insolubility of

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Experimental and theoretical evaluation of the solubility/ insolubility of spruce Xylan (Arabino Glucuronoxylan) Saina Kishani, Alfredo Escalante, Guillermo Toriz, Francisco Vilaplana, Paul Gatenholm, Per Hansson, and Lars Wågberg Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01686 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on February 6, 2019

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

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Biomacromolecules

Experimental and theoretical evaluation of the solubility/insolubility of spruce Xylan

(Arabino Glucuronoxylan) Saina Kishani*a b, Alfredo Escalantee, Guillermo Torize f, Francisco Vilaplanab c, Paul Gatenholmd f, Per Hanssong, Lars Wagberg*a b

a

School of Chemical Science and Engineering, Fibre and Polymer Technology, Royal

Institute of Technology, Teknikringen 56-58, SE-10044 Stockholm, Sweden.

b Wallenberg

Wood Science Centre (WWSC), Teknikringen 56-58, SE-10044

Stockholm, Sweden.

c School

of Biotechnology, Division of Glycoscience, Royal Institute of Technology,

Albanova University Centre, SE-10691 Stockholm, Sweden.

ACS Paragon Plus Environment

1

Biomacromolecules 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

d

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Chemical Biological Engineering/Biopolymer, Chalmers University of Technology,

Goteborg, Sweden.

e

Wood, Cellulose and Paper Research, Universidad de Guadalajara, Guadalajara

Jalisco, Mexico.

f

WWSC, Chalmers University of Technology, Gothenburg, Sweden.

g Department

Keywords:

of Pharmacy, Uppsala University, Box 580, 75123 Uppsala, Sweden.

Hemicellulose,

Isolation.

Characterization,

Solubility,

Flory-Huggins

Simulation, Phase separation.

Abstract


2

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The molecular solubility of softwood arabinoglucuronoxylan (AGX) has been thoroughly investigated, and it has been shown that the chemical and physical structures of the extracted hemicellulose are not significantly influenced by different purification steps, but a transient molecular solubility of AGX was observed in aqueous media at low concentrations (1 g/L), when the dissolved macromolecules had a hydrodynamic diameter of up to 10 nm. A phase separation was detected when the concentration was increased to 15 g/L leading to an association of the smaller molecules into fractal structures with a considerably larger diameter, even though the dispersions were still transparent to ocular inspection. Dynamic Light Scattering, and Cryo-Transmission Electron Microscopy showed dimensions in the range of 1000 nm. The phase separation of the sample was further characterized by estimating the -interaction parameter of AGX in water using the Flory-Huggins theory, and the results supported that water is a poor solvent for AGX. This behavior is crucial when films and hydrogels based on these biopolymers are made, since the association will dramatically affect barrier and mechanical properties of films made from these materials.


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1. Introduction In a bioeconomy context, the forest is one of the largest assets for renewable resources. The structural biopolymers at the plant are intimately associated in the cell wall1, and in order to liberate them, the deconstruction of plant cells and their fractionation is necessary. These deconstruction procedures are nowadays commonly referred to as biorefinery processes. Cellulose, hemicelluloses and lignin are the structural components of plant cell walls, and hemicelluloses are the second most abundant source of biomass after cellulose2. These carbohydrate polymers have therefore been the subject of numerous studies regarding their isolation, physicochemical properties, chemical modification and material properties2-14. Hemicelluloses incorporate a variety of sugar units, glucose, xylose, mannose, galactose, arabinose, fucose, and glucuronic acid, the contents of which depend on the origin of the hemicellulose3. Xylans are the most abundant hemicelluloses in nature and are present not only in wood, but also in cereals and grasses3. They have a backbone of ß-(1-4) D-xylopyranose (Xylp) units, often substituted with glycosyl and acetyl groups depending on the plant type, tissue and developmental stage15. Xylans in conifers in general, and particularly in Norway spruce,


4

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have -(1-2) 4-O-Me-D-glucuronic acid (mGlcA) as pendant groups at C2 in xylose, and at C3 in -(1-3)-L-arabinofuranose (Araf)5. The presence of regular intramolecular patterns in spruce arabinoglucuronoxylan has recently been demonstrated, with major domains having an even spacing of Araf and mGlc groups on the xylan backbone and with minor domains having clustered mGlcA substituents on consecutive Xylp units16. In softwoods, xylan constitutes 5-15% of the dry weight and it has been shown to be an ideal material for practical applications such as films, barriers and hydrogels5, 17. In spite of the great potential of these biopolymers for high value applications, there is a lack of basic knowledge of their physicochemical behavior in aqueous media, but an understanding of the manner in which xylan macromolecules disperse or associate in water will facilitate the advanced design of materials based on wood biopolymers. The solubility of polysaccharides depends on their composition, degree of branching and pattern of substitutions, which depend in turn on the biosynthetic processes in planta and by the isolation/purification

processes18-19.

We

have

quantitatively

isolated

spruce

arabinoglucuronoxylan and subjected the material to different purification/fractionation steps in order to identify the factors that affect the solubility of the material. The solubility


5


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and association of the extracted xylans were investigated using light scattering and microscopies techniques. The results showed that the purification technique did not affect the physico-chemical structure of the extracted samples, although the association of the molecules at high concentrations indicated that all the samples had a low solubility in aqueous media. Such a behavior suggests a phase transition, which has been further investigated using Flory-Huggins theory.

2. Materials and methods 2.1 Isolation and purification of spruce arabinoglucuronoxylan 2.1.1 Extraction of spruce arabinoglucuronoxylan (Xylan 1) Arabinoglucuronoxylan was extracted from Norway spruce using the method reported by Wise and Timell20-21 and adapted by Escalante5. Briefly, spruce flour (150 g) was subjected to sodium chlorite delignification using 15 mL glacial acetic acid and 45 g sodium chlorite at a wood to liquor ratio of 1:25, adjusted with water at 70-80 °C followed by the addition of the same amount of chemicals every 12 h four more times. The holocellulose obtained (about 100 g) was extracted with KOH (24 %wt. in a ratio 1:7 w:v)


6

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Biomacromolecules

at room temperature. Hemicelluloses were then precipitated by pouring the supernatant into acidic ethanol (supernatant: ethanol: glacial acetic acid 1: 4: 0.4 v:v:v,), after which the precipitate was filtered, dried and redissolved in KOH 10 wt.% (1:10 w:v) at room temperature. The galactoglucomanans were further separated from the hemicellulose mixture by complexation with Ba(OH)2, after which the supernatant was poured into an acidic ethanol solution to precipitate arabinoglucuronoxylan (Xylan 1) with an approximate yield of 7 wt. % from the original wood. 2.1.2 Xylan 2: Removal of ionic impurities by DTPA Xylan 1 was further purified with diethylenetriamine pentaacetic acid (DTPA) to give an ion-free sample (Xylan 2). In brief, 2g of Xylan 1 was added to 40 mL of deionized (DI) water and 24 mg of DTPA was added. The mixture was stirred for 24 h and then precipitated in a mixture of 160 mL ethanol (96%) and 1 mL acetic acid. The precipitate was filtered through a #2 Whatman filter paper and washed with ethanol and vacuum dried overnight. The yield was 91.3%. 2.1.3 Xylan 3: Dialysis of Xylan 1 to remove low-molecular mass impurities


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Xylan 3 was obtained by dialyzing Xylan 1 against DI water through a 1000 MWCO membrane of standard regenerated cellulose tubing (spectra/Por, Sigma-Aldrich, US). 3 g Xylan 1 was added to 100 mL water and placed in the dialysis tubing which was then submerged in 4L-deionized water and stirred. Each 24 h for 72 h, the DI water was replaced with fresh water and kept under stirring. Dialyzed Xylan 1 was precipitated in 96% ethanol (1:4) with one mL of acetic acid. The precipitate was filtered on a #2 Whatman filter, washed with ethanol and vacuum dried overnight (Yield was 77.5%).

2.1.4 Xylan 4: Bleaching Xylan 1 to remove lignin impurities Xylan 1 was bleached with H2O2 to prepare Xylan 4 as follows: 3.34 gr of Xylan 1 was added to 70 mL of deionized water, 40 mg of DTPA was added and the mixture was stirred overnight. Sodium silicate was then added (75 mg) and the temperature was raised to 50 °C. H2O2 then was added (2 mL of 50% v:v) and the pH was adjusted to 10.5 with potassium hydroxide. Bleaching was carried out for 3 h, and the bleached Xylan 1 was then precipitated in 96% ethanol (1:4) with one mL of acetic acid. The precipitate was


8

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Biomacromolecules

filtered on a #2 Whatman filter, washed with ethanol and vacuum dried overnight. The yield was 90%. 2.1.5 Xylan 5: Dialysis of Xylan 4 to remove low-molecular mass impurities after bleaching Xylan 5 was obtained by dialyzing Xylan 4 against DI water through a 1000 MWCO membrane of standard regenerated cellulose tubing (spectra/Por, Sigma-Aldrich, US). 3 g of Xylan 4 was added to 100 mL water and placed in the dialysis tubing, which was submerged in 4L-deionized water under stirring. Water was replaced with fresh DI water every 24 h during 72 h. The dialyzed Xylan 4 was precipitated in 96% ethanol (1:4) with 1 mL of acetic acid, and the precipitate was filtered through a #2 Whatman filter and washed with ethanol and vacuum dried overnight. The yield was 95%.

2.2. Characterization


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2.2.1 Carbohydrate Composition The carbohydrate composition of the purified Xylan samples was determined after acid hydrolysis with 2 M trifluoroacetic acid (TFA) at 120 º C for 3 h22. The hydrolyzed samples were filtered through a nylon filter (0.2 µm) and transferred to special vials and analyzed using high-performance anion exchange chromatography with a pulsed amperometric detector (HPAEC-PAD). The monosaccharide components of the hydrolyzates were analyzed with HPAEC (IC-3000, Dionex, Sunnyvale, CA, USA) and separated with a CarboPac PA-1 column (4×250mm) (Thermo Fisher Scientific, USA). Different gradients were used for the separation and quantification of neutral sugars and uronic acids as reported by McKee23. For neutral sugars, the chromatographic system was equilibrated using 260 mM sodium hydroxide for 7 min, and 170 mM sodium acetate for 7 min, followed by MQ water for 6 min. The eluent was MQ water (1 mL/min), and the temperature was 22 º C. A 300 mM sodium hydroxide solution was added to the column effluent at a flow rate of 0.5 mL/min, before PAD detection. The data were processed using Chromeleon 7.1 software. The analysis was duplicated. Monosaccharide standards, supplied by Sigma-Aldrich, were used to determine the response factors. 4-


10

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Biomacromolecules

O-methylglucuronic acid was quantified using the relative response factor reported in the literature24.

2.2.2. Average molar mass (Mw) The average molar mass distributions of the Xylan samples were determined using a size exclusion chromatography (SEC) system (Agilent Technologies 1260 Infinity, from PSS, Germany) equipped with a refractive index (RI) detector (G1362A 1260 RID Agilent Technologies) and a multi-angle laser light scattering (MALLS) detector (SLD 7000 PSS, Germany) using water and 0.1 M NaNO3 as eluent. The MALLS signal at 90 º was used to detect aggregates or mass separation in the samples. The system was calibrated with pullulan standards of narrow dispersity (PSS, Germany). Samples were dissolved in the SEC eluent at 2 g/L and left in a thermo-mixer (Eppendorf thermomixture comfort, Germany) at 60 °C overnight. All the samples were transferred to the special SEC vials without filtration. A volume of 100 µL of each sample was injected into a PSS GRAM guard column with a flow rate of 0.5 mL/min, and two other 100 Å (300 × 8 mm, 10 μm particle size) and 10000 Å (300 × 8 mm, 10 μm particle size) columns (PSS, Germany)


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thermostated at 40 º C. The specific refractive index increment (dn/dc) values were measured using an automatic refractometer (J457, Rudolph, USA), and the numberaverage (Mn) and weight-average (Mw) molar masses were calculated with the WinGPC UniChrom GPC/SEC software (PSS, Germany) using both standard and light scattering calibrations.

2.2.3. Size determination Dynamic light scattering (DLS) (Zetasizer, ZEN 3600, Malvern, U.K.) was used to determine the hydrodynamic size distributions of the Xylan samples in water. Samples were dispersed in Milli-Q water (18.2 MΩ) and mixed at different concentrations and temperatures to investigate the effects of temperature and concentration on the solubility of the different Xylan samples. The mixing was carried out with a thermo-mixer (Eppendorf thermomixture comfort, Germany) for at least 6 h, and measurements were made at the preparation temperature after filtration through a 0.45 µm nylon filter. The hydrodynamic diameter (dH) of the samples in water was calculated using the StokesEinstein equation:


12

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Biomacromolecules

𝑘𝑇

𝑑𝐻 = 3𝜋ƞ𝐷

[1]

Where 𝑘 is the Boltzmann’s constant, 𝑇 the absolute temperature, 𝐷 the translational diffusion coefficient, and ƞ the viscosity. The number distribution was used to evaluate the degree of association in the samples at different concentrations and temperatures. The shape and the decay of the autocorrelation function curves with time were used to determine the diffusion coefficient and the mean size of the particles25.

2.2.4. Charge and zeta potential The purified Xylan samples were dispersed in MQ water to a concentration of 1 g/L. The pH values were measured, and the cationic demand was determined by polyelectrolyte

titration

(PET)26

using

polydiallyldimethylammonium

chloride

(PDADMAC). The average consumed amount was calculated from three independent


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measurements. The zeta potential ξ of the samples was determined in MQ water (1g/L) using a Zatasizer, ZEN 3600, (Malvern Ltd., U.K.).

2.2.5. Morphology projection The conformation of the molecules at a low concentration (1 g/L) and the aggregates formed at a high concentration (15 g/L) were visualized using atomic force microscopy (AFM) and cryo-transmission electron microscopy (cryo-TEM), respectively. At the low concentration, molecules were adsorbed onto a mica surface using an anchoring polymer solution PAH (polyallylamine hydrochloride with a specific molar mass of 17.5 kDa, Sigma-Aldrich). A multimode 8 AFM (Bruker, USA) in ScanAsyst mode with NTESP tips (Bruker, USA) was used for imaging in air, and the images were processed using DIPimage software (Delft University of Technology, The Netherlands). The aggregates formed at high concentration (15 g/L) were probed using Cryo-TEM (Zeiss EM 902 Ltd. Welwyn. UK). An acceleration voltage of 80 keV was used, and the images were captured digitally with a CCD camera. Specimens were made according to the method developed by Almgren27, using copper grids (200 mesh) coated with holey


14

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Biomacromolecules

polymer film. About 2 µl of the sample was placed on the specimen in a Controlled Environment Vitrification System (CEVS). The specimen was then quenched in liquid ethane and stored under liquid nitrogen until measurements were made.

3. Results 3.1 Carbohydrate sugar composition The purity and chemical structures of the extracted and differently purified Xylan samples were analyzed by HPAEC-PAD. Arabinoglucuronoxylan was the main constituent of all the samples consisting of xylose, 4-O-methylglucuronic acid and arabinose. Galacturonic acid and small traces of glucose are also present. The results indicated that the purification process did not affect the chemical structure of the samples. The small differences detected in the samples were negligible and within the error limits (Figure 1).


15

Biomacromolecules

80

Carbohydrate sugar compositions

70 60

Xylan1 Xylan2 Xylan3 Xylan4 Xylan5

50

%

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

Page 16 of 37

40 30 20 10 0

e e e ose cos nos lactos bin am Glu Ara Ga Rh

cA ose nnose c acid -Gl Xyl ni Ma -Me uro O t c 4 la Ga

--

Figure 1. Carbohydrate compositions of the extracted and differently purified xylan samples from Norway spruce.

3.2. Molar mass and size The molar mass distributions of the samples were investigated using SEC. The distributions were not significantly influenced by the choice of purification step. Xylan 1 showed a large peak at a low molar mass that could be due to impurities removed during the DTPA treatment, dialysis or precipitation steps, i.e. once Xylan 1 is re-dissolved and re-precipitated in acidic ethanol (mixture of glacial acetic acid and ethanol), there will probably be a loss of the low molar mass fraction of the xylan. In samples Xylan 2 and Xylan 4, small peaks appeared just before the main peak, showing the presence of entities with lower molar mass


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removed by dialysis in Xylan 3 and Xylan 5 (Figure 2). The number average (Mn) and weight average (Mw) molar mass values, detected using both standard calibration and light scattering calibration, are listed in Table 1. The results obtained with light scattering calibration reveal a virtual monodisperse molar mass distribution for the xylan samples compared with the standard pullulan calibration. This is plausible because the multi-angle laser light scattering (MALLS) detector measures the absolute molar mass at each slice of retention volume during SEC separation, which is unaffected by band broadening during the separation and extrapolation of molar masses assumed during standard pullulan calibration. On the other hand, the average results from the standard calibration are more susceptible to interferences from the contaminants that appear as tails in the low molar mass range (Figure 2). Therefore, the elimination of these low molar mass contaminants resulted in increasing the average Mn and Mw data from the standard calibration with pullulans. Xylan1 Xylan2 Xylan3 Xylan4 Xylan5

100

Refractive Index (RI)

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

Biomacromolecules

80

60

40

20

0 2,5

3,0

3,5

4,0

4,5

5,0

5,5

Log Mw

Figure 2. Molar mass distributions of the extracted and purified Xylan samples.


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Table 1. Number average (Mn) and weight average (Mw) molar masses of Xylan samples obtained by SEC calibrated with Pullulan standard (CS) and light scattering (LS).

CS

CS

LS

LS

Sample

Mn(kDa)

Mw(kDa)

Mn(kDa)

Mw(kDa)

Xylan1

11.41

42.89

17.33

17.69

Xylan2

17.76

45.22

14.37

16.08

Xylan3

24.51

49.35

17.34

17.56

Xylan4

19.58

47.88

18.42

18.72

Xylan5

27.62

50.71

14.57

15.18

The size distribution was also evaluated using DLS for all the samples at different concentrations and temperatures. At low concentrations, all the samples showed hydrodynamic diameters below 10 nm. Between 30 to 60 °C the temperature had no significant influence on the hydrodynamic size distributions of the samples in aqueous media (Figure 3).


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Xylan1

35

35

20 15 10 5

25 20 15

2

4

6

8

0

10 12 14 16 18 20

Size(d.nm)

35

Number(%)

20 15

4

6

5 0

4

6

8

10 12 14 16 18 20

Size(d.nm)

10 12 14 16 18 20

0

2

4

6

8

10 12 14 16 18 20

Size(d.nm)

Xylan5

15

5 2

8

20

10

0

15

25

10

0

2

30

25

20

0

0

35

Xylan4

25

5

Size(d.nm)

30

30C 50C 60C

30

10

10 5

0

Xylan3

35

Number(%)

25

0

Xylan2

30

Number(%)

Number(%)

30

Number(%)

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

Biomacromolecules

0

2

4

6

8

10 12 14 16 18 20

Size(d.nm)

Figure 3. Number distributions of Xylan samples dispersed in aqueous media at 1 g/L as functions of hydrodynamic diameter (dH) determined by dynamic light scattering.

An increase in xylan concentration to 15 g/L resulted in a significant increase in the hydrodynamic size of the samples as shown in Figure 4, due to the association of smaller molecules. Increasing the temperature from 30 °C to 60 °C caused Xylan 1 and Xylan 3 to disaggregate, although association occurred again when the temperature was reduced.


19

Biomacromolecules

It was concluded that increasing the concentration led to aggregation and a further phase separation of the Xylan samples. Similar behavior was observed in our previous study of acetylated-galactoglucomannans extracted from Spruce19.

Number(%)

30

Number(%)

Xylan2

25

Xylan1

25 20 15 10

Xylan3

30C 50C 60C

35

20 15 10

30 25 20 15 10

5

5

40

Number(%)

35

5

0

0,1

1

10

100

0

1000 10000100000

0,1

1

10

Size(d.nm)

100

1000 10000100000

Size(d.nm)

Xylan4

25

0

0,1

1

10

100

1000 10000100000

Size(d.nm)

Xylan5

40 35

Number(%)

20

Number(%)

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

Page 20 of 37

15 10

0,1

25 20 15 10

5 0

30

5 0 1

10

100

1000 10000100000

0,1

1

10

100

1000 10000100000

Size(d.nm)

Size(d.nm)

Figure 4. Number distributions of Xylan samples dispersed in aqueous media at 15 g/L as a function of hydrodynamic diameter (dH).


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The formation of aggregates was also shown by comparing the autocorrelation function curves obtained at low (1 g/L) and high (15 g/L) concentrations (Figure 5). The curves obtained at a low concentration showed a very fast decay due to the fast Brownian motion of small molecules (Figure 5a), but tailing was observed for all the samples at longer delay times showing the presence of larger particles. Although there are some larger objects in the samples, molecules with dH below 10 nm predominate at low concentrations. The curves at higher concentrations, shown in Figure 5b, show that the decay of the larger associated structures is much slower.

a)

b) 1,0

Correlation coefficient

0,8

0,6

0,4

0,2

0,0 0,1

High concentration 15(g/L)

Low concentration 1(g/L)

1,0

Correlation coefficient

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

Biomacromolecules

Xylan1 Xylan2 Xylan3 Xylan4 Xylan5

0,8 0,6 0,4 0,2 0,0

1

10

100

1000 10000 1000001000000 1E7

0,1

1

10

100

1000

10000 100000 1000000

1E7

Time(s)

Time(s)


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Figure 5. Autocorrelation curves for the Xylan samples at, a) low (1 g/L) and b) high (15 g/L) concentrations.

A comparison between the size distribution and the autocorrelation function data showed that associated structures were present in all the samples. The curves also indicate that the extracted and purified Xylan samples show a molecular solubility at low concentrations, but the molecules undergo a phase separation and associate into larger structures when the concentration is increased. 3.3. Charge and zeta potential The cationic demand and zeta potential of the extracted and purified samples are shown in Table 2. The negative charges increased slightly as a result of the treatments. The negative charges on these samples are due to the presence of anionic moieties in the spruce glucuronoxylan assigned to uronic acid (4-O-methyl glucuronic acid) substitutions. For example, the DTPA treatment to remove impurities from xylan 2 slightly increased the negative charges and decreased the zeta-potential. In xylan 2 (dialysis of xylan 1) not


22

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only were small molecular fragments removed but also ions. Bleaching increased the negative charges even more, due to the oxidizing effect of the bleaching (Xylan 4 and 5). The pH was lowered during treatment, which means that the detected change in the charge was somewhat underestimated due to protonation of the carboxyl groups in this pH-range, emphasizing that the different treatments led to significant changes in the charge of the xylans.

Table 2. Zeta potential, cationic demand and pH of Xylan samples from DLS and PET measurements.

Zeta potential

Charge from PET

(mV)

(meq/g)

5.2

-17

-0.96

Xylan2

5.2

-18

-1.08

Xylan3

4.2

-19

-1.09

Xylan4

4.6

-22

-1.14

Xylan5

4.1

-19

-1.12

sample

pH

Xylan1

3.4. Morphology


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The morphology of the molecules and associated structures were investigated at low and high concentrations using AFM and Cryo-TEM techniques, respectively. AFM images, captured for the samples at low concentration (1 g/L) show the adsorbed molecules on model mica surfaces (Figure 6). The width of the adsorbed entities in the samples was measured to be 50-70 nm for all the samples except Xylan 5, which had larger particles in the 70-100 nm range. This indicates an increase in size (compared to the DLS data) which probably is due to the slight concentration increase close to the surface, due to the adsorption of the xylan at the solid liquid interface, and hence a further increase in the degree of association of the molecules, in combination with a flattening after the adsorption and drying steps before imaging of the associates. Further image analysis on Xylan 5 showed that the clusters are mostly formed by association of two or three smaller groups (See Figure 1, SI). In comparison, the autocorrelation curve (Figure 5a) obtained for Xylan 5, also showed a slightly slower decay, which was probably due to the association of the smaller molecules after dialysis of the bleached sample (Xylan 4). The detected width of the samples needs to be analyzed with care due to tip broadening


24

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Biomacromolecules

with AFM but considering the dimensions of the tip (nominal tip radius < 8 nm) and the size of the samples this factor is believed to have a minor effect on the measured results.

a)

b)

d)

e)

c)

Figure 6. AFM images of the different Xylan samples adsorbed on to mica surfaces at 1 g/L concentration, a) Xylan 1, b) Xylan 2, c) Xylan 3, d) Xylan 4, e) Xylan 5.


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A Cryo-TEM study was performed on the extracted Xylan at a higher concentration (15 g/L) since it was impossible to obtain images with a good contrast at lower concentrations. Figure 7 shows the association of smaller molecules to larger fractal structures up to 1000 nm in size, which support the DLS data. a)

b)

Figure 7. Two separate Cryo-TEM images of Xylan 1 at 15 g/L. (The DLS data were similar for all the xylan samples).

4. Phase separation of hemicellulose solutions as described by the Flory-Huggins theory


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Biomacromolecules

All the extracted hemicellulose samples showed similar size and molar mass distributions in aqueous media, indicating that these hemicelluloses were soluble in water, but at 15 g/L a large fraction of the molecules were found to be in an associated state indicating that the sample is in two-phase state, although the dispersions were still transparent. The association was shown by DLS and Cryo-TEM imaging. The larger hydrodynamic diameter of the samples at higher concentrations shows that water is a poor solvent for these polymers and the associates found with both DLS and Cryo-TEM indicate a phase transition with increasing polymer concentration28. This phase transition is typical for polymers in poor solvents and the solubility parameter for the polymer/solvent system was investigated using the Flory-Huggins theory which is commonly used to describe the phase equilibrium of polymer solutions taking into consideration both the enthalpy and entropy of mixing of both polymer segments and solvent molecules according to29:

∆𝐺𝑚𝑖𝑥 kT

= {𝑁𝐴ln ɸ𝐴 + 𝑁𝐵ln ɸ𝐵 } + {𝜒𝑁𝑡𝑜𝑡ln ɸ𝐴 ɸ𝐵 }

[2]


27


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where the first term within brackets on the right-hand side is the entropy contribution ― ∆𝑆𝑚𝑖𝑥 𝑘

, and the second term is the enthalpy of mixing

∆𝐻𝑚𝑖𝑥 𝑘𝑇

, ɸ𝐴 and ɸ𝐵 are the volume

fractions of the solvent and polymer respectively, χ is the interaction parameter, and 𝑁𝐴 and 𝑁𝐵 are respectively the numbers of solvent and polymer molecules. Then 𝑁𝑡𝑜𝑡 is given by: [3]

𝑁𝑡𝑜𝑡 = 𝑁𝐴 +𝑀𝑁𝐵 where M is the degree of polymerization of the polymer.

The chemical potential of the solvent (A) and the polymer (B) can be derived from eq. 2 30: ∂𝛥𝐺

[

(

1

) ]

[4]

∆µ𝐴 = µ𝐴 ― µ0𝐴 = ∂𝑁𝐴 = 𝑘𝑇 𝜒ɸ2𝐵 + ln (1 ― ɸ𝐵) + 1 ― 𝑀 ɸ𝐵 ∂𝛥𝐺

[5]

∆µ𝐵 = µ𝐵 ― µ0𝐵 = ∂𝑁𝐵 = 𝑘𝑇[𝜒𝑀(1 ― ɸ𝐵)2 + ln ɸ𝐵 ― (𝑀 ― 1)(1 ― ɸ𝐵)] The condition for phase equilibrium between two phases I and II can be written:

{

µ𝐼𝐴 = µ𝐼𝐼 𝐴 µ𝐼𝐵 = µ𝐼𝐼 𝐵

[6]


28

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The solution to the equation system [6] for M=113 is shown in Figure 8 by plotting 𝜒 as a function of ɸ𝐵. The details of the calculations are attached in the supplementary information.

Semi-dilute Dilute

0,67 0,66 0,65 0,64



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Biomacromolecules

0,63 0,62 0,61 0,60 0,59 0,0

0,1

0,2

0,3

0,4

0,5



Figure 8. Phase diagram (𝜒 as a function of ɸ𝐵) calculated from the Flory-Huggins theory for polymers with degree of polymerization M=113.

The critical point is {𝜒𝑐 = 0.598; ɸ𝐵 = 0.087 }. For 𝜒 < 𝜒𝑐 the system is one-phase for all ɸ𝐵. For 𝜒 > 𝜒𝑐, in the area above the plotted curves, a dilute solution (left branch, red) is in equilibrium with a semi dilute solution (right branch, blue).


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The experiments show that the system is phase separated when the polymer concentration is at 15 g/L. The corresponding polymer volume fraction is 0.0095, calculated from the molar mass of a segment in the chain (with average value of 150 g/mol) and setting the molar volume of a segment in the xylan chain equal to 5.3 times the molar volume of water (0.0180 L/mol). According to the phase diagram in Fig. 8, 𝜒 must be larger than 0.64 for that composition to be located in the two-phase region. Such a phase separation behavior has also been observed for acetylatedgalactoglucomannan (A-GGM) hemicelluloses extracted from Spruce19. The association of the small A-GGM molecules was found to be a reversible process, since the aggregates easily dissociate when the concentrated samples are diluted. The differences in the chemical or physical structures, i.e. substitution patterns, of these hemicelluloses can naturally be a driving force increasing or decreasing the molecular solubility of these samples, but all the investigated samples showed a poor or very poor solubility in water. This will no doubt have a major impact on the properties of films and gels made from these hemicelluloses since most of the films and gels are formed by


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Biomacromolecules

concentrating aqueous solutions of the hemicelluloses. The results also indicate that the molar mass of these materials have a large impact on their solubility.

5. Conclusions Arabinoglucuronoxylans extracted from Spruce and purified using different ion separation, dialysis and bleaching procedures showed similar solubility and association behavior at different concentrations. Although all the samples were optically transparent at the measured concentrations, they showed an overall poor solubility in water. Molecular solubility was only achieved at low concentrations. Increasing the concentration led to a phase transition of the solutions and the Flory-Huggins fitting of the data gave a value about χ= 0.64 at 15 g/L, showing the poor solubility of this hemicellulose in aqueous media. These fundamental findings are of crucial importance for developing a more explicit path for extracting hemicelluloses and improving the products made from these biopolymers in aqueous media by procedures such as hydrodynamic processing, film formation from casting and by using chemical modification to improve the solubility in water.


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Supporting Information

Morphology investigation of the AFM image of Xylan 5, i.e. width and height measurements of the associated structures. Different calculation steps for Flory-Huggins theory and phase separation. An image showing the clear dispersion obtained at high concentration (15 g/L).

Author information

Corresponding authors

*Email:

[email protected]

*Email:

[email protected]


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Author contribution

The manuscript was written through contribution of all authors. All authors have confirmed the final version of the manuscript.

Acknowledgement

The Knut and Alice Wallenberg foundation is gratefully acknowledged for financial support through the Wallenberg Wood Science Centre. The authors would also like to thank Jonny Eriksson at Uppsala University, Uppsala, Sweden for excellent help and support in the Cryo-TEM imaging.

References

1.

Albersheim, P.; Darvill, A.; Roberts, K.; Sederoff, R.; Staehelin, A., Plant cell

walls. Garland Science: 2010.


33


2.

Page 34 of 37

Puls, J., Chemistry and biochemistry of hemicelluloses: Relationship between

hemicellulose structure and enzymes required for hydrolysis. Macromolecular Symposia 1997, 120 (1), 183-196. 3.

Ebringerová, A.; Heinze, T., Xylan and xylan derivatives – biopolymers with

valuable properties, 1. Naturally occurring xylans structures, isolation procedures and properties. Macromolecular Rapid Communications 2000, 21 (9), 542-556. 4.

Deutschmann, R.; Dekker, R. F. H., From plant biomass to bio-based chemicals:

Latest developments in xylan research. Biotechnology Advances 2012, 30 (6), 16271640. 5.

Escalante, A.; Gonçalves, A.; Bodin, A.; Stepan, A.; Sandström, C.; Toriz, G.;

Gatenholm, P., Flexible oxygen barrier films from spruce xylan. Carbohydrate Polymers 2012, 87 (4), 2381-2387. 6.

Timell, T. E., Isolation of galactoglucomannans from the wood of gymnosperms.

Tappi 1961, 44, 88-96. 7.

Timell, T. E., Recent progress in the chemistry of wood hemicelluloses. Wood

Science and Technology 1967, 1 (1), 45-70. 8.

Wise, L. E.; Murphy, M.; D’Addieco, A. A., Chlorite holocellulose, its fractionation

and bearing on summative wood analysis and on studies on the hemicelluloses. Paper

Trade J. 1946, 122 (2). 9.

Grondahl, M.; Eriksson, L.; Gatenholm, P., Material properties of plasticized

hardwood Xylans for potential application as oxygen barrier films. Biomacromolecules 2004, 5 (4), 1528-1535. 10.

Linder, A.; Roubroeks, J. P.; Gatenholm, P., Effect of ozonation on assembly of

xylans. Holzforschung 2003, 57 (5), 496-502. 11.

Stepan, A. M.; King, A. W. T.; Kakko, T.; Toriz, G.; Kilpelainen, I.; Gatenholm, P.,

Fast and highly efficient acetylation of xylans in ionic liquid systems. Cellulose 2013, 20 (6), 2813-2824. 12.

Westbye, P.; Kohnke, T.; Gatenholm, P., Fractionation and characterization of

xylan rich extracts from birch. Holzforschung 2008, 62 (1), 31-37.


34

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

Biomacromolecules

13.

Westbye, P.; Svanberg, C.; Gatenholm, P., The effect of molecular composition

of xylan extracted from birch on its assembly onto bleached softwood kraft pulp.

Holzforschung 2006, 60 (2), 143-148. 14.

Zhang, Y. J.; Li, J. B.; Lindstrom, M. E.; Stepan, A.; Gatenholm, P., Spruce

glucomannan: Preparation, structural characteristics and basic film forming ability.

Nordic Pulp & Paper Research Journal 2013, 28 (3), 323-330. 15.

Wyman, C. E.; Decker, S. R.; Himmel, M. E.; Brady, J. W.; Skopec, C. E.; Viikari,

L., Hydrolysis of cellulose and hemicellulose. Polysaccharides: Structural diversity and

functional versatility 2005, 1, 1023-1062. 16.

Martínez-Abad, A.; Berglund, J.; Toriz, G.; Gatenholm, P.; Henriksson, G.;

Lindström, M.; Wohlert, J.; Vilaplana, F., Regular motifs in xylan modulate molecular flexibility and interactions with cellulose surfaces. Plant physiology 2017, pp. 01184.2017. 17.

Kuzmenko, V.; Hagg, D.; Toriz, G.; Gatenholm, P., In situ forming spruce xylan-

based hydrogel for cell immobilization. Carbohydrate Polymers 2014, 102, 862-868. 18.

Whistler, R. L., Solubility of polysaccharides and their behavior in solution.

Advances in Chemistry Series 1973, (117), 242-255. 19.

Kishani, S.; Vilaplana, F.; Xu, W.; Xu, C.; Wågberg, L., Solubility of Softwood

Hemicelluloses. Biomacromolecules 2018, 19 (4), 1245-1255. 20.

Wise, L. E.; Murphy, M.; D’Addieco, A. A., Paper Trade J., 122, No. 2, 35 (1946).

Tech. Assoc. Papers 1946, 29, 210. 21.

Timell, T., Polysaccharides From Ferns- (Filicineae). Soc Chemical Industry 14

Belgrave Square, London SW1X 8PS, England: 1961; pp 474-474. 22.

De Ruiter, G. A.; Schols, H. A.; Voragen, A. G.; Rombouts, F. M., Carbohydrate

analysis of water-soluble uronic acid-containing polysaccharides with high-performance anion-exchange chromatography using methanolysis combined with TFA hydrolysis is superior to four other methods. Analytical biochemistry 1992, 207 (1), 176-185. 23.

McKee, L. S.; Sunner, H.; Anasontzis, G. E.; Toriz, G.; Gatenholm, P.; Bulone,

V.; Vilaplana, F.; Olsson, L., A GH115 α-glucuronidase from Schizophyllum commune contributes to the synergistic enzymatic deconstruction of softwood glucuronoarabinoxylan. Biotechnology for biofuels 2016, 9 (1), 2.


35


24.

Page 36 of 37

Chong, S.-L.; Koutaniemi, S.; Virkki, L.; Pynnönen, H.; Tuomainen, P.;

Tenkanen, M., Quantitation of 4-O-methylglucuronic acid from plant cell walls.

Carbohydrate polymers 2013, 91 (2), 626-630. 25.

Instruments, M., Dynamic light scattering: an introduction in 30 minutes.

Technical Note Malvern, MRK656-01 2012, 1-8. 26.

Wågberg, L.; Ödberg, L., Polymer adsorption on cellulosic fibres. Nordic Pulp

and Paper Research Journal 1989, 4, 71-76. 27.

Almgren, M.; Edwards, K.; Karlsson, G., Cryo transmission electron microscopy

of liposomes and related structures. Colloids and Surfaces A: Physicochemical and

Engineering Aspects 2000, 174 (1), 3-21. 28.

Holmberg, K.; Jönsson, B.; Kronberg, B.; Lindman, B., Surfactants and polymers

in aqueous solution. Wiley Online Library: 2002. 29.

Holmberg, K.; Jönsson, B.; Kronberg, B.; Lindman, B., Surfactants and polymers

in aqueous solution. Wiley Online Library: 2003; Vol. 2. 30.

Hill, T. L., An introduction to statistical thermodynamics. Courier Corporation:

1986.

Table of Contents graphic


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insolubility of

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