Optimisation of preparation conditions for Ti nanowires and suitability as an antibacterial material

Abstract

Ultrafine titanium dioxide (TiO₂) nanowires were synthesised using a hydrothermal method with different volumes of ethylene glycol (EG) and annealing temperatures. It shows that sodium titanate nanowires synthesised using 5 and 10 ml EG, which annealed at 400°C produced TiO₂ nanowires that correspond to a photochemically active phase, which is anatase. The influences of annealing temperatures (400–600°C) on the morphological arrangement of TiO₂ nanowires were evident in the field emission scanning electron microscopy. The annealing temperature of 500°C led to agglomeration, which formed a mixture of TiO₂ nanoparticles and nanowires. High thermal stability of TiO₂ nanowires revealed by thermogravimetric analysis and Fourier transform infrared spectroscopy spectrum showed the presence of the Ti–O–Ti vibrations as evidenced due to TiO₂ lattices. An antibacterial study using TiO₂ nanowires toward Escherichia coli and Klebsiella pneumoniae showed large zones of inhibition that indicated susceptibility of the microbe toward TiO₂. Growth kinetic analysis shows that addition of TiO₂ has reduced optical density (OD) suggesting an inhibition of the growth of bacteria. These results indicate TiO₂ nanowires can be effectively used as an antimicrobial agent against gram‐bacteria. The TiO₂ nanowires could be exploited in the medical, packaging and detergent formulation industries and wastewater treatment.

1 Introduction

Biomedical devices related infections are of considerable importance as they increase the morbidity and healthcare costs [1, 2, 3, 4]. Infectious diseases are the leading cause of death worldwide, with microbial infections contributing significantly to the high rate of mortality. Control or destroy of microorganisms is very important as it increases the risk of infections causing patient's pain and functional loss [5]. On the basis of some research studies, Staphylococcus aureus is the leading cause of both the surgical site infections and periprosthetic joint infection [6]. The central venous catheter is another device that has been associated with a high number of bloodstream infections [7, 8, 9]. It was estimated that in the USA alone at least 80,000 catheter‐related bloodstream infections occur annually. The infections rate in the orthopaedic surgical field is occurring continuously even though research scientists are finding solutions to minimise the risk of infections [10]. It was estimated that 2.5% of primary hip and knee arthroplasties and 20% of arthroplasties are complicated by periprosthetic joint infection [11].

In the attempt to address the device‐related infection issues due to bacterial colonisation and proliferation, the photocatalytic properties of titanium dioxide (TiO₂) nanowires have been investigated and reported in the literature. Many nano‐dimensional structures have been studied such as nanowires, nanotubes, nanorods and nanofibres. One‐dimensional (1D) Ti‐oxide nanowires and nanorods have attracted the attention of researchers, because of their excellent electron transport efficiency and chemical stability. TiO₂ nanowires have an extraordinary property among some nanostructured materials that make them attractive for the fabrication of novel analytical devices that have advantages over traditional ones [12, 13, 14]. The bactericidal activity of 1D ultrafine TiO₂ nanowires has demonstrated a reduced bacterial proliferation due to the photocatalytic capability of TiO₂.

Exploring the novel applications of functional nanostructured materials is a great challenge. It is critical to produce nanowires with a smaller diameter of large diameters has low specific surface areas. Besides that, there are many factors such as particle size, surface area and crystallinity that affect the properties of TiO₂ nanowires [15, 16]. The TiO₂ photocatalytic reaction was studied by the effectiveness of photocatalytic oxidation in water with many microorganisms including Lactobacillus acidophilus (Gram‐positive bacteria), Saccharomyces cerevisiae (yeast), Escherichia coli (Gram‐negative bacteria) and Chlorella Vulgaris (green algae) which contributed to its biocidal property [17].

There have been studies incorporating nanowires to test the antibacterial activity against Gram‐positive and Gram‐negative bacterias such as Klebsiella, whereby low bacterial adhesion resulted due to low surface energy [18]. The underlying mechanisms of bactericidal effects of TiO₂ photocatalyses are due to the reaction of bacteria and the reactive hydroxyl radical of the photocatalytic products.

This paper is focusing on the production of ultrafine TiO₂ nanowires with a larger surface area, which contributes to its antimicrobial activity. The hydrothermal method is used to synthesise the TiO₂ nanowires. The influences of different volumes of ethylene glycol (EG) and different annealing temperatures were investigated in the formation of TiO₂ nanowires. The surface morphology of TiO₂ nanowires and the Gram‐negative and Gram‐positive bacterias treated with TiO₂ nanowires showed the effectiveness of the antimicrobial activity.

2 Experimental

2.1 Synthesis of TiO₂ nanowires

The TiO₂ nanowires were synthesised by a method modified from a previous report [19]. About 1.0 g of TiO₂ nanopowder (Sigma Ultra) was dissolved in 100 ml of deionised water and added with different volumes of 2 M Na hydroxide (NaOH) and stirred for 30 min. For set A, 1.0 g of TiO₂ nanopowder was dissolved in 35 ml of NaOH, whereas for set B 1.0 g of TiO₂ nanopowder was dissolved in 30 ml of NaOH. A mixture of EG (Sigma‐Aldrich) and distiled water was prepared in 50:50 ratios. The sets A and B were added with 5 and 10 ml of a mixture of EG, respectively. Both the mixtures were stirred for an hour to attain adsorption–desorption equilibrium. After stirring, the solutions were transferred to 50 ml of the teflon‐lined autoclave. The mixtures were subjected to 16 h of the heating process for 160°C that led to the formation of sodium (Na) titanate. The mixtures were centrifuged at 6000 rpm for 15 min each time, while it was subjected to acid washing using 0.4 M of HCl and ultrapure water. The Na titanate nanowires were allowed to dry overnight in an incubator at 40°C. The dried nanowires were annealed thermally overnight with the respective temperatures (400, 500 and 600°C).

2.2 Characterisation of TiO₂ nanowires

2.2.1 Functional group analyses

The pristine TiO₂ and TiO₂ nanowires (0.2 mg) were used to record the wavelength from 600 to 4000 cm⁻¹ by using Fourier transform infrared (FT‐IR) spectrophotometer (model Varian 640‐IR).

2.2.2 Thermal analysis

The thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were performed on a thermal analysis system (METTLER TOLEDO, TGA/DSC 1 STAR^e, USA). About ∼5 mg of the samples were analysed over the temperature range from 30 to 600°C, at a heating rate of 10°C min⁻¹ under the flow of nitrogen at the rate of 50 ml min⁻¹.

2.2.3 Antimicrobial activity

Modified Bauer–Kirby disc diffusion method was followed to study antimicrobial activity of TiO₂ nanowires. Discs used were made up of sterilised filter paper and had a diameter of 5 mm. Six bacterial strains were chosen: two Gram +ve and four Gram −ve clinical pathogens, which included methicillin‐resistant S. aureus (Gram +ve, American Type Culture Collection (ATCC) 700699), Enterococcus faecalis (Gram +ve, ATCC 2921), Klebsiella pneumoniae (Gram −ve, ATCC 10031), Proteus mirabilis (gram−ve, ATCC 12453), E. coli (gram−ve, ATCC 25922) and Pseudomonas aeruginosa (gram−ve, ATCC 10145). These discs were then impregnated with 20 μl TiO₂ nanowires solution and 20 μl of pristine TiO_2. The nutrient broth required for the overnight bacterial cultures was prepared by dissolving 3.7 g of Merck nutrient broth (Kenilworth, NJ, USA) in 100 ml of 18.1 MΩ distiled water. The nutrient broth was sterilised by autoclaving at 121°C for 15 min and cooling down to 37°C. A 5 ml of nutrient broth was poured into six 15 ml centrifuge tube along with 1 ml inoculum of the glycerol stock of bacterial strains and incubated at 37°C for 18 h at 200 rpm. The agar plates were prepared by specifically dissolving 13.5 g of Merck nutrient agar (Kenilworth, NJ, USA) in 500 ml of 18.1 MΩ distiled water. The nutrient agar was sterilised by autoclaving at 121°C for 15 min, cooling it down to 37°C and 20 ml was poured into each Petri dish and allowed to form an agar gel. The Petri dishes were inoculated with each bacterial strain by spread plate method. About 100 µl of the overnight culture was pipetted onto each plate and spread evenly using a sterile L‐rod, followed by the placement of the TiO₂ nanowires, pristine TiO₂ and ten units of penicillin antibiotic sensitivity discs onto the Petri dishes. The Petri dishes were labelled accordingly and incubated at 37°C for 24 h to obtain the inhibition zone. Large zones of inhibition around the disc indicated susceptibility of microbe toward TiO₂ nanowires, while small zones or no zones of inhibition indicated resistive microbes [20].

2.2.4 Growth kinetic analysis

Effects of microbial viability are studied by following the growth kinetics of methicillin‐resistant S. aureus (gram+ve, ATCC 700699), E. faecalis (gram+ve, ATCC 2921), K. pneumoniae (gram−ve, ATCC 10031), P. mirabilis (gram−ve, ATCC 12453), E. coli (gram−ve, ATCC 25922) and P. aeruginosa (gram−ve, ATCC 10145) in the absence and the presence of TiO₂ nanowires. The mother cultures of test organisms were prepared in nutrient broth taking loop full of bacteria from the specified slant culture, and cultured overnight at 37°C and 150 rpm agitation. The TiO₂ nanowire stock solution was prepared by dispersing TiO₂ nanowire in sterilised nutrient broth followed by ultraviolet (UV) radiation sterilisation before use. The reaction mixtures without nanowires were taken as controls. Briefly, 200 μl of bacterial mother cultures were added to the 20 ml nutrient broth. The kinetic growth studies were performed by measuring the optical density (OD) at 600 nm using a plate reader (TECAN Spark^® 10M) at regular time interval. At approximately mid‐log phase of bacterial growth (0.6 OD), 50 mg final concentration of TiO₂ nanowire in 20 ml nutrient broth was added to the culture. The kinetic growth studies were performed by measuring OD at 600 nm using the plate reader at the regular time interval for 24 h [21].

2.2.5 Evaluation of bacterial survivability by colony‐forming units (CFUs) measurement

The number of viable cells was quantified by measuring CFUs for E. coli and K. pneumoniae. For CFU measurement, 10 μl of sample from the stationary phase of growth kinetics was taken from 24 h growth kinetics study and spread on the nutrient agar plates after 10⁻⁴ times of dilution in autoclaved distiled water. The plates were incubated overnight at 37°C. The number of viable cells after treatment with TiO₂ nanowire was quantified, and compared with positive control (culture without nanowires) to evaluate the antimicrobial property of TiO₂ nanowire [21].

2.2.6 Field emission scanning electron microscopy

The morphology of the TiO₂ nanowires obtained in the present work was investigated by a field emission scanning electron microscopy (FESEM) (SU8010) operated at an accelerated voltage of 15–30 kV and the energy dispersive X‐ray analysis (EDX) was carried out as well.

FESEM (SU8010) was also used to study the shape, size and interaction of bacteria (E. coli and K. pneumoniae) with nanowires for both the bacterial sample in the presence and the absence of TiO₂ nanowires. Each of the samples was incubated for 60 min with 2.5% concentration of glutaraldehyde [22, 23] in phosphate buffer for fixation. Furthermore, the samples were rinsed with phosphate buffer and dehydrated in 50–100% ethanol dehydration series by incubating for 30 min in each of 50, 60, 70, 80, 90 and 100% ethanol. Finally, the air‐dried slides were fixed on stubs using double‐faced conductive carbon adhesive tape and coated with a thin layer of platinum using a Quorum (Q150RS) division FESEM sputter coating system for 15 min before observing at various desired magnifications.

3 Results and discussion

3.1 Synthesis of TiO₂ nanowires

Na titanate nanowires were the first product obtained from a hydrothermal process, which was subjected to heating for 16 h at 160°C. Na titanate nanowires were synthesised using two different volumes of EGs of 5 and 10 ml. The effects of different volumes of EG on the light absorption in UV range of Na titanate nanowires were studied by performing the spectrophotometric analysis. Graphs were plotted to show the absorbance maximum of Na titanate nanowires for different volumes of EG.

On the basis of Fig. 1 a, Na titanate nanowires, which were synthesised using 5 and 10 ml EG, show the absorbance maximum in the UV range from 400 to 450 nm. As for peak absorption, Na titanate nanowires synthesised using 5 ml EG demonstrate the absorbance maximum at 420 nm, whereas the 10 ml EG shows at 400 nm. The difference in absorbance maximum proves that the products obtained were not of pure TiO₂ nanowires. This could be due to the presence of micron range size primary particles, which will later lead to agglomeration of TiO₂ nanowires after the thermal annealing process. Besides that, the presence of Na and some other elements such as chlorine after acid washing could also be one of the reasons for obtaining the difference in the absorbance maximum. To acquire pure TiO₂ nanowires without the presence of Na, thermal annealing is acquired. Thus, Na titanate nanowires of 5 and 10 ml EG were subjected to thermal annealing at three different temperatures: 400, 500 and 600°C.

Fig. 1.

UV–visible absorption spectra of TiO₂ nanowires

(a) Obtained with 5 and 10 ml of EG, (b) Obtained with 5 and 10 ml EG at an annealing temperature of 400°C, (c) At annealing temperatures of 400, 500 and 600°C obtained with 5 ml of EG, (d) Obtained with 10 ml of EG

According to Fig. 1 b, it shows that Na titanate nanowires synthesised using 5 and 10 ml EGs which were annealed at 400°C produced TiO₂ nanowires with the absorbance maximum at a range of 200–350 nm. As for TiO₂ nanowires, which were produced by annealing temperatures at 500 and 600°C, the absorbance maximum drastically varied from the nanowires obtained at 400°C. Fig. 1 c shows the absorbance maximum of nanowires annealed at 500°C was at a range of 400–450 nm, whereas for 600°C was at a range of 450–550 nm. The TiO₂ exists in polymorphs, namely anatase, rutile and brookite. The most photochemically active phase is anatase corresponding to UV wavelength absorption of 385 nm, and rutile has UV wavelengths absorption at 410 nm. The reason for this higher activity should be attributed to the combined effect of the higher surface adsorptive capacity of anatase and its higher rate of hole trapping. Recently, studies have shown that mixtures of anatase and rutile or brookite–anatase were more active than anatase alone [24]. As for Na titanate nanowires synthesised using 10 ml EG (Fig. 1 d), which were annealed at 400, 500 and 600°C produced TiO₂ nanowires, which have an absorbance maximum of 250, 400 (anatase phase) and 500 nm (transformation to rutile phase), respectively [25]. The absorbance maximum of TiO₂ nanowires varied according to the annealing temperatures. The absorbance maximum increased with the increase in the annealing temperature.

Annealing temperature influences the morphology of the TiO₂ nanowires and leads to the changes in the UV absorbance maximum. The bell curve for the annealing temperatures of 500 and 600°C has a blue shift compared with the curve of annealing temperature at 400°C, which has a red shift due to the difference in the absorbance rate. The differences in light absorption of nanowires are caused by the changes of morphological order, which were induced by different annealing temperatures.

3.2 FT‐IR and thermal analysis

FT‐IR spectral analysis is an essential tool to predict the formation of functional groups in the prepared TiO₂ nanowires at 500°C. Fig. 2 a shows the characteristic peak for TiO₂ nanowires at 1400 cm⁻¹ that correspond to symmetric and asymmetric stretching vibrations of the adsorbed carboxylic group co‐ordinating to Ti.

Fig. 2.

FT‐IR and thermal analysis

(a) FT‐IR spectra for pristine TiO₂ and TiO₂ nanowires annealed at 400°C, (b) TGA thermograms of the TiO₂ nanowires, (c) DSC thermograms of the TiO₂ nanowires

The hydroxyl peaks intensity at 3600–3100, 1640 and 950 cm⁻¹ were found to be representing the H–O–H bending for water, oxonium ions and O–H bending for a hydroxyl group, respectively. The intensities of the peaks are weak indicating the removal of adsorbed interlayer water, oxonium ions and hydroxyl groups from pristine TiO₂ as well as in TiO₂ nanowires during the calcination process that has dehydrated and recrystallised into the more stable form, which could be TiO₂ anatase phase [26, 27]. Also, the broad, intense bands below 1000 cm⁻¹ in both spectrums are due to Ti–O–Ti vibrations in the TiO₂ lattices, to which the formation of TiO₂ is attributed. The broad peak at 400–800 cm⁻¹ in both spectra shows the mixture of polymorphic phases of amorphous and anatase of TiO₂ [28].

Fig. 2 b shows TGA thermogram of the pristine TiO₂ and synthesised TiO₂ nanowires that correspond to thermal degradation. At temperature 40–50°C, weight loss of pristine TiO₂ was observed. This could be due to the reduction of its moisture content. Furthermore, at 120–600°C heating, the sample shows a gradual deceleration in weight loss which occurred due to loosely bound water. The pristine TiO₂ curve has shown a peak below 50°C, indicating the evaporation of lower volatile components during the initial phases of heating.

As for TiO₂ nanowires, the initial weight loss was observed at 60–100°C which was possibly due to evaporation of water. A notable weight loss of TiO₂ nanowires was observed between 100 and 150°C. This could be attributed to residual EG and degradation of organic‐containing starting material [26]. It is evident that the TiO₂ nanowires produced were more thermally stable compared with pristine TiO₂. This corresponds to previous findings that TiO₂ is inert and thermally stable with non‐inflammable characteristics due to different polymorphic forms of anatase, rutile and brookite [28]. Fig. 2 c shows the DSC curve for the synthesised TiO₂ nanowires. The shallow endothermic peak is at 60–140°C and weight loss corresponds to the dehydration of water. The continuous slow progression of exothermic peaks from 160 to 600°C could be attributable to the phase transformation from anatase to rutile [29].

3.3 Field ESEM

During the process of acid washing, it was noted that agglomeration occurred highly in Na titanate nanowires from set A (35 ml of NaOH and 5 ml of EG) compared with set B (30 ml of NaOH and 10 ml EG), which is a normal phenomenon. Fig. 3 a shows the image of agglomeration of TiO₂ nanowires under a light microscope, which was similar for both 5 and 10 ml of volumes of EGs, and annealing temperature at 400°C. As discussed by Schilling et al. [30], in all production processes of particulate materials, there will be a distribution of primary particle sizes around the average value, and it is likely that a small fraction of the primary particles was <100 nm. In practise, all these particles tend to agglomerate into the micron (μm) size range as schematised in Fig. 3 b. Scientifically primary particles are strongly bound or fused together by chemical bonds to form aggregates. These aggregates further agglomerate via van der Waals attractive forces to form particles in the micron (μm) size range. It was further clarified that volume of EG is one of the factors, which cause agglomeration in the production of TiO₂ nanowires.

Fig. 3.

Image of agglomeration of TiO₂ nanowires under a light microscope

(a) Image of agglomeration of TiO₂ nanowires under light microscope at 10× magnification, (b) Agglomeration process reported by Schilling et al. [30]

Agglomeration was first noted in dried Na titanate nanowires (first product obtained after acid washing). To rectify the further reasons of agglomeration and to study the morphology of agglomerated nanowires, the annealed samples were observed under FESEM with different magnifications. Fig. 4 a shows the pristine TiO₂ in the form of nanoparticles. The annealing temperature influences surface morphology of the TiO₂ nanowires.

Fig. 4.

FESEM micrograph of

(a) Pristine TiO₂, (b) TiO₂ nanowire annealed at 400°C at 30× magnification, (c) TiO₂ nanowire annealed at 500°C at 50× magnifications, (d) EDX‐FESEM for TiO₂ nanowire annealed at 400°C

Fig. 4 b illustrates the morphology of the prepared TiO₂ nanowires annealed at 400°C. This correlates with the results reported by Park et al. [31] that morphology of TiO₂ remained as nanowires at an annealing temperature of 400°C. The morphology changes when the annealing temperature increases. Fig. 4 c shows the agglomerated TiO₂ nanowires, which annealing temperature at 500°C contributes to the formation of a mixture of nanoparticles and nanowires merged. According to Visai et al., annealing at 600°C would have resulted in a mix of grain‐like objects and short rods, which look similar as nanobelts. In this experiment, EDX spectroscopy was used for the elements analysis presence of TiO₂ nanowires. According to the EDX analysis as in Fig. 4 d, there were two peaks for Ti in the spectrum for TiO₂ nanowires. The highest peak was at 4.508 keV and a small peak at 0.452 keV. Both the peaks represent the Lα and Kα lines of Ti [32].

According to Sedach et al., the growth of nanowires are highly sensitive to several number of factors such as effects of stirring, solvent volume, substrate pre‐treatment as well as calcination temperature. It was evident that calcination temperatures above 500°C led to the transformation of onset rutile phase. Binding of Ti‐oxide molecular clusters to the base‐treated surface is required as the first step of growth mechanism which was found to increase the affinity of nucleation sites for the surface. The nucleation growth mechanisms of TiO₂ nanowires show aggregation of clusters at the initial stage of the reaction. Further with subsequent hydrolysis steps, it is evident that the wires originate from nucleation sites on the substrate surface. This proves that the wire growth appears irregular, thus driving wire growth in different directions [33].

Broad zones of inhibition around the disc indicated susceptibility of microbe toward TiO₂ nanowires while small zones or no zones of inhibition for specific bacterial strains. Variable zone of inhibition of penicillin was observed. A large zone of inhibition for TiO₂ nanowires was observed with E. coli and K. pneumoniae. Minimal zone of inhibition TiO₂ nanowires was observed with P. mirabilis and P. aeruginosa. Pristine TiO₂ also produced a zone of inhibition in E. coli and K. pneumoniae. Table 1 shows the zone of inhibitions for the bacteria under investigation. It was observed that the zones of inhibitions were prominent for Gram− bacteria, whereas the Gram+ bacteria showed no zones of inhibition.

Table 1.

Zone of inhibition of TiO₂ nanowires against different pathogenic bacterias

	Microorganism
Zone of inhibition, mm	P. mirabilis Gram−	P. aeruginosa Gram−	Methicillin‐resistant S. aureus Gram+	E. faecalis Gram+	E. coli Gram−	K. pneumoniae Gram−
antibiotic susceptibility disc (penicillin)	10	6	—	10	10	8
pristine TiO2	6	—	—	—	8	6
TiO2 nanowires	8	9	—	—	11	10

3.4 Antimicrobial activity

The antimicrobial activity exerted by TiO₂ nanowires can be attributed to the fact that the TiO₂ nanowires bear a net negative charge, which helps in the interaction of the TiO₂ nanowires with the bacterial outer membrane. No activity for Gram+ was observed as the Gram+ bacteria has a thick peptidoglycan layer which is absent in Gram− and hence the antimicrobial activity was shown only in Gram− bacteria [34]. Fig. 5 demonstrates the difference in the outer membrane makeup of the Gram− and Gram+ bacteria. The TiO₂ nanowires interact with the lipopolysaccharide and proteins which are held together by electrostatic interactions with divalent cations that are required to stabilise the outer membrane helping the TiO₂ nanowires to form a molecular linkage at the cell surface allowing it to disturb membrane function of the bacteria leading to the lysis of the bacteria [24, 35]. Whereas in the case of Gram+ bacteria, no antimicrobial activity was witnessed as there might be no interaction of TiO₂ nanowires with lipotheoic acid, which is present in the outer membrane of Gram+ bacteria. Generally, TiO₂ has a photocatalytic surface that is self‐disinfecting and shown capable of killing the Gram− than Gram+ bacteria [24].

Fig. 5.

Antimicrobial activity of pathogenic Gram +ve and Gram –ve bacteria against TiO₂ nanowires, pristine TiO₂ and penicillin antibiotic discs

(a) P. mirabilis (Gram−, ATCC 12453), (b) P. aeruginosa (Gram−, ATCC 10145), (c) Methicillin‐resistant S. aureus (Gram+, ATCC 700699), (d) E. faecalis (Gram+, ATCC 2921), (e) E. coli (Gram−, ATCC 25922), (f) K. pneumoniae (Gram−, ATCC 10031)

3.5 Growth kinetic analysis

The growth curve of the bacteria was analysed in order to determine the efficacy of the TiO₂ nanowires in suspension. Fig. 6 shows the growth curve of S. aureus (Gram+, ATCC 700699), E. faecalis (Gram+, ATCC 2921), K. pneumoniae (Gram−, ATCC 10031), P. mirabilis (Gram−, ATCC 12453), E. coli (Gram−, ATCC 25922) and P. aeruginosa (Gram−, ATCC 10145) treated with TiO₂ nanowires and untreated. The TiO₂ nanowires were added to the broth after ∼3 h for the treated bacteria group, but the untreated bacteria were allowed to grow for 15 h without the addition of TiO₂ nanowires. TiO₂ nanowires provide an increased surface area compared with the TiO₂ nanoparticles. As the surface increases, O₂ concentration in the solution increases that destroys the cell wall of bacterial spores. This is due to the OH⁻ surface functionalisation of TiO₂ nanowires. Also, increased superoxide ion concentrations in the solution react with the carbonyl group in the peptide linkages leading to degradation of the proteins on the bacterial cell wall. A similar effect is also reported in magnesium oxide nanowires [36].

Fig. 6.

Kinetics growth curves of different pathogenic bacterial strains in the absence and the presence of TiO₂ nanowires

It was observed that OD of the treated gram‐bacteria reduced eventually with the progression of incubation time until 15 h, whereas the growth curve for Gram+ bacteria continued to stationary phase at 15 h. The results indicate that the TiO₂ nanowires can be effectively used as an antimicrobial agent against Gram− bacteria.

3.6 Evaluation of bacterial survivability

Adhesion and proliferation assay methods are performed to evaluate the killing efficacy of the TiO₂ nanowires. The CFU was analysed by taking count of the number of individual colonies on the agar plates after 24 h of incubation at 37°C. Fig. 7 shows the CFU for untreated E. coli, E. coli treated with TiO₂, untreated K. pneumoniae and K. pneumoniae treated with TiO₂ nanowires. It was observed that the CFU for E. coli reduced from 1.4 × 10⁵ to 4 × 10³, whereas for K. pneumoniae CFU was reduced from 5.3 × 10⁵ to 1.2 × 10⁵. The data shows that TiO₂ nanowires effectively act as an antimicrobial agent byreducing the number of CFUs.

Fig. 7.

Cell viability of E. coli and K. pneumoniae on treatment with TiO₂ nanowires

3.7 Field ESEM

The FESEM images were captured for E. coli and K. pneumoniae as shown in Fig. 8. Figs. 8 a and c show the images of the untreated E. coli and K. pneumoniae. The images show the lysis of the outer membranes of E. coli in Fig. 8 b and K. pneumoniae in Fig. 8 d, which can be differentiated by the images of untreated E. coli (Fig. 8 a) and K. pneumoniae (Fig. 8 c). Both of the E. coli and K. pneumoniae are Gram− bacteria treated with TiO₂ nanowires showed various degrees of disruptions. Severe disruption was observed for E. coli, whereby the remnant of the degradation loses its rod shape. For K. pneumoniae, the cell wall disruption was observed, and the remnant of the degradation still has the rod shape. A similar observation has been made subjecting bacteria to TiO₂ with the aid of UV radiation [24].

Fig. 8.

FESEM micrograph depicted the size and structure of

(a) E. coli before, (b) After treatment with TiO₂ nanowires; (c) K. pneumoniae before, (d) After treatment with TiO₂ nanowires

4 Conclusion

In this paper, ultrafine TiO₂ nanowires were synthesised using a hydrothermal method using 5 and 10 ml EG, which were annealed at 400°C produced TiO₂ nanowires with the absorbance maximum at a range of 200–350 nm, which corresponds to phase; anatase. The absorbance maximum increased with the increase in the annealing temperatures (400, 500 and 600°C). As for Na titanate nanowires synthesised using 10 ml EG, which were annealed at 400, 500 and 600°C produced TiO₂ nanowires, which have an absorbance maximum at 250, 400 (anatase phase) and 500 nm (transformation to rutile phase), respectively. The absorbance maximum increased with the increase in the annealing temperature. The annealing temperature influences surface morphology of the TiO₂ nanowires, whereby TiO₂ nanowires annealed at 400°C remained as nanowires, and at an annealing temperature of 500°C the agglomerated TiO₂ nanowires formed a mixture of nanoparticles and merged nanowires. High thermal stability of the TiO₂ nanowires revealed by TGA analysis and FT‐IR spectrum of the TiO₂ nanowires showed the presence of the Ti–O–Ti vibrations evidenced due to the TiO₂ lattices. An antibacterial study using TiO₂ nanowires toward E. coli and K. pneumoniae by disc diffusion method showed large zones of inhibition around the disc indicated susceptibility of the microbe toward TiO₂. The P. mirabilis and P. aeruginosa showed a minimal zone of inhibition toward TiO₂ nanowires. The growth kinetic analysis curve showed that addition of the TiO₂ had reduced the OD suggesting the inhibition of growth of the bacteria. Evaluation of bacterial survivability by CFU measurement showed a reduction in CFU counting for E. coli and K. pneumoniae. These antimicrobial test results indicate that the TiO₂ nanowires can be effectively used as an antimicrobial agent against Gram− bacteria. The TiO₂ nanowires could be exploited in the industries such as medical area as a disinfectant, in packaging, detergent formulation and wastewater treatment.