**4. The supramolecular organization of polysaccharide fibrils: from cell release to giant gel networks**

Diatoms excrete large quantities of extracellular polymeric substances, both as a function of their motility system and as a response to environmental conditions [63,64]. The natural occurrence of EPS is closely linked to diatom biomass, a pattern consistent over both macro (km) and micro (µm) scales. We will focus on diatom exopolysaccharides produced in cell culture and from large aggregates they form in marine environment [3]. Those polysaccharides stand out by their supramolecular organization and gelling capacity due to hydrogen bonding and electrostatic interaction resulting in formation of reversible physical gel. Unlike chemical gels that are formed by chemical reaction using a crosslinking agent, the characteristics of polysaccharide electrolytes is to form gels by physical bonds through intermolecular forces among polymer chains [65,66].

Intermolecular interactions are the basis of life, and an extremely important part of biological research, so an enormous range of techniques have been applied to their study. Among the new methodologies (experimental and theoretical) developed and applied to polysaccharide conformation and dynamics, solution properties, chain aggregation and gelation, the results obtained using atomic force microscopy (AFM) have been pointed out among those giving the most striking results [67]. AFM connects the nanometer and micrometer length scales utilizing a sharp probe tip that senses interatomic forces acting between the surface of a sample and the atoms at the apex of the tip. The physical basis behind AFM and its ability to "feel" the surface, make AFM a versatile tool in biophysics allowing high resolution imaging, nanomechanical characterization and measurements of inter and intramolecular forces in living and non-living structures [68]. Thanks to the simple principle on which it is based, the AFM is a surprisingly small and compact instrument. Its use includes electronic control unit, computer and usually two monitors for simultaneous checking of image and imaging parameters. The probe which scans the sample surface consists of a cantilever and the tip located at the free end of a cantilever. The deflection of the cantilever is measured by an optical detection system. Registered values of cantilever deflection are electronically converted into pseudo 3D image of a sample. AFM is a nondestructive method which gives real 3D images of the sample with a vertical resolution of 0.1 nm and lateral resolution of 1 nm. Measured forces range from 10-6 N to 10-11 N.

356 The Complex World of Polysaccharides

diatoms, as well [62].

**release to giant gel networks** 

intermolecular forces among polymer chains [65,66].

depths.

dependence of hydrodynamic diameters in three seawater samples taken at different

Although the aggregation kinetics was the same (asymptotic shaped curve in Figure 7) the highest value (1 µm) of hydrodynamic diameter was significantly lower than values reported by Verdugo group (several micrometers) for oceanic samples [60,61] and cultured

**Figure 7.** Hydrodynamic diameter in dependence on time for Adriatic seawater samples.

**4. The supramolecular organization of polysaccharide fibrils: from cell** 

Diatoms excrete large quantities of extracellular polymeric substances, both as a function of their motility system and as a response to environmental conditions [63,64]. The natural occurrence of EPS is closely linked to diatom biomass, a pattern consistent over both macro (km) and micro (µm) scales. We will focus on diatom exopolysaccharides produced in cell culture and from large aggregates they form in marine environment [3]. Those polysaccharides stand out by their supramolecular organization and gelling capacity due to hydrogen bonding and electrostatic interaction resulting in formation of reversible physical gel. Unlike chemical gels that are formed by chemical reaction using a crosslinking agent, the characteristics of polysaccharide electrolytes is to form gels by physical bonds through

Intermolecular interactions are the basis of life, and an extremely important part of biological research, so an enormous range of techniques have been applied to their study. Among the new methodologies (experimental and theoretical) developed and applied to polysaccharide conformation and dynamics, solution properties, chain aggregation and gelation, the results obtained using atomic force microscopy (AFM) have been pointed out among those giving the most striking results [67]. AFM connects the nanometer and micrometer length scales utilizing a sharp probe tip that senses interatomic forces acting Here we will cover the recent achievements using AFM as the principal method in revealing the supramolecular organization of diatom exopolysaccharide fibrils beyond the chemical composition.

Polysaccharide samples for AFM imaging [69-72] and polysaccharide gels [70,73-77] are usually spread on freshly cleaved mica surface. The imaging of hydrated samples is preferably conducted in air to inhibit the unfavourable motion of polysaccharides in liquid medium. Such AFM studies have been validated against data obtained directly under buffers, transmission electron microscopy (TEM) studies and cryo-AFM. Balnois and Wilkinson [78] showed that when AFM is operated under ambient conditions, the thin water layer both sorbed to the biopolymers and present on the mica surface maintains molecular structure during AFM imaging.

Protocols for marine sample AFM imaging have been developed only recently: single diatom cells and released polymers [79]; isolated polysaccharides from diatom cultures [3,79]; marine gel polymers and networks [80]. The samples were prepared using the drop deposition method (5-10 µL aliquots) and mica as a substrate. Mica sheets were placed in enclosed Petri dishes for approximately 30–45 min to allow biopolymers and cells to settle and attach to the surface. Samples were then rinsed in ultrapure water (three times for 30 s) and placed in enclosed Petri dish to evaporate the excess of water on the mica. The rinsing step was necessary to remove the salt crystals that would hamper imaging. AFM imaging was performed in air at room temperature and 50–60% relative humidity. In contact mode the force was kept at the lowest possible value in order to minimize the forces of interaction between the tip and the surface. For imaging in tapping mode the ratio of the set point amplitude was maintained to the free amplitude (A/A0) at 0.9 (light tapping).

### **4.1. Exopolysaccharide production at a single cell level**

The ubiquitous marine diatom *Cylindrotheca closterium*, isolated from the northern Adriatic Sea, was used in AFM studies of exopolysaccharide production at a single cell level.

**Figure 8.** (**a**) AFM image of the whole *C. closterium* cell presented as deflection data. Arrow indicates the position of the polymer excretion site; (**b**) The released polymers still attached to the apex of the cell rostrum, deflection data, scan size 5 µm × 5 µm; (**c**) Released polymers presented as height data, scan size 4 µm × 4 µm and vertical scale shown as the color bar [3].

Figure 8a revealed the general features of a live *C. closterium* cell with the two chloroplasts and its drawn-out flexible rostra. Arrow indicates the position of polymer release shown in Figures 8b and 8c. The bundles of polymer fibrils extended up to10 µm from cell surface. Their heights are 5–7 nm at the position close to the site of excretion. At a distance of 1 µm the dense network is observed with fibril heights of 2–3 nm. At even larger distances the network is less dense with the fibril heights in the range of 0.4 to 1.2 nm. The lower value of fibril height corresponds to the single monomolecular polysaccharide chains [71]. At this

**Figure 9.** AFM images (tapping mode) of polysaccharides isolated from the *C. closterium* culture medium and dissolved in ultrapure water: (a) single fibrils (concentration 5 mg/L) vertical scale 2.5 nm; (b,c) fibril networks (concentration 10 mg/L). Vertical scales: 5 nm (b) and 10 nm (c) [79].

larger distance, the network appeared with incorporated spherical nanoparticles–globules. The globules are found to interconnect two or more fibrils. The globules may represent positively charged proteins whose function before the release is efficient intracellular packing of negatively charged polysaccharide fibrils, in line with molecular crowding in living cells [81].

## **4.2. Extracted and purified polysaccharides of C. closterium**

358 The Complex World of Polysaccharides

**Figure 8.** (**a**) AFM image of the whole *C. closterium* cell presented as deflection data. Arrow indicates the position of the polymer excretion site; (**b**) The released polymers still attached to the apex of the cell rostrum, deflection data, scan size 5 µm × 5 µm; (**c**) Released polymers presented as height data, scan

Figure 8a revealed the general features of a live *C. closterium* cell with the two chloroplasts and its drawn-out flexible rostra. Arrow indicates the position of polymer release shown in Figures 8b and 8c. The bundles of polymer fibrils extended up to10 µm from cell surface. Their heights are 5–7 nm at the position close to the site of excretion. At a distance of 1 µm the dense network is observed with fibril heights of 2–3 nm. At even larger distances the network is less dense with the fibril heights in the range of 0.4 to 1.2 nm. The lower value of fibril height corresponds to the single monomolecular polysaccharide chains [71]. At this

**Figure 9.** AFM images (tapping mode) of polysaccharides isolated from the *C. closterium* culture medium and dissolved in ultrapure water: (a) single fibrils (concentration 5 mg/L) vertical scale 2.5 nm;

(b,c) fibril networks (concentration 10 mg/L). Vertical scales: 5 nm (b) and 10 nm (c) [79].

size 4 µm × 4 µm and vertical scale shown as the color bar [3].

The polysaccharide fraction isolated from the axenic *C. closterium* culture medium [7] was used to test the capacity of photosynthetically produced polymers to self-assemble into gel phase.

The isolated polysaccharides for AFM imaging were prepared in two concentrations (5 and 10 mg/L). Single fibrils prevail in samples prepared with polysaccharide concentration of 5 mg/L. Dissolved polysaccharides resumed a flexible fibrillar structure (Figure 9a) with fibril heights of 0.4 and 0.9 nm. The value of 0.4 nm corresponds to polysaccharide single molecular chain. AFM imaging of samples prepared from the solution containing 10 mg/L revealed fibrillar networks varying in the degree of fibril associations (Figures 9b,c). The fibril heights in the networks span over the same range (0.9–2.6 nm) and the mode of fibril association follows the same pattern. However, the segments forming the network shown in Figure 9c are significantly wider (50 vs. 140 nm), suggesting side-by-side associations once the maximum height of individual fibrils is reached. If we take the value of 2.6 nm observed using AFM analysis as the maximum fibril height, then up to six monomolecular fibrils can constitute a single polysaccharide fibril [79]. The absence of globules in the AFM images of isolated polysaccharides also indicates that the globules, which appeared as a constitutive component in the EPS of single cells (Figures 9b,c), are not polysaccharides.

### **4.3. Evolution of marine gel polymer networks**

The fact that the isolated polysaccharide fraction has the capacity to self-assemble into a gel network in pure water is an important finding with implications on the mechanism of the macroscopic gel phase formation in marine systems. The marine gel is characterized as a thermoreversible physical gel and the dominant mode of gelation as crosslinking of polysaccharide fibrils by hydrogen bonding which results in helical structures and their associations [80]. This mechanism contrasts a more generally established view [82,83] that marine gel phase formation proceeds via cross-linking of negatively charged biopolymers (namely polysaccharides) by Ca2+ ions. Only recently, Ding *et al*. [62] reported that diatom EPS can spontaneously self-assemble in calcium-free artificial sea water, forming microscopic gels of 3–4 µm. They pointed out an overlooked issue of crosslinkers other than calcium ions in the formation of marine polymer networks.

Figure 10 represents the evolution of polymer networks of the macroscopic gel phase in the northern Adriatic Sea [84,85]. Samples were prepared from the macroaggregates with different residence time in the water column, from early stage of gel phase formation to the

**Figure 10.** Evolution of polymer networks in the macroscopic gel phase from (**a**) to (**c**): early stage of gel phase formation to condensed gel network of older macroaggregate. AFM images are acquired in contact mode and presented as height data, scan size 4 µm×4 µm [3].

condensed (mature) gel network of an older macroaggregate. The long polymer strands with small patches of initial fibril associations (Figure 10a) coexisted with the continuous gel network shown in Figure 10b. With the prolonged residence time (one month) the more condensed network is formed as presented in Figure 10c. The analysis of fibril heights for early and mature gel state is given in Table 6. The fibril heights for the early stage of gel network correspond to the fibril heights produced by Adriatic *C. closterium*.


**Table 6.** Comparison of polysaccharide fibril heights [3].

## **4.4. Exopolysaccharides interactions with nanoparticles**

Diatom EPS production increases as a feedback response to the presence of NPs and may thus contribute to detoxification mechanisms [86,87]. Specifically, in the study of Ag NPs toxicity to the marine diatoms *C. closterium* and *C. fusiformis* [88] an increase of EPS production was documented and the incorporation of Ag NPs was clearly demonstrated. Although Ag NPs integrated in EPS–gel network are beneficial to the diatom cell (detoxification), their accumulation and persistence in microenvironments prolong their presence in the water column and make NPs available to higher organisms. Residing in a gel environment, particles are prevented from aggregation and export from the water column.

The diatom EPS component responsible for the NPs interaction was identified by bringing in direct contact polysaccharide fraction isolated from *C. closterium* EPS with Ag and SiO2 NPs.

The polysaccharide fibril network was prepared by dissolving the polysaccharide fraction isolated from the *C. closterium* culture medium in ultrapure water at concentration of 20 µg/mL and stirred for 45 minutes before adding NPs, 10 µg/mL Ag-citrate coated NP, nominal size ~ 25 and SiO2 NP (LUDOX® HS-40, Sigma), nominal size 15nm were used.

**Figure 11.** Interaction of polysaccharide network and nanoparticles visualized by AFM. The polysaccharide network was preformed in 20 mg/L of isolated polysaccharides in ultrapure water before addition of 10 mg/L NPs.

The Ag and SiO2 NPs were detected exclusively on polysaccharide fibrils as single spherical particles or their agglomerates as shown by AFM images (Figure 11). The NPs did not induce cross-linking of fibrils nor change the fibril heights. Rather, particles are imbedded into the preexisting polysaccharide network. Besides the significance for the environment, such interaction of NPs and diatom polysaccharides can be applied for the design of composite materials, such as biocompatible gels with new properties [89]. Diatom extracellular polysaccharides could be also used as a capping agent giving rise to the stability of NPs in liquid environments over a broad range of ionic strength and pH.

#### **4.5. Force spectroscopy as future prospective**

360 The Complex World of Polysaccharides

**Figure 10.** Evolution of polymer networks in the macroscopic gel phase from (**a**) to (**c**): early stage of gel phase formation to condensed gel network of older macroaggregate. AFM images are acquired in

condensed (mature) gel network of an older macroaggregate. The long polymer strands with small patches of initial fibril associations (Figure 10a) coexisted with the continuous gel network shown in Figure 10b. With the prolonged residence time (one month) the more condensed network is formed as presented in Figure 10c. The analysis of fibril heights for early and mature gel state is given in Table 6. The fibril heights for the early stage of gel

**analyzed** 

Attached to the diatom cell 120 0.85 ± 0.32 0.4 - 1.8 Marine gel network: early stage 189 0.92 ± 0 .40 0.4 - 2.0 Marine gel network: mature gel 178 3.58 ± 0.76 1.6 - 5.0

Diatom EPS production increases as a feedback response to the presence of NPs and may thus contribute to detoxification mechanisms [86,87]. Specifically, in the study of Ag NPs toxicity to the marine diatoms *C. closterium* and *C. fusiformis* [88] an increase of EPS production was documented and the incorporation of Ag NPs was clearly demonstrated. Although Ag NPs integrated in EPS–gel network are beneficial to the diatom cell (detoxification), their accumulation and persistence in microenvironments prolong their presence in the water column and make NPs available to higher organisms. Residing in a gel environment, particles are prevented from aggregation and export from the water column. The diatom EPS component responsible for the NPs interaction was identified by bringing in direct contact polysaccharide fraction isolated from *C. closterium* EPS with Ag and SiO2 NPs. The polysaccharide fibril network was prepared by dissolving the polysaccharide fraction isolated from the *C. closterium* culture medium in ultrapure water at concentration of 20 µg/mL and stirred for 45 minutes before adding NPs, 10 µg/mL Ag-citrate coated NP, nominal size ~ 25 and SiO2 NP (LUDOX® HS-40, Sigma), nominal size 15nm were used.

**Fibril height /nm**  Mean value Range

contact mode and presented as height data, scan size 4 µm×4 µm [3].

**Polysaccharide fibrils Number of fibrils** 

**4.4. Exopolysaccharides interactions with nanoparticles** 

**Table 6.** Comparison of polysaccharide fibril heights [3].

network correspond to the fibril heights produced by Adriatic *C. closterium*.

In AFM force spectroscopy mode a single molecule or fiber is stretched between the AFM flexible cantilever tip and a flat substrate mounted on a highly accurate piezoelectric positioner. Polysaccharide molecule, protein or other biopolymer, is either adsorbed to the substrate or linked to it through the formation of covalent bonds. When the tip and substrate are brought together and then withdrawn, one or more molecules can attach to the tip by adsorption. As the distance between the tip and substrate increases, extension of the molecule generates a restoring force that causes the cantilever to bend. The deflection of the cantilever measures the force on the polymer with an accuracy of ~5 pN, while the piezoelectric positioner records the changes in the molecule's end-to-end length with an accuracy of 0.1nm.

AFM force spectroscopy is widely used method in polymer biophysics allowing measuring mechanical properties of single molecules, and with a possibility to directly quantify the forces involved in both intra- and inter-molecular polymer interactions [90-93]. It is also adopted in advancing diatom research into the nanotechnology era [94]. Most of the work done so far on measuring forces with AFM has distinguished non adhesive and adhesive EPS components and discovered the adhesive properties and designs that give explanation as to why diatoms have the great tendency to attach to surfaces. However, force spectroscopy has not yet been performed on single diatom polysaccharide fibrils or their networks. The data that follows are the results of exploratory experiments conducted on diatom polysaccharide molecules (single fibrils, Figure 12) and on marine gel polysaccharide network (Figure 13) in filtered seawater.

**Figure 12.** Force approach (in red) and extension (in blue) curves acquired for polysaccharide single fibrils in filtered seawater. Two typical curves are shown.

The extension curve with the two rupture events in Figure 12 could result from the two individual fibrils of different length (0.9 and 1.5 µm) simultaneously attached to the tip. The force spectrum signature for polysaccharide fibrils assembled in marine gel network is by far more complex (Figure 13) [95]. Assigning the underlying disentanglement events is in progress.

The force spectra can provide the critical piece of information that will allow us to characterize and quantify physical forces in polysaccharide network assemblies. Further developments will contribute to the new field of nanoecology and open the possibilities for rational design of polysaccharide gels with desired properties.

**Figure 13.** Force approach (in red) and extension (in blue) curves of polysaccharide fibrils assembled in marine gel network acquired in filtered seawater.
