Article pubs.acs.org/Langmuir
Structure and Dynamics of a Polysaccharide Matrix: Aqueous Solutions of Bacterial Levan Elizabeta Benigar,† Iztok Dogsa,‡ David Stopar,‡ Andrej Jamnik,† Irena Kralj Cigić,† and Matija Tomšič*,† †
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana, Slovenia Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana, Slovenia
‡
S Supporting Information *
ABSTRACT: The polysaccharide levan is a homopolymer of fructose and appears in nature as an important structural component of some bacterial biofilms. This paper reports the structural and dynamic properties of aqueous solutions of levan of various origin obtained from dynamic rheological, small-angle X-ray scattering, static and dynamic light scattering, as well as density and sound velocity measurements, determination of polymer branching after per-O-methylation, and microscopy. Besides samples of commercially available levan from Zymomonas mobilis and Erwinia herbicola, we also isolated, purified, and studied a levan sample from the biofilm of Bacillus subtilis. The results of dynamic rheological and light scattering measurements revealed very interesting viscoelastic properties of levan solutions even at very low polymer concentrations. The findings were complemented by small-angle X-ray scattering data that revealed some important differences in the structure of the aqueous levan solutions at the molecular level. Besides presenting detailed dynamic and structural results on the polysaccharide systems of various levans, one of the essential goals of this work was to point out the level of structural information that may be obtained for such polymer systems by combining basic physicochemical, rheological, and various light scattering techniques.
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INTRODUCTION Polysaccharides and their aqueous systems continue to attract much attention in scientific research due to their high potential applicability in various fields of technology and also due to their high structural variety and complexity.1 However, it is this structural complexity that is usually the factor limiting the level of detail resolvable by modern physicochemical research techniques. A lot of structural studies of such systems are consequently prone to a phenomenological level of structural details. Static and dynamic light scattering techniques, which are in one variant well-established for direct detailed structural studies of particulate systems,2 seem to be somewhat more tedious when applied for studies of polymer solutions. In these cases they need to be employed with some precaution but can still provide some detailed effective molecular parameters of the polymer chains in solutions and gels.1,3−9 Modeling approaches to resolve the structural details of polysaccharide systems are scarce10,11 and increasingly hard to apply for branched polymers with high molecular mass. As is shown in the present paper in the case of dilute and semiconcentrated aqueous solutions of the nonionic bacterial polysaccharide levan of various origin, a combination of carefully chosen complementary techniques can nevertheless lead to detailed insight into the complex nature of such polymer systems. A biofilm is usually formed as a response of bacterial cells to adverse and stressful living conditions. Its basic structure is governed by various biopolymers, i.e., extracellular polymeric substances (EPS) forming the EPS-matrix. Such a matrix embraces bacterial cells, water molecules, ions, lipids, nucleic acids, and other products of cell metabolism. It physically © 2014 American Chemical Society
protects the cells against external influences but still enables intercellular communication via the transport of nutrients and signaling molecules. As such biofilm structures may be quite persistent,12 in practice we usually try to avoid their formation.13 Knowledge of the structure of the biofilm matrix and identification of the external factors that affect it represent an important contribution toward understanding biofilm formation and prevention of its occurrence. There is thus current interest in the structural properties of biofilms of the bacterium Bacillus subtilis subs. subtilis NCIB 3610. In a sucrose-rich liquid medium this organism forms a complex biofilm with the polysaccharide levan as its prevailing component.14 Levan is an extracellular β-fructan composed of fructofuranosyl rings, connected by β-(2,6) glycosidic linkages and occasional branching through the β-(2,1) glycosidic linkages. Our attention was drawn to the structure of this biofilm because levan of the genus Bacillus sp. has been reported to exhibit rather atypical nongelling behavior in aqueous solutions in comparison to other common polysaccharides; it is soluble in water even up to 60 wt %, with the aqueous solution behaving as a Newtonian fluid up to about 30 wt % of levan in the system.15,16 On the other hand, we estimated that the concentration of levan in a firm gel-like biofilm of B. subtilis subs. subtilis NCIB 3610 is only about 8 wt %. Obviously the bacteria affect the intermolecular interactions, structure, and rheological properties of levan with the products Received: March 2, 2014 Revised: March 19, 2014 Published: March 21, 2014 4172
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DLS, SLS, and Refractive Index Increment. The correlation times τc were determined from the DLS data20 utilizing the equation τc = 1/(Deffq2), where Deff is the effective diffusion coefficient, q is the length of the scattering vector equal to (4πns/λ)sin(ϑ/2), ns the refractive index of the solvent, ϑ the scattering angle, and λ the wavelength of the light. From this equation it follows that the reciprocal correlation time 1/τc is a linear function of q2 in the case of relaxation processes with diffusive character. The diffusive character of the observed process can therefore be easily tested by the slope of the line in the plot of ln(1/τc) vs ln(q), which equals 2 in the case of a diffusive process and differs from 2 in cases of nondiffusive processes.21−23 On the basis of the measured values of the refractive index increments (see Appendix A, Supporting Information), SLS data were put on an absolute scale and as such enabled assessment of the trend of the polymer molecular masses of the levan samples studied. Density and Sound Velocity Measurements. On the basis of these measurements the adiabatic compressibility values were obtained (see Appendix A, Supporting Information), and then the Pasynski model24,25 was used to calculate the hydration numbers nh.14,15 Even though there are some known weaknesses connected to the absolute values of nh obtained by this model, the trends are fully reliable, which is probably the reason why this approach has nonetheless been applied to numerous electrolyte, nonelectrolyte, and even polymer systems.25 We used it in order to facilitate a comparison of the results with the available literature data. Rheological Measurements. Due to the small amounts of levan samples available, the oscillatory and rotational rheological measurements could only be performed with the rheometer utilizing the cone− plate measuring system. In this mode approximately 1 mL of the sample was sufficient for measurement, but the sensitivity of the instrument in the rotation mode was somewhat compromised (see Apendix A, Supporting Information). In order to obtain reliable intrinsic viscosity values the dilute levan solutions were also measured utilizing an Ubbelohde viscometer. Small-Angle X-ray Scattering. Full details of SAXS measurements are given in Appendix A, Supporting Information. For interpretation of the SAXS curves we followed the classical approach for homogeneous polymer solutions based on the Ornstein−Zernike function yielding an expression for the scattering intensity of Lorentzian form3,8
of its metabolism and secretion in such a way that a compact gel can be formed even at very low levan concentrations. Hence, the long-term goal was to recognize and highlight the causes that lead to formation of compact gel-like structures of levan in aqueous biofilms from the structural point of view. However, in order to reach that goal knowledge of the structure in plain aqueous levan solutions is first needed. In the present study we therefore focused on the structure of aqueous solutions of levan of three very important bacteria: Bacillus subtilis (B. subtilis), Zymomonas mobilis (Z. mobilis), and Erwinia herbicola (E. herbicola). Bacillus subtilis is a soil bacterium important in the synthesis of numerous antibiotics (more than 25).17 The bacterium Z. mobilis is known to cause spoilage of beer and cider,18 whereas the bacterium E. herbicola is a plant pathogen, causing huge economic losses.19 The plain aqueous levan samples were studied in the concentration range up to 10 wt % of polymer in the system, i.e., at polymer concentrations comparable to those that appear during the process of biofilm formation. We aimed to test if the selected techniques were sensitive enough to detect the structural changes found in the plain nongelling aqueous levan samples. Therefore, we used three different levan samples originating from three different bacteria that already show considerable differences in their appearance. Levan samples were investigated by rheological, small-angle X-ray scattering (SAXS), static and dynamic light scattering (SLS, DLS), density and sound velocity measurements, as well as microscopy. Determination of polymer branching after per-O-methylation analysis and basic chemical and spectrophotometric analysis of the purity of the original levan samples was also performed. Besides gaining dynamic and structural information on plain aqueous levan systems that should aid in future investigation of structural phenomena relevant to biofilm formation, one of the aims of this paper was also to point out the level of dynamic and structural information that can be obtained for such polymer systems by combination of the chosen techniques. Although it might seem surprising, this is to our knowledge one of the first detailed structural studies of levan samples utilizing the small-angle X-ray scattering technique.
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I(q) =
C 1 + q 2ξ 2
(1)
where C is a constant and ξ the dynamic correlation length corresponding to the distance to which the movement of the flexible polymer chains is correlated. In the case of long-lived elastic polymer entanglements in the polymer system, i.e., static correlations, an additional term is needed in eq 1 to account for the excess scattering in the low-q region of the scattering curve. This term is usually expressed in terms of the Debye−Bueche formalism4,5 (squared Lorentzian) leading to the following expression for the overall scattering intensity6−8
EXPERIMENTAL METHODS
Due to the large number of research techniques used in this study we only give a very brief description of them in this section. For a more detailed description the reader is directed to Appendices A and B, which can be found in the Supporting Information. Materials. In this study three samples of the polysaccharide levan of three different bacterial origins were used. Two of them were commercially available levans made by Z. mobilis (Sigma Aldrich) and E. herbicola (Sigma Aldrich), but the third was obtained from the biofilm of the bacterial organism B. subtilis subs. Subtilis strain NCIB 3610. Great efforts were made to grow enough of its biofilm, to isolate EPS, and to purify the couple of grams of pure levan need for these studies. The details of this isolation are given in Appendix A, Supporting Information. Levans originating from bacteria B. subtilis, Z. mobilis, and E. herbicola are hereinafter referred to as BS, ZM, and EH levans, respectively. Detailed Chemical Analysis, Polymer Branching Analysis, and Microscopy. All three levan samples were analyzed for their contents of levan, nucleic acids, and proteins. The results are presented in Appendix A, Supporting Information, and show that the purity of isolated BS levan was comparable to the purity of commercial EH and ZM levans. Similarly, the experimental details of the polymer branching analysis and microscopy can also be found in Appendix A, Supporting Information.
I(q) =
A B + 1 + q 2ξ 2 (1 + q2 Ξ2)2
(2)
where A and B are constants and Ξ is the static correlation length, which is larger than ξ and corresponds to the correlations within the long-lived entanglements or their average size. These and similar equations for the scattering intensity have been successfully tested on numerous small-angle neutron scattering (SANS) and SAXS data on gels and solutions.6−8,26−28 As discussed in detail in Appendix B, Supporting Information, in the case of the experimentally smeared SAXS data additional transformation of eq 1 and eq 2 is needed prior to fitting them the experimental data.29 Even though the studied liquid samples were not gels in appearance, they do show considerable elasticity in their structure; therefore, use of eq 2 is considered justified. 4173
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RESULTS AND DISCUSSION
Levan-based biofilms play a key role in various biological processes; therefore, a better understanding of biofilm formation and its structure is of the utmost practical and theoretical importance.13 Aiming to highlight the structural aspects of levan-based biofilm formation in the long term, we initially sought to deepen our insight into the structure of nongelling aqueous solutions of levan samples from three different bacteria (BS, ZM, and EH) utilizing various physicochemical techniques. Rheological Results. It has already been reported in the literature that some polysaccharide levan samples of bacterial origin showed rather surprising nongelling behavior in plain aqueous solutions.15,16 Therefore, this study began with measurement of the rheological response of the three BS, ZM, and EH levan samples in the form of their viscosity dependence on shear rate γ̇. The corresponding experimental results obtained at 20 °C are shown in Figure 1. The curves for pure water and samples with the lowest concentrations of levan are somewhat noisier at low shear rates γ̇, which is due to the fact that even with the state-of-the-art cone−plate measuring system (used of necessity for low sample volumes) we were close to the sensitivity limit of the instrument in the case of low-viscosity samples. Nonetheless, one can clearly see that these levan samples also showed Newtonian-like behavior at low enough polymer concentrations. Only a weak shear thinning was observed in BS levan at concentrations higher than 4 wt %, indicating that these samples were actually nonNewtonian. Similarly, the limiting concentration for evident shear thinning behavior of levan seems to be around 2 and 1 wt % for the ZM and EH levan systems, respectively. In the latter two cases the pseudoplastic nature of the sample was much more pronounced than in the case of BS levan. As the shear thinning phenomenon occurs when polymer molecules orient themselves in the shear direction and disentangle to a certain degree, these differences in rheological behavior could be attributed to possible differences in polymer branching, packing in space, and/or differences in polymer molecular weight of the three levan samples. Figure 1d shows a comparison between 1 and 8 wt % BS, ZM, and EH levan solutions. Interestingly, the viscosity curves for all 1 wt % levan solutions were very similar, while those for 8 wt % levan solutions varied significantly. In any case, these results showed that at higher concentrations of levan there was a much greater difference in the viscosity and rheological behavior between BS levan and the two other studied levan samples (about 1000 fold) than between ZM and EH levan (about 10-fold). The fact that these macroscopic rheological differences were much more expressed at higher polymer concentrations where the interparticle interactions were stronger implies some important differences between these three samples on a molecular level. The increase of viscosity with increasing concentration is due to the increasing sample volume fraction occupied by the polymer and due to concomitant increasing strength of the interparticle interactions that eventually lead to entanglements among adjacent polymer chains when the critical chain overlap concentration is reached. Further dynamic rheological results were obtained by oscillatory tests, where the storage G′ and loss G″ moduli dependence on strain γ was measured at a moderate angular frequency of 10 s−1. The experimental results of oscillatory measurements at 20 °C for aqueous BS, ZM, and EH levan
Figure 1. Double logarithmic plot of viscosity η vs shear rate γ̇ for aqueous levan solutions measured at 20 °C: (a) BS, (b) ZM, and (c) EH levan concentration series. (d) Comparison of 1 and 8 wt % BS, ZM, and EH levan solutions.
solutions are shown in Figure 2. The results for samples with low concentrations of levan showed relatively constant G′ and G″ moduli with increasing strain. Furthermore, the storage modulus G′ was higher than the loss modulus G″ in all levan samples, i.e., at polymer concentrations below 8 wt % for BS levan, below around 3 wt % for ZM levan, and below around 4174
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light scattering measurements that are presented into detail in the next subsection. The most interesting aspect of these results is the fact that the value of the storage modulus G′ was more or less independent of the levan concentration in these concentration ranges, although G″ increased with concentration. On the basis of this fact it can be concluded that the observed elasticity phenomenon reflects an intrinsic property of the polysaccharide levan. This can be explained as a consequence of the polymer chain entanglements that are most probably mainly intramolecular at these low polymer concentrations; this is also in accordance with the rather low viscosities found in these concentration regimes. However, in the case of BS levan samples, which are in general much less viscous than ZM and EH levan samples, it is evident that with increasing levan concentration the G′ and G″ values became more or less equal at around 8 wt % of levan but at higher concentrations G″ eventually predominated. The latter trend was much more obvious in the case of the ZM and EH levan samples which in addition showed a strong strain dependence of the two moduli (see Figure 2). This is in agreement with their strong non-Newtonian behavior at these moderate levan concentrations. In Figure 2d the dynamic rheological curves of 1 and 8 wt % samples are compared, and very similar conclusions can be drawn as from Figure 1d, namely, all these rheological results indicate a great difference in the macroscopic structural response of BS, ZM, and EH levan solutions, especially at higher levan concentrations, but surprisingly a rather similar response in the case of 1 wt % solutions. In attempting to understand and explain the differences in the macroscopic rheological behavior of the studied samples we collated these results in the following subsections with results on the microscopic structure of these systems. However, in order to obtain and interpret such structural details, knowledge of the basic parameters of the polymer molecules, such as the level of polymer branching, extent of hydration, and at least an estimate of the polymer molecular mass, was needed. Dynamic Light Scattering Results. To investigate the nature of the dynamics in the levan samples on the microscale and to support the macroscopic dynamic rheological findings on the elastic character of the samples, 3D-DLS measurements were performed on diluted BS, ZM, and EH levan solutions at scattering angles between 50° and 140°. Dilution of the samples was made in order to reduce the sample turbidity to acceptable levels and ensure reliable 3D-DLS measurements. The highest concentration of BS levan used for these measurements was therefore 2 wt %, but correspondingly lower concentrations were taken for ZM and EH levan samples. Effective values of the correlation times τc were obtained by the method of cumulants and further used to construct the plots of ln(1/τc) vs ln(q) that are shown in Figure 3a. This DLS data revealed the nondiffusive nature of the relaxation dynamics (slope > 2) in the case of all three levan samples, meaning that these processes correspond to nondiffusive structural relaxations. This finding is therefore consistent with the dynamic rheological results on the strong elastic character of levan solutions at very low polymer concentrations. The slope of these lines was practically independent of concentration and also of type of levan sample, being around 4.35 in all cases. This shows that on the microscopic scale the nature of the dynamics is quite similar in these samples, which is probably due to the fact that the samples are composed of the same homopolymers although from different sources. As can be seen in Figure S2a in
Figure 2. Double logarithmic plot of G′ and G″ vs strain γ for aqueous levan solutions measured at 20 °C and an angular frequency of 10 s−1: (a) BS, (b) ZM, and (c) EH levan concentration series. (d) Comparison of the curves for 1 and 8 wt % BS, ZM, and EH levan solutions.
2.5 wt % for EH levan. This is interesting because from Figure 1 it can be seen that roughly in the same concentration ranges these systems exhibited Newtonian-like behavior, but these results in parallel also clearly showed the strong elastic character of the sample. To complement and support these dynamic rheological results we also performed the dynamic 4175
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Figure 3. Scattering angle-dependent DLS results: (a) Plot of ln(1/τc) vs ln(q) for diluted BS, ZM, and EH levan samples. (b) Averaged intensity autocorrelation function G2(τ) for 0.1 wt % BS, ZM, and EH levan solutions at 90° scattering angle.
Appendix C, Supporting Information, the spread of the relaxation times is rather broad in all these samples. Figure 3b depicts the raw DLS results in the form of the intensity autocorrelation function for 0.1 wt % BS, ZM, and EH levan solutions. This data shows that even though the nature of the observed relaxation processes on the microscale seems to be rather similar (similar slopes of lines in Figure 3a), the relaxations seem to be the fastest in BS, only slightly slower in ZM, and evidently slower in EH levan solution. This conforms with the macroscopic rheological results at low concentrations of levan depicted in Figure 1d; even though these three curves are alike, the result for EH levan solution is evidently somewhat higher with respect to the other two. The speed of such relaxations is expected to depend on the branching and size of the polymer molecules; therefore, these DLS results indicate that one or both mentioned molecular parameters increase in the direction from BS to ZM and to EH levan. Branching of Levan Molecules. In the next step we made efforts to determine the extent of molecular branching in the studied BS, ZM, and EH levan samples. This was achieved by the per-O-methylation protocol with subsequent reductive cleavage and acetylation of the cleaved monomers, which were further analyzed by GC-MS. The corresponding total ion current chromatograms (TIC) for BS, ZM, and EH levan samples are shown in Figure 4. These chromatograms show a number of peaks each representing an individual residue identified according to the mass spectra. The peaks numbered from 1 to 5 are important for determination of levan branching; the remaining peaks were recognized as background arising from the column. The details of these peaks are given in Appendix C, Supporting Information. The mole fractions of three types of monomers in the levan molecule were obtained
Figure 4. TIC chromatograms of partially methylated anhydroalditol acetates of (a) BS, (b) ZM, and (c) EH levan, normalized to the highest peak. Numbered peaks: (1) terminal monomers; (2 + 3) β(2,6)-linked monomers in the main levan chain; (4 + 5) branching points.
according to the peak area: one should be aware that the mole fractions of terminal and branching monomers are practically the same in the case of long polymer molecules and that the remaining mole fraction corresponds to the secondary monomers in the polymer chain. The mole fraction of branching fructose monomers was found to be (10.5 ± 0.7)% in BS, (11.0 ± 0.7)% in ZM, and (10.2 ± 0.3)% in EH levan. Surprisingly, within the uncertainty limits of these values branching is obviously alike in all three studied levan samples. As comparable values were also obtained for levan samples of bacterial origin in some previous studies (e.g., levan from Streptococcus salivarius had 10%, from Bacillus polymixa 12%, and from Aerobacter levanicum 9% of branching points),16,30,31 these results indicate that the polymer branching of levan molecules of bacterial origin is very similar. This means that the branching of levan molecules in this case is certainly not such 4176
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an important factor that it could alone lead to the observed differences in the macroscopic rheological behavior of the studied samples; the latter are therefore more likely related to possible differences in molecular mass of the polymer molecules. However, one should be aware that these results unfortunately do not provide any information on the length of the sections between the branching points nor on the length of the branches in the polymer molecule. Theoretically these could of course still differ significantly for the three studied levan samples, but due to the biocatalytic nature of levan synthesis by the enzyme levansucrase there might not be much difference between the studied levan samples even in this respect, namely, it has been reported that levansucrases from gram-positive bacteria differ widely in their biochemical characteristics, but levansucrases from gram-negative bacteria are similar in their molecular mass and substrate-independent expression.32 Since ZM and EH are gram negative and BS is gram positive, bigger differences in this respect were therefore anticipated between BS levan and the other two levans than between ZM and EH levan. This feature was actually confirmed by the results presented in this work. Nevertheless, possible differences in the length of branches of the studied polymers could be reflected in the degree of polymer hydration, which is the topic of the following subsection. Hydration of Levan Molecules. Density and sound velocity measurements of aqueous BS, ZM, and EH levan solutions were performed at 20 and 25 °C, as shown in Figure S3, Supporting Information, in Appendix C. In this respect there was also a bigger difference between the trends obtained for BS levan and the other two studied levan samples than between ZM and EH levan. Using the Laplace equation the adiabatic compressibility of levan solutions was calculated on the basis of these experimental data. Its concentration dependence is presented in Figure 5a and shows a decreasing trend with increasing polymer concentration. Similarly, a decrease in compressibility was also observed with an increase in temperature. In the case of strongly hydrated polymer solutions a substantial fraction of water molecules is restrained by the hydration of the polymer molecules and therefore cannot contribute to the compressibility of the system. The compressibility of such a polymer solution is consequently significantly lower than the compressibility of water and for a similar reason shows a decreasing trend with increasing polymer concentration. The effect of increasing temperature on adiabatic compressibility is however more complex, as it reduces hydration due to the increased thermal molecular motion, increases the sound velocity for the same reason, and reduces the sample density due to the volume expansion, the overall effect thus being a reduction of compressibility with increasing temperature. According to eq S2, Supporting Information, see Appendix A, the hydration number can be calculated from the adiabatic compressibility results. The concentration dependence of the hydration numbers for BS, ZM, and EH levan samples is shown in Figure 5b. In all three bacterial levan samples the number of water molecules per monomer unit of levan is around 6. This value seems to be in the upper half of the range of values reported for other polysaccharides, e.g., previous results of a hydration number of 9 per sodium hyaluronate monomer, 1 per N-acetylglucosamine monomer, 11.3 per sodium glucuronate monomer, 3.4 for carboxymethyl cellulose monomer, and 1.8 for carboxymethyl dextran monomer with a degree of
Figure 5. Concentration dependence of (a) adiabatic compressibility βS,sol and (b) hydration number nh at 20 °C (open symbols−dashed lines) and 25 °C (full symbols−solid lines) for aqueous BS, ZM, and EH levan solutions.
substitution equal to zero were reported.33,34 The hydration number is also somewhat reduced by temperature increase. Nevertheless, even though no large differences can be observed between these hydration results, they do suggest that levan molecules are slightly more hydrated in BS than in ZM and EH levan solutions. This could indicate slightly shorter branches in the case of BS levan compared to ZM and EH levan (as discussed in the previous subsection), or it could simply be the consequence of a somewhat better solubility of BS levan on a molecular length scale in comparison to ZM and EH levan, as discussed in the following subsection. Static Light Scattering and Microscopy Results. In the case of 1 wt % levan solutions of BS, ZM, and EH the differences in sample turbidities were obvious already to the naked eye (e.g., see the graphical abstract). The most turbid was the solution of EH levan, and the most transparent was the solution of BS levan. The same trend was also confirmed in the dilute regime by the SLS results for three transparent 0.01 wt % solutions of BS, ZM, and EH levan as shown in Figure 6. Sample turbidity is closely related to the size and refractive index of the scattering particles. The scattering curves in Figure 6 show increasing scattering power of the samples arising from the increasing size of the scattering particles in solution in the order BS to ZM to EH levan. It has already been suggested in the literature that the nongelling behavior of levan originates in weak attractive interparticle interactions and predominant intraparticle interactions. The latter lead to the low intrinsic viscosity values of levan and the appearance of spherical microscopic levan particles that have been observed in some levan samples.15,35,36 Therefore, we took images for 1% BS, ZM, and EH levan samples utilizing differential interference contrast microscopy. 4177
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kDa to 50 MDa37,38 and for EH levan from 29.7 MDa to 125 MDa.39,40 A broad molecular weight distribution was also observed for samples of levan produced by acetic acid bacteria. Gluconobacter f rateurii and Gluconobacter cerinus produced polymer fractions from 4 MDa to 98 MDa and from 6 MDa to 98 MDa, respectively, whereas Neoasaia chiangmaiensis and Kozakia baliensis produced portions of comparatively higher molecular weight levan molecules ranging from 100 to 575 MDa or even from 1000 to 2000 MDa, respectively.35 Nevertheless, it seems likely that the levan particles observed in our samples and similar ones are indications that these levan particles might actually be aggregates of at least several levan molecules, but it is not at all easy to separate them in a nondestructive way; a more detailed study of this issue is currently underway but is beyond the scope of this paper. Nevertheless, a higher molecular mass of the polymer leads to greater entanglement possibilities, and therefore, the size of the levan particles should correlate to the molecular mass. Hence, we made a very rough estimate of Mw of the studied levans based solely on the SLS curves of 0.01 wt % levan solutions depicted in Figure 6 and their extrapolation to zero scattering angle:41 The estimate for BS levan was (31 ± 2) MDa, for ZM (226 ± 4) MDa, and for EH levan (276 ± 44) MDa. These values can of course be taken only qualitatively to confirm the trend of increasing molecular mass (scattering particles) in the direction from BS, to ZM, to EH levan. To summarize, these results clearly demonstrate that the difference in polymer solubility, i.e., the difference in concentration and sizes of the microscopically observed levan particles that are related to a different polymer molecular mass (see Figure 7), is the most significant difference between the studied levan samples. It seems that it is mainly this difference that contributes to the considerable differences in the macroscopic rheological properties of these systems. Other results on these polymer systems at the molecular level conform with this conclusion. Determination of Intrinsic Viscosity Values. At this point attention is drawn to another detail arising from the rheological measurements. Extrapolating the so-called viscosity number, i.e., the ratio of the specific viscosity to the concentration, to zero concentration (see eq S3, Supporting Information, Appendix A) the intrinsic viscosity of the polymer may be determined. The corresponding plot for the BS, ZM, and EH levan samples is shown in Figure 8. Such Huggins plots are usually made at polymer concentrations low enough to show linear dependence.42 In our case such a linear regime was observed at much lower levan concentrations in the case of ZM and EH than in the case of the BS levan sample. We therefore show in Figure 8a a broad concentration regime for all 3 samples, even though the linear fits were performed strictly in the linear range. Due to the specific behavior of the curve for the BS levan sample at very low levan concentrations we plot the initial part of these curves on an expanded scale in Figure 8b, namely, in the case of BS levan sample an upturn in the values of the viscosity number is observed that is found to be highly reproducible and much greater than the measurement uncertainty. Unfortunately we found no reports of a similar trend in the literature, which might be connected to the fact that we also would not have observed this trend at all if the viscosity measurements had not been performed at such low concentrations of BS levan. Thus, for BS levan a perfectly linear trend in the Huggins plot was obtained at higher concentrations that yielded an intrinsic viscosity value within the range
Figure 6. Experimental SLS scattering curves of 0.01 wt % BS, ZM, and EH levan solutions on an absolute scale. Measurement uncertainty is well within the symbol size.
These images are depicted in Figure 7, and indeed, some larger compact globular levan particles were observed in these plain
Figure 7. DIC image of (a) solvent, (b) 1 wt % BS levan, (c) 1 wt % ZM levan, and (d) 1 wt % EH levan plain aqueous solutions.
aqueous solutions. The largest particles of (0.5 ± 0.3) μm were observed in EH levan solution, where these particles are also strongly polydisperse, followed by ZM levan particles of (0.48 ± 0.09) μm and BS levan particles of (0.34 ± 0.09) μm in diameter. At least 60 particles were considered in each case to obtain these values. It should be stressed that a strong decrease in particle concentration could also be observed in the same order (see Figure 7); in the case of BS levan their concentration was so low that they were hard to observe at all. The latter fact indicates that BS levan is much more soluble than ZM and EH levans on this length scale. This is probably the reason for the slightly larger hydration numbers observed for BS levan (see Figure 5b). In any case these results show that in this respect these levan systems are microphase separated, which is somewhat similar to hydrocolloid microgel systems. There are some reports of extremely large molecular masses of levan samples of bacterial origin in the literature. For example, it was reported that Mw for ZM levan ranges from 75 4178
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higher in the case of levan that is able to dissolve on a molecular level (see Figure 7). Small-Angle X-ray Scattering Results. The SAXS technique has previously been successfully applied in structural studies of various polysaccharides,47−49 but there are few structural SAXS studies on the polysaccharide levan in the literature. In this work we followed the classical evaluation of scattering data on polymer solutions according to eqs 1 and 2 or better their forms adapted for SAXS smearing effects (see eqs S6 and S7, Supporting Information, in Appendix B). The experimental SAXS scattering data for 1 and 8 wt % BS, ZM, and EH levan samples are shown in a double logarithmic plot in Figure 9 (open symbols). As evident from the course of these
Figure 8. Huggins plot for determination of intrinsic viscosities of BS, ZM, and EH levan samples. (a) Experimental data are presented with the symbols; gray lines are used only to guide the eye. Colored solid lines represent fits in the linear regime. (b) Curves at low concentrations of levan on an expanded scale.
of values already reported in the literature. The only reasonable explanation for such behavior is that there is some qualitative change in the mode that BS levan molecules pack in space at very low concentrations compared to higher concentrations. This trend is closely connected to the large levan particles observed in Figure 7, which significantly decrease in number when the concentration of levan is decreased. It is also reasonable that this trend was observed only in the case of BS levan, because there the concentration of large levan particles was very low and consequently the contribution of the completely dissolved levan molecules large enough to be observed. Nevertheless, various bacterial levans were usually reported to have very low intrinsic viscosities (from only 0.07 to 0.38 dL/g)15,43 in comparison to some other polysaccharides such as cellulose, carrageenan, xanthan, and guar (in the range from 5 to 50 dL/g).44−46 Such low values of intrinsic viscosity clearly indicate that levan occupies a relatively small space per unit mass and that the molecules are inherently compact. This is in accordance with the fact that in aqueous solution levan can be found mainly in the form of large levan particles (see Figure 7).15,35,36 On the basis of measurements in the linear range of Huggins plots we obtained an intrinsic viscosity of (0.35 ± 0.04) dL/g for BS levan, (0.36 ± 0.01) dL/g for ZM levan, and (0.45 ± 0.01) dL/g for EH levan. From the results presented in this paper, we presume that these low values correspond to levan in the form of large particles. The results for BS levan further indicate that the intrinsic viscosity is actually much
Figure 9. Log−log plots of experimental SAXS curves on absolute scale (open symbols) of BS (blue), ZM (green), and EH (red) levan: (a) 1 and (b) 8 wt % levan scattering curves. For the sake of clarity, the curves corresponding to ZM an EH levan samples are multiplied in a by the factor 22 and 400 and in b by the factor 5 and 30, respectively. Black lines indicate the slopes corresponding to q−1, q−2, q−3, and q−4 behavior. Yellow lines represent best fits to the data based on eq S7, Supporting Information.
scattering curves there seems to be a much greater difference between BS levan and the other two samples than between ZM and EH levan. Something very similar could also be observed in the case of the macroscopic rheological, sound velocity, and hydration results, which indicates mutual dependence of the microscopic and macroscopic structures. Inspecting the black lines in Figure 9, indicating the characteristic slopes corresponding to q−1, q−2, q−3, and q−4 behavior, it is apparent that in the regime of high q values the data fall somewhere between q−1 and q−2 behavior, in good agreement with theoretical expectations. It is important to understand that for the Gaussian chain, which is a common 4179
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Table 1. Parameters of the Fits to the Experimental SAXS Data for BS, ZM, and EH Levan BS parameters A [cm−1] ξ [nm]
1 wt % 0.167(4) 1.43(2)
ZM 8 wt % 0.836(8) 1.060(7)
1 wt % 0.091(3) 0.96(2)
model used for polymer molecules in a good solvent, q−2 behavior is expected in the regime of high q values (see eq 1), but in the case of ideally smeared experimental data, i.e., smeared by an infinitely long primary X-ray beam, q−1 behavior of such a chain is expected in this regime. The length profile in our case was not infinitely long (see Figure S1, Supporting Information, in Appendix B); therefore, the experimental data decrease with a slope between the expected limits. According to similar reasoning and eq 2, the behavior of the scattering curve at low values of q should follow q−4 or in the case of ideally smeared SAXS data q−3 behavior. The central part of the experimental scattering curves of 1 wt % ZM and EH levan samples (at very low q values) shows a steep increase that is very close to the expected value of q−3 (since smearing is the strongest at low values of q) but does not even come close to that value in the case of the BS levan sample and all three 8 wt % samples. The most probable explanation is that the method is unable to provide results at low enough values of q, where such behavior could be observed. The fact that all these scattering curves show a persisting steep upturn even at the lowest values of q indicates that at least one dimension of the scattering particles is much above the resolution of the SAXS experiment (around 50 nm in this case). In fact, the microscopy results in Figure 7 showing very polydisperse macroscopic levan particles in these systems clearly confirm that. In evaluating the experimental SAXS data shown in Figure 9 in more detail we first tested the fit to eq S6, Supporting Information, given in Appendix B (based on eq 2). Since this equation was unable to describe these experimental SAXS results sufficiently well, these results are not presented in this paper in detail; we only show the corresponding fits in Figure S4, Supporting Information, in Appendix C. It is obvious that the problems with these fits were in describing the central parts of the scattering curves where the upturn corresponding to the scattering contribution of the static polymer entanglements is expected. With the exception of the BS levan sample and the 8 wt % levan samples, the resulting values of the static correlation length Ξ went to infinity but the upturn of all these fits was still much too small in the central part in all cases (see Figure S4, Supporting Information). The reason why we nevertheless feel it is worth briefly discussing these results lies in the fact that these fits clearly show that there is an additional scattering contribution present in the central part of the scattering curves that cannot be described by the contributions of eq 2, causing these curves to deviate from the expected course of eq 2, i.e., preventing its smooth transition between the Lorentzian and squared Lorentzian term. This additional scattering contribution most probably represents the tail of the scattering contribution of the highly polydisperse large levan particles with finite dimensions observed in Figure 7 or could also arise from the possible internal nanostructure of these large particles and/or structural features of their surface. As it was not possible to obtain reasonable information on the static correlation length Ξ for the studied samples as discussed in the previous paragraph, we decided to use a cutoff technique taking into account only the Lorentzian term of eq 2
EH 8 wt % 0.573(5) 0.832(6)
1 wt % 0.076(3) 0.94(3)
8 wt % 0.503(6) 0.778(7)
in the SAXS data analysis. Here it is well known that the scattering contribution of the static polymer entanglements significantly affects the scattering curves only at q values above the limit of ξ·q > 1, but below these values the Lorentzian term fits the data rather well and provides a very good estimate of the dynamic correlation length ξ.26 The resulting fits to the experimental SAXS data according eq S7, Supporting Information, are shown as yellow lines in Figure 9. The corresponding values of parameters A and ξ are presented in Table 1. On the basis of these values of ξ it can be concluded that they show a reasonable trend as they decrease with increasing concentration of levan in all three cases. This decrease of ξ corresponds to the fact that with increasing polymer concentration the volume fraction of the sample occupied by the polymer increases; therefore, the regions of static polymer entanglements also increase and lead to shorter dynamic intermediate segments of the polymer molecule. It is also interesting to notice a decrease in parameter ξ in the direction from BS through ZM to EH levan samples even at a constant value of the concentration. This indicates that the structure of levan is the least entangled in the case of BS and the most entangled in the case of EH levan samples. Again, it is noticeable that there is a much greater difference between BS levan and the other two samples than between ZM and EH levan. Obviously the structure of BS levan is somewhat more flexible than the structure of other two levan samples, which could also be a consequence of the fact that there is much less BS levan present in the form of large levan particles than in the other two samples. It can therefore be concluded that these results on the levan structure at the molecular level conform well to the trends observed from the results on its dynamics at the macroscopic scale.
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CONCLUSIONS The present study demonstrated the level of detail of macroscopic and microscopic sample dynamics and structure that can be obtained by a careful combination of various basic physicochemical techniques on the interesting specific example of aqueous solutions of BS, ZM, and EH levans. The macroscopic rheological results interestingly showed Newtonian-like behavior of aqueous solutions of levan at low concentrations but at the same time surprisingly pointed out the strongly elastic character of these solutions. This elastic character was supported by the DLS results showing strong nondiffusive relaxation processes which could be explained by the strong intramolecular interactions of levan at such low concentrations. The latter also manifest themselves in the presence of large levan particles that were confirmed by microscopy. The relaxation times of the dynamic response of these samples on the microscopic scale were in accordance with the sample viscosities. The extent of branching and the hydration of the levan samples of different origin were also investigated, but they did not show any considerable mutual differences. Slightly more intensive hydration was observed only in the case of BS levan samples that were found to consist of the most soluble levan. Structural details on the nanoscale in 4180
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terms of correlation lengths were obtained utilizing the SAXS technique and well complemented the macroscopic rheological and other results. We therefore conclude that the results provide some important answers about the origin of the dynamic, viscoelastic, and structural properties of the levan samples studied. Furthermore, successful combination of a number of research techniques presented in this paper demonstrates the level of details available on the structure and dynamics of branching polymer systems. In addition, it provides concrete results on the structure and dynamics of the specific levan samples of present interest, which it is believed will be relevant in future studies connected with formation of BS biofilms.
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ASSOCIATED CONTENT
* Supporting Information S
Appendices A, B, and C. 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]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are most grateful to Professor Otto Glatter for his generous contribution to the instrumentation of our laboratory for light scattering methods. We thank Alenka Ž agar and Urša Kristan from Anton Paar and Dr. Janez Orehek from JUB for access to their rheometers. We also thank Simon Sretenovič for his help with the microscopy. We acknowledge the Slovenian Research Agency for financial support (programmes P1-0201, P1-0153, and P4-0116).
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