Structure and Dynamics of a Model Polymer Mixture Mimicking a

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Structure and Dynamics of a Model Polymer Mixture Mimicking a Levan-Based Bacterial Biofilm of Bacillus subtilis Elizabeta Benigar, Andreja Zupancic Valant, Iztok Dogsa, Simon Sretenovic, David Stopar, Andrej Jamnik, and Matija Tomsic Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02041 • Publication Date (Web): 20 Jul 2016 Downloaded from http://pubs.acs.org on July 30, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Structure and Dynamics of a Model Polymer Mixture Mimicking a Levan-Based Bacterial Biofilm of Bacillus subtilis Elizabeta Benigar,a Andreja Zupančič Valant,a Iztok Dogsa,b Simon Sretenovic,b David Stopar,b Andrej Jamnik,a Matija Tomšič.a,* a

University of Ljubljana, Faculty of Chemistry and Chemical Technology, Večna pot 113, SI-

1000 Ljubljana, Slovenia *Correspondence e-mail: [email protected] b

University of Ljubljana, Biotechnical Faculty, Večna pot 111, SI-1000 Ljubljana, Slovenia

In this paper, we report on the structure and dynamics of biologically important model polymer mixtures that mimic the extracellular polymeric matrix in native biofilm of Bacillus subtilis. This biofilm is rich in nonionic polysaccharide levan, but also contains other biopolymers as DNA and proteins in small concentrations. Aiming to identify the contribution of each component to the formation of the biofilm, our investigations encompassed dynamic rheology, small-angle X-ray scattering, dynamic light scattering, microscopy, densitometry and sound velocity measurements. As it turned out, this very powerful combination of techniques is able to provide solid results on the dynamical and structural aspects of the microbiologically and chemically complex biofilm formations. Macroscopic rheological measurements revealed that the addition of DNA to levan solution increased the viscosity, pseudoplasticity and 1 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 37

elasticity of the system. The addition of protein contributed similarly, but also increased the rigidity of the system. This confirms that the presence of minor biofilm components is essential for biofilm formation. DNA and proteins appear to confine levan molecules within their supramolecular structure and, in this way, restrict the role of levan to merely a filling agent. These findings were complemented by small-angle X-ray scattering data, which provided insight into the structure on a molecular scale. One of the essential goals of this work was to compare the structural properties of the native biofilm and synthetic biofilm mixture. KEYWORDS: Levan, Biofilm, Rheology, DLS, SAXS, String-of-beads model.

1. INTRODUCTION Biofilms are gel-like multicellular microbial communities encased in a slimy extracellular polymeric matrix (EPS matrix) that protects them from harmful environmental conditions.1 They can occur on virtually any surface (e.g. human tissue, medical devices, water systems etc.);2,3 therefore, they represent a biointerface that is problematic in many fields of industry and medicine. Nonetheless, the formation of biofilms can also offer advantages, e.g. in wastewater treatment; in fact, biofilms show potential as energy sources for microbial fuel cells.4-6 Although natural biofilms are mainly composed of various microbial species, the monomicrobial biofilms are usually used in systematic investigations of model systems. There are numerous studies of biofilms that are formed by the opportunistic gram-negative human pathogenic bacterium Pseudomonas aeruginosa,7,8 as several of those are also formed by the gram-positive bacteria Staphylococcus sp..9,10 However, the gram-positive soil bacterium Bacillus subtilis has also emerged over the last decade as an alternative model for studying the molecular mechanisms of biofilm formation. Bacillus subtilis is one of the best-characterized organisms, and it serves as a model organism for important gram-positive pathogens, such as 2 ACS Paragon Plus Environment

Page 3 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Staphylococcus aureus, Bacillus anthracis, Listeria monocytogenes, etc. Moreover, B. subtilis is highly amenable to genetic manipulation and is used in biotechnology for commercial products. Bacterial biofilms are very interesting from a functional microbiological as well as a structural physico-chemical point of view. The latter still presents a great scientific challenge due to the large number of biofilm components and also because of the great difficulty involved in isolating and purifying them in adequate quantities.11 In the case of B. subtilis biofilm, we have already made important initial steps in our previous detailed study on the structure and dynamics of bacterial nonionic polysaccharide levan in aqueous solution.12 Levan is a common polysaccharide from the group of fructans that can be found in many bacterial biofilms. Because of the complex hierarchical structure of biofilms, it is also not straightforward to find physico-chemical techniques that are capable of properly describing them at all levels. Lightscattering methods have proven to be a promising tool in this respect.12-18 Sticky-gel-like extracellular polymeric substance (EPS) matrix, which is composed of polysaccharides, proteins, nucleic acids and other biopolymers produced by the cells, is structurally the most important biofilm constituent.19 It plays an important role in establishing and maintaining biofilm structure, and also mediates cell-to-cell and cell-to-surface interactions.20,21 The structure of EPS matrix has already been studied in various model systems by a variety of spectroscopic techniques, such as scanning and transmission microscopy, optical rotation spectroscopy and atomic force microscopy.17,18,22,23 Small-angle X-ray scattering (SAXS) was also found to be an effective way to study the influence of pH on the structure of EPS matrix.15 The major carbohydrate component of the B. subtilis EPS matrix is either polysaccharide EpsA-O, which is composed of glucose, galactose and N-acetylgalactosamine, or polysaccharide levan. When it is grown in a medium without sucrose (e.g. MSgg, based on glycerol and glutamate), polysaccharide EpsA-O represents a major polymer fraction of the

3 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 37

EPS matrix.24,25 But when B. subtilis grows in a sucrose-rich medium, a major component of the EPS matrix is the polysaccharide levan (89.52 wt.%).12,24 Levan is a nonionic extracellular β-fructan, composed of fructofuranosyl rings. Monomers in the main chain are connected by β(2,6)-glycosidic linkages, while occasional branches occur through β-(2,1)-glycosidic linkages. As we showed in our recent paper, levan from B. subtilis contains around 10.5 % of such branching points.12 If the genes for polysaccharide EpsA-O (epsA-O) and protein TasA (tasA) are absent, B. subtilis does not form a biofilm, even in the presence of levan.24 This indicates that levan alone is not sufficient for biofilm formation. Nonetheless, it contributes significantly to the physico-chemical properties of the biofilm, as the total mass of EPS matrix is 50-fold higher in biofilms that are grown in a sucrose-rich medium.24 In contrast to most other common polysaccharides, levan exhibits non-gelling behavior in aqueous solutions.26,27 Such solutions show extremely low intrinsic viscosity, even though levan can form rather compact biofilms at concentrations as low as 8 wt.%.12 All these data suggest that DNA (5.74 wt.%) and TasA protein (4.74 wt.%), while only minor polymer components of the EPS matrix, play an important role in biofilm formation. Apparently, the interparticle interactions in such mixed systems significantly change the solubility of levan, so that it forms less-soluble structures.28 Our goal is to identify and investigate the structural contribution of an individual biofilm component to the formation of a compact gel-like structure. For this purpose, we initially investigated basic aqueous levan solutions12 and now continue our research on more-complex aqueous mixtures of various polymers found in the biofilm of B. subtilis. To mimic the structural situation in native biofilm as closely as possible, we made great efforts to use the polymers isolated directly from the biofilm of B. subtilis. Levan and DNA were successfully isolated; however, for the protein TasA, which is known to form the fibrils,21,29,30 we eventually had to compromise and use the commercially available collagen.


Page 5 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

2. EXPERIMENTAL METHODS In the present study, the minor components of biofilm, such as DNA and protein, were added to aqueous solutions of the major component levan in the same concentrations as were found in B. subtilis biofilm. To study the effects of simple electrolytes on such polymer systems, similar mixtures were also prepared in solutions of simple electrolytes from the sucrose-yeast growth medium (SYM electrolytes).24 The polymers levan and DNA that were used in this study were isolated directly from B. subtilis biofilm. Protein TasA, which is known to form the fibrils21,29,30 was unable to be isolated in sufficient quantities from the native biofilm, and was therefore replaced in our study by the commercially available collagen (Sigma Aldrich), which is also a fibril-forming protein. By adding B. subtilis cells from the native biofilm to the mixture of levan, DNA, and protein, we obtained our synthetic biofilm mixture. The samples were investigated by rheology, small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), density and sound velocity measurements, as well as by microscopy. One of the aims of this paper was also to compare the structural properties of a native and synthetic biofilm.

2.1. Materials Levan and DNA were isolated directly from natural biofilm of Bacillus subtilis, while collagen was purchased from Sigma-Aldrich (Colagen from calf skin; Bornstein and Traub Type I; Bioreagent, suitable for cell culture; CAS 9007-34-5; Lot # SLBH0466V). Since the main interest of our latest studies is to explore the structural developments in the natural B. subtilis biofilm, it was logical to prepare and study the polymer samples with the same concentrations that build up in nature during the natural growth of the B. subtilis biofilm


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 37

(mature biofilm: 8 wt.% of polysaccharide levan, 0.52 wt.% of DNA, and 0.43 wt.% of protein Tas A).24 In this manner, in the samples containing polymer mixtures, the polymers were mixed in the same proportions as were found in the native biofilm. Further details about the materials used can be found in Appendix A, Supporting Information. The samples in this paper are designated by the following abbreviations given in the brackets: 8 wt.% levan (L); 0.52 wt.% DNA (D); 0.43 wt.% collagen (C); synthetic EPS matrix (sEPS), which is a mixture of 8 wt.% levan, 0.52 wt.% DNA and 0.43 wt.% collagen; synthetic biofilm mixture (sBF), which is an sEPS sample that also contains added bacterial cells; native biofilm (nBF1); and homogenized native biofilm (nBF2), which is mildly ultrasonicated nBF1 sample. The majority of the samples were prepared in aqueous SYM electolytes consisting of: K2HPO4 (70 mM), KH2PO4 (30 mM), (NH4)2SO4 (25 mM), MgSO4 (0.5 mM), MnSO4 (0.01 mM), and ammonium iron (III) citrate (22 mg/L).24,31 The pH value of SYM electrolyte medium was arround 7.

2.2. Rheological Measurements Rheological measurement details are presented in Appendix A, Supporting Information. Viscoelastic materials show both elastic and viscous components in their dynamic rheological response, which are reflected in the storage G ' and loss moduli G '' , respectively. An important characteristic of elastic behavior is reversibility; after the perturbing force is removed, the structure of an ideally elastic material returns to its original state. In contrast, in the case of ideally viscous behavior, deformation of the material accumulates as a function over time, leading to permanent change in the sample’s structure. For gel-like samples, it is characteristic to show prevailing elastic character through very high values of the store modulus G ' and low values of the loss modulus G '' . The peak that appears on the curve of the G '' modulus in the region of the yield point is also its typical feature. The yield point is the point where the G ' and G '' curves intersect. It also provides the value of the yield stress, i.e. the stress that has to be


Page 7 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

reached for the sample to start exhibiting flow behavior. A strong indication of gel-like behavior of a predominantly elastic sample is also a parallel horizontal course of curves G ' and G '' with increasing angular frequency ω .

2.3. Dynamic Light-Scattering Measurements DLS experimental and data interpretation details are given in Appendix A, Supporting Information. Since the investigated samples were rather turbid, the use of 3D cross-correlation DLS technique was necessary to eliminate the possible effects of multiple scattering. The DLS results were interpreted by means of the so-called mode-coupling theory,32-34 which deals with systems in which the scattering moieties strongly interact with their surroundings. A slow, nondiffusive relaxation mode is typical for such coupling effects. It can easily be recognized in the DLS results from the plot ln (1 τ c ) vs. ln ( q ) , where τ c represents the correlation time obtained by a well-known method of cumulants and q the scattering vector. The unconstrained relaxation processes, which is diffusive exhibits in this plot the slope of the line equal to the value of 2, but in case of the scattering moieties that strongly interact with their surroundings the nondiffusive relaxation mode appears and yields much larger slope. The stronger is the coupling effect the larger value of this slope can be expected and in case of very strong coupling the slow relaxation mode can prevail the DLS signal.32-34

2.4. Density, Sound Velocity, Microscopy and Gel-Electrophoretic Measurements The experimental and the data evaluation details for these techniques can be found in Appendix A, Supporting Information. Utilizing the density and sound velocity measurements, the adiabatic compressibility values were obtained. The Pasynski model35,36 was further used to calculate the hydration numbers nh .


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 37

2.5. SAXS Measurements and Interpretation In our previous study of bacterial levan solutions, we showed that the classical OrnsteinZernike37,38 and additional Debye-Bueche38-40 terms fail to satisfactorily describe the SAXS intensities in the innermost regime of the scattering curves.12 For this reason, in the present study, the more detailed, but still rather robust »string-of-beads« model was applied for interpreting the SAXS curves. The string-of-beads model is described in detail elsewhere,15,4143

but for convenience, a schematic representation is given in Figure 1 and brief description is

made in Appendix A, Supporting Information. The structure of the gel can be considered as hierarchically organized, as depicted in Figure 1a, with a pervading polymer network. The network contains some denser inhomogeneities, i.e. the regions where the polymer molecules overlap or connect to form longer-lived regions that introduce some elasticity into the structure. On a molecular scale, even these denser regions are not homogeneous scattering moieties but, rather, are more or less structured on the nanoscale themselves. Such nanostructure represents an additional level in the hierarchical structure of such systems and obviously introduces an additional scattering contribution to the SAXS signal, which cannot be described by a classical approach to the SAXS scattering intensities of polymer solutions.12,45 The goal of the string-of-beads model is to describe such an additional scattering contribution through the introduction of the nanostructure’s form factor P ( q ) into the general scattering equation of the polymer gels.41 These nanostructures are modeled as the strings of spherical beads, each string representing the polymer molecule and each bead a monomer in the polymer chain. The position of the bead relative to its predecessor in the polymer molecule is described by the specific bond angle Θp and torsion angle Φp , where index p indicates that these angles are set in the model according to

the

probability

p*

for

random

variation

of

these

two


Page 9 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 1: (a) Schematic representation of the hierarchical gel structure containing dense Debye-Bueche inhomogeneities that are nanostructured on a molecular level. This nanostructure can be described by various sets of the uncorrelated modeled polymer segments. (b) In the string-of-beads model, the monomer units of the polymer are presented as spherical homogeneous beads, which are lined up as if on a string. The scheme was partially prepared by the VMD program (http://www.ks.uiuc.edu/Research/vmd/).44 (c) Levan monomer unit, i.e. fructose. (d) Schematic representation of the conformational space of the string-of-beads model: the 3D cube with the fourth dimension expressed by the grayscale represents an


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 37

individual set of four shape parameters Θp , Φp , Θplim and p * obtained by fitting experimental SAXS data.

angles. The parameter Θplim represents the upper limit of the bond angle Θp and reflects the stiffness of the polymer chain. A specific choice of four shape parameters Θp , Φp , Θplim and p * fully determines a characteristic set of similar molecular conformations of the modeled polymer molecule. An average scattering contribution P ( q ) is calculated for each such characteristic set.14 Accordingly, P ( q ) represents the intramolecular scattering contribution and is therefore designated as the characteristic average form factor of the polymer molecule (or its modeled segment). The string-of-beads model allows a broad variety of molecular conformations. Depending on the length of the polymer molecule, the whole molecule or only its shorter segment can be modeled in this way; accordingly providing different detailed information on the hierarchical structure. For example, levan is an uncharged polysaccharide with fructose as its basic monomer unit. Such a monomer is schematically shown in Figure 1c and is presented within this model as a string of spherical beads with a constant electron density, as indicated in Figure 1b. Furthermore, the scheme of nanostructured Debye-Bueche inhomogeneities with the correlation length of Ξ m is shown in Figure 1a. Note that this model does not distinguish between the polymer molecules (or their parts) situated within such denser inhomogeneities and those polymer molecules (or their parts) situated within the low-polymer-density areas surrounding them. The model therefore provides unified effective structural results that also depend on the actual extent of the molecules residing inside and outside such denser inhomogeneities. Since this model allows for a broad variety of possible molecular conformations, the set of resulting adequate solutions can also be broad. Moreover, as this


Page 11 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

model greatly simplifies the monomer units as homogeneous spheres, and is in this respect not very detailed, it can also be used in the case of the polymer mixtures, where one of the polymer types prevails. However, in such a case, the presence of the minority component in the polymer mixture is reflected only through its influence on the modeled conformations of the major polymer component within the mixture. The classical polymer intermolecular scattering contribution, i.e. the contribution due to the interparticle correlations, is within this model treated very similarly as in the classical polymer theory.37-40 This is done utilizing the following scattering function:41-43

 8π δϕ 2 Ξ ______  v m I ( q ) = ∆ρ  + v ⋅ P ϕ ( q ) , v 0  (1 + q2Ξ 2 )2  m   2

(1)

that combines the modelled intramolecular polymer form factor P ( q ) with the terms introducing the consideration of the intermolecular interaction effects. The symbol ∆ρ 2 represents the scattering contrast, v0 the polymer chain or polymer segment volume, Ξ m the correlation length of the Debye-Bueche inhomogeneities that cause the fluctuations δϕ v2

in

the local volume fraction of the polymer ϕv . Accordingly, within this model the information regarding the polymer molecular conformations is obtained through the values of four shape parameters Θp , Φp , Θplim and p * , and regarding the intermolecular correlations through the parameters

K,

ξm , Ξ m , δϕ v2

ϕv

2

, N and ξS , where N represents the number of the

monomers in the modeled segment and ξS is calculated as:

ξS =

2 ⋅ N

N

∑r

2 j

,

(S8)

j =1

where rj represents the distance of the monomer unit from the center of segments gravity. Prior to fitting the SAXS data the smearing effect needs to be considered on eq 1.15 Details of the


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 37

SAXS measurements as well as more detailed explanation of the model is given in Appendix A, Supporting Information.

3. RESULTS AND DISCUSSION The results presented in this section deal with the dynamics and the structure that develops in the Levan-based biofilms, which play a key role in various biological processes (e.g. in the development of plant diseases,46 in dental caries and periodontal disease,47,48 in basic functioning of bacterial populations, etc.). Since there is still a need for a greater understanding of the physicochemical interactions underlying the structure of the biofilms, and a number of questions remain regarding the intermolecular interactions during the biofilm formation,13,49,50 we provide and discuss some answers through investigation of the formation and structure of levan-based biofilms from Bacillus subtilis. For this purpose, we apply dynamic rheological, SAXS, DLS, density and sound velocity measurements, and microscopy. The following results provide insight into the origin of the shear-thinning, viscoelastic and structural properties of these model biofilms and the specific contributions of their individual components.

3.1 Rheological Results Levan samples of bacterial origin show non-gelling behavior in binary aqueous solutions. For example, levan from genus Bacillus sp. is soluble in water even up to about 60 wt.%, with its aqueous solutions behaving as Newtonian fluids up to about 30 wt.% of levan in the system.26,27 On the other hand, biofilm has a firm, gel-like structure, whereas the estimated concentration of levan in biofilm of B. subtilis subs. subtilis strain NCIB 3610 is only about 8 wt.%.12 Bacteria affect the intermolecular interactions, structure and rheological properties of levan and its metabolism in such a way that a compact gel can form even at low levan 12 ACS Paragon Plus Environment

Page 13 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

concentrations. We assume that the interaction of minor biofilm components (nucleic acids and proteins)24 with levan significantly changes the solution viscosity and the solubility of levan so that it forms less soluble structures. Aiming to highlight the structural aspects of biofilm formation, we now investigate the effects of DNA28 and protein (minor polymer components of biofilm) on levan solutions. The rheological responses of the samples with biofilm components and their mixtures in the form of viscosity η vs. shear rate γ& curves are shown in Figure 2. The polymer concentrations in these samples resemble those found for an individual component in the native biofilm. To facilitate the interpretation of the effects that an individual component exerts in more complex polymer mixtures presented later in the text, we first examine the rheological behavior of individual polymeric biofilm components and their less complex mixtures in solution. Since the biofilms studied in this paper were grown on medium containing SYM electrolytes, these electrolytes are also present in the biofilm. Therefore, in Figure 2a, the rheological behavior of solutions of EPS matrix components in pure water is compared with that of the same solutions containing SYM electrolytes. Due to the fact that levan is a nonionic polymer, and also that the viscosities of water and SYM electrolytic aqueous solution are very similar, one would expect that incorporating levan into each of these two solvents would lead to very similar viscosity curves. However, the results show that the viscosity of levan in SYM electrolytes is slightly lower than in water. This small difference may be due to some ionic impurities in the levan sample (DNA, proteins), which represent up to 2 wt.% of levan’s dry mass.12 In the SYM electrolyte solutions, the long-range electrostatic interparticle interactions are screened, which is expected to result in slightly lower viscosities of these samples. Interestingly, the viscosity curve of protein collagen was similar in the two solvents, which is probably due to the fact that collagen’s structure is fibrils in both cases, and thus, it is not significantly influenced by the electrostatic screening. On the other hand, the viscosities of the DNA samples differed a lot in


Langmuir

the two solvents. Namely, the slope of the viscosity curve in SYM electrolytes was higher; also, the curve settled at lower viscosity values at higher shear rates than in water. This means

(a) 1

(b) 10 L+D+C (= sEPS)

Medium: water SYM electrolytes

D

Medium: SYM electrolytes

L+D

1 0,1 C

L+C

η [Pa s]

η [Pa s]

L

0,01

0.1

0.01 SYM

1E-3

0,1

L

SYM

1E-3

water

1

10

100

γ. [1/s]

1000

0.1

1

10

γ. [1/s]

100

1000

(c) nBF1


10 sBF

η [Pa s]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 37

1 nBF2 0.1 0.01

sEPS SYM

1E-3 0.1

1

10

γ. [1/s]

100

1000

Figure 2. Double logarithmic plot of viscosity η vs. shear rate γ& for biofilm components and their mixtures measured at 20 °C: (a) Comparison of EPS matrix components in water and in SYM electrolytes, (b) polymer mixtures in SYM electrolytes, and (c) comparison of synthetic EPS matrix, synthetic biofilm, native biofilm, and homogenized native biofilm. For ease of comparison, the same curve may appear on several plots. The following abbreviations are used: 8 wt.% levan (L); 0.52 wt.% DNA (D); 0.43 wt.% collagen (C); synthetic EPS matrix (sEPS), which is a mixture of 8 wt.% levan, 0.52 wt.% DNA and 0.43 wt.% collagen; synthetic biofilm mixture (sBF), which is the sEPS sample containing also the added bacterial cells; native biofilm (nBF1); and homogenized native biofilm (nBF2), which is mildly ultrasonicated nBF1 sample.


Page 15 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

that DNA molecules dissolved in SYM electrolytes may be more compact and therefore able to rearrange themselves well in the flow, whereas DNA molecules dissolved in water exhibit more extended conformations and yield higher viscosities and sample rigidity. Very similar effects were observed when DNA was added to levan solutions (curves not shown). Newtonian-like behavior is exhibited by 8 wt.% levan samples in water and in SYM electrolytes (see Figure 2a), but samples of levan, collagen, and DNA show shear-thinning (pseudoplastic) behavior. Shear thinning occurs when polymer molecules orient themselves in the direction of the shear flow and disentangle to a certain extent, reducing the flow resistance in the sample and consequently decreases its viscosity. The viscosity values and the viscosity curves of all 4 samples varied significantly in this case. Differences in the rheological behavior of different biofilm components could be attributed to differences in their charge, shape, spatial packing, monomer composition, and polymer molecular weight. In the following, more attention will be placed on solutions in SYM electrolytes, because this is the basic electrolytic media in which the native biofilms are grown. In Figure 2b, the effects of adding minor EPS components (protein, DNA) to the SYM electrolytes solutions of major EPS component (levan) are shown. Adding DNA to levan solution significantly increased the solution’s viscosity and its already strong pseudoplastic character, while adding collagen had a similar effect, which was, in this case, accompanied by a notable increase in the rigidity of the system. The former effect can be clearly seen in the strong shearrate dependence of viscosity, while the increase in rigidity can be seen in the higher viscosity values of the plateau, which is reached at higher shear rates than for the initial aqueous levan sample. Also, the sample containing levan, DNA and collagen (designated also as the synthetic EPS) shows significantly increased viscosity with the pseudoplastic behavior and also increased rigidity of the mixed system. Comparing these viscosity curves to the corresponding


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 37

curves of individual components in SYM electrolytes from Figure 2a, one can clearly see that the presence of levan in mixtures merely increases the system’s viscosity. Interestingly, these results therefore already suggest that levan acts as a filling agent in these samples. The long, flexible DNA molecules seem to interconnect the levan moieties in the system and introduce a strong pseudoplastic behavior, while the less-flexible pseudoplastic collagen fibrils contribute to the structural rigidity of the system. Figure 2c compares synthetic EPS, synthetic biofilm mixture, native biofilm, and homogenized native biofilm. The SYM-electrolyte-based sample designated as the synthetic biofilm mixture sBF contained levan, DNA, collagen and additional bacterial cells. The bacterial cells were harvested during the levan isolation procedure from the native biofilm.12 These components were mixed in the same proportions as were found in the native biofilm, with the compromise that the model protein collagen was used instead of the native biofilm protein TasA. The curve for the native biofilm (nBF1) corresponds to the first measurement that was recorded immediately after the sample was loaded between the cone and the plate of the rheometer, but the curve for the homogenized native biofilm (nBF2) corresponds to the result obtained after consecutively repeated measurements, when the response of the system stabilized in the rheometer. As expected, the viscosity of the native biofilm is much higher than the viscosity of the homogenized native biofilm. This is due to the local density inhomogeneities found in the intact native biofilm; and/or because the connections between the living cells and their surroundings are irreversibly destroyed with biofilm homogenization between the cone and plate of the rheometer during the consecutive measurements. The viscosity curve of a homogenized native biofilm nBF2 was very similar to the viscosity curve of a synthetic biofilm sBF, composed of levan, DNA, collagen and bacterial cells, thus indicating that we managed successfully to mimic the viscous pseudoplastic response of homogenized native biofilm with a complex EPS structure in these mixtures. Slight differences


Page 17 of 37

might also arise because the fibril-forming protein TasA has been replaced by the structurally similar protein collagen in the present study. Viscoelasticity is another important rheological property that can be studied by making dynamic oscillatory rheological measurements. The experimental viscoelastic results on samples of polymeric biofilm components and their mixtures are presented in Figure 3 in the form of strain dependence of the storage G ' and loss G '' moduli. The effect of different solvents

(water

G', G'' [Pa]

D

1 D

0.1

Medium: SYM electrolytes G' G''

(b)

C

G', G'' [Pa]

Medium: SYM electrolytes - G' SYM electrolytes - G'' water - G' water - G''

(a)

0,1

L

0.01 0.1

1

γ [%]

10

100

0,1


(c) 10

L+C L+D

γ [%]

10

100


sBF 10 nBF1 sEPS

G', G'' [Pa]

1

1

(d)

L+D+C (= sEPS)

G', G'' [Pa]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

nBF2

1

0.1

0.1 0.1

1

γ [%]

10

100

0.1

1

γ [%]

10

100

Figure 3. Double logarithmic plots of G ' and G '' vs. strain γ for biofilm components and their mixtures measured at 20 °C: (a) The effect of the solvent on DNA solution, (b) individual biofilm components in SYM electrolytes, (c) polymer mixtures in SYM electrolytes, and (d) comparison of synthetic EPS matrix, synthetic biofilm mixture, native biofilm, and homogenized native biofilm.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 37

vs. SYM electrolytes) on DNA samples is shown in Figure 3a. DNA seems to exhibit gel-like behaviour in both solvents. Its elastic structure in water breaks down at much smaller strains than in SYM electrolytes. This is in accordance with the difference in the storage moduli of these two samples and also with the difference in their viscosities observed in Figure 2a. Very similar differences due to the solvent effect were also observed for the samples containing DNA and levan (curves not shown), but the results for the collagen samples were again rather independent of the solvents used (curves not shown). All these results are in accordance with expectations based on the electrostatic screening of interparticle interactions in the case of SYM electrolytes. DNA molecules are more extended in pure water due to repulsive electrostatic interactions, and therefore DNA contributes more strongly to the elasticity of the sample than it does in the SYM electrolytes. Similarly, due to electrostatically pre-stressed DNA structures in water, the structure can be broken at lower strain. In Figure 3b the dynamic rheological results obtained for collagen and levan in the SYM electrolyte medium are shown. From Figures 3a and 3b it can be seen that, in the SYM electrolyte medium, the levan and collagen show strong viscous character, whereas the sample with DNA shows predominantly elastic character. Such predominant elastic character was also observed for levan+collagen, levan+DNA, synthetic EPS, synthetic biofilm and the homogenized native biofilm samples that are shown in Figures 3c and 3d. A typical gel-like G '' curve was only identified in the SYM electrolytes and water-based DNA samples, and in the DNA+levan sample. The results obtained for plain collagen solution in SYM electrolytes clearly shows its predominating viscous character, but the result for the L+C curve in Figure 3c clearly indicates that the presence of collagen in the levan solution also significantly contributes to the elasticity of the system ( G ' > G '' ). Surprisingly, at the polymer concentrations used in the present study, the presence of collagen in the levan solution increases the elasticity of the system even more


Page 19 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

strongly than the presence of DNA (see G ' values in Figure 3c). Nonetheless, inferred from the clear peak in the G '' curve of the L+D sample in the vicinity of the yield point, DNA seems to be the component that actually initiates the characteristic gel-like behaviour of the samples. The gel-like nature of the samples can be more precisely analysed by the dependence of the storage G ' and loss G '' moduli on the angular frequency ω. For a typical gel-like sample, the prevailing elastic nature of the sample can be observed as G ' > G '' , both moduli changing at a similar rate with the change of frequency. The results from frequency-sweep measurements of biofilm components and their mixtures measured at 20 °C are shown in Figure S3 in Appendix B, Supporting Information. Because of the sensitivity limit of the instrument, it was not possible to measure the storage modulus, G ' , of levan. From this rheological test we could only conclude that plain levan and collagen solutions do not show gel-like behavior. To summarize the rheological results: even a very low concentration of additives can dramatically change the rheology of a levan solution. We found that levan mainly acts as a filling agent in these systems, basically increasing the sample’s viscosity, whereas the presence of DNA and collagen also significantly increase the system’s pseudoplasticity and elasticity, with DNA inducing gel-like behavior and the collagen enhancing the system’s rigidity. Of course, this is a somewhat simplified view of the roles of the components, because in reality their contribution is not additive; rather, they mutually affect and dictate the samples’ behaviour. Nonetheless, the minor biofilm components are essential for biofilm formation in this model system. In the following sections, we supplement these results on the macroscopic behaviour, as seen by rheology, with the results obtained by DLS, which probes the dynamics of the systems on a microscopic scale and also further with the microscopic structure as seen by SAXS.

3.2 Dynamic Light Scattering


Langmuir

To investigate the dynamics of the polymer solutions on the micro scale, the scatteringangle-dependent 3D-DLS measurements were performed on diluted levan, collagen, and DNA samples, as well as their mixtures at scattering angles between 50° and 140°. These results are presented in the form of ln (1 τ c ) vs. ln ( q ) plots in Figure 4a. The samples analysed in this way had to be considerably diluted (8 times) due to high turbidity, and contained the following concentrations of polymers: 1 wt.% levan (L), 0.06 wt.% DNA (D), and 0.05 wt.% collagen (C). As shown in our previous investigation, such dilution does not practically affect the slope of the line in ln (1 τ c ) vs. ln ( q ) plot.16 The sample containing solely collagen could not be investigated in this way, because it was too polydisperse to provide reliable DLS results. Figure 4a

reveals

the

strongly

non-diffusive

6

of

the

relaxation

100

• L, slope = 4.39 ± 0.02 • D, slope = 4.63 ± 0.05 • L+D, slope = 4.46 ± 0.02 • L+C, slope = 4.47 ± 0.04

polydispersity index

7

nature

(b)

(a)

ln(1/τc)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 37

• L+D+C, slope = 4.48 ± 0.04

5 4 3

80

60

40 -4,4

-4,2

-4,0

-3,8

0.015

0.020

0.025

q [nm-1]

ln(q)

Figure 4. (a) Scattering angle dependent DLS results as a plot of ln (1 τ c ) vs. ln ( q ) for 1 wt.% levan, 0.06 wt.% DNA, 0.05 wt.% collagen and their mixtures, dissolved in SYM salts; and (b) the corresponding q dependence of the obtained polydispersity index.

dynamics in the case of all measured samples (slope > 2), i.e. levan, DNA, levan+DNA, levan+collagen, and levan+DNA+collagen solutions. The slope of the line was found to be

( 4.39 ± 0.02 )

for levan; somewhat higher for DNA ( 4.63 ± 0.05) and for the polymer mixtures 20 ACS Paragon Plus Environment

Page 21 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

approximately mid-way between these values (around ( 4.48 ± 0.04 ) ). In terms of the modecoupling theory,32-34 this indicates that in Figure 4a we only observe the slow-relaxation mode, and that the structural coupling is somewhat more intense in pure DNA than in pure levan, but is rather similar in other samples, generally lying somewhere between the values observed here. Such strong coupling on a micro scale is in accord with the strong pseudoplastic and also strong viscoelastic behaviour observed on the macro scale. Figure 4b shows the polydispersity index, which reflects the considerable range of relaxation times in all these samples. The least polydisperse samples are the plain levan sample and the mixture of all three polymeric components, whereas the DNA sample shows a very strong polydispersity in the relaxation times, which can already be seen from the broad decrease of the corresponding intensity correlation function shown in Figure S2a in Supporting Information. Turning our attention from the nature of the relaxation processes in these samples to their speed: the relaxations are fastest and most polydisperse in the DNA sample, slightly slower in levan and the levan+DNA mixture, and even slower in the levan+collagen and levan+DNA+collagen mixtures. The speed of such structural relaxations can be directly correlated to the observed macroscopic viscosities measured at high shear rates depicted in Figure 2b; hence, those rheological results nicely complement these results obtained on the micro scale at high frequencies of the red laser light. Furthermore, in Figure S2b in Supporting Information, the intensity autocorrelation functions of levan and DNA solutions in water and in SYM electrolytes medium are shown and also seem to be consistent with the macroscopic rheological results. The two solvents used do not seem to have a significant effect on the levan, which is also true for the DLS results, but show considerable differences in the speed of relaxation of DNA—the relaxation is much faster in SYM electrolytic medium than in water. This indicates that DNA molecules are more compact in SYM electrolytic medium than in 21 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 37

water, as could be expected due to the electrostatic screening effects in the former. This feature is probably also expressed in the possible differences in polymer hydration, which is the topic of the following section. To conclude, the DLS results indicate highly non-diffusive structural relaxations in these samples on the micro scale. These results complement the dynamic rheological results showing strong viscoelastic character on the macro scale. Similarly, the results also nicely conform to the measured microscopic viscosities of the samples.

3.3 Hydration of Levan, DNA and Collagen Molecules Hydration of the polymer molecules is an important issue in polymeric solutions that influences the overall structure and dynamics of the system from the fundamental molecular point of view. We present here results of our investigation into the hydration of the polymers in water and in SYM electrolytes medium. The results of the density and sound-velocity measurements of levan, DNA, and collagen solutions are shown in Figure 5. The density and sound-velocity Using eq S2 in the Supporting Information, the adiabatic compressibility of levan, DNA, and collagen solutions was calculated. Its concentration dependence is presented in Figure 5c. The adiabatic compressibility decreases with increasing polymer concentration. A decrease in compressibility was also observed when SYM salts were added to the polymer solutions. When polymer solutions are strongly hydrated there is a substantial fraction of water molecules restrained by the hydration of the polymer molecules. Therefore, they cannot contribute to the compressibility of the system. Consequently, the compressibility of such polymer

solution

is


Page 23 of 37

(a) 1.05

1.02 1.01

C

L

L

1.03

D

L+D+C

1540

L+D+C

1530

v [m/s]

ρ [g/mL]

(b) Medium: water SYM electrolytes

1.04

1520 CD

1510

1.00

1500 0

20

40

60

80

0

γ [mg/mL]

20

40

60

80

γ [mg/mL]

(c)

(d)

4.5

30

4.4

D C

4.3

D

nh

βS,sol [10-10 Pa-1]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

20

4.2 C

10

4.1

L

L

L+D+C

4.0 0

20

40

60

γ [mg/mL]

80

0 0

20

40

60

80

γ [mg/mL]

Figure 5. Concentration dependence of: (a) density ρ , (b) sound velocity v , (c) adiabatic compressibility β S,sol , and (d) hydration number nh at 25 °C in water (open symbols – dashed lines) and SYM salts (full symbols – solid lines) for levan, DNA, and collagen monomer units.

significantly lower than the compressibility of water and it shows a decreasing trend with increasing polymer concentration. The hydration also exhibits a strong influence on the activity values were significantly lower for the samples in water than for the samples in SYM electrolytes medium. of the water molecules.35,51 The effect of the salt solution on the adiabatic compressibility is to reduce the hydration due to the reduced fraction of free water molecules, to increase the sound velocity for the same reason, and to increase the sample density. The overall effect is therefore a reduction of the compressibility with added salt solution.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 37

The hydration number can be calculated according to eq S3, which is based on the adiabatic compressibility. Concentration dependence of the hydration numbers for the levan, DNA, and collagen samples are shown in Figure 5d. Larger hydration numbers mean easier access of water molecules into the polymer structure. The number of water molecules per fructose monomer unit of levan is between 5 and 6, the number of water molecules per base pair in the DNA samples is between 25 and 27, and the number of water molecules per tripeptide in the collagen samples is between 12 and 13. These values are consistent with previous reports: the hydration number for the fructose monomer of levan from B. subtilis has been reported at around 6,12 the hydration number for a base pair of a DNA molecule was found to be between 19 and 26,52,53 while that of a collagen tripeptide was around 11.54 In all cases the hydration number of polymer monomer units was somewhat reduced in SYM electrolytes medium, which is consistent with more-condensed molecular conformations of the ionic polymers caused by electrostatic screening effects in SYM electrolytes medium.

3.4 Light Microscopy and Gel Electrophoresis Microscopy is a powerful tool that can usually bridge the gap between the macroscopic supramolecular structure of the system (which is usually much above the resolution of the SAXS experiment) and the microscopic molecular structure, which can be studied by using SAXS. The differential interference contrast (DIC) microscopy images of 8 wt.% levan, 0.52 wt.% DNA, 0.43 wt.% collagen, and their mixtures, dissolved in SYM electrolytes medium polymers are depicted in Figure 6. In the levan solution, spherical particles were observed that were similar to those seen in our previous study of 1 wt.% levan solutions.12 The DNA molecules were too small to be visible under the light microscope (image not shown), while the collagen solution revealed just a few visible collagen fibrils. The image of the levan+DNA solution was very similar to the plain levan solution, which may be due to the very small size of


Page 25 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

the DNA isolated from B. subtilis. In the levan+collagen solution, some levan particles were visible and were caught in the collagen fibrous network. It is possible that levan reduces the solubility of collagen. The image of the levan+DNA+collagen solution was similar to the image of levan+collagen, although the former solution seemed to be more viscous. Also, there appeared to be more clusters of collagen fibrils in the mixture of levan+DNA+collagen. Another interesting feature of the DNA samples was revealed by a combination of microscopy and gel electrophoresis. Results from the DNA samples from B. subtilis and from salmon are shown in Figure S4 in Appendix B, Supporting Information,. When we added levan to these two DNA solutions, the salmon DNA “embraced” the levan particles and created many clusters of levan (see Figure S4b). The same phenomenon was observed by Stojković et al. at high DNA concentrations.55 On the other hand, the DNA from B. subtilis did not show any such effect on levan particles (see Figure S4a). Gel electrophoresis (see Figure S4c) showed that the salmon DNA segments were much larger (> 1 kbp) than the B. subtilis DNA segments (< 0.5 kbp). These results demonstrate that the size of the DNA molecule is important for interaction with levan particles.

3.5 Small-Angle X-ray Scattering Applying SAXS in research of polymer and similar systems with large scattering units, one has to bear in mind that due to the limited experimentally accessible range of the scattering vector SAXS gives meaningful information for structures of approximately 1 nm up to a few tens of nm. Therefore, this technique can successfully reveal the structural information on the microscopic molecular level, whereas for information on the macroscopic structure of polymer systems, other methods need to be applied. In combination with an appropriate molecular model of the scattering system or theoretical scattering equation, one can usually extract


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 37

Figure 6. DIC image of (a) solvent (SYM salts), (b) 8 wt.% levan, (c) 0.43 wt.% collagen, (d) 8 wt.% levan + 0.52 wt.% DNA, (e) 8 wt.% levan + 0.43 wt.% collagen, and (f) 8 wt.% levan + 0.52 wt.% DNA + 0.43 % collagen.


Page 27 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

important geometrical parameters of the macromolecules from the SAXS data. While there are several reports of the use of SAXS in structural studies of a few polysaccharides,56-58 SAXS studies on polysaccharide levan and polymer mixtures are scarce.12,15,43,59 Polymer mixtures of levan, DNA and collagen are very complex. This complexity is reflected in the macromolecular structure and dynamics of these systems, as revealed by the rheological results and microscopic images. The SAXS data from levan, DNA, collagen and their mixtures are shown in a double logarithmic plot in Figure 7. In Figure 7a, the solutions in pure water are compared with those in SYM electrolytes medium. The steeper course of the scattering curves in the regime of low q values indicates that the polymers dissolved in pure water contain larger or more expanded molecular structures. The greatest difference between the two solvents is seen in the case of the DNA samples; most likely because the DNA molecules bear a high net charge that causes strong electrostatic interactions, which are successfully shielded in the SYM medium. These findings are in accordance with the macroscopic effects observed; i.e., the rheological, DLS, sound velocity and hydration results. In Figure 7b, the various mixtures of levan, DNA and collagen show rather similar scattering curves, which is a first indication that the nanostructure of these systems might be rather similar. The latter is confirmed by the modelling results presented below. Interestingly, the scattering curves of the samples containing also the bacterial cells (sBF and nBF1) in Figure 7c do show considerable differences in comparison to the one obtained for the polymer mixture without the cells (sEPS). Introducing bacterial cells to the polymer mixture does influence the scattering contrast in these systems. Furthermore, the cells are rather complex nanostructures themselves, and they correspondingly also influence the SAXS curves. Nevertheless, there are


Langmuir

(b)

Medium: water SYM electrolytes

(a) 1

L+D


L+D+C (= sEPS) L+C

-1

I(q) [cm ]

0,1 D

-1

I(q) [cm ]

L

C

0,01

0,1

1E-3 0,01 1E-4 0,1

-1

0,1

1

-1

q [nm ]

1

q [nm ]

(d)

(c)

1



1

sEPS

-1

I(q) [cm ]

1

0,1

0,1

0,8 I(q)

I(q)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 37

nBF1

0,6

sBF

0,01

sEPS

0,1

-1

L

0,01

1

0,1 q [nm-1] 0,2

0,1

q [nm ]

0,3 0,4

-1

1

q [nm ]

Figure 7. Log-log plots of experimental SAXS curves of biofilm, its individual components and their mixtures: (a) The effect of the solvent on individual biofilm components, (b) polymer mixtures in SYM electrolytes, (c) comparison of synthetic EPS matrix (), synthetic biofilm () and native biofilm (), and (d) comparison of SAXS curves of levan () and sEPS (), normalized to 1 at

qmin ;

the yellow lines represent best fits to the data according to eq S6; Inset:

Enlargement of the innermost part of the scattering curves. For the sake of comparison, the same curve appears on several plots. Absolute scaling was used on these experimental curves, but they still reflect the experimental smearing effect.

some notable differences between the SAXS results for sBF and nBF1; especially the broad peak at around q = 0.2 nm-1 in the scattering curve of nBF, which is absent in that of sBF. Since the SAXS curves for (non-homogenized) nBF1 and homogenized nBF2 (data not shown) were practically the same, the differences in the structural correlations between the cells and


Page 29 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

EPS in the two samples obviously do not exhibit themselves in the regime of our SAXS measurements. Therefore, the difference in the SAXS curves of nBF and sBF is also not expected to origin in possible differences in the structural correlations between the cells and EPS in the samples nBF and sBF. This difference in the SAXS curves can therefore be attributed to the fact that the protein in nBF1 (TasA) is different from that in the sBF sample (model protein collagen). It is also important to realize that the sBF sample contains only three purified main biofilm components, whereas in the natural nBF1 mixture, other bacterial metabolic components are also present in low concentrations. For detailed interpretation of the SAXS data of 8 wt.% levan and sEPS, the string-ofbeads model was applied based on eq S6 given in Supporting Information. The best fits to the SAXS data are depicted with full lines in Figure 7d. As explained above, the results reflect the structure of the medium through the structure of levan, which is influenced by the presence of some DNA and collagen in the sEPS system. The resulting intervals of modelling parameters obtained for the scattering curves presented in Figure 7d are given in Table 1. The intervals of the shape parameters are very similar or even overlap for these two samples. Since, in this * model, the parameter p represents the level of randomness during the growth of the model

string-of-beads and its value is low in both two cases, it indicates rather extended molecular conformations in both samples. These values namely suggest that during the growth of the model polymer string, approximately every fourth bead is oriented randomly, but still considering the excluded volume of other beads. The largest differences within the interaction parameters given in Table 1 are seen in the values of N and consequently also in are somewhat smaller in the case of sEPS. The value of

ξS

ξS , which

represents the characteristic size of

the modeled molecular segment that sufficiently well describes the nanostructure of the Debye-


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 37

Table 1. (a) Shape and (b) interaction parameters of the acceptable fits to the experimental SAXS data of 8 wt.% levan and sEPS solutions. The parameters of the best fits are given in bold. Parameters are explained into details in Appendix A, Supporting Information. SHAPE PARAMETERS

8 % levan

sEPS

θ plim θp Φp

1.60–1.75-2.00

1.75–2.00

1.10–1.20

1.00–1.10-1.90

1.25–1.50

1.30–2.30

p*

0.19-0.22–0.30

0.24–0.38

INTERACTION PARAMETERS

8 % levan

sEPS

N

40–50

30–35

〈 δϕ

2 v 〉/〈

ϕ v〉 0.040–0.045-0.100 2

Ξ m [Å] ξ S [Å]

> 0.030 (0.035)

80–165-220

>120 (270)

52–66

40–46

Bueche inhomogeneities. A slightly lower value of N or

ξS

obtained for the sEPS sample can

therefore be interpreted as a somewhat higher degree of nanostructuring of the inhomogeneities, or a somewhat higher degree of entanglement in the case of sEPS. Two of the resulting characteristic modeled conformations of a molecular segment are depicted in Figure S5 in Appendix B, Supporting Information,. These conformations also reflect the slightly * smaller values of parameter p in the case of levan solution. Based on these model results,

further visualization of the effective Debye-Bueche inhomogeneities is presented in Figure S6 in Appendix B, Supporting Information. Nevertheless, these results clearly indicate that the structure of these two samples is very similar on the SAXS scale (up to approximately 100 nm). This means that the considerable differences in the dynamic behavior of these systems revealed by rheological experiments must arise from the intermolecular interactions on the macroscale, while there are no significant structural differences on the nanoscale. Such a conclusion is reasonable, because both samples are based on the polysaccharide levan; therefore, the nanostructure is naturally mainly 30 ACS Paragon Plus Environment

Page 31 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

governed by levan, whereas the long DNA and protein molecules enhance the differences on a macroscale.

3.6 The model of the synthetic EPS network The results of this study indicate pseudoplastic rheological behavior of all studied polymer mixtures. Levan acts as a filling agent that increases viscosity of these samples, the long, flexible DNA molecules interconnect the levan moieties in the network and introduce a strong pseudoplastic behavior, while the less-flexible pseudoplastic collagen fibrils contribute to the structural rigidity of the system. There were no specific structural elements observed by microscopy. These findings were complemented with oscillatory rheological results that show strong elastic nature of all studied solutions with the exception of pure collagen sample. The storage modulus G ' was higher tham the loss modulus G '' . Strong elastic nature of the samples causes strong coupling and nondiffusive relaxation processes on the microscopic scale as observed by DLS technique. The strongest coupling effect was observed in the DNA sample that had the highest G ' / G '' ratio. On the other hand the lowest coupling was observed in levan sample. There was a good agreement found between the trends in the DLS intensity autocorrelation functions and the viscosities, even though high polydispersity was observed in the relaxation times. Furthermore, the molecular polymer structure of levan and synthetic EPS sample was also studied through the results of the string-of-beads model and SAXS data. Interestingly, very similar molecular structure was found in both samples, meaning that the considerable differences in the dynamic behavior of these systems revealed by rheological experiments must arise from the intermolecular interactions on the macroscale. As levan is a predominant polymer molecules in both samples the long DNA and protein molecules may enhance the differences on a macroscale. revealed.

The importance of the interparticle


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 37

interactions in these systems is eventually reflected also in the small differences in the hydration number obtained for the samples in pure water and in SYM electrolyte medium.

4. Conclusions In this study, we demonstrated the level of details on the macroscopic and microscopic sample dynamics and structure that can be addressed by a combination of various physicochemical techniques on levan, DNA, collagen solutions, their mixtures, as well as on synthetic and native biofilm. We were mainly interested in the role of the individual components in the development of the gel-like structure that is observed in the biofilm of B.

subtilis subsp. subtilis NCIB 3610. For this purpose, the rheological, small-angle X-ray scattering, dynamic light scattering, density, sound velocity measurements, and light microscopy were combined with the modeling of the resulting SAXS data by the string-ofbeads model. As it turned out, this very powerful combination of techniques is able to provide solid results on the dynamical and structural aspects of the microbiologically and chemically complex biofilm formations. Similar studies of the biofilms are scarce; therefore, we believe these results will be of broad interest to microbiological and colloidal community. The rheological results showed that adding DNA to levan solution greatly increases the viscosity, introduces the pseudoplasticity and raises the elastic nature of the system, while adding protein causes very similar effects and also enhances the system’s rigidity. Dynamic rheological results were confirmed by the DLS results, which showed non-diffusion relaxation processes in all samples studied. Utilizing microscopy, we were able to visualize the structure of some systems on a macroscopic level and, by applying other physicochemical techniques, also get an insight into the structure on a molecular level. Based on all these results, we can conclude that minor biofilm components (nucleic acids, proteins, simple salts) are essential for formation of the biofilm’s compact gel structure. DNA and proteins appear to represent some kind of elastic


Page 33 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

framework that confines the levan molecules. Levan molecules therefore behave like a filling agent and contribute mainly to the increase in the samples’ viscosity. The strong elasticity of such frameworks mainly arises from the molecular entanglements on the macroscopic level exerted by the extended molecular conformations of DNA and protein fibrils; whereas its rigidity is mainly gained by the presence of the protein fibrils.

Associated Content Supporting Information. Appendix A (materials, experimental and methods) and Appendix B (side results and discussion) are available free of charge via the Internet at http://pubs.acs.org.

Author Information Corresponding Author. Matija Tomšič; e-mail: [email protected]

Acknowledgment Sadly, Andreja Zupančič-Valant passed away on October 28, 2015. We dedicate this article to the memory of Andreja. We thank to Slovenian Research Agency for its financial support through grants P1-0201, P4-0116 and N1-0042. We are most grateful also to prof. Otto Glatter for his generous contribution to the instrumentation of our laboratory for the light scattering methods.

References (1) Davey, M. E.; O'Toole, G. A. Microbial biofilms: From ecology to molecular genetics. Microbiol. Mol. Biol. Rev. 2000, 64, 847–867. (2) Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Bacterial biofilms: from the natural environment to infectious diseases. Nat. Rev. Microbiol. 2004, 2, 95–108. (3) Stewart, P. S.; Franklin, M. J. Physiological heterogeneity in biofilms. Nat. Rev. Microbiol. 2008, 6, 199–210. (4) Erable, B.; Duteanu, N. M.; Ghangrekar, M. M.; Dumas, C.; Scott, K. Application of electro-active biofilms. Biofouling 2010, 26, 57–71. 33 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 37

(5) Logan, B. E. Exoelectrogenic bacteria that power microbial fuel cells. Nat. Rev. Microbiol. 2009, 7, 375–381. (6) Singh, R.; Paul, D.; Jain, R. K. Biofilms: implications in bioremediation. Trends Microbiol. 2006, 14, 389–397. (7) Hall-Stoodley, L.; Stoodley, P. Evolving concepts in biofilm infections. Cell. Microbiol. 2009, 11, 1034–1043. (8) Monds, R. D.; O’Toole, G. A. The developmental model of microbial biofilms: ten years of a paradigm up for review. Trends Microbiol. 2009, 17, 73–87. (9) Rohde, H.; Burandt, E. C.; Siemssen, N.; Frommelt, L.; Burdelski, C.; Wurster, S.; Scherpe, S.; Davies, A. P.; Harris, L. G.; Horstkotte, M. A.; Knobloch, J. K. M.; Ragunath, C.; Kaplan, J. B.; Mack, D. Polysaccharide intercellular adhesin or protein factors in biofilm accumulation of Staphylococcus epidermidis and Staphylococcus aureus isolated from prosthetic hip and knee joint infections. Biomaterials 2007, 28, 1711-1720. (10) Mack, D.; Davies, A. P.; Harris, L. G.; Rohde, H.; Horstkotte, M. A.; Knobloch, J. K. M. Microbial interactions in Staphylococcus epidermidis biofilms. Anal. Bionanal. Chem. 2007, 387, 399-408. (11) Flemming, H. C., Wingender J. The biofilm matrix. Nat. Rev. Microbiol. 2010, 8, 623– 633. (12) Benigar, E.; Dogsa, I.; Stopar, D.; Jamnik, A.; Kralj Cigić, I.; Tomšič, M. Structure and Dynamics of a Polysaccharide Matrix: Aqueous Solutions of Bacterial Levan. Langmuir 2014, 30, 4172−4182. (13) Garnett, J. A., Matthews S. Interactions in Bacterial Biofilm Development: A Structural Perspective. Curr. Protein Pept. Sci. 2012, 13, 739–755. (14) Wanga, L. L., Wanga L. F., Yeb X. D., Lia W. W., Shenga G. P., Yu H. Q. Spatial configuration of extracellular polymeric substances of Bacillus megaterium TF10 in aqueous solution. Water Res. 2012, 46, 3490–3496. (15) Dogsa, I., Kriechbaum M., Stopar D., Laggner P. Structure of bacterial extracellular polymeric substances at different pH values as determined by SAXS. Biophys. J. 2005, 89, 2711–2720. (16) Orehek, J.; Dogsa, I.; Tomšič, M.; Jamnik, A.; Kocar, D.; Stopar, D. Structural investigation of carboxyinethyl cellulose biodeterioration by Bacillus subtilis subsp subtilis NCIB 3610. Int. Biodeter. Biodegr. 2013, 77, 10-17. (17) Yun, U. J., Park H. D. Physical properties of an extracellular polysaccharide produced by Bacillus sp. CP912. Lett. Appl. Microbiol. 2003, 36, 282–287. (18) Ridout, M. J., Brownsey G. J., Gunning A. P., Morris V. J. Characterisation of the polysaccharide produced by Acetobacter xylinum strain CR1/4 by light scattering and atomic force microscopy. Int. J. Biol. Macromol. 1998, 23, 287–293. (19) Branda, S. S.; Vik, A.; Friedman, L.; Kolter, R. Biofilms: the matrix revisited. Trends Microbiol. 2005, 13, 20–26. (20) Branda, S. S.; Chu, F.; Kearns, D. B.; Losick, R.; Kolter, R. A major protein component of the Bacillus subtilis biofilm matrix. Mol. Microbiol. 2006, 59, 1229–1238. (21) Romero, D.; Aguilar, C.; Losick, R.; Kolter, R. Amyloid fibers provide structural integrity to Bacillus subtilis biofilms. PNAS 2010, 107, 2230–2234. (22) Gamini, A., Paoletti S., Toffanin R., Micali F., Michielin L., Bevilacqua C. Structural investigations of cross-linked hyaluronan. Biomaterials 2002, 23, 1161–1167. (23) Villain-Simonnet, A., Milas M., Rinaudo M. Comparison between the physicochemical behaviour of the two microbial polysaccharides: RMDP17 and rhamsan. Int. J. Biol. Macromol. 1999, 26, 55–62. (24) Dogsa, I.; Brložnik, M.; Stopar, D.; Mandič-Mulec, I. Exopolymer Diversity and the Role of Levan in Bacillus subtilis Biofilms. PLoS ONE 2013, 8, 1–10. 34 ACS Paragon Plus Environment

Page 35 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(25) Chai, Y.; Beauregard, P. B.; Vlamakis, H.; Losick, R.; Kolter, R. Galactose metabolism plays a crucial role in biofilm formation by Bacillus subtilis. MBio 2012, 3, e00184–00112. (26) Arvidson, S. A.; Rinehart, B. T.; Gadala-Maria, F. Concentration Regimes of Solutions of Levan Polysaccharide from Bacillus sp. Carbohyd. Polym. 2006, 65, 144–149. (27) Han, Y. W.; Clarke, M. A. Production and Characterization of Microbial Levan. J. Agric. Food Chem. 1990, 38, 393–396. (28) Stojkovic, B.; Sretenovic, S.; Dogsa, I.; Poberaj, I.; Stopar, D. Viscoelastic Properties of Levan-DNA Mixtures Important in Microbial Biofilm Formation as Determined by Micro- and Macrorheology. Biophys. J. 2015, 108, 758-765. (29) Romero, D.; Vlamakis, H.; Losick, R.; Kolter, R. An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms. Mol. Microbiol. 2011, 80, 1155–1168. (30) Chai, L.; Romero, D.; Kayatekin, C.; Akabayov, B.; Vlamakis, H.; Losick, R.; Kolter, R. Isolation, characterization, and aggregation of a structured bacterial matrix precursor. J. Biol. Chem. 2013, 288, 17559–17568. (31) Shida, T.; Mukaijo, K.; Ishikawa, S.; Yamamoto, H.; Sekiguchi, J. Production of longchain levan by a sacC insertional mutant from Bacillus subtilis 327UH. Biosci. Biotechnol. Biochem. 2002, 66, 1555-1558. (32) Bartsch, E.; Frenz, V.; Baschnagel, J.; Schartl, W.; Sillescu, H. The glass transition dynamics of polymer micronetwork colloids. A mode coupling analysis J. Chem. Phys. 1996, 106, 3743-3756. (33) Rajagopal, A. K.; Ngai, K. L.; Teitler, S. Theoretical Aspects of Coupling Model Schemes of Slow Relaxation in Complex Correlated Systems. J. Non-Cryst. Solids 1991, 131, 282-288. (34) Kjoniksen, A. L.; Nilsson, S.; Thuresson, K.; Lindman, B.; Nystrom, B. Effect of Surfactant on Dynamic and Viscoelastic Properties of Aqueous Solutions of Hydrophobically Modified Ethyl(hydroxyethyl)cellulose, with and without Spacer. Macromolecules 2000, 33, 877–886. (35) Glinski, J.; Burakowski, A. Modification of the Pasynski Method for Determining the Hydration Numbers of Nonelectrolytes. Chem. Phys. Lett. 2013, 566, 21–24. (36) Burakowski, A.; Glinski, J. Hydration Numbers of Nonelectrolytes from Acoustic Methods. Chem. Rev. 2012, 112, 2059−2081. (37) de Gennes, P. G. Scaling Concepts in Polymer Physics. Cornell University Press: Itacha, New York, 1979; p 324. (38) Hecht, A. M.; Horkay, F.; Schleger, P.; Geissler, E. Thermal Fluctuations in Polymer Gels Investigated by Neutron Spin Echo and Dynamic Light Scattering. Macromolecules 2002, 35, 8552−8555. (39) Geissler, E.; Horkay, f.; Hecht, A.-M.; Rochas, C.; Lindner, P.; Bourgaux, C.; Courrazze, G. Investigation of PDMS gels and solutions by small angle scattering Polymer 1997, 38, 1520. (40) Horkay, F.; Hecht, A. M.; Geissler, E. Fine Structure of Polymer Networks as Revealed by Solvent Swelling. Macromolecules 1998, 31, 8851−8856. (41) Dogsa, I.; Štrancar, J.; Laggner, P.; Stopar, D. Efficient modeling of polysaccharide conformations based on Small-Angle X-ray Scattering experimental data Polymer 2008, 49, 1398-1406 . (42) Dogsa, I.; Tomšič, M.; Orehek, J.; Benigar, E.; Jamnik, A.; Stopar, D. Amorphous supramolecular structure of carboxymethyl cellulose in aqueous solution at different pH values as determined by rheology, small angle X-ray and light scattering. Carbohyd. Polym. 2014, 111, 492-504.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 37

(43) Benigar, E.; Tomšič, M.; Sretenovic, S.; Stopar, D.; Jamnik, A.; Dogša, I. Evaluating SAXS Results on Aqueous Solutions of Various Bacterial Levan utilizing the String-of-Beads Model. Acta. Chim. Slov. 2015, 62, 509-517. (44) Humphrey, W.; Dalke, A.; Schulten, K. VMD - visual molecular dynamics. J. Molec. Graphic 1996, 14, 33-38. (45) Cerar, J.; Jamnik, A.; Tomšič, M. Testing Classical Approach to Polymer Solutions on SAXS Data of λ-Carrageenan, κ-Carrageenan and Methylcellulose Systems. Acta. Chim. Slov. 2015, 62, 498-508. (46) Koczan, J. M.; McGrath, M. J.; Zhao, Y.; Sundin, G. W. Contribution of Erwinia amylovora Exopolysaccharides Amylovoran and Levan to Biofilm Formation: Implications in Pathogenicity. Phytopathology 2009, 99, 1237–1244. (47) Mukasa, H.; Slade, D. H. Mechanism of the Adherence of Streptococcus mutans to Smooth Surfaces. Infect. Immun. 1974, 10, 1135–1145. (48) Manly, R. S.; Richardson, D. T. Metabolism of Levan by Oral Samples. J. Dent. Res. 1968, 47, 1080–1086. (49) Donlan, R. M.; Costerton, J. W. Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms. Clin. Microbiol. Rev. 2002, 15, 167–193. (50) Characklis, W. G.; Wilderer, P. A. Structure and Function of Biofilms. John Wiley & Sons: Berlin, 1989; p 387. (51) Bell, W.; Pethrick, R. A. Adiabatic Compressibility of Polymer Solutions. Eur. Polym. J. 1972, 8, 927–931. (52) Chalikian, T. V.; Sarvazyan, A. P.; Plum, G. E.; Breslauer, K. J. Influence of base composition, base sequence, and duplex structure on DNA hydration: apparent molar volumes, and apparent molar adiabatic compressibilities of synthetic, and natural DNA duplexes at 25°C. Biochemistry 1994, 33, 2394–2401. (53) Buckin, V. A. Experimental studies of DNA-water interaction. Mol. Biol. (Mosk.) 1987, 21, 512–525. (54) Fullerton, G. D.; Amurao, M.; Rahal, A.; Cameron, I. L. Micro-CT dilatometry measures of molecular collagen hydration using bovine extensor tendon. Med. Phys. 2011, 38, 363–376. (55) Stojković, B.; Sretenovic., S.; Dogsa, I.; Poberaj, I.; Stopar, D. Viscoelastic Properties of Levan-DNA Mixtures Important in Microbial Biofilm Formation as Determined by Micro- and Macrorheology. Biophys. J. 2015, 108, 758-765. (56) Robitzer, M.; David, L.; Rochas, C.; Di Renzo, F.; Quignard, F. Nanostructure of Calcium Alginate Aerogels Obtained from Multistep Solvent Exchange Route. Langmuir 2008, 24, 12547–12552. (57) Shtykova, E.; Dembo, A.; Makhaeva, E.; Khokhlov, A.; Evmenenko, G.; Reynaers, H. SAXS Study of ι-Carrageenan−Surfactant Complexes. Langmuir 2000, 16, 5284–5288. (58) Gawronski, M.; Aguirre, G.; Conrad, H.; Springer, T.; Stahmann, K.-P. Molecular Structure and Precipitates of a Rodlike Polysaccharide in Aqueous Solution by SAXS Experiments. Macromolecules 1996, 29, 1516–1520. (59) Khorramian, B. A.; Stivala, S. S. Assessment of Branching in Hydrolysates of S. salivarius Levan and L. mesenteroides Dextran from Small-Angle X-Ray Scattering. Carbohyd. Res. 1982, 108, 1–11.


Page 37 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Graphical Abstract:


Structure and Dynamics of a Model Polymer Mixture Mimicking a

Recommend Documents