Ultrastructure of Ulvan: A Polysaccharide from Green SeaweedsAudrey Robic,1,2 Cedric Gaillard,2 Jean-Francois Sassi,1 Yannick Lerat,1 Marc Lahaye2

1 Centre d’Etudes et de Valorisation des Algues, Presqu’ıle de Pen Lan, BP3, 22610 Pleubian, France

2 Institut National de la Recherche Agronomique, Unite Biopolymeres, Interactions, Assemblages, UR1268, Rue de la

Geraudiere, BP 71627, 44316 Nantes, France

Received 17 February 2009; revised 26 March 2009; accepted 26 March 2009

Published online 7 April 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.21195

This article was originally published online as an accepted

preprint. The ‘‘Published Online’’date corresponds to the preprint

version. You can request a copy of the preprint by emailing the

Biopolymers editorial office at biopolymers@wiley. com

INTRODUCTION

Ulvan is a complex water-soluble acidic polysaccha-

ride extracted from the cell-walls of the green sea-

weeds belonging to Ulvales (Ulva and Enteromorpha

sp.). This biomass is easily available from proliferat-

ing algae in eutrophicated coastal waters1 or from

culture.2–5 The polysaccharide displays physico-chemical and

biological features of potential interest for diverse applica-

tions.6 It is mainly composed of rhamnose, glucuronic and

iduronic acids, and xylose7,8 mostly distributed in disaccha-

ride repeating units. The two major disaccharides are the

ulvanobiuronic acid 3-sulfate Type A (b-D-GlcA (1 ? 4) a-L-

Rha 3S ? 1) and Type B (a-L-IdoA (1 ? 4) a-L-Rha 3S ? 1)

(see Figure 1). Other minor repeating units contain glucu-

ronic acid as a branch on O-2 of rhamnose 3-sulfate or par-

tially sulfated xylose replacing the uronic acid (see Figure 1).

The unusual polyelectrolyte develops low intrinsic viscosities

of the order of 95–285 ml g21 in saline solutions6 and forms

gels with borate and divalent cations at pH 7 by yet an

unclear mechanism.9,10 Solid-state 13C NMR data obtained

from partially hydrated ulvan showed that the polysaccharide

displays an unexpected lower affinity for water than expected

for a water-extracted biopolymer.11

To obtain a better insight into the structure of ulvan and

to further our understanding on its solution properties and

its mechanism of gelation, high molecular weight ulvan from

Ulva rotundata was fractionated by anion exchange chroma-

tography prior to microscopic observations. The chemical

composition, chemical structure, and macromolecular prop-

erties of the different fractions were determined. The phys-

ico-chemical parameters of ulvan fractions were modified

prior to electron microscopy and AFM observation to high-

light polysaccharide interactions behavior and to examine

the gelling mechanism of ulvan.

RESULTS

Composition of Raw Ulvan Extract

The chemical composition and macromolecular properties

of high molecular weight ulvan from U. rotundata stored at

2808C have been characterized previously.12 This extract

designated as ulvan in Table I was composed mainly of rham-

Ultrastructure of Ulvan: A Polysaccharide from Green Seaweeds

Correspondence to: Jean-Francois Sassi; e-mail: jean-francois.sassi@ceva.fr and

Marc Lahaye; e-mail: lahaye@nantes.inra.fr

ABSTRACT:

Ultrastructural analysis of the gel forming green seaweed

sulfated polysaccharide ulvan revealed a spherical-based

morphology (10–18 nm diameter) more or less

aggregated in aqueous solution. At pH 13 in TBAOH

(tetrabutyl ammonium hydroxyde) or NaOH, ulvan

formed an open gel-like structure or a continuous film by

fusion or coalescence of bead-like structures, while in

acidic pH conditions, ulvan appeared as dispersed beads.

Low concentrations of sodium chloride, copper or boric

acid induced the formation of aggregates. These results

highlight the hydrophobic and aggregative behavior of

ulvan that are discussed in regard to the peculiar gel

formation and the low intrinsic viscosity of the

polysaccharide in aqueous solution. # 2009 Wiley

Periodicals, Inc. Biopolymers 91: 652–664, 2009.

Keywords: atomic force microscopy; biopolymers; electron

microscopy; nanoheterogeneity; solution properties

Contract grant sponsors: French National Association for Research and Technology

(ANRT); the General Council of Cotes d’Armor

VVC 2009 Wiley Periodicals, Inc.

652 Biopolymers Volume 91 / Number 8

nose, xylose, glucuronic acid, iduronic acid, and sulfate.

Minor amounts of glucose and protein were also measured.

The HPSEC refractive index profile of the extract showed

that ulvan molecular weight distribution was asymmetrical

(Figure 2a). The UV trace showed a weak response all over

the fractionation range of ulvan, which was indicative of

minor amounts of proteins co-eluting with ulvan (data not

shown). The macromolecular characteristics of the material

eluted from 16.5 to 20.7 min (Peak A) and from 20.7 to 25.7

min (Peak B) were analyzed separately (Table I). The average

molecular weights of ulvan from Populations A and B were

500 3 103 and 180 3 103 g mol21, respectively. Population A

represented 80% of ulvan. The intrinsic viscosity varied from

460 to 270 ml g21 for Populations A and B, respectively. For

Population A, the radius of gyration Rg used to characterize

polymer chain extension was 35 nm and the exponent ‘‘a’’

from the Mark-Houwink-Sakurada relation ([g] 5 KMa,

MHS) was 0.07.

Fractionation of Ulvan

Most ulvan was eluted by 0.9–2.0M NaCl on anion exchange

chromatography (Figure 2b). The acidic polysaccharides

were split into six fractions: (UA), a minor uronic acid rich

polysaccharide eluted with about 0.5M NaCl and a broad

population subdivided in Fractions U1–U5 according to

their elution order. A protein peak eluted from the anion

exchanger before the gradient and a second peak was

observed with about 0.6M NaCl (data not shown).

Chemical Composition. Fractions U2–U5 were made of 52.6

and 58.8% dry weight (dw) sugars (Table I). No major differ-

ences were observed for the individual sugar proportions.

Rhamnose amounted to 53.2–54.2 mol% of the fractions

whilst uronic acids contents ranged from 34.7 to 37.9 mol%.

Fraction U5 was slightly poorer in xylose (7.1 mol% 6 0.0

mol%) whereas the Fraction U2 was slightly poorer in uronic

acids (34.7 mol% 6 1.8 mol%). Sulfate was the lowest in

Fraction U3 (16.4% dw 6 0.5% dw) and the highest in Frac-

tion U5 (19.6% dw 6 0.0% dw). All fractions were low in

protein content (1.0–1.9% dw).

Macromolecular Properties. As for the native ulvan, Frac-

tions U1–U5 were composed of two major macromolecular

populations (A, B) in varying proportions (Figure 2c). The

macromolecular characteristics of the polysaccharide Popula-

tions A and B are given in Table I.

The proportion of the Population A increased in fractions

from U2 (56%) to U5 (77%) while that of Population B

decreased. The molecular weight of the two polysaccharide

populations increased from Fraction U2 (respectively, 350 3

103 and 80 3 103 g mol21) to Fraction U5 (respectively, 470

3 103 and 1403 103 g mol21).

For each fraction, the radius of gyration of the Population

A varied between 28 and 32 nm and the exponent ‘‘a’’ from

the Mark-Houwink-Sakurada relation was slightly higher

than that of raw ulvan for Fractions U1–U4 at about 0.13

and was lower for Fraction U5 (0.06). The intrinsic viscosity

of Population A varied from 400 to 520 ml g21. The lowest

value was obtained for U1 and the highest for U5.

Fraction UA was composed of a wide distribution of poly-

saccharides eluting close to the low molecular weight limit of

detection of the MALLS detection system. It also differed

from the other samples by a strong UV absorbance in the low

molecular weight range (data not shown).

FIGURE 1 Structure and nomenclature of the main repeating disaccharides in Ulva ulvan: ulvano-

biuronic acids A3S and B3S and ulvanobioses U3S and U20S,3S.

Ultrastructure of Ulvan 653

Biopolymers

Fractions U2 and U5 which contained the highest pro-

portions in Populations B and A, respectively, were taken as

representative molecular weights extremes of the ulvan

structures.

Chemical Structure. 13C NMR spectra of Fractions U2 and

U5 yielded typical ulvan carbon chemical shifts with signals

for the ulvanobiuronic acid 3-sulphate Types A and B13

(see Figure 3). Several minor signals likely reflected other

TableI

FractionationYield,Chem

icalComposition,an

dPhysico-Chem

icalCharacteristicsofRaw

Ulvan

andFractions

Sam

ple

s

Yie

ld

(%)

To

tal

Su

gars

(%d

w)

Sulf

ate

(%d

w)

Pro

tein

(%d

w)

Suga

rs(m

ol%

)A

B

Rh

aX

ylG

alG

lcU

rAa

Mw3

10

3%

Rg

(nm

)a

[g]

Mw3

10

3%

ulv

an5

5.6

14

.96

0.4

2.86

0.3

50

.26

0.6

7.06

0.7

0.06

0.0

0.96

0.2

41

.96

0.1

50

08

03

50

.07

46

01

80

20

UA

0.7

nd

bn

dn

dn

dn

dn

dn

dn

dn

dn

dn

dn

dn

dn

dn

d

U1

3.1

nd

nd

nd

nd

nd

nd

nd

nd

42

01

92

80

.18

40

07

08

1

U2

16

.05

8.86

0.0

18

.66

0.0

1.96

0.7

53

.96

0.0

7.96

0.0

1.56

0.0

2.06

0.0

34

.76

1.8

35

05

62

90

.12

47

08

04

4

U3

25

.35

2.66

0.2

16

.46

0.5

1.36

0.1

54

.26

0.0

8.36

0.1

0.06

0.0

0.76

0.1

36

.96

1.6

31

06

02

80

.14

46

09

04

0

U4

34

.35

5.96

0.3

18

.16

0.3

1.06

0.1

53

.96

0.7

7.66

0.4

0.06

0.0

0.66

0.2

37

.96

0.2

33

06

52

90

.10

48

01

10

35

U5

20

.65

7.56

0.0

19

.66

0.0

1.66

0.6

53

.26

0.0

7.16

0.0

2.16

0.0

0.96

0.0

36

.76

0.0

47

07

73

20

.06

52

01

40

23

Mo

lecu

lar

wei

ght

(Mw

)w

asex

pre

ssed

ing

mo

l21,

%co

rres

po

nd

edto

the

Fra

ctio

ns

Aan

dB

pro

po

rtio

ns,

[g]

toth

ein

trin

sic

visc

osi

tyin

ml

g21,

Rg

toth

era

diu

so

fgy

rati

on

and

‘‘a’’

toth

e

Mar

k-H

ou

win

k-S

aku

rad

aex

po

nen

t.a

UrA

:u

ron

icac

ids.

bn

d:

no

td

eter

min

ed.

FIGURE 2 (a) High pressure size exclusion chromatogram of

ulvan and plots of the radius of gyration (r) against elution volume.

(b) Purification of ulvan by anion exchange chromatography. NS:

neutral sugars (mg ml21), UrA: uronic acids (mg ml21), % corre-

spond to the recovered fraction weight on the total recovered matter

weight. (c) High pressure size exclusion chromatographic profiles of

ulvan purified fractions.

654 Robic et al.

Biopolymers

linkages between rhamnose, uronic acids, and sulfate as well

as the presence of other sugars residues such as xylose, galac-

tose, and glucose. The resonance at 83 ppm for contiguous

1,4-linked b-D glucuronic acids (Gg4 for GlcA C-4) in ulvan or

from contaminating glucuronan observed in the raw ulvan was

absent on the spectra of U2 and U5 (see Figure 3). This signal

became minor after the purification step. Signals attributed to

R1x(s) at around 98 ppm for C-1 in rhamnose 3-sulphate

linked to xylose or xylose 2-sulphate, to X5 at 65 ppm for C-5

of xylose and to X5s/C6 for C-5 of xylose 2-sulphate and for

C6 of different hexoses at around 60 ppm were always present

on the two spectra. No major differences were observed

between the spectra of Fractions U2 and U5 (data not shown).

Fractions U2 and U5 were degraded by an ulvan-lyase to

characterize their fine chemical structure. They were totally

degraded and yielded identical oligosaccharides chromato-

graphic profiles composed of three peaks with Kav 0.1 (Peak

1), 0.4 (Peak 2), and 0.6 (Peak 3). For the two oligo-ulvan

preparations the proportion of the different peaks were very

close with about 84.8% for Peak 1, 4.3% for Peak 2, and 10.9%

for Peak 3. The two ulvan fractions had similar fine structures.

Microscopy Characterization and Microstructure

Figure 4 shows the typical micrographs of raw ulvan

observed by TEM (Figure 4a), cryo-TEM (Figure 4b), AFM

(Figure 4c), and SEM (Figure 4d). Although the specimens

were prepared using different routes according to the micros-

copy used, and each technique may have induced various

artifacts, ulvan systematically appeared as small spherical-

shaped particles and elongated filaments. These two types of

nano-objects appeared as more or less independent from one

another and mainly arranged in a network. One can notice

some classical differences between cryo- and negatively-

stained TEM images. Indeed, the filaments appear thinner in

TEM (Figure 4a) than in cryo-TEM (Figure 4b), that is

expected due to shrinkage occurring during the water evapo-

ration of ulvan solution while drying (negative staining

protocol). This effect is more visible for the lyophilized speci-

mens prepared for SEM investigation (Figure 4d). Negative

staining is also well-known for fixing soft samples when

applied before a complete drying of the sample14 and may

influence the ionic strength and pH of the sample. However,

in this work, it was applied on the evaporated sample and

was rapidly dried to limit its influence. The molecules were

also submitted to a strong shrinkage during the drying step

for AFM observations. All these artifacts may explain the

differences observed between the TEM, cryo-TEM and AFM

images (see Figure 4).

Ulvan Fractions U2 and U5 showed solely spherical-

shaped particles with no more filaments (see Figure 5). For

the low molecular Fraction U2, these particles appeared poly-

dispersed with a mean diameter of 10.5 nm (Figures 5a–5c).

Some of these were associated but no large aggregates were

found. The high molecular Fraction U5 showed only larger

various shaped aggregates that ranged from ten nanometers

to several hundred nanometers (Figures 5d and 5e). The fine

morphology of these aggregates can be seen as the strong

association of smaller particles.

Influence of Salts and pH on the Morphology and

Assembling of Ulvan Fractions

To better understand the driving mechanisms of ulvan aggre-

gation and gel formation, Fractions U2 and U5 were further

studied in different physicochemical environments.

pH and counter-ions were found to affect markedly ulvan

morphology (see Figure 6). At pH 13 a very dense thick film

was formed for both U2 and U5 fractions in presence of

0.1M NaOH. It resulted from a massive aggregation of the

individual particles of Fraction U2 and of the larger preag-

gregates of the Fraction U5. This film was revealed on Figures

6a and 6b through a variation of contrast between the bright

carbon support film and the dark aggregated ulvan layer that

demonstrated a notable surface roughness. A dense film was

not achieved when NaOH was replaced by tetrabutylammo-

FIGURE 3 13C NMR spectra of raw ulvan and Fraction U2. R

refers to nonreducing end rhamnose 3-sulfate linked to glucuronic

acid, R0 refers to nonreducing end rhamnose 3-sulfate linked to idur-

onic acid, G and I refer to glucuronic and iduronic acids, respectively.

X5 and X5s refer to xylose C-5 in ulvanobiose repeating sequences.

Gg signals corresponded to carbons in glucuronan segments in ulvan

as well as in contaminating 1,4-linked b-D-glucuronan. R1x(s) refer

to C1 of rhamnose and C6 to C-6 of hexoses like glucose or galactose.

Ultrastructure of Ulvan 655

Biopolymers

nium hydroxide (TBAOH): a porous network composed of

interconnected cylindrical-shaped nanorods took place

(Figures 6c and 6d). The dimension of the transversal rods

ranges from 20 to 50 nm in diameter and 50 to 250 nm in

length. Neither spherical-shaped particles nor surface rough-

ness were observed in these rods.

In acidic conditions, two assembling behavior were distin-

guished depending on pH (Figures 6e–6h). In weak acidic

condition (pH 4.5) and in presence of salts (NaCl), spheri-

cal-shaped smaller particles associated as large aggregates

with random shapes (Figures 6e and 6f). The size range of

the aggregates apparent diameter ranged from 50 to 1200 nm

and the diameter of the spherical-shaped sub-structures at

around 10 nm was very close to the starting particles diame-

ter of the ulvan Fraction U2 (Figure 5c). At pH 2.8, only iso-

lated spherical particles were produced from ulvan Fractions

U2 or U5 (Figures 6g and 6h). The corresponding mean

diameter (9 nm with a broad dispersion) and the global mor-

phology of Fraction U2 at this low pH were close to those of

Fraction U2 in deionized water (pH 5). Fraction U5 was also

composed of isolated spherical-shaped particles with a larger

mean diameter (18 nm) and broader size distribution (inset

FIGURE 4 Microscopy characterization of raw ulvan. (a) Negative stained TEM (1 mg ml21 in

water); (b) cryo-TEM of a vitreous thin film of the ulvan solution (5 mg ml21 in water); (c) Height

AFM image, scalebar is 100 nm; (d) SEM from a freeze-dried ulvan solution. Insets: higher magnifi-

cation views.

656 Robic et al.

Biopolymers

Figure 6g). The particles in Fraction U5 were all clearly sepa-

rated while they only existed as aggregates in deionized water

at pH 5 (Figures 5d and 5e).

The effect of ions in acidic conditions on the assembly of

ulvan Fraction U2 was also studied by varying the nature of

the salts in solutions (see Figure 7). The ions chosen for this

investigation corresponded to the ones involved in ulvan gel

formation (H3BO3 and/or CuSO4). Progressive states of

aggregation were seen when using only H3BO3 salt (Figures

7a and 7b), only CuSO4 (Figures 7c and 7d), and the mixture

of H3BO3 and CuSO4 (Figures 7e and 7f). H3BO3 induced

the association of the spherical U2 particles. The CuSO4 ions

FIGURE 5 Negatively stained TEM of (a,b) ulvan Fraction U2 and (d,e) ulvan Fraction U5 with

(c) particle size distribution corresponding to the ulvan Fraction U2.

Ultrastructure of Ulvan 657

Biopolymers

FIGURE 6 Effect of pH on the morphology of ulvan Fraction U2. Negative stained images of ulvan in (a,b) a 0.1M NaOH solution (pH

13); (c,d) a 1M TBAOH solution (pH 13); (e,f) a 0.1M NaCl solution (pH 4.5); (g,h) a 0.1M citrate phosphate buffer (pH 2.8). Insets: TEM

images of ulvan Fraction U5 recorded for the same conditions of pH.

Biopolymers

were able to assemble the U2 particles in a fractal network in

which the ulvan spheres were strongly associated. The combi-

nation of both H3BO3 and CuSO4 ions produced a massive

aggregation of the particles resulting in a very dense film.

The same effect was observed on Fraction U5 but the net-

works appeared to be denser.

DISCUSSIONUlvan is a lightly-branched polysaccharide of broad distribu-

tion in term of charge density and molecular weight.6,7 It

contains two types of negatively charged groups: sulfate

esters and carboxylate groups that are essentially distributed

in repeating disaccharides consisting of glucuronic or idur-

FIGURE 7 Effect of ions on the morphology of ulvan Fraction U2. Negative stained images of

ulvan in (a,b) a 0.03M H3BO3 solution (pH 3.8); (c,d) a 0.007M CuSO4 solution (pH 4); (e,f) a

0.03M H3BO3 1 0.007M CuSO4 solution (pH 4). Insets: TEM images of ulvan Fraction U5 recorded

from solutions with the same ions.

Ultrastructure of Ulvan 659

Biopolymers

onic acid and rhamnose sulfate. It is characterized by a high

mean average molecular weight (5.3 3 105 to 3.6 3 106 g

mol21)15 which distributed in U. rotundata and U. armori-

cana into high (1200 3 103–300 3 103 g mol21) and

medium (180 3 103–85 3 103 g mol21) molecular weight

populations.12,16 Ulvan develops low viscous solutions and

gels with borate and divalent cations.6 In this work, the

chemical structure and the macromolecular features of these

populations in U. rotundata ulvan were investigated further.

Ulvan Fractions (U1–U5) varying in the proportion of the

two macromolecular populations were prepared by anion

exchange chromatography. A polyuronan attributed to

b-1,4-linked glucuronan17 and other protein rich fractions,

some coeluting with ulvan were also obtained. Ulvan Frac-

tions U5 and U2 were further studied, as they were, respec-

tively, the richest in the high molecular weight (470 3 103 g

mol21, A) and medium molecular weight (80 3 103 g

mol21, B) populations and had close mean chemical compo-

sitions and fine structure.

Ulvan in aqueous solutions were observed by different

microscopic techniques. Prior to fractionation, ulvan

occurred on transmission electron micrographs as aggregates

of spherical-shaped structures of varying diameters and

partially linked by strands and filament-like material. These

structures were also observed by cryo-TEM, scanning elec-

tron microscopy, and atomic force microscopy observations.

The linear relation between the radius Rg and the elution

volume demonstrated that these structures were homogene-

ously blended in ulvan. An exponent of 0.07 in the Mark-

Houwink-Sakurada (MHS) relation of molecular weight (M)

and intrinsic viscosity [g] of the Population A was in good

agreement with the compact spheres18 observed by micros-

copy. Ulvan ultrastructure departs from the classical rod-

shape or fiber-shape structures of gel-forming polysaccha-

rides.19,21 It rather resembles necklace-type structures formed

by a polyelectrolyte in a poor solvent condition,22 such as

those observed for DNA, hyaluronan, or synthetic polyelec-

trolytes in solutions containing solvent perturbing com-

pounds.23–26 Water is therefore a poor solvent for ulvan.

Although the polysaccharide is highly charged, it has a strong

hydrophobic character leading to highly condensed confor-

mation of the polysaccharide. The methyl group of rhamnose

may be responsible for this peculiar behavior. The low in-

trinsic viscosity of ulvan in aqueous saline solutions can be

explained by the dispersion of condensed bead-like structures

in the solvent rather than as fully dissolved extensively rami-

fied chains.

After anion exchange chromatography, Fraction U2 was

composed of small bead-like particles whereas Fraction U5

was mainly composed of aggregates of bead-like particles.

The two purified fractions were free of fiber-like and linking

strand materials. The increase in the molecular weight of

ulvan in Fraction U5 appears to relate with aggregation. The

lower MHS exponent calculated for this fraction supports

the densification of aggregated spheres observed by micros-

copy but this fraction also demonstrated a higher intrinsic

viscosity. The latter is likely related with the impact of the

nature of the solvent on the polysaccharide interactions.

TEM images of Fractions U2 and U5 were recorded under

different conditions to investigate the behavior of ulvan

in solution and in conditions leading to gel formation.

Counter-ion condensation on the charge of polyelectrolyte

contributes to chain expansion or condensation depending

on the type and concentration of salts and on the solvent

quality. In poor solvent, polyelectrolytes chain contraction

increases with increasing counter-ion condensation and

counter-ion valence and lead to phase separation.22 In 0.1M

NaCl at pH 4.5, the condensed ulvan particles in Fractions

U2 and U5 aggregate. This behavior is in agreement with the

aggregative properties of ulvan at low NaCl concentration

observed by light scattering or rheological measurements.6,27

One consequence of such aggregation properties can be at

the basis of the molecular weight distribution of ulvan into

the two broad Populations A and B. It is proposed that such

distribution results from the dispersion of different sized

nanospheres aggregates to ultimately elementary nanobeads

(see Figure 8). Algal handling and storing prior extraction is

known to markedly affect ulvan molecular weight distribu-

tion12 probably due to polysaccharide degradation leading to

different sizes of ulvan beads aggregates and to the presence

of different chemical species.

The solubility of polymers can be affected by its charge

content. Decreasing the pH of ulvan U2 and U5 solutions

FIGURE 8 The origin of ulvan Fractions U2 and U5 polymolecu-

larity observed by HPSEC elution time and refractometric detection

(Y axis): ulvan nanospheres aggregates dissociate into low molecular

weight substructures and elementary nanoparticles.

660 Robic et al.

Biopolymers

below the pKa of glucuronic acid (3.28)28 protonated the car-

boxylate group while the sulfate esters remain ionized. It led

to the dispersion of aggregates as free condensed bead-like

structures. It thus appears that ulvan particles association

implies ionic interactions and notably carboxylate groups.

Increasing the pH was expected to promote ionic interac-

tions through charged carboxylate and sulfate groups and to

reduce hydrogen bonding. Bead-like structures in U2 and U5

in 0.1M NaOH (pH 13.0) collapsed into a dense homogene-

ous network. Replacing NaOH by 1.0M TBAOH (pH 13.0)

that submitted the ulvan chains to a steric hindrance from

the butyl lateral chains of the counter-ions led to the forma-

tion of an open network. The denser network obtained with

Fraction U5 compared with Fraction U2 indicates a molec-

ular weight contribution to the assembly formation. The dif-

ferent size of the counter-ion probably affects their integra-

tion within the polymer network. The small sodium ion is

likely localized within the chain volumes leading to a densely

packed film resulting from the coalescence of bead-like struc-

tures. TBAOH is a strong base able to readily deprotonate all

the carboxylic acid moiety and form carboxylate salts. The

bulky and hydrophobic nature of the lateral aliphatic chains

of this cation reduces intermolecular attractions thus maxi-

mizing the probability of the resulting ulvan salt being a

compact film.29 The topology of polymer networks depends

on the type of bonding or interaction that provides the cross-

links and on conformational constraints of the polymer

chains. The open gel network resulting from the interaction

between ulvan TBAOH alkyl chains may be related with the

ability of the four constituting aromatic structural units of

the cation to arrange in rigid, linear structure in solution.30

Such structuration may have guided the organization of

ulvan in linear block assemblies and may have prevented fur-

ther ulvan aggregation as dense films. The resulting impact

of TBA on ulvan contrasts with what is observed for other

gelling polysaccharides. Bulky quaternary ammonium ions

are known to prevent carrageenan aggregation31 but in the

case of ulvan, it was ineffective in promoting dispersion of

individualized extended chains.

Although low ionic strength sodium chloride solution can

lead to ulvan aggregation, ulvan gel formation is usually pro-

moted by the presence of boric acid and divalent cations at

preferably slightly alkaline pH.6–10 The individual contribu-

tion of boric acid and copper sulfate solutions in concentra-

tions used for gel formation was observed with U2 and U5

fractions. The very low concentration (30 mM) of H3BO3 at

pH 3.8 promoted aggregation of the bead-like structures.

Boric acid and borate are in equilibrium in solution and

borate ions are known to form esters with vic-diols, which

under favorable stereochemical circumstances can cross-link

polysaccharides.32,33 Concerning ulvan the site and the

mechanism of boric acid/borate interaction has yet to be

identified and is by itself insufficient to promote ulvan gel

formation.9,10,17 The very low concentration of CuSO4

(7 mM) at pH 4.0 induces a dense ulvan film. Copper is well

known to form complexes with neutral sugars or uronic acids

and the strong interactions can even lead to polysaccharides

precipitation.34 When both copper and boric acid are present

a very dense continuous film is formed. Thus, by themselves,

boric acid and copper contribute to aggregate ulvan beads

irrespective of the fraction molecular weight. Together, they

densify the very tight association of bead like structures. The

contribution of boric acid would be to favor ionic interac-

tions between ulvan beads mediated by divalent cations in

creating additional charges on the surface of the beads

through ester formations with exposed hydroxyl. However,

the failure of U2 and U5 fractions to gel in the presence of

boric acid and copper indicates that additional interconnect-

ing structure present in the initial ulvan extract are required

to strongly link bead aggregates. The thermoreversible nature

of ulvan gel can be explained by thermal destructuration of

boric acid/borate esters and ionic interactions. Thus, unlike

classical polysaccharide hydrogels, which involve tight junc-

tion zones of more or less extended helices or flat buckled

ribbons,35,36 ulvan gels likely result from the aggregation of

bead-like structures interconnected by more hydrophilic ulvan

and/or contaminating polymeric fractions (see Figure 9).

CONCLUSIONThe microscopy data and macromolecular characteristics of

ulvan show that this polyelectrolyte has unusual water solu-

tion properties in agreement with previous interpretations of

solid-sate 13C NMR data.11 Dispersion of ulvan in water

appears to be as ‘‘raspberry-like’’ aggregates of elementary

nanospheres more or less connected by fiber-like constitu-

ents. Further definition of the macromolecular properties of

this unusual polyelectrolyte requires the identification of

solvent conditions able to swell ulvan beads and to expand

the polysaccharide chains. Work is also needed to define the

impact of extraction and purification of ulvan on its confor-

mation. Questions remain on whether ulvan is present in the

seaweed cell walls as interconnected bead aggregates or as

extended chains that are prevented to collapse in the aqueous

and ionic wall environment by a peculiar organization and/

or the presence of other molecular partners such as glucur-

onan, xyloglucan, and cellulose. The removal of the latter

during extraction and purification and the drying step of

ulvan may favor the modification of the polysaccharide

conformation and properties.

Ultrastructure of Ulvan 661

Biopolymers

MATERIALS AND METHODS

ExtractionUlvan from frozen ground Ulva rotundata at 2808C was extracted

by reflux for 2 h at 858C in 0.05M sodium oxalate in deionized

water (dry solid content: 6.8% w/w). The suspension was then

diluted one-fold with 0.05M sodium oxalate and stir 1 h at room

temperature. After filtration, the residues were reextracted with

water (dry initial solid content: 1% w/w) for 3 h at room tempera-

ture. The combined extracts were concentrated by ultrafiltration

(Mw 10 kDa, Amicon) and diafiltered with 5 vol. of deionized water,

and freeze-dried. This sample corresponded to ulvan Fraction F8

reported previously.12

FractionationUlvan in deionized water (10 mg ml21, inj vol: 5 ml) was applied to

a HiLoadTM 16/10 Q Sepharose High Performances—FPLC (Fast

Protein Liquid Chromatography, Ge Healthcare) equilibrated in

deionized water on a AKTATM Purifier 10XT coupled to a UV detec-

tor at 280 nm (Ge Healthcare). The column was eluted by a step-

wise gradient of NaCl from 0 to 2.0M (0–0.5M: 1 column volume,

0.5–1.0M: 2 column volumes, 1.0–2.0M: 1 column volume, 2.0M:

1 column volume) at a flow rate of 1 ml min21. Fractions (1 ml)

were collected and checked for sugars and for uronic acids by

colorimetric methods.37,38 Fractions were pooled, dialyzed against

deionized water, and freeze-dried.

Chemical AnalysisSulfate content was measured according to Quemener et al.8 Protein

content was estimated by the Folin-Lowry method.39 Total uronic

acid content was measured by colorimetry38 using glucuronic acid

as standard. Quantification of neutral sugars was performed by

gas-liquid chromatography (GC) after sulfuric acid degradation and

derivation as alditol acetates.40,41 Sugars were identified and quanti-

fied by comparison with reference sugars.

Structural Analysis13C NMR spectra were recorded on a Bruker ARX 400 spectrometer.

About 70-mg sample was dissolved in 1 ml of D2O (99.9%) and

1 ml of H2O with trace of acetone as internal reference (31.45 ppm).

Fine structural analysis by enzymatic degradation was carried

out with partially purified ulvan-lyase as described elsewhere.11

Size Exclusion Chromatography and Molecular

Weight DeterminationsUlvan solution (4 mg ml21) in 50 mM NaNO3 containing 0.02%

NaN3, passed through 0.22-lm membrane filter was injected at

258C on a high-performance size exclusion chromatography system

(HPSEC) consisting of two Shodex OH-pack SB HQ 804 and 805

columns (8 3 300 mm2, Guard columns: OHpak SB-800P, 6 3 50

mm2, Shodex, Showa Denko KK, Miniato, Japan) eluted at 0.7 ml

min21 with 50 mM NaNO3 containing 0.02% NaN3. On-line molar

mass and intrinsic viscosity ([g]) determinations were performed at

room temperature using a multi-angle laser light scattering

(MALLS) detector (mini-Dawn1, Wyatt, operating at three angles:

418, 908, and 1388), a differential refractometer (ERC 7517 A) (dn/dc

5 0.146 ml g21) and a differential viscometer (T-50A, Viscotek,

USA). The weight average molecular weight and radius of gyration

(Rg) were determined using ASTRA 1.4 software (Wyatt, USA).

Intrinsic viscosity [g] and the Mark-Houwink-Sakurada parameter

‘‘a’’ were determined using the TRISEC software (Viscotek, USA).

Protein elution was followed by UV absorbance at 280 nm.

Oligosaccharides from ulvan enzymatic degradation were dis-

solved in 50 mM NaNO3 (0.02% NaN3) and chromatographed on a

column SHODEX OH Pak SB-802.5 HQ (8 3 300 mm2) eluted by

NaNO3 (50 mM, 0.7 ml min21). Elution was followed by a differential

refractometer (ERC 7517 A). Elution of chromatographic peaks (Ve)

was expressed according to the void (V0) and total volumes (Vt) of

the column using the following relation: Kav 5 (Ve 2 V0)/(Vt 2 V0).

MicroscopyPreparation of Ulvan Solutions and Gels. Aqueous solutions

from raw ulvan and Fractions U2 and U5 were prepared by dissolu-

FIGURE 9 Schematic representation of the ulvan gelation mechanism. The line represents fiber-

like material, which can be constituted by proteins, glucuronan and/or extended ulvan segments.

Interactions between the fiber-like material and ulvan bead-like structures and assembly of bead-like

structures may involve similar ionic interactions.

662 Robic et al.

Biopolymers

tion of 1 mg of the corresponding lyophilized samples into 1 ml of

distilled water (pH 5). To study the effect of the pH on the mor-

phology of ulvan, solutions of 1 mg of U2 and U5 fractions were

dissolved, respectively, in 1 ml of 0.1M NaOH (final pH 13), 1 ml of

a 1M tetrabutylammonium hydroxide (final pH 13), and 1 ml of

0.1M citrate phosphate buffer (pH 2.8). Solutions of ulvan Fractions

U2 and U5 (1 mg ml21) were also prepared in the presence of 0.1M

NaCl (pH 4.5), 0.03M H3BO3 (pH 3.8), 0.007M CuSO4 (pH 4), and

0.03M H3BO3/0.007M CuSO4 (pH 4) and investigated by micros-

copy without further dilution. For practical reasons, negative

staining TEM observation was preferred to characterize the struc-

ture of the different ulvan samples, especially for the ones prepared

with ions. Under these conditions ulvan solutions appeared highly

viscous and would have required important dilution for cryo-TEM

and AFM observations with the risk of altering the biopolymer

behavior. Although one can expect some influence of the prepara-

tion method (flattening, shrinkage, associations under drying),

negative staining remains a suitable technique to characterize the

overall morphology of the samples.14,42

TEM. A drop of each aqueous solution was first placed on a car-

bon-coated TEM copper grid (Quantifoil, Germany) and allowed to

air-dry. The sample was then negatively stained with uranyl acetate

(Merck, Germany). For that, glow-discharge-treated carbon-coated

TEM grids were successively placed on a drop of the sample solution

and of an aqueous solution of uranyl acetate (2% w/w) for few sec-

onds and rinsed with a drop of distilled water. The grid was then

air-dried before introducing them in the electron microscope. The

samples were viewed using a JEOL JEM-1230 TEM (JEOL, Tokyo,

Japan) operating at 80 kV and equipped with a LaB6 filament. All

the micrographs were recorded on a Gatan 1.35 K 3 1.04 K 3 12

bit ES500W CCD camera.

Cryo-TEM. Specimens for cryo-TEM observation were prepared

using a cryoplunge cryo-fixation device (Gatan, USA) in which

a drop of the aqueous suspension was deposited on to glow-

discharged holey-type carbon-coated grids (Ted Pella, USA). The

TEM grid was then prepared by blotting the drop containing the

specimen to a thin liquid layer which remained across the holes in

the support carbon film. The liquid film was vitrified by rapidly

plunging the grid into liquid ethane cooled by liquid nitrogen. The

vitrified specimens were mounted in a Gatan 910 specimen holder

(Gatan, USA) that was inserted in the microscope using a CT-3500-

cryotransfer system (Gatan, USA) and cooled with liquid nitrogen.

TEM images were then obtained from specimens preserved in

vitreous ice and suspended across a hole in the supporting carbon

substrate. The samples were observed under low dose conditions

(\10 e-/A2), at 21788C.

SEM. The lyophilized samples were observed in a JEOL JSM

6300 scanning electron microscope (JEOL, Tokyo, Japan) using

low-voltage conditions to avoid any gold sputtering step.

AFM. The atomic force microscope (AFM) used was a MFP3D Bio

system (Asylum Research UK). Samples were prepared by dissolving

them in water to a concentration of 1 lg ml21. About 3 lg of solu-

tion were drop-deposited onto freshly cleaved mica and allowed to

dry. Images were recorded in AC mode in air using AC160 cantile-

vers (Olympus, Japan).

Image Analysis. The software ImageJ (Research Services Branch

NIMH & NINDS http://rsb.info.nih.gov/ij/) has been used to estab-

lish the size distribution of the ulvan Fraction U2 particles and to

estimate the dimension of the larger aggregates of the U5 fraction.

The authors are grateful to Andrew Kirby and Vic Morris from the

Institute of Food Research, UK for the AFM images. The authors

wish to thank J. Vigouroux and M.-J. Crepeau from INRA-BIA for

the technical assistance. Access to the microscope and NMR

facilities of the BIBS platform (Biopolymeres Interactions

Biologie Structurale) of INRA-Nantes was greatly appreciated by

the authors.

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Reviewing Editor: C. Bush

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