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Научни трудове на Съюза на учените в България - Пловдив Серия В. Техника и технологии, том XIII., Съюз на учените, сесия 5 - 6 ноември 2015 Scientific Works of the Union of Scientists in Bulgaria-Plovdiv, series C. Technics and Technologies, Vol. XIII., Union of Scientists, ISSN 1311-9419, Session 5 - 6 November 2015.
ФИЗИЧЕСКИ ХАРАКТЕРИСТИКИ НА ПОЛИЗАХАРИД СИНТЕЗИРАН ОТ ХАЛОФИЛНИ МИКРООРГАНИЗМИ ОТ ЩАМ
Chromohalobactercanaden sis И. Панчев^М. Кунчева2,Д. Ковачева2, М. Ка мбурова3, Н. Радченков а3, И. Бояджиева3 Университет по хранителни технологии,Пловдив, България1; Институт по обща и неорганична химия, БАН, Софил, България2 Институт по м икробиолония, БАН, София, Бългалия3,
PHYSICAL CHARACTERISTICS OF EXOPOLYSACCHARIDE SYNTHESIZED FROM HALOPHILIC MICROORGANISMS OF1 THE Chromohalobactercanadensis °TRAIN I. Panchev1, M. Kuncheva2, D. Kovacheva2, M. Kamburova3, N. Radchenkova3, I. Boyadzhieva3 University of FoodTechnologies, Plovdiv, Bulgaria1; Institute of General and Inorganic Chemisiry-BAS, Sofia, Bulgaria2; Institute of Microbiology-BAS, Sofia, Bulgaria3
Rbsiraci:
This work presents experimental daia from DTR-TG, SEM, XOD, FT-IO analyses, intrinsic and dynamic viscosity, optical and surface activities of a newly synthesized biopolymer of the Caromoaalbactre caeadeesys strain. It was found that the biopolymer is water-soluble, has a surface activity, but does not have optical activity. Its intrinsic viscosity is [ =3.26 dl.g-1 The thermal stability of its macromolecule is retained up to 170 °С after which pyrolisis processes of degradation take place. Key words: biopolymer, Caromoaalobactercaeadeesis, DTR-TG, SEM, XOD, FT-IO
Introduction:
In the recent decades worldwide there is an acute need for industrial produciton of polysaccharides with new properties, which determines the interest in a study of the possibilities for biosynthesis of exopolysaccharides from microorganisms [1]. In Bulgaria, the extraordinary properties of extremophilic bacteria and the polymers synthesized therefrom are subject of study at the Extremophilic bacteria Laboratory with the Institute of Microbiology, BAS [2]. Microbial diversity in Bulgarian halophilic niches has not been the subject of study until now, nor are known their capacity for biosynthesis of polysaccharides. Unfamiliar are also the physical characteristics of newly synthesized biopolymers.
The aim of this work is to obtain experimental data on some essential optical, rheological and thermophysical characteristics of EPS synthesized from Chromohalobactercanadensis which are halophilic microorganisms isolated from the Atanasovsko lake in Bulgaria.
Materials and methods:
Diversity and biosynthetic ability for exopolysaccharide production of heterotrophic moderately halophilic and halotolerant bacteria isolated from three hypersaline ecosystems at the southern Black See coast, Bulgaria, were investigated. TheChromohalobactercanadensis 66
strain was observed as a perspective EPS producer. The optimal temperature for synthesis was 30 °C, the optimal pH was 7.3 and the optimal concentration of NaCl was 15 %.
The FT-IR spectra of EPS were implemented using KBr tablets on a spectrometer Nicolet Avatar 330 FT-IR, Thermor Electron Corporation, Madison, USA.
The surface tension was determined for aqueous solutions at concentrations of 0.025, 0.05, 1.5, 2.0% (w/v) and was measured with a tensiometer by Kruss, Germany. Shishkovski's equation (1) was used for the analytical description of surface activity [3]
where: a0 - surface tension of water; a - surface tension of solutions; c - concentration; a, b - constants.
The rheological properties of aqueous solutions of EPS were determined using an Ubbelohde type of capillary viscosimeter with a capillar diameter of 0.54 mm and Rheoviscometer Reotest 2, Germany with measuring cylinder N within velocity gradient change range 1.5 <y<1312 s-1. The numerical processing of the experimental data was carried out using software approximating by the least squares method [4], the Huggins equations (2) and the Oswald-de Waele power law (3)
where KH is the Huggins constant, r|ri is the relative viscosity, c is the concentration of solution, t is the shear stress and y is the shear rate, K is the flow consistency index (Pa.s) which coincides with dynamic viscosity at the power index n = 1 for Newtonian fluids.
The optical activity of EPS was identified by the automatic polarimeter POLAMAT A, Carl Zeiss, Jena, Germany.
DTA and TG measurements: The methods were implemented using the LABSY Sevo apparatus by SETARAM, France in the temperature region of 10 - 300 °C, as the samples were heated at a rate of 5 °C min-1by gas carrier -synthetic air passing through the measurement chamber at a speed of 20 ml min-1. The samples weighing 10 - 20 mg were placed in a corundum crucible.
SEM:The EPS samples under study were observed on a scanning electronic microscope JSM T 200 (Jeol, Japan) at 15 kV accelerating voltage of electrons in modes of secondary electrons (SEI - secondary electron imaging) and backscattered electrons ( BEI -backscattered electron imaging). The second mode offers possibilities for registering the image in modes of topography (TOPO) and qualitative differences in the ingredients of the items under study (COMPO).
XRD were collected at room temperature 25 °C on a Bruker D8 Advance instrument with CuKa radiation and LynxEye detector within the 2D range from 5.3 to 80 °C,2D degrees at counting time 1 s/step. Data evaluation was made with the use of a Software package EVA phase identification was made with the use of data base ICDD-PDF2.
Results and Discussion:
Valuable information and an important feature of the ingredients and structure of polysaccharides is provided by their IR-spectrum. Fig. 1 presents the infrared spectrum with Furrier transformation (FT-IR) of the assayed EPSproduced from the Chromohalobactercanadensis strain. There are characteristic absorption bands characteristic of plant polysaccharides. The broad asymetric and intensive band at 3300 - 3600 cm-1 with a peak at 3420 cm-1 is characteristic of the valentoscillations of -OH groups involved in the formation of intramollecular hydrogen bonds. The absorption band at 2924 cm-1 is characteristic of the valent oscillations of the C-H bond in a pyranose ring, while the morenarrow band at 1374 cm-1 is indicative of deformation oscillations of C-H bonds. The band at 1653cm-1 can be attributed to S(H2O). As other authors have also observed [5], the band at 1645 - 1654cm-1 for plant glucomanan is due to the in plane deformation of the water molecule. This water is the strongly bound water of crystallization. Kacurakova and Wilson [6] have also registered this band for polysaccharides and also attribute it to structured water.
(1)
n?=№ + KH[v]2c
(2)
T = KYn
(3)
The authors note that the film was free of the broad adsorbed water deformation band at 1645 cm-1typical for KBr-spectra. The absorption peaks at 1452 cm-1 and at 1028 cm-1 are due to water bands and v(C-C) and v(C-O) in the C-OH groups of pyranose ring.
Figurel. FT-IR spectra of EPS, produced from the Chromohalobactercanadensis
strain.
The experimental data from the measurements with the capillary viscosimeter of dilute aqueous solutions of EPS after a mathematico-statistical processing of equation (1) allow allow to establish that the intrinsic viscosity [n] is (3.26 +/- 0.02) dl.g-1, while Huggins constant KH = 3.65.
The measurements of the rotation angle of polarized light made via the Polamat A при polarimeter at a wave length of 546 nm reveal that EPS does not manifest an optical activity (rotation angle a = +0.05 for a 0.5 % aqueous solution)
The rheological profile of concentrated aqueous solutions measured via Rheotest 2 was adjusted to the Oswald-de Waele power law (3), then computer processing of by the least squares method yielded the following values of К and n.
K = 73.7 mPa.sn n = 0.55 at the correlation coefficient R=0.9332 Quantitatively, the surface activity was determined by the change in the surfacetension of the aqueous solutions containing EPS in the concentration range of 0.025 - 2.5 %. The concentration relationship of surface tension is shown in Fig. 2. Figure 2 was used to identify the critical point in micelle formation concentration (CMC) for the EPS under study and it was 0.05 %.
л
4 60 I
z Y01
0 so D
| 40
1 30
J '"2 20 ¿O
10
0,5 1 Ц5 2
EPS concentration, %
о L o
Figure 2. The concentration relationship of surface tension
After treatment of experimental data for a from an equation(1) it received the nextnumber values for material constants a and b: a = 1.16 N.m-1b = 1.07.10-3 kg-1.m3
Figure 3 presents the thermophysical relations of EPS obtained from DTA and TG measurements.
Figure3. DTA-Tg analysis of EPS
It can be seen from the graphics that weight loss up to 150 °C are due to the capillary bound and by the EPS macromolecule adsorbed water, while after 166 °C pyrolisis processes of degradation took place whose peak was at 230 °C. The loss of weight during heating up to 270 °C was 25 %. This suggests that in future applications of EPS in cosmetic, pharmaceutical and food technologies,temperature effects of up to 170 °C will not affect the size of its macromolecule.
The photos below show CEM images ofpowder samples of EPS produced at various
c) d)
Figure 4. CEM of power pattern of EPS
The X-ray diffraction pattern of EPS is shown in Fig. 5 The sample shows only few broad humps at approximately 16.3, 29.6 and 40.1 degree 20, corresponding to the interplanar distances of 5.43, 3.01 and 2.14 Â.The result of the XRD indicates that the sample exhibits mostly amorphous nature with low crystallinity.
2-Theta - Scale
fflFile 62raw - Type 2Th/Th locked - Start 5 300 ■ - End 79 995 ■ - Step 0029 ■ - Step time 52 5 s - Temp 25 -C (Room) - Time Started 10 s - 2-Theta 5 300 ■ - Theta 2650 ■ - Chi 0 00 ■ - Phi 0 00 ■ - X 00 m Operations Import
Figure 5. X-ray diffraction spectra of EPS
Asknowledgements
The authors wish to thank the National Science Fund of the Ministry of Education for the Funds provided for research project B 02/26 under which this work was carried out.
References:
1. Yun U., Park H. (2003) Physical properties of an extracellular polysaccharide produced by Bacillus sp. CP912. Letters in Applied Microbiology, 36: 282 - 287
2. Radchenkova N, Vassilev S, Panchev I, Anzelmo G, Tomova I, Nicolaus B,Kuncheva M, Petrov K, Kambourova M (2013) Production and properties of two novel exopolysaccharides synthesized by a thermophilic bacterium Aeribacillus pallidus 418. ApplBiochemBiotechnol 171:31 - 43
3. Ludger O. Figura, Arthur A. Teixera (2007) Food Physics, p.218, Springer, Berlin
4. Les Kirkup (2012) Data Analysis for Physical Scientists, p.226, Cambridge University Press, Cambridge
5. Zhang H., M.Yoshimura, K.Nishinari, M.Williams, T.Foster, I.Norton, (2001) Gelation behaviour of konjac glucomannan with different molecular weights, Biopolymers 59, 38 - 50.
6. Kacurakova M., R.H.Wilson, (2001), Developments in mid-infrared FT-IR spectroscopy of selected carbohydrates, Carbohydrate Polymers, 44, 291 - 303.
