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Article

Research article

J. Microbiol. Biotechnol. 2018; 28(10): 1664-1670

Published online October 28, 2018 https://doi.org/10.4014/jmb.1807.07005

Copyright © The Korean Society for Microbiology and Biotechnology.

TiO2 Nanoparticles from Baker’s Yeast: A Potent Antimicrobial

MMK Peiris 1, TDCP Gunasekara 1, PM Jayaweera 2 and SSN Fernando 1*

1Department of Microbiology, Faculty of Medical Sciences, University of Sri Jayewardenepura, Nugegoda 10250, Sri Lanka, 2Department of Chemistry, Faculty of Applied Sciences, University of Sri Jayewardenepura, Nugegoda 10250, Sri Lanka

Received: July 9, 2018; Accepted: August 20, 2018

Abstract

Titanium dioxide (TiO2) has wide applications in food, cosmetics, pharmaceuticals and
manufacturing due to its many properties such as photocatalytic activity and stability. In this
study, the biosynthesis of TiO2 nanoparticles (NPs) was achieved by using Baker’s yeast. TiO2
NPs were characterized by X-ray Diffraction (XRD), UV-Visible spectroscopy, Scanning
Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Energy Dispersive
X-ray analysis (EDX) studies. The XRD pattern confirmed the formation of pure anatase TiO2
NPs. According to EDX data Ti, O, P and N were the key elements present in the sample. SEM
and TEM revealed that the nanoparticles produced were spherical in shape with an average
size of 6.7 ± 2.2 nm. The photocatalytic activity of TiO2 NPs was studied by monitoring the
degradation of methylene blue dye when treated with TiO2 NPs. TiO2 NPs were found to be
highly photocatalytic comparable to commercially available 21 nm TiO2 NPs. This study is the
first report on antimicrobial study of yeast-mediated TiO2 NPs synthesized using TiCl3.
Antimicrobial activity of TiO2 NPs was greater against selected Gram-positive bacteria and
Candida albicans when compared to Gram-negative bacteria both in the presence or absence of
sunlight exposure. TiO2 NPs expressed a significant effect on microbial growth. The results
indicate the significant physical properties and the impact of yeast-mediated TiO2 NPs as a
novel antimicrobial.

Keywords: Titanium dioxide, Yeast, X-ray diffraction, photocatalytic activity

Introduction

Titanium dioxide (TiO2) nanoparticles (NPs) have a wide range of applications in industry and medicine. TiO2 NPs have unique optical, electronic, photocatalytic and antimicrobial properties [1] which can be exploited towards novel drug delivery systems and also for water purification [2], dye-sensitized solar cells, photonics and food preservation [3]. Three main phases of TiO2 are anatase, brookite and rutile [4].

Synthesis of TiO2 NPs with controlled particle size can be reached using energy-consuming, sophisticated, conventional chemical and physical synthesis methods. However, this approach has intrinsic drawbacks due to high cost and risk to the environment. Comparatively, eco-friendly, cost-effective and non-toxic NP synthesis techniques using microorganisms and plant sources are in demand [5]. This green synthesis has the ability to produce higher yields of NPs under mild laboratory conditions facilitating large-scale production. Microbial TiO2 NP synthesis has been reported using several microorganisms including Bacillus cereus [6], Fusarium oxysporum [7], B. subtilis [8, 9], Aspergillus species [10, 11], B. mycoides [12] and Lactobacilli [13]. Since these are renewable sources, it is environmentally less harmful and generates less amount of toxic waste.

TiO2 NP biosynthesis is thought to arise as a result of the reduction of the metal salts to nanosized metal particles mediated by the enzymatic and chemical interactions. Jha et al. [14] suggested that the negative electro-kinetic potential of microbes can attract cations triggering the series of events leading to biosynthesis of NPs. The presence of reducing agents such as glucose reduces the redox potential thereby enhancing NP formation.

TiO2 NPs have good antimicrobial potential, where they deactivate cellular enzymes and DNA and also form pores in bacterial membranes leading to fluid loss and cell death [15]. This potential is further supported by their photocatalytic activity. When TiO2 NPs are treated with visible light [16] or UV light [12], reactive oxygen species are produced resulting in enhanced microbial killing [17].

Yeast cells (Class Ascomycetes) can act as a template inducing biomineralization [16]. The present investigation was undertaken to synthesize anatase TiO2 NPs by using Baker’s yeast (Y-TiO2) and to characterize their antimicrobial and photocatalytic properties.

Materials and Methods

Biosynthesis of TiO2 NPs

Baker’s yeast 0.8 g was cultured in 77 ml of sterile filtered 5%(w/v) glucose (SISCO, India) solution for 24 h [18]. Three milliliters of TiCl3 solution (TiCl3 12% in HCl, Sigma Aldrich, USA) was added slowly to the 24 h old yeast culture, while stirring until a clear purple solution was observed. Then the solution was allowed to stand at room temperature for three days under dark conditions. The precipitate was centrifuged at 6,000 rpm for 20 min and washed several times using distilled water. Following washing the obtained precipitate was allowed to dry at room temperature. The precipitate was then heated at 250°C, 350°C, 450°C, 550°C, 650°C, and 750°C for 2 h time intervals each to obtain TiO2 NPs as described by Bao et al. [18].

Characterization of Biosynthesized TiO2 NPs

The obtained Y-TiO2 powder was subjected to X-Ray Diffraction (XRD), Energy Dispersive X-ray analysis (EDX) and UV-Visible spectroscopy to confirm the formation of TiO2 NPs. The size and morphology were determined using SEM and TEM.

X-Ray Diffraction (XRD)

XRD analysis was carried out using an X-ray diffractometer (RigakuUltima-IV). The air-dried nanoparticles were coated onto an XRD grid and diffracted intensities were documented from 20 to 90 of 2θ angles with a scan speed of 2°/min at 40 kV and 30 mA.

Energy Dispersive X-Ray Analysis

UV-Visible spectroscopy. Y-TiO2 NP solution was prepared using distilled water. Then 1 ml of the NP solution was diluted with 9 ml of distilled water and UV-Visible spectrum was obtained in the range of 200-800 nm using a PerkinElmer Lambda 35 UV-Visible spectrophotometer. Data obtained were compared with 21 nm TiO2 NPs (Sigma Aldrich, USA).

Scanning electron microscopy (SEM). NP powder was coated onto a coverslip and the morphology of the synthesized NP powder was studied using SEM imaging (ZEISS Sigma Scanning Electron Microscope, 10 kV accelerating voltage). EDX was also obtained (Ametek EDAX Z230, USA).

Transmission electron microscopy (TEM). The NP sample was coated onto a coverslip which was then placed onto a sample stub. TEM imaging (JEOL JEM 2100, Japan) was performed using Drop casting method on a holey carbon-coated Cu grid (Accelerating voltage 200 kV).

Photocatalytic Activity

To study the photocatalytic activity, a modified method was used as described by Periyat et al. [19]. Two mililitres of 25 mg/ml TiO2 NP solution was dispersed in 50 ml of 1 × 10-5 M methylene blue (HiMedia, India) solution. Before UV exposure, the suspension was stirred in the dark for 30 min. Then the suspension was exposed to 150 mJ/s of UV radiation (GS Gene Linker UV Chamber, Bio-Rad). Photodegradation was monitored at 5 min time intervals by withdrawing 3 ml aliquots of TiO2 added methylene blue solution. The aliquots were filtered using 0.22 μm syringe filters and absorbance was taken using a UV-Visible spectrophotometer. Percentage of photodegradation was plotted against exposure time.

Antimicrobial Activity Using the Plate Coating Method

The selected microorganisms Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Candida albicans ATCC 10231, Methicillin resistant Staphylococcus aureus (MRSA) (confirmed clinical strain) and Acinetobacter baumannii (confirmed clinical strain) were obtained from the culture collection at the Department of Microbiology, University of Sri Jayewardenepura, Sri Lanka. According to the method described by Peiris et al. [20], sterile 6 cm petri dishes were coated with (A) Y-TiO2 NPs (2 ml) and (B) bacterial/fungal culture supernatant (2 ml) as the negative control. NP concentration was 20 mg/ml. NP solution was prepared in distilled water and sonicated at 35 kHz for 1 h followed by autoclaving for 15 min at 121°C. The coated plates were allowed to dry completely for 24 h inside a class II biological safety cabinet. Subsequently 2 ml of standardized bacterial suspension (0.5 McFarland standard) was added to each petri dish and incubated for different time intervals (30 min, 45 min, 1 h, 2 h, 3 h, 4 h) at room temperature. To determine the photo-enhanced antimicrobial activity, one set of NP-coated plates containing bacterial cell suspension was exposed to sunlight (test and control) for 30 min and another set of NP-coated plates containing bacterial cell suspension (test and control) was kept in the dark for 30 min. Viable cell counts at different contact time points were obtained by spread plate method. The number of colony forming units per milliliter (CFU/ml) was calculated at each incubation time. All experiments were carried out in triplicate.

Statistical Analysis

The percentage of photodegradation was calculated using the following equation:

Percentage photodegradation=AOAtAO×100

AO is the initial maximum absorbance of methylene blue; At is the absorbance at time t.

The viable counts obtained at different contact times were used to calculate the percentage reduction using the following equation:

Average percentage reduction=CFU.ml in control .CFU.ml in TiO2 NPsCFU.ml in control×100

For graph preparation and statistical analysis, OriginPro 9.0 SR2 software was used.

Results and Discussion

In this study, the yeast Saccharomyces cerevisiae was used successfully to synthesize green anatase TiO2 NPs of less than 12 nm that also showed strong antimicrobial activity. The milky white yeast culture obtained at 24 h was used for adding TiCl3 which resulted in a purple-colored suspension with a pH of 5.4 due to the acidity of TiCl3. A greyish white deposit was observed at the bottom of the flask after 72 h which was used for further purification of TiO2 nanoparticles. Biosynthesis of TiO2 NPs occurs through redox reactions mediated by reducing agents such as glucose [14], alpha amylase [21] and urease [22] in the medium. In this study, we have synthesized Y-TiO2 NPs using the surface of yeast cells as the site for Ti3+ nucleation [18]. The porous surface of yeast cells provides a template where the surface-attached macromolecules and the cells themselves induce TiO2 NP nucleation [16]. Yeast cells stay attached together during nucleation and subsequent treatment at high temperature results in formation of a porous lamellar structure containing TiO2 NPs and inorganic matter [18]. Heat treatment is a critical factor determining the phase of TiO2 NPs. During this process, yeast cells are removed, spilling over CO2 gas and forming a unique lamellar mesophase structure [18].

Characterization of Y-TiO2 NPs

The presence of Y-TiO2 NPs was confirmed using XRD. XRD results indicated predominant formation of crystalline anatase TiO2-NPs having characteristic peak positions at 25.30, 37.78, 48.03, 54.45, 62.80, 70.34, 74.91, and 82.81 degrees. Characteristic peaks for rutile phase were undetected. Narrow peaks indicate the crystalline structure. Prime orientation was the (101) plane as observed at the highest peak position at 25.3 degrees (Fig. 1).

Figure 1. X-ray diffraction pattern of Y-TiO2 NPs. Peaks appear when the X-ray beam has been diffracted by the crystal lattice. Characteristic peaks indicating crystalline structure of Y-TiO2 NPs are shown here.

Fig. 2 demonstrates the detailed EDX analysis of TiO2 NPs showing the surface element composition of Y-TiO2 NPs. The measurements show that the Y-TiO2 NP sample consists of Ti (20.89%), O (70.95), P (5.78%) and N (2.38%) by atomic percentage. According to EDX analysis, other than titanium and oxygen, nitrogen and phosphorous inherited from yeast cells were also detected in the spectrum. Carbon was undetected due to the oven drying process, where organic matter gets destroyed. Chlorine was also undetected showing that no residual chlorine is present in the sample after thorough washing prior to oven drying. Fig. 3 depicts the optical absorption of yeast-mediated TiO2 NPs compared with chemically synthesized 21 nm TiO2 NPs. The absorption spectra of both NPs exhibit strong absorption below 400 nm. The UV-Visible spectrum obtained was comparable to chemically synthesized TiO2 NPs as previously reported [17].

Figure 2. EDX spectrum of Y-TiO2 NPs. For titanium, two K-shell lines are resolved, indicated as Kα and Kβ. This spectrum shows characteristic distribution of major elements in the YTiO2 NP sample.

Figure 3. UV absoprtion spectra for Y-TiO2 NPs and the chemical TiO2 NPs. Both samples have shown a strong absorption below 400 nm.

To study the surface morphology of Y-TiO2 NPs, TEM and SEM techniques were used. Fig. 4 indicates that TiO2 NPs were spherical in shape with rough surfaces and were embedded in a thin lamellar structure (Figs. 4A, 4B, and 4D). Dense dispersion of NPs was evident. A narrow particle size distribution was observed in the range of 3.6–12.0 nm with a mean particle size approximately 6.7 ± 2.2 nm (Fig. 4C). The TEM images of the Y-TiO2 NPs demonstrated a unique lamellar structure as previously reported [18]. This lamellar mesophase occurs when inorganic layers are produced with semi conducting materials such as TiO2 NPs placed in between them [23].

Figure 4. (A, B) TEM images of Y-TiO2 NPs, (C) Particle size distribution of Y-TiO2 NPs (D) SEM image of Y-TiO2 NPs (Scale bars are given). Particle size distribution was 3.6-12.0 nm with the average size of 6.7 ± 2.2 nm.

TiO2 NP nucleation and growth depend on multiple factors relying on physical, chemical or biological conditions employed in the synthesis process [4]. The particle size, shape and photocatalytic activity are important determinants of potent antimicrobial action. This study reports a narrow particle size range of 3.6–12.0 nm as compared to previous reports of TiO2 NPs by He et al. [16] which yielded 10-12 nm particles while 8-35 nm particles were reported from yeast by Jha et al. [14] (10-12 nm) with the smaller-sized particle suggesting strong antimicrobial potential.

The photocatalytic activity of synthesized Y-TiO2 NPs was determined by monitoring the changes in methylene blue concentration using UV-Visible spectroscopy after TiO2 NP treatment and UV exposure. Both the chemically synthesized 21 nm TiO2 NPs and Y-TiO2 NPs decolorized methylene blue dye within 30 min (Table 1). Fifty percent dye reduction was observed within 15 min of exposure for both TiO2 NP samples. This was confirmed by the reduction of the intensity of the characteristic peak position of untreated methylene blue with time (Fig. 5). Initially at 0 min absorbance measurements, there was a slight reduction of absorbance in Y-TiO2-treated methylene blue and a noticeable reduction in 21 nm TiO2 NP-treated dye due to adsorption of NPs into the dye.

Table 1 . Percentage of degradation of methylene blue..

% Degradation

Time interval (min)21-nm TiO2 NPsY-TiO2 NPs
022.71.8
538.124.0
1071.940.2
1581.650.0
2084.864.6
2597.293.3
3099.099.0


Figure 5. Photodegradation of methylene blue dye by (A) Y-TiO2 NPs, (B) Chemical TiO2 NPs, monitored by UV-Visible spectroscopy. Reduction of the peak intensity indicated that both the chemically synthesized (Sigma Aldrich) 21-nm TiO2 NPs and Y-TiO2 NPs decolorized methylene blue dye within 30 min.

In the study on photocatalytic activity, the initial absorption of Y-TiO2 NPs into methylene blue dye was negligible when compared to absorption of 21 nm TiO2 NPs. The photocatalytic property of TiO2 is dependent on the dopant concentration, optical absorption range, phase, size range and number of electrons produced [24-26]. The anatase form of TiO2 reported in this study is known to have maximum photocatalytic activity compared to the rutile and brookite forms of TiO2 NPs [27]. TiO2 is efficient as a photocatalyst due to high rate of mass transfer, strong light absorption and high rate of H2O2 decomposition [16]. TiO2 NPs act as indirect semiconductors and absorb light through direct and indirect electron transitions [28]. According to Diffey [29], on a typical summer day, 6% of UV consists of UVB (290-320 nm) which is responsible for more than 80% of photocatalytic damage and 94% has UVA which contributes to 20% of harmful effect. When exposed to sunlight, electrons are released through band gap forming holes in the valence band and move onto the surface of NPs. The holes react with oxygen forming oxygen radicals where they react with water to produce reactive OH radicals [19, 30, 31]. These reactive oxygen species exert potent antimicrobial activity during their very short life span.

Antimicrobial Activity of TiO2

TiO2 NPs strongly inhibited Gram-positive bacteria and C. albicans when compared to Gram-negative bacteria (E. coli, A. baumannii, and P. aeruginosa). The percentage reduction of colony-forming units (CFU/ml) after exposure to Y-TiO2 NPs following 30 min of sunlight exposure resulted in significant reduction of S. aureus (77%), MRSA (97%), and C. albicans (95%) compared to the control without TiO2 indicating enhanced antimicrobial activity due to the photocatalytic properties of TiO2 following sunlight exposure (Supplementary material). In contrast, the percentage reduction of CFU/ml for gram-negative bacteria P. aeruginosa, E. coli and A. baumannii were 58%, 46%, and 50%, respectively, after exposure to sunlight (Fig. 6A). Y-TiO2 NPs themselves were seen to have antimicrobial activity in the absence of sunlight exposure when incubated under room conditions (Fig. 6B). After 30 min of contact with Y-TiO2 NPs, percentage inhibition of S. aureus (20%), MRSA (25%), C. albicans (74%), P. aeruginosa (30%), E. coli (26%), and A. baumannii (23%) were seen but at a lower percentage than following exposure to sunlight (Supplementary material). TiO2 NPs were also seen to have strong antimicrobial activity against C. albicans in the presence or absence of sunlight suggesting that they have much potential as an anti-Candida agent for future applications.

Figure 6. Colony count after 30 min of contact time (A) with sunlight exposure (B) without sunlight exposure (p values are indicated as follows; “*” for p value between 0.01-0.05, “**” for 0.01-0.001 and “ns” for not significant). Y-TiO2 NPs were more sensitive to Gram-positive bacteria and Candida species (C. albicans) while Gram-negative bacteria were less susceptible to Y-TiO2 NPs with or without sunlight exposure

In this study, Y-TiO2 NPs were found to be more sensitive to Gram-positive bacteria (S. aureus and MRSA) and Candida species (C. albicans) while Gram-negative bacteria (P. aeruginosa, A. baumannii, and E. coli) were found to be less susceptible to Y-TiO2 NPs with or without sunlight exposure. The phase of TiO2 NPs also affects their biocidal properties. Anatase form is reported to show the highest antimicrobial activity [32]. Recent studies suggest several mechanisms of photo-enhanced antimicrobial activity. Reactive oxygen species (ROS) released by UV-irradiated TiO2 NPs attack microbial phospholipids causing cell damage [31]. Direct contact of bacterial cells with TiO2 NPs facilitates ROS contact with microbial cell membranes. According to Singh [9], TiO2 NPs have a positive charge on their surface and microbial cell membranes have a negative charge enhancing attachment. According to Carré et al. [33] TiO2 NPs show high affinity to membrane proteins and alter their structures. Photocatalysis also influences TiO2 NPs to bind with phospho-proteins and phosphor-lipids [34]. Moreover, Ahmad et al. [21] reported that TiO2 NPs invade bacterial cells by damaging the cell membrane in both Gram-negative and positive bacteria causing cell leakage and death. The peptidoglycan layer of Gram-positive bacteria, which is cross-linked by a large number of short peptides, is found to be more sensitive to ROS which explains the higher antimicrobial activity against this group of bacteria.

In conclusion, Y-TiO2 NP synthesis, as reported in this study, is a cost-effective approach yielding pure, highly stable, anatase TiO2 NPs with small particle size. Due to their small size and photocatalytic activity, they have a significant antimicrobial effect on the selected pathogens. The results suggest the possibility of using green TiO2 NPs for future antimicrobial investigation.

Supplemental Materials

Acknowledgments

The authors wish to thank the University of Sri Jayewardenepura, Sri Lanka, for providing financial support (ASP/01/RE/MED/2016/42). KAATSU International University, Sri Lanka, is also acknowledged for providing laboratory facilities.

Conflict of Interest


The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.X-ray diffraction pattern of Y-TiO2 NPs. Peaks appear when the X-ray beam has been diffracted by the crystal lattice. Characteristic peaks indicating crystalline structure of Y-TiO2 NPs are shown here.
Journal of Microbiology and Biotechnology 2018; 28: 1664-1670https://doi.org/10.4014/jmb.1807.07005

Fig 2.

Figure 2.EDX spectrum of Y-TiO2 NPs. For titanium, two K-shell lines are resolved, indicated as Kα and Kβ. This spectrum shows characteristic distribution of major elements in the YTiO2 NP sample.
Journal of Microbiology and Biotechnology 2018; 28: 1664-1670https://doi.org/10.4014/jmb.1807.07005

Fig 3.

Figure 3.UV absoprtion spectra for Y-TiO2 NPs and the chemical TiO2 NPs. Both samples have shown a strong absorption below 400 nm.
Journal of Microbiology and Biotechnology 2018; 28: 1664-1670https://doi.org/10.4014/jmb.1807.07005

Fig 4.

Figure 4.(A, B) TEM images of Y-TiO2 NPs, (C) Particle size distribution of Y-TiO2 NPs (D) SEM image of Y-TiO2 NPs (Scale bars are given). Particle size distribution was 3.6-12.0 nm with the average size of 6.7 ± 2.2 nm.
Journal of Microbiology and Biotechnology 2018; 28: 1664-1670https://doi.org/10.4014/jmb.1807.07005

Fig 5.

Figure 5.Photodegradation of methylene blue dye by (A) Y-TiO2 NPs, (B) Chemical TiO2 NPs, monitored by UV-Visible spectroscopy. Reduction of the peak intensity indicated that both the chemically synthesized (Sigma Aldrich) 21-nm TiO2 NPs and Y-TiO2 NPs decolorized methylene blue dye within 30 min.
Journal of Microbiology and Biotechnology 2018; 28: 1664-1670https://doi.org/10.4014/jmb.1807.07005

Fig 6.

Figure 6.Colony count after 30 min of contact time (A) with sunlight exposure (B) without sunlight exposure (p values are indicated as follows; “*” for p value between 0.01-0.05, “**” for 0.01-0.001 and “ns” for not significant). Y-TiO2 NPs were more sensitive to Gram-positive bacteria and Candida species (C. albicans) while Gram-negative bacteria were less susceptible to Y-TiO2 NPs with or without sunlight exposure
Journal of Microbiology and Biotechnology 2018; 28: 1664-1670https://doi.org/10.4014/jmb.1807.07005

Table 1 . Percentage of degradation of methylene blue..

% Degradation

Time interval (min)21-nm TiO2 NPsY-TiO2 NPs
022.71.8
538.124.0
1071.940.2
1581.650.0
2084.864.6
2597.293.3
3099.099.0

References

  1. Rajakumar G, Rahuman AA, Roopan SM, Khanna VG, Elango G, Kamaraj C, et al. 2004. Fungus-mediated biosynthesis and characterization of TiO2 nanoparticles and their activity against pathogenic bacteria. Spectrochim. Acta A Mol. Biomol. Spectrosc. 91: 23-29.
    CrossRef
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