3.1. X-Ray Diffraction Analysis of Extracellular Polymeric Substance
X-ray diffraction (XRD) is a rapid analytical and spectroscopic technique most widely used for phase identification of a crystalline material. A polymeric materials can be considered partly crystalline and partly amorphous. Extracellular polymeric substances (EPS) are the prolongable, green and the important group of polymeric materials. These type of biological polymers have a wide range of high molecular weight molecules like polysaccharides, proteins, nucleic acids, phospholipids, uronic acids, humic substances, lipids, etc [27]. Due these functional groups intra- and intermolecular H bonding can be important role in EPS and formation of crystalline forms.
XRD analysis is used for the structural analysis of EPS to determine the crystallinitiy degree and crystalline/amorphous character of the substance. Fig. 1 showed the XRD pattern of the cyanobacterial extracellular polymeric substance in the range of 2–70°. The XRD diffractogram of EPS exhibited the characteristic mainly diffraction peaks at 9.12, 18.2, 29.1, 31.8, 37.7, 45.5 and 56.5° with inter-planar spacing (d-spacing) 9.719, 4.927,3.164, 2.920, 2.521, 2.159 and 1.847 A°, respectively (Fig. 1). XRD pattern also predicts that EPS is just about the amorphous in nature with %13.47 percent crystallinity. In the diffractogram, crystalline parts can be recognized as sharp narrow peaks while amorphous phase gives a broad peak with low-crystalline structure. It could be difficult to interpretation of broad peaks of several amorphous polymers in X-ray scattering diffractogram and therefore the ratio between crystalline and amorphous intensities is used to calculate the amount of crystallinity in the material.
3.2. Differential Thermal Analysis (DTA) of Extracellular Polymeric Substance
Thermal stability and weight loss of EPS with Thermogravimetric Analysis (TGA) was analysed and publish our previous study [16]. EPS of cyanobacterial origin showed the degradation in three steps and with the temperature range 30 and 800 oC. Thermal degradation of EPS of cyanobacterial can be identified as in three main steps and with in the temperature range 30 and 800 oC. Thermal stability can be ascribed to the existence of sulfate groups, uronic acids and calcite crystal that prevent whole of decomposition. Degradation rates arranging in a decreased order from amide I, amide II and polysaccharides-like substances to polysaccharide can be given [16]. Tg is the glass-rubber transition that occurs in amorphous polymers and Tm (melting point) occurs when the ordered regions of a polymer fall apart upon heating can be seen From the DTA curve. When the temperature increases of macromolecule, an amorphous solid will become less viscous. Then a particular temperature the molecules obtain enough freedom of motion to spontaneously arrange themselves into a crystalline state [28]. This transition from amorphous solid to crystalline solid is an exothermic process and DTA analysis showed a significant thermal transition of EPSs (Fig. 2).
From the DTA thermogram of the extracellular polymeric substance, the first broad peak can be defined as glass transition temperature, Tg, at around 50.0°C. DTA thermogram also showed characteristic exothermic transition of exopolymer with melting of crystalline regions at 97.8°C followed by Tg transition. This transition peak can be explained as water molecules due to the hydrophilic groups of cyanobacterial EPS such as carboxyl, sulfate and hydroxyl.
Theoretically, water may induce the glass-rubbery transition (Tg) of amorphous regions of EPS before the melting of crystals. A broad endothermic transition at 200°C can be attributed as melting of crystal regions of EPS due to the α-1-4-glucose linkage (C-O-C) of polysaccharides units [16]. Releasing absorbed water and chain conformational change that could be associated with inter- and/or intramolecular interactions of polymer chain give two endothermic processes at 97.8°C and the other at 200°C [29]. Besides to the water release of absorbed water, these endothermic processes also can be resulted from the movements and interactions of lateral groups and associated with movements of the main skeleton at 350°C and 480.4°C, respectively. At the corresponding DTA thermogram (Fig. 2) exhibit endothermic relaxations whose temperature to higher temperatures and the fixed area confirmed the existence of strong inter–intra chain interactions [30].
3.3. Dynamic Mechanical Analysis of Extracellular Polymeric Substance
Dynamic mechanical analysis is one of the most important techniques used to investigate the viscoelastic properties of polymeric materials. In particular, the dynamic mechanical viscoelastic behaviors of biological materials, natural polymers and polysaccharides are very important to describe their basic properties and behaviors. In this technique, sinusoidal oscillating stress is applied and the response of a polymeric material, sinusoidal deformation, is mainly measured at a certain fixed frequency and a temperature range. Viscoelastic properties of the polymeric materias can be described by parameters obtained from the stress response due to the sinusoidal deformation [31].
The dynamic mechanical properties provide information of one of the most fundamentals properties of polymers, the glass transition temperature (Tg). The Tg can be obtained by storage modulus (SM) onset, loss modulus peak (LM), or tan δ peak.
Figure 3 showed the storage modulus (SM) and derivative storage modulus (dSM) curves of the EPS in the temperature range of 30-250 °C. The storage modulus indicated the elastic nature of the polymeric material and related with the stored energy representing elastic part in a deformation. The storage modulus showed a typical shape for polymeric material that decreased with increasing temperature. Four transition temperatures were observed from the dSM-T curve at 48.00 °C, 91.53 °C, 204.72 °C and 219.85 °C. Up to 48.00 °C the EPS segments were rigid and chain segments were frozen in a fixed positions (glassy region) [32]. The first transition temperature, 48.00 °C were defined as the glass transition temperature for the EPS and obtained from the temperature of the midpoint of the inflectional tangent at SM-T curve [33] and peak minimum at dSM-T curve. The second observed peak at 91.53 °C may due to the removal of water molecules. Because EPS contains functional groups such as carboxyl, sulfate and hydroxyl, and these groups can removed from the structure with the increasing temperature. The other two peaks observed at higher temperatures were related with the degradation/decomposition.
Figure 4 showed the loss modulus and tan δ plots depending on temperature. The loss modulus (LM) demonstrated the viscous nature of the EPS and related to the loss energy representing the viscous part of molecule. Tan δ (loss tangent) is another dynamic mechanical parameter measures damping in the polymeric material which is equal to the ratio of the loss modulus to storage modulus. It was observed that the loss modulus and tan delta followed the similar behavior with the storage modulus. The loss modulus and tan delta curves reached a maximum with the increase in temperature and then decreased due to the energy loss caused by the free movement of the segments [34]. The three peaks were observed from both of these curves with a small shoulder at higher temperature region.
The main transitions were seen at 56.95 °C, 94.50 °C, 225.11 °C and 59.99 °C, 94.50 °C, 226.61 °C from LM-T and tan δ-T curves, respectively. All transition temperatures obtained from DMA parameters (SM, LM and tan δ) were in good agreement with each other. The first peak maximum values at lower temperatures were associated with the glass transition temperature of EPS. The second and the third peak maximum values were originated from the removal of small groups and degradation/decomposition of main chains of the EPS. Although the glass transition temperature is generally reported as peak in the tan delta in the literature, the glass transition temperatures found from other parameters are also used as long as it is specified from which parameter they are found.
Figure 5 and 6 showed the behavior of dynamic force, complex modulus, dynamic and complex viscosity with increasing temperature at a constant frequency. The complex modulus is the total resistance of a polymeric material to deformation, however, whether the deformation of the polymeric material is elastic or viscous [35]. The dynamic force and complex modulus gradually decreased with increasing temperature up to ≈ 200 °C in two steps and decreased sharply after this temperature region. The dynamic viscosity plots showed three peaks at 55.14 °C, 94.50 °C, 225.11 °C with a shoulder at 200.98 °C. While the complex viscosity initially decreased slightly with increasing temperature in the low temperature region, it decreased sharply after reaching the high temperature. The reason of this behavior can be interpreted as the thermal transition of polymeric EPS from solid state to soft state with increasing temperature.
The storage modulus, complex viscosity and dynamic force values at a certain temperatures (30 °C, 50 °C, 100 °C, 150 °C, 200 °C and 250 °C) were summarized in Table 1 for the cyanobacterial EPS.
All parameters showed slow decreasing behavior up to about 200 °C, and after this temperature they showed a sharp decreasing trend due to the degradation/decomposition of the main chain cleavage of the polysaccharide component in cyanobacterial EPS.