stabilized ZnO nanofluids which are in spherical in shape. Gum arabic stabilized the agglomerated ZnO nanofluids and slow down the growth rates resulted in the spherical-shaped. A smaller size of ZnO nanofluids providing greater surface area and higher amount of zinc atoms produced toxicity effect to the bacteria [38]. As the size of the ZnO nanofluids decreases their antibacterial activity increases.
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The antibacterial activities of stabilized ZnO nanofluids were evaluated by measuring the inhibition zones of the Gram-positive bacterium S. aureus and Gram-negative bacterium E. coli. As seen in Fig. 8, both stabilized and unstabilized ZnO nanofluids have demonstrated an inhibition zone indicating the antibacterial activities on S. aureus and E. coli. The sizes of the inhibition zones were reported in Table 1. The disks with ZnO nanoparticles stabilized with 1.5 % gum arabic displayed diameter of inhibition zone of 1.8 (± 0.06) cm for S. aureus and 1.6 (± 0.08) cm for E.coli. Stabilized ZnO nanofluids demonstrated a bigger size of the inhibition zone compared to unstabilized ZnO nanofluids. This result was similar to many pieces of research finding that reported the smaller size of nanofluids will have a higher antibacterial effect [39]. The S. aureus was more sensitive against ZnO nanofluids compared to Gram-negative bacterium E. coli. However, the disk diffusion method depends on the solubility and infusibility of the test material as well. Hence it may not exhibit its full potential [40]. Therefore the evaluation of antibacterial activities of ZnO nanofluids was investigated more in killing kinetics study.
ZnO nanofluids
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Unstabilized ZnO nanofluids (1020 nm, starlike shape)
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Table 1. Inhibition zones against S. aureus and E. coli using stabilized and unstabilized ZnO nanofluids. E.coli
1.4 (± 0.04) cm
1.2 (± 0.12) cm
1.8 (± 0.06) cm
1.6 (± 0.08) cm
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Stabilized ZnO nanofluids (1.5 % gum arabic) (240 nm, spherical in shape)
S.aureus
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Experiments are in triplicates and the results are presented as a mean ± standard deviation.
Using a light source wavelength of 600 nm, OD assessment indicated the level of light scattering caused by the bacteria turbidity. OD measurements of S. aureus and E. coli were conducted hourly up to 12 h. Growth curve studies of S. aureus and E.coli were first conducted to determine the bacterial growth patterns and the mid-log phase. ZnO nanofluids were added in the bacteria culture at mid-log phase. Fig. 8 and Fig. 9 show the effect of ZnO nanofluids stabilized with 1.5 % gum arabic and unstabilized ZnO nanofluids on the growth
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of S. aureus and E.coli. ZnO nanofluids that stabilized by gum arabic demonstrated higher growth inhibition compared to unstabilized ZnO nanofluids. From both graphs, a similar result in the disk diffusion method was observed where S. aureus was more sensitive against ZnO nanofluids compared to E. coli.
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Fig. 8. Killing kinetics of stabilized (1.5 % gum arabic) and unstabilized ZnO nanofluids on S.aureus. (The arrow is indicated the time of ZnO nanofluids was added).
Fig. 9. Killing kinetics of stabilized (1.5 % gum arabic) and unstabilized ZnO nanofluids on E.coli. (The arrow is indicated the time of ZnO nanofluids was added).
The structures of Gram-negative bacteria are more complex, their outer membranes consist of a high concentration of lipids, polysaccharides, and protein. The exterior part of the central membrane contains the periplasmic region and the peptidoglycan region surrounded by additional membrane [17]. The lipopolysaccharide layer (LPS) found in Gram-negative bacterial cells could protect the cell membrane from toxic molecules. As compared to Grampositive bacteria, Gram-negative bacteria contain higher amounts of lipids and lipoproteins. Furthermore, the outer membrane exists in Gram-negative bacteria only [41], which make them more resistant to ZnO nanofluids. Thus, these might be the reason S. aureus more sensitive to ZnO nanofluids compared to E.coli.
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Previous research showed that ZnO nanoparticles less than 100 nm in size have more obvious growth inhibitory effects than particles greater than 1 μm [42]. Additionally, factors such as particles size and morphology also play a role in determining the antibacterial potential of a material [43]. It has been shown that when the nanoparticle sizes decrease, the microbial interaction between the cell membrane and the Zn2+ released from the ZnO nanofluids becomes strong, thus increasing the antibacterial activity.
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4. Conclusion
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In this study, green synthesis of ZnO nanofluids using gum arabic as a natural stabilizing agent by microwave heating method is demonstrated. Gum arabic most likely contributes to colloid stability via steric stabilization. Long-term stability (6 months) of the gum arabic stabilized nanofluids complemented by “green” synthesis methods for more sustainable processes produced in this work will support the increasing demand of worldwide ZnO nanofluids. Stabilized ZnO nanofluids at 1.5 % gum arabic have better antibacterial properties compared to unstabilized ZnO nanofluids resulted by a smaller size of ZnO nanofluids.
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5. Acknowledgment
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We acknowledge the financial support from a research university Grant number RDU 150333 and PGRS 1803106 of University Malaysia Pahang (UMP) as well as Mybrain15 scholarship under Ministry of Higher Education Malaysia. Thanks for providing all facilities to carry out this research work and for awarding financial support through Doctoral Scholarship Scheme.
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S2213-3437(19)30454-3 https://doi.org/10.1016/j.jece.2019.103331 103331
Reference:
JECE 103331
To appear in: Received date: Revised date: Accepted date:
18 January 2019 27 July 2019 27 July 2019
Please cite this article as: Pauzi N, Zain NM, Yusof NAA, Gum Arabic As Natural Stabilizing Agent In Green Synthesis Of ZnO Nanofluids For Antibacterial Application, Journal of Environmental Chemical Engineering (2019), https://doi.org/10.1016/j.jece.2019.103331 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Gum Arabic As Natural Stabilizing Agent In Green Synthesis Of ZnO Nanofluids For Antibacterial Application. Norlin Pauzi1 , Norashikin Mat Zain1 and Nurul Amira Ahmad Yusof1 Faculty Of Chemical and Natural Resources Engineering. University Malaysia Pahang, Lebuhraya Tun Razak, 26300 Gambang, Kuantan, Pahang MALAYSIA
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Corresponding author: Tel: +6017-3281653, Fax: +609-549 3190, Email:[email protected]
Highlights
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Zinc oxide (ZnO) nanofluids were synthesized by a precipitating method assisted with microwave heating method. Agglomerated size of ZnO nanofluids was found to be around 200 to 350 nm compared to unstabilized ZnO nanofluids was 1020 nm. These ZnO nanofluids relatively stabilized for at least 6 months using gum arabic as a stabilizer. Stabilized ZnO nanofluids have better antibacterial properties compared to unstabilized ZnO nanofluids.
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Abstract. This work is part of a responsible green approach for the development of nanotechnology. Zinc oxide (ZnO) nanofluids are known as one of the versatile inorganic materials used in health-related applications with effective antibacterial activities. In this study, ZnO nanofluids were synthesized by a precipitating method assisted with microwave heating method. Zinc nitrate had been used as zinc salt, sodium hydroxide as reducing agent and gum arabic as a stabilizing agent. The objective of this study was to investigate the effect of gum arabic concentration on the size, optical properties, and stability of ZnO nanofluids. Nanosizer was used for the measurement of the particles size and the average size of ZnO nanofluids was found to be around 200 to 350 nm compared to unstabilized ZnO nanofluids was 1020 nm. The UV-vis absorption spectra were found in the range of 340 nm. The FTIR spectra showed peaks range from 424 to 475 cm-1 which indicated a standard peak of ZnO nanofluids. These ZnO nanofluids relatively stabilized for at least 6 months. The antibacterial activities of ZnO nanofluids were tested against Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Escherichia coli. Our results showed that stabilized ZnO nanofluids have better antibacterial properties compared to unstabilized ZnO nanofluids.
Keywords ZnO nanofluids; Gum arabic; Microwave heating; Antibacterial
1.
Introduction
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Since nanoparticles possess unique chemical, physical and biological properties, several kinds of nanofluids are being produced and commercialized. Hence, ZnO nanofluids have attracted marketable interest in various different areas of antibacterial applications such as paint industry [1], wastewater treatment [2], biomedical materials [3] and detergents [4]. ZnO nanofluids are less prone to bacterial resistance, thus possess greater effectiveness on resistant strains of bacteria [5]. The absorption 3.37 eV band gap by ZnO nanofluids can result in the generation of reactive oxygen species (ROS) [6]. ROS and Zn ions that are released from ZnO nanofluids will disrupt the integrity of the cell wall and destroy the bacteria [7].
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In general, ZnO nanofluids are not stable when dispersed in water [8] because of ZnO agglomeration in water due to high polarity of water which leads to the deposition. Thus, the industrialists use different surface modifying agents to improve the stability of ZnO nanofluids [1].
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Several methods are used to synthesize ZnO nanofluids, these include direct precipitation, homogeneous precipitation, solvothermal method, sonochemical method, reverse micelles, the sol-gel method, hydrothermal, and thermal decomposition [9]. Nevertheless, most of the identified methods utilized sophisticated equipment, toxic chemicals and generate environmentally risky by-products [10]. Frequently, the chemical synthesis can lead to the presence of some toxic substances adsorbed on the surface which is not suitable for clinical use [11]. Furthermore, the poisonous chemicals such as hydrazine, sodium citrate, amines, diethylenetriamine, sodium hypophosphite, and sodium borohydride are used as reducing and capping agents to create uniform suspensions [3]. These toxic substances are absorbed on the nanofluids' surfaces and contribute to higher toxicities are not safe for the environment and biomedical purposes.
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Thus, this drawback can be resolved using “green” synthesis methods to achieve more sustainable processes, focussing on reducing or preventing the use of toxic and risky substances. It is essential to reduce the potential risk associated with nanofluids during the design stage rather than downstream when manufacturing or at the end product [4, 5]. Recently, the stabilization of metallic nanofluids through natural polymers is widely studied. Stabilization is important in metallic nanofluids synthesis to ensure the shape control and increase the yield of monodisperse metallic nanoparticles. Nanofluids have the tendency of aggregating and agglomerating due to the high surface area and surface activity [12]. The agglomeration will not only cause settlement and clogging but decline the thermal conductivity of nanofluids [13]. Controlling the nanofluids by simplicities and costeffectiveness methods are the challenging drawbacks facing the preparation of stable nanoparticles. However, ZnO nanoparticles possess high surface energy which has a
tendency to agglomerate leading to the formation of bulk structures with lower reactivity [14]. This fastens the process that can affect the active surface area of the nanofluids [15]. On the other hand, a study had shown that the agglomeration of nanofluids was relevant to the drying methods [16]. Moreover, ZnO nanofluids are easily agglomerated by coalescence, this can be prevented by incorporation with a stable polymer [17]. Therefore, studying stability is an important issue influencing the properties of nanofluids applications.
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Moreover, the significant process needed to improve the stability of nanofluids using surfactants. In this study, gum arabic has been proposed as a stabilizing agent for green synthesis of ZnO nanofluids. Gum arabic is a complex polysaccharide, obtained as sticky exudates from the stems and branches of Acacia trees [18]. It is hydrophilic, non-toxic, phytochemical glycoprotein, biocompatible and biodegradable polymer. It is a weak polyelectrolyte that carries charged groups of carboxylate and amine groups. When adsorbed on a particle surface, it creates steric stabilization [19].
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A study had reported that gum arabic-mediated chemical synthesis was carried out to obtain ZnO nanoparticles (particle size of ZnO ≈ 40 nm) which were used for preparing ZnO–biopolymer nanocomposites used in the antibacterial application [19]. In addition, a study stated that the optimal amount of gum arabic to produce ZnO nanoparticles of 16 nm was 1.50 wt % [20]. It was used as a superb dispersant in the preparation of silver microspheres [18]. Highly stable gold nanoparticles have been successfully synthesized at room temperature to a little higher temperature by using gum arabic simultaneously as the reducing and stabilizing agents [21]. Furthermore, the hydroxyl groups in the gum arabic network hold the nanoparticles through hydrogen bonding, this helps them to stay separate and provide stability to the nanoparticles.
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Thus, the novelty in this study compared to previous studies is the use of natural gum arabic as a stabilizing agent through microwave-assisted synthesis method as a green approach for antibacterial application development in nanotechnology. In this study, Grampositive bacterium, S. aureus and Gram-negative bacterium E. coli were selected as model bacteria for most common pathogenic bacteria obtained in a clinical setting.
Materials and experimental methods
2.1
Materials
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Zinc nitrate hexahydrate Zn(NO3)2.6H2O and sodium hydroxide (NaOH, 98.9%, Bendosen). of the analytical grade used without further chemical treatment and purification was used as the zinc cation and the hydroxide anion precursors, respectively. Gum arabic were functioning as stabilizing agents, were purchased from Sigma chemicals and used freshly after dissolving in distilled water to obtain reproducible results. The bacterial strain of S. aureus ATCC 6538 and E.coli ATCC 8739 were provided from Central Lab of University Malaysia Pahang.
2.2
Synthesis of ZnO nanofluids
2.3
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The synthesis of ZnO nanofluids was carried out using precipitation method by using gum arabic as a stabilizer. 0.5, 1, 1.5, 2, 2.5 and 3 % of gum arabic was dissolved in 100 mL of distilled water and subjected to the microwave at 450W for 2 minutes to completely dissolve the gum arabic. 2.974 g of solid Zn(NO3)2.6H2O zinc nitrate was added into gum arabic solution and continue stirring. Then the obtained solution was heated in the microwave at 450W for 2 minutes. The initial pH of gum arabic-zinc nitrate solution was 4.5. NaOH solution was added drop-wise into the zinc nitrate and gum arabic solution under vigorous stirring until got pH value 10. The mixture solution was subjected to microwave heating for 5 minutes at 450 W. After 5 minutes, the mixture was left to cool at room temperature. The milky white solution of ZnO nanofluids was centrifuged for 2 minutes and the precipitated were washed two times with distilled water for removing excess zinc nitrate. These ZnO nanofluids were used for the characterization process.
Stability evaluation of ZnO nanofluids
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Sedimentation photograph of nanofluids was taken by a camera was used as a method for observing the stability of nanofluids. Zeta potential analysis is another method for evaluating the stability of nanofluids, 30 µL of ZnO solution was diluted in 2 mL of distilled water. The zeta potential in liquid suspension at pH 10, 25 °C was measured using Zeta potential (Malvern Instruments).
Characterization of ZnO nanofluids
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30 µL of ZnO nanofluids were diluted in 2 mL of distilled water. The nanoparticle sizes in liquid suspension at 25 °C were measured using Zetasizer Nano ZS (Malvern Instruments (ZEM5002, Malvern Instrument Ltd., Malvern, UK). Hydrodynamic diameter of ZnO nanofluids were measured. The optical properties of ZnO nanofluids were studied by diluting 0.1 mL of ZnO nanofluids in 1.9 mL of distilled water. The UV–vis absorption spectra of ZnO nanofluids were analyzed using the UV-vis spectrophotometer (U-1800 UV/VIS Spectrophotometer, Hitachi, Berkshire, United Kingdom) in the range of 250 nm to 800 nm. The chemical composition of the synthesized nanoparticles was studied using the FTIR spectrometer in the range of 400 cm-1- 4000 cm-1 using FTIR (iD7 ATR Nicolet iS5 Spectrometer, Thermo Fisher, USA). The size and the morphology of the ZnO nanofluids were examined using the field emission scanning electron microscope (FESEM)(JSM-7800F, JEOL, USA). 2.3.2. The ZnO nanoparticles were dispersed in distilled water and sonicated for 15 minutes. Next, the dispersed solutions of ZnO nanoparticles were dried on the carbon tape. The sample was coated with platinum in order to avoid charging during the observation.
2.5
Antibacterial assays (disk diffusion method)
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The antibacterial activity of ZnO nanofluids synthesized with and without gum arabic as a stabilizing agent was evaluated using the disk diffusion method. The antibacterial test was achieved using Kirby-Bauer antibiotic testing, also called disc diffusion antibiotic sensitivity testing [22]. ZnO nanofluids were sterilized by autoclaving at 121 °C for 2 h. A single colony of E.coli and S.aureus was grown overnight in a sterile Tryptic Soy Broth (TSB) in incubator shaker at 37 ̊ C and 200 rpm until late log phase. 0.5 McFarland suspension (approximately cell density 1X108 CFU in sterile Phosphate buffer saline (PBS)) of E. coli and S. aureus were prepared in a tube and were vortexed to make sure the bacteria suspension were well mixed. A sterile cotton-tipped swab was dipped into the suspension and the excess liquid was removed from the swab by pressing it against the side of the tube. 50 μL of the bacteria samples were spread onto a solid Tryptic soy agar medium in Petri dishes using a sterile swab. 15 μL of the ZnO nanofluids were pipetted on the 1 cm of equal size autoclave-sterilized filter paper disk and left at room temperature to dry. The paper disks containing the ZnO nanoparticles samples were placed on the surface of the inoculated plates and incubated, lid down, at 37 °C for 24 hours. Triplicate experiments were carried out for each sample. The diameter of the inhibition zone will be measured after 24 hours of incubation.
Killing kinetics study
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The killing kinetics study was performed according to [23] with modifications. The inoculum culture of S. aureus and E. coli were prepared by transferring a single colony of E.coli and S.aureus into sterile Tryptic Soy Broth (TSB) at 37 ̊ C until late log phase. One mL of 0.5 McFarland of S. aureus and E. coli culture was transferred into the sterile Tryptic Soy Broth (TSB). The cultures were incubated in an incubator shaker at 37 ̊ C and 200 rpm. Optical density (OD) measurements from each flask were taken every 1 h to record the growth of the microbes. The growth rate of microbial cells interacting with the nanofluids was determined from a plot of the optical density versus time. ZnO nanofluids at 125 μg/mL were added into each flask at the mid-log phase of incubation for S. aureus and E. coli ( the mid-log phase time was decided before this experiment). Experiments were performed using flask without ZnO nanofluids as a control to track the normal growth of the microbial cells without nanofluids.
3.0
Results and discussion
ZnO nanofluids were synthesized by a precipitating method assisted with microwave heating method. Reaction mechanism involved in ZnO nanofluids synthesis is given below. i) Zinc nitrate dissolves in gum arabic solution [Eq. (1)]. Zinc salt molecules are then formed in water and stabilized using the gum arabic solution [Eq. (2)].
Zn NO3 (aq) Zn2 (aq) 2 NO₃ˉ (aq) 2
(1)
2
Zn2 (aq) GA(aq) Zn GA (aq)
(2)
ii) NaOH is added to the zinc salt (stabilized by gum arabic solution). Zinc nitrate reacts with NaOH to form zinc hydroxide, sodium nitrate and water molecules [Eq. (3 and 4)]. [Zn(GA)](2 ) (aq) 2 NaOH (aq) [Zn(OH )2 ]GA(s) 2 NaNO3 (aq) 2H 2O(aq) (3)
Zn OH GA (aq) 2NaOHˉ(aq) Zn OH GA(s) 2NaNO (aq) 2H ₂O(aq) 2
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2
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(4)
3
Zn OH ₄ ²ˉ GA(aq) ZnO GA (s) 2HOˉ(aq) H ₂O(aq)
(5)
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Nanoparticle, i.e. intermediate zinc nitrate hydroxide hydrate, is formed [24]. It is having a similar structure as that of structure Zn (OH) 2 [Eq. (6)]. 5Zn2 (aq) 8OHˉ(aq) 2 NO3ˉ(aq) 2 H 2O(aq) Zn5 NO3 OH .2 H ₂O( s) 2
8
(6)
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Zinc nitrate hydroxide hydrate contains a positive ion denoted as [Zn₅ (OH)₈ ]²⁺ in the unit blocks of hydroxyl double salts. This positive layer is interrelated by water and nitrate anions to counter the positive charge [25]. Similar to Zn(OH)2, Zinc nitrate hydroxide hydrate nanofluids can easily decompose some surface nitrate ions and become positively charged. Therefore, it can then interact with the gum arabic molecules that are negatively charged [24]. Under the hydrothermal condition, Zinc nitrate hydroxide hydrate and Zn(OH)2 are unstable. Subsequently, they have transformed into ZnO nanoparticles [Eq. (7)] (Pholnak et al. 2014). Zn5 NO3 OH .2H 2O(aq) 5ZnO( s) 2 HNO3 (aq) 5H ₂O(aq) 8
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(7)
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Sedimentation method was used to evaluate the stability of ZnO nanofluids. Stability results in Fig. 1 show that the colloidal suspensions of ZnO nanofluids stabilized by gum arabic possessed greater stability upon storage in aqueous media (almost 6 months) and better dispersion of nanoparticles in water. The stability was obtained due to the protective role of gum arabic, as it slows down the growth and agglomeration of nanofluids by the steric effect. However with aging, the ZnO nanoparticle size increases and leads to the formation of the non-uniform and in the homogenous cluster, which may affect the stability of ZnO nanofluids after 6 months. From Fig. 1, it also can be seen that unstabilized ZnO nanofluids were easily coagulated within 30 minutes. The bare ZnO nanofluids stabilized themselves either by absorption of molecules from surroundings or by lowering the surface area through
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coagulation and agglomeration [27]. The settling behavior of dispersions may be influenced by the size and density of the dispersed particles [28]. Nanoparticles in the dispersion may stick together and form aggregates of the increasing size which may settle out due to gravity.
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Fig. 1. Stabilized ZnO nanofluids using gum arabic as stabilizing agent (left) compared to unstabilized ZnO nanofluids (right). (color should be used)
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The stability of colloidal suspensions can be assessed by measuring their zeta potential [29]. Colloids with high zeta potential (negative or positive) are electrically stabilized, while colloids with low zeta potentials tend to coagulate or flocculate. In general, a value of 25mV (positive or negative) can be taken as the arbitrary value that separates lowcharged surfaces from highly charged surfaces. The colloids with zeta potential from 40 to 60 mV are believed to be good stable, and those with more than 60 mV have excellent stability [30]. The magnitude of zeta potential determines the stability of the dispersion.
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To decrease the agglomeration among the smaller particles, gum arabic molecule containing many –OH functional groups was used to bind on the surface of nanofluids at the initial nucleation stage. Fig. 2, shows the zeta potential of unstabilized ZnO nanofluids and stabilized ZnO nanofluids at different gum arabic concentration. From this figure, it can be seen that zeta potential of the stabilized ZnO nanofluids was improved drastically with the increasing trend when gum arabic concentration increased. The zeta potential of the resultant dispersion was - 36 mV for stabilized ZnO nanofluids with 1.5 % gum arabic increased to 47 mV in 3.0 % gum arabic concentration indicated the stabilized condition of the nanofluids. It was far better than unstabilized ZnO nanoparticles which are – 8 mV. This might be due to the higher level of adsorption of gum arabic molecules on ZnO nanofluids surface.
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Fig. 2. Zeta potential of ZnO nanofluids at different gum arabic concentration.
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Agglomerated ZnO nanofluids of varying size are reported in Fig. 3. The results from DLS indicated that the ZnO nanofluids stabilized by gum arabic formed smaller agglomerates compared to the unstabilized ZnO nanofluids. The measurement of the average size of agglomerates ZnO nanofluids was found to be around 200 to 350 nm compared to 1020 nm of unstabilized ZnO nanofluids (not included in the graph).
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Fig. 3. ZnO nanofluids size at different gum arabic concentration based on DLS analysis.
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The rapid increase in the nanofluids size when the concentration of gum arabic is above 1.5 % proved that the mean size of nanofluids was sensitive to gum arabic concentration. The steric effect of gum arabic as a stabilizer is determined by the concentration of the stabilizer which will directly affect the size of nanoparticles. Selfassociation and aggregation properties of gum arabic over a large concentration range also can be the factor of the increasing size of agglomerated ZnO nanofluids above 1.5 %. However, at low concentration (0.5 %), agglomerated size of ZnO nanofluids increased. This is due to ZnO nanofluids are not enough to stabilize and the nanoparticles agglomerate more and influence the size of the nanofluids. Therefore, fewer gum arabic molecules are needed to covering the nanofluids surfaces sufficiently to provide a steric barrier against aggregation
[31]. In this study, 1.5 % of gum arabic concentration synthesized smallest ZnO nanofluids compare to other concentration. Therefore 1.5 % of gum arabic was selected to being investigated more for its characterization and its antibacterial study as it may serve as potential alternatives of antibacterial agent.
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Fig. 4 shows the DLS narrow size distribution of the best 1.5 % concentration of gum arabic is compared to unstabilized ZnO nanofluids. ZnO nanofluids stabilized by gum arabic 1.5 % displayed a narrow hydrodynamic particle size distribution averaging about 240 nm. The smaller size of the nanofluids produced using polymeric gum arabic indicates that gum arabic better passivate the nanoparticle against core growth. Nanofluids may grow by the aggregation of nanofluids of similar size, or by the addition of single ZnO atoms or small clusters to comparatively large particles. It may be accepted that polymeric gum arabic are better passivants against particle aggregation.
Fig. 4. Size distribution by the intensity of ZnO nanofluids a) synthesis at a concentration of gum arabic 1.5 % and b) synthesis without gum arabic stabilization.
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The size distribution in the intensity of the unstabilized ZnO nanofluids presents a multimodal distribution with several bumps in the range 100 nm –1 μm. Fig. 4b shows a few peaks at the different size of nanoparticles which are 200 nm, 1000 nm, and 8000 nm. The multimodal distributions might result from a process involving breakup of large particles, multiple sources of particles or variable growth mechanisms in the system [32]. Without stabilizer, the kinetics of nucleation and accretion increased rapidly, particle-particle interaction will be much higher, accounting the growth of large particles resulted in instability of the sample. The UV absorption spectra of ZnO nanofluids synthesized at different concentration of gum arabic were examined by UV- vis in Fig. 5. The absorbance increased from 0.351 to 0.478 with the increased in gum arabic concentration from 0.5 % to 1.5 %. However, when the concentration was higher than 1.5 %, the absorbance decreased. The maximum absorbance value for 2 %, 2.5 % and 3 % gum arabic concentration was 0.379, 0.344 and 0.299 respectively. A sharp peak was formed in the range of 340 nm – 350 nm, representing ZnO [33].
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Fig. 5. Uv-vis spectra of ZnO nanofluids synthesis at different concentration of gum arabic. GA= Gum arabic. (color should be used)
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FTIR measurement was conducted at room temperature in the acquired range of 4000400 cmˉ1. Fig. 6 shows the compositional analysis of the structure–bonding of 1.5 % gum arabic biopolymer stabilized ZnO nanofluids. The FTIR spectra of unstabilized ZnO nanofluids reveal main absorption bands at 419 and 412 cm-1 attributed to the Zn-O stretching vibration mode [34]. For the ZnO nanoparticles synthesized with gum arabic as a stabilizing agent, the absorption band has been split to a few bands. Gum arabic and ZnO nanofluids mainly interact through the surface hydroxyl groups of the gum arabic as biopolymer stabilizer. The broad band at 3447 cmˉ1 corresponded to the O-H mode of vibration within the hydroxyl groups [35]. The absorption band at 1352 cm-1 might be originated from the carboxylate group of the gum arabic [19,33]. The absorption bands near 1033-835 cm-1 are due to NO3 bonding on the ZnO surface which might be due to the absorption of the nitrate ion from the precursor. The absorption band in the region between 424 to 475 cm−1 is allotted to Zn–O stretching [37].
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Fig. 6. FTIR spectra of stabilized ZnO nanofluids synthesis with 1.5 % gum arabic concentration and unstabilized ZnO nanofluids. GA=Gum arabic
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The unstabilized ZnO nanofluids are branched star-shaped commonly referred to as nano-stars or nano-urchins with dimensions fluctuating in the range of 1000–1500 nm. Without capping agent or stabilizing agent, the nucleation and growth are continuously ongoing developing cluster aggregation and secondary formation of ZnO leading to a chemically unstable condition. Though stars are formed, the length of the spines is not uniform at some places. They are not well separated from each other. Some of the fully grown stars like structures with well-separated spines are observed in Fig. 7a. The formation of ZnO star-like particles is due to fourlings in ZnO. ZnO has different morphologies due to its hexagonal structure and its special atomic arrangement.
(a) (b) Fig. 7. FESEM image of the a) unstabilized ZnO nanofluids and b) Stabilized ZnO nanofluids with 1.5 % gum arabic concentration
Fig. 7b shows the morphology of uniformly dispersed stabilized ZnO nanofluids which are in spherical in shape. Gum arabic stabilized the agglomerated ZnO nanofluids and slow down the growth rates resulted in the spherical-shaped. A smaller size of ZnO nanofluids providing greater surface area and higher amount of zinc atoms produced toxicity effect to the bacteria [38]. As the size of the ZnO nanofluids decreases their antibacterial activity increases.
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The antibacterial activities of stabilized ZnO nanofluids were evaluated by measuring the inhibition zones of the Gram-positive bacterium S. aureus and Gram-negative bacterium E. coli. As seen in Fig. 8, both stabilized and unstabilized ZnO nanofluids have demonstrated an inhibition zone indicating the antibacterial activities on S. aureus and E. coli. The sizes of the inhibition zones were reported in Table 1. The disks with ZnO nanoparticles stabilized with 1.5 % gum arabic displayed diameter of inhibition zone of 1.8 (± 0.06) cm for S. aureus and 1.6 (± 0.08) cm for E.coli. Stabilized ZnO nanofluids demonstrated a bigger size of the inhibition zone compared to unstabilized ZnO nanofluids. This result was similar to many pieces of research finding that reported the smaller size of nanofluids will have a higher antibacterial effect [39]. The S. aureus was more sensitive against ZnO nanofluids compared to Gram-negative bacterium E. coli. However, the disk diffusion method depends on the solubility and infusibility of the test material as well. Hence it may not exhibit its full potential [40]. Therefore the evaluation of antibacterial activities of ZnO nanofluids was investigated more in killing kinetics study.
ZnO nanofluids
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Unstabilized ZnO nanofluids (1020 nm, starlike shape)
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Table 1. Inhibition zones against S. aureus and E. coli using stabilized and unstabilized ZnO nanofluids. E.coli
1.4 (± 0.04) cm
1.2 (± 0.12) cm
1.8 (± 0.06) cm
1.6 (± 0.08) cm
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Stabilized ZnO nanofluids (1.5 % gum arabic) (240 nm, spherical in shape)
S.aureus
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Experiments are in triplicates and the results are presented as a mean ± standard deviation.
Using a light source wavelength of 600 nm, OD assessment indicated the level of light scattering caused by the bacteria turbidity. OD measurements of S. aureus and E. coli were conducted hourly up to 12 h. Growth curve studies of S. aureus and E.coli were first conducted to determine the bacterial growth patterns and the mid-log phase. ZnO nanofluids were added in the bacteria culture at mid-log phase. Fig. 8 and Fig. 9 show the effect of ZnO nanofluids stabilized with 1.5 % gum arabic and unstabilized ZnO nanofluids on the growth
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of S. aureus and E.coli. ZnO nanofluids that stabilized by gum arabic demonstrated higher growth inhibition compared to unstabilized ZnO nanofluids. From both graphs, a similar result in the disk diffusion method was observed where S. aureus was more sensitive against ZnO nanofluids compared to E. coli.
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Fig. 8. Killing kinetics of stabilized (1.5 % gum arabic) and unstabilized ZnO nanofluids on S.aureus. (The arrow is indicated the time of ZnO nanofluids was added).
Fig. 9. Killing kinetics of stabilized (1.5 % gum arabic) and unstabilized ZnO nanofluids on E.coli. (The arrow is indicated the time of ZnO nanofluids was added).
The structures of Gram-negative bacteria are more complex, their outer membranes consist of a high concentration of lipids, polysaccharides, and protein. The exterior part of the central membrane contains the periplasmic region and the peptidoglycan region surrounded by additional membrane [17]. The lipopolysaccharide layer (LPS) found in Gram-negative bacterial cells could protect the cell membrane from toxic molecules. As compared to Grampositive bacteria, Gram-negative bacteria contain higher amounts of lipids and lipoproteins. Furthermore, the outer membrane exists in Gram-negative bacteria only [41], which make them more resistant to ZnO nanofluids. Thus, these might be the reason S. aureus more sensitive to ZnO nanofluids compared to E.coli.
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Previous research showed that ZnO nanoparticles less than 100 nm in size have more obvious growth inhibitory effects than particles greater than 1 μm [42]. Additionally, factors such as particles size and morphology also play a role in determining the antibacterial potential of a material [43]. It has been shown that when the nanoparticle sizes decrease, the microbial interaction between the cell membrane and the Zn2+ released from the ZnO nanofluids becomes strong, thus increasing the antibacterial activity.
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4. Conclusion
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In this study, green synthesis of ZnO nanofluids using gum arabic as a natural stabilizing agent by microwave heating method is demonstrated. Gum arabic most likely contributes to colloid stability via steric stabilization. Long-term stability (6 months) of the gum arabic stabilized nanofluids complemented by “green” synthesis methods for more sustainable processes produced in this work will support the increasing demand of worldwide ZnO nanofluids. Stabilized ZnO nanofluids at 1.5 % gum arabic have better antibacterial properties compared to unstabilized ZnO nanofluids resulted by a smaller size of ZnO nanofluids.
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5. Acknowledgment
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We acknowledge the financial support from a research university Grant number RDU 150333 and PGRS 1803106 of University Malaysia Pahang (UMP) as well as Mybrain15 scholarship under Ministry of Higher Education Malaysia. Thanks for providing all facilities to carry out this research work and for awarding financial support through Doctoral Scholarship Scheme.
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