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Научни трудове на Съюза на учените в България-Пловдив. Серия В. Техника и технологии, естествен ии хуманитарни науки, том XVI., Съюз на учените сесия "Международна конференция на младите учени" 13-15 юни 2013. Scientific research of the Union of Scientists in Bulgaria-Plovdiv, series C. Natural Sciences and Humanities, Vol. XVI, ISSN 1311-9192, Union of Scientists, International Conference of Young Scientists, 13 - 15 June 2013, Plovdiv.
APPLICATION OF ELECTRON-MICROGRAPH IMAGE ANALYSIS TO NANOSTRUCTURES OF BIOGENIC ORIGIN
R. Angelova(12), E. Balabanova(2), L. Slavov(2), M. Iliev(1), M.V. Abrashev(3V I.
Nedkov(2), V. Groudeva(1)
(1): Faculty of Biology, Sofia University "St. Kliment Ohridski", Dragan
Tzankov Blvd. 8, BG-1164 Sofia
(2): Institute of Electronics, Bulgarian Academy of Sciences, Tsarigradsko
Chaussee blvd. 72, BG-1784 Sofia
(3): Faculty of Physics, Sofia University "St. Kliment Ohridski", J. Boucher
blvd. 5, BG-1164 Sofia
e-mail(1): angelova87@abv.bg
Abstract
The further development of biotechnologies necessitates the application of reliable methods for data processing to improve the research efficiency and the bioprocess control and management. This work is focused on electron micrograph processing as applied to the study of bionanomaterials. The latter were obtained from the metabolic activity of iron oxidizing bacteria from the Sphaerotilus-Leptothrix group cultivated under laboratory conditions. The identification of the isolates gathered from natural sources was performed by classical and molecular taxonomy methods. Raman analysis was used to determine the bio products' chemical composition; SEM and microscopic image analysis were employed to study and characterize the shape and size of the different bacterial product formations and the bacteria themselves. The formations had tubular shape and characteristics typical for the Leptothrix genus. The image analysis method applied allowed us to optimize the genus Leptothrix bacteria cultivation and the biogenic production of
iron hydroxides.
Introduction
The need of reliable information on the structural characteristics (e.g. mean size, size distribution, shape etc) of nanosized materials naturally leads one to using electron-micrograph image analysis, which has been proven to be a very successful method in this respect. TEM or SEM micrograph processing (image analysis) allows one to study the structure of different kinds of nanomaterials (powders, thin films, composites, carbon nanotubes etc.). The electron micrograph image analysis is a universal method giving one the opportunity to analyze all types (hard and soft) of solid-state materials. The SEM micrograph technique has been applied successfully to the observation of biological samples [1].
The iron oxidizing bacteria from the Sphaerotilus-Leptothrix group are of great interest
because during their growth they produce extracellular formations consisting of Fe-oxides and hydroxides. In a natural environment, the process takes place in free-flowing water streams at an altitude over 1000 meters above sees level. The microbial biomass in such habitats is abundant; however, cultivating these bacteria under laboratory conditions does not usually result in forming large quantities of iron oxides/hydroxides; this still constitutes a major research problem.
The formations produced differ in size and diameter. Studying the formations is very important because their shape depends on the bacteria type. It is known that Leptothrix ochracea yield hollow microtubes (sheaths), while Gallionella ferruginea produce twisted stalks, and, sometimes, perfect spheres [2]. Therefore, describing the morphology of the formations observed could help one in finding the best cultivating conditions for each specific type of bacteria. Moreover, knowledge of the formations' size provides valuable information needed in view of the products application. One possible, and a very attractive, application is fabrication of thin film consisting of ordered spherical Fe-oxides formations injected with a magnetic material. Such "natural" films can be used as recording media, industrial catalysts, adsorbents, and pigments [3], or after a suitable heat treatment, for producing magnetite nanocomposites [4].
Aim
The main purpose of this work was to analyze the structural characteristics of the products of iron-oxidizing bacteria pure cultures by using SEM micrograph image analysis, namely, determine the size of the formations produced and obtain their size distribution. Furthermore, we used Raman spectroscopy to obtain information on the type of iron content in the biogenic oxides produced.
Materials and methods
Bearing in mind the specific environmental requirements of the target group of microorganisms, a habitat located in the Vitosha Mountain was chosen as the main area of sampling (Fig. 1). The altitude of the location is 1783 m with coordinates 42°35'15"N/23°14'55"E. The average temperatures measured at this altitude are relatively low (average monthly temperature in January is -4°C, in June, +13°C). The region is characterized by a large number of small water sources. To mimic the natural conditions for iron bacteria cultivation, we used three nutrients media (described in Table 1), and two types of cultivation vessels as described below (Fig. 2). The specific shape of the selected tubes provides high volume and surface for aeration of the culture medium in order to allow the optimal conditions for bacterial growth. The pH was maintained at about 7. The cultivation was carried out at 16 °C under static conditions.
(Fig. 1). Typical deposits in the water flow. (Fig. 2) Cultivation vessels:
Roux Flasks Fehrenbach Flasks
The pure cultures were obtained from enriched samples on an isolation medium (IM) following a standard procedure. The taxonomic status of the pure cultures was determined by the methods of the classical taxonomy according to Bergey's identification key (Bergey's Manual of Determinative Bacteriology, 8-th ed., 1989) on the basis of the morphological, physiological and biochemical characteristics. For the confirmation of the status of the isolates to genus Leptothrix, a molecular taxonomy method (PCR detection assay) was used. The bacterial cells were harvested by centrifugation (4 500 rpm/10 min), the cell pellet was washed with PBS and subjected to DNA isolation with Prep Mini Spin Kit (GE Healthcare).
The published sequence of the mofA gene (GenBank № Z25774.3) was chosen as a specific target for PCR detection of Leptothrix spp. The specific primers were constructed with Primer-Blast Software. F1_ thrix e 5'-TGT-TCG-AGC-CGG-TGT-TCG-GC-3', and R1_ thrix 5'-GAA-TCG-ATC-GCG-ACC-GGC-GT-3'. The PCR mixture contained 1 ^M of each primer (Sense and Antisense), 0,2 mM dNTPs, Taq buffer 1x (Invitrogen), 1,5 mM MgCl2, 2,5 U Taq polymerase and 5 ^l (10-100 ng) total DNA (Ready-To-Go PCR kit (GE Healthcare). The PCR program consisted of an initial denaturating step lasting 95°C/5min, followed by 35 cycles (95°C/1min; 54°C/1min; 72°C/1min) and a final extension step at 72°C for 5 min. All reactions were carried out on an Eppendorf Thermocycler (Eppendorf).
The cultures cultivated on different media were investigated by SEM using a scanning electron-microscope JEOL JSN-5510, JEOL, Japan; magnification x10 000.
The statistical analysis of the objects observed on the microphotographs was performed by a program for image analysis.
Table 1. Content of the media used.
Sample Culture media Contents
1 "Fedorov" (NH4)2SO4 - 1,5g; MgSO4.7H2O - 0,05g; KCl - 0,05g; Ca(NO3)2 - 0,03g; dH2O - 1000 ml; Fe-source: Fe-grit, Fe-sulfate; pH 7,0
2 "Adler" Na-lactate - 40,0 mg; Yeast extract - 1,0 g; Vitamin C - 0,1 g; MgSO4.7H2O - 0,2 g; K2HPO4 - 0,01 g; FeNH4SO4 - 0,01 g; dH2O -1000 ml; pH 7,0
3 "Isolation media" Glucose - 0,150 g; (NH4)2SO4- 0,5 g; Ca(NO3)2 - 0,01 g; K2HPO4 -0,05 g; MgSO4.7H2O - 0,05 g; KCl - 0,5 g; Vitamin B12 - 0,00001g; Thiamin - 0.0004g; dH2O - 1000 ml; Fe-source: Fe-grit, Fe-sulfate; pH 7,0
We used Raman spectroscopy to identify the different iron bioproducts; this technique allows one to characterize many types of samples without any specific preliminary preparation [5]. Glass slides with different biofilms depending on the media used were prepared from FeOB by three drop-wise depositions of biomass. Each layer was dried at room temperature.
Thus prepared, the samples were examined by Raman spectroscopy using a LabRAM HR Visible single spectrometer equipped by a microscope and a Peltier-cooled CCD detector. The 633-nm He-Ne laser-line was used for excitation. The power was adjusted using a set of neutral filters and was kept low (> 0,1 mW); the laser beam was focused on the surface by a ><20 long-working-distance objective. The spectral slit width was 1 cm-1. The acquisition time ranged between 3 and 300 s (three accumulations for each scan) for all optical excitation intensities. The Raman measurements were performed at room temperature and atmospheric pressure.
Results and Discussion
After identification by the classical taxonomic scheme of Bergey Manual of Determinative Bacteria, it was found that the isolated pure cultures belonged to genus Leptothrix. This finding was confirmed by the molecular method used. All nine strains tested were positive for product size 781 bp corresponding to the expected one (Figure 3). The results obtained confirmed that the selected primers were sufficiently informative for detecting bacteria of this genus. In addition, this type of PCR allows proving the bacteria of the genus in complex samples.
Figure 3. Amplification profile of mofA - PCR.
Micrographs of the samples are shown in Figures 4a-c. As it is seen from the SEM analysis in Figures 4a-c, the objects have a tubular shape and are thus characterized by two dimensions - a diameter and a length. We evaluated the distributions of both dimensions. Figures 4a-c present the the tubular objects investigated; the respective distributions (diameters, lengths) are shown in Figures 5a-c.
Figure 4a SEM of sample 1
Figure 5a Size distributions for sample 1
JL
Da
average diameter 0,477 pm average length 0,852 pm
Mil
0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 tube- diameter (white), length (black), pm
Figure 4b. SEM of sample 2
Figure 5b. Size distributions for sample 2
0,0
In what concerns the studies reported here, Raman spectroscopy is a useful tool as it determines the phase composition and provides clear and unambiguous information about the state of the iron in the material obtained. Thus, the Raman measurements revealed that the iron (III) (oxy) hydroxide is the basic phase found in the biogenic materials. Figure 6 presents the Raman spectra alongside optical microscope photographs of the places where the laser beam was focused (at x = 0 and y = 0).
Figure 6. Raman spectra and optical microscope photographs of bacteria-derived biogenic material
600 800 Raman shift (cm-1 )
Figure 6a. Raman spectra of sample 1: a) FedoroVs medium - (a) goethite (a-FeO(OH)); _(b) lenidocrncite (v-FeQ(OH)); (c) magnetite (Fe^OJ_
I8 Adler (Fehrenbach)
§ JS"*"""'"'^ (a)
Si s s ... (b)
(c) S
(d)
400 600 800 1000
Raman shift (cm-1)
Figure 6b. Raman spectra of sample 2: b) Adler's medium (Fehrenbach) - a) and (b) _lepidocrocite (Y-FeO(OH)); (c) and (d) magnetite (FeO)._
200
Intensity (a.u.) Isolation medium (Fehrenbach) 1 (a) J^ ^ (b)
200 400 600 800 1000 1200 Raman shift (cm-1)
Figure 6c. Raman spectra of sample 3: c) Isolation medium (Fehrenbach) - (a) h (b) lepidocrocite (y-FeO(OH)).
Conclusions
The results obtained show that the variation of the substrate content and concentrations of the different components leads to a variation of the size and diameter of the cultures grown. The type of iron-bearing compounds produced is also dependent on the content of the culture medium. Raman spectroscopy revealed that the major compounds found in the samples were the two iron oxyhydroxides (goethite and lepidocrocite), with the unexpected presence of magnetite in the samples from Fedorov and Adler's medium. Further investigations are needed to clarify the connection between these variations, the medium components and the hydroxides formed.
Acknowledgments
This work was supported in part by the National Science Fund at the Ministry of Education and Science of Republic of Bulgaria under project DID 02/38/2009.
References
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[3] Sawayama M. et al., Curr. Microbiol. 63 (2011) 173 -180.
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