Catalytic degradation of methylene blue by biosynthesized Au nanoparticles on titanium dioxide (Au@TiO2)

The degradation of methylene blue is a critical procedure in its wastewater remediation and thus has inspired wide catalysis research with semiconductors such as titanium dioxide (TiO2) and rare metals such as gold (Au). In this study, we report bacterial cells assisting biosynthesis for Au@TiO2 as an efficient catalyst for the catalytic degradation of methylene blue. Multiple complementary characterization for bio-Aux@TiO2 evidenced the evenly distributed Au nanoparticles (NPs) on the bio-TiO2 layers. Meanwhile, bio-Au2@TiO2 displayed the superior catalytic activity in the degradation of methylene blue with the highest kinetics constant (kapp) value of 0.195 min−1. In addition, bio-Au2@TiO2 keeps stable catalytic activity for up to 10 cycles. The origin of the catalytic activity was investigated by the hydroxyl radical fluorescence quantitative analysis and optical band gap analysis. In the bio-Au2@TiO2 catalytic system, Au NPs decreased the band gap energy of TiO2 and enabled the generation of abundant photogeneration hydroxyl radicals, resulting in an enhanced photocatalytic activity. Our microbial synthesized bio-TiO2 and bio-Aux@TiO2 study would be useful for developing green synthesis catalyst technology.


Introduction
The elimination of organic pollutants from wastewater is a major challenge for the ecosystem. (Cai et al. 2022;Lee et al. 2016). Due to their synthetic origin and aromatic nature, azo dyes are stable, nonbiodegradable, and carcinogenic. (Routoula and Patwardhan 2020) Even microquantities of these dyes lead to acute toxicity in aquatic life and thus pose a health risk to human, vis., the food chain. (de Luna et al. 2014;Mittal 2020) As a cationic dye, methylene blue (MB) is used broadly in the textile industry for dyeing cloth, cotton, and silk, and as a pollutant, it can cause nausea, vomiting, diarrhea, and eye burning sensation (Moosavi et al. 2019). The removal dynamics and mechanisms of MB using chemical and physical technologies have been discussed with drawbacks of the generation of iron sludge, secondary pollution of adsorbents, membrane fouling, and high cost (Bedin et al. 2016;Wolski and Ziolek 2018;Mittal 2021). In this case, the photocatalytic degradation of methylene blue is an efficient and feasible treatment method for wastewater (Miranda et al. 2015;Saeed et al. 2022;Gupta et al. 2012).
Titanium dioxide (TiO 2 ) is widely applied in environmental remediation owing to its non-toxicity, chemical stability, high photocatalytic activity, and recyclability (Chong et al. 2010;Forgacs et al. 2004;Jain et al. 2007). TiO 2 materials were most commonly used photo-catalyst and have been successfully employed in the degradation of organic contaminants (Chen et al. 2010;Pelaez et al. 2012;Gupta et al. 2007). However, TiO 2 can only be excited in the UV region (200-400 nm) because of its wide band gap (3.0~3.2 eV) (Chen and Mao 2007). Photogenerated electron-hole recombination rate of pristine TiO 2 was high, which resulted in decreased catalytic activity. It was found that loading noble metal nanoparticles (silver, gold, platinum, and palladium) with surface plasmon resonance effect could effectively Yanan Wang and Tieliang Zhang contributed equally to this work.
Responsible Editor: George Z. Kyzas * Wenjing Liu liuwenjing@caas.cn enhance the optical absorption and accelerate the interfacial charge transfer (Kochuveedu et al. 2013;Sarina et al. 2013;Zhang et al. 2012). Au NPs are one of the most promising and widely used materials in a variety of heterogeneous catalytic reactions (Liu et al. 2018b). The several applications of Au/TiO 2 nanocomposite have been reported in the photocatalytic removal and degradation of dyes, including methylene blue, orange 16, rhodamine-B, and tartrazine under visible or UV light (Angthararuk et al. 2015;Ayati et al. 2014). A laser flash photolysis study of Au-capped TiO 2 demonstrated an increase of 40% in hole transfer efficiency (Dawson and Kamat 2001). It has been observed that the photocatalytic activity of Au/TiO 2 materials was higher than that of Au nanoparticles or pure TiO 2 materials (Liu et al. 2013).
Hence, it has attracted great attention to synthesize Au@ TiO 2 nanocomposite materials with outstanding catalytic properties (Alsaidi et al. 2022;Green et al. 2014). Due to its low-cost, eco-friendliness, and mild synthetic environment, the microorganism-assisted green synthesis is considered as an alternative to synthesizing composite materials (Bhargava et al. 2016;Dahl et al. 2007;Nasrollahzadeh and Sajadi 2016;Nasrollahzadeh et al. 2020;Singh et al. 2016). Several microorganisms, notably bacteria, have been investigated for synthesizing Au NPs (Reith et al. 2006;Southam et al. 2009). Previous studies have shown that Pantoea sp. IMH, a strain that has multiple metal resistance genes (Liu et al. 2018b), was able to reduce Au(III) to form Au NPs (Liu et al. 2018a). Consequently, earlier results inspired us to synthesize Au@TiO 2 nano-catalyst by employing biomineralized Au NPs.
The objectives of this study were to propose a green synthesis method for Au@TiO 2 using bacterial strain IMH and to employ in the catalytic degradation of methylene blue. Multiple complementary methods were utilized to characterize the biosynthesized Au@TiO 2 . In addition, hydroxyl radicals formed in the degradation reaction and optical band gap of Au@TiO 2 materials were quantitatively analyzed to explore the catalytic mechanism of Au@TiO 2 . The insights obtained from our work enhance the deep understanding on the microbial synthesis and mechanism of TiO 2 -based environmental technologies.

Bacterial strains and culture conditions
Pantoea sp. IMH (JX861130) was cultured in Luria−Bertani (LB) broth at 30 °C. Bacterial cells were incubated to late-exponential phase at 160 rpm for 8 h on a rotary shaker. Cells were harvested by centrifugation at 4000 g for 5 min and were washed three times with phosphate-buffered saline (PBS buffer). Then the harvested cells were re-suspended and incubated in PBS buffer.

Synthesis of bio-TiO 2 and bio-Au x @TiO 2
A 3 mM and 100 mL TiO(OH) 2 solution was prepared and stirred in a water bath (60 °C, 1 h). After the TiO(OH) 2 solution was cooled to room temperature, the above cell suspension (10 mL) was mixed with TiO(OH) 2 solution at 30 °C for 3 h. The resulting bacterial suspension was centrifuged at 8000 g for 10 min and washed thoroughly with sterile water. The obtained precipitated solid substance was dried in vacuum (60 °C, 2 h) and then calcined in a tube furnace at 400 °C for 2 h to get TiO 2 (namely, Bio-TiO 2 ). Meanwhile, partial reaction production suspension without drying treatment was used in the next step. In addition, commercial titanium dioxide (TiO 2 , P25) particles as control sample were obtained from Beijing Baoruyi Biotechnology Co., Ltd (China).
The above TiO 2 suspension was further mixed with the cell suspension (10 mL). Then, Au(III) was added to a final concentration of 0.5 mM, 0.75 mM, 1 mM, and 2 mM, respectively. The mixtures were incubated at 30 °C for 5 h. Then, the resulting suspension was centrifuged at 8000 rpm for 10 min, and the precipitate was washed with sterile water. And the products was dried (60 °C, 2 h) and then calcined (400 °C, 2 h) to get Au/TiO 2 (namely, Bio-Au x @TiO 2 , where x is the final concentration of Au(III) in the solution).

Characterization
X-ray diffraction (XRD) technology was used to analyze the crystal structure of the obtained materials with X-ray powder diffractometer (Rigaku D/Max2500PC). The powder samples were mounted on aluminum stubs using a doublesticky tape and analyzed with field-emission scanning electron microscopy (FE-SEM) with an Oxford energy dispersive X-ray spectroscopy (EDS) analyzer (Hitachi SU 8020). The high-resolution transmission electron microscopy (HR-TEM) and EDX elemental mapping of the samples were carried out with the transmission electron microscope (FEI Tecnai G 2 F20). X-ray photoelectron spectroscopy (XPS) spectra were obtained on the X-ray photoelectron spectrometer (Thermo Scientific ESCALab 250Xi). Infrared spectra (IR) were collected by a FTIR spectrometer (Thermo-Nicolet Nexus 6700) using 256 scans and a 4 cm −1 spectral resolution.

Catalytic degradation of methylene blue
The catalytic degradation of methylene blue experiments was conducted in an Erlenmeyer flask with 0.0375 mM methylene blue in a suspension (50 mL) containing 30 mg bio-TiO 2 and bio-Au x @TiO 2 . The samples were firstly stirred in the dark for 3 h to achieve adsorption equilibrium. Then A mercury UV lamp (CEL-M500, wavelength 275 nm) was used as the light source. UV-Vis spectra of the reaction solutions at different times were acquired using a UV-2450 spectrophotometer (Shimadzu, Japan) to record the catalytic degradation progress.

Fluorescence analysis
The measurement of OH radical was detected by the fluorescence probe (terephthalic acid) method on a FLS-1000 fluorescence spectrometer (Edingurgh Instruments, UK) with an excitation wavelength at 312 nm (Nakabayashi and Nosaka 2015). Terephthalic acid (TA) could readily react with OH radical to form 2-hydroxy terephthalic acid (TAOH), which emits a fluorescence light at 426 nm. Before irradiation, the 50 mL mixed suspension including 1 mM TA and 30 mg of bio-TiO 2 /bio-Au x @TiO 2 was magnetically stirred in the dark for 3 h to establish the adsorption-desorption equilibrium. The light irradiation and sampling conditions were the same as those in catalytic degradation experiments.

Material characterization
The morphology and structure of bio-TiO 2 and bio-Au x @ TiO 2 materials synthesized by the IMH strain were characterized using multiple complementary techniques. XRD spectra (Fig. 1) (Tran et al. 2022). And other major peaks from bio-Au x @ TiO 2 spectra corresponded to the planes of standard anatase (JCPDS-01-071-1168) (Nassar et al. 2017). In addition, the peak positions of TiO 2 did not shift from bio-TiO 2 to bio-Au x @TiO 2 , indicating that bio-TiO 2 still kept the anatase crystalline phase during the deposition of Au NPs. XRD spectra results demonstrated that the IMH strain could successfully synthesize bio-TiO 2 and bio-Au x @TiO 2 materials. The FE-SEM (Fig. 2a) micrograph and EDX spectrum showed that bio-TiO 2 consists of aggregated irregular spheres with diameters of about 50-100 nm. The SEM images (Fig. 2b-e) of bio-Au x @TiO 2 exhibited the Au NPs as bright dots were dispersed on the surface of bio-TiO 2 support. Meanwhile, the Au NPs of bio-Au 0.5 @TiO 2 and bio-Au 0.75 @TiO 2 were spheres with particle size of 5~10 nm; the diameter of Au NPs in bio-Au 1 @TiO 2 was mainly 10 nm; and the Au NPs of bio-Au 2 @TiO 2 were spheres with a diameter of 5~15 nm. With the increase of concentration of Au ions in cell solution, the quantity and dimension of biosynthesized Au NPs notably increased, and the nanoparticle distribution tends to be even.
HR-TEM in Fig. S1 showed that Au NPs (5-20 nm) were evenly dispersed in the bio-TiO 2 layers. The EDS elemental mapping in Fig. S1c has shown that the bio-Au 2 @TiO 2 nanocomposite synthesized by the IMH strain consisted of elemental Au, Ti, O, and C.
The valence state of Au and Ti in bio-Au x @TiO 2 was analyzed using XPS as shown in Fig. 3. Specifically, the peaks at 83.7~84.4 eV and 87.4~87.9 eV were ascribed to the 4f 7/2 and 4f 5/2 of Au(0), respectively (Liang et al. 2012). XPS results further confirmed that the IMH strain could completely reduce Au(III) to synthesize Au NPs. The high-resolution XPS scan resolved two peaks of Ti 2 p electrons at 459.3 eV and 465.05 eV, which were, respectively, assigned to Ti 2p 3/2 and Ti 2p 1/2 of oxidized state Ti 4+ , respectively (Khalid et al. 2016). The energy difference (5.7 eV) between the peaks of Ti 2p 3/2 and Ti 2p 1/2 is corresponding to the Ti 4+ state of anatase phase TiO 2 , which is consistent with the previous XRD results (Trino et al. 2018).
The FTIR spectra of the IMH strain, bio-TiO 2 , and bio-Au x @TiO 2 were shown in Fig. S2. The IMH strain spectra displayed typical bacterial characteristic peaks, which were mainly located at 3310 cm −1 (-OH stretching), 1610 cm −1 (C=C stretching), 1540 cm −1 (N-H bending), and 1040 cm −1 (C-N stretching) (Bosch et al. 2008;Fischer et al. 2006;Schmitt and Flemming 1998). In the FTIR spectra of bio-TiO 2 and bio-Au x @TiO 2 , the broad band around 1000 cm −1 represented the stretching vibration of Ti-O-Ti bond (Li et al. 2022). Meanwhile, the bands at 3310 cm −1 and 1610 cm −1 , which were ascribed to -OH group and C=C bond stretching, were also appeared in the spectra of bio-TiO 2 and bio-Au x @TiO 2 .
Our previous research reported that the strain IMH used diverse strategies to reduce Au(III) to Au NPs (Lengke et al. 2006;Liu et al. 2018a), including reduction of Au(III) by exopolysaccharides in extracellular polymeric substances (EPS) and protein/enzymatic reduction in the cytoplasm involving fucO and glutathione-relevant proteins (Liu et al. 2018b). According to the fundamental chemical principles and previous literature (Ni et al. 2021), bio-TiO 2 material was formed by dehydration reaction from TiO(OH) 2 . In this work, cells reduce Au(III) to form Au NPs, and then Au NPs were deposited on the surface of bio-TiO 2 , thus successfully synthesizing bio-Au x @TiO 2 .

Catalytic performance
The bio-TiO 2 and bio-Au x @TiO 2 materials were applied to the degradation of methylene blue. After adding the bio-TiO 2 and bio-Au x @TiO 2 materials, the absorbance at 664.5 nm (Abs664.5 nm) indicative of methylene blue decreased (Zou et al. 2016). As shown in Fig. 4a, the bio-TiO 2 degraded 79% of methylene blue molecules within 60 min, but the degradation does not continue with the further increase of reaction time to 90 min. Increasing concentrations of Au(III) in bio-Au x @TiO 2 from 0.5, 0.75, 1, to 2 mM bleached the blue color of the methylene blue in 50, 50, 40, and 30 min, respectively, indicating the completion of the reaction. For a quantitative comparison of kinetics, the first-order kinetics rate constant k app was calculated to evaluate the catalytic activity of bio-TiO 2 -relevant materials. Among others, bio-Au 2 @TiO 2 exhibited the highest degradation efficiency on methylene blue with the highest k app (0.195 min −1 ) (Fig. 4f). In addition, as shown in Figs  S3 and S4, the optimum condition of activity of bio-Au 2 @ TiO 2 was at the acidic or neutral environment and at room Fig. 4 UV-Vis absorption spectra showing the degradation of methylene blue by bio-TiO 2 (a), bio-Au 0.5 @TiO 2 (b), bio-Au 0.75 @TiO 2 (c), bio-Au 1 @TiO 2 (d), and bio-Au 2 @TiO 2 (e). Plot of ln(C t /C 0 ) against the reaction time for the degradation of methylene blue by bio-TiO 2 and bio-Au x @TiO 2 materials (f). k represents the value of k app temperature of 22 °C. Meanwhile, bio-TiO 2 and other bio-Au x @TiO 2 materials also have good photocatalytic degradation effect on methylene blue. And the degradation effect is directly proportional to the loading contents of Au in bio-Au x @TiO 2 system. Moreover, compared with Au@TiO 2 -containing catalysts synthesized by chemical or biological methods reported in the previous literature (Table S2), the bio-Au x @TiO 2 material, particularly bio-Au 2 @TiO 2 , had comparable or even higher catalytic performance in the degradation of methylene blue. More importantly, for up to 10 cycles, no remarkable decay can be discerned, which evidenced the excellent stability of the present bio-Au 2 @ TiO 2 material (Fig. S7).

Hydroxyl radical analysis
Highly reactive oxygen species, especially hydroxyl radical (•OH), plays a vital role in the organic pollutant degradation procedure in TiO 2 /UV system. To evaluate the content of ·OH in our study, the terephthalic acid (TA) fluorescent probe method was employed (Nakabayashi and Nosaka 2015). The TAOH concentration in the experiments could be calculated on the basis of the linear relationship between the concentration of TAOH and its fluorescence intensity at 426 nm. Since OH radical is the only reactant for the formation of TAOH, the concentration of ·OH was calculated through the formula [·OH] = [TAOH]/0.45. Fluorescence experiment result (Fig. 5) shows that signal intensity for ·OH was the Fig. 5 The production of •OH in the bio-TiO 2 and bio-Au x @ TiO 2 materials at different UV irradiation times Scheme 1 Proposed mechanism of bio-TiO 2 and bio-Au x @ TiO 2 for the degradation of methylene blue under UV light irradiation highest for bio-Au 2 @TiO 2 . Meanwhile, the amount of ·OH produced in solution followed the order of bio-Au 2 @TiO 2 > bio-Au 1 @TiO 2 > bio-Au 0.75 @TiO 2 > bio-Au 0.5 @TiO 2 > bio-TiO 2 , which is in accordance with the catalytic degradation speed of methylene blue.
In the photocatalytic process, electron-hole pairs are created by the excitation of electron from valence band to the conduction band. The photogenerated electrons (e − ) and hole (h + ) pairs could react with adsorbed O 2 and H 2 O to generate •OH. In the irradiation of UV light, biosynthesized TiO 2 can be photoexcited and produce electron-hole pairs to form hydroxyl radicals, thereby realizing the degradation of methylene blue (Scheme 1).
Furthermore, Au NPs in bio-Au x @TiO 2 play a vital role in the photocatalytic effect. The UV irradiation by the Au NPs resulted in the positive charges in the lower 5d band of Au, which can capture electrons from the organic pollutants adsorbed on Au NPs, ensuing in the oxidation of methylene blue. Meanwhile, the biosynthesized materials, particularly Au NPs, are nanoscale with large specific surface area, which are capable to adsorb more methylene blue molecules, contributing to the high catalytic activity.
More importantly, assuming an indirect transition between band gaps, the band gaps of TiO 2 can be estimated by extrapolating the tangent line in the plot of (αhυ) 1/2 against hυ, where α is the absorption coefficient and the hυ is the photon energy. As shown in Fig. S6, the bio-Au 2 @TiO 2 possessed an indirect band gap of 2.25 eV, which is the lowest value among these five materials. Meanwhile, the band gap energy of the bio-Au x @TiO 2 is determined as 2.79 eV (bio-Au 0.5 @TiO 2 ), 2.56 eV (bio-Au 0.75 @TiO 2 ), 2.50 eV (bio-Au 1 @TiO 2 ), and 2.25 eV (bio-Au 2 @TiO 2 ), which are lower than the value of 3.07 eV for bio-TiO 2 and bulk TiO 2 (3.2 eV for anatase and 3.0 eV for rutile). Therefore, as Au is added to TiO 2 , its band gap energy decreases below the values in bio-TiO 2 and bulk TiO 2 (anatase and rutile TiO 2 ). The decrease in the band gap energy of TiO 2 resulted in the stronger interaction between Au and TiO 2 and increased charge separation between the excited electron (e − ) and hole (h + ), causing the enhancement of photocatalytic activity of TiO 2 .
Thus, bio-Au x @TiO 2 materials, especially bio-Au 2 @ TiO 2 , displayed the superior catalytic activity in the degradation of methylene blue.

Conclusion
In sum, bio-TiO 2 and bio-Au x @TiO 2 were synthesized via dehydration reaction and Au(III) bioreduction. Multiple complementary characterizations verified that IMH strain could successfully synthesize bio-TiO 2 and bio-Au x @ TiO 2 materials, and Au NPs were uniformly distributed in the bio-TiO 2 layers in the bio-Au x @TiO 2 system. The green-synthesized bio-TiO 2 and bio-Au x @TiO 2 exhibited elevated catalytic activity for the degradation of methylene blue. In particular, bio-Au 2 @TiO 2 showed the highest k value of 0.195 min −1 . The optimum condition of activity of bio-Au 2 @TiO 2 was at the acidic or neutral environment and at room temperature of 22 °C. Moreover, the bio-Au 2 @TiO 2 keeps stable catalytic activity for up to 10 cycles. The Au NPs with large surface area adsorbed more target molecules and decreased the band gap energy of TiO 2 , resulting in the enhanced generation of hydroxyl radicals and pronounced photocatalytic activity of Au x @ TiO 2 material. The bio-Au x @TiO 2 material could be the promising catalytic materials, and the green synthesis provides a route to the recovery of noble metals such as gold and silver and synthesis of metal-semiconductor catalysts for organic pollutant degradation.