1 Introduction

In recent years, microbial cells as biotemplates have attracted burgeoning interests in a new research field of micro-nano materials synthesis, due to the major advantages, including abundant resources, easy accessibility, renewable and environmentally friendly materials, easy to remove, special and highly repetitive configurations and so on [1–3]. For example, nano-capsid, Escherichia coli, Deinococcus radiodurans and yeast cells have been used as biotemplates in the fabrication of micro-nano materials [4–7]. Especially, the photocatalytic materials synthesized by microbial cell templates have represented excellent comprehensive properties. Bo Bai et al. [8] reported the synthesis of novel TiO2@yeast composite particles with raspberry-like morphology by a simple single-step electrostatic process. The TiO2@yeast composite particles with selective bio-sorption and photocatalytic function have been applied into the decolourization of methylene blue and shown great effectiveness. Han Zhou et al. [9, 10] developed a novel and general bacteria-based bottom-up approach to synthesize biomorphic porous PbS and ZnS hollow nanostructures using Streptococcus thermophilus and Lactobacillus bulgaricus as morph-biotemplates.

Bacillus subtilis, known as a member of the genus Bacillus, is an extremely common bacterium found in soil, water, air, and decomposing plant matter [11, 12]. Due to abundance and the special physicochemical/biological properties [13], B. subtilis exhibits an ideal candidate as a microbial cell template for the formation of micro-nano photocatalytic composite materials. To be specific, B. subtilis cells are rod-shaped around 2 μm in length and 400 nm in width with a rigid cell wall structure [14]. The cell wall, comprised virtually of 46 % peptidoglycan and 54 % teichoic acids, is a peritrichous structure with abundant hydroxyl (–OH), acylamino (–CONH2), carboxyl (–COOH), amino (–NH2) and phosphate (–OPO3H2) functional groups [15, 16]. Therefore, such coexistence of peritrichous structure and the hydrophilic functional groups in the framework of B. subtilis cell wall provides plentiful absorption sites [17] and excellent absorbility [18, 19], which makes a good reason to utilize B. subtilis as a biotemplate to synthesise micro-nano photocatalytic composite materials. Moreover, the excellent suspension, dispersibility, easy availability and pretreatment of natural B. subtilis support the application as well. However, there are no articles on the facilitation of micro-nano photocatalytic composite materials with B. subtilis cells and TiO2 nanoparticles.

In the present study, we report the manufacture of novel TiO2@Bacillus subtilis composite particles using B. subtilis cells as biotemplates via a simple single-step electrostatic process. The product maintained the shape of the primitive B. subtilis cores and simultaneously integrated the particular natures of each participator (i.e., good suspensibility, dispersibility, large contact area and bio-sorption capacity from B. subtilis cells and photocatalytic activity from TiO2 nanoparticles). The structures of samples were characterized and the formation mechanism was proposed. The bio-sorptions and photocatalytic degradations of rhodamine B (RB) by composite materials were investigated further.

2 Experimental Section

2.1 Preparation of TiO2@Bacillus subtilis Composite Particles

The preparation of TiO2@Bacillus subtilis composite particles was based on self-assembly driven heterocoagulation. Briefly, 350 mg spores B. subtilis cell powders (air-dried) were suspended in 100 ml distilled water and the mixed solution was adjusted to pH 9–10 approximately by adding drop-wise sodium hydroxide solution. In a separate vessel, 50.0 mg TiO2 nanoparticles (AEROXIDE-TiO2 Degussa P25, primary particle size, 20–30 nm by TEM; specific surface area 52 m2/g by BET; composition 78 % anatase and 22 % rutile by X-ray diffraction) were dispersed in 100 ml distilled water and the TiO2 suspension was adjusted to pH 3–4 by adding drop-wise sulphuric acid solution. Next, the two suspensions were simultaneously sonicated in an ultrasonic bath for 10 min to facilitate de-aggregation of the particles and then respectively centrifuged to obtain alkali-treated B. subtilis cells and acid-treated TiO2 nanoparticles at a rate of 5000 revolutions per minute. After that, B. subtilis cells and TiO2 nanoparticles were re-dispersed again in 75 ml distilled water respectively and mixed for 1.0 h by magnetic stirring to promote the electrostatic attachment of TiO2 nanoparticles onto the surface B. subtilis cell wall. The resultant pH was approximately 4.5, which promoted self-assembly of TiO2 nanoparticles onto B. subtilis cell wall. The above mixture was left for 2.0 h at ambient temperature and pressure to form the TiO2@Bacillus subtilis composite particles. The resulting particles were collected by centrifugation from the mixture, followed by two cycles of distilled water and ethanol rinsing. The obtained TiO2@Bacillus subtilis composite particles were dried in air at 60 °C for 5 h and stored in a sealed bottle at ambient temperature for further use.

2.2 Characterization

The morphology of primitive B. subtilis and final TiO2@Bacillus subtilis microparticles were studied using Hitachi S4800 scanning electron microscopy (SEM) on the double sided adhesive conductive carbon tape with thin layers of platinum sputtered onto the samples before imaging. The detailed composition characterization of the TiO2@Bacillus subtilis composite particles was carried out with energy-dispersive spectroscopy (EDS) analysis. An X-ray diffraction (XRD) study was carried out using Cu Kα radiation (λ = 0.15418 nm) at a scanning rate of 0.02° per min. FTIR spectra of the samples were recorded with a Nicolet FT-IR spectrometer using a KBr disc containing 10 % of each finely ground sample in the range of 400–4000 cm−1. The supernatants of solutions were analyzed on a UV–Vis spectrophotometer (TU 4100) to determine the residual RB concentration (λmax = 552 nm).

2.3 Decolourization of RB

The TiO2@Bacillus subtilis composite particles were applied to degrade the concentration of the cationic dye, rhodamine B, to further investigate the TiO2@Bacillus subtilis removal effeciency of water contaminants. The bio-absorptive capacity and photocatalytic activities of TiO2, bare B. subtilis and TiO2@Bacillus subtilis composite particles were evaluated by the following experimental procedures: 200 mg per sample was dispersed in 100 mL RB solution (140 mg L−1 initially). The suspensions were magnetically stirred in the dark for 90 min to establish absorption–desorption equilibrium respectively. Then the suspensions were treated by UV irradiation, which was produced by a 500 W high pressure Hg lamp placed 8 cm above the beakers. Before and after UV irradiation, 4 mL suspension of each mixture was collected every 30 min and centrifuged to separate the catalyst from the suspension. And the supernatants were analyzed colorimetrically on a UV–visible spectrophotometer (TU-4100) to determine the concentrations of the residual RB. The bio-sorption and photoreduction ratio (%) could be calculated from the following equation:

$$\eta_{1} = \frac{{c_{s} - c_{o} }}{{c_{o} }} \times 100\%$$
(1)
$$\eta_{2} = \frac{{c_{o} - c_{e} }}{{c_{o} }} \times 100\% ,$$
(2)

where η 1, η 2 are bio-sorption and photoreduction ratios, c s , c o and c e are concentrations of RB before adsorbing, before and after irradiation, respective.

3 Results and Discussion

Figure 1a–c shows the SEM images of naked B. subtilis and TiO2@Bacillus subtilis composite particles. In Fig. 1a, the primitive B. subtilis cells were ordered rods with the length of around 1.3 ± 0.1 μm and width of 400 ± 50 nm. In Fig. 1b, The TiO2@Bacillus subtilis composite particles maintained the shape of the primitive B. subtilis cores and possessed relatively good monodispersity. Due to the nanoparticles on the surface of B. subtilis, the composite particles were 1.4 ± 0.1 μm in length and 450 ± 50 nm in width. As can be seen in Fig. 1c, parts of the B. subtilis surface was covered with TiO2 nanoparticles under high magnification, which roughened the surface of cell wall in comparison with primitive B. subtilis. Figure 1d presented the EDS analysis of TiO2@Bacillus subtilis composite particles. Obviously, Ti element was observed on the cell wall surface, which provided an evidence of the existence of TiO2 nanoparticles on the surface of the B. subtilis cells.

Fig. 1
figure 1

SEM images of a naked Bacillus subtilis, b general observation of the TiO2@Bacillus subtilis composite particles, c typically TiO2@Bacillus subtilis composite particles observed under high magnification, and EDS spectrum of the TiO2@Bacillus subtilis composite particles

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XRD spectra of the bare B. subtilis, TiO2 nanoparticles and TiO2@Bacillus subtilis composite particles were exhibited in Fig. 2. In Fig. 2a, the unconspicuous and broad peak around 2θ = 20° was attributed to the amorphous phase of B. subtilis. The sharp diffraction peaks at 25.30°, 37.82°, 48.10°, 53.94°, 55.08°, 62.61°, 69.00°, 70.22° and 27.45°, 75.00° of the TiO2@Bacillus subtilis composite particles (Fig. 2b) corresponded well with the reflections of anatase and rurile phase respectively (JCPDS NO. 21-1272), which were similar to the characteristic peaks of TiO2 nanoparticles (Fig. 2c). The intensity change of TiO2@Bacillus subtilis composite particles (Fig. 2b) around 2θ = 20° was different with that of TiO2 nanoparticles (Fig. 2c), due to the amorphous structure of B. subtilis. The results above demonstrated that TiO2 nanoparticles attached onto the B. subtilis cell wall and the crystalline phase of TiO2 nanoparticles remained unchanged.

Fig. 2
figure 2

XRD patterns of (a) the original Bacillus subtilis, (b)TiO2@Bacillus subtilis composite particles and (c) TiO2 nanoparticles

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Figure 3 exhibited the FT-IR spectra of original B. subtilis, pristine TiO2, TiO2@Bacillus subtilis composite particles, and their intermediate synthesis stages. In Fig. 3a, the broad peaks of B. subtilis at 3298, 2927, 2854, 1654, 1544, 1380, 1240 and 1078 cm−1 were assigned to the stretching vibration of O–H stretching vibration, symmetric and asymmetric stretching vibration of methylene, amide I, amide II, C=O stretching vibration, amide III and P=O stretching vibration, respectively [20, 21]. The characteristic peaks of alkali-treated B. subtilis cells in Fig. 3b were similar to that of B. subtilis cells. In Fig. 3d, e, the intense peaks at 3417 cm−1 (O–H stretching vibration) and 900–500 cm−1 (Ti–O–Ti) were both observed in pure TiO2 and acid-treated TiO2 nanoparticles. After self-assembly interaction of alkali-treated B. subtilis cells and acid-treated TiO2 nanoparticles, as can be seen in Fig. 3c, the characteristic peaks of the B. subtilis composite particles at 1380 and 1240 cm−1 were shifted to 1386 and 1242 cm−1, respectively. The vibration intensities of –OH bond became stronger, and that of amide III and O=P bonds became weaker in comparison to bare B. subtilis cells, suggesting that –OH, –CONH2, –COO− and –OPO3 2− functional groups are major contributors for the anchoring of the TiO2 nanoparticles. The peaks ranging from 1000 to 500 cm−1 were increased, due to Ti–O-Ti absorption typical of TiO2 type phase, which indicated the successful fabrication of TiO2@Bacillus subtilis composite particles. The results were in good agreement with XRD analysis.

Fig. 3
figure 3

FT-IR spectra of (a) the original Bacillus subtilis, (b) Bacillus subtilis treated by NaOH solution, (c) TiO2@Bacillus subtilis composite particles, (d) pristine TiO2 and (e) TiO2 nanoparticles treated by H2SO4 solution

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Based on the above analysis, the possible formation mechanism of TiO2@Bacillus subtilis composite particles synthesized by electrostatic self-assembly were proposed in Scheme 1. The materials with sufficiently high opposite zeta-potentials drove self-assembly via electrostatic interaction and achieved stable guest–host particles suspensions [22]. Specifically, the isoelectric point of the TiO2 nanoparticles was approximately pI of 6.8 [23, 24]. When dissolved in water, there generated surface hydroxyl groups (≡Ti–OH) on TiO2 nanoparticles and produced the positively charged surface (≡Ti–OH2 +) after proton association or dissociation reaction in acid solution [8]. Below pH of 6.8, the TiO2 nanoparticles were positively charged and there was a gradual increase of positively charged groups as the pH was lowered [8, 25, 26]. Similarly, the isoelectric point of the B. subtilis cells was about pI of 2.19 [27]. The complicated components of B. subtilis cell wall, including peptidoglycan, teichoic acids, etc., endowed the cell wall with abundant hydrophilic anionic groups, including OH−, –CONH− and –COO−, which provided ideal binding sites for the protonated hydroxyl groups. Therefore, it can be speculated that TiO2 nanoparticles and B. subtilis cells have opposite zeta-potentials in the range of pH from 2.2 to 6.8. At pH of approximately 4.5, the electrostatic driving force reached a maximum to improve the self-assembly of TiO2 nanoparticles onto B. subtilis cells. Futher low-temperature heating resulted in the formation of –O–O– bonds due to partial dehydration [28]. Hence, the mechanical stability and strength of adhesion were enhanced.

Scheme 1
scheme 1

Formation mechanisms of the TiO2@Bacillus subtilis composite particles and the synergetic effect in the removal of dyes aqueous solution

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To further examine the application of TiO2@Bacillus subtilis composite particles in the removal of water contaminants, the aqueous solution of RB, was taken as an example. As shown in Fig. 4, the bio-sorption and photocatalytic degradation of RB by pure B. subtilis, TiO2 nanoparticles and TiO2@Bacillus subtilis composite particles were performed respectively via the pretreatment of 1.5 h dark phase and the UV irradiation of 3.5 h. Clearly, the absorption–desorption equilibrium was established during the 1.5 h dark phase preceding the irradiation of the dyes solutions. Compared with B. subtilis, TiO2@Bacillus subtilis composite particles exhibited more favourable bio-sorption capacity. The increase was assigned to the enhanced negatively charged surface by alkalization treatment, which facilitated the capture of the cationic RB dye through electrostatic attraction. The removal ratio of RB by B. subtilis was about 64 %, while the removal ratio of RB by TiO2@Bacillus subtilis composite particles was approximately 89 %. The increase was attributed to combination of the bio-sorption ability of B. subtilis cells and photocatalytic activity of TiO2 nanoparticles. What is more, the well suspension property and dispersibility, large contact area facilitate the bio-sorption capacity of TiO2@Bacillus subtilis composite particles, which may provide TiO2 nanoparticles with RB molecule for photocatalytic degradation on the surface of B. subtilis cells. In return, the photocatalytic degradation of RB molecule on the surface can refresh the bio-sorption capacity of TiO2@Bacillus subtilis composite particles. These two mutually enhancing processes significantly increase the removal ratio of RB. All the results above suggested that TiO2@Bacillus subtilis composite particles were effective for dye removal, due to the integrated combined properties originated from their hybrid components, such as well suspension property and dispersibility, large contact area, favourable bio-sorption capacity and photocatalytic activity.

Fig. 4
figure 4

Changes in relative concentration of RB due to photolysis, photocatalysis by TiO2 nanoparticle, photolysis and adsorption on naked Bacillus subtilis and degradation by TiO2@Bacillus subtilis composite particles. [RB]0 = 140 mg L−1

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4 Conclusions

In summary, we have successfully developed a simple, economical and friendly environmental route for the manufacture of TiO2@Bacillus subtilis composite nanomaterials by a single-step strategy based on electrostatic interaction driving self-assembling heterocoagulation. The samples were characterized by SEM, EDS, XRD and FT-IR spectroscopy analyses. The possible formation mechanism of TiO2@Bacillus subtilis composite particles was proposed. The effectiveness of the TiO2@Bacillus subtilis composite particles for the removal of water contaminants was investigated by decolourizing RB and the removal ratio was approximately 89 %. The obtained materials with biotemplates possess integrated combined properties of well suspension and dispersibility, large contact area, favourable bio-sorption capacity and photocatalytic activity and have potential applications in the treatment of different waste water.