Water-floating nanohybrid films of layered titanate–graphene for sanitization of algae without secondary pollution

Abstract

A novel efficient and safe methodology to sanitize algae in natural water without secondary pollution is developed by fabricating floating graphene–inorganic hybrid films. Two kinds of floating freestanding hybrid films of layered titanate–graphene with efficient algae-killing functionality are fabricated by vacuum-assisted filtration of mixed colloidal suspensions of reduced graphene oxide (rG-O) nanosheets and exfoliated layered titanate nanosheets. Both the titanate nanosheets with lepidocrocite- and trititanate-type structures form homogeneous colloidal mixtures and hybrid freestanding films with rG-O nanosheets. The incorporation of a layered titanate nanosheet enhances the algae-killing activity of the graphene freestanding film, highlighting the beneficial role of photocatalytically-active titanate nanosheet. In comparison to the trititanate nanosheet, the lepidocrocite-type titanate nanosheet is more effective as a building block for enhancing the algae-killing activity of graphene film and for forming a novel nanoblade structure on the surface of the graphene film. The observed high sterilization functionality of the present layered titanate–graphene hybrid films is attributable to both the formation of the novel sharp nanoblade structure and the photocatalytic activity of layered titanate. The present result underscores that hybridization between graphene and photocatalytically-active inorganic nanosheets can provide a powerful way to explore pollution-free recoverable matrix efficient for removing harmful microorganisms in natural water.

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Introduction

As an emerging member of the low-dimensional nanostructured materials, exfoliated two-dimensional (2D) nanosheets of layered materials like graphite, layered transition metal oxides, and layered metal chalcogenides attract a great deal of research interest because of their unique physicochemical properties.^1–4 Of prime importance is that the freestanding films of exfoliated 2D nanosheets can be easily fabricated by vacuum filtration of the corresponding colloidal suspensions, which facilitates the practical use of these exfoliated nanosheets.^5–7 The obtained freestanding films of 2D nanosheets boast many valuable functionalities for diverse application fields such as energy- and environmental technologies.^8–12 In one instance, the freestanding film of reduced graphene oxide (rG-O) nanosheet shows promising antibacterial activity for Escherichia coli in terms of non-reactive oxygen species (non-ROS)-dependent oxidation.^10–12 This antibacterial property of the graphene film suggests its applicability for the sanitization of biological pollutions. One of the most serious biological pollutions of the aquatic system is algal bloom that frequently arises worldwide in lakes and rivers.¹³ Even though the algae are essential microorganisms in the aquatic ecosystem as fundamental nourishment and self-purification species, some of algae such as Microcystis, Anabaena, Aphanizomenon, and Oscillatoria produce toxin in eutrophic lake and static river.¹⁴ This harmful algal bloom involving toxic or harmful phytoplankton often causes serious problems for ecosystems and human society.¹⁴ One of the most widely-used solutions for reducing algal bloom is to spray algicide such as copper sulfate.¹⁵ Although the use of such chemical can induce the irreversible deconstruction of algae cells, it suffers from serious side effects such as the creation of algal tolerant species, the destruction of zooplankton, and the release of toxins upon cell lysis.¹⁶ Alternatively many researchers report the application of nanoparticles for killing algae.^17–22 However, the spraying of these nanoparticles still causes serious secondary pollution in natural water.^23,24 To avoid the secondary pollution caused by contamination with inorganic material, it is demanded to develop recoverable matrix with high anti-microorganism activity and high chemical stability. Taking into account the water-floating property and antibacterial activity of graphene film, it is applicable for the readily-recoverable matrix efficient for removing harmful microorganisms in natural water. Unfortunately, however, microorganisms like algae are much more resistant to the non-ROS-dependent oxidation caused by the graphene in comparison with bacteria, leading to the low activity of the pure graphene film for the sanitization of algae.²⁵ An effective way to circumvent this limitation of graphene film is the hybridization with photocatalytically active metal oxide nanosheet, leading to the enhancement of the oxidative sanitization activity of graphene.^26,27 Of noteworthy is that the algae-killing functionality of the obtained graphene–metal oxide hybrid film can be further improved by tailoring its surface structure, because the cell disturbance of microorganism occurs on the surface of the hybrid film. Taking into account the fact that the surface of the hybrid freestanding film is composed of exposed component nanosheets, the surface structure of the hybrid freestanding film of graphene–layered titanate can be tailored by controlling the crystal structure of titanate nanosheet incorporated. There are several structure types of layered titanates depending on the linkage pattern of TiO₆ octahedra.^28,29 In one instance, the lepidocrocite-type titanate shows flat layered structure composed of edge-shared TiO₆ octahedra whereas the trititanate phase has puckered layered structure composed of corner-sharing of three edge-shared TiO₆ octahedral units, as illustrated in Fig. 1.^28,29 Thus the incorporation of these different titanate nanosheets into graphene film can provide useful opportunity to fabricate efficient algae-killing hybrid film with controlled surface structure and optimized functionality. Yet, at the time of the publication of the present study, we are aware of no study about the application of floating graphene–inorganic hybrid films for sanitizing algae in water as well as the optimization of their algae-killing functionality through the control of the crystal structure of titanate nanosheet incorporated.

Fig. 1 Crystal structures of layered titanates with (a) lepidocrocite- and (b) trititanate-type structure.

In the present study, two kinds of floating hybrid films of layered titanate–graphene are fabricated by the vacuum-assisted filtration of the mixed colloidal suspensions of rG-O and layered titanate nanosheets with lepidocrocite- and trititanate-type structures for killing harmful algae of Microcystis aeruginosa. The influences of the structure type of layered titanate nanosheet on the crystal structure, surface morphology, and physicochemical properties of the resulting hybrid film are systematically investigated. The mechanism responsible for the observed algae-killing effect of the obtained hybrid film is also systematically investigated.

Experimental

Sample preparation

The layered titanate 2D nanosheets with lepidocrocite- and trititanate-type structures were prepared by soft-chemical exfoliation of the corresponding pristine layered titanates, as reported previously.^28,29 Another precursor of rG-O was synthesized by the reduction of Hummers' modified graphene oxide precursor.^5,30,31 The colloidal mixtures of rG-O and layered titanate nanosheets were obtained by mixing the suspensions of rG-O (0.0025 wt%, 12.00 mL) and the layered titanate nanosheet (0.00820 M, 20.29 mL for lepidocrocite-type layered titanate and 0.00154 M, 108.0 mL for trititanate-type layered titanate, respectively). Prior to the fabrication of the hybrid films, residual ionic species in the precursor suspensions were removed by dialysis using deionized water. The layered titanate–rG-O hybrid films were fabricated by vacuum-assisted filtration of the mixed colloidal suspension through an Anodisc membrane filter (Whatman, diameter = 47 mm, pore size = 0.02 μm). As a reference, pure rG-O film was also prepared by applying the same fabrication process using pure rG-O suspension.

Characterization

The surface charges of the colloidal nanosheets were measured with zeta potential measurements. The crystal structures and stacking morphologies of the obtained films were found with X-ray diffraction (XRD) and field emission-scanning electron microscopy (FE-SEM), respectively. The nature of the chemical bonding of the hybrid films was investigated with micro-Raman spectroscopy. The local crystal structures and orientations of layered titanates in the present hybrid films were examined with Ti K-edge X-ray absorption near edge structure (XANES) analysis. All the present spectra were measured at beam line 10C in the Pohang accelerator laboratory (PAL). The effect of titanate incorporation on the surface roughness of graphene film was examined with contact angle measurement. The concentration of titanium ion in algal solution was determined with inductively coupled plasma-mass spectrometry (ICP-MS).

Test of algae-killing activity

The algae-killing activity was investigated with the modified guideline of the Organization for Economic Co-operation and Development (OECD).^32,33 Microcystis aeruginosa (=KCTC AG20763) algae were obtained from the Korean collection for type cultures of the Korea Research Institute of Bioscience and Biotechnology. The films (0.01 g, 2.83 cm²) were soaked in BG-11 medium (10 mL) containing algae. The algal solutions with films were placed in 25 °C for 7 days in an incubator under 2000 lux illumination with fluorescent lamp. The lamp was alternatively turned on and off every 12 h. The initial number of living algae was 1.325 × 10⁷ cells per mL, which was estimated by counting colonies in a hemocytometer.³⁴ The changes in the number of living algae was monitored by quantification of chlorophyll A with the spectroscopic method reported by Lorenzen.^35,36 The generation of ROS by the present films was probed by monitoring the absorption of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT, Sigma-Aldrich, USA).^12,36 The morphological change of algae during the activity test was examined with FE-SEM.^6,36 Statistical analysis was performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California USA, http://www.graphpad.com). The differences in the mean values were evaluated using unpaired t-tests. Differences were considered significant when the p value was less than 0.05 or 0.0005 (marked as * or **), respectively.

Results and discussion

Fabrication of the hybrid films and floating test

The mixed colloidal suspensions of layered titanate and rG-O nanosheets displayed good colloidal stability without any phase separation for several weeks, which was confirmed by the maintenance of Tyndall phenomenon (see the Fig. S1 in ESI†). According to zeta potential measurements, both of the colloidal suspensions have average zeta potentials of −43 to −33 mV, which are similar to those of the precursor suspensions of rG-O, lepidocrocite-type titanate, and trititanate-type titanate nanosheets (−44, −29, and −44 mV). Due to the similar surface charges of these nanosheets, all the precursor nanosheets can be homogeneously mixed with each other and maintain negative surface charge. Vacuum-assisted filtration of both the colloidal suspensions yielded two kinds of freestanding hybrid films of rG-O–layered titanate with lepidocrocite- (GLT) and trititanate-type (GTT) layered titanate, as shown in the left-top panel of Fig. 2. There was no significant difference between the morphologies of these hybrid films and pure rG-O film. In comparison with the GLT and pure rG-O films, the GTT film showed inferior stability and flexibility (see the Fig. S2 in ESI†). As shown in the left-bottom panel of Fig. 2, all three of the films floated on the surface of water, which made it easy to restore them after the test in water without secondary pollution. No distinct destruction or swelling occurred for all the present films after the floating test.

Fig. 2 (Left, top) Photoimages, (left, bottom) floating ability test, and (right) XRD patterns of (a) pure rG-O film and the rG-O–layered titanate hybrid films of (b) GLT and (c) GTT.

The XRD patterns of the freestanding hybrid films of GLT and GTT are plotted in the right panel of Fig. 2, together with that of pure rG-O film. Broad (00l) peaks appear at 2θ = ∼12° and ∼24° for the pure rG-O film. The GLT film showed a series of well-developed (00l) reflections, clearly demonstrating the highly-ordered stacking structure of layered titanate and rG-O nanosheets. Since the (00l) reflections become discernible only for the well-ordered stacking of nanosheets, the absence of the XRD peaks related to the restacked rG-O nanosheet strongly suggests homogeneous mixing of the rG-O and layered titanate nanosheets without any phase separation.⁶ Conversely, two kinds of Bragg reflections corresponding to the rG-O and layered titanate phases were discernible for the GTT film, indicating the separated restacking of rG-O and layered trititanate nanosheets and non-uniform mixing between these nanosheets. In comparison with the pure rG-O film, the GTT film exhibits a slight shift of (001) reflection of graphene phase, which would be attributed to the different concentration of intercalated water molecules in the interlayer space of restacked rG-O nanosheets coupled with layered titanate nanosheet. From least-squares fitting analysis, the basal spacings of the present hybrid films were determined to be c = 9.49 and 10.08 Å for GLT and GTT, respectively, which are slightly larger than the basal spacings of the restacked titanate nanosheets.^6,29 Both the hybrid films showed similar c-axis lattice parameters that lie midway between the sum of the theoretical thickness of layered titanate monolayer (∼7.00 Å for lepidocrocite-type titanate and ∼7.29 Å for trititanate-type titanate) with the covalent diameter of carbon atom (∼1.52 Å) and that with the van der Waals diameter of carbon atom (∼3.70 Å). This suggests partial incorporation of graphene nanosheets in-between the stacked nanosheets of layered titanate.

Chemical bonding nature and local atomic structure of the hybrid films

The chemical bonding nature and local atomic structure of the hybrid films were investigated with micro-Raman and Ti K-edge XANES spectroscopies. As presented in Fig. 3, the Raman features of titanate phases in the GLT and GTT films below 800 cm⁻¹ are notably broader and more diffusive than those of the references of lepidocrocite-type titanate and trititanate, which is attributable to the shielding effect of the electromagnetic fields of conductive graphene species. However, the overall spectral features of titanate-related phonon lines in the hybrid films remain somewhat similar to those of the corresponding reference titanate, confirming the incorporation of the layered titanate in these hybrid films. In the high wavenumber region, both of the present hybrid films exhibit characteristic D and G peaks of graphene species, confirming the incorporation of rG-O nanosheets in the films.⁶ The D band at ∼1318 cm⁻¹ is attributed to the κ-point phonon of A_1g symmetry while the G band at ∼1592 cm⁻¹ is usually assigned as the zone center phonon of E_2g symmetry.^37,38 Since the D mode becomes active in the presence of disorder, the observed high intensity ratio of I_D/I_G indicates the partially disordered crystal structure of rG-O nanosheets.^37,38 The intensity ratio of I_D/I_G was estimated to be 1.14 and 1.12 for the GLT and GTT hybrid films, respectively, which is larger than that of pure rG-O film (I_D/I_G = 1.01), reflecting the enhanced disorder of the stacking structure of graphene nanosheets upon the incorporation of layered titanate nanosheets. The GTT hybrid film exhibited a smaller intensity ratio of I_D/I_G than did the GLT hybrid film, indicating the less intimate mixing between rG-O and titanate nanosheets in GTT hybrid film.

Fig. 3 Micro-Raman spectra of (a) pure rG-O film, the rG-O–layered titanate hybrid films of (b) GLT and (c) GTT, (d) lepidocrocite-type titanate, and (e) trititanate-type titanate.

Fig. 4 presents Ti K-edge XANES spectra of the present hybrid films with several reference spectra. Both of the present hybrid films of rG-O–layered titanate display similar edge energies to those of TiO₂ references, indicating the maintenance of a tetravalent Ti oxidation state upon film fabrication. All of the present hybrid films and reference layered titanates exhibited typical pre-edge features (P₁, P₂, P′₂, and P₃) of layered titanate phase, which are related to the dipole-forbidden 1s → 3d transitions. The observed spectral features, including a strong intensity of the peak P₂, are clearly distinguishable from those of anatase and rutile TiO₂ references, providing strong evidence for the maintenance of a layered titanate structure after the film formation. In the main-edge region, there are several spectral features A, B, and C which correspond to the dipole-allowed 1s → 4p transitions.^39,40 Among them, the intensity ratio of peak B/C provides a sensitive measure for the orientation of layered titanate, since peaks B and C correspond to the transition of out-of-plane 4p_z orbital and in-plane 4p_x,y orbital, respectively.^6,39 While nearly identical intensity of the peaks B and C was observed for the powdery lepidocrocite-type titanate, the GLT film displayed a larger spectral weight for peak C than for peak B, as observed for the c-axis-polarized XANES spectra of layered titanate film.⁶ Thus the higher intensity of peak C strongly suggests the parallel alignment of layered titanate nanosheets to the planar direction of the freestanding films.⁶ Conversely, there is no remarkable difference in the intensity ratio of peaks B and C for the GTT film and powdery trititanate reference, which can be understood from the crystal structure of trititanate lattice with a puckered layer. In contrast to the lepidocrocite-type titanate having a flat layered structure composed of edge-shared TiO₆ octahedra, the trititanate phase has a puckered layered structure composed of corner-sharing of three edge-shared TiO₆ octahedral units, as illustrated in Fig. 1. In other words, the layer of trititanate nanosheet was somewhat tilted with respect to the planar direction of the freestanding films, which was responsible for the observed weak angle-dependence of the XANES feature of the GTT film upon the layer-by-layer-ordering of the trititanate nanosheets.

Fig. 4 (Left) Ti K-edge XANES spectra of (a) anatase TiO₂, (b) rutile TiO₂, (c) powdery lepidocrocite-type cesium titanate, (d) powdery trititanate-type Na₂Ti₃O₇, and the rG-O–layered titanate hybrid films of (e) GLT and (f) GTT, and (right) their expanded view for pre-edge region.

Stacking and surface structures of the hybrid films

The effect of titanate incorporation on the surface structure and stacking structure of graphene film was examined by FE-SEM analysis. As illustrated in Fig. 5, the cross-sectional FE-SEM images of the pure rG-O freestanding film and the GLT hybrid film clearly demonstrated the layer-by-layer-ordered stacking of graphene and lepidocrocite-type layered titanate nanosheets. The incorporation of a lepidocrocite-type titanate nanosheet had little influence on the stacking structure of graphene film, suggesting effective mixing between the two types of nanosheets. Conversely the cross-sectional FE-SEM image of the GTT hybrid film demonstrates the significant disruption of the layer-by-layer-ordered stacking of graphene nanosheets upon the incorporation of trititanate-type layered titanate nanosheets, reflecting less homogeneous mixing of graphene with the trititanate nanosheet. Such poor ordering of GTT film makes additional contribution to the observed spectral similarity in the XANES spectra of the GTT film and trititanate powder (Fig. 4).

Fig. 5 (Top) Cross-sectional and (bottom) top-view FE-SEM images of (a, d) pure rG-O film and the rG-O–layered titanate hybrid films of (b, e) GLT and (c, f) GTT. The arrows in (e) indicate the sharp edges on the surface of the GLT film.

In the top-view FE-SEM images of Fig. 5, a flat and clean surface appears for the pure rG-O freestanding film whereas the GLT hybrid film displays sharp edges (i.e. nanoblades) on its surface. The formation of nanoblade structure is ascribable to stacking faults between intimately mixed rG-O and layered titanate nanosheets.⁶ Conversely, such a nanoblade structure is hardly formed on the surface of the GTT hybrid film. Instead, this film exhibits several flat domains composed of stacked nanobelt-like crystallites corresponding to trititanate phase. The absence of the nanoblade structure in the GTT film can be interpreted as a result of the non-uniform mixing between rG-O and trititanate nanosheets, as evidenced by the powder XRD results. The different mixing of rG-O and layered titanate nanosheets in both the GLT and GTT films can be understood from the dissimilar morphology of the precursor nanosheets (see the Fig. S3 in ESI†). While the lepidocrocite-type layered titanate shows wide 2D sheet-shaped morphology like the rG-O nanosheet, the trititanate-type layered titanate has quite different belt-shaped lateral morphology. Such a significant difference in the lateral morphology is responsible for the poor mixing between trititanate and rG-O nanosheets.

The effect of the crystal structure of layered titanate component on the surface property of the present hybrid films is also studied with contact angle measurement. As illustrated in Fig. 6, the GLT film shows a smaller contact angle of water droplet (∼40°) than does the pure rG-O film (∼85°), reflecting the enhanced surface roughness caused by the presence of sharp nanoblades. There is only a little spatial variation of contact angles in the entire surface of the GLT film, confirming the homogeneous mixing of lepidocrocite-type titanate and rG-O nanosheets. In contrast, the hybrid GTT film exhibits a significant variation of contact angle (64–85°) depending on the spatial region of this film, reflecting the inhomogeneous mixing between rG-O and trititanate nanosheets. This finding provides clear evidence for control of the surface property of hybrid film by changing the crystal structure of layered titanate nanosheet incorporated.

Fig. 6 Contact angle images of water droplet on the (a) pure rG-O film and the rG-O–layered titanate hybrid films of (b, c) GLT and (d, e) GTT.

Algae-killing activity of the hybrid films

The obtained hybrid films as well as the pure rG-O film were used for the removal of algae to investigate the influence of the crystal structure of layered titanate on the algae-killing effect of graphene film. As shown in the left panel of Fig. 7, the growth of algae under illumination was dramatically inhibited in the presence of hybrid films of GLT and GTT, while the significant growth of algae occurs in the presence of pure rG-O film and for the positive control without these films. This provides strong evidence for the remarkable merit of titanate incorporation in enhancing the algae-killing activity of the graphene film.

Fig. 7 (Left) Photoimage and (right) quantitative results of algae growth under illumination for the rG-O–layered titanate hybrid films of (a) GLT, (b) GTT, and (c) pure rG-O film. The result of algae growth in the absence of any film was used as (d) a positive control. The data are presented as the mean ± standard deviation of three independent experiments (n = 3). The * or ** on the bar graph indicates the significance (*: p < 0.05, **: p < 0.0005) of the activity of the hybrid film with respect to the pure rG-O film.

The amount of algae growth was determined via the quantification and normalization of the content of the chlorophyll A component in algae. As plotted in the right panel of Fig. 7, the inhibition efficiency of algae growth was 93.7 ± 2.7, 87.5 ± 2.1, and 24.4 ± 7.7% for GLT, GTT, and pure rG-O films, respectively. For the same loading amounts of the films, the activities of the rG-O–layered titanate hybrid films of GLT and GTT were much stronger than that of pure rG-O films, as confirmed by low p value of <0.0005. The better performance of the GLT film than the GTT film was proved by the low p value of <0.05, highlighting the optimization of the algae-killing activity of the hybrid film by controlling the crystal structure of layered titanate nanosheet incorporated. To further verify the efficient algae-killing ability of the present hybrid film, we studied the correlation between the loading amount of hybrid film and the inhibition efficiency of algae growth. As can be seen clearly from the Fig. S4 in ESI,† the addition of more GLT film leads to a notable increase of inhibition efficiency for algae growth, confirming that the observed inhibition of algae growth is surely attributable to the intrinsic sterilization functionality of the rG-O–layered titanate hybrid film.

Since we fabricated both the GLT and GTT hybrid films with the same amount of titanate nanosheet, the observed higher algae-killing activity of the GLT film than the GTT one can be regarded as evidence for a better role of lepidocrocite-type titanate nanosheet than trititanate one. To further confirm the important role of titanate nanosheet in the algae-killing functionality of the present hybrid films, we fabricated additional hybrid films with variable titanate/rG-O ratios. As plotted in the Fig. S5 in ESI,† an increase of lepidocrocite-type titanate content induces marked enhancement of the inhibition efficiency of the GLT film for algae growth, confirming the critical role of lepidocrocite-type titanate nanosheet in the improved algae-killing functionality of the GLT film. This result indicates that the titanate/rG-O ratio of 0.664 is an optimal value for optimizing the sterilization functionality of the GLT hybrid film. Conversely, the addition of more trititanate nanosheet into the GTT film does not improve significantly the algae-killing activity of this film, suggesting a weakly efficient role of trititanate nanosheet in the algae-killing functionality of the hybrid film. Of noteworthy is that, for all the ratios of layered titanate/rG-O employed here, the GLT films show better sterilization activities than do the GTT ones, clearly demonstrating a higher efficiency of the addition of lepidocrocite-type titanate nanosheet than that of trititanate nanosheet for optimizing the algae-killing activity of graphene film.

The practical applicability of the present hybrid films for the prevention of algae blooming was probed by algae-killing test at higher concentration of algae (7.1 × 10⁷ cells per mL) (see the Fig. S6 in ESI†). Even at this ∼5 times higher concentration, the GLT film still exhibits high algae-killing efficiency, underscoring the usefulness of this film for the sanitization of practical environment.

All the floating films were readily restored from the algal solution after the test of algae-killing effect. The ICP-MS analysis for the remaining algae solutions demonstrated no dissolution of titanium ions from the hybrid films during the algae-killing test, highlighting the high chemical stability of the hybrid films. This finding clearly demonstrated the present hybrid films were free from secondary pollution problem caused by the precipitation or excess supply of algicide.

Mechanisms responsible for the algae-killing activity of the hybrid films

There were five kinds of sterilization mechanisms contributing to the algae-killing activity of the semiconductor, that is, (1) a ROS-dependent oxidative stress (the damage of the cell caused by an imbalance between the systemic manifestation of ROS and ability of a biological system to detoxify ROS), (2) a physical disruption of cell membrane (the direct physical contact between nanostructured particles and cell that leads to membrane perturbation and to the release of intracellular contents), (3) an aggregation stress (the interruption of metabolism of the cell by wrapping the cell of nanoparticles), (4) a shading effect (the inhibition of cell growth by blocking illumination), and (5) a toxicity by the dissolution of metal (the toxicity provided by excessive supply of minerals).^17–20 The much higher algae-killing activity of the titanate-containing hybrid films than that of the pure rG-O film provides strong evidence for the crucial contribution of the photocatalytic activity (i.e. ROS-dependent oxidative stress) of layered titanate to the sanitization of algae by the present hybrid films.

To confirm the contribution of the mechanism (1), the generation of superoxide radical anion (O₂˙⁻) by the films was probed by monitoring the absorption of XTT. XTT was reduced by the ROS of superoxide radical anion (O₂˙⁻), leading to the formation of water-soluble formazan species showing a strong absorption peak at 470 nm. As presented in Table 1, both the rG-O–layered titanate hybrid films of GLT and GTT showed greater activity for the generation of superoxide radical anion (O₂˙⁻) than did the pure rG-O film, indicating a significant contribution of ROS-dependent oxidative stress to the removal of algae by the hybrid films of GLT and GTT. Between them, the hybrid GLT film induced a greater generation of superoxide radical anion (O₂˙⁻) compared with the hybrid GTT film, indicating the higher photocatalytic activity of the former film. This is interpreted as a result of the better mixing of rG-O with the lepidocrocite-type titanate nanosheet than with the trititanate nanosheet. Conversely, the pure rG-O film generated only a small amount of superoxide radical anion (O₂˙⁻), reflecting the negligible contribution of the ROS-dependent oxidative stress to the algae-killing activity of the pure rG-O film. The present findings highlight that the generation of ROS of the rG-O freestanding film was surely enhanced by the incorporation of the layered titanate nanosheet.

Table 1 Generation test of ROS for the rG-O–layered titanate hybrid films of GLT, GTT, and pure rG-O film

Sample	Absorbance at 470 nm	Relative quantity for the generation of ROS (%)
Hybrid GLT film	0.1055	100
Hybrid GTT film	0.0980	92.9
rG-O film	0.0570	54.0

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The presence of nanoblade-shaped surface structure of hybrid film of GLT shown in Fig. 5 suggests the significant contributions of the surface-dependent mechanisms of (2) the physical disruption of cell membrane and (3) aggregation stress on the algae to the algae-killing activity of this film. The effects of nanoblade structure on the surface areas of the GLT and GTT films were examined by measuring the amount of methylene blue (MB) adsorbed to probe the effect of surface area on the algae-killing activities of the hybrid films.^41,42 The GLT film possesses larger surface area of 24.3 m² g⁻¹ than that of the GTT film (9.5 m² g⁻¹), which is in good agreement with the formation of nanoblade structure on the surface of the former. This result confirms the important contribution of the enhanced physical contact of algae cells to the superior algae-killing activity of the GLT film, which originates from the formation of nanoblade structure and the accompanying surface expansion.

To further confirm the roles of these surface-dependent mechanisms, the morphological change of algae upon the attachment to the present films was examined with FE-SEM analysis. As presented in the top panel of Fig. 8, a huge number of algae were anchored on the sharp surface edges of the hybrid film of GLT. This finding clearly demonstrates that both mechanisms (2) and (3) made significant contributions to the highest algae-killing activity of the GLT hybrid film. Conversely, the surface adhesion of algae on the GTT and rG-O films was less effective than that on the GLT film, suggesting the weak contribution of both the mechanisms (2) and (3) to the sanitization of algae by the GTT hybrid film and pure rG-O film. As shown in the bottom panel of Fig. 8, algae attached on the surface of all the present films demonstrated hollow defects on their surface, which reflects the damage of algae by the oxidative stress.¹¹

Fig. 8 FE-SEM images of algae on the rG-O–layered titanate hybrid films of (a, d) GLT, (b, e) GTT, and (c, f) pure rG-O film with (top) low and (bottom) high magnification. The arrows indicate the algae attached on the surface of the film.

Taking into account the fact that the present floating films of GLT, GTT, and pure rG-O can block illumination of the algae, mechanism (4) of the shading effect must contribute to the algae-killing activity of all the films.^17,20 However, since all of the films block the illumination in the same area, the remarkable enhancement of the algae-killing activity of graphene film upon the incorporation of titanate nanosheet is not attributable to this shading effect. Also, the contribution of mechanism (5), i.e. toxicity originating from dissolved metal ion, can be ruled out for the present hybrid films, because both the titanate nanosheets showed high stability in aqueous media without the leaching of Ti ions as confirmed by ICP-MS analysis.

Conclusions

In conclusion, an efficient and safe methodology to sanitize harmful microorganism in natural water was developed by fabricating the layered titanate–rG-O hybrid films via vacuum-assisted filtration of the colloidal mixture of layered titanate and rG-O nanosheets. Regardless of the crystal structure of layered titanate, the incorporation of layered titanate nanosheet was fairly useful in enhancing the algae-killing activity of the graphene freestanding film. Between both the titanate nanosheets, the incorporation of lepidocrocite-type titanate nanosheet was more effective in enhancing the algae-killing activity of graphene film. The beneficial role of lepidocrocite-type titanate nanosheet was interpreted as a result of the formation of novel sharp nanoblade structure and the photocatalytic activity of layered titanate. Of prime importance is that the water-floating nature of the obtained hybrid films facilitates the restoration of these films after use in water environment. The present experimental findings underscore the usefulness of the present synthetic strategy in exploring new recoverable matrix efficient for purifying biological pollutants in natural water. Our current project is the optimization of the algae-killing efficiency of graphene–metal oxide hybrid films through the enhancement of the photocatalytic and redox catalytic activities of component nanosheets with chemical substitution.

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. NRF-2014R1A2A1A10052809). The experiments at PAL were supported in part by MOST and POSTECH.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: The detailed experimental method for extraction of chlorophyll A, detection of ROS generation, monitoring of the morphological change of algae, photoimages of the homogeneous colloidal mixtures, photoimages for the flexibility tests, FE-SEM images of the precursors, and algae-killing activity tests for the hybrid films with variable loading amount, several compositions, and high algae concentration. See DOI: 10.1039/c6ra24140a

‡ These authors contributed equally to this work.

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