High-Performance Filaments from Fractionated Alginate by Polyvalent

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High performance filaments from fractionated alginate by polyvalent crosslinking: A theoretical and practical approach Martin Sterner, and Ulrica M Edlund Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00619 • Publication Date (Web): 29 Jun 2018 Downloaded from http://pubs.acs.org on July 2, 2018

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High performance filaments from fractionated alginate by polyvalent crosslinking: A theoretical and practical approach Martin Sterner, Ulrica Edlund*

Fiber and Polymer Technology, KTH Royal Institute of Technology, Teknikringen 56, SE100 44 Stockholm, Sweden

KEYWORDS: Filament, fiber, alginate, polyaluminum, aluminum, calcium, gel, macroalgae

ABSTRACT A series of alginate fractions with significant differences in molecular weight and uronic acid compositions were produced by consecutive fractionation and converted to thin and strong crosslinked polymer filaments via extrusion into calcium, aluminum, or polyaluminum (PolyAl) polyvalent solutions followed by drawing and drying. Models were elaborated to relate the alginate uronic acid composition to the tensile performance, both in the wet gel filament and in the dry filament state. The wet gel model were compared to the theory of the unidirectional elongation of charged polyelectrolyte gels based on the classical rubber elasticity of dilated polymer networks extended to include the contributions of non-Gaussian chain extensions and the effect of electrostatic interactions. The theory of equilibrium swelling pressure was applied to describe the observed shrinkage of the alginate gels following immersion in a polyvalent solution. Congruent with the theoretical model of charged gels, the tensile performance of the gel filaments prepared from CaCl2 depended on the compositional ratio of guluronic acid dyads in the alginate fraction multiplied by the alginate concentration, while the tensile behavior of wet gel filaments prepared by AlCl3 instead resembled that of elastic solid materials and depended only on the alginate concentration. The dry filament tensile properties were greatly dependent on the preparation

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conditions, particularly the ‘stress to alginate concentration’-ratio and the nature of the ions present during filament drawing. The PolyAl solution effectively caused shrinkage of alginate to a strong extent, and the resulting filaments behaved as highly stiff materials able to withstand stresses of approximately 500 MPa and having elastic moduli as high as 28 GPa.

1.

INTRODUCTION

To gain momentum in the transition to a more sustainable economy, new strategies and enabling technologies are needed to utilize biomass as a platform to generate chemicals and materials as viable alternatives to fossil-based products. Macroalgae have the potential to generate large quantities of valuable algal biomass. Sea-based cultivations can generate just as much biomass as the same area in a cultivated forest.1,2 Aerials can be used that are not occupied by current cultivations and with no need for continuous nourishment, instead accumulating valuable elements from the sea that could be brought back to land plantations.3 A large-scale cultivation plant of Saccharina latissima algae has been established along the Swedish west coast. This algae is grown on algae-seeded ropes in seawater, but it also grows naturally in the same waters.1,4,5

Present in all brown algal species, alginate constitutes approximately one fifth of the algal dry weigh.6 Alginate is a polyelectrolyte polysaccharide composed of mannuronic (M) and guluronic (G) structural units. Alginate forms a gel when exposed to multivalent ions, with a gel-network junction motif referred to as the egg-box structure.7,8 Alginate has found diverse applications as a viscous solution, as a gel, and in the dry state: as food texturizer, textile bath viscosity aid, emulsifier, hemostatic woven fibers, skin healing gels and cell immobilization gels, to name a few.9–13 The alginate composition determines its quality. A high share of G has a direct strengthening effect of most metal ion based hydrogels.13 A high molecular weight is beneficial for slow release purposes and to effectively bind large protein.14,15 A high share of MG sequences gives chain flexibility that can aid for purposes such as protein binding.16 To enable broader and new applications of alginate and make this renewable resource a competitive alternative to fossil-based counterparts, two often requested features are i) a higher stress load capacity (especially for medical applications such as scaffolds) and ii) the possibility to have more consistent properties in gels and solutions for applications with demand for very precise reproducibility.17


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We developed a direct property based alginate fractionation,18 in which alginate is liberated under close to neutral conditions in smaller portions in several consecutive extractions. With this method, it is possible to regenerate the extraction solution with ion exchange resin and recycle it, and the method also generated other valuable fractions of cellulose, proteins and soluble carbohydrates. A major benefit of the cyclic fractionation is that a number of alginate fractions can be recovered with different uronic acid compositions and molecular weights. In addition, ultra-high molecular weight alginate may be recovered, which we herein aim to utilize for material purposes. Fractionation of alginate has been performed for instance by19 when pre-extracted alginate was fractionated, but alginate fractionation during extraction is, to our knowledge, not performed in other studies. It is possible to epimerize alginate with enzymes to change the uronic acid composition, an efficient tool to increase the share of G which may be costly in large scale and do not give the sequence length of G found in native alginate.20

Our aim is to elaborate and demonstrate a practical and theoretical framework for the preparation of cross-linked alginate filaments. Filaments are processed by extrusion into polyvalent solutions of calcium or aluminum, and in addition, a polyaluminum (PolyAl) ion solution is herein explored in the making of stiff alginate filaments. There are several studies on alginate filament formation in calcium solution,21–24 where the tensile behavior and structure is characterized. Aluminum ions have long been known to form gel with alginate10 but have not been applied for filament applications and PolyAl has hitherto not been used in connection to alginate.

We further aim to provide a systematic investigation of alginate filaments with equal effort put into understanding tensile behavior in the wet the gel state as in the dry filament state. Herein, we develop an empirical model to predict the tensile properties of the filaments based on the alginate concentration and the uronic acid composition as determined by 1H-NMR. Earlier studies have revealed that the gel stiffness of a given alginate is linearly dependent on the square of the alginate concentration and is highly dependent on the amount of G in the alginate.25,26 Our approach, in contrast to previous models, is to explain the model based on the unidirectional elongation of charged polyelectrolyte gels after shrinkage to equilibrium27,28 and to focus on the square of the G dyad content, rather than on G content, to model the filament behavior for more accurate property predictions.



2.

EXPERIMENTAL

2.1. Materials Cultivated Saccharina latissima brown algae were harvested on May 17, 2016, from a cultivation site at the Sven Lovén Centre for Marine Science (University of Gothenburg) on the Swedish west coast (Scheme 1, step A). The wet algae where frozen and kept at -20 °C until freeze drying (Scheme 1, step B). The freeze-dried algae was finely ground into a powder in a KG40 - De'Longhi Coffee Mill (Scheme 1, step C), and stored in a desiccator with dry silica gel (Scheme 1, step D) until further use. Sodium alginate, sodium citrate dihydrate ACS reagent ≥99%, sodium hydroxide ≥97% ACS reagent and hydrochloric acid 37% ACS reagent 98% where purchased from Sigma Aldrich. Deuterium oxide (D2O) (DLM4-100) 99.9% was purchased from Cambridge Isotope Laboratories, Inc. Ethanol 96% (v/v) was purchased from VWR. Aluminum chloride, anhydrous, sublimed, ≥98%, was purchased from Honeywell Fluka. Polyaluminum chloride solution (ACH PAX-XL19) was provided by Kemira AB and is denoted PolyAl in this paper.

Scheme 1. Experimental scheme for the fractionation of Saccharina latissima yielding several fractions of alginate precipitated by acid.

2.2. Extraction procedure of alginate

The preparation of the powdered algal sample followed by a fractionation procedure is schematically illustrated in Scheme 1. The extraction solutions were prepared from sodium citrate 0.01 M (Na3C6H5O7) dissolved in water. Samples were prepared in 600 mL centrifugation bottles to which 25.00 g algae powder and 475 g extraction solution were added for each bottle. The extraction was performed in 4 replicates. The fractionation of the algal material was performed by 18 consecutive extractions. In the first 15 extractions, 5%


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algae were extracted with 0.01 M sodium citrate. For the last 3 extractions, 2.5% algae were extracted with 0.05 M sodium citrate. Centrifugation bottles with extraction solution and algae were fixed in a standing position on a shaking board with a shake frequency of 300 revolutions/min and a shake radius of 0.5 cm, (Scheme 1, step E), shaking for 1.5 h for the first 15 fractions and for 5, 2 and 16 h, respectively, for the last three extractions. After each extraction cycle, the bottles were centrifuged for 20 min at 2680×g (Scheme 1, step F). The insoluble algal material that pelleted in the centrifuge bottles was removed, (Scheme 1, step G), re-dispersed in new extraction solution and subjected to a new extraction cycle (Scheme 1, step E). This cycling was performed 18 times to gradually extract increasing amounts of the alginate from the algae and thus fractionate it. For each extraction, the supernatant from the initial centrifugation was collected (Scheme 1, step H). The solution was cooled to 4 °C and alginate was precipitated by the addition of hydrochloric acid to pH 1. The precipitation time was 30 min, (Scheme 1, step I). The precipitated alginate was then concentrated by centrifugation, with 5 min runs at 2680×g. The first two runs were to gather all the alginate in one bottle and then a final time with addition of 25% ethanol was to form a more compact pellet (Scheme 1, step K). The alginate was further purified by neutralization in 1 L water and then precipitated in hydrochloric acid (pH 1) (Scheme 1, step I) two more times. The next step of purification was to disperse the alginate pellet from centrifugation in 50% ethanol in a water solution, (Scheme 1, step I). The alginate was shaken with 10 times the original alginate pellet volume at 200 rounds per minute for 15 min. This purification step was repeated twice. The purified alginates were finally carefully pH neutralized by the addition of sodium hydroxide and air dried in petri dishes (Scheme 1, step M). The first 6 fractions were pH neutralized before the ethanol purification step, a procedure that separated a top fraction. The pH values of the solutions were measured with a pH electrode, (Section 2.8). The fractions as well as materials made from alginate were named as follows: FX_Y% where X represents the fraction number and

Y

represents the alginate concentration in preparation (% w/w). For the

commercial alginate, Com was used instead of F, giving the name Com_Y%. Some examples include F8, F4_1%, F16_2%, Com_5%. 2.3. Alginate filament production

Samples of 5%, 2% or 1% (w/w) sodium alginate were prepared by mixing deionized water and dry sodium alginate, either commercial or from the fractionations (described in section 2.2). Each sample solution was prepared in a 50 mL Falkon tube and shaken over night to



reach complete dissolution. The solutions were centrifuged at 3850×g for 30 min to remove air bubbles. Each alginate solution was filled into a 10 mL plastic syringe with a diameter of 15 mm. A metal-nozzle with an end diameter of 1 mm was connected to the syringe and the syringe was placed in a ProSense NE300 screw syringe pump, (Scheme 2, step A). The syringe was placed with the nozzle approximately 1 cm below the liquid level of a 20 cm high conical flask filled with CaCl2 solution for crosslinking of the extruded alginate filament. The extrusion speed was altered for each alginate fraction, alginate concentration and CaCl2 concentration, to give a similar linear concentration to the formed gel filament. The extrusion speed was set low enough to make the solutions act as fluids and was in the range 0.2 - 5 mL/min (Table 1). The extruded alginate filaments were left in the flask for 2.5 h. Next, the alginate filaments were drawn by mounting them on 34.4 g steel plummets, with an apparent weight of 30 g in water, (Scheme 2, step B). The filaments were drawn in measurement cylinders filled with CaCl2 solution. The specific solutions are found in Table 1.

Scheme 2. Schematic illustration of the preparation of alginate filaments.

In the experiments with highly concentrated solutions, a tube filled with the solution was instead mounted directly on the thread acting both as an immersion bath and a weight of 30 g (Scheme 2, step E). The filament was in this case attached to the bottom of the tube by


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attaching it to a screw bolt that was fixed to the bottom of the tube by a small magnet. The alginate filaments were drawn for 2.5 h, then moved into another solution by replacing the measurement cylinder (Scheme 2, step C) and left for another 2.5 h. Finally, the liquid was removed, and the alginate filaments were drawn over night to dry (Scheme 2, step D) or (Scheme 2, step F). The dry alginate filaments where cut into 4 cm long pieces and dried in a desiccator for 3 days before weighing. Some specimens were made to test the tensile properties in the wet gel state; these specimens were prepared by immersing the extruded never-dried filaments into beakers filled with CaCl2 or AlCl3 solution with the two ends clamped above the surface of the solution. This setup was set to rest for 24 h so that both ends dried and formed two attachment zones for tensile testing (Scheme 2, step G).

Table 1. List of solutions used for the filament preparation outlined in Scheme 2. Alginate sources – Alginate conc. % (w/w) of sodium alginate in water – 5%, 2% Coma – 2%, 1% F16 & F17 F4, F8 & F12 – 2%, 1%b – 2%b, 1%b F3 F18 – 1%b Solutions step A M in mol/L and % (w/w) of aluminum in PolyAl solution CaCl2: 0.02 M, 0.09 M, 0.25 M, 0.015 M b Solutions step B Solutions step E Solutions step G CaCl2: 0.02 M, 0.09 CaCl2: 0.09 M, 0.25 M CaCl2: 0.015 M M AlCl3: 0.015 M, 0.06 M AlCl3: 0.01 M Solutions step C AlCl3: 0.015 M PolyAl: 12.4%, 6.2%, 0.8% PolyAl: 0.8% PolyAlc: 6.2%, 3.1%, 0.8% c PolyAl : 0.8% a. Commercial alginate form Sigma-Aldrich b. Only for step G c. PolyAl with lowered pH

2.4. Tensile testing The 4 cm long filaments, prepared as described in section 2.3, were conditioned at 50% relative humidity for 3 days before tensile testing. Tensile testing was performed with an Instron 5566 instrument and Bluehill software was used for test control and collection of data.



The filaments were fixed by gluing 1 cm of each end into a glass pipe filled with epoxy glue and then clamping the pipes into the grips of the instrument. A 500 N load cell was used for the testing. The elongation speed was set to 10%/min and applied after a slower pre-test that ran until a force of 0.5 N was reached. The alginate gel filaments were also tensile tested in the wet gel state. The wet samples had dry ends, prepared as described in section 2.3, which were mounted in the grips of the instrument. A 50 N load cell was used for the wet gel samples and the tensile test proceeded with an elongation speed of 50%/min, which started after a slower pre-test that ran until a force of 0.01 N was reached. 2.5. Alginate shrinkage in PolyAl solution A test was performed to determine to which extent alginate shrinks in a PolyAl solution. Alginate filament samples were formed by extruding a solution of 5% commercial alginate, 5 mL/min into a 0.25 M CaCl2 solution in which the formed filaments were immersed for a day. The alginate filaments were then cut into pieces that were quickly weighed, and then immersed in PolyAl solutions of different concentrations. The alginate samples were removed from the solution after 10, 40 and 130 min. The surfaces were gently and quickly dried with cotton swabs; the filaments were weighed, and then put back into the solution. The solutions tested were AlCl3 and PolyAl with different amounts of added HCl. The pH values of the solutions were measured with a pH electrode, (section 2.8). For comparison, some non-dried filaments from the dry filament manufacturing were also submerged in a PolyAl solution to determine the difference in shrinkage for different alginate fractions and concentrations.

2.6. Nuclear magnetic resonance, NMR 1

H-NMR was used to determine the uronic acid composition of the extracted alginate

fractions (Scheme 1, step M). The samples were dissolved in water, and the pH was adjusted to 3 via small additions of hydrochloric acid. Then, the samples were heated to 100 °C for 1 h. After cooling, the solutions were neutralized to pH 7 with sodium hydroxide, stirred until the alginate was completely dissolved, and then left to dry under an airflow at room temperature for approximately one day. Samples of 1% (w/v) dried material were dissolved in D2O and transferred to NMR tubes with an outer diameter of 5 mm. 1H-NMR spectra were recorded at 500 MHz on a Bruker DMX-500 NMR spectrometer. MestReNova software was used for data acquisition. 2.7. Rheology measurements


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Samples of extracted alginate 1% (w/v) were dissolved in deionized water and frequency sweeps were run in a rheometer of the TA Instruments Discovery HR-2 model. The settings were 25 mm diameter steel plate, 100 µm plate gap and 4 Pa pressure. The storage and loss moduli were recorded between 10-1.2 – 101.5 Hz. The complex viscosity was calculated with Trios v.4.21 software. For the sample solutions of lower viscosity, the storage and loss moduli showed an oscillating trend at higher frequencies, in these cases a trend line was drawn in the middle of the data points. 2.8. pH measurements A VWR SympHony SB70P pH-meter equipped with a Hamilton Biotrode electrode was used to measure the pH of the extraction solutions before and after extraction. CertiPur® disodium hydrogen phosphate/potassium dihydrogen phosphate (pH 7.0) and CertiPur® potassium hydrogen phthalate (pH 4.01) solutions from Merck were used for calibration.

3.

RESULTS AND DISCUSSION

We have devised a method for macroalgal valorization spanning from alginate fractionation from crude biomass to gel extrusion and the preparation and characterization of oriented dry filaments. The tensile properties in the gel state and in the final dry state were thoroughly evaluated to shed light on the important factors governing the generation of high performing alginate filament materials. Our in-house developed fractionation process allowed us to isolate a series of alginate fractions with significant differences in molecular weight and uronic acid compositions shown by compositional analyses (section 3.3), a necessity to draw conclusions about the properties in the gel and the structure-property relationships. A solid understanding of the mechanical properties in the gel state is the key to understanding the behavior in the dry state. Calcium alginate is the most studied form of alginate and is hence a good starting point when studying alginate. The properties of aluminum alginate are less known and studied. In this work, we also studied the behavior of aluminum alginate as well as if the shrinking effect was caused by the PolyAl solutions.

This work covers the process from alginate fractionation, gel filament extrusion, and filament drawing to generate dry filaments. Rheology and uronic acid composition analyses are performed on the alginate fractions and the tensile properties of the gel and dry filaments were ACS Paragon Plus Environment


tested. The alginate samples were prepared in fractions with significant differences in molecular weight and uronic acid composition, a necessity to draw conclusions about the properties in the gel state filaments. The knowledge of the tensile properties in the gel state was then used to understand the behavior in the dry state.

A first finding was that the mechanical performance of the filaments will depend on whether the crosslinking was achieved by rapid diffusion into the gel material or if it was done by slow deposition of calcium to make the material. The rapid diffusion method, which was used in this work, resulted in denser gels since they shrink after the gelation, while a slow deposition gives less dense gels with almost no shrinkage on a macroscopic scale. The degree of shrinkage will have a significant impact on the tensile properties, which may be explained by the fact that the first part of a tensile extension is walking in the reverse path of the shrinkage, and the extension is thus mediated by the same forces that stopped the shrinkage. For our gels, rapid diffusion was the preferred approach since it is a straight-forward method for making long gel filaments by continuous extrusion. Accordingly, our stress-strain model for the gel behavior is developed with gels formed by rapid diffusion in mind, based on the consideration that thin gel filaments form in a relatively high concentration of Ca2+. Calcium alginate is the most studied form of alginate and is a good starting point when studying alginate. Less studied are the properties of aluminum alginate, while alginate interaction with PolyAl ions has not yet been reported in the literature.

3.1. Extraction yields of the alginate fractions The multiple extractions of alginate described in section 2.2 generated 18 individual fractions. The yields and cumulative yields of these are found in Figure 1. To visualize the difference in extraction conditions F16-F18, the distance between these steps are ten times longer since they utilized ten times more sodium citrate for the extractions.


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Figure 1. Yield and cumulative yield of extracted alginate fractions. Rings represent the cumulative yield while bars represent the yield of each fraction. Fractions 16-18 were extracted at more effective extraction conditions, with ten times more sodium citrate per amount of algae and the distance between these data points was also increased ten times. 3.2. Rheological performance of the alginate fractions The rheometry results (Figure 2) expressed as tan δ indicate that there is a significant difference in molecular weight between the extracted alginate fractions. Storage modulus (G´) and loss modulus (G´´) vs. shear rate profiles for all extracted fractions of alginate are available in Supporting information, Figure S1. The apparent viscosities (η) as a function of shear rate (1/s) for each fraction are shown in Figure 2 (right). For each fraction, the zero shear viscosity (η0) and a calculated approximation of the alginate molecular weight (Mw) are also given. The apparent viscosity is approximately equal to the complex viscosity calculated from the frequency sweeps according to a relationship referred to as the Cox-Merz rule. The correlation is valid when the complex viscosity is expressed as the angular frequency (rad/s) where 1 Hz = 2π rad/s. The zero shear viscosity was calculated from the apparent viscosity at the lowest shear rate of our measurements where the curves levelled out the most. Furthermore, molecular weights of the alginate fractions were approximated from the zero shear viscosities using the power law equation proposed by Mancini et al 29. In the study of Mancini et al,29 the apparent viscosity of alginate in the concentration range of 0.125-1.5% and in the temperature interval 5-35 °C was correlated to alginate samples with determined Mw. The Mw values of Mancini et al29 were determined from measurements of intrinsic viscosities and by utilization of the Mark-Houwink equation.29 From the calculated values of Mw of the extracted alginates (Figure 2, right) it can be concluded that the later fractions appear to have higher molecular weights than fractions recovered early in the process. However, the last fractions, F17 and F18, showed lower values, which are most likely due to



an incomplete purification, something that is supported by the fact that gels made by F17 had lower concentrations than expected (Section 3.3). A shear thinning effect was observed for fractions F4 and above. F1 and F2 had such low viscosities that these fractions did not generate meaningful data of tan δ or complex viscosity at higher frequencies. The reference commercial alginate, Com, displayed a rheology profile very similar to F3, one of the earliest extracted alginate fractions.

Figure 2. Left: tan δ of a frequency sweep of the extracted alginate fractions. The striped line estimates the middle when the results oscillate at higher frequencies. F3 and Com were only plotted at low frequencies, since the data were too noisy at higher frequencies. Right: Apparent viscosity and zero shear viscosity (η0) of the extracted alginate fractions as well as a calculated approximation of Mw of each alginate fraction.29

3.3. Alginate composition The extracted alginate fractions were analyzed by 1H-NMR to determine the composition ratio of the two uronic acid building blocks, G and M in the alginate (Figure 3). 1H NMR spectra of all extracted alginate fractions are provided in Supplementary information, Figure S2. The uronic acid compositions were calculated from the peak areas of a, b, and c in the 1H


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NMR spectra using Equations 1 and 2. Equation 1 was used to determine the share of G and Equation 2 to determine the share of GG dyads. The areas a, b and c were calculated after first smoothing the NMR curve by a ‘centered simple moving average’ method to limit the impact of noise and differences in spectral resolution. The period for the moving average was 11 data points, which approximately equaled 0.003 ppm. After smoothening, five minimum points were calculated in the intervals: 1. (5.13-5.07 ppm), 2. (4.90-4.84 ppm), 3. (4.68-4.62 ppm), 4. (4.52-4.46 ppm) and 5. (4.32-4.26 ppm). The baseline and area delimiter for peak a was drawn between minimum 1 and 2. Likewise, the baseline was extended from minimum 3 to 4 for peak b, and from minimum 4 to 5 for peak c (Figure 3).

Figure 3. Left: The percent of G (dark gray) and GG dyads (light gray) in the alginate fractions calculated according to Equations 1 and 2. Right: Areas of peaks a, b and c in the 1

H-NMR spectrum of alginate showing the five minimum points in-between which the areas

are calculated. An alginate sequence of GGMG illustrates the peak assignments to uronic acids and the uronic acid dyads in the spectrum are indicated as well as the positions of the peak-generating hydrogens. a Equation 1 c

Equation 2

The alginate fractions extracted early in the process had greater shares of G and especially GG dyads. The G content decreased gradually for the fractions in the middle and then increased



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again for the final fractions. This could indicate that the G content is higher in the low molecular weight alginate fractions that are extracted first. Later in the process, when alginates with higher molecular weights are extracted, the alginate with less G is liberated first since it is soluble at higher concentrations of divalent ions in the extraction solution. In the final three extractions, when a more concentrated sodium citrate extraction solution is also utilized, the highest molecular weight alginate, with a higher G content, is finally extracted. 3.4. Tensile properties of alginate filaments in the wet gel state Alginate filaments were extruded starting with alginate solutions of 1% (w/w) and 2% (w/w) into a solution of 0.015 M CaCl2 solution. The exception was the reference commercial alginate, which did not form filaments at such low CaCl2 concentrations and instead was extruded in 0.09 M CaCl2, but then immersed in a 0.015 M CaCl2 solution, similar to the other threads. Even though the starting concentration of each fraction was known, the final concentration in the filaments changed due to shrinkage of the extruded gel. The shrinking effect was further explored by immersing some filaments into an AlCl3 solution. The filaments were weighed before and after drying to quantify the final alginate concentration. The filaments analyzed in the wet gel state were dried at their end zones according to (Scheme 2, step G) while keeping the mid-section wet. The dry ends were clamped in the grips of the tensile instrument to allow for measurements. The filament extrusion settings for the filaments assessed in the wet state are given in Table 2, together with the resulting filament diameters and alginate concentrations. In addition, Table 2 gives the compositional ratios of the G and GG dyads as a percent of total uronic acids, since these parameters were shown to impact the behavior of the wet state. Table 2. Alginate fractions and extrusion settings for the gel filaments assessed by tensile testing in the wet state. Commercial alginate, Com, was used as a reference sample. Alginate Com F3 F8 F12 F16 F17 G (% w/w) 57.9 75.7 67.4 71.9 79.2 81.9 GG (% w/w) 35.8 69.5 48.7 44.8 55.3 58.6 2 1 2 1 2 1 2 1 2 1 2 Alginate initial 1 conc. (% w/w) Final alginate 2.37 3.65 1.69 2.30 2.35 3.62 2.71 3.70 2.61 3.81 2.64 3.30 conc. (% w/w) 5 1 2 1 2 0.6 2 0.6 2 0.4 1.5 0.2 Extrusion (mL/min)


F18 80.1 57.9 1

2.71 1

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Filament diameter 1.67 1.41 1.64 1.35 1.46 1.41 1.39 1.41 1.36 1.28 1.47 1.37 (mm) 8 5 8 8 18 14 7 7 11 Samples in CaCl2 10 18 9 (0.015 M) 5 8 13 7 13 11 6 5 6 Samples in AlCl3 8 13 7 (0.010 M)

1.23 7 7

The observed change in alginate concentration between the initial solution to the final calcium alginate gel was in accordance with the results from a study on alginate spheres that were allowed to shrink in calcium solution.26 The concentration changes for the alginate spheres were similar to that of the filaments in our study with a concentration increase in the span of 0.75-2% for initial concentrations of 1-2% sodium alginate. In both studies, alginates with a high share of G and GG, (as in our fraction F3), showed the least degree of shrinkage.

Figure 4. Average stress-strain curves of gel filaments made by extrusion of alginate solutions into a CaCl2 bath. Left: filaments prepared from 2% (w/w) alginate solution, and right: filaments prepared from 1% (w/w). Error bars are not shown over the entire curves for clarity but are given at the end of each curve where the middle of each error-zone represents the average stress at break. The vertical direction of the error-zone indicates the double-sided T-distribution of stress at break (90% confidence level), and the lateral direction indicates the corresponding strain of the upper and lower value of the interval.



It has been shown that the Young’s modulus (E) is dependent on the square of the alginate concentration and that E strongly depends on the G concentration.25,26 It has also been shown that the bulk of the ‘the egg-box structure’ crosslink junctions motifs are stable over time.8,30 According to the classic rubber elasticity theory, the elastic modulus is solely dependent on the number of active chain segments per volume, which is dependent on the number of crosslinks. Since G units are involved in the crosslinking ‘egg-box structural motif’ it is reasonable to believe that there is a correlation between the elastic modulus of the gel and the amount of GG dyads. The concentration of G in the GG dyad positions (cGG) was calculated as the total concentration of alginate (calg (%w/w)) multiplied by the compositional ratio of GG dyads. Indeed, a trend was found for the stress of elongated filament to the individual cGG2 (R2 ≈ 0.90). The poorer trend of stresses for elongated filaments vs calg2 resulted in (R2 ≈ 0.60). The linear trend is visualized as the stress at extensions 50%, 100% and 150% plotted versus cGG2 for all the samples (Figure 5, top). The fact that the linear regression is intersecting at zero stress for zero concentration confirms our belief that it is a valid measure of the gel stiffness.

Next, we attempted to derive a general relationship to describe the stress in a calcium alginate gel; we plotted stress as a function of cGG vs strain. The samples that passed 130% elongation were chosen to draw a general curve (Figure 5, bottom). By using the value from a point in this curve and multiplying it with cGG2, it will give the stress at the given strain. The following linear relationship was seen at strains > 40%: 1972ε − 723" Equation 3

The relation can more closely be described as a linear function guided by two exponential functions. #1972ε − 723 e-&&' -723 − 1972(" e-)' 1446 − 1972(", Equation 4


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Figure 5. Left: Stress vs (cGG)2 for all samples at the strains 50%, 100% and 150% with the

R2-value of the linear trend line for each case. Right: σ/cGG2 vs strain for all samples and combined to an average curve described by Equation 4.

The tensile behavior of alginate gel filaments crosslinked in CaCl2 shows that higher amounts of GG dyads as well as higher concentrations of alginate result in stiffer filaments. The tensile νs·RT·(λ-1/λ2), where νs is the active chain segments and λ is the stretch ratio. We hypothesize

stress vs strain curve (Figure 5, bottom) differed from the classical rubber equation: σ =

that the stiffness can be explained by using the rubber network theory of charged gels, which is discussed thoroughly in section 3.5.

The gel filaments immersed in AlCl3 were also subjected to tensile testing and data analysis in the same way as the calcium samples (Figure 6).



Figure 6. Average stress-strain curves of gel filaments made by extrusion of alginate solutions into a CaCl2 bath but transferred to and immersed in an AlCl3 solution. Left: filaments prepared from 2% (w/w) alginate solution, and Right: filaments prepared from 1% (w/w). Error bars are not shown over the entire curves for clarity but are given at the end of each curve, where the middle of each error-zone represents the average stress at break. The vertical direction of the error-zone indicates the double-sided T-distribution of stress at break (90% confidence level) and the lateral direction indicates the corresponding strain of the upper and lower values of the interval.

The gel filaments prepared in the AlCl3 solution became much stiffer than the analogous filaments prepared in CaCl2. Their calcium counterparts also broke at lower elongations (Figure 6). We used a similar approach as when deriving the relationship between stress and CGG for Ca2+ crosslinked alginate gel filaments. First, we plotted stress vs. alginate concentration (Figure 7, top). In this case, the stress values measured at the elongations 15%, 30% and 45% were chosen for modeling and the best correlation was achieved when stress was plotted vs. calg2 (R2 ≈ 0.85). In this case, a poorer fit (R2 ≈ 0.55) was achieved when stress was plotted vs. cGG. These results will be further discussed in section 3.5. To derive a general relationship for the stress in the Al3+ crosslinked alginate gel, stress as a function of calg was


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plotted vs. strain (Figure 7, bottom). Samples that passed 40% without breaking were chosen to make the general curve. At strains > 10% the following relationship was observed: 012 1188ε 150" Equation 5

Similar to the gel crosslinked with Ca2+, the relationship can also be described as a linear function guided by two exponential functions.

012 #1188ε 150 e-67' 150 1188( " e-&8' -300 1188( ", Equation 6

Figure 7. Left: Stress vs (calg)2 for all samples at the strains of 50%, 100% and 150%. The

equations result from a linear regression of the fitted data for each case. Right: σ/calg2 vs 9 for all samples and combined to an average curve described by Equation 6.

The substantial difference between the alginate gels made by crosslinking with Ca2+ or Al3+ can be explained by a difference in the network structure. The egg-box structure is the most common network junction motif for alginate crosslinked by polyvalent metal ions such as Ca2+. Due to the high charge and small size of Al3+ ions, the crosslinking motif is different in this case: it not as dependent on G and with more possible binding sites in the alginate compared to Ca2+.31–34 Alginate forms gels at low pH and AlCl3 solutions are by nature acidic, however the pH of approximately 4 that is found for the concentration ranges used in this study are well above pH ~2.5 when alginate start to form gels.35,36



3.5. Theoretical explanation of the empirical data model To explain the behavior of the gels, a calculation model was applied in which a gel is first crosslinked at the initial concentration (the concentration of alginate that is extruded from the syringe into the CaCl2 solution). The crosslinking causes the gel to shrink and, causing an increase of the alginate concentration in the gel. The gels, after reaching equilibrium shrinkage, are then uniaxially elongated which induces a stress acting to reverse the elongation. Our model is based on the classical rubber elasticity of dilated polymer networks

but also includes the contributions of non-Gaussian chain extensions (Ø; ) and the effect of

electrostatic interactions (Ø< ).27,28,37 In the model, the modulus of unidirectional elongation (G) is related to the amount of active gel network chain segments.

The calculations are based on the theoretical framework presented by Hasa et al, and Alenichev et al, with necessary changes made since we use polyvalent counter ions instead of monovalent. Since alginate has a dense gel network with short repeating motifs and we only have a moderately high ionic strength in our system, the most commonly used approximation of electrostatic contributions to the free energy of flexible polyelectrolytes works poorly for short changes of the polymer chain end-to-end distances.37 Therefore, an extra parameter was added that was derived from the better approximation of the free energy for the low salt situation and presented in the appendix of Katchalsky et. al.37 Without this parameter, the model often predicts negative tensile curve slopes. The theory of equilibrium swelling pressure was utilized to describe the shrinking effect in the alginate gels that were formed after a rapid crosslinking due to immersion in the CaCl2 or AlCl3 solutions. The contributing pressures were Gaussian and non-Gaussian rubber elasticity

(=>1 ), electrostatic forces (=>1? ) and osmotic forces (=@?A ).27,28 The pressure caused by

polymer mixing is sometimes included in similar calculations on other polyelectrolyte systems but was omitted here since it is said to be negligible for alginate.13,38 We

hypothesized that the crosslinked gels were dense enough to behave partly as solid materials and, therefore, the shape of a solid material test curve was fitted to a Boltzmann superposition integral curve of dampened springs as of a solid material (σ?@1CD ".39 The curves of the

different models are plotted and compared to the average experimental data (Figure 8).

σ2>1 G ∙ 1 Ø< Ø; "λ − λ- " Equation 7 =G@G =>1 =>1? =@?A Equation 8


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σ?@1CD Hεt" Bεt" J1 −

KL" M

N Equation 9

Figure 8. a) Ptot for alginate gels starting with 1% or 2% alginate concentration for three different effective degree of ionization (i), from top to bottom i = 0.20, 0.25 and 0.30. The curves go from the initial alginate concentration towards the concentration at zero pressure (equilibrium). The full and striped curves represent the increase in calcium alginate concentration resulting from shrinkage, (1% → 2.6%) and (2% → 3.6%), respectively, while the dotted curve represents the increase in aluminum alginate concentration resulting from shrinkage, (2% → 4.2%). b) Stress vs. unidirectional strain of the calcium alginate gels with a concentration change (1% → 2.6%) for different i-values, compared to the average empirical data at a 2.6% alginate concentration and 50% GG dyads. c) Equivalent to b but for a concentration change of 2% → 3.6%. d) Stress vs. unidirectional strain of the aluminum alginate gels with a concentration change of 2% → 4.2% for different i-values, compared to the average empirical data of 4.2% alginate concentration, a fitted curve of a solid behavior and the lowest curve of tested solid material with its stress values divided by five. e) The best fitted curve of calcium alginate gels with a concentration change of 1% → 2.6%, i = 0.30, a fitted curve for the solid behavior fitted based on the first 20% of elongation and the addition



of the two; as well as a comparison to the average empirical data. f) Equivalent to e but for a concentration change of 2% → 3.6%.

The gel shrinkage (Figure 8a) is the result of conflicting omnidirectional forces that act on the gels as pressures. A schematic illustration of the gel shrinkage as a result of these omnidirectional forces is shown in the supporting information, Figure S3. The elastic pressure (Pel) acts in the direction to shrink the gel while the electrostatic (Pels) and osmotic pressures (Pos) act in the opposite direction. An equilibrium is reached, which is very much governed by i) the parameter effective degree of ionization, which increases Pels and Pos, and ii) the chain segment length (Z), which decreases Pel. To predict shrinkages similar to the ones that were observed in our experiments, the parameters i and Z were set so that calcium alginate with an initial concentration of 1% changed to 2.6% after shrinkage while alginate with an initial concentration of 2% changed to 3.6% after shrinking. The gel shrinkage in AlCl3 solution system changed concentration from 2% to 4.2% after shrinkage, to simulate a moderately higher shrinkage than with CaCl2. The fitting to realistic shrinkages supplies us with ratios of Z and i that could give such shrinkage effect. To test which of these ratios that comply with our data, we compared the plot of the calculated unidirectional stress performed on the gels after shrinkage to our real tensile test data. A previous study showed that in terms of creep compliance, both M-rich and G-rich alginate behave as almost linear materials for 2% alginate gels.40 A Poisson ratio of 0.5 was used to calculate the modulus, since gels mostly behave as rubber materials.41 Comparisons to the average data for gel filaments with concentration changes from 1% to 2.6% and from 2% to 3.6% are shown in Figures 8b and 8c, respectively. A gel with i = 0.30 gives the best fitted tensile curve, with Z = 80 or 68 for the gels with 2.6% or 3.6% final concentration, respectively. The magnitude of these Z-values agree with the literature. For example, A. Haug and O. Smidsrød show that alginate with a DP of 65 still can form gels (albeit weaker) which suggests that the chain segment length was in that order of magnitude.25 The lower Z-value for higher alginate concentrations that we predicted is in agreement with literature results showing that the gel mesh size is in the order of 5-15 nm (10-30 uronic acid units) and decrease with increased alginate concentration.42


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The scenario for the pressure/concentration plot of 2% alginate in AlCl3 (dotted line in Figure 8a) is a simplification since our gels were first made in CaCl2 and later transferred to AlCl3. Still the predicted tensile curve deviates so significantly from the actual data curve that it can safely be said that the charged gel model is not applicable in the case of the gels made with Al3+ at the concentrations used. The curve shape instead resembles that of a solid material, as described by Equation 9. The calcium alginate gels also exhibited a certain element of the solid behavior. The solid part of the behavior is visualized in Figures 8e and 8f by fitting a curve based on Equation 9 to the first 20% of the average curves of calcium gels with 2.6% and 3.6% alginate content, respectively. By combining the curves for the solid behavior with the curve of the charged gel behavior it is possible to visualize how a charged gel expressing a partly solid behavior can be fitted to our empirical data. The contribution of non-Gaussian effects is a common explanation to the initial drop and later upturn in stress/strain-curves of soft gel materials. The non-Gaussian effect is included in our calculations of the elasticity and is evident as the somewhat bent shape of the calculated test curve. The curve would linearize from approximately 60-80% elongation if only the electrostatic effect and the Gaussian extension were considered. The amount of non-Gaussian contribution depends on the persistence length of the polymer and, in the case of alginate, the persistence length decreases with increasing concentration. At the concentrations reached by the gels in our experiments, alginate behaves as a flexible polymer with a persistence length of approximately 1.5-1.75 times the length of a monomer unit, which equals a flexible chain with low amounts of non-Gaussian contribution. However, the curves representing our experimental results show no upturn at higher elongations, which implies that non-Gaussian effects are still overestimated in the model. The effective degree of ionization is correlated to the activity of the polyelectrolyte counter ions and determines the degree of osmotic pressure related to the thermodynamically defined charging parameter ( ⁄OPQ 7 )), which is the

that can be exerted by a volume of polyelectrolytes. The effective degree of ionization is

distance between charges (b0) and the permittivity of the solvent (D). A charge screening effect can occur in the interaction between a specific polyelectrolyte and its counter ions which affect the effective degree of ionization. Multivalent counter ions contribute more to charge screening and always exhibit lower values than the same polyelectrolyte with monovalent ions. When analyzing systems with multivalent counter ions, it is common to measure below predicted values of the effective degree of ionization due to the formation of aggregates, which keeps parts of the charges screened from detection.37,43,44 In the presence of



calcium ions, alginate forms small individual aggregates at low alginate concentration and a gel network of fibrous aggregates at higher concentrations.44–46 In both cases, the effective degree of ionization is difficult to quantify. The exception is alginate with a high M/G-ratio, which have a lower tendency to form aggregates and where the effective degree of ionization thus can be more accurately determined. Measurements of the effective degree of ionization on an M-rich alginate resulted in a value of i = 0.28 for normal to high molecular weight alginate44,47 while alginate with a higher share of G was estimated to have i = 0.30.48,49 Both M and G in alginate have a charge distance below that of the Bjerum length (lB) (7.1 Å at room temperature), which is the limit where theoretically a decrease in charge distance have low impact on the effective degree of ionization.43,50,51 It has also been shown that the polymer concentration and reasonable excess concentration of divalent salt only have a low impact on effective degree of ionization.28,52–55 We found that an effective degree of ionization of i = 0.30 fitted best to the empirical data, which is reasonable since it is close to the literature values of alginate. It is important to note that this fit concerns a gel with a compositional ratio of 50% GG-dyads. A weaker gel (lower ratio of GG-dyads) with less stress at a certain strain would require a slightly lower effective degree of ionization to give a similar curve shape (Figure 8 b and c). Our experiments show that the curve shape was more or less retained for gels with different GG-dyad ratios, which enabled us to derive the empirical Equation 4. The retention of curve shape could be due to the fact that the effective degree of ionization increases with increasing ratio of G. It has been shown that increased backbone rigidity of charged polysaccharides increases the effective degree of ionization.48 In addition, it has theoretically been shown that the backbone rigidity is highest for GG sequences, second highest for MM sequences and lowest for MG repeating sequences in alginate.56,57 An experimental study from 2006 indicated that there was no or very little difference in the effect of rigidity between the different uronic acid sequences.58 Another study later indicated that there is indeed a difference in rigidity.57 The alginates prepared in our experiments were rich in G compared to M. MM segments were scarce and not contributing much to the overall rigidity of alginate, instead an increase in G increases the amount of GG sequences (highest backbone rigidity) at the expense of MG sequences (lowest backbone rigidity). The gels made from F3 showed less shrinkage than the other gels, possibly since F3 had the highest share of GG, a high share of MM and a very low share of MG compared to other fractions, and therefore the most rigid alginate backbone. The extra high rigidity of F3 and possibly also its’ lower molecular weight are two factors that have been attributed to give less shrinkage.39,59


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Biomacromolecules

In summary, it appears that the tensile properties of calcium crosslinked alginate gels can be described by material properties largely described as charged gels that has undergone shrinkage after crosslinking and, to a smaller degree, behave as a solid material. The aluminum gels on the other hand seemingly behave more similar to a solid material. The tensile curves of the aluminum crosslinked samples with the lowest molecular weights, F3 and Com, both lack some of the initial upturn characterizing a solid material tensile curve. F3 and Com are of different ends in the scale of M/G ratios so the effect of different uronic acid compositions can be excluded. Instead, the lack of long distance chain entanglements may give rise to the softer behavior. We did not observe any difference based on molecular weight for our metal ion crosslinked hydrogels when comparing the stress resistance of gels made from high to very high molecular weight fractions, This observation correlates well to the results of other studies.25,26 The difference between high and very high molecular weight alginate is more clear when alginate interacts with large charged species such as proteins.14 The fact that stress resistance of alginates seemingly depend on the amount of GG dyads can be understood by an increase of the number of crosslinks, which increases the elastic modulus of the gels. This does not, however, explain why the shape of the curves seems to be fairly similar even when the number of crosslinks increases. One hypothesis is that the system is partly self-regulated: more GG dyads leads higher rigidity of the alginate backbone which is observed to increase effective degree of ionization. The equations used to calculate the behavior of the charged gels are found in section 3.6. 3.6. Calculations of the behavior of alginate charged gel filaments To be able to apply Equation 7 and Equation 8 in the calculations of the calcium/aluminum alginate gel behavior, the following properties of alginate are required: The persistence length density of alginic acid (S", and the initial concentration of alginate at gelation (calg). The

(lp), the monomer unit length (b0), the effective degree of ionization of alginate gels (i), the

following parameters of the gelation solution are also needed: The inverse Debye length (κ),

the concentrations (cion) and activity constants (fion) of the free ion species (Ca2+, Al3+ and Cl-) in the solution. The monomer length (b0), one polyelectrolyte chain segment, is different for the two uronic acids units in alginate. M units have a length of 5.15 Å, while G units have a length of 4.35 Å in the crystalline state56. We used a value of 5 Å for the rubber elasticity model as the average



of the ab initio calculated lengths of G (4.64 Å) and M (5.50 Å) units in the amorphous state, respectively.60

The persistence length (lp) is concentration dependent and is calculated from theories of polyelectrolyte stretching with two factors, one for charge independent length (l0) and one for the contribution of electric charges (le). A small angle X-ray study showed that sodium alginate solutions behave according to the model with a dependence on the square of the

inverse Debye length (κU& ".61 The same study showed that the charge independent length

approximately equals the length of a monomer. Since the segment length (b0) is shorter than

the Bjerum length (lB), the segment lengths are replaced by the Bjerum length in the equation.

The concentration of ions (?01G,C ) refer to the polymer charge concentration multiplied by the

effective degree of ionization plus the concentration of salt molecules multiplied by the square of their charge number (zi). For the number of monomer units in the statistical segment (W):

Gas constant: R = 8.315 kg m2 s−2 K−1 mol−1

Permittivity of water: O 80 ∙ 8.85 ∙ 10U& Y/[. (at 20 °C)

Elemental charge: 1.6022 ∙ 10U&\ ] .

The density of alginic acid: S 1600 P^/[) .

Molecular weight of alginate uronic acid sub-unit _7 0.194 P^/[` .

Concentration of ion species from salt: C@a 15 × 3 [`/[) for CaCl2 and C@a 10 ×

4 [`/[) for AlCl3.

Concentration of ion species from salt: C@a 15 × 3 [`/[) for CaCl2 and C@a 10 ×

4 [`/[) for AlCl3.

Boltzmann’s constant: P 1.38 ∙ 10U) .

Temperature: Q 293 c

`d

Equation 10 4eOPf a

-&

jk κU& g4eld i

l ?01G,C ∙ mC no Equation 11 _7 `p `7 l> 7

C

ld U 1

7 [" Equation 12 κ jk 4ld 4 eld

∑aC C@a,C ∙ mC " _7 ACS Paragon Plus Environment

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The fully extended chain length (L) was calculated from the chain segment length (Z) and the monomer length (b0):

r s 7 Equation 13 The average mean square end-to-end distance of the polymer chain was calculated from the assumption that the Kratky-Parod (wormlike chain) model applies62,63, which was also shown to fit the X-ray data61 well:

〈 〉 2r`p − 2`p #1 − Uv/p , Equation 14

The volume fraction of alginate (ν " can be calculated from the concentration of alginate by utilizing the density of dry alginic acid (S012 " and the density of water (Sxy 7 ".

ν 012 ∙

SDz{ Equation 15 Sxy 7

The dilation factor 〈∝7 〉 of the dry state can be calculated as the mean square end-to-end

distance in the reference state 〈7 〉 and the mean square end-to-end distance of the dry state

〈7 〉. The mean square distances are calculated according to equation 14. 〈∝7 〉

〈7 〉 〉 Equation 16 〈7

The dilation factor of the swollen state 〈∝? 〉 can further be calculated as follows:

〈∝? 〉 〈∝7 〉⁄ν ") Equation 17

The active chain segment concentration (νs) can be calculated from the weight concentration (calg) multiplied by the monomer molecular weight (M0) and divided by the chain segment length (Z):

ν}

012 ∙ _7 Equation 18 s

The unidirectional stretching (σ) can then be described as proposed by Dusek and Alenichev , with the electrostatic contribution (Ø< ) and the effects from non-Gaussian effects (Ø; )

27,28

taken into account. The most common approximate solution of the equation of the

electrostatic free energy in randomly kinked macromolecules gives flawed values at low



Page 28 of 50

elongations and for small values of (κ·h0) (dilute solutions), as is the case of our study.37 We

therefore included a parameter (A ", which we calculated from a better approximation of the

free energy for the low salt situation, which is found in the appendix of Katchalsky et. al.37.

A is given by equation 19.3 which was derived from a derivation of equation 19.4 in analogy

with the derivation of the expression for Ø< 27 and with an additional term in the second part

of the parenthesis.25

σ ν? RT〈∝? 〉1 Ø< Ø; "λ − ν λ- " Equation 7 (introduced in section 3.5) To calculate the electrostatic contribution and non-Gaussian effects, the following additional parameters are needed:

Effective degree of ionization: i (discussed in section 3.5)

Number monomer units a chain segment: s (discussed in section 3.5)

Number of monomer units in the statistical segment: W

Ø<

- s & ) H& y )〈∝? 〉 〈7 〉λ 2ν λ-& ") − `1 H& " A Equation 19.1 OPf 1 H&

&

2〈∝? 〉λ 2λ-& " H& Equation 19.2 〉κ 〈7

〉λ 〈∝? 〉〈7 〉λ 2λ-& "y 〈∝? 〉〈7 s

2λ-& "y A

∙ κ√e − Equation 19.3 OPf λ 2λ-& "〈∝? 〉〈7 〉κ y -& 3 〈∝? 〉〈7 〉κ #), 〈∝? 〉y 〈7 〉y λ 2λ "y

F<

s 6 1 √e 1 ` 1 − − Equation 19.4 OPf 2 κ κ 6 ⁄7 κ7

Ø;

) 〈∝ 〉λ ?

s 〈7 〉/ 7 s&. " Equation 20 -&

-

\\ ? &〈∝? 〉 #λ ν λ ν λ- , ?

ν λ-& " Equation 21 &) -)

8〈∝? 〉) #λ6 ν λ) ν ν) λ-) , ?

To also cover the initial shrinkage due to elastic and electrostatic forces arising from crosslinking with multivalent ions, a pre-shrinkage was performed with the same physical parameters as used for the later unidirectional elongation. A model of volumetric


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swelling/shrinkage with many similarities to the unidirectional stretching previously described was applied. The force is equally distributed from all directions and hence acts as a pressure. If a gel has a negative pressure at a certain alginate concentration, it means that the gel will shrink. Conversely, a positive value indicates swelling. The pressure can then be divided into three individual parameters: i) electrostatic interaction in the polyelectrolyte gel (Pels), ii) the elastic stretching of the gel (Pel), and iii) osmotic effects in the charged gel (Posm).

=G@G =>1 =>1? =@?A Equation 8 (introduced in section 3.5) To calculate the osmotic pressure, the activity constants (fion) of the free ion species must be known. The average activity constant at 0.1 ionic strength is 0.68 for CaCl2 and 0.66 for AlCl3.64 Posm is calculated as follows: &

Q Sν Posm

Sν − 2C@a C@a _7 i¡1 ¢ − 1n £ Equation 22 _7 _7 C@a C@a

The pressure arising from elastic contributions of both normal and non-Gaussian elastic stretching Pel is calculated as follows: P¤¥

3 99 513 J-¦ ν} 1 N -) -" - # , #-, -& 〈∝7 〉) ν ? − 〈∝ 〉 ν Equation 23 Q ν − 〈∝7 〉ν − 〈∝7 〉 ν ? − 5 175 875 7 ? ν 2

The pressure arising from electrostatic interactions Pels is calculated as follows: # ,

ν} ν §¨ s 2.5H P>1?

− `1 H " Equation 25.1 3O〈 〉y 〈∝ 〉y 1 H 7

7

y 9〈∝? 〉OPQ_7 H Equation 25.2 e§¨ 〈7 〉2_7 C@a Sν "

The model described herein is more complicated than the standard elasticity rubber equation but still necessary to describe the tensile behavior of our charged calcium alginate gels and achieve theoretical data in proximity to empirical data. A very important factor is the dilation factor of the swollen state 〈∝? 〉 which depends on the dilation factors of the dry state 〈∝7 〉 and contributes to the model by generating higher stress values per amount of alginate in the gels


Biomacromolecules

compared to the same amount of strained dry alginate (theoretically, if alginate would act as

rubber in the dry state). Another very important factor is the electrostatic contribution (Ø< )

which contributes to the model by adding an initial delayed stress response (in accordance

with our observations) unlike the classical elasticity theory. The theory of charged gels (Equation 7) combined with a small contribution of solid material theory (Equation 9) describes the empirical data of calcium alginate gels well (Figure 8). The aluminum alginate which behaved more like solids were best described by Equation 9 alone. 3.7. Tensile properties of alginate filaments in the dry state Filaments were extruded, starting with 1% (w/w), 2% (w/w) and in one case 5% (w/w) alginate from different fractions, into CaCl2 and are drawn by a fixed weight in containers with either AlCl3, PolyAl or CaCl2 (Section 2.3). The filaments in the wet and dry state were characterized with respect to the dimensions in the gel and dry state, their alginate content and their elongation with a fixed weight of 30 g. The exact preparation parameters as well as the alginate characteristics, extrusion rate and elongation during drawing are given in Table 3. The alginate solutions were extruded into a CaCl2 solution, and the filaments were then drawn with a fixed weight in containers of other solutions of either AlCl3, PolyAl, or CaCl2 (Section 2.3). The filaments were characterized with respect to cross-sectional dimension in the gel and dry states, the alginate content and the degree of elongation upon drawing with a fixed weight of 30 g (Table 3).

Alginate properties

Table 3. Characteristics of the alginate grades used, preparation parameters, and the resulting alginate filaments.

Gel filament properties

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

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Alginate

Com

F4

F8

F12

F16

F17

G (%)

57.9

74.0

67.4

71.9

79.2

81.9

GG (%)

35.8

58.0

48.7

44.8

55.3

58.6

Viscosity, 1% (Pa·s)

0.012

0.078

1.05

3.76

6.35

1.63

Initial conc. (% w/w)

2

5

2

2

2

1

Final conc. (% w/w)

3.59

6.75

2.83

3.35

3.51

2.28

3.63

4.29

a

Extrusion (mL/min) 5.0

2.0

1.0

0.8

0.6

5.0

0.4

0.2

0.2

0.09

0.25

0.02

0.02

0.02

0.02

0.02

0.25

0.02

Wet diameter (mm) 1.44

1.63

1.34

1.44

1.37

1.51

1.28

0.98

1.37

50

70

68

75

71

68

79

a

2

2

b

Extrusion CaCl2 (M) Elongation upon drawing (%)

85


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Sample name

Com_

Com_5%

F4_2%

F8_2%

F12_2%

0.02

0.09

0.02

0.02

Dry diameterc (µm) 163

256

124

2%

CaCl2 (M) Immersion Solutions

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

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AlCl3 (M)

0.015 0.015 / 0.06

F16_1 F16_2 F16_2%_t

F17_2%

%

%

hin

0.02

0.02

0.02

0.02 / 0.09

0.02

140

140

125

135

99 / 97

125

0.015

0.015

0.015

0.015

0.02

PolyAl (% w/w)

0.8

0.8 / 6.2 / 12.4

0.8

0.8

0.8

0.8

0.8

PolyAld (% w/w)

0.8

0.8 / 3.2 / 6.2

0.8

0.8

0.8

0.8

0.8

a) The value was not determined. b) The CaCl2 solutions to which the alginate is extruded. c) The diameter of filaments that have been immersed in the new CaCl2 solution and subsequently dried. d) PolyAl solutions with addition of HCl to decrease pH. The pH of each solution is given in Figure 10.

Figure 9. Average stress-strain curves of alginate extruded into CaCl2 solutions. Left: A comparison of samples with varying filament preparation conditions. Right: A comparison of samples from different alginate fractions. Some samples are displayed in both figures. Sample specifications are given in the inset table to the right.



From the initial tests with varying filament preparation conditions, certain trends were observed (Figure 9, left): The filaments became softer and the stress-at-break decreased with higher concentrations of CaCl2, as explained by the apparent volume change. This was also reflected by a difference in the filament dry weight when using higher concentrations of CaCl2. When using 0.09 M and 0.25 M CaCl2 solutions instead of a 0.02 M solution, the difference in the filament dry weight was approximately +18% and +45%, respectively, for a filament made from 2% (w/w) alginate (final concentration ≈ 3.5%). The extra CaCl2 salt content most likely also caused moisture absorption during the conditioning of the filaments at 50% relative air humidity for 3 days prior to tensile testing, with the absorbed water acting as a plasticizer. A lower CaCl2 concentration in the drawing bath during preparation clearly led to increased stiffness and stress-at-break for the dry filaments. The stiffness and stress-atbreak increased with lower alginate concentrations and with decreasing gel diameter, indicating that the drawing stress per amount of alginate is an important parameter. The drawing stress seemingly impacted the end stiffness and stress-at-break more than the actual filament elongation. As an example, Com_2% was 17% more elongated than F16_2% but still had lower stiffness and stress-at-break. The relationship between drawing stress and the parameters elastic modulus, stress-at-break and stress-at-yield will be discussed later in this section. No clear difference was observed when comparing the dry filaments made under similar conditions but from different alginate fractions.


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Figure 10. Average stress-strain curves of alginate drawn in AlCl3 solutions. Left: A comparison of samples with varying filament preparation conditions. Right: A comparison of samples from different alginate fractions. Some samples are displayed in both Figures. Sample specifications are given in the inset table to the right, stars indicate that the value was not determined. The general shape of the stress-strain curves for alginate crosslinked by Al3+ was different from the curve shape generated by the analogous Ca2+ crosslinked alginate filaments. The most obvious difference was that the stress-at-yield increased as well as the stiffness to some extent. This can be related to the greater relative stiffness of Al3+ crosslinked wet gel filaments compared to the Ca2+crosslinked wet gel filaments. Just as with CaCl2, it was beneficial to have a lower concentration of AlCl3 during the preparation of the filaments. Filaments made in the 0.06 M AlCl3 solution were less stiff and went to break earlier than their counterparts prepared in 0.015 M AlCl3 solution. The filament also became slightly more unpredictable in their breakage, which was revealed by the larger error zones. Conversely, the thinnest filaments (with diameters below 100 µm) with the highest stiffness and stress-at-break (Figure 10, sample G) all had very similar tensile profiles, indicating that this is the way to control the preparation of aluminum alginate filaments in a good way. The relative weight gain recorded for the Al3+crosslinked filaments in comparison to the Ca2+ crosslinked filaments is included in Figure 6. The relative weight gain can be explained by the behavior in the wet gel state.



When elongated filaments were immersed in AlCl3, the ion exchange from Ca2+ to Al3+ made the gels somewhat stiffer. The contraction forces that arise from this transfer is enough to pull the 30 g plummet somewhat upwards and contract the gel, a change that resulted in slightly higher weight per length of filament (Scheme 2, preparation step C). Interestingly, it appeared that this effect was governed by the molecular weights of the alginate fractions since both samples Com and F4, respectively, contracted the least. The thinnest filament, F16, which had higher drawing stress is seemingly less affected by this expansion since there is a relatively higher stress from the plummet to overcome. The assumption that the gel actually contracted and is not simply an observation of a weight increase due to the uptake of salt ions, is sustained by the fact that the AlCl3 solution had a lower concentration than the CaCl2 solution and an exchange would only result in a small weight loss.


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Figure 11. Average stress-strain curves of alginate drawn in PolyAl solutions. a) Filaments from 5% commercial alginate made with varying PolyAl concentration, b) a comparison of samples from different alginate fractions drawn in 0.8% (w/w) PolyAl concentration, c) a comparison of samples from different alginate fractions drawn in 0.8% (w/w) PolyAl concentration at pH 4.4, d) a comparison of samples from different alginate fractions drawn in 0.8% (w/w) PolyAl concentration at pH 4.1 and e) sample specification table, stars indicate that the value was not determined. Some samples were allowed to shrink in a PolyAl solution instead of the AlCl3 solution to particularly induce high shrinkage of the gel filaments. The shrinkage process is further elaborated in section 3.8. Gel filaments prepared from 5% (w/w) commercial grade alginate showed increasing stress-at-break when prepared with the PolyAl solution of lower concentrations (Figure 11a, samples A-E). Since we were aiming for stiff filaments with high stress-at-break, the lowest concentration of the PolyAl solution of the ones tested (0.8% (w/v)) was used for the remainder of the tests. We did not further dilute the PolyAl because we wanted to explore the phenomenon of a high amount of induced shrinkage in the gel state, which was still very present at the concentration of 0.8% (w/v) but started to be less impactful at even lower concentrations (Section 3.8). We observed in the initial experiments (Figure 11, A-E) that filaments prepared by extrusion of 5% alginate in PolyAl with a lower pH (due to



the addition of HCl) performed better in terms of stress-at-break. The improvement of stress at break with the decreased pH in the PolyAl solution was not seen for the samples prepared from 1-2% alginate, and more often the reverse trend was seen. The character of the experiments with PolyAl was fail vs. not fail in the sense that some filaments performed really well in terms of high stress-at-break, high stiffness and low variation between the tensile results within the same sample group, while others behaved poorly in that sense. The main factor that governed the poor characteristics was the weight increase compared to the similar dry filament that was crosslinked only with Ca2+-ions. In contrast to the ‘filament weight gain’-scenario that was discussed previously for the Ca2+ filaments drawn in the AlCl3 solution, the total salt concentration in the surrounding PolyAl solution is far greater than that inside the gel filament. Per volume unit, the dry mass content of the PolyAl solution is in the same order as the alginate gels. The observed weight increases of the filaments drawn in the PolyAl solution are therefore caused by an uptake of PolyAl salt. The samples with a less than 10% weight increase were at the same time the samples that performed well in terms of stressat-break (Figure 11, samples E, D, J, K, L, M, O, and R) and the factors that they had in common were as follows: i) higher concentration, ii) higher compositional ratio of GG dyads, and iii) highest pH of the PolyAl solution. Our hypothesis is that the limited uptake of PolyAl is related to a dense alginate network and the nature of the PolyAl ions. The size of the alumina species in the PolyAl solution was shown to decrease with decreasing pH.65–67 Large species, such as Al30O8(OH)56(H2O)2618+ and Al13O4(OH)24(H2O)127+ (both being in the nm range), are more common at higher pH values, while species such as Al2(OH)51+ become more frequent at lower pH values. Alginate fraction F16 extruded into 0.25 M CaCl2 solution resulted in thin filaments with high alginate concentration, (Figure 11, samples K-M), with high stress-at-break and the highest elastic modulus of all the tested filament samples. The applied stress during the gel filament drawing was indeed an influential factor governing the resulting stress-at-break, stress-at-yield and elastic modulus of the gel filaments. We found that a logarithmic scale on the axis representing drawing stress in the gel filament state led to linear rising trends for the plotted parameters for the samples prepared only in CaCl2 (Figure 12). A similar rising trend was observed for the samples prepared in AlCl3, but the data were more dispersed, and the linearity was not so evident. For filaments prepared in the PolyAl solution, in all cases, when the relative weight gain compared to the corresponding filaments prepared in CaCl2 was above 105%, the stress values and elastic modulus values were multiplied with the relative weight gain to compensate for the presence of salt in the


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Biomacromolecules

samples (since the salt does not contribute at all to the increase the stress resistance). Even with such compensation, these samples were still weaker than the samples without weight gain.

Figure 12. Stress-at-break, stress-at-yield and elastic modulus for the upper percentile of all tests for all filaments prepared from different fractions of alginate. The data are grouped with respect to solutions used during filament preparation: a) CaCl2, b) AlCl3, c) PolyAl pH 4.1 and d) PolyAl pH 4.4. X-axes are given as logarithmic scales.

The most evident trend, taking all the above data into account, is that the drawing stress in the wet gel state has a significant effect on the tensile performance of the dry filaments. In the wet gel state, the observed differences between filaments were due to the variations in the uronic acid composition and the molecular weight of the alginate fraction. In the dry state, these differences were not observed. The dependence of stress-at-break on the drawing stress in the wet gel state was most clear-cut for the CaCl2 drawn filaments and is described by Equation 26 (R2 = 0.895).

® WfWW Stress- atbreak 189 405 ∙ log&7 ¡ ¢ Equation 26 ff ®f ^`

Another interesting observation was that the stress-at-break was the highest for filaments prepared in Ca2+, while the stress-at-yield was always highest for filaments prepared in Al3+. The elastic modulus was higher for filaments prepared with Al3+ compared to filaments prepared in Ca2+. The highest elastic moduli were observed for the samples prepared in the



PolyAl solution, regardless of the pH of the latter. The difference was small between filaments prepared in PolyAl and those prepared in AlCl3. Still, the acquired understanding of suitable preparation conditions allows for the process and the resulting filament properties to be further optimized. The observed stress-at-break (550 MPa) and elastic modulus (25 GPa) for the stiffest dry calcium alginate filaments surpass values previously reported for alginate filaments (270-340 MPa and 10 GPa, respectively).68–70 The highest reported value of stress-at-break for a dry calcium alginate filament was 450 MPa.22 The stress-at-break and elastic modulus are in addition comparable to values of other natural polysaccharide filaments, and to filaments made from synthetic polymers. Literature values of stress-at-break for dry filaments of cotton cellulose and Nylon 6.6 are 400-820 MPa and 470-510 MPa, respectively.70 Literature values of elastic modulus for dry filaments of cotton cellulose and Nylon 6.6 are 5.5-12.6 GPa and 3.5 GPa, respectively.68–70 The increase of the stress-at-break, stress-at-yield and elastic modulus in the dry state with increasing ‘drawing stress to alginate concentration’ is most likely due to the increased chain orientation of alginate. The uronic acid composition differs between different alginate fractions and, hence, the filaments with the highest ‘stress to alginate concentration’-ratio were not always the ones which elongated the most during drawing. A higher G content increases the gel network density, hence, the chain segments between crosslink junctions are shorter and more extended even before the elongation. The orientation of the alginate polymers could thus be of similar extent, even with less visible filament elongation. 3.8. Shrinkage in PolyAl solution To systematically explore the effect of the PolyAl solution on alginate, an experiment was set up in which a piece of alginate filament was first weighed, then immersed in PolyAl solutions of varying concentrations and basicity. The filaments were withdrawn from the solution at fixed time intervals to be quickly dried, weighed and then put back into solution. The recorded relative shrinkage values are presented in Figure 13. The basicity concept relates to the percentage of the three charges on the Al3+ ion that are neutralized by OH- ions. As an example, a basicity of 2.5 equals 2.5/3 = 83% of the charges neutralized by OH- ions. In all cases, the alginate shrinkage was greater when immersed in a PolyAl solution with a higher basicity (and thus also pH).


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Biomacromolecules

The alginate filaments made from 5% alginate solution (Com_5%) were used for the systematic examination of shrinkage in PolyAl solution. The shrinking caused by crosslinking in Ca2+ solution during the production of the filaments changed the alginate concentration from 5% to 6.75%, hence the remaining weight after Ca2+-induced shrinkage was 5/6.75 = 74% of the original weight. After reaching equilibrium in Ca2+, the crosslinked alginate filaments were transferred to a PolyAl solution and their weights, respectively, at the time of transfer were set as the starting point, 100%, for the additional shrinkage occurring in the PolyAl solution (Figure 13). The total shrinkage of a filament, that is the shrinkage occurring in the Ca2+ solution + the shrinkage occurring in the PolyAl solution, may thus be calculated by multiplying the relative shrinkage value in Figure 13 (left) with 0.74. The swelling ratio of a filament corresponds to the values in Figure 13 (left) subtracted by 100%. The data in Figure 13 (right) are recorded from samples with varying initial alginate concentrations and Ca2+ shrinking histories (details are given in Table 3) and can be processed in analogy with the values in Figure 13 (left).

Figure 13. Left: The observed wet gel weight change after shrinkage of Com_5% alginate filaments for 130 min in PolyAl solutions of different concentrations and basicity values. The pH values of the PolyAl solutions at four different concentrations and for each basicity value are indicated. Right: The observed wet gel weight change for a set of wet alginate filaments after 130 min of shrinkage in the two PolyAl solutions, having pH 4.4 (filled columns) and pH 4.1 (striped columns), used for the generation of dry filaments.



In summary, the results show that cyclic fractionation is a method that successfully produce a series of alginate fractions with varying G/M ratios and molecular weights. The extracted alginate can readily be converted to thin filaments by extrusion followed by drawing in di- trior multivalent ion solutions. We derived equations to model the tensile properties of the wet gel filaments and found that the tensile stress and strain of filaments prepared in CaCl2 solution depend on the compositional ratio of G dyads in the alginate fraction multiplied by the alginate concentration while the tensile stress and strain of filaments prepared in AlCl3 solution depend only on the alginate concentration. In general, the produced alginate filaments behaved as stiff, solid materials in the dry state while their tensile behavior in the wet gel state is more accurately described by a crosslinked charged gel model. PolyAl cause effective shrinkage of alginate filaments and produce dry filament materials with Young’s moduli around 25 GPa and stress-at-break as high as >500 MPa.

4.

CONCLUSIONS

Alginate from brown algae Saccharina latissima was directly fractionated during extraction into alginate fractions with different uronic acid compositions and average molecular weights. Aqueous solutions of alginate (1-2% (w/w)) were good starting points for making CaCl2 drawn filaments since they mostly behaved as flexible gels that could be strained to high extensions and further used as frameworks for incorporating other ions, aluminum or polyaluminum (PolyAl) in the already oriented gel, resulting in stiffer filaments. Wet gel filaments were prepared by extrusion from the alginate fractions into a CaCl2 solution and in some cases followed by immersion in an AlCl3 solution. The structural characteristics of the alginate fraction had a significant impact on the material properties. For the wet gel filaments

prepared by CaCl2, the stress ( ) at a strain (( ) was observed to depend on the compositional ratio of guluronic acid (G) dyads in the alginate fraction multiplied by the alginate concentration ( ):

#1972ε − 723 e-&&' -723 − 1972(" e-)' 1446 − 1972(",

The tensile performance of wet gel filaments prepared by AlCl3 instead depends only on the alginate concentration (calg):


Page 40 of 50

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Biomacromolecules

012 #1188ε 150 e-67' 150 1188(" e-&8' -300 1188(",

The tensile properties of the CaCl2 wet gel filaments in our model are described as that of crosslinked charged gels that have shrunk to equilibrium and with a small contribution from the behavior of a solid material. The stiffer wet gel filaments prepared in AlCl3 are almost solely described as a soft solid material. A molecular weight dependency was observed for the gels prepared in AlCl3. Interestingly, there was no non-Gaussian behavior at high elongation rates, elongation ranges that are seldom explored for gels due to experimental limitations.

Dry filaments were prepared by extrusion of the fractionated alginate into CaCl2 solution, followed by filament drawing and transfer to CaCl2, AlCl3, or PolyAl polyvalent solutions. The tensile properties of dry filaments were less dependent on the alginate fraction than the properties of gel filaments. Instead, an increasing ‘stress to alginate concentration’-ratio had a great influence and increased the filament stiffness and stress-at-break. The nature of the ion in solution during filament drawing also had a major role in determining the material properties of the dry filaments. The dependence of stress-at-break on the drawing stress in the wet gel state was most clear-cut for the CaCl2 drawn filaments and could be described as:

® WfWW Stress- atbreak 189 405 ∙ log&7 ¡ ¢ ff ®f ^` The maximum stress-at-break of the dry filaments was achieved when CaCl2 was used as the immersion solution. The stress-at-break was slightly lower for filaments drawn in AlCl3 but the stress-at-yield and elastic modulus increased. The highest elastic modulus was observed for filaments immersed in PolyAl-solution during drawing. High alginate concentration, high compositional ratio of G units and the use of PolyAl solutions with higher basicity (which relate to higher pH) hindered the PolyAl from migrating into the filaments. The PolyAl solution effectively alginate to shrink to a high extent, and the resulting filaments behaved as very stiff materials in the tensile tests. Alginate filaments containing 6.5% alginate shrank to one fifth of their initial volume in a concentrated PolyAl solution. The simultaneous extraction and fractionation of alginate enables many different material applications from a single source of brown algae. The fractionation into alginates with different uronic acid compositions enables multiple uses of alginate in the wet gel state. The



developed method for filament preparation and the empirical model to predict dry and wet filament behavior can both aid in generating drawn dry filaments with a strong tensile performance. The effect of different polyvalent ions can further aid in custom-making filaments for diverse applications.

ACKNOWLEDGEMENTS The authors thank FORMAS (project number 2013-92, SEAFARM) for their financial support. Göran Nylund, Gunnar Cervin and Henrik Pavia at The Sven Lovén Centre for Marine Sciences, Gothenburg University, are thanked for harvesting the Saccharina latissima. Jean-Christian Zirignon is thanked for preparing the NMR samples. Kemira AB is thanked for kindly supplying the PolyAl solution.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. G’ and G’’ curves, 1H NMR spectra of all extracted alginate fractions, schematic illustration of gel shrinkage.

AUTHOR INFORMATION Corresponding author E-mail: [email protected] ORCID Ulrica Edlund: 0000-0002-1631-1781 Notes The authors declare no competing financial interest.

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(2)

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Table of Content graphic



Figure 2. Left: tan δ of a frequency sweep of the extracted alginate fractions. The striped line estimates the middle when the results oscillate at higher frequencies. F3 and Com were only plotted at low frequencies, since the data were too noisy at higher frequencies. Right: Apparent viscosity and zero shear viscosity (η0) of the extracted alginate fractions as well as a calculated approximation of Mw of each alginate fraction. 216x261mm (300 x 300 DPI)


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Figure 2. Left: tan δ of a frequency sweep of the extracted alginate fractions. The striped line estimates the middle when the results oscillate at higher frequencies. F3 and Com were only plotted at low frequencies, since the data were too noisy at higher frequencies. Right: Apparent viscosity and zero shear viscosity (η0) of the extracted alginate fractions as well as a calculated approximation of Mw of each alginate fraction.29 216x261mm (300 x 300 DPI)



Table of Content graphic 82x33mm (600 x 600 DPI)


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High-Performance Filaments from Fractionated Alginate by Polyvalent

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