Dilute Solution Viscosity of Red Microalga Exopolysaccharide

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P e r g a m o n

Chemical Engineering Science Vol. 51, No. 9, pp. 1487 1494, 1996

Copyright © 1996 Elsevier Science t d

Printed in Great Britain. All rights reserved

0009 2509/96 15.00 + 0.00

0009-2509(95)00305-3

D I L U T E S O L U T I O N V I S C O S I T Y O F R E D M I C R O L G

E X O P O L Y S C C H R I D E

EDWARD ETESHOLA, MOSHE GOTTLIEB*t and SHOSH ANA (MALIS) ARAD *

tDepartment of Chemical Engineering and *Department of Life Sciences and The Institutes for Applied

Research, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

First received 4 November

1994;

revised manuscript received and accepted

24

April

1995)

Abstract The

red microalga

Porphyridium sp P.

sp) is encapsulated in a sulphated polysaccharide. The

external part of this capsule dissolves n the growth medium. This extracellular polysaccharide is a hetero-

polyelectrolyte with molecular weight of ~ 6 x 106 Da. The effect of solvent, counterion and pH on chain

flexibilityand structural features in dilute solution of the exopolysaccharide was investigated by intrinsic

viscometry. From the dependence of the intrinsic viscosity [q] on ionic strength, it was estimated that the

stiffness of P. sp polysaccharide chains is in the same range as that of xanthan and DNA. The effect of the

counterion on [q] is found to be specific and dependent on the type and valence of the counterion. The

polyelectrolyte behaviour of the polymer is confirmed by the decrease of [q] with the addition of salt

without any observable order-disorder conformational transition in aqueous salt solutions in the com-

monly used range of ionic strength (0.01 1.0). At considerably lower ionic strength (< 0.01) there is an

indication of a transition in the P. sp polyion conformation, most likely reflecting a contraction of the

polymer chain from a highly stretched to a stiff, wormlike chain. It is hypothesized from the overall dilute

solution features that the P. sp biopolymer chain molecules adopt stiff ordered conformation in solution.

I N T R O D U C T I O N

P o r p h y r i d i u m

sp. is a red microalga which is encap-

sulated in a sulfated polysaccharide. The external part

of this capsule dissolves in the growth medium. This

extracellular mucil aginous material (hereafter referred

to as PspP, the second P standing for polysacchar-

ide/polymer) carries carboxyl and half ester sulfate

groups o n its glycosidic backbo ne which confers on it

the properties of a biopolyelectrolyte. The exocellular

polymer of P. sp contains different sugars, including

xylose, galactose, glucose, mannose, arabinose,

methyl hexoses and methyl pentoses in various

amou nts and ratios (Jones, 1962; Ramus, 1973; Med-

calf et

al.,

1975; Heaney-Kiera s and Chapm an, 1976;

Heaney-Kieras

et al.,

1976; Percival and Foyle, 1979;

Geresh et al., 1992). In addition, the polysaccharide

conta ins ca 9 glucuroni c acid, ca 10 half ester

sulfate, and a protein moiety (Heaney-Kieras

et al.,

1976). The molecular weight of the P. sp polysacchar-

ide was estimated as 5-7 x 106 Da (Simon

et al.,

1992).

The polysaccharide produced by the red microalga

P. sp is of great interest because at low polymer

concentrations it yields highly viscous aqueous solu-

tions with unique theological properties comparable

with those of xanthan and carrageenan, and thus

suitable for technological applications (Savin, 1978;

Ramus, 1986; Ramus

et al.,

1989; Geresh and Arad,

1991). For example, dilute aqueous solutions of the

polymer have been shown to be effective in drag

*Corresponding author.

CE

5 1 : 9 -

reducti on in capillary pipe flow (Ramus, 1986; Ramus

et al.,

1989). Due to its special properties, PspP aque-

ous solutions have been reported to be compatible

with various salts and stable under varying pH, high

temperature, and accelerated flow rates (Savin, 1978;

Ramus, 1986; Ramus

et al.,

1989; Geresh and Arad,

1991). Preliminary studies in our laboratory indicate

that P. sp polysaccharide has also interesting surface

activity properties and has potential use as a pharma-

ceutical material.

Most of the work published in the literature on this

polysaccharide is concerned with glycosyl content

and chemistry and hardly touches upo n the relation-

ships between conformat ional structure and rheologi-

cal properties (Geresh and Arad, 1991). In order to

develop the extracellular polysaccharide of P. sp, we

need to u nderstand their structure- function relation-

ships, which presupposes a detailed knowledge of

their molecular behaviour and physico-chemical

properties (Lee and Chandrasekaran, 1992). Accord-

ingly, in the work described in this paper we examined

the dilute solutio n properties of PspP as a function of

the nature of the solvent (water, aqueous salt solu-

tions, pH), and the type of counterion. Our purpose

was to determine the influence of these experimental

variables on the chain flexibility and on the conforma-

tional features of the polymer in dilute solutions as

manifested by intrins ic viscosity values.

EXPERIMENTAL

A l g a a n d g r o w t h c o n d i t i o n s


sp. (UTEX 637) was obtained from

the culture collection of algae at the University of

1487



1488

E ETESHOLAet a l .

Texas, Austin. The cells were grown in batch culture

in 1-1 column s 6 cm in diameter at 24 + I°C in artifi-

cial seawater according to Jones e t a l . (1963). The

cultures were illuminated cont inuou sly with fluor-

escent cool-white lamps at a n ir radiance of 150 micro-

einsteins m- 2 s- ~. The med ium was aerated with ster-

ile air cont aini ng 3% COg (Arad e t a l . , 1988). At the

stationary phase of growth (after 2 weeks), the cells

were separated from the growth medium by centrifu-

gation at 10,000 rpm for 20 min using a Sorvall centri-

fuge model RC2-B (Sorvall Instruments Du Pont).

The super natant medium cont aining the polymer was

collected and dialyzed in Visking size 98 32/32 dialy-

sis tubing (Medicell International Ltd., London)

against distilled water and then exhaustively against

bidistilled water at 4°C. The resultant solution was

then freeze dried in order to isolate the water soluble

polysaccharide fraction.

P r e p a r a t i o n o f p o l y s a c c h a r i d e s o l u ti o n s

The •eze- drie d exopolysaccharide was dissolved

in bidistilled water or in aqueous salt solution of the

desired concentration by prolonged gentle stirring

with a magnetic stirrer. For studies on the effect of

pH, test solutions were made by judicio us adjustment

of the pH with a few drops of either 8 M HC1 or

NaO H solutions so as to mai ntain constant polysac-

charide concentration.

I n t r i n s i c v i s c o s i t y

The viscosities of the polymer solutions were meas-

ured with a size 75 Cannon- Fens kesemi-micro capillary

viscometer (Cannon Instrument Co., State College, PA,

USA). The shear rate experienced by the polymer solu-

tions in this viscometer was estimated to be in the range

of 175-670 s - 1. The effect of shear ra te was examined by

the use of a narrower capillary (size 50, 110-450 s). The

difference in [~/] values was insignificant.

The viscometer was suspended in a thermostati-

cally controlled water bath maintaine d at the required

temperature to within + 0.2°C, in the temperature

range 25-85°C. Equilibration time of 15-20 min was

allowed before measurements were made since the

setup was experimentally found to reach thermal

equili brium within 10 min.

The relative viscosity of a given so lut ion qr¢l (de-

fined as the ratio between the solution viscosity and

the solvent viscosity

q / q s )

was determined by measure-

ment of the relative efflux times in the capillary (Mays

and Hadjchristidis, 1991; Van Krevelen and Hoftyzer,

1976). Viscosity values were based on at least 2-3

efflux time readings taken for any given sample in-

serted into the viscometer. Variation between con-

secutive readings was lower than 1.5% and typically

around 0.5%. At least two independent viscosity de-

termi natio ns were performed for each concentration.

Four experimental points were used for extrapolation

to obtain the intrinsic viscosity [~/]. The intrin sic

viscosity is usually obtain ed from either the extrapola-

tion of In

? ] r e l / C

(Kraemer relation)

o r ? ] r e l -

1 ) / c

(Huggins relation) to zero polymer conce ntrat ion, c.

However, (~/rel -- 1 ) / C - - r h p / C where ~/sp s the spe-

cific viscosity r hp = q - q , ) / q s ) of flexible polyelec-

trolyte solutions in pure water exhibits a unique de-

pendence on concentration, i.e. it diverges rapidly

with dilut ion. This effect, due to polymer chain expan-

sion, makes it extremely inaccurate to extrapolate

q s p / C to infinite dilution. Several equations have been

proposed in the literature to describe the concentra-

tion dependence of the viscosity of polyelectrolyte

solutions and to satisfactorily handle the extrapola-

tion of the experimental data (Fuoss and Strauss,

1948; Fuoss, 1951; Liberti and Stivala, 1966; Yuan

e t a l . , 1972). However, some quest ions have been

raised concerning the validity of these methods and

their ability to determine intrinsic viscosities with

precision (Yuan e t a l . , 1972). Furthermo re, it has been

shown that for flexible polyelectrolytes the apparent

divergence in r lsp /C as c ~ 0 is actually a ma ximum

followed by a decrease in q s p / C at very low c values.

The maxi mum is attributed to configurational cha-

nges due to dissolved gases and other impurities (Co-

hen and Priel, 1988). Consequently, in the present

study viscosity data were extrapolated to zero concen-

tration by the combined standard Huggins and

Kraemer treatments. The choice of these latter

methods was also made because they appear to be

more widely used in the literature and thus facilitate

comparison of [q] obtained in the present study with

similar ones for other biopolyelectrolytes and with [q]

values for salt solutions which do not present any

extrapolation difficulty.

R S U L T S

A typical set of data for the viscosity of PspP

solutions as a function of polymer concentration is

depicted in Fig. 1. The data for solutions in water and

in four different aqueous NaC1 solut ions at 25 °C are

plotted in terms of r h p / C vs c. The reported (Cohen

and Priel, 1988; Yamanak a e t a l . , 1990) divergence of

r h p / C as e -~ 0 in water is not observed in the concen-

trati on range examined here. The effects of salt con-

centration, type of counterions, and temperature on

the intrinsic viscosity of the PspP are summarized in

Table 1. The reported values are averages of at least

two ind epende nt determina tions of [~/] with the vari-

ance always below 5% of the reported mean.

The intrins ic viscosity in NaCI solution s at 25°C is

plotted in Fig. 2 against I, the ionic strength of the salt

solution, I = ~ c i Z ~ ) / 2 p , where c i is the ion molar

concentration, Zi the nu mber of charges on the ion

and p the solvent density. The plot shows an initial

sharp decrease of [~/] as the amount of salt is

increased at low NaC1 concent rat ion s (I < 0.025),

followed by a very moderate decrease in [q] at higher

NaC1 concentrations.

The effect of the cation used to prepare the salt

solutions is shown in Fig. 3 (here [q] is plotted against

1 - ° 5 for reasons discussed in the following section).

As clearly observed in this figure the type of cation

and not only its valence are of importance. Sodium



Dilute solution viscosity of red

5 0 . . . . t . . . . i . . . .

0

v

40

i1

~ 0 M

N a I

i

• _e . . . . . • . . . .

~ 6 ~ 2 5 e 4 M

~ ~ 1 - . 2 5 e M

•

3 0

~ - ~ ~ -

v . v . . . . v i f

J

0 . 0 0 0 . 0 1 0 . 0 2

P o l y s a c c h a r i d e c o n c e n t r a t i o n g / d l )

0.03

Fig. 1. Specific viscosity of Porphyrid ium sp. polymer solu-

tions at 25°C. Solvents: (e ) bidistilled water, (11)

6.25 x 10- 5 M, ( ) 1.25 x 10 -4 M, (T) 0.01 M, and (4,) 0.1 M

NaC1 aqueous solutions. Each data point represents the

average of at least two independent determinations. The

lines represent the best fit linear regression.

salt is less effective in reducing molecular dimensi ons

than potassium. N o significant difference is observed

between intrinsic viscosity values for Ca ++ and

Mg + +, the two divalent c ations used in this study,

and at identical ionic strength [r/] is considerably

smaller for divalent cations than for mono vale nt ones.

Figur e 4 s hows the effect of pH on the [-~/] of Psp P

solutions prepared in water and in the presence of

microalga exopolysaccharide 1489

0.5 M aqueous NaC1. For all test solutions in water,

there was a decrease in the [r/] values after the first

adjustment to acidic or basic pH. These lower Jr/]

values were very similar for all pH values. It can also

be seen from Fig. 4 that in the prese nce of external salt

the l-r/] of test solutions is unaffected by the pH

adjustments remaining essentially constant in the

2.0-11.0 pH range. This demonstrates once again the

dimensio nal stabilization effect of salt solutions.

DISCUSSION

Ef f e c t o f i o ni c s t re n g th

A decrease in intrinsic viscosity is observed when

the ionic strength increases (see Table 1). For a salt-

free solution, electrostatic interactions due to the

charges on the polymer favour a stretched chain con-

formation as a result of long-range electrostatic ef-

fects. This behavi our results in higher intrinsic viscos-

ity. Addition of a simple electrolyte screens these

intermolecular electrostatic interactions and causes

the polyme r to assu me a more flexible configurat ion

(wormlike chain), resulting in reduced intrinsic viscos-

ity (Robinson e t a l . 1991; Smidsrod, 1970; Smidsrod

and Haug, 1971; Tinland and Rinaudo, 1989; Shatwell

et al. 1990; see Figs 1 and 3). Thus, recorded differ-

ences in viscosity between solutions of two different

batches of Ps pP in water can stem from differences in

the amounts of salts present as impurities in the two

samples (due for example, to ineffective dialysis pro-

cedure), and need not necessarily be related to differ-

ences in molecular characteristics such as molecular

weight and its distribution or branching mode.

E f f e c t o f t h e n a t u r e o f c o u n t e ri o n

The fact that the different cations gave different [r/]

values (Table 1 and Fig. 3) suggests that the effect of

the counterion on Psp P is specific and dependent on

the type of counterion. The KC1 salt (compared to

NaC1) was mo re effective in redu cing [r/] at very low

Table 1. Intrins ic viscosity [-~/] (dl g- 1) of Porphyrid ium sp polysaccharide as a function of temperature and salt concentra-

tion

Salt concentrat ion (M)

Temp.

(°C) Salt type 0 6.25 × 10 -5 1.25× 10 -4 0.01 0.0 25 0.05 0.1 0.5 1.0

25 NaC1 42.7 35.0 33.2 28.7 24.4 24.6 24.8 23.2 22.8

CaC12 28.3 25.4 23.8 23.2 20.3 - -

MgClz'6HzO 27.1 26.6 24.3 20.0 19.7 - -

KC1 32.0 31.0 25.3 24.0 - - - -

45 36.0

55 35.5

65 NaCI 36.5 26.7 - - - - 23.7 22.8 22.3

CaC12 23.7 - - - - 23.4 - - - -

MgCI2 •6H20 24.7 -- -- 21.1 -- --

75 NaC1 34.6 26.7 - - - - 23.4 22.3 22.5

CaC12 22.9 - - - - 24.0 - - - -

24.5 -- -- 20.3 - - --

85 NaC1 32.4 26.6 - - - - 22.0 20.7 21.5

CaCIz 25.8 23.0 - - - -

MgClz • 6HzO 23.5 22.3 - - - -

Note: The [q] values given represent the average of at least two independent determinations.



1490

E . E T E S H O L A

et a l

A

0

0

9

4 5

I = 0

o l

: 3 5 ,

3 0

2 5

2 0

) . 0 0

4 0

3 5

3 0

2 5

2 O

1 . e - 5

i r ' ' ' ' 1 ' ' . . . . 1 ' ' ' ' . . . . I

- . . ° . ° .

_ . . .

- . . &

i i q i i i L i l

1 . e - 4

- - - . . • . . . .

• - - • - . . . • . •

° ° .

i ~ 1 1 I L l i i i J l l l

1 . e - 3 1 . e - 2

, , I , , L , I i ~ , ~ I

0 . 2 5 0 . 5 0 0 . 7 5

I o n i c S t r e n g t h ( N a C I )

1 . 0 0

Fig. 2. Intr insic viscosity of

Porphyridium

sp. polysaccharide as a fu nct ion of the ionic s t rength in aq ueous

NaC I a t 25°C. The inser t i s an enlargem ent of the low ionic s t rength range .

A

i,.,

0

0

._~

.o

C

° ~

C

3 5

3 0

2 5

2 0

i i t I i i ~ i i i I

I ' X ,

° i i i i i i i • • . . . . . . . . .

i i v

i i i

°

. • ° ' •

I I I I

1

i i i i ~ i i i I

.

.

• , D

N a C I . - KCI

. - '

. ' . -

• - • - • . * '

•

• o .

. - . . .

C a C I 2

M g C I 2

[ I I L I I I I I

1 0 1 0 0

[ - 0 . S

Fig. 3. In trins ic viscosity of

Porphyridium

sp. polymer for several m ono valen t and diva lent ca t ions . The

lines represent the best fit l inear regression of the data.

a n d m o d e r a t e l y l o w s a l t c o n c e n t r a t i o n ( < 1 × 1 0 - 2 M ) .

T h e d i v a l e n t c a t i o n C a + + r e d u c e d Jr /] e v e n f u r t h e r i n

c o m p a r i s o n w i t h t h e m o n o v a l e n t c a t i o n N a + . A s t h e

h y d r a t i o n l e ve l o f K + i s h i g h e r t h a n t h a t o f N a + th e

v a l u e o f [ q ] i s l o w e r f o r t h e f o r m e r b e c a u s e t h e h y -

d r a t e d i o n s o f K + a r e m o r e t i g h t ly b o u n d ( P a s i k a ,

1 97 7) . T h e d i v a l e n t c a t i o n s , w h i c h g i v e e v e n l o w e r J r/ ]

v a lu e s, m o s t p r o b a b l y f o r m i o n p a i r s to a m u c h

g r e a te r e x t e n t t h a n d o t h e m o n o v a l e n t c a ti o n s w i t h

t h e c a r b o x y l a n d t h e h a l f e s t er s u l f a te g r o u p s o f P s p P .

S m i d s r o d ( 1 9 7 0 ) s u g g e s t e d t h a t M g + ÷ i o n s , w h Jc ~

g a v e l o w e r I -r /] t h a n N a ÷ i o n s p r o b a b l y f o r m i o n p a i r s

w i t h t h e c a r b o x y l g r o u p s o f a lg i n a t e . A l t e r n a ti v e l y ,

t h e g r e a t e r e f fe c ti v en e s s o f t h e d i v a l e n t c a t i o n s m a y b e

d u e t o m o r e e f fe c ti v e s h i e l d i n g o f t h e P . s p p o l y i o n

c h a r g e s b y b i v a l e n t i o n s ( S c h n e i d e r a n d D o t y , 1 95 4) .

Determinat ion of the f lexibi l i ty parameter B

F o r m a n y y e a r s i t h a s b e e n k n o w n t h a t , fo r f le x i b le

p o l y e l e c t r o l y t e s , i n t r i n s i c v i s c o s i t y v a r ie s w i t h 1 - 0 . 5



5

' ' ' l ' ' ' I

4

. _ _ >

8

= o

u

3

Dilute solut ion viscosi ty of red micro alga exopolysaccharide

, I , , , I ~ , ,

8 1 0 1 2

2 0 , , , I , = , I , , , I ,

0 2 4 6

p H

Fig. 4. Va riatio n of the Psp P intrinsic viscosity as a function

of the pH in water (0 ) and in 0.5 M NaC1 solut ion (11).

1491

( w i t h I t h e i o n i c s t r e n g t h ) , a n d t h a t t h e s l o p e o f t h i s

p l o t , S , is a n i n d i c a t o r o f c h a i n f l e x ib i l i ty ( S h a t w e l l

e t a l .

1990).

S m i d s r o d a n d H a u g (1 97 1) d e v e l o p e d a s i m p l e

s e m i - e m p i r i c a l m e t h o d b a s e d o n t h i s o b s e r v a t i o n .

T h i s i s t h e s o - c a l l e d B v a l u e m e t h o d , in w h i c h t h e

p a r a m e t e r B i s g i v e n b y :

B s / l - r / ] o . 1 ) 1 .3 1 )

w h e r e [ r/ ]o .1 i s t h e i n t r i n s i c v i s c o s i t y i n 0 .1 M N a C 1 ,

a n d t h e p e r s i s t e n c e l e n g t h a i s :

a = 0 .2 6 /B ( 2)

( f o r [ r / l in d l g - 1 a n d a i n n m ) . T h e c o n s t a n t ( 0 .2 6 ) w a s

o r i g i n a l l y f o u n d b y c o n s t r u c t i n g a c a l i b r a t i o n p l o t f o r

a n u m b e r o f s e m i - fl e x i b l e b i o p l o y m e r s . T h e p e r s i s t -

e n c e l e n g t h i s a m e a s u r e o f t h e l e n g t h o v e r w h i c h t h e

c h a i n ' p e r s i s t s' i n t h e d i r e c t i o n o f th e f ir s t b o n d o f t h e

c h a i n . F o r s t if f w o r m l i k e c o i l s t h e K u h n s t e p l e n g t h , l,

i s g i v e n b y ( A n t h o n s e n e t a l . 1993)

l = 2a. (3)

F o l l o w i n g t h is t y p e o f a n a l y s is , t h e i n t r i n s i c v i s c o s it y

d a t a f o r P s p P i n N a C 1 s o l u t i o n s a r e p l o t t e d i n F i g . 5

a s f u n c t io n o f I - ° ' s . F o r c o m p a r i s o n p u r p o s e s d a t a

f o r s e v e r a l o t h e r c o m m o n p o l y s a c c h a r i d e s a r e a l s o

s h o w n i n t h e s a m e f i g u r e . T h e l i n e a r d e p e n d e n c e o f

I-r /] o n i - o . s i n t h e s a l t c o n c e n t r a t i o n r a n g e o f

0 . 0 1 -1 . 0 M N a C 1 i s c l e a r l y o b s e r v e d . B o t h q u a l i t a t -

i v e ly a n d q u a n t i t a t iv e l y t h e b e h a v i o r o f P s p P i s i n

o

>

.2

t - .

t -

1 6 0

1 5 0

1 4 0

5

4

3

2

1

i i n } i i i i I i i n I

r h a m s a n

¢

[ ] ~

xanthan

[ ]

_ - - ~

welan

P s p

~ . . ~ - , a l g i n a t e

h l i l

K - c a r r a g e e n a n

I i ~ i i [ ~ i i I

5 1 0 1 5

..o.s

Fig. 5. Va riatio n of intrinisc viscosity with ionic strength, I ,

for


sp. polysa char ide (O) , rham san (V) , xan-

than (D) , welan ( i) , a lginate (A) , and x-carrageenan (O) .

Data for a lginate , welan and rhamsan were taken f rom

Robinson e t a l . (1991), for x-carageenan from Slootmaekers

e t a l . (1988) and for xan than f rom Shatwell e t a l . (1990):

a c c o r d w i t h t h a t o b s e r v e d f o r o t h e r p o l y s a c c h a r i d e s .

A l i n e a r d e p e n d e n c e o f [r /] o n 1 - 0 . 5 i s a ls o o b s e r v e d

f o r a l l o t h e r s a l t s e x a m i n e d h e r e ( F i g . 3 ) .

A t h i g h i o n i c s t re n g t h , c h a r g e s c r e e n i n g r e d u c e s t h e

i n fl u e nc e o f c o u l o m b i c i n t e ra c t i o n s o n p o l y m e r c o n -

f o r m a t i o n , w h e r e a s a t l o w i o n i c s t r e n g t h t h e s e i n t e r a c -

t i o n s b e c o m e i m p o r t a n t a n d d o a ff ec t c o n f o r m a t i o n .

T h i s f ac t m a y b e d e d u c e d f ro m t h e n o n l i n e a r i t y o b -

s e r v e d in t h e p l o t i n F i g . 6, w h i c h i n c l u d e s d a t a o n t h e

e ff e ct o f N a C 1 a t v e r y l o w i o n i c s t r e n g t h a n d o v e r

a c o n s i d e r a b l y b r o a d e r c o n c e n t r a t i o n r a n g e t h a n t h a t

s h o w n i n F i g . 5 . T h e d e c r e a s e i n [ ~ /] w i t h i n c r e a s i n g

i o n i c s t r e n g t h i m p l i e s t h e e x i s t e n c e o f a t t ra c t i v e i n t e r -

a c t i o n s b e t w e e n c h a i n e l e m e n t s , p o s s i b l y o f e l e c t r o s -

t a t i c o r i g i n . T h e s l o p e c h a n g e o b s e r v e d i n t h e p l o t

a r o u n d I ~ 0.0 1 M m a y h i n t a t a t r a n s i t i o n i n t h e

P . s p p o l y i o n c o n f o r m a t i o n , m o s t l i k e l y r e f le c t in g

a c o n t r a c t i o n a n d c o n f o r m a t i o n a l o r d e r i n g ( w i th i n -

c r e a s i n g i o n i c s tr e n g t h ) o f th e p o l y i o n c h a i n f r o m

a h i g h l y s t r e t c h e d c o n f o r m a t i o n t o a s t if f b u t n e v e r -

t h e l e ss w o r m l i k e c h a i n . S i m i l a r t r a n s i t i o n s h a v e b e e n

r e p o r t e d i n t h e l i t e r a t u r e ( H o l z w a r t h , 1 98 1; S l o o t -

m a e k e r s

e t a l .

1988).

W e w i l l e x a m i n e th e P s p P J r/] d a t a w i t h i n t h e

c o n c e n t r a t i o n r a n g e 0 . 0 1 -1 . 0 M N a C I i n g r e a t e r d e ta i l

( F i g . 5 ). A p p l y i n g e q s (1 ) - (3 ) t o t h e d a t a p r e s e n t e d i n

F ig . 5 , w e obt a in B = 0 .0074 f or the s ti f fnes s pa r am ete r ,



1492

4 5

4

~ ~ 35

0

u

.~_

~ 3 0

° -

2 5

. . . . I I l l l l l l l l l l l l l l l l I

1 = 0

2

~ 1 ~ I ~ 1 ~ l l ~ I

0 2 5 5 0 7 5 1 0 0 1 2 5

I - .0 .5

Fig. 6 . The depende nce of the in t r ins ic v i scos i ty of P o r -

p h y r i d i u m s p . po l ym e r on t he c onc e n t r a t i on o f a dde d s od i um

chloride at 25°C.

a = 3 5 n m f o r t h e p e r s is t e n c e le n g t h a n d 7 0 n m f o r t h e

K u h n s te p l e n g t h fo r P s p P . T h e s e v a l u es a r e r e p o r t e d

i n T a b l e 2 i n c o m p a r i s o n w i t h o t h e r b i o p o l y e l e c -

t r o ly t e s . T h e v a l u e o f 0 . 0 07 4 e s t i m a t e d f o r B , w h i c h i s

a m e a s u r e o f c h a i n f l e x i b i li t y ( h i g h e r B v a l u e c o r r e s -

p o n d s t o a m o r e f l e x i b l e c h a i n ) , is c l o s e t o v a l u e s

E ETESHOLAe t a l .

o b t a i n e d f o r ri g i d h e l ic e s s u c h a s x a n t h a n a n d D N A ,

b u t l es s t h a n t h o s e o b t a i n e d ( S m i d s r ~ d a n d H a u g ,

1 97 1) f o r c a r b o x y l a t e d p o l y s a c c h a r i d e s w i t h c h a r a c -

t e r is t ic a l l y m o r e f l ex i b le b a c k b o n e s , s u c h a s a l g i n a t e

a n d c a r b o x y m e t h y l c e l l u l o s e ( A x e l o s a n d T h i b a u l t ,

1 9 9 1 ; S l o t m a e k e r s e t a l . 1 98 8; s e e T a b l e 2 ) . T h e r e f o r e

t h e m o d e s t c h a n g e i n [ r/ ] a s a f u n c t i o n o f i o n i c

s t r e n g t h ( F i g . 5 ) m a y b e t a k e n t o a t t e s t t o t h e s t i ff n e s s

o f t h e P s p P c h a i n s t r u c t u r e . A p o s s ib l e e x p l a n a t i o n

f o r t h is p r o p e r t y i s t h a t t h e P . s p b i o p o l y m e r h a s

a c o n f i g u r a t i o n w h i c h m a i n t a i n s t h e c h a i n m o l e c u l e i n

a s ti ff f o r m s i m i l a r t o t h e s i t u a t i o n r e p o r t e d f o r D N A

( C o x , 1 96 0). W e n o t e t h a t t h e q u e s t i o n o f w h e t h e r t h e

P s p P m o l e c u l e is a s i n g le o r a m u l t i p l e s t r a n d e d h e l ix

i n a q u e o u s s o l u t io n m a y n o t h a v e b e e n a n s w e r e d b y

t h e e x p e r i m e n t s r e p o r t e d h e r e . H o w e v e r , p r e l i m i n a r y

X - r a y d i f f r a c t i o n s t u d i e s ( E t e s h o l a

e t a l .

s u b m i t t e d )

i n d i c a t e t h e p o s s i b i l i t y o f a t w o - f o l d h e l i c a l s tr u c t u r e .

I n t e r m s o f p r o p e r t y - s t r u c t u r e r e l a t io n s h i p s , t h e lo w

e x p a n s i o n c o e f f i c ie n t o b s e r v e d f o r t h e P s p P c h a i n ( t h e

l i n e a r i t y o b s e r v e d i n F i g . 5 ) c o u l d a l s o b e d u e t o

a c o m p l e x b r a n c h i n g m o d e ( P a i n t e r , 1 9 8 3 ; F l a i b a n i

e t a l .

1989) . Y ua n

e t a l .

( 1 9 7 2 ) h a v e r e p o r t e d t h a t

b r a n c h i n g c a n c u r t a i l t h e a b i l it y o f a p o l y i o n t o

e x p a n d .

T h e p e r s i s t e n c e l e n g t h w a s e s t i m a t e d a s a l r e a d y

i n d i c a t e d a b o v e . I n a d d i t i o n , t h e K u h n s t e p l e n g t h

w a s e s t im a t e d ( d a ta n o t s h o w n ) b y i n t e r p o l a ti o n f r o m

F i g u r e 1 0 i n t h e p a p e r b y S m i d s r o d a n d C h r i s t e n s e n

(1 99 1), w h i c h m e n t i o n s a n e m p i r i c a l c o r r e l a t i o n b e -

t w e e n B a n d t h e K u h n l e n g t h . T h i s r e l a ti o n s h i p a l s o

r e v e a l s t h a t t h e P s p P h a s a c h a i n s t if fn e s s c o m p a r a b l e

t o t h at o f D N A a n d x a n t h a n . T h u s , f r o m t h e v a ri o u s

s t i f f n e s s i n d i c e s r e p o r t e d i n T a b l e 2 , i t m a y b e c o n -

c l u d e d t h a t t h e o v e r a ll m o l e c u l a r c o n f o r m a t i o n a n d

d i m e n s i o n s o f t h e P . s p b i o p o l y m e r c h a i n s a s r e f le c t e d

b y t h e i n t r i n s i c v i s c o s i t y b e a r s o m e s i m i l a r i t i e s t o

Table 2. St iffness indices for several biopolyelectrolytes

Polyelec t rolyte B a (nm) l (nm)

P o r p h y r i d i u m sp. 0.0074 35.1 70.2

R ha m s a n* 0 .003 88 - -

Xa ntha n 0.00525 t 50* 100

Xanthan* 7 * (coil)

40** (helix)

Xan than ps . PXO61~ 42.2 84.4

Xa nth an ps. 556 ~ 39.2 78.4

DN A* 0.0055 45 90

Pect in (C72)~ 0.017 15.3 30.6

Alginates-gu luronate- r ich * 0 .031 7 .8 1

-man nuron ate- r ich* 0 .040 6 .5 - -

x-carrageenan~ 6.8 t*

3.7**

C a r boxym e t hy l c e l lu l o s e* 0.065 4.1 - -

* R ob i ns on e t a l . 1991.

t Tinlan d and Rinaud o, 1989.

S l oo t m a e ke r s e t a l . 1988.

~Shatwell e t a l . 1990.

~Axelos and Thibaul t , 1991.

** By vi scomet ry .

t t By l ight scat tering.



Dilute solut ion vi scos i ty of red microalga exopolysacchar ide

t h o s e o f D N A a n d x a n t h a n . F r o m t h e a b o v e r e su l ts

a n d o b s e r v a t i o n s , i t i s h y p o t h e s i z e d t h a t t h e P . s p

p o l y s a c c h a r i d e c h a i n m o l e c u l e s a d o p t r e l a t iv e l y s t if f

c o n f o r m a t i o n i n d il u t e s o l u t io n s o v e r a r e l a t iv e l y w i d e

s a l t c o n c e n t r a t i o n r a n g e .

E f f e c t o f p H a n d i o n i c s t re n g t h

T h e i n t r in s i c v i s c o s i t y d e c r e a s e a s a r e s u l t o f p H

c h a n g e s ( F i g . 4 ) c a n b e r e g a r d e d a t m o s t a s m o d e r a t e ,

a n d s o m e o f th e d e c r e a s e , e s p e c ia l l y u n d e r a c i d i c

c o n d i t i o n s , i s n e g l ig i b l e . T h e P s p P s e e m s t o b e s o m e -

w h a t m o r e s t a b l e u n d e r a c i d i c t h a n b a s i c c o n d i t i o n s .

T h e d e p e n d e n c e o f t h e i n tr i n s i c v is c o s i t y o f P s p P

s o l u t i o n s o n t h e p H i n t h e p r e s e n c e o f e x t e rn a l s a l t is

c h a r a c t e r i s t i c f o r a p o l y e l e c t r o l y t e s o l u ti o n . T h e l a r g e

c o n f o r m a t i o n a l c h a n g e s d u e t o s t r o n g s c r e e n i n g o f

c o u l o m b i c i n t e r a c t i o n s b y t h e s al t m a s k s t h e s m a l l

c h a n g e s d u e t o t h e p H e f fe c ts d i s c u s s e d a b o v e . S i m i l a r

o b s e r v a t i o n s h a v e b e e n m a d e f o r t h e p o l y e l e c t r o l y t e s

N a - x a n t h a n a n d N a - c a r b o x y m e t h y l - c e l lu l o s e ( R i n a u d o

a nd Mi l a s , 1978) .

CONCLUSION

T h i s p a p e r r e p r e s e n t s t h e f i r s t a t t e m p t t o s t u d y

c h a i n f l e x i b il i ty a n d c o n f o r m a t i o n a l f e a t u r e s f o r P o r -

p h y r i d i u m s p . p o l y s a c c h a r i d e i n d il u t e a q u e o u s a n d

s a l t s o l u t i o n s b y i n t r i n s i c v i s c o m e t r y .

P o l y e l e c t r o l y t i c b e h a v i o u r i s c o n f i r m e d b y t h e d e -

c r e a s e o f I -q ] w i t h t h e a d d i t i o n o f n e u t r a l s a l t. C o m -

b i n e d d a t a f o r v e r y lo w t o m o d e r a t e i o n i c s t r e n g t h

i n d i c a t e a c h a n g e i n s l o p e i n t h e p l o t o f E ~ /] v s 1 - ° 5 ,

w h i c h m a y h i n t a t a t ra n s i t i o n i n P s p P c h a i n c o n -

f i r m a t i o n f r o m a h i g h l y s t r e tc h e d t o a s ti ff , w o r m l i k e

c h a in . H o w e v e r , n o c l e ar o r d e ~ d i s o r d e r c o n f o r m a -

t i o n a l t r a n s i t i o n a s a r e s u lt o f i o n i c s t re n g t h c h a n g e s

w a s d e t e c t e d . T h e s t if fn e s s p a r a m e t e r w a s d e d u c e d

f r o m t h e d e p e n d e n c e o f [ q ] o n i o n i c s t re n g t h ; f r o m t h e

v a l u e o b t a i n e d i t i s c o n c l u d e d t h a t t h e s t if fn e s s o f

P s p P c h a i n s is i n t h e s a m e r a n g e a s x a n t h a n a n d

D N A . D u e t o t h e st if f c o n f i g u r a t i o n a d o p t e d b y t h e

c h a i n m o l e c u l e s i n s o l u t i o n , a r e l a t i v e l y lo w s e n s i ti v i ty

t o i n c r e a s i n g i o n i c s t r e n g t h i s o b s e r v e d . T h e l o w t h e r -

m a l e x p a n s i o n c o e f f ic i e n t d i s p l a y e d m a y b e r e l a t e d t o

t h e d e t a il e d p r i m a r y a n d c h e m i c a l s tr u c t u r e o f t h e

p o l y m e r . T h e p o l y m e r s h o w e d s p e c if i ci ty t o c o u n t e r -

i o n t y p e a n d v a l e n c y .

A c k n o w l e d g e m e n t - - M G a c know l e dge s t he s uppor t o f the

Is rae l Science Foundat ion adminis tered by the I s rae l Acad-

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