Induced transformation of amorphous silica to cristobalite on bacterial surfaces

Abstract

Extreme conditions such as high temperature and/or pressure are usually required for the transformation of amorphous silica to crystalline polymorphs. In this article, we present our results that amorphous silica can be deposited on a bacterial surface and transformed to cristobalite at a relatively low temperature and ambient pressure. The phase transformation of amorphous silica to cristobalite under thermal treatment was investigated by a variety of methods including X-ray diffraction, electron microscopy, and Fourier transform infrared spectroscopy. Results show that amorphous silica on a bacterial cell surface exhibits a direct phase transformation to cristobalite structure at a relatively low temperature (800 °C). The surface charge of the bacterial cells does not affect the phase transformation. Three Gram-negative bacteria and three Gram-positive bacteria have been tested in the present study. All these bacteria have been found to facilitate the phase transition of amorphous silica into cristobalite. The observation of amorphous silica transformation on bacterial surfaces to cristobalite highlights the use of bacteria in the synthesis and structure control of silica minerals.

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Introduction

Silica is with broad applications such as in semiconductors, ceramics, high performance thin-film transistors, solar cells, and electro-optical devices. Quartz, cristobalite and tridymite are the main phases in silica bricks. Elastic properties of cristobalite at room temperature have been extensively studied for their auxetic behavior such as in the fabrication of microelectronic devices.¹ However, the applications of cristobalite are restricted due to extreme crystallization conditions such as high temperatures, high pressures, and/or the use of alkaline chemicals.^2–10 There have been many attempts for controlling the crystallization of silica. Metal-induced crystallization (MIC) method for preparing polycrystalline silica from amorphous silica was developed based on observations that the phase transition temperature was significantly reduced in the presence of metals.^5,11–13 Template-induced crystallization (TIC) is another approach that improves crystallization conditions on different templates. For example, the synthesis of polycrystalline cristobalite balls has been achieved using colloid-imprinted carbon as a template.¹⁴ Both MIC and TIC approaches require extensive interactions between precursors and inducers (or templates) for initiating and promoting crystallization. In biosilicification, proteins act as inducers or templates for silica crystallization. A recent report showed that the protein silicatein induced the crystallization of amorphous silica at a relatively low temperature and ambient pressure.¹⁵ Roles of silicatein rely on interactions between silicatein and crystallization precursors of silica. Theoretically, it is possible to promote the transformation of amorphous biosilica by substituting silicatein with organic molecules. These organic molecules are either chemically synthesized polymers or biologically produced recombinant proteins that are carefully designed to function as silicatein. However, these methods are rather complicated or technologically difficult, and limited by interacting specificities between inducers and precursors.

The present study aims to provide a straightforward approach to control and improve phase transformation of amorphous silica at a relatively low temperature and ambient pressure. We propose to take advantage of bacterial cells for inducing the transformation of amorphous silica to cristobalite. Deposition of amorphous silica on a bacterial surface was facilitated by drying treatment. Multidisciplinary analysis tools such as Fourier transform infrared (FTIR) spectroscopy and electron microscopy were used to study the transformation of amorphous silica to cristobalite structure at ambient pressure and a relatively low temperature (800 °C).

Experimental

Bacterial cultivation and collection

Single bacterial colony was inoculated in 5 mL of Luria–Bertani (LB) medium, followed by shaking overnight at 37 °C at 220 rpm. The cell suspension was inoculated in a Luria–Bertani (LB) medium at a ratio of 1 : 50, followed by shaking at 220 rpm at 37 °C for 5 hours of cultivation until cell growth reached a stationary phase. Cells were then harvested by centrifugation at 8000g for 5 minutes at 4 °C.

Amorphous silica deposition and transformation on bacterial cell surface

SiO₂ Ludox silica colloidal was diluted in PBS (8 g L⁻¹ NaCl, 0.2 g L⁻¹ KCl, 1.15 g L⁻¹ Na₂HPO₄·7H₂O and 0.2 g L⁻¹ KH₂PO₄, pH 7.3) at the final silica concentration of 2 mg mL⁻¹, followed by ultrasonic wave dispersion for 10 minutes. The deposition of silica on cells was initiated by mixing cells with the silica solution (5 × 10⁸ cells per mL) and incubating at 37 °C at 220 rpm for 12 hours, followed by centrifugation at 8000g at 4 °C for 5 minutes and washing three times with distilled water to remove excess silica. Precipitates were resuspended in a small volume of distilled water and subjected to overnight treatment of vacuum freeze-drying or heat drying at 60 °C. The phase transition of bio-deposited silica was completed by heating at 800 °C or other desired temperatures in a muffle furnace for 360 minutes, followed by controlled cooling.

Encapsulation of cells with polyelectrolytes

Bacterial cells were coated with polyelectrolytes in a layer-by-layer (LbL) fashion by alternating the deposition of positively charged poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) complexes (noted as PAH/PAA) onto the surface of cells.^16,17 The LbL procedure was initiated by coating cells with a positively charged PAH, because the surface charge of the cells is negative. Polyelectrolytes were dissolved in PBS and mixed with the same volume of bacterial cells, followed by incubation at room temperature for 15 minutes. Excess polyelectrolytes were washed and removed with PBS. Polycations and polyanions were sequentially coated on cells by repeating the procedure, resulting in encapsulated cells with PAH/PAA multilayers (3/2) (3 layers of PAH and 2 layers of PAA).

Characterizations of silica transformation

X-ray powder diffraction (XRPD) patterns were acquired with a Bruker AXS D8 Advance diffractometer with Cu Kα (λ = 1.5418 Å) irradiation operating at 40 kV and 20 mA. Samples were mashed with an agate mortar and placed in a PP holder. Data was acquired from a 2θ range of 10° to 80° at a rate of 0.05° s⁻¹. FT-IR/ATR spectra were recorded with a Bruker Alpha spectrometer. Measurements were performed in triplicate and samples were scanned 64 times at a 4 cm⁻¹ resolution in the region of 4000–450 cm⁻¹ for each measurement. The surface morphology of the samples was observed and characterized with a scanning electron microscope (SEM S-4800).

Results and discussion

Effects of E. coli surface on silica transformation

Deposition of amorphous silica on E. coli surfaces was facilitated by drying overnight. The substance significantly changed from light yellow loose powder to white loose powder upon drying. Silica deposition on E. coli surfaces was investigated with SEM (Fig. 1, panel A) and energy dispersive spectrometry (EDS) analysis (Fig. S1, panels A and A′, see ESI†). SEM images revealed a change in the roughness of the E. coli cell surface from smooth to rough. EDS data showed that Si (Kα 1.74 keV) and O (Kα 0.52 keV) are the major inorganic compositions in cell surface deposits. These observations implied silica deposition on E. coli surfaces.

Fig. 1 Transformation of silica on E. coli surfaces. Panel (A), SEM images of surface deposits on E. coli. Panel (A′), SEM images showing morphology changes of the deposited silica. Panel (B), XRD spectra of silica deposits before and after thermal treatment. Inset shows the HR-TEM SAED analysis of silica deposits after thermal treatment. Panel (C), FTIR spectra of silica deposits before and after thermal treatment.

The phase transformation of amorphous silica to crystalline polymorphs is known to occur only at a high temperature and/or pressure.^18,19 It can be promoted by a surface directing agent and/or with highly porous preordered silica (known as zeolites).¹⁹ The first transformation from α-quartz to α-tridymite occurs at 870 °C and from quartz to β-cristobalite at 1600 °C.^20,21 In the present study, the phase transformation of amorphous silica on E. coli surfaces was induced at 800 °C. This temperature is lower than the typical temperature for the phase transformation of amorphous silica. After heating at 800 °C for 360 minutes, SEM demonstrated morphological changes in the deposited silica (Fig. 1, panel A′). The phase transformation of silica was examined with XRD and FTIR spectroscopy (Fig. 1). The X-ray diffraction pattern (Fig. 1, panel B) of the thermally treated samples showed characteristic peaks at 21.96°, 28.44°, 31.40°, and 36.09°. These peaks correspond to the spacing of the 101, 111, 102, and 200 crystal planes of SiO₂ α-cristobalite (JCPDS PDF no. 39-1425).²² An X-ray diffraction peak was observed at 20.69°, which is related to quartz SiO₂ (JCPDS PDF no. 46-1045). The phase transformation was also supported by HR-TEM, selected areas electron diffraction (SAED) patterns (Fig. 1, inset of panel B) and FTIR spectroscopy (Fig. 1, panel C). The cristobalite structure was identified in the FTIR spectrum with a peak at 620 cm⁻¹ and a knee at 1200 cm⁻¹.²³ These observations suggest that the transformation of amorphous silica to cristobalite on E. coli surfaces was achieved at a relatively low temperature.

We further investigated the critical phase transition temperature of amorphous silica on E. coli surfaces. When the sample was subjected to heating treatment at different temperatures for six hours, the XRD spectra (Fig. 2) showed that the phase transformation of amorphous silica to cristobalite starts at 650 °C. There is a significant improvement in the crystallization when the temperature was increased to 700 °C. Complete crystallization is observed at 800 °C. In subsequent experiments, 800 °C was used as the temperature for heating treatment to induce the phase transformation of amorphous silica to cristobalite.

Fig. 2 XRD spectra of silica deposits heated at different temperatures.

Effects of bacterial surface charge on inducing amorphous silica transformation

Bacteria can concentrate elements and act as nucleation sites for authigenic minerals.²⁴ Surface charges of bacteria and precursors may establish interactions between them through electrostatic adsorption. Studies on calcium carbonate suggested that polymorph selection is largely controlled by the surface charge density of a macrocyclic monolayer.^25,26 Therefore, we investigated effects of E. coli surface charge on inducing amorphous silica transformation. E. coli cells were coated with polyelectrolytes in a layer-by-layer (LbL) fashion with alternating deposition of PAH/PAA onto cell surfaces. Cell surface charge was then changed from negative to positive. Coated and uncoated E. coli cells were compared for inducing silica transformation. No significant difference was observed in the X-ray diffraction patterns of polyelectrolytes coated and uncoated E. coli cells (Fig. 3). Both types of cells were observed in inducing the transformation of amorphous silica to α-cristobalite. PAH alone or PAH and PAA together did not show influences in inducing silica transformation. These results suggested that E. coli surface charge did not contribute to the phase transformation of amorphous silica.

Fig. 3 XRD spectra of silica deposited on E. coli (with or without polyelectrolytes coating) or polyelectrolytes after thermal treatment.

Silica transformation on different bacterial surfaces

Because the surface charge of bacterial cells did not affect the phase transformation of amorphous silica, we further investigated the specificity of bacterial strains with different surfaces in inducing amorphous silica transformation. Bacterial cells were distinct in their envelopes and belonged to two major categories: Gram-positive type and Gram-negative type. In addition to the Gram-negative bacterium E. coli, five more representatives of the two types of bacteria, including two Gram-negative bacteria (Salmonella typhimurium and Proteus vulgaris) and three Gram-positive bacteria (Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes), were tested for their surfaces for amorphous silica transformation (Fig. 4). Silica deposition on cell surface was observed for two types of bacteria after drying (Fig. S1, see ESI†). SEM revealed the morphological changes of bacteria after silica deposition and subsequent thermal treatment. Most bacterial cells collapsed after silica deposition promoted by drying treatment. After thermal treatment at 800 °C, porous structure with average pore size of 50–300 nm was generated from amorphous silica deposits on bacterial cells. Phase transformation of amorphous silica was confirmed by XRD. Characteristic peaks of α-cristobalite were identified at 21.83°, 28.33°, 31.29°, and 36.00° (JCPDS PDF no. 39-1425).²² Phase transformation of amorphous silica was also demonstrated by FTIR spectra with a peak at 620 cm⁻¹ and a knee at 1200 cm⁻¹. These peaks correspond to cristobalite.²³ These data proved that the phase transformation of amorphous silica was achieved on surfaces of both Gram-positive bacteria and Gram-negative bacteria.

Fig. 4 Transformation of silica on bacterial surfaces. Panels (A–F), SEM images of vacuum silica deposits on bacterial surfaces. Panels (A′–F′), SEM images showing morphological changes of the deposited silica after thermal transformation. Panel G, XRD spectra of transformed silica on bacterial surfaces. Panel H, FTIR spectra of transformed silica on bacterial surfaces. EC, Escherichia coli; ST, Salmonella typhimurium; PV, Proteus vulgaris; BC, Bacillus cereus; SA, Staphylococcus aureus; LM, Listeria monocytogenes.

Discussion

Phase transformation from amorphous precursors to crystals occurs in the mineralization of many minerals such as CaCO₃, TiO₂ and SiO₂.^18,27–30 This process has been investigated in different biological environments or biomimetic conditions.^31–35 Various biological molecules have been tested for inducing the phase transformation of amorphous precursors. Examples of these molecules are amino acids, polysaccharide, and proteins.^31–35 These molecules interact with amorphous precursors and affect polymorph selections. In the present study, the surfaces of both Gram-positive and -negative bacteria are observed to promote the transformation of amorphous silica. Although the two types of bacteria show different envelopes, both bacteria have phospholipids as the common component to form cell membranes. The phospholipids membranes play an important role in amorphous silica transformation. Studies on calcium carbonate suggested that polymorph selection is largely controlled by the surface charge density of a macrocyclic monolayer.^25,26 However, a recent report of calcium carbonate mineralization on a phospholipid monolayer showed that the surface energy appears to be the ultimate determinant in polymorph selection.³⁶ Because we also observed that bacterial surface charge did not affect amorphous silica transformation, it is possible that the surface energy of phospholipid membranes promotes the phase transformation of amorphous silica.

Conclusion

In summary, the present study showed that amorphous silica can be deposited on bacterial cell surfaces. The transformation of silica deposits on bacterial surfaces to cristobalite was achieved at a relatively low temperature (800 °C) and ambient pressure. The surface charge of the bacterial cells did not affect phase transformation. In addition, the phase transformation of amorphous silica can be induced on the cell surface of both Gram-positive and -negative bacteria. The observation of induced silica transformation on bacterial surfaces opens a new avenue to take the advantage of bacteria in the synthesis and structure control of silica minerals.

Acknowledgements

This study was financially supported by the National Natural Science Foundation of China (51161140399), the Ministry of Science and Technology of China (S2010GR0771), and the China Scholarship Council (CSC).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra13619a

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