Chapter 51
Effect of ZnO Nanostructured Thin Films on Pseudomonas Putida Cell Division I. Ivanova, A. Lukanov, O. Angelov, R. Popova, H. Nichev, V. Mikli, Doriana Dimova-Malinovska, and C. Dushkin
Abstract In this report we study the interaction between the bacteria Pseudomonas putida and nanostructured ZnO and ZnO:H thin films prepared by magnetron sputtering of a ZnO target. The nanostructured ZnO and ZnO:H thin films possess some biological-active properties when in contact with bacteria. Our experimental data show that these films have no destructive effect on the cell division of Pseudomonas putida in poor liquid medium and can be applied in biosensor devices. Keywords Pseudomonas putida ZnO nanostructured film Live/dead test
Introduction The bactericide effect of some metal oxide nanoparticles, in particular ZnO, has recently received significant attention due to their potential application in pharmacology and medicine as antibacterial agents [1, 2]. It was found that the toxicity of ZnO particles and of ZnCl2 is essentially due to dissolved Zn2+ ions [3]. Other
I. Ivanova and A. Lukanov Faculty of Biology, Department of Microbiology, Sofia University “St. Kl. Ohridski”, Sofia, Bulgaria O. Angelov, H. Nichev (*), and D. Dimova-Malinovska Central Laboratory of Solar Energy and New Energy Sources, Bulgarian Academy of Sciences, 72 Tzarigradsko Chaussee, 1784 Sofia, Bulgaria e-mail:
[email protected] R. Popova and C. Dushkin Faculty of Chemistry, Department of General and Inorg. Chemistry, Sofia University “St. Kl. Ohridski”, Sofia, Bulgaria V. Mikli Centre for Materials Research, Tallinn Technical University, Ehitajate tee 5, 19086 Tallinn, Estonia J.P. Reithmaier et al. (eds.), Nanotechnological Basis for Advanced Sensors, NATO Science for Peace and Security Series B: Physics and Biophysics, DOI 10.1007/978-94-007-0903-4_51, # Springer Science+Business Media B.V. 2011
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group has reported that the concentration of ZnO nanoparticles is more important than the particle size, and the reason for the toxic effect is damaging of the cell membrane wall [4]. In this study results for the nondestructive influence of ZnO thin films on the cell division of Pseudomonas putida are reported encouraging the development of biosensors on the basis of ZnO thin films.
Experimental Preparation of ZnO Nanostructured Thin Films Two set of ZnO samples were used. ZnO and ZnO:H thin films were deposited on glass substrates by r.f. magnetron sputtering of a ceramic ZnO target in atmospheres of Ar (0.5 Pa) or Ar (0.5 Pa) + H2 (0.1 Pa) at substrate temperatures Ts of 500 C and 400 C and a r.f. power of 180 W. The thickness of the films was about 600 nm. The optical and structural properties of the films were studied by transmittance and reflectance measurements, atom force microscopy (AFM), scanning electron microscopy (SEM) and X-ray diffraction (XRD).
Toxicity Test With Bacteria To study the influence of the nano-size grained ZnO thin films on the bacterial activity of Pseudomonas putida cells two types of nutrient media were used: the rich natural and the poor synthetic medium ISO 12072. The ZnO and ZnO:H films were sterilised by UV light for 30 min and put in the bacterial suspension in an exponential growth phase. The bacteria were prepared by inoculation in the solid rich medium and adapted to the poor mineral medium, as described in the ISO standard by three consecutive subcultivations. They were cultivated in aerated suspension in dark and light conditions at 25 C using an orbital benchtop shaker operating at 220 rounds/min. The light conditions were created by illumination with a tungsten lamp (100 W) at a distance of 1 m. The optical density of the bacterial suspension was measured. Then immediately tenfold dilutions were prepared, inoculated and cultivated on the rich medium. A suspension of 0.1 ml was formed and analysed by a fluorescence microscope. The samples were collected after 3, 6, 9, 12 and 24 h. The bacterial abundance ranged from 105 to 108 cells/ml in the poor and rich media. The ZnO toxicity was measured by comparison of the ratio of live/dead cells by applying the special kit from Promega Live/Dead® BacLight (TM) with propidium iodide and SYTO 9. A fluorescence microscope (Leica DM5500B) was used to observe the samples. The image analysis was based on a two steps procedure: the recorded micrograph of
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each microscopic field was first numerated and stored in the data base folder. In the second step the digitalized images were submitted an to the Image J. software. The background was subtracted manually. The manual counting of bacterial cells was performed by two independent operators. The percentage of living active cells was determined from the images by counting the green (live) and red cells (considered as dead or inactive). The quantity of living cells was determined by the most probable number method in the rich solid medium and by measurement of the optical density by Specol 11 at l ¼ 600 nm in the poor medium.
Results and Discussion The optical properties of ZnO and ZnO:H films were studied by transmittance and reflectance measurements. The spectral dependence of (aE)2 versus the energy E is presented in Fig. 51.1 where a is the coefficient of absorption. The calculated energy gaps Eg between 3.27 and 3.33 eV are typical for ZnO. Our previously published data [5] show that hydrogen changes the stoichiometry of the sputtered species, so the growth conditions lead to a decrease of defects. The XRD spectra in Fig. 51.2 reveal the polycrystalline structure of the films with a preferential crystallographic (002) orientation with the c-axis perpendicular to the substrate surface. The AFM pictures in Fig. 51.3a (ZnO) and b (ZnO:H) show rough top surfaces of the films. The grain size is about 100 nm for ZnO and 150 nm for ZnO:H films. We observed that in all experiments the optical density of the suspensions with nanostructured ZnO and ZnO:H increases faster than that in the control experiment; thus the cells division of Pseudomanas putida is not inhibited (Fig. 51.4). This phenomenon was proven by classical cultivation methods. In the solid medium we counted only the active cells after 18–24 h of cultivation at 25 C. Under these conditions it seems clear that only the active dividing cells create visual colonies. The bacterial growth in contact with the samples is comparable with those for rich and poor liquid media. Cells exposed to ZnO in the poor medium divide faster than
Fig. 51.1 Spectral dependence of (aE)2 versus the energy E of ZnO and ZnO:H thin films
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Fig. 51.2 XRD spectra of ZnO (Ts ¼ 500 C) and ZnO: H (Ts ¼ 400 C)
Fig. 51.3 AFM pictures (3D images, 2 2 mm) of a ZnO film (a) and a ZnO:H film ( b)
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Fig. 51.4 Density of Pseudomonas putida bacteria grown in (i) poor medium as control experiment, (ii) poor medium with nanostructured ZnO thin films and (iii) rich medium
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those from the control experiment under the same condition. However, the rates are close to the cell division rate in the rich medium. It was observed that the percentage of living cells having contact with the nanostructured thin film is more than 90% of the total amount of cells. In the control experiment the living cells were about 65%. This difference could be due to the activation effect of the ZnO nanofilms. We did not observe any significant difference between ZnO and ZnO:H nanofilms. The reason may be that the amount of Zn2+, which is dissolved into the suspension, is equal. The presence of zinc ions even at very low concentrations in this poor medium influences on the cell division rate, leading to its increase. If the concentration is higher the effect is opposed, i.e., toxic. This hypothesis was tested by an additional control experiment. Our expectations are in congruence with the results from the image analysis. We also found that in spite of the similarity between the division rates in the presence of ZnO and ZnO:H, the treated cells are much smaller in size than the cells in the rich medium. This fact may be explained by the presence of enough amounts of nutrients in the rich medium, which lack in the poor medium with the ZnO nanofilm.
Conclusions This study report the influence of nanostructured ZnO and ZnO:H thin films on the cell division rate of Pseudomanas putida. Our experiments prove unambiguously that the presence of ZnO thin films is not toxic for the bacterial microsuspension. They activate the rate of the cell division and increase the percentage of live cells. There is no significant difference regarding effect between ZnO and ZnO:H nanostructured thin films. These results are encouraging for the development of ZnO based biosensor devices. Acknowledgement This work has been supported by the Bulgarian Ministry of Science and Education, National Sci. Fund, Project: D002-207/2008 and by the Operative program “Human resources” (project BG 051PO001/07/3.3-02/58/17.06.2008).
References 1. A. Kroll, M. H. Pillukat, D. Hahn, J. Schnekenburger, Eu. J. Pharmaceutics and Biopharmaceutics 2, 370 (2009). 2. O. Yamamoto, Int. J. Inorg. Materials 3, 643 (2001). 3. H. Karlsson, J. Gustaffson, P. Cronholm, L. M€ oller, Toxicology Letters 188, 112 (2009). 4. N. M. Franklin, N. Rogers, S. Apte, G. Batley, G. Gad, P. Casey, Environ. Sci. Technol. 41, 8484 (2007). 5. D. Dimova-Malinovska, O. Angelov, H. Nichev, J.C. Pivin, JOAM 9, 248 (2007).
