In Situ Synthesis of Photocatalytically Active ... - ACS Publications

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In Situ Synthesis of Photocatalytically Active Hybrids Consisting of Bacterial Nanocellulose and Anatase Nanoparticles Falko Wesarg,† Franziska Schlott,‡ Janet Grabow,† Heinz-Dieter Kurland,† Nadine Heßler,‡ Dana Kralisch,‡ and Frank A. Müller*,† †

Institute of Materials Science and Technology (IMT), Friedrich-Schiller-University of Jena, Löbdergraben 32, 07743 Jena, Germany Institute of Technical Chemistry and Environmental Chemistry (ITUC), Friedrich-Schiller-University of Jena, Lessingstrasse 12, 07743 Jena, Germany

‡

S Supporting Information *

ABSTRACT: Bacterial nanocellulose (BNC) is an extraordinary biopolymer with a wide range of potential technical applications. The high specific surface area and the interconnected pore system of the nanofibrillar BNC network suggest applications as a carrier of catalysts. The present paper describes an in situ modification route for the preparation of a hybrid material consisting of BNC and photocatalytically active anatase (TiO2) nanoparticles (NPs). The influence of different NP concentrations on the BNC biosynthesis and the resulting supramolecular structure of the hybrids was investigated. It was found that the number of colony forming units (CFUs) and the consumption of glucose during biosynthesis remained unaffected compared to unmodified BNC. During the formation of the BNC network, the NPs were incorporated in the whole volume of the accruing hybrid. Their distribution within the hybrid material is affected by the anisotropic structure of BNC. The photocatalytic activity (PCA) of the BNC-TiO2 hybrids was determined by methanol conversion (MC) under UV irradiation. These tests demonstrated that the NPs retained their PCA after incorporation into the BNC carrier structure. The PCA of the hybrid material depends on the amount of incorporated NPs. No alteration of the photocatalyst’s efficiency was found during repeated PCA tests. In conclusion, the in situ integration of photocatalytically active NPs into BNC represents an attractive possibility to extend its fields of application to porous filtering media for drinking water purification and air cleaning.

1. INTRODUCTION Heterogeneous photocatalysis is a versatile and effective process to clean drinking water and polluted air.1,2 It can be defined as the surface induced acceleration of a photoreaction in the presence of a solid catalyst.3−5 Further technically relevant applications utilizing the photocatalytic effect include selfcleaning surfaces,6 photochemical hydrogen production,7,8 and solar energy conversion.9 Usually, semiconductors are used as photocatalysts. Optical excitation with energies exceeding the semiconductor’s band gap results in the formation of conduction band electrons and valence band holes. Both represent powerful reductants and oxidants, respectively, for the reaction with surface adsorbed molecules.3 The ideal photocatalyst has to meet various requirements including a high photocatalytic activity (PCA) in the range of visible or near-ultraviolet (UV) radiation,6,10 applicability on a large scale,10 stability toward photocorrosion,6,7 biological and chemical inertness,5 lack of toxicity,8 and low costs.10 Accordingly, titania (TiO2) is described to be the most suitably and technically most widely applied photocatalyst.8 Titania occurs in three different crystalline polymorphs, namely, anatase, rutile, and brookite. With respect to photocatalytic applications, the activity of rutile and anatase can overlap.11 It © 2012 American Chemical Society

depends on their structural and physical characteristics (e.g., specific surface area).12 However, usually the photocatalytical activity of the anatase polymorph is described to exceed that one of rutile.13 One opportunity to enhance the effectiveness of a photocatalyst is to maximize its specific surface area. This can be realized by using nanoparticles (NPs). Their high surface-areato-volume ratio leads to an increased probability of reactions between adsorbed molecules and conduction band electrons or valence band holes.14 To exploit the high reactive surface area, three-dimensional fibrous structures were applied to carry the NPs.14 In this context, electrospinning was described as an appropriate method.14,15 In the case of TiO2, fibrous networks can be prepared directly using a precursor solution such as titanium alkoxide (Ti(OR)4) and poly(vinyl pyrrolidone).16 However, the resulting polycrystalline fibers are very brittle and consequently not suitable for technical applications.14 Other studies utilized sol−gel techniques for the coating of porous organic templates with titania NPs obtained from a precursor Received: July 11, 2012 Revised: August 27, 2012 Published: August 27, 2012 13518

dx.doi.org/10.1021/la302787z | Langmuir 2012, 28, 13518−13525

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Figure 1. SEM micrographs of the top surfaces (a, d, g, and j), the cross sections (b, e, h, and k), and the bottom surfaces (c, f, i, and l) of pure BNC (BNC-T00, a−c) and the BNC-TiO2 hybrids (BNC-T05, d−f; BNC-T10, g−i; and BNC-T20, j−l). μm, laser power 2 kW). Expanding into the flowing process gas (air at normal pressure, total volume flow rate 14.5 m3/h), the vapor instantly cools down. The fast gas-phase condensation leads to the formation of nanoscale particles which are spherically shaped and merely softly agglomerated by weak van der Waals forces. The average particle size and the specific surface area of the resulting anatase NPs amount to 21 nm and 41 m2/g, respectively.30 For the synthesis of BNC, the Gluconacetobacter xylinus (GX) strain AX-DSM 14666 (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) was cultivated in an established culture medium proposed by Hestrin and Schramm (HSM).31 The HSM consists of 20 g of anhydrous D-glucose (Roth, Karlsruhe, Germany), 5 g of bacto yeast extract (Fluka, Munich, Germany), 5 g of bacto peptone (Becton Dickinson, Heidelberg, Germany), 3.4 g of disodium hydrogen phosphate dihydrate (Fluka, Munich, Germany), 1.15 g of citric acid monohydrate (Roth, Karlsruhe, Germany), and 1 L of deionized water. The culture medium had to be adapted in order to stabilize the dispersion of the NPs. For this purpose, the concentration of the buffer components and the content of yeast were reduced to 25% and 50%, respectively, of the initial concentrations. The dispersions were prepared applying an Ultra Turrax rotor-stator disperser (T25 Basic, IKA, Staufen, Germany) for 10 min at 11 000 rpm. A 7 day old preculture of bacteria AX-DSM 14666 was used to inoculate the sterile dispersions at a volume ratio of 1:20. The biosynthesis proceeded for 14 days at a temperature of 28 °C under dark conditions to prevent a light-induced inactivation or apoptosis of the bacteria cells by the photocatalytically active NPs.32,33 Samples with an anatase mass concentration of 0 g/L (BNC-T00), 0.5 g/L (BNC-T05), 1.0 g/L (BNC-T10), and 2.0 g/L (BNC-T20) were synthesized. After incubation, the samples were boiled in a 0.1 N aqueous sodium hydroxide solution for 30 min. Subsequently, they were washed with distilled water until the rinsing agent was neutralized. One half of the samples was f reeze-dried at a pressure of 1 Pa and at a condenser temperature of −85 °C for 48 h (Alpha 2-4 LSC, Martin Christ, Osterode, Germany). The other half was autoclaved at 121 °C for 20 min and stored in distilled water (neverdried). 2.2. Characterization. The consumption of glucose was evaluated reflectometrically (Reflectoquant Analysis System, Merck, Darmstadt, Germany) each day during the first 5 days of cultivation and once again after 7 and 14 days. The number of colony forming units (CFUs) was determined during biosynthesis after 1, 2, and 7 days. For this purpose, the culture broth was diluted in the range from 10−1 to 10−5. Subsequently, 100 μL of each dilution was spread onto an agar plate. The samples were incubated at 28 °C for 7 days. The colonies

(e.g., titanium isopropoxide) at an acidic pH.17 In particular, cellulose-based carriers proved to be suitable due to the high availability of hydrogen bonds.17−20 When looking for an effective porous cellulosic template with a high specific surface area, bacterial nanocellulose (BNC) seems to be a promising material. BNC represents a highly crystalline hydrogel that is produced by acetic acid bacteria of the genus Gluconacetobacter.21 It is synthesized at the interface between culture medium and air and consists of ultrafine cellulose ribbons with diameters of 40−60 nm which is approximately 100 times thinner than the fibers of plant derived pulp celluloses.22−24 Consequently, the specific surface area gains up to 200 m2/ g.25,26 Sun et al.27 described the coating of BNC with anatase NPs by surface hydrolysis of titanium butoxide in an ethanolic solution and presented the remarkable capability of BNC for orienting TiO2 NP arrays. Moreover, an enhanced PCA has been achieved with mesoporous titania networks synthesized from a solution of tetra-n-butyl titanium and BNC.28 However, in both cases, the prepared composites had to be calcined at a temperature of about 500 °C. At this temperature, the BNC carrier is pyrolyzed and as a consequence the favorable hydrogel character of the BNC is destroyed.27,28 In this study, we present a new in situ method to synthesize a hybrid material consisting of BNC and anatase NPs. This innovative method allows the direct incorporation of the NPs into the accruing BNC carrier during its biosynthesis. Thus, no further treatments are necessary to achieve a photocatalytically active hybrid structure. Consequently, the beneficial hydrogel character remains unaffected. The influence of the photocatalytically active NPs on the biosynthesis of BNC and on the resulting network structure was investigated. The PCA of the BNC-TiO2 hybrids was evaluated using the methanol conversion (MC) method.

2. EXPERIMENTAL SECTION 2.1. Biosynthesis of BNC-TiO2 Hybrids. Phase pure anatase NPs were prepared by the CO2 laser vaporization technique (LAVA) as described earlier.29 Briefly, a coarse anatase raw powder (GPR Rectapur 20732.298, VWR, Darmstadt, Germany) was vaporized in the intense focus of a continuous CO2 laser beam (wavelength 10.59 13519


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Table 1. Summary of the Evaluated Data for Pure BNC (BNC-T00) and the BNC-TiO2 Hybrids (BNC-T05, BNC-T10, and BNC-T20)a BNC-T00 fiber diameter [nm] content of the NPs in the top surface [%] content of the NPs in the cross section [%] content of the NPs in the bottom surface [%] dry mass [mg] OH• concentration [mmol/L]

BNC-T05

64 ± 19

33 ± 2 0.013 ± 0.002

66 2.7 0.2 0.5 30 0.033

± ± ± ± ± ±

20 0.2 0.1 0.4 1 0.003

BNC-T10 69 6.0 0.9 5.6 28 0.049

± ± ± ± ± ±

39 0.8 0.2 0.8 2 0.003

BNC-20 76 6.3 1.3 14.1 26 0.047

± ± ± ± ± ±

38 0.9 0.1 0.2 2 0.004

a Mean fiber diameters in the cross sections evaluated from SEM micrographs (Figure 1a, d, g, and j) using AxioVision, contents of anatase NPs in the SEM micrographs of the top surfaces (Figure 1d, g, and j), the cross sections (Figure 1e, h, and k), and the bottom surfaces (Figure 1f, i, and l) of the samples using ImageJ, dry masses of pure BNC and the BNC-TiO2 hybrids after 14 days of cultivation, and hydroxyl radical concentrations determined by the MC tests.

which were formed after this time were counted, and the CFUs were calculated using the method of weighted average.34 The investigations of the CFUs and the glucose consumption were performed in 50 mL Erlenmeyer flasks. The dry masses of the hybrids which were synthesized in 12-well plates were weighed after each day of cultivation (accuracy of 10−4 g, CPA224S, Sartorius, Göttingen, Germany). The supramolecular structure and composition of the BNC samples were characterized by scanning electron microscopy (SEM) in combination with energy dispersive X-ray spectroscopy (EDX) (S440i, Leica, Wetzlar, Germany). In this context, the top surface, the bottom surface, and cross sections of the f reeze-dried samples were investigated. The cross sections were prepared by using a razor blade. All samples were sputtered with gold. The SEM investigations were performed at an acceleration voltage of 15 kV. The micrographs were recorded at a working distance of 10 mm which was increased to 25 mm for the EDX analyses. The distribution of the NPs within the hybrid material and the BNC fiber diameters were determined from the SEM micrographs. The digital image data were processed and evaluated using the software packages AxioVision LE 4.4 (Carl Zeiss, Jena, Germany) and ImageJ 1.45 (Wayne Rasband, Bethesda, MD). X-ray diffraction (XRD) patterns of pure BNC, the LAVA NPs, and the prepared hybrids were measured (D8 Discover, Bruker, Karlsruhe, Germany) using f reeze-dried samples. Data were collected at diffraction angles 2θ ranging from 12° to 60° applying monochromatic Cu Kα radiation, an accelerating voltage of 40 kV, a beam current of 40 mA, a scan speed of 0.5°/min, and a step size of 0.02°. The PCA of the never-dried hybrids was evaluated by MC tests.35,36 For this purpose, the solvent was exchanged from distilled water to 1 vol % methanol solution (VWR, Darmstadt, Germany). In order to activate the samples photocatalytically, they were irradiated for 15 min at a distance of 0.5 m from a near-UV radiation source (wavelength 360 nm, xenon arc lamp XBO 450 W, Mueller Elektronik GmbH, Moosinning, Germany). At this position, the irradiance amounts to 18 W/m2 for the UVA-range (UVM-CP UV hand-held measuring instrument, UV-Consulting Peschl e.K., Mainz, Germany). Analyses were performed by high pressure liquid chromatography (HPLC) (Dionex Ultimate 3000 pump equipped with a Dionex Ultimate 3000 autosampler, a Dionex UVD 340 U detector, and a Kromasil 100 C18 column, Dionex Softron, Germering, Germany). The eluent consisted of 70 vol % acetonitrile (HPLC-grade, VWR, Darmstadt, Germany) and 30 vol % deionized water. A flow rate of 1800 μL/min and an injection volume of 20 μL were applied.

The SEM micrographs of pure BNC demonstrate an anisotropic structure that is representative for BNC obtained under static cultivation conditions.37 The cross section of the fleece is characterized by the typical nanofibrillar and highly porous network architecture (Figure 1b). The individual fibers are randomly entangled. This is caused by the unhindered motion of bacteria during cultiviation.38 At the top surface, the fleece is in contact with the culture medium and air. This interface ensures access to oxygen for the aerobic bacteria. However, because the top surface of BNC is exposed to air, it is partially air-dried.37 Consequently, the ultrafine ribbons aggregate and form a denser layer (Figure 1a). In contrast, the loose bottom surface of the fleece shown in Figure 1c represents a residue of BNC that is synthesized within the first 2 cultivation days. The aerobic bacteria synthesize a small amount of BNC to reach the interface between culture medium and air by using oxygen that is dissolved in the culture medium.37 This kind of BNC is more disordered. The supramolecular architecture of the BNC-TiO2 hybrids confirms that the generation of the anisotropic BNC structure during biosynthesis remains unaffected in the presence of anatase NPs. All hybrids consist of a dense top surface, a loose bottom surface, and an ultrafine network in the inner core. The fiber diameters of the BNC network range from 60 to 80 nm for all samples (Table 1). The diameters slightly increase by the incorporation of an increasing amount of TiO2 NPs. The corresponding SEM micrographs suggest that aggregation of a number of BNC fibers occurs in the presence of the NPs (Figure 1b, e, h, and k). EDX analyses (Figure S1) reveal the characteristic peaks of Ti (EKα1 = 4.511 keV and EKβ1 = 4.932 keV), of O (EKα1 = 0.525 keV), and of the gold coating (EMx = 1.661 keV, EMα12 = 2.123 keV, and ELα1 = 9.713 keV).17,36 XRD analyses (Figure 2) of pure BNC indicate reflexes at 14.4°, 16.8°, and 22.6° which correspond to the (1−10), (110), and (200) reflections of cellulose I, respectively.27,39 For the BNCTiO2 hybrids, additional reflections corresponding to anatase occur at 25.3° (101), 37.0° (103), 37.8° (004), 38.6° (112), 48.1° (200), 53.9° (105), and 55.1° (211).28,30 The intensity of the anatase peaks increases with an increasing concentration of the anatase NPs in the culture medium. However, the amount of incorporated anatase NPs that was calculated by the graphical analyses of SEM micrographs (Figure S2) differs as a function of their location within each sample. For all samples, the lowest content of incorporated NPs was found in the inner core of the BNC-TiO2 hybrids (table 1). In order to evaluate the influence of anatase NPs during biosynthesis, the consumption of glucose, the alteration of the CFUs, and the obtained dry masses were determined. During

3. RESULTS SEM micrographs reveal the supramolecular structures of the f reeze-dried fleeces of the pure BNC (BNC-T00: Figure 1a−c) and of the BNC-TiO2 hybrids prepared in culture media with anatase concentrations of 0.5 g/L (BNC-T05: Figure 1d−f), 1.0 g/L (BNC-T10: Figure 1g−i), and 2.0 g/L (BNC-T20: Figure 1j−l). Micrographs were taken from the top surfaces (Figure 1a, d, g, and j), the cross sections (Figure 1b, e, h, and k), and the bottom surfaces (Figure 1c, f, i, and l) of the fleeces. 13520


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content decreases rapidly, and after 5 days of cultivation 90% of the glucose is consumed (Figure 3a). Similar values have also been reported in literature.37 The glucose consumption is in good agreement with the amount of synthesized cellulose within this period. After 7 days, lower glucose contents lead to a deceleration of the synthesis as well as to a reduction of the number of active cells (Figure 3b). After approximately 9 days, the biosynthesis is completed (Figure 4a: day 7 to day 14). The results confirm that the addition of anatase NPs to the culture medium has no effect on the consumption of glucose and the alteration of the CFUs. However, the dry masses of the BNCTiO2 hybrids are slightly reduced by increasing mass concentrations of anatase NPs in the culture medium (Table 1). In order to evaluate the PCA of the prepared samples, the hydroxyl radical concentration under near-UV irradiation was measured with the MC method (Figure 5a). For pure BNC (BNC-T00), a minor activity was found. The incorporation of anatase NPs in the hybrid material results in an increase of the hydroxyl radical concentration from 0.013 mmol/L (BNCT00) to 0.033 mmol/L (BNC-T05) and to 0.049 mmol/L (BNC-T10) (Figure 5a). A further increase of the amount of incorporated NPs (BNC-T20) does not lead to a further increase of the PCA (Table 1). Similar results have been obtained by evaluating the photocatalytic activity of the BNCTiO2 hybrids using the decomposition of methylene blue under near-UV irradiation (see Supporting Information, Figure S3). Also, the stability of the photocatalyst during the implementation of the tests was investigated. For this purpose, the MC test was performed 10 times for the sample that produced the highest hydroxyl radical (OH•) concentration (BNC-T10). For each revision, the test solution was renewed. The OH• concentrations vary only marginally and randomly around an average value of c = 0.049 ± 0.005 mmol/L (Figure 5b). These results show that no consumption or degradation of the photocatalytically active hybrid material occurs even after a 10fold revision of the test.

4. DISCUSSION 4.1. BNC Formation in the Presence of Anatase NPs. The extracellular synthesis of BNC is performed under aerobic conditions by using a carbon source (HSM). The initial stage of the BNC formation implies the growth of the bacteria population. After the inoculation (Figure 4a: day 0), the population proliferates by consuming oxygen that is dissolved in the culture medium.37 As a consequence, a small amount of cellulose is synthesized by bacteria within the first cultivation day although the bacteria have not yet reached the interface between culture medium and air (Figure 4a: day 1). Carbon dioxide, a gaseous product of the carbon metabolism of the bacteria, is integrated in the accruing material.42 In this manner, bacteria integrated in the BNC rise to the surface of the culture medium (Figure 4a: day 2). Looking at the unchanged number of CFUs, it can be concluded that this mechanism is not affected by the integration of anatase NPs into the culture medium (Figure 3b). However, increasing the mass concentration of anatase NPs increases also the number of NPs that can be integrated into the BNC during the first day of cultivation. Consequently, the density of the hybrid material increases, and the buoyancy of the fleece is reduced. Thus, although the number of bacteria is almost equal for all hybrids after 2 days, bacteria in a highly NP loaded medium have a delayed access to the interface between culture medium and air.

Figure 2. Normalized XRD diagrams of the TiO2 NPs from the LAVA process (TiO2) and (a) the top surfaces, (b) the cross sections, and (c) the bottom surfaces of pure BNC (BNC-T00) and the BNC-TiO2 hybrids (BNC-T05, BNC-T10, and BNC-T20); indices of anatase and BNC taken from the JPDCS database.

an adaption period of 2 days, the initial glucose content of 20 g/L (Figure 3a) and the initial number of 105 CFU/mL (Figure 3b) remains almost unchanged. This is in good agreement with data found in literature,37,40 and it also corresponds to the measured dry masses within the first 2 days of cultivation (Figure 3c). Within this period, only a small amount of cellulose is synthesized by bacteria which have to reach the interface between medium and air (Figure 4a: day 1). After 2 days of cultivation, the majority of the aerobic bacteria have reached the interface (Figure 4a: day 2). Consequently, the number of CFUs rises from 105 to 106/mL (Figure 3b). This means that an increasing number of cells metabolizes an increasing amount of glucose.37,41 Consequently, the glucose 13521


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Figure 3. Biosynthesis of pure BNC (BNC-T00) and the BNC-TiO2 hybrids (BNC-T05, BNC-T10, and BNC-T20): (a) glucose consumption, (b) number of the CFUs, and (c) dry masses in dependence of the cultivation time in days (d).

Figure 4. BNC formation in the presence of anatase NPs: (a) schematic illustration of the hybrid formation under static conditions in dependence of the cultivation time, (b) synthesis of the BNC fibers by AX-DSM 14666 (GX), and (c) the integration of the anatase NPs into the BNC structure.

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Figure 5. Photocatalytic activity: (a) hydroxyl radical concentration obtained from MC tests of pure BNC (BNC-T00) and the BNC-TiO2 hybrids (BNC-T05, BNC-T10, and BNC-T20) in dependence of the mass concentration of the incorporated anatase NPs and (b) hydroxyl radical concentration of the BNC-TiO2 hybrid sample BNC-T10 after repeated MC tests in renewed test solutions.

emit the originating subelementary fibrils (diameter 1.5 nm) from their terminal complexes (TCs) to the medium (Figure 4b).21,44,45 About 50 to 80 TCs are located longitudinally on the outer membrane of a cell.42 Thus, neighboring subelementary fibrils can assemble.44 Due to the short distances between each TC (3.8 pores per 100 nm),46 no NPs can be integrated between the subelementary fibrils. The selforganization and crystallization processes continue and lead to the formation of microfibrils (diameter 3−6 nm) which subsequently form the BNC fibers (diameter 40−60 nm).45,20 During this process, the accruing high specific surface area network can strongly interact with the surrounding environment.38 Therefore, large amounts of water are integrated inside the BNC fibers as well as around them (solvate shells).23,47 In a similar manner, the NPs can interact with the hydroxyl groups or with the interfaces of intermolecular hydrogen bonds (O6H−O3 interface of adjacent anhydroglucose units) of the microfibrils.48,49 Accordingly, the anatase NPs are integrated at the surface of BNC fibers or between adjacent microfibrils during the self-organization of the nanofibrillar network by hydrogen bonds (Figure 4c). This is confirmed by the increasing fiber diameter of the hybrids with increasing content of TiO2 NPs in the culture medium. An increasing amount of NPs in the medium increases the probability of an interaction with the hydroxyl groups of the microfibrils. Consequently, more NPs will be integrated which leads to the increase of the fiber diameters (Figure 1b, e, h, and k; Table 1). It was described that the main reason for the incorporation of the inorganic material is related to the high specific surface area of the BNC fibers and hence to the enormous number of hydrogen groups available on the cellulose chains.39 4.2. Distribution of the NPs within the BNC-TiO2 Hybrids. As mentioned before, pure BNC as well as all hybrids in which the nanofibrillar BNC network acts as a carrier for the incorporation of TiO2 NPs are characterized by an anisotropic morphology (Figure 1). This anisotropic structure of the BNC affects the distribution of the NPs within the final hybrid material (Figure S2). At the beginning of the synthesis, the NPs are homogenously distributed within the whole culture medium (Figure 4a: day 0). During the first 2 days of cultivation, numerous NPs are integrated into the BNC which is synthesized within the medium (Figure 4a: day 1 and 2). Therefore, the NP concentration in the medium is reduced. This kind of BNC forms the top surface of the accruing BNC-

The increase of the dry mass is delayed and consequently not as steep as for pure BNC (Figure 3c). Bacteria that are located at the interface between medium and air can maintain their activity and continue to synthesize BNC. The BNC formation takes place in the oxygen-rich region below the top surface which protects the bacteria from competitors, desiccation, harmful chemicals, and environmental influences.42 During the synthesis of new layers, the previously synthesized ones are steadily displaced downward in the medium (Figure 4a: day 7).37 As a consequence of the increasing thickness, nutrients are hindered to diffuse through the fleece toward the interface and the glucose content at the interface is reduced. The synthesis decelerates and finally stops after 9 days of cultivation (Figure 4a: day 7 to day 14). The maximum yield of this biotechnological process is reported to be in the range from 35% to 40% for the GX strain AX-DSM 14666.37 For the modified medium, it amounts to 33% ± 2%. This reduced yield is caused by the reduced amount of yeast which displays a nutrient source for the bacteria. The dispersion of NPs in the culture medium leads to a further slight decrease (Table 1). Taking into account the course of the hybrid dry masses vs time (Figure 3c), it becomes evident that the increase of these masses between day 2 and day 5 is delayed compared to pure BNC. However, after bacteria reached the surface, the synthesis continues in a similar manner as for pure BNC. This is also confirmed by the number of CFUs and the glucose content which are almost equal for all samples after 1, 2, and 7 days of cultivation. Figure 4c illustrates the manner of the NP integration into the nanofibrillar BNC network. The NPs attach to the BNC fibers and are located between adjacent microfibrils. This might be explained by the hierarchical structuring of the hydrogel during its biosynthesis. By adding anatase NPs to the culture medium water molecules adsorb to the TiO2 surface by dative bonds between the oxygen of the water and titanium ions as well as by hydrogen bonds between the hydrogen of the water and the bridging titania oxygen.13,43 Consequently, the water covered NPs do not affect the metabolism of the bacteria during cultivation under dark conditions (Figure 3a and b). The absence of a photocatalytic inactivation corresponds to findings of Rincón and Pulgarin.32,33 They show that no disinfection of E. coli occurs in the presence of TiO2 after stirring under dark conditions. Hence, all bacteria (length about 1 μm) are active in the presence of the titania photocatalyst and 13523


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4. CONCLUSION Nanofibrillar hybrid materials consisting of BNC and photocatalytically active anatase NPs from the LAVA process were prepared by an in situ biosynthesis process. The accruing high specific surface area network of BNC acts as a carrier for the NPs. The formation of the BNC network is not disturbed in the presence of the NPs. The NPs are distributed within the whole volume of the resulting BNC network. The photocatalytic activity of the hybrid material depends on the mass concentration of incorporated anatase NPs. The prepared photocatalytically active and stable BNC-TiO2 hybrids might be of specific interest for environmental applications like the purification of drinking water and the cleaning of polluted air. Furthermore, this innovative in situ synthesis has the potential to be utilized for a wide range of functionalized NPs in order to extend the properties of native BNC.

TiO2 hybrid. The high number of NPs in this layer is confirmed by the amount of NPs evaluated from SEM micrographs (Figure S2a, d, and g). This NP content depends on the mass concentration of the NPs primarily dispersed in the culture medium (Table 1 and Figure 2a). The NPs are homogenously distributed within the top surface (Figure S2a, d, and g). During the progress of the biosynthesis, the layers are steadily displaced downward into the medium and the thickness of the hybrid material increases. NPs in the medium as well as those that can diffuse into the oxygen-rich layer are integrated into the inner core. However, their content is below that of NPs integrated in the top surface (Figure S2b, e, and h). This is a result of the reduced concentration of NPs in the medium which is caused by the incorporation of numerous NPs into the BNC during the first cultivation days. However, their amount increases with increasing concentration of anatase NPs in the medium (Table 1 and Figure 2b). The bottom surface of the fleece belongs to the oldest synthesized layer (Figure 4a). It consists of NPs that were integrated during the first steps of the synthesis. Additionally, this surface picked up NPs that remained in the medium as well as NPs from the bottom part of the cultivation container which were not incorporated during the first days of cultivation. As a result, a relatively large amount of NPs is integrated into the bottom surface. Their content and distribution depends on the mass concentration of NPs dispersed in the medium (Table 1 and Figure S2c, f, and i). For higher concentrations like in BNC-T20, agglomerates become visible. The concentration of NPs in the bottom surface exceeds that one in the top surface (Table 1). This can be explained by the high mass concentrations of the prepared anatase NP dispersions. An increasing NP concentration increases the probability for the agglomeration of the NPs. Thus, a higher amount of NPs is located in the bottom part of the cultivation container and is incorporated in the bottom surface instead of the top surface (Figure 1j and l). However, the allover amount of NPs that is incorporated in each layer and in the resulting hybrid increases with increasing mass concentration of NPs in the culture medium. These results correspond with the results of the XRD measurements (Figure 2). Furthermore, very similar results were described for the incorporation of silica NPs during the biosynthesis of BNC.39 The amount of silica NPs in the hybrid could be increased by increasing the content of silica sol in the culture medium. 4.3. Photocatalytic Activity of the BNC-TiO2 Hybrids. The PCA of the BNC-TiO2 hybrids depends on the mass concentration of incorporated anatase NPs. The corresponding supramolecular structures confirm that the NPs are homogeneously distributed within each layer of the hybrid material. Their content and distribution depends on the corresponding layer. Nevertheless, with an increasing mass concentration of NPs in the culture medium the overall content of incorporated NPs in the hybrids increases. This is equivalent to an increased photocatalytically active surface area. As a result, an increasing number of hydroxyl radicals can be created (Figures 5a). Sample BNC-T20 has the highest content of incorporated anatase NPs (Table 1). However, Figure S2 suggests that the NPs are mainly agglomerated. Consequently, the reactive surface area is reduced and the PCA is not further increased compared with sample BNC-T10. The stability of the photocatalytically active BNC-TiO2 hybrids was demonstrated by the repeated performance of the MC test. No consumption or degradation of the photocatalyst occurred after the 10-fold revision of the MC test (Figure 5b)

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S3. EDX diagram measured from the cross-section of the BNC-TiO2 hybrid sample BNC-T10 (Figure S1). Distribution of the NPs within the hybrids evaluated using ImageJ (Figure S2). Decomposition of methylene blue under near-UV irradiation (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +49-3641-947750. Fax: +49-3641-947702. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.G., H.-D.K., and F.A.M. thank the German Research Foundation (Deutsche Forschungsgemeinschaft − DFG) for financial support (MU1803/8-2). F.W.’s contribution was supported by the scholarship program of the German Federal Environmental Foundation (Deutsche Bundesstiftung Umwelt − DBU). The authors thank Elena Pfaff (ITUC Jena) and Heidrun Garlipp (IMT Jena) for kinetic investigations and SEM micrographs, respectively.

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REFERENCES

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