Growth inhibition of bacterial pathogens by photo-catalyst process of nano-alloys FeCuNi doped TiO2 under ultraviolet irradiation.

This study reports the application of FeCuNi nano-alloy doped TiO2 synthesized via the sol-gel method as an antibacterial with a sterilization rate greater than 95% under ultra-violet (UV) irradiation. The performance was characterized using X-ray diffraction (XRD), thermal analysis (TG-DTA), scanning electron microscope (SEM-EDX), and transmission electron microscope (TEM). The results showed that the sterilization process of FeCuNi-TiO2 in cell suspension of Escherichia coli, Staphylococcus aureus and Bacillus subtilis increased the effectiveness of UV irradiation at wavelength (λ) ≥ 385 nm after 120 min. The optimum growth inhibition of FeCuNi-TiO2 was observed in the concentrations 1.5 g/L of E. coli, 1.5 g/L of S. aureus and 2.0 g/L of B. subtilis. The highest antimicrobial efficiency of FeCuNi-TiO2 powder was provided by a particle size of 16.8 nm, surface area of 70.98 m2/g. The increased antimicrobial activity in multiplied-three doped ions was related to the increase of illumination energy of UV absorption in the photo-catalyst process. The inhibition mechanism reaction of the three species of bacteria cell affects the lipid peroxidation process at the microbe cell's wall. This was indicated by the formation of malondialdehyde (MDA). Lipid oxidation was based on the reaction of 2-thiobarbituric acid (TBARS) as an indicator of primary and secondary oxidation.

Introduction

In the last few decades, titanium dioxide (TiO2) has been used as an alternative catalyst for the sterilization process of several pathogenic bacteria. The photo-catalyst performance of TiO2 is proven to be more effective in degrading several types of organic matter contaminants than that of the conventional method of chlorination [1]. Chlorination is an in efficient process as it can cause environmental problems that require further treatment [2]. When organic matter contaminants decompose in water containing TiO2, the photo-catalyst surface becomes much more effective after irradiation with ultra-bandgap light with UV radiation (λ ≥ 385 nm) [3, 4, 5, 6]. TiO2 photo-catalyst can decompose organic materials such as dyes, peptides and microbes through a series of oxidation processes initiated by the formation of holes (h+) in the valence band and hydroxyl radicals (·OH), while in the conduction band they form radicals (·O2) in oxidized water.

TiO2 photo-catalyst activity is determined by several parameters including crystal structure, surface area, size distribution, porosity and hydroxide density [6, 7]. This performance will affect the electron hole recombination time (e-h+) and the adsorption of organic matter contaminants on the surface of the TiO2 photo-catalyst [8]. TiO2 has three crystal structures namely brookite, rutile, and anatase. The last two crystal structures are thermodynamically more stable [9] with the band gap energy (Eg) of ±3.0–3.2 eV. When compared to the rutile structure, the anatase TiO2 phase structure has more excellent photo-catalytic properties including electron transfer rate that is 89 times greater, chemically and biologically inert, mechanical toughness, low cost and non-toxic [10, 11, 12, 13, 14]. The photocatalytic process requires photon energy. The anatase phase structure (Eg: 3.2 eV) requires UV light energy with λ ≥ 385 nm, which is the energy required to produce illumination energy in the anatase TiO2 photo-catalyst process. The photo-catalytic process is strongly influenced by the electron-hole recombination time (e-h+), whereas the recombination can be extended if the doping process is carried out using transition metal ions on the TiO2 surface [15].

Synthesis of TiO2 has been carried out via different methods such as sol-gel process [16], non-hydrolytic sol-gel route [17], ultrasonic technique [18], chemical vapor deposition [19], microemulsion or reverse micelles and hydrothermal process [20]. High calcination temperatures above 450 °C are usually required to form its crystal structures. Up to the present time, no method has been reported without calcination to produce anatase TiO2 particles [21]. The nano-sized TiO2 particles doped with metal alloys are of great interest for further development because they can increase the photo-catalytic activity of TiO2 [22, 23]. It is important to note that powder obtained synthetically by sol-gel has several advantages including low temperature, simplicity, microstructure morphology with different phase compositions can be obtained by varying parameters such as temperature, pressure, process duration, chemical species concentration, solution concentration and pH [24, 25, 26].

Sterilization is the rate required at cell suspension to inactivate broad-spectrum microbes such as Escherichia coli, Staphylococcus aureus and Bacillus subtilis. The application of TiO2 photo-catalysts to inactivate microbes has been widely reported. Some examples are explained in the following. Using a doped 1% Pd+3 ion on TiO2, it was found that E. coli was inhibited by ± 98% after 2 h of UV radiation [27]. Floating TiO2 photo-catalyst has been used for inactivation of E. coli [28], S. typhimurium [29], and inactivation and inhibition of P. aeruginosa virulence factor expression [30]. TiO2 coated with polystyrene foam has been used for inactivation of E. coli bacteria [31], and antibacterial against A. baumannii [32]. Pt powder doped TiO2 has been used to inactivate L. acidophilus, S. cerevisiae and E. coli, and it was found that TiO2 can be used to replace conventional disinfectant compounds such as chlorination, ozone, and chloride oxides [33, 34, 35, 36].

This paper reports a synthesis of FeCuNi nano-alloy doped TiO2 via the sol-gel method. As has been mentioned previously, dopants from the transition metal group have several advantages because they are the catalysts, low energy levels so that they are easy to capture electrons and hence they can inhibit electron hole recombination [23]. Photo-catalyst activity needs photon energy, one of which from UV irradiation, which has the same energy as the energy gap from TiO2 anatase [37]. Based on this consideration, the FeCuNi doped alloy on TiO2 has antibacterial activity with a higher sterilization rate when synergized with UV irradiation. To study the mechanism in which TiO2 can inactivate microbes is based oxidation process and lipid peroxidation [38, 39], which is usually based on the analysis of refractive index, peroxide value (PV) through the formation of malondialdehyde (MDA) compounds as indicators [40].

Materials and methods

Synthesis of TiO2 doped FeCuNi nanoparticles

The synthesis of FeCuNi doped into TiO2 Nanoparticles consists of several phases, initially by preparation of titania sol using titanium isopropoxide (TIP) as the basic element, then mixed in the isopropanol solvent. Diethanolamine (DEA) was used as the additive with a ratio of 1:2 TIP to DEA. TIP addition was done by using nitrogen gas flow. The sol was homogenized for ±15 min and then acetate salt was added from Fe, Cu and Ni metals with different composition ratios. The total concentration number was 4 % mol to TIP matrix. Sol solution was then homo-genized for ±2 h at room temperature. Then, the sol was oven-heated at 100 °C–110 °C for ±15 h to allow dry gel formation. To obtain FeCuNi–TiO2 powder, dry gel was burned in the furnace at 400 °C–600 °C under nitrogen gas flow of 100 psi for ± 2–3 h to prevent oxidation of metal Fe, Cu and Ni. FeCuNi–TiO2 was characterized using XRD (X’ Port PAN Analytical, Rigaku RINT–2400), SEM-EDX (JEOL JSM 6360 LA), TEM (Philips CM 12 Analysis Docuversion 3.2 image) and TG-DTA (Quantachrome, Serial 1089111903. Model: AS-68).

Cell suspension preparation

In this experiment, E. coli (Gram −), S. aureus and B. subtilis (Gram +) were used as models. Nutrient Broth (NB) was used as a media for bacterial culture stock preparation. Pure bacterial culture of dense slant gelatin Nutrient Agar (NA) of 24–48 h was inoculated into the NB as sterile liquid medium. Aerobic base was incubated inside a rotary shaker for 24 h, at 37 °C and 120 rpm speed. Then, cell production was continued in a medium in the same condition. Cells were harvested after 8 h of incubation process and then centrifuged at 8000 rpm, for 15 min. Cell sedimentation was rinsed with sterile aquadest twice repeatedly and then centrifuged again at 8000 rpm for 15 min. The sedimentation was given cell suspension by adding phosphate buffer (pH: 7.0) at 1:10 ratio. Cell suspension preparation for photo-catalyst reaction samples was made by dilution treatment using sterile phosphate buffer up to 103–106 cell/mL cell concentration.

Diffusion media preparation

To determine the inhibition power of FeCuNi–TiO2 against the growth of bacteria, a diffusion medium which consisted of NA media for bacteria was prepared. ± 15 mL NA was poured into a Petri dish after the medium was frozen, then the surface of the medium was lubricated evenly by the following: bacterial cell suspension with 105 cell/mL cell concentration, at volume of 0.1 mL. A stainless steel cup was used for the addition of 15 mg of FeCuNi–TiO2. Incubation was performed inside a chamber with a vertical lamp radiating UV (λ ≤ 385 nm) at temperature of 37 °C for 24 h. The intensity of UV radiation was monitored by a detector (Blue Light Safety Detector UV) with intensity set at 3.25 mW/cm2. The results of the inhibition zone diameter were measured in millimeters. The processing was done in an aerobic manner, duplo and aseptic. Controlling was done without addition of FeCuNi–TiO2. Using the diffusion method, inhibition efficiency was determined as the optimum sensitivity boundary of FeCuNi–TiO2 to bacteria cells by adding 0–3.5 g/L of FeCuNi–TiO2 powder. Additionally, the inhibition zone was used as a preparation sample to examine the physical injury of microbes with the use of SEM. Sample preparation was done by applying the freeze drying method. Inhibition zones resulting from FeCuNi–TiO2 in bacteria were called the outer inhibition zone and inner inhibition zone, whereas the growing zone is used as a control. These parts were cut into 5 × 5 mm size, and were steamed with 2 % osmium tetroxide (OsO4). The sample was then dipped into liquid nitrogen steam at −210 °C and placed into the freeze dryer (Emitech K 750) for ±10 h. The sample was then coated in gold plating to the size of 5–10 mm and then monitored by SEM.

Colony plate count agar (PCA)

A total of 0.1 mL bacteria cell supernatant was transferred into NA media inside a petri-dish which was then lubricated evenly (spread plate) on the surface of the media, and incubated at 37 °C for 24 h. After 24 h of incubation, the growth of the colony was examined and counted with a colony counter equipment. The count was then converted using the following Eq. (1) which results in a percentage value reported.

Bacteria cell sample from photo-catalyst was used to determine the malondialdehyde (MDA). A 2 mL sample was transferred into a test tube, added with 4 mL of 10% trichloroacetic acid (TCA), then homogenized and centrifuged at a speed of 11,000 rpm for 45 min. 6 mL of 0.67% 2-Thiobarbituric acid (TBA) was added into supernatant, incubated for 30 min in a hot water bath, then cooled down in an iced cup for 30 min. Next, it was re-centrifuged at speed of 11,000 rpm for 45 min. Supernatant was used in absorbance measurement using spectrophotometric at λ ≤ 400 nm.

Photo-catalyst reaction media preparation

The preparation of photo-catalyst media were transferred into 1 mL a beaker glass, with the initial bacteria cell suspension of 103–106 cell/mL, and into 9 ml of sterile NB media and 15 mg of FeCuNi–TiO2 powder. The variations in conditions were: Varied radiation system of UV λ ≤ 365 nm and without UV radiation, incubation time of 30–210 min, and 0–3.5 g/l of FeCuNi–TiO2 concentration. As a control, another experiment was performed without added FeCuNi–TiO2 powder. Then, the mixture of photo-catalyst reaction was stirred in a magnetic stirrer or sonicator (50 kHz ultrasonic wave frequency). Radiation intensity was vertically controlled by putting a beaker glass surface in a 30 cm distance from the radiation source. The intensity was monitored by using a detector (Blue Light Safety Detector UV) of 3.25 mW/cm2. The process was done in an aerobic duplo and aseptic manner. The inhibition percentage of bacteria was quantitatively determined by applying 2 measurement methods namely plate count agar (PCA) that is based on the calculation of the number of colonies and TBARs that is based on the number of MDA product formation as a result of peroxide lipid.

Results and discussion

The XRD patterns of FeCuNi–TiO2 powder synthesized using varying calcination temperatures (400 °C, 500 °C, and 600 °C) are presented in Figure 1. All the XRD patterns are indexed according to an anatase TiO2 standard diffraction pattern with the tetragonal I41/amd space group (ICSD-154604) and rutile TiO2 with the tetragonal P42/mnm space group (ICSD-97277). All three XRD patterns matched well with the standard XRD of the TiO2 phase without any additional peaks, confirming the formation of single-phase products. No peaks corresponding to oxides of each Fe, Cu, and Ni metals we reobserved in the doped TiO2 samples, which thus demonstrates that the substitution of all metals in TiO2 host lattice was successful.

Figure 1

XRD Pattern of FeCuNi–TiO2 powders prepared by the sol-gel method with different calcination temperatures.

At calcination temperatures of 400 °C and 500 °C, the observed XRD peaks at 2θ = 24.8°, 37.3°, 47.4°, 53.6°, 54.7°, 62.1° corresponding to reflection planes (101), (004), (200), (105), (211), (204) confirmed the formation of single anatase phase of TiO2. When the calcination temperature was raised to 600 °C, the major peaks characteristic of the rutile phase peaks shown at 2θ: 27.4°, 35.7°, and 40.9° appeared, as highlighted in Figure 1. This suggests that a phase transition from anatase to rutile initially occurred around 600 °C, which agrees with previous reports. It was revealed that the calcination process at 600 °C initially led to the structural transformation from anatase to rutile (A → R). Higher temperatures can cause all crystal position turns to defect crystal wherein the cutting-off in M-TiO2 atoms occurs. Consequently, the FeCuNi–TiO2 structure experienced restructuring and a transformation occurred on the structure. Also, the structure of anatase becomes unstable thermodynamically at high temperatures which causes the anatase particles to stick together to form larger particles and the interface of the anatase particles will become the rutile phase nucleation, resulting in the transformation from anatase to rutile phase [41, 42]. The formation of both anatase and rutile phases was further confirmed by the refinement analysis discussed below.

It was also noticeable that the XRD peaks became sharper as the calcination temperature was increased, indicating an increase in crystallinity. Using the full width at half maximum value (FWHM), the crystallite size of the particles was estimated using the Debye–Scherrer’s equation [43]. The average crystallite size was approximately 13.9 nm, 16.8 nm, and 20.2 nm, which increased with increasing calcination temperature. It is expected that the calcination temperature plays a crucial role to accelerate the crystal growth, leading to an increase in larger crystallite size and rising intensity of the anatase phase.

XRD data were then refined using the Le Bail refinement technique using Rietica software [27] to determine the phase formations and crystal structure in detail. The initial refinements considered the structural parameters of anatase TiO2 with a tetragonal I41/amd space group (a = b = 3.7862 Å, c = 9.4951 Å; α = β = γ = 90°) (ICSD-154604). All structural parameters were then automatically refined to obtain the best fits between the refinement patterns and optimize the value of reliability factors (R, R, and χ2). Figure 2 shows the Le Bail fits of the XRD patterns of FeCuNi–TiO2 samples. For sample calcined at 400 °C and 500 °C, the refinement was done with a single-parameter system, since the XRD peaks only show the presence of a single anatase phase. The profile plots in Figure 2a and b show good fits between experimental and calculated patterns for both samples and all peaks matched well with the Bragg reflection of the anatase phases, indicating the existence of both phases. The refinement results confirm that the synthesized FeCuNi–TiO2 samples at 400 °C and 500 °C were a single phase of anatase TiO2 without any formation from rutile or brookite phases, which adopts a tetragonal symmetry with a I41/amd space group.

Figure 2

Le Bail fits of the XRD patterns of FeCuNi–TiO2 samples, (a) 400 °C; (b) 500 °C; (c) 600 °C.

Considering the formation of mixed anatase and rutile phase in the sample with a calcination temperature of 600 °C (as highlighted in Figure 2), we therefore refined the XRD data using the multiphase refinement system. Refinement was done accordingly using the parameter of anatase TiO2 phase as the major phase and added parameter of rutile TiO2 with a tetragonal P42/mnm space group (a = b = 4.6257 Å, c = 2.9806 Å; α = β = γ = 90°) as the secondary phase. As a result, the profile of refinement plots displayed in Figure 2c shows a good fit of all XRD patterns and provides clear evidence for the formation of mixed-phase of anatase and rutile TiO2, according to the Bragg reflection of each phase. The phase fractions obtained from the refinement data were approximately 82.3% for the anatase phase and 17.7% for the rutile phase.

The refined lattice parameters and unit cell volumes are shown in Table 1. All lattice parameters essentially increased as the calcining temperature increased, leading to an increase in cell volume. As expected, the increase in crystal volume can be correlated to the increased crystallite size occurring due to varying calcination temperatures. The appropriate value of reliability factors (R, R, and χ2) justified the accuracy of refinement results. Since the focus of this study was on the stable anatase TiO2 for higher photo-catalytic activity than that of rutile or mixed phases, the sample calcined at a temperature of 500 °C exhibiting pure anatase phase and higher crystallinity was chosen for subsequent analysis.

Table 1

Refined structural parameters of FeCuNi–TiO2 sample obtained from XRD.

Calcination temp.	FeCuNi–TiO2
	400 °C	500 °C	600 °C
Space Group	I41/amd	I41/amd	I41/amd	P42/mnm
Cryst Struct	Tetragonal	Tetragonal	Tetragonal	Tetragonal
Phase	Anatase	Anatase	Anatase	Rutile
a(Å)	3.7750(7)	3.7819(8)	3.7946(5)	4.6082(6)
b(Å)	3.7750(7)	3.7819(8)	3.7946(5)	4.6082(6)
c(Å)	9.4563(6)	9.4775(3)	9.5317(6)	2.9742(7)
V(Å3)	134.764(3)	135.562(1)	137.252(2)	63.162(4)
Z	4	4	4	2
Rp(%)	4.14	3.40	3.88
Rwp(%)	5.68	4.89	5.22
χ2	1.217	1.101	1.168

TG-DTA analysis of FeCuNi–TiO2

Thermal analysis of nanoalloys FeCuNi–TiO2 under nitrogen atmosphere was performed to study the effect of mass reduction with the increasing temperature. The mass reduction from the TG analysis and the DTA pattern displayed the effects of temperature on the change of the nano structural phase of TiO2 [44]. The reduction in mass at certain temperatures resulted in a change in the phase structure of FeCuNi–TiO2 into two structural phases, namely the anatase phase and the rutile phase. This can be understood since, thermodynamically, the rutile phase is formed at temperatures above 500 °C and the rutile structure is more stable than the anatase phase [45].

Figure 3 shows the TG-DTA FeCuNi–TiO2 pattern, where there are four exothermic patterns that fluctuate in the temperature range of 200 °C to 500 °C. The first pattern in the temperature range of 25 °C–200 °C indicates a reduction in mass of FeCuNi–TiO2 due to the release of water or organic solvents from the precursor and additive mixtures used in the synthesis process. The second stage at a temperature of 300 °C, in which in this condition the mass reduction is greater, indicates degradation of the organic residue. The exothermic pattern at a temperature of 300 °C–400 °C shows crystal growth and a transformation of the FeCuNi–TiO2, phase in the anatase phase structure. At temperatures ≥500 °C, there is a transformation of the anatase structure pattern to the rutile phase structure with greater weight loss and stability at high temperatures [46].

Figure 3

TG-DTA Pattern of Mass stability FeCuNi–TiO2 at different temperature.

SEM-EDX analysis of FeCuNi–TiO2

The SEM pattern of FeCuNi–TiO2 resulting from calcinations at 500 °C is shown in Figure 4a. Each produced a rough surface like that of a piece of rocky stone where ion dopant particles were distributed evenly and homogeneously on the surface of FeCuNi–TiO2 with different sizes. FeCuNi–TiO2 powder surface shown by SEM indicated similarities. However, using EDX measurements, their different chemical compositions were identified: FeCuNi–TiO2 1:2:1, 97.06 % at 4.5 keV (Figure 4b).

Figure 4

SEM (a)-EDX (b) morphology of FeCuNi–TiO2 (1:2:1) at 500 °C.

TEM characterization of FeCuNi–TiO2

The TEM pattern from the FeCuNi–TiO2 powder is shown in Figure 5a. It is observed that the highest photo-catalytic activity occurs in FeCuNi composition with a 1:2:1 ratio. This FeCuNi–TiO2 nanoparticles form three-dimensional crystals that are regularly structured in a spherical shape. The result of TEM measurement shows that most FeCuNi–TiO2 particle sizes are of 10–15.7 nm in size (Figure 5b). The particles are distributed evenly as much as 45%. The results of the TEM measurement on particle size leads to the correlation with the particle size in Debye-Scherrer formula (Eq. (1)).

Figure 5

(a) TEM the form of FeCuNi–TiO2 particle; (b) The percentage of particle distribution at temperature of 500 °C.

Bioactivity examination of FeCuNi–TiO2

The free radical ·OH attack from the process of electron-hole photo-generation of FeCuNi TiO2 powder at the bacteria cell partition can be indicated as Malondialdehyde compound (MDA) formation. MDA is the final product from the result of an oxide saturated process in the cell membrane. Figure 10 shows the numbers of MDA product which were formed from a series of ·OH radical attacking process in the bacteria cell partition that was determined by applying the TBARs method [47].

Figure 10

MDA Product (Malondialdehyde) from interaction FeCuNi–TiO2 with E. Coli, S. aureus, B. Subtilis.

When TiO2 nano particles exist in a medium containing moisture or water and then receive UV radiation at appropriate wavelength with energy as needed by TiO2 semiconductor, electron-hole photo-generation will occur that produces free hydroxyl radicals ·OH. The ·OH radical is extremely effective as a toxic compound which kills microorganisms. When the ·OH radical interacts with a microbe cell wall, the DNA chromosome of the microbe will develop a thymine dimer that allows knots among thymine base inside the similar DNA strand. This thymine dimer will obstruct the formation of the double helix and disturbs the normal replication of DNA. Cell growth is obstructed and eventually leads to cell death. The inhibition of FeCuNi–TiO2 photo-catalyst at microbe can be explained by the attack from O2 radicals and ·OH of photo-generated electron holes on the catalyst surface. Among the three types of species, ·OH radical is the most reactive because it has very effective oxidation capabilities for various kinds of organic compounds, such as microbe cells [47].

The doping process can increase photo-catalytic activity of FeCuNi–TiO2. Doping can stimulate free radical formation with a high hydroxyl series density through a redox reaction on the FeCuNi–TiO2 surface. Doping of transition metal ions which have been multiplied by three can significantly increase the photo-biocatalytic activity in inhibiting microbes. Based on this consideration, the FeCuNi doped alloy on TiO2 has antibacterial activity with a higher sterilization rate when synergized with UV irradiation. The mechanism of TiO2 inactivation against microbes can be studied from oxidation process and lipid peroxidation. Lipid oxidation is usually based on the analysis of refractive index, peroxide value (PV) through the formation of malondialdehyde (MDA) compounds and 2-thiobarbituric acid reactive substances (TBARS) as indicators, as shown in Figure 6 [38, 39, 40].

Figure 6

Schematic illustration of photo-catalytic process of FeCuNi doped TiO2 for growth inhibition of bacterial cell with oxidation of lipids and formation of malondialdehyde.

FeCuNi doped by multiple of three is more effective against bacteria E. coli (+++), S. aureus (++) and B. subtilis (+). E. coli, S. aureus, B. subtilis microbes that were chosen as models for the examination of microbe inhibition of FeCuNi–TiO2. These are pathogens in nature when they interact with humans directly or indirectly. E. coli is a negative gram bacterium, having a thinner layer of peptidoglycan cell wall when compared to positive gram bacteria such as S. aureus, and B. Subtilis [46]. Figure 7b shows the inhibition power of FeCuNi–TiO2 at E. coli greater than S. aureus greater than B. Subtilis.

Figure 7

Antimicrobial activity of FeCuNi–TiO2 at bacteria E. coli and without FeCuNi–TiO2 as control (a), photography zone inhibition of E. coli and control (x) and (b) colony counter of E. coli and control (y) with UV radiation for 120 h.

Microbe cells which were given inhibition treatment with FeCuNi–TiO2 powder that have a wide inhibition zone was used to examine the effect of FeCuNi–TiO2, whereas the zone of microbe growth without the FeCuNi–TiO2 treatment was used as a control (Figure 7b). The reaction effect of photo-biocatalyst was observed based on the interaction between bacteria cells and FeCuNi–TiO2 that were blended continuously and each bacterium was given vertical UV radiation as a function of time. As a result of photo-biocatalytic reaction, a number of bacteria cells died off, thus causing the reduction in the number of initial cells by 104 to 105 cell/mL for each bacterium. This germicide action was examined by Upreti, et al (2018), to inhibit the E. coli bacteria inside Luria Bertani culture which was radiated by UV and nanoparticle composite Nd+3 doped TiO2. A longer time of UV radiation caused a reduction in concentration of E. coli bacteria [48].

The effectiveness of FeCuNi–TiO2 powder as an antimicrobial is determined by the performance of FeCuNi–TiO2 photo-catalyst. In these pictures (Figure 7), it can be seen that the inhibition response of FeCuNi–TiO2 to the three bacteria species as a function of time can reduce the number of bacteria colonies following the extension of inhibition time. Using Eq. (1), inhibition percentage was counted based on the reduction of the number of initial colonies at 0 h and at the end of photo-catalyst reaction time between bacteria cells and FeCuNi–TiO2 powder. Figure 7b indicates the zone inhibition of E. coli bacteria cells and the control. Species as the function of time can reduce the number of bacteria colonies following the extension of inhibition time. Using Eq. (1), the inhibition percentage was counted based on the reduction of the number of initial colonies at 0 h and at the end of the photo-catalyst reaction time between bacteria cell and FeCuNi–TiO2 powder.

The inhibition percentage of bacteria cells by UV radiation was analyzed based on measurements of cell turbidity during UV radiation at 120 min Figures 8 and 9 display physical changes of microbe cells as a result of FeCuNi–TiO2 powder and UV radiation as observed by SEM to ensure the effect of these treatments on bacterial cell damage. Figure 8a, b and c shows the physical appearance pattern of SEM: E. coli; S. aureus; B. subtilis before inhibition by FeCuNi–TiO2 powder, and Figure 8d and e shows the physical appearance pattern of SEM: E. coli; S. aureus; B. subtilis before inhibition by FeCuNi–TiO2 powder. E. coli is 61.6% in Figure 9a, S. aureus is 52.8%, in Figure 9b and B. subtilis is 34.3% in Figure 9c. Without the addition of FeCuNi–TiO2 and UV radiation, the inhibition percentage for cells are as follows: E. coli is 12.2 %, S. aureus is 8.8 % and B. subtilis is 6.5%. Potential synergy was found when FeCuNi–TiO2 was combined with UV radiation in the application of stirring system, where by an increase in photo-biocatalyst activity occurred for each bacterium: E. coli 96.4%, S. aureus 92.8% and B. subtilis 78.3%.

Figure 8

The physical appearance pattern of SEM: (a) E. coli; (b) S. aureus; (c) B. subtilis before inhibition by FeCuNi–TiO2 powder, and (d) E. coli; (e) S. aureus; f) B. subtilis after inhibition by FeCuNi–TiO2 powder.

Figure 9

The inhibition response of FeCuNi–TiO2 to varied Concentration vs UV irradiation time for (a) E. coli; (b) S. Aureus; and (c) B. subtilis.

When mechanism treatment was applied by means of ultrasonic wave from sonicator at 50 kHz frequency, there occurred an increase in inhibition efficiency by 6% for 120 min application time. The optimum inhibition efficiency of FeCuNi–TiO2 in each bacterium was noted as follows: E. coli 1.0 g/L, S. aureus 1.5 g/L and B. subtilis 1.5 g/L. This inhibition efficiency was determined by applying Diffusion Method (spread plate) based on the calculation of colony.

The free radical ·OH attack from the process of electron-hole photo-generation of FeCuNi–TiO2 powder at the bacteria cell partition can be indicated as Malondialdehyde compound (MDA) formation. MDA is the final product from the result of an oxide saturated process in the cell membrane. Figure 10 shows the numbers of MDA product which were formed from a series of ·OH radical attacking process in the bacteria cell partition that was determined by applying the TBARs method [49].

Conclusion

In this work, the performance of TiO2 as an antimicrobial agent has been increased via structural modification and particle size using dopant FeCuNi with a ratio of 1:2:1. The performance of FeCuNi–TiO2 is related to the inhibition efficiency improvement against bacteria in the concentration range of E. coli: 1.5 g/L, S. aureus: 1.5 g/L, B. subtilis: 2.0 g/L. The FeCuNi–TiO2 with particle size of 16.8 nm and surface area of 70.98 m2/g provided more effective inhibition activity based on the measurement of the inhibition zone using diffusion method. The inhibition activity from the highest to the lowest is for E. coli followed by S. aureus and B. Subtilis. The photo-catalytic activity of FeCuNi–TiO2 powder as the antimicrobial agent was more effective when it was irradiated using UV with λ = 365 nm, which provided an inhibition percentage in the range of 78.2%–96.4%. The final product from the series of chemical inhibition processes indicated the formation of MDA product from a series of ·OH free radical attacking process in the bacteria cell partition that was determined by TBARs method.

Declarations

Author contribution statement

Yetria Rilda: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Syukri Arief: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.

Anthoni Agustien: Performed the experiments

Eti Yerizel, Nofrijon Sofyan: analyzed and interpreted the data, analysis tools or data.

Hilfi Pardi: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Funding statement

This work was supported by (T/9/UN.16.17/PT.01.03/IS-RPBQ/2022, 7 Juli 2022).

Data availability statement

Data included in article/supplementary material/referenced in article.

Declaration of interests statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.