Exocellular polysaccharides from cyanobacteria and their ...

Exocellular polysaccharides from cyanobacteriaand their possible applications

Roberto De Philippis, Massimo Vincenzini *Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Universitaé degli Studi di Firenze,

and Centro di Studio dei Microrganismi Autotro¢, CNR, Piazzale delle Cascine 27, I-50144 Florence, Italy

Accepted 6 August 1998

Abstract

Cyanobacteria are photoautotrophic prokaryotes which include a large variety of species of widespread occurrence and withdiverse morphological, physiological and biochemical properties. Many cyanobacteria are known to be able to synthesiseoutermost slimy investments and to release polysaccharidic material into the culture medium during cell growth. These releasedpolysaccharides (RPSs), being easily recoverable from the culture medium, are attracting much interest in view of their possibleuses in several industrial applications. In this paper, an overview of the current knowledge on both RPS-producingcyanobacterial strains (including the possible roles of the exopolysaccharides) and chemical characteristics of thecyanobacterial RPSs is given, with particular emphasis on RPS properties and possible industrial applications. On the whole,cyanobacterial RPSs are characterised by a great variety in both number (from two to 10) and type of constitutivemonosaccharides (various arrangements of acidic and neutral sugars). Most polymers show an anionic nature due to thepresence of uronic acids and/or other charged groups such as pyruvyl or sulfate. Polypeptide moieties as well as acetylsubstituents have also sometimes been found, causing additional structural complexity. All the cyanobacterial RPSs so fartested showed a pseudoplastic behaviour, but with marked differences in both viscosity values and shear thinning. In terms ofRPS production, the responses of cyanobacteria to changes of culture conditions appear strain-dependent. RPS productivitiesshown by some cyanobacteria are well comparable with those reported for other photosynthetic microorganisms proposed forpolysaccharide production, but very low in comparison with those of heterotrophic microorganisms. Nevertheless,cyanobacteria may be regarded as a very abundant source of structurally diverse polysaccharides, some of which may possessunique properties for special applications, not fulfilled by the polymers currently available. However, much work has still to bedone to bridge the wide gap existing between data on the biology of the RPS-producer strains and information concerningtechnological and other useful properties of the cyanobacterial RPS. z 1998 Federation of European MicrobiologicalSocieties. Published by Elsevier Science B.V. All rights reserved.

Keywords: Cyanobacterium; Cyanobacterial outermost cell investment; Exopolysaccharide release; Polysaccharide production; Potential

application of cyanobacterial polysaccharide

0168-6445 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.PII: S 0 1 6 8 - 6 4 4 5 ( 9 8 ) 0 0 0 1 2 - 6

FEMSRE 613 15-10-98 Cyaan Magenta Geel Zwart

* Corresponding author. Tel. : +39 (55) 3288309; Fax: +39 (55) 330431; E-mail: [email protected]

FEMS Microbiology Reviews 22 (1998) 151^175

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1522. Possible roles of exopolysaccharides in cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1563. Cyanobacterial exopolysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1584. Exopolysaccharide release and factors a¡ecting polymer production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1635. Properties and possible applications of cyanobacterial RPSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1656. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

1. Introduction

Cyanobacteria are a major and phylogeneticallycoherent group of Gram-negative prokaryotes pos-sessing the unifying property of performing oxygenicplant-like photosynthesis with autotrophy as theirdominant mode of nutrition [1]. However, in spiteof their typically aerobic photosynthetic nature,some of the cyanobacterial species can grow in thedark on organic substrates [2,3] and others underanaerobic conditions with sul¢de as electron donorfor photosynthesis [4]. Certain strains have the abil-ity to ¢x atmospheric dinitrogen into organic nitro-gen-containing compounds, so displaying the sim-plest nutritional requirements of all microorganisms[5,6]. Cyanobacteria are also characterised by a greatmorphological diversity, unicellular as well as ¢la-mentous species being included with a cell volumeranging over more than ¢ve orders of magnitude[7]. Representatives of the group have been found,frequently in abundance, in most of the natural illu-minated environments examined so far, both aquaticand terrestrial, including several types of extremeenvironments [7]. This widespread distribution re-£ects a large variety of species, covering a broadspectrum of physiological properties and toleranceto environmental stress [8].

Owing to their ecological and biochemical diver-sity, cyanobacteria, as well as several species of mi-croalgae, have been regarded as good candidates forvarious biotechnological applications and their po-tential in the conversion of light energy into renew-able forms of useful chemicals for food, feed, phar-maceutical and other industries has often beenclaimed and assessed. Indeed, since the ¢rst majorvolume edited by Burlew in 1953 [9], extensive stud-

ies on the practical exploitation of these microorgan-isms have been carried out and reviewed [10^14].

A relatively new ¢eld of possible exploitation ofcyanobacteria has arisen in the last decade by thegrowing industrial interest towards polysaccharidesof microbial origin, that often show advantagesover the polysaccharides extracted from plants ormarine macroalgae. As a result, a wide search forbacterial strains able to produce good yields of newpolysaccharides with potentially useful properties hasbeen undertaken, also involving cyanobacteria be-cause of the well-known capability of some strainsto excrete mucilaginous material [15^18]. Indeed,several cyanobacterial strains possess, outside theirouter membrane, additional surface structures,mainly of a polysaccharidic nature, that comprise awide variety of outermost investments di¡ering inthickness, consistency and appearance after staining.These structures, in spite of the rather arbitrary ter-minology sometimes used, can be referred to as threedistinct types, namely sheaths, capsules and slimes.The sheath (Fig. 1) is de¢ned as a thin, electron-dense layer loosely surrounding cells or cell groupsand is usually visible by light microscopy withoutstaining; the capsule (Fig. 2) generally consists of athick and slimy layer intimately associated with thecell surface with sharp outlines and structurally co-herent to exclude particles (e.g. India ink); the slime(Fig. 3) refers to the mucilaginous material dispersedaround the organism but not re£ecting the shape ofthe cells. During cell growth in batch cultures, ali-quots of the polysaccharidic material of both capsu-les and slimes may be released as water-soluble ma-terial into the surrounding medium, causing aprogressive increase of its viscosity. These water-soluble released polysaccharides (RPSs), being easily


R. De Philippis, M. Vincenzini / FEMS Microbiology Reviews 22 (1998) 151^175152D

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Fig. 1. Nomarski di¡erential interference contrast photomicrographs of sheathed cyanobacterial strains. Top: Chroococcus sp. (1000U) ;bottom: Phormidium sp. (1000U).

R. De Philippis, M. Vincenzini / FEMS Microbiology Reviews 22 (1998) 151^175 153D

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Fig. 2. Photomicrographs of negatively stained cyanobacterial strains with capsule. Top: Cyanothece CE 4 (775U ; bright ¢eld); bottom:Nostoc sp. (480U ; Nomarski di¡erential interference contrast).


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Fig. 3. Nomarski di¡erential interference contrast photomicrographs of cyanobacterial strains with slime. Top: Cyanothece PCC9224(775U) ; bottom: Nostoc PCC7906 (194U).


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recoverable from liquid cultures, are currently at-tracting much interest because of their suitabilityfor a variety of industrial purposes and make cyano-bacteria one of the most attractive sources of newpolymers.

The aim of this paper is to give an overview of thecurrent knowledge on exocellular polysaccharides re-leased by cyanobacteria, with particular emphasis onRPS properties and possible industrial applications.

Throughout the text, cyanobacterial strains will bedesignated as they were cited in the original litera-ture; to get a useful correspondence between strainnames as they will appear in the text and currentcyanobacterial taxonomy as well as between strainnumbers in the di¡erent culture collections, the read-er is referred to the strain catalogue of the PasteurCulture Collection [19].

2. Possible roles of exopolysaccharides incyanobacteria

The synthesis of exocellular investments of a poly-saccharidic nature by bacterial cells is generally con-sidered to be directly related to environmental con-straints on the producing microorganism [20].Therefore, the main function attributed to capsulesor other polysaccharidic investments is to serve as aboundary between the bacterial cell and its immedi-ate environment. More speci¢cally, it could ful¢l aprotective role against desiccation, antibacterialagents (e.g. antibiotics, antibodies, bacteriocins,phages, phagocytic cells, surfactants) or predationby protozoans [21^23]. Moreover, exocellular poly-saccharides may furnish microorganisms with the ca-pability to form bio¢lms on solid surfaces [21^23].

It has been hypothesised that the synthesis of exo-cellular polysaccharides in microorganisms, includ-ing cyanobacteria, plays a major role in protectingcells from stress in extreme habitats and from otherharmful conditions. Many studies have focused onthe capability of some polysaccharide-producing cy-anobacteria to overcome stress due to desiccation orto low water activity in desert or saline environ-ments. For a desiccation-tolerant Nostoc communestrain, Hill et al. [24] proposed that the secreted gly-can provides a repository for water, thereby acting asa bu¡er between cells and the atmosphere and repre-

senting the key component of the mechanism usedby this cyanobacterium to tolerate desiccation. In afollowing study [25], performed with the polymerreleased by N. commune strain CHEN, it was foundthat the addition in vitro of the polysaccharide toarti¢cial membrane vesicles prevented membrane fu-sion, which is the main damage process occurringwhen cells desiccate and subsequently rehydrate.Thus, the authors concluded that the polysaccharide,together with the synthesis of a mixture of trehaloseand sucrose, may represent the key mechanism in thestabilisation in vivo of N. commune cells when theyare dried in air. Mazor et al. [26], studying the re-lease of polysaccharides by some cyanobacterialstrains isolated from sand dunes in the Negev Desert(Israel), concluded that these polymers play an im-portant role in maintaining the moisture in desertmicrobial crusts where, for many months, the onlysource of water was, occasionally, the morning dew.A role as bu¡er compound for the accumulation andthe slow release of water has also been suggested forthe polysaccharide released by a Chroococcidiopsisstrain [27].

Recently, in a review on the ecology of the genusNostoc, Dodds et al. [28] pointed out that the densemucilage surrounding the trichomes of many strainscould make them less preferred food in comparisonwith other microalgae that are devoid of capsules.

In benthic cyanobacteria, it has been suggestedthat the attachment of cells to the sediment is modu-lated by cell hydrophobicity [29], which is usuallydetermined by extracellular polymeric substances(EPS). These are polysaccharides bearing non-sugarcomponents. For instance, Phormidium J-1 synthe-sises a sulfated heteropolysaccharide, namedemulcyan, which contains fatty acids and proteinsthat confer variable degrees of hydrophobicity onthe macromolecule [30]. The attachment of Phormi-dium J-1 as well as of several other benthic cyano-bacteria is also enhanced by the co-£occulation ofpolysaccharide-producing cells with sedimentaryclay particles [31,32].

For cyanobacterial exopolysaccharides, other spe-ci¢c roles have been proposed as well. Since the en-ergetic metabolism of cyanobacteria depends on theavailability of light, it has been suggested that therelease of emulcyan by Phormidium J-1 also plays arole in £occulation of suspended clay particles, there-



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by clearing the water column and increasing theamount of light available for the cyanobacteriumgrowing as a mat on the bottom sediments [33]. InNostoc sp., the slimy shrouds surrounding trichomeshave been suggested to facilitate the homogeneousdispersion of trichomes into the liquid medium, soimproving light utilisation and nutrient uptake [15].

Another possible role of cyanobacterial polysac-charides could be to protect nitrogenase from harm-ful e¡ects of oxygen. Indeed, the thick mucilaginousenvelope surrounding the heterocysts of a strain ofNostoc cordubensis appeared to be essential for theprotection of nitrogenase activity from the inactiva-tion due to the atmospheric oxygen [34]. However, it


Table 1Monosaccharide composition of RPSs produced by cyanobacterial strains belonging to subsection I (Chroococcales) [1]

Species Hydrolyticprocedure

Monosaccharides (molar ratios) Ref.

Ara Fuc Gal Glc Man Rha Rib Xyl GalA GlcA UrAa Others

Aphanocapsa halophyticaMN11

A,B 3 26.5 1.5 12.5 7.5 1.0 3 1.5 3 3 3 3 [44]

Anacystis nidulans C 3 3 1.0 4.7 1.4 3 3 3 3 3 3 3 [45]Chroococcusminutus B 41.79

ns 5.1 4.6 9.5 19.1 10.6 10.0 3 10.7 1.0 2.8 b [46]

Cyanothece sp. CA 3 D 9.2 2.0 3 1.4 tr 1.0 3 3 + + 66.8 3 [47]Cyanothece sp. CE 4 D 0.4 0.3 tr 1.3 3 1.0 3 0.5 + 3 80.1 3 [47]Cyanothece sp. CE 9 D 3 1.1 0.3 2.8 0.7 1.0 3 3 + 3 35.7 3 [47]Cyanothece sp. CH 1 D 3 3.1 1.4 3 0.6 1.0 3 0.9 + 3 27.4 3 [47]Cyanothece sp. ET 2 D 5.8 1.5 1.4 1.3 0.8 1.0 3 3 + + 63.1 3 [47]Cyanothece sp. ET 5 D 3 2.1 2.2 3.1 1.8 1.0 3 3.3 + + 29.4 3 [47]Cyanothece sp. IR 20 D 3 1.5 0.1 0.1 2.3 10.0 0.1 3 3 + 9.8 3 [47]Cyanothece sp. PCC 8801 D 3 0.2 0.6 1.0 0.2 1.2 3 0.9 + + 35.0 3 cCyanothece sp. PE 13 D 3 3.8 11.6 22.0 5.9 1.0 0.3 5.9 3 + 20.9 3 [47]Cyanothece sp. PE 14 D 6.1 0.2 3 0.5 0.3 0.1 3 3 + + 21.7 3 [47]Cyanothece sp. TI 4 D 3 2.7 1.2 2.9 0.4 1.0 3 0.7 3 + 58.2 3 [47]Cyanothece sp. TP 5 D 0.4 1.2 3 0.9 3 1.0 3 0.3 + 3 40.4 3 [47]Cyanothece sp. TP 10 D 1.8 0.8 3 0.6 0.1 1.0 3 3 + + 31.3 3 [47]Cyanothece sp. VI 13 D 3 1.5 0.1 2.0 0.5 1.0 3 1.5 + + 32.1 3 [47]Cyanothece sp. VI 22 D 3 1.8 0.2 2.8 1.0 1.0 3 1.8 3 + 40.8 3 [47]Cyanothece sp. 16Som2 E 3 1.6 2.4 6.8 4.8 3 3 2.9 2.0 1.0 3 [48]Cyanothece sp. 16Som2 D 3 1.0 0.1 1.8 0.4 1.0 3 1.2 + + 20.6 3 [47]Gloeothece sp. PCC 6909 F 3 3 4.2 4.9 2.6 1.6 3 1.0 2.4d e [49]Microcystisaeruginosa K 3A

ns + + + + + + 3 + + 3 3 f

Microcystis£os-aquae C3-40

G,H 3 3 1.0 1.0 3.0 3.0 3 2.0 43.0 3 3 [50]

Synechocystissp. PCC 6714g

I 5.5 2.1 6.0 34.8 3.8 2.8 3 2.8 16.7 h [51]

Synechocystissp. PCC 6803g

I 3 6.0 1.0 6.7 3.9 3.6 3 3.5 16.4 i [51]

Abbreviations: Ara = arabinose; Fuc = fucose; Gal = galactose; Glc = glucose; Man = mannose; Rha = rhamnose; Rib = ribose; Xyl = xylose;GalA = galacturonic acid; GlcA = glucuronic acid; UrA = uronic acid (not identi¢ed); + = present (not quanti¢ed); 3= absent; ns = notspeci¢ed; tr = traces.Notes: A = 2 N TFA (tri£uoroacetic acid) at 100³C for 6^12 h; B = 4 N HCl at 100³C for 6^12 h; C = 2 N TFA at 121³C for 2 h; D = 2 NTFA at 120³C for 45 min; E = 2 N TFA at 120³C for 10 and for 60 min; F = 1 N HCl at 100³C for 10 h; G = 0.5, 1, 2 N H2SO4 at 100³C(time not stated); H = 2 N H2SO4 at 121³C (time not stated); I = 2 N HCl at 100³C for 2 h. a: Expressed as percent of total carbohydrates. b:Deoxyhexose, 6-deoxy-2-O-methylhexose, 2-O-methylhexose; 3-O-methylhexose, glucosamine. c : Our unpublished data. d: Expressed asmolar ratio. e : 2-O-Methylhexose. f: Nagakawa et al. (1987) in [52]. g: Composition of RPSs from young cultures (15 days of batchcultivation); in older cultures (2 months of batch cultivation) the quantitative composition changed. h: 3-O-Methylpentose, glucosamine,galactosamine. i : 3-O-Methyldeoxyhexose, 4-O-methylhexose, methylhexose, glucosamine, galactosamine.


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is worth mentioning that the nitrogenase activity of asheathless mutant of the unicellular cyanobacteriumGloeothece proved to be as sensitive to oxygen asthat of the sheathed strain [35]. More recently,Reddy et al. [36], discussing the positive e¡ect onaerobic nitrogen ¢xation by the mucilaginous invest-ments that surround the colonies of CyanotheceBH68 growing on agar plates, suggested thatthe polysaccharide, in addition to acting as a phys-ical barrier to the atmospheric oxygen, couldalso serve as a chelator for iron and calcium, whichare both essential for nitrogen ¢xation. In severalother cases, it has been suggested that cyanobacterialpolysaccharides, which are mostly characterisedby their anionic nature, play an important role inthe sequestering or immobilisation of metal ions,which are respectively essential or harmful to bacte-rial life [37^40]. In particular for cyanobacteria likeMicrocystis £os-aquae C3-40, living in alkalinehabitats, the polysaccharide capsule that surroundsthe cells seems to play a useful function in nutrition,allowing the accumulation of both iron and man-ganese, metals essential for cyanobacterial growthbut relatively insoluble in aerobic alkaline waters[41].

Finally, for some cyanobacteria that live in asso-ciation or symbiosis with higher plants, it has beensuggested that the EPS may act as an adhesive forcyanobacterial cells. For instance, the release of anexopolysaccharide by the cyanobiont Anabaena azol-lae has been considered essential for the attachmentof this cyanobacterium to the surfaces and the cav-ities of the fronds of the host plant Azolla ¢liculoides[42]. More recently, Gantar et al. [43] showed thatthe ¢rm association of the ¢laments of a Nostocstrain to the roots of wheat are due to the bindingof the polysaccharide matrix that surrounds the tri-chomes. In the same study, it was noted that thepolysaccharide surrounding the cells of an Anabaenastrain was not able to bind to the root surface, sothat only a loose association was established betweenthe cyanobacterium and the roots.

The only conclusion that can be drawn from theabove reported studies is that cyanobacterial exopo-lysaccharides may ful¢l a variety of di¡erent roles,depending on the strain and on the physico-chemicalcharacteristics of the natural habitat in which theorganisms thrive.

3. Cyanobacterial exopolysaccharides

Since the beginning of the 1950s, many cyanobac-teria have been reported to be capable of synthesis-ing exocellular polysaccharides and, in some cases, ofreleasing them into the surroundings. To date, about70 strains have been studied with regard to theirproduction of RPSs, most of the studies being de-voted to the determination of the sugar compositionof the polymers. All these studies deal with RPS-producer strains that belong to subsections I, III orIV of the cyanobacterial classi¢cation (Tables 1^3),with the single exception of a recent paper, reportingthe composition of a polysaccharide released by astrain of Mastigocladus laminosus [52]. No data areavailable on the production of RPSs by strains in-cluded in subsection II.

On the whole, only 10 di¡erent monosaccharideshave been found in the cyanobacterial RPSs: thehexoses glucose, galactose and mannose, the pento-ses ribose, xylose and arabinose, the deoxyhexosesfucose and rhamnose and the acidic hexoses glucur-onic and galacturonic acid. In a few cases, the pres-ence of additional types of monosaccharides (i.e.methyl sugars and/or amino sugars) has been re-ported [40,46,49,51,53,55]. However, a precise iden-ti¢cation of these sugars concerns only the RPSsproduced by one Chroococcus [46], one Gloeothece[49] and two Synechococcus [51] strains. The mono-saccharide most frequently found in the RPSs is glu-cose (in more than 90% of the polymers), followedby galactose, mannose and rhamnose (80^85% of thepolymers). In a large number of RPSs, glucose isalso the most abundant monosaccharide, but thereare also polymers where other sugars, like arabinose,galactose or fucose, are present at higher concentra-tions than glucose; ribose has only been found in asmall number of polymers (about 9% of the RPSsanalysed). The presence of acidic sugars in the mac-romolecules accounts for the anionic nature observedfor almost all the polysaccharides studied so far.When identi¢cation of uronic acids was carriedout, only glucuronic and/or galacturonic acid wasfound, the simultaneous presence of both of themhaving been observed in about half of the polymersanalysed.

Information from papers giving only a partial de-scription of the monosaccharide composition of cy-



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Tab

le2

Mon

osac

char

ide

com

posi

tion

ofR

PSs

prod

uced

bycy

anob

acte

rial

stra

ins

belo

ngin

gto

subs

ecti

onII

I(O

scill

ator

iale

s)[1

]

Spec

ies

Hyd

roly

tic

tech

niqu

eM

onos

acch

arid

es(m

olar

rati

os)

Ref

.

Ara

Fuc

Gal

Glc

Man

Rha

Rib

Xyl

Gal

AG

lcA

UrA

Oth

ers

Lyn

gbya

conf

ervo

ides

S9g

Atr

tr17

.840

.23.

5tr

3tr

38.6

a[5

3]M

icro

cole

ussp

.(2

stra

ins)

B3

++

++

+3

33

3[2

6]O

scill

ator

iaam

phib

iaP

CC

7105

Atr

tr16

.033

.021

.74.

53

12.3

6.7

b[5

3]O

scill

ator

iaco

ralli

nae

CJ1

A5.

11.

715

.029

.615

.63.

43

4.0

24.2

a[5

3]O

scill

ator

iasp

.cC

35.

49.

918

.38.

03

d4.

7d5.

51.

01.

0e

[40]

Pho

rmid

ium

ecto

carp

iC

86A

33

3.1

34.7

23.2

tr3

8.0

29.9

a[5

3]P

horm

idiu

mec

toca

rpi

K5

Atr

tr12

.636

.610

.91.

83

7.3

28.7

f[5

3]P

horm

idiu

mec

toca

rpi

ME

3A

trtr

4.1

59.1

8.7

2.7

36.

518

.93

[53]

Pho

rmid

ium

ecto

carp

iN

182

A3

tr4.

152

.115

.23

3tr

28.7

3[5

3]P

horm

idiu

mec

toca

rpi

PC

C73

75A

tr7.

88.

325

.88.

12.

63

3.2

41.5

a[5

3]P

horm

idiu

mfo

veol

arum

gD

,E,F

1.0

2.0

4.0

8.0

2.0

2.3

31.

5+

+3

[54]

Pho

rmid

ium

fove

olar

umC

52A

0.6

0.5

3.4

43.0

15.3

1.5

35.

529

.4f

[53]

Pho

rmid

ium

fove

olar

umM

EU

A1.

77.

028

.237

.712

.02.

83

3.8

0.5

f[5

3]P

horm

idiu

mm

inut

umD

5A

30.

934

.833

.611

.2tr

31.

117

.1f

[53]

Pho

rmid

ium

min

utum

NB

5A

5.0

37.

232

.718

.0tr

39.

524

.4f

[53]

Pho

rmid

ium

min

utum

RT

6A

18.9

6.0

7.4

19.1

12.7

1.1

312

.020

.1b

[53]

Pho

rmid

ium

sp.

CC

AP

1463

/4A

2.1

tr13

.449

.27.

16.

73

7.4

13.0

a[5

3]P

horm

idiu

msp

.C

CA

P14

64/3

Atr

310

.557

.44.

1tr

31.

626

.1b

[53]

Pho

rmid

ium

sp.g

D,E

,F1.

02.

02.

04.

02.

02.

53

1.5

++

9.0h

3[5

4]P

horm

idiu

msp

.J-

1G

33

0.5

32.

01.

03

334

.0h

3[3

0]P

horm

idiu

msp

.P

NG

91A

2.5

2.1

12.9

30.3

22.0

3.5

tr13

.6tr

f[5

3]P

horm

idiu

msp

.90

-14/

1A

tr5.

89.

439

.929

.2tr

39.

43.

0f

[53]

Spi

rulin

apl

aten

sisi

H3

0.7

2.7

2.0

tr0.

33

1.3

++

40.0

hl

[55]

Spi

rulin

apl

aten

sism

I3

3+

33

+3

+3

20.0

hn

[56]

Abb

revi

atio

ns:

Ara

=ar

abin

ose;

Fuc

=fu

cose

;G

al=

gala

ctos

e;G

lc=

gluc

ose;

Man

=m

anno

se;

Rha

=rh

amno

se;

Rib

=ri

bose

;X

yl=

xylo

se;

Gal

A=

gala

ctur

onic

acid

;G

lcA

=gl

ucur

onic

acid

;U

rA=

uron

icac

id(n

otid

enti

¢ed)

;+

=pr

esen

t(n

otqu

anti

¢ed)

;3

=ab

sent

;tr

=tr

aces

.N

otes

:A

=2

NH

Cla

t10

0³C

for

2h

;B

=3

NH

Cla

t10

0³C

for

4h

;C

=2

NT

FA

at12

1³C

for

2h

;D

=90

%ac

etic

acid

;E

=72

%H

2SO

4;

F=

2N

TF

Aat

95³C

for

16h

;G

=2

NT

FA

at12

0³C

for

2h

;H

=4

NT

FA

at10

0³C

for

4h

;I=

4N

H2SO

4at

100³

Cfo

r6

h.a

:U

nkno

wn

suga

r,pr

obab

lym

ethy

lsu

gar.

b:

Hex

osam

ines

.c:

Not

axen

iccu

ltur

e.d

:U

ncer

tain

iden

ti¢c

atio

n;

auth

ors

give

the

two

poss

ibili

ties

,R

ibor

Rha

.e:

Unk

now

nsu

gar

and

unkn

own

amin

osu

gar.

f:H

exos

amin

esan

dun

know

nsu

gar,

prob

ably

met

hyl

suga

r.g

:C

apsu

lar

poly

sacc

hari

de(C

PS)

extr

acte

dw

ith

hot

wat

erat

100³

Cfo

r3

h.h

:E

xpre

ssed

aspe

rcen

tof

tota

lca

rboh

ydra

tes.

i:C

PS

extr

acte

dw

ith

bu¡

erat

100³

Cfo

r20

min

.l:

An

unkn

own

met

hyls

ugar

and

anun

know

nur

onic

acid

.m:

CP

Sex

trac

ted

wit

hho

tw

ater

;th

epo

lym

eris

com

pose

dof

thre

efr

acti

ons

wit

hth

esa

me

com

posi

tion

,wit

hth

eex

cept

ion

ofon

ela

ckin

gur

onic

acid

s.n

:U

nkno

wn

suga

r.


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Tab

le3

Mon

osac

char

ide

com

posi

tion

ofR

PSs

prod

uced

bycy

anob

acte

rial

stra

ins

belo

ngin

gto

subs

ecti

ons

IV(N

osto

cale

s)an

dV

(Sti

gone

mat

ales

)[1

]

Spec

ies

Hyd

roly

tic

Mon

osac

char

ides

(mol

arra

tios

)R

ef.

tech

niqu

eA

raF

ucG

alG

lcM

anR

haR

ibX

ylG

alA

Glc

AU

rAO

ther

s

Subs

ecti

onIV

Ana

baen

acy

lindr

ica

CC

CA

P14

03/2

A1.

03

1.0

5.0

31.

03

4.0

34.

03

[57]

Ana

baen

acy

lindr

ica

B3

tr1.

08.

74.

73

34.

73

3[5

8]A

naba

ena

£os-

aqua

eA

37C

33

388

.03

33.

039

.03

1.0

3[5

9,60

]A

naba

ena

£os-

aqua

eA

37a

D3

33

8.0

33

31.

03

3[6

1]A

naba

ena

£os-

aqua

eA

37b

D3

33

6.0

33

1.0

1.0

10.0

3[6

1]A

naba

ena

sp.

C5

E3

++

++

33

351

.5c

3[4

3]A

naba

ena

sp.

10C

F3

1.0

1.0

2.5

1.5

1.0

33.

13

1.7c

d[6

2]C

yano

spir

aca

psul

ata

AT

CC

4319

3G

1.0

1.0

31.

01.

03

33

2.0

33

[63]

Nos

toc

calc

icol

a79

WA

01H

1.0

2.8

3.8

5.9

1.7

1.0

36.

13.

02.

83

[64]

Nos

toc

com

mun

eU

TE

X58

4eH

1.6

1.7

6.5

2.0

1.3

1.0

32.

84.

06.

73

[64]

Nos

toc

insu

lare

54.7

9ns

22.9

11.1

0.2

53.2

2.9

1.0

30.

23.

625

.33

[46]

Nos

toc

linck

iaf.

mus

coru

mns

+3

++

3+

3+

+3

fN

osto

csp

.P

CC

7423

I3

0.5

4.2

10.0

1.7

0.1

34.

03

+18

.8c

3g

Nos

toc

sp.

PC

C79

36I

30.

611

.010

.08.

03

33

3+

51.1

c3

gN

osto

csp

.W

V2

G3

3+

+3

+3

++

+3

[65]

Nos

toc

sp.

221h

L3

33

1.0

33

31.

03

1.2

3[6

6]N

osto

csp

.2S

9BE

31.

03

2.0

1.0

33

33

1.0

3[4

3]N

osto

csp

.ns

33

++

3+

3+

++

il

Nos

toc

sp.

C+

+3

+3

33

33

+3

[60]

Subs

ecti

onV

Mas

tigo

clad

usla

min

osus

M+

++

++

+3

++

3[5

2]

Abb

revi

atio

ns:

Ara

=ar

abin

ose;

Fuc

=fu

cose

;G

al=

gala

ctos

e;G

lc=

gluc

ose;

Man

=m

anno

se;

Rha

=rh

amno

se;

Rib

=ri

bose

;X

yl=

xylo

se;

Gal

A=

gala

ctur

onic

acid

;G

lcA

=gl

ucur

onic

acid

;U

rA=

uron

icac

id(n

otid

enti

¢ed)

;+

=pr

esen

t(n

otqu

anti

¢ed)

;3

=ab

sent

;ns

=no

tsp

eci¢

ed;

tr=

trac

es.

Not

es:

A=

2.5%

H2SO

4at

97³C

for

24h

;B

=2

NH

2SO

4at

110³

Cfo

r1

h;

C=

1N

H2SO

4at

100³

Cfo

r6

h;

D=

1N

H2SO

4at

100³

Cfo

r2

h;

E=

1N

H2SO

4at

100³

Cfo

r18

h;

F=

0.5

or2

NT

FA

at80

³Cfo

r16

h;

G=

2N

TF

Aat

120³

Cfo

r45

min

and

3h

;H

=1

NH

2SO

4at

98³C

for

12h

;I=

2N

TF

Aat

120³

Cfo

r45

min

;L

=2.

5%H

2SO

4at

100³

Cfo

r24

h;

M=

met

hano

l/1N

HC

lat

80³C

for

24h.

a:

Neu

tral

frac

tion

.b

:A

cidi

cfr

acti

on.

c:E

xpre

ssed

aspe

rcen

tof

tota

lca

rboh

ydra

tes.

d:

Unk

now

nU

rA.

e:C

apsu

lar

poly

sacc

hari

deex

trac

ted

wit

h0.

1M

ED

TA

at22

³Cov

erni

ght.

f:K

okyr

sta

and

Che

koi(

1972

)in

[66]

.g:

Our

unpu

blis

hed

resu

lts.

h:

Ext

ract

edw

ith

hot

wat

er.i

:U

nkno

wn

suga

r.l:

Hou

ghet

al.

(195

2)in

[66]

.


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anobacterial RPSs has not been included in Tables1^3. In this connection, Reddy et al. [36] reportedthe presence of glucuronic acid in addition to sixunidenti¢ed neutral sugars in the polymer producedby Cyanothece sp. ATCC 51142 and Bar-Or andShilo [30] described the bio£occulant released byAnabaena circularis PCC 6720 as a polymer not con-taining uronic acids. Polymers obtained from naturehave also not been included in the tables, owing tothe di¤culty of establishing which is the cyanobac-

terium that produced the polysaccharide. In anycase, the monosaccharide composition of these poly-mers appears to be consistent with those reported inTables 1^3. Indeed, Gloaguen et al. [67] describedthe composition of the capsular polymers extractedby hot water treatment from £oating and benthicmats of a thermal spring, respectively dominatedby Mastigocladus laminosus and Phormidium sp.They found rhamnose, glucose, xylose, mannose,galactose and fucose as the major components, ara-


Table 4Polysaccharide daily production by cyanobacterial strains

Species Culture device and duration (days) Crude RPS production (mg RPS l31 day31) Reference

Subsection IA. halophytica MN11 4-l £ask, 20 days 32.0 [44]A. nidulans 3-l £ask, 21 days 20.0 [45]C. minutus B 41.79 8-l £ask, 39 days 12.7 [46]C. minutus B 41.79 250-l photobioreactor, 50 days 8.6 [46]Cyanothece BH68K 2-l £ask, 16 days 8.0 [36]Cyanothece sp. CA 3 0.5-l £ask, 8 days 19.0a [47]Cyanothece sp. CE 4 0.5-l £ask, 8 days 43.0a [47]Cyanothece sp. CE 9 0.5-l £ask, 8 days 67.0a [47]Cyanothece sp. CH 1 0.5-l £ask, 8 days 60.0a [47]Cyanothece sp. ET 2 0.5-l £ask, 8 days 38.0a [47]Cyanothece sp. ET 5 0.5-l £ask, 8 days 19.0a [47]Cyanothece sp. IR 20 0.5-l £ask, 8 days 80.0a [47]Cyanothece sp. PE 13 0.5-l £ask, 8 days 62.0a [47]Cyanothece sp. PE 14 0.5-l £ask, 8 days 47.0a [47]Cyanothece sp. TI 4 0.5-l £ask, 8 days 33.0a [47]Cyanothece sp. TP 5 0.5-l £ask, 8 days 17.0a [47]Cyanothece sp. TP 10 0.5-l £ask, 8 days 27.0a [47]Cyanothece sp. VI 13 0.5-l £ask, 8 days 21.0a [47]Cyanothece sp. VI 22 0.5-l £ask, 8 days 20.0a [47]Cyanothece sp. 16Som2 0.5-l £ask, 8 days 25.0a [47]Cyanothece sp. 16Som2 0.5-l £ask, 21 days 29.0 [48]Synechocystis sp. PCC 6803 ns, 90 days 3.0 [51]Synechococcus sp. BG0011 4-l £ask, 30 days 33.3 [88]Subsection IVA. cylindrica 10C 1-l fermenter, 21 days 15.0 [62]A. £os-aquae A37 1-l £ask, 7 days 36.0b [61]A. £os-aquae A37 1-l £ask, 12 days 20.0 [87]A. £os-aquae A37 2-l Pyrex column, 12 days 46.4 [60]Anabaena sp. C5 10-l £ask, 30 days 4.7 [43]C. capsulata ATCC 43193 6-l open pond, 28 days 116.0 [39]C. capsulata ATCC 43193 3-l fermenter, 21 days 144.0 [65]N. insulare 54.79 8-l £ask, 52 days 47.0 [46]N. insulare 54.79 12-l photobioreactor, 70 days 18.4 [46]Nostoc sp. 2-l Pyrex column, 12 days 34.6 [60]Nostoc sp. 221 0.25-l £ask, 20 days 45.4 [66]Nostoc sp. 2S9B 10-l £ask, 30 days 1.4 [43]

Abbreviations: ns=not speci¢ed.Notes: a: Expressed as soluble carbohydrates. b: Mixotrophic growth with glucose.


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binose at very low concentrations and variableamounts of uronic acids that were identi¢ed, in alater paper [68], as glucuronic and galacturonicacids.

From the data on the monosaccharide composi-tion of cyanobacterial RPSs, it is possible to drawsome peculiar features of these polymers in compar-ison with those released by other microbial sources.(i) Most cyanobacterial polysaccharides are charac-terised by an anionic nature, many of them contain-ing two di¡erent uronic acids, a feature rarely foundin the polymers released by strains belonging to oth-er microbial groups [69]. (ii) Cyanobacterial RPSsoften show the presence of one or two pentoses,sugars that are usually absent in other polysacchar-ides of prokaryotic origin [69]. (iii) Most RPSs syn-thesised by cyanobacteria (about 80% of the poly-mers) are quite complex, being composed of six ormore monosaccharides. This is a striking di¡erencefrom the polymers synthesised by other bacteria orby macroalgae, in which the number of monomers isusually less than four [70], and it has been suggestedthat it is due to the low position of cyanobacteria inthe evolutionary scale [17]. The large number of dif-ferent monosaccharides present in a polymer and thevariety of linkage types usually produce quite com-plex repeating units and a broad range of possiblestructures and architectures of the macromolecules[71]. The few studies available con¢rm this picturealso for cyanobacterial RPSs: for the polysacchar-ides produced by Spirulina platensis [55] and Masti-gocladus laminosus [52], repeating units of 15 sugarshave been suggested, while for the RPS from Cyano-spira capsulata a decasaccharide [72] or octasacchar-ide repeating unit [73] has been proposed.

Very few studies have been devoted to verifying ifRPS-releasing cyanobacterial strains produce poly-mers with a stable chemical composition, not chang-ing from batch to batch and una¡ected by growthconditions, a very important feature for microbialstrains proposed for industrial applications. Insome cases, it has been reported that the composi-tion changes during cell growth: two Synechocystisstrains modi¢ed the molar ratio among the RPS con-stitutive monosaccharides with culture age [51], oneof them also producing an additional polymer with adi¡erent qualitative and quantitative compositionfrom that produced by the younger culture; Spirulina

platensis PCC8005 [55] showed only a slight modi¢-cation of the RPS quantitative composition, decreas-ing the relative amount of galactose with culture age-ing, but this change was attributed to a di¡erentratio among the individual polymers that were simul-taneously synthesised by the cyanobacterium duringthe growth period. On the other hand, the RPS pro-duced by Cyanospira capsulata showed a stablemonosaccharide composition throughout growthphases and under di¡erent growth conditions. In-deed, it was shown that RPS samples taken fromdi¡erent phases of cell growth possessed the samemonosaccharide composition [63]. Moreover, the uti-lisation of di¡erent light regimens (continuous lightand light-dark cycles) to grow the cyanobacterialstrain did not a¡ect the monosaccharide compositionor the relative proportions among sugar units [74]. Itis noteworthy that the composition of the RPS syn-thesised by C. capsulata is still the same after morethan 10 years of cultivation (our unpublished obser-vations). These ¢ndings also demonstrate that thiscyanobacterium proved to be stable as a RPS pro-ducer, since its capability to synthesise the capsuleand to release the RPS has been maintained throughthe years. This feature is of great technological sig-ni¢cance, considering that many other microorgan-isms, accumulating signi¢cant amounts of exopoly-saccharides in natural habitats, lose this propertywhen cultivated under laboratory conditions, wherethe environmental pressure has been removed [21],and thus need to be tested with regard to their stabil-ity of RPS production [75].

The composition of the polymer released by Ana-baena cylindrica 10C was slightly modi¢ed by culti-vating the strain with di¡erent nitrogen sources orunder phosphorus starvation [62], whereas the RPSsynthesised by C. capsulata showed the same compo-sition also when various nutritional de¢ciencies (Mg,Ca, P) or salinities were tested (our unpublisheddata). In some cases, di¡erent chemical compositionshave been reported for RPSs produced by the samestrain. One example of this, from both a qualitativeand a quantitative point of view, is the polymer pro-duced by Cyanothece 16Som2 (Table 1): the polysac-charide was ¢rst described as composed of ¢ve neu-tral sugars and galacturonic and glucuronic acids[48], whereas a di¡erent batch, carried out 5 yearslater, showed the additional presence of rhamnose



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and rather di¡erent molar ratios among the mono-saccharides [47]. Even if the hydrolytic conditionswere not the same, the observed discrepancy couldbe most likely ascribed to changes in the syntheticcapability of the strain during the long period ofcultivation and conservation under laboratory con-ditions.

As a concluding remark to this section, it must bestressed that qualitative and quantitative analyses ofthe monosaccharides can be strongly a¡ected by theconditions utilised for the hydrolysis of the polymers.In particular, the determination of uronic acidsseems to be very critical: the presence of a carboxylgroup stabilises the glycosidic linkage betweenmonosaccharides and thus an incomplete acid hy-drolysis may occur [76,77]; otherwise, the uronicacids can undergo some degradation [54,78] or lacto-nisation [76], once a complete hydrolysis has beenachieved. Consequently, the presence of uronic acidsshould be excluded only after a careful check madeby means of speci¢c colorimetric analysis (e.g. themethods described by Galambos [79], Blumenkranzand Asboe-Hansen [80] or Taylor and Buchanan-Smith [81]). In this connection, the absence of uronicacids in the RPSs produced by Aphanocapsa halophy-tica MN11 [44] and by Anabaena cylindrica [58]should be regarded with a certain caution. Further-more, since other sugars, particularly pentoses, canbe degraded in the presence of strong acids [57], thehydrolytic conditions should be carefully selected foreach polymer in order to give sound results.

4. Exopolysaccharide release and factors a¡ectingpolymer production

Usually, polysaccharide-releasing cyanobacteriaare characterised by the presence of capsules orslimes enclosing cells or cell groups. Consequently,cyanobacterial RPSs are generally believed to be or-iginated from the mere solubilisation of these exter-nal layers of the mucilaginous outermost invest-ments. The few available data seem to support thishypothesis: indeed, at least for A. £os-aquae [59,66],some Phormidium strains [53] and C. capsulata [63],the capsular and the released polysaccharidesshowed the same monosaccharide composition; inthe case of C. capsulata, the two polymers also

showed quite similar molecular masses (our unpub-lished results). With regard to the morphologicalchanges that may occur during polysaccharide re-lease, it was observed that, in C. capsulata [63] andCyanothece 16Som2 [48], the thickness of the capsulesurrounding the cells remained almost constantthroughout growth phases and under all the cultureconditions tested, in spite of the large amounts ofpolysaccharide released into the culture medium.Thus, the processes of synthesis and release of thepolymer very likely occurred at the same rate. On thecontrary, the RPS-producing red microalgae Por-phyridium and Rhodella showed a quite di¡erent be-haviour, the capsule thickness increasing with cultureageing [82,83]. Gantar et al. [43], on the other hand,reported that the mucilaginous sheath of Nostoc2S9B was mainly synthesised in the aseriate stageof the developmental cycle of the cyanobacteriumand then released, as empty shells, when hormogoniawere liberated. In Anabaena C5, the same authorsfound a quite di¡erent behaviour, the sheath beingcontinuously synthesised and released so that cell¢laments appeared devoid of any polysaccharide en-velope [43].

In any case, apart from the origin of the RPS, itseems that there is not a common behaviour of poly-mer release among the RPS-producing cyanobacteri-al strains described so far. In A. halophytica [44], S.platensis [55] and C. capsulata [63], polysaccharideproduction parallels biomass production so that thepolymer may be considered a primary metabolite, asde¢ned by Filali Mouhim et al. [55] in the case of S.platensis. Among these strains, C. capsulata, a het-erocystous, akinete-forming cyanobacterium isolatedfrom the alkaline soda lake Magadi (Kenya) [84], isthe most extensively studied with regard to the pro-cess of polymer release. In batch cultures, this cyano-bacterium showed an almost constant speci¢c rate ofpolysaccharide release (expressed as mg of RPS permg of protein per day) and maintained the samecapsule thickness throughout growth phases, matureakinetes being the only cells completely devoid ofcapsule [63,85]. This kinetics of polysaccharide re-lease has been considered by Vincenzini et al. [85]to be the result of a complex dynamic equilibriumamong di¡erent processes: trichome elongation andakinete germination, in which the polysaccharidesynthesis is mainly directed towards the formation



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of the capsule, and, as opposed processes, trichomefragmentation and akinete di¡erentiation, whichcause the release of the polymer into the culturemedium. Other cyanobacteria produce the polysac-charide as a typical secondary metabolite: Cyano-thece BH68K showed a signi¢cant release of poly-saccharide only starting from the late exponentialgrowth phase [36], like N. calcicola [64] and Phormi-dium J-1 [33,86]. On the other hand, in A. £os-aquaeA37 [60,87] and A. cylindrica 10C [62], RPS produc-tion occurred during all growth phases, but the high-est production rates were observed starting from thelate exponential or from the stationary phase. Con-versely, in the case of a Nostoc strain, the highestrates of polysaccharide synthesis and release wereachieved by young cultures [66].

All these ¢ndings point out that each strain shouldbe carefully tested in order to envisage the right cul-ture strategies aimed at optimising RPS production.In this connection, many data are now available onRPS daily production by cyanobacterial strains;these are reported in Table 4. Although most strainshave only been tested in small culture devices and instudies not oriented to maximising RPS production,it appears that almost all strains attained very lowRPS productivities, apart from C. capsulata andsome Cyanothece strains. In particular, the mostpromising productivities have been shown by C. cap-sulata, with daily RPS productions of 116 mg l31 bycultures run in open ponds and 144 mg l31 by cul-tures grown in fermenters. These data are quite com-parable with those reported for other photosyntheticmicroorganisms proposed for polysaccharide pro-duction. Indeed, strains of the red microalga Por-phiridium have been reported to release a sulfatedpolysaccharide with a daily production of 55^75mg l31 in 2.5-m2 open ponds [89] or 133 mg l31 in1-m2 open ponds [90], while a Botryococcus brauniistrain produced a polysaccharide with interestingrheological properties at a daily productivity of130^145 mg l31 in 1-l columns [91]. On the otherhand, it has to be stressed that the photosyntheticorganisms tested so far are characterised by polysac-charide productivities very low in comparison withthe heterotrophic microorganisms industrially uti-lised for biopolymer production: for instance, in atypical batch for xanthan gum production, Xantho-monas campestris can reach productivities of 7^10 g

(PS) l31 day31 [75]. This serious drawback of photo-synthetic microorganisms compared to the RPS-pro-ducing heterotrophic bacteria could be partially dis-regarded in view of some advantages, of bothenvironmental and economic impact, that cyanobac-teria could achieve: (i) they are capable of utilisingrenewable and cheap substrates, being photoauto-trophs and, many of them, nitrogen ¢xers; (ii)many strains can grow in brackish or in wastewaters; (iii) it is possible to utilise as carbon sourcethe CO2 emitted by industrial plants; (iv) the econ-omy of the process could be enhanced by recoveringmore than one useful compound, with a multiprod-uct strategy as already proposed for microalgae [92].

The possibility of stimulating polysaccharide re-lease by means of an optimisation of the cultureconditions has been poorly considered. Most of theavailable studies were mainly devoted to assaying thee¡ects of nitrogen de¢ciency, which has been shownto stimulate polysaccharide synthesis in some RPS-producing microalgae [93^95]. However, the re-sponse of cyanobacteria to nitrogen starvation isnot univocal. Some strains, like A. nidulans [45]and several Cyanothece [47,48], released largeramounts of polysaccharides under conditions of ni-trogen limitation and others, like A. cylindrica [62]and A. £os-aquae [87], varied the amount of polymerproduced depending on the nitrogen source used. Onthe other hand, in strains like Synechocystis [51],some Cyanothece [47], C. capsulata [96] and Phormi-dium [33], nitrogen starvation did not a¡ect the exo-cellular production of polysaccharides. In the nitro-gen-¢xing cyanobacterium C. capsulata, when themetabolic carbon £ux was a¡ected by cultivatingthe organism under conditions of nitrogen de¢ciencydue to the presence of an argon atmosphere or to theuse of inhibitors of nitrogen assimilation (like O-di-azoacetyl-L-serine, D,L-7-azatryptophan or methio-nine-D,L-sulfoximine) [96], an accumulation of intra-cellular carbon reserves instead of an increase in theproduction of RPS was observed. In contrast, whenthe metabolic carbon £ux was directly stimulated bythe addition of glyoxylate, a stimulator of both CO2

and nitrogen ¢xation rates in Anabaena cylindrica[97], the cyanobacterium released larger amounts ofpolysaccharide, roughly corresponding, in terms ofcarbon balance, to the amount of the organic com-pound added. It is worth stressing that the possibility



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of increasing the amount of polysaccharide releasedwithout a¡ecting growth, as it occurs in C. capsulatacultures carried out with the addition of glyoxylate,is very promising owing to the actual enhancementof the ¢nal yield of the polymer achieved by thisway.

In some cases, the e¡ects of other nutrient de¢-ciencies or variations of growth parameters (e.g. lightintensity, salinity, pH, etc.) have also been tested. Intwo Synechocystis strains, the release of polysacchar-ide was not a¡ected by the addition of 0.5 M NaCland glyoxylate nor by decreasing light intensity [51].On the other hand, Cyanothece 16Som2 increasedthe exopolysaccharide release under conditions ofphosphorus starvation, being una¡ected by magnesi-um, calcium or potassium de¢ciencies as well as bysalinities up to 2 M [48]. The release of the polysac-charidic bio£occulant by Phormidium J-1 was stimu-lated by calcium starvation but not by phosphate orsulfate de¢ciencies [33]. In C. capsulata, magnesiumstarvation, but not calcium or phosphate de¢ciencies,stimulated polysaccharide release, the amount ofRPS being about 17% higher than in cultures grownon the standard medium [39]. In A. cylindrica 10Cphosphorus shortage as well as the addition of ace-tate, propionate, valerate, citrate or glucose to theculture medium reduced RPS production [62].

In spite of the phototrophic nature of cyanobac-teria, almost no studies are available on the in£uenceof light on RPS production, apart from two studiescarried out with C. capsulata [65] and SynechococcusBG0011 [88] that were grown under light-darkcycles. These two strains produced smaller amountsof exocellular polysaccharide in comparison with thecontrol cultures carried out under continuous light,this reduction roughly corresponding to the shorter

light period experienced by the cells. These ¢ndingspoint out that the processes of polymer synthesis andrelease are tightly light-dependent.

The above data demonstrate that, in terms of RPSproduction, the responses of cyanobacteria to alter-ations of culture conditions are strain-dependent.This behaviour possibly re£ects di¡erent physiologi-cal roles played by the exocellular polysaccharides indi¡erent strains. For instance, when signi¢cant en-hancements of RPS release are achieved by theshortage of some metallic ion, a role of the polymeras chelating agent for cations essential for cell life, assuggested for some algal and cyanobacterial anionicpolysaccharides [23,37,98], may be envisaged. On theother hand, when polymer release is stimulated bynitrogen starvation, the polymer could act as a prod-uct of over£ow metabolism, so that it is excreted toallow cells to get rid of the carbon excess [99].

5. Properties and possible applications ofcyanobacterial RPSs

Most cyanobacterial polysaccharides described inthe previous sections have not yet been studied withregard to their physical, structural or rheologicalproperties, so that the assessment of their potentialuse for various industrial purposes or speci¢c appli-cations is a quite arduous task. In such a situation,in order to have useful guidelines to envisage thepossible applications of these polymers, one shouldrefer to some recent overviews on properties anduses of polysaccharides from other microbial sources[69,70,100^103]. In any case, the main features ofpossible industrial interest of cyanobacterial RPSswill be brie£y considered.


Table 5Molecular masses of the exopolysaccharides released by cyanobacteria

Species Apparent molecular mass (kDa) Technique for MW determination Reference

C. minutus B 41.79 1200^1600 GPC [46]Oscillatoria sp. 200 GPC [40]Phormidium J-1 1200 GPC [30]S. platensis 81^98 GPC [56]A. circularis PCC 6720 s 1200 GPC [30]C. capsulata ATCC 43193 1400^1900 LALLS; GPC [74,78]N. insulare 54.79 540^1300 GPC [46]

Abbreviations: GPC = gel permeation chromatography; LALLS = low angle laser light scattering.


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One of the most important prerequisites of a poly-saccharide, which determines many of the properties

generally considered useful for its industrial utilisa-tion (i.e. high viscosity of its aqueous solutions, ca-


Table 6Substituent groups and protein content of the exopolysaccharides released by cyanobacteria

Species Substituent groups (% on RPS dw) Protein content(% on RPS dw)

Reference

Acetate Pyruvate Sulfate

Subsection IA. halophytica MN11 nd nd 11.9 10.3 [44]C. minutus B 41.79 nd nd nd 3.2 [46]Cyanothece sp. CA 3 0.62 2.72 tr nd [47]Cyanothece sp. CE 4 0.66 0.36 tr nd [47]Cyanothece sp. CE 9 3 1.17 tr nd [47]Cyanothece sp. CH 1 0.52 1.04 tr nd [47]Cyanothece sp. ET 2 4.2 2.28 3 nd [47]Cyanothece sp. ET 5 2.5 0.39 3 nd [47]Cyanothece sp. IR 20 0.75 2.11 + nd [47]Cyanothece sp. PCC 8801 1.40 0.50 nd nd aCyanothece sp. PE 13 3 2.08 tr nd [47]Cyanothece sp. PE 14 0.32 0.17 tr nd [47]Cyanothece sp. TI 4 0.98 1.37 tr nd [47]Cyanothece sp. TP 5 3 1.10 + nd [47]Cyanothece sp. TP 10 nd 3.86 + nd [47]Cyanothece sp. VI 13 3 0.34 + nd [47]Cyanothece sp. VI 22 0.55 0.23 + nd [47]Cyanothece sp. 16Som2 3 3 + 1.4 [47,48]Gloeothece sp. PCC 6909 nd nd 13.8 6.2 [49]M. aeruginosa K3A nd nd nd + bM. £os-aquae C3-40 nd nd nd 3 [50]Synechocystis sp. PCC 6714c nd 3 1.2 20.0 [51,106]Synechocystis sp. PCC 6803c nd 3 1.0 40.0 [51,106]

Subsection IIIMicrocoleus sp. (2 strains) nd nd nd 6.0 [26]Phormidium sp. nd nd 3 13.0 [54]Phormidium sp. J-1 nd nd 1.6 4.4 [30]S. platensisd nd nd 5.0 nd [55]

Subsection IVA. circularis PCC 6720 nd nd 3 3 [30]A. cylindrica nd nd nd 5.0e [58]A. cylindrica 10C nd nd + nd [62]Anabaena sp. C5 3 3 3 0.6 [43]C. capsulata ATCC 43193 3 1.5 3 2.0 [63,72]N. calcicola 79WA01 nd nd nd 7.9 [64]N. commune UTEX584 nd nd nd 16.7 [64]N. insulare 54.79 nd nd nd 3.5 [46]Nostoc sp. PCC 7423 12.9 3.2 nd nd aNostoc sp. PCC 7936 3.1 5.9 nd nd aNostoc sp. 2S9B 3 3 3 2.8 [43]

Abbreviations: nd = not determined; dw = dry weight; + = present (not quanti¢ed); 3= absent; tr = traces.Notes: a: Our unpublished results. b: Nakagawa et al. (1987) in [50]. c: Composition of RPSs from young cultures (15 days of batchcultivation); in older cultures (2 months of batch cultivation) the quantitative composition changed. d: CPS extracted with bu¡er at 100³C for20 min. e: Expressed as amino components.


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pability of forming gels with good tensile strength,stabilising emulsions, etc.), is that it must possess,together with an adequate composition and structure[104], a high molecular mass (MW) [105]. In thisrespect, only seven cyanobacterial polysaccharideshave been tested, ¢ve of them showing MWs higherthan 1000 kDa (Table 5). The highest MW values, inthe range of 1400^2800 kDa, depending on the ana-lytical techniques utilised for MW determination,have been reported for the RPS produced by C. cap-sulata [74,78].

Another important feature that contributes to thechemical and physico-chemical properties of the poly-saccharides is the presence of a polypeptide moietyor other non-saccharidic components such as organic(e.g. acetyl, pyruvyl, succinyl groups) or inorganic(e.g. sulfate or phosphate groups) substituents [69].However, systematic investigations of the presence ofthese non-saccharidic components in cyanobacterialRPSs have been undertaken only recently, so thatdata are available only for almost half of the poly-saccharides described (Table 6). A proteinaceousmoiety has been found in almost all the polymersinvestigated, but only for C. capsulata [72] and N.calcicola [64] its amino acid composition is available;in the other cases, only the protein content has beendetermined, by colorimetric or Kjeldahl methods.For C. capsulata, an amino acid composition richin glycine, alanine, valine, leucine, isoleucine andphenylalanine has been reported. Large amounts ofthe same amino acids have also been found in thepolypeptide moiety of the RPS produced by N. cal-cicola, where they are reported to contribute signi¢-cantly to the hydrophobicity of the macromolecule[64].

It must be stressed that, in those cyanobacterialRPSs reported to have a very high protein content,contamination due to cell lysis could have been oc-curred. However, most authors agree in consideringamino acidic nitrogen not reducible below a certainvalue (usually in the range of 1^3% of RPS dryweight), even after repeated puri¢cations, thus point-ing out that it is a true component of the polymers.On the other hand, since the removal of the protein-aceous moiety drastically reduced the viscosity ofaqueous solutions of the polysaccharides producedby some microalgae, a structural role of this constit-uent in or among the macromolecules has been sug-

gested [107,108]. In the case of the polysaccharidereleased by Nostoc 2S9B, it was observed that theprotein removal from the polymer signi¢cantly re-duced the adhesive capacity of the polysaccharideto the roots of Triticum vulgare L., so underliningthe importance of the polypeptide moiety in theprocess of root colonisation by the nitrogen-¢xingcyanobacterium [43].

For many years in the past, only eukaryotic cellswere believed to be capable of producing sulfatedpolysaccharides, the sulfation of the polymer occur-ring in the Golgi apparatus [109^112]. In the last 10years, this opinion has been falsi¢ed because of theincreasing number of cyanobacterial RPSs producedby strains isolated from both fresh water and salineor hypersaline environments that showed the pres-ence of sulfate groups (Table 6). The current opinionis that sulfate groups may be present also in someprokaryotic polysaccharides, but only in those pro-duced by strains belonging to cyanobacteria or ar-chea [69]. The occurrence of phosphate residues, fre-quently found in many polysaccharides synthesisedby other bacterial groups, has never been investi-gated in cyanobacterial RPSs, although phosphate-containing polysaccharides are attracting much inter-est because of their possible immunological signi¢-cance [70]. In this connection, a certain biologicalactivity could be also displayed by cyanobacterialRPSs bearing sulfate groups, since it is known thatsulfated polysaccharides possess inhibitory propertiesagainst various types of viruses [113,114] and tu-mours [115,116]. With regard to the organic substitu-ents, pyruvyl and acetyl groups, as shown in Table 6,have been frequently found, whereas succinyl resi-dues have been reported only for the capsular poly-saccharides obtained from cyanobacterial mats of athermal spring [67]. The frequent presence of theseinorganic and organic constituents introduces a fur-ther cause of structural complexity in cyanobacterialRPSs, but, on the other hand, makes these polymersexcellent markers of biodiversity. However, a recentinvestigation [117], carried out on the RPSs pro-duced by several Cyanothece strains isolated fromsaline environments, demonstrated that the chemicalcomposition of the RPSs cannot be utilised for clus-tering the strains into more homogeneous sub-groups and thus it is not a reliable chemotaxonomicmarker.



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As already noted, any correlation between chem-ical composition and physical properties of the poly-saccharides is generally hazardous in the absence ofinformation about secondary and tertiary structuresof the macromolecules, but, in spite of this, somebehaviours can be expected. A signi¢cant presenceof ester-linked acetyl groups (see Table 6) as well

as of deoxy sugars like fucose and rhamnose (seeTables 1^3) has been suggested to give an appreci-able contribution to the emulsifying properties of thepolysaccharides, owing to a certain lipophilic char-acter introduced by these small molecules in the mac-romolecules that otherwise would be highly hydro-philic [105,118]. However, some polysaccharides


Table 8E¡ect of changes in pH, temperature and NaCl concentration on viscosity of 0.1% (w/v) aqueous solutions of RPS from C. capsulata andof commercial xanthan gum (Kelco Keltrol, commercial grade) at two shear rates

Viscosity (log mPa s)

Xanthan gum C. capsulata RPS

shear rate = 0.1 s31 shear rate = 10.2 s31 shear rate = 0.1 s31 shear rate = 10.2 s31

pH2.2 nd 1.40 nd 1.503.7 2.30 1.56 2.78 1.785.6 2.70 1.80 3.05 1.957.5 2.95 1.90 3.32 2.088.4 2.30 1.65 nd 2.0510.0 nd nd 2.95 1.9011.8 nd 1.48 nd 1.60Temperature25³C 2.95 1.92 3.40 2.0840³C 2.78 1.83 3.23 2.0560³C 2.10 1.61 3.08 1.9925³Ca 2.48 1.80 3.30 2.08[NaCl]0.1 M 1.82b 1.40 1.60b 1.400.5 M 1.80b 1.42 1.65b 1.401.0 M 1.83b 1.42 1.70b 1.48

Abbreviations: nd = not determined.Notes: a: Determined at the end of a thermal cycle (from 25³C to 60³C and back to 25³C). b: Measured at shear rate = 1.0 s31.

Table 7Anionic charge density (determined according to [123] as modi¢ed by [30] ; our unpublished results), uronic acid, pyruvate and sulfatecontent of the exopolysaccharides released by some cyanobacterial strains

Strain Uronic acid(% on crude RPS dry weight)

Pyruvate(% on crude RPS dry weight)

Sulphate Anion densitya

Cyanothece CA 3 18.8 2.72 tr 1.1Cyanothece CE 4 24.8 0.36 tr 1.4Cyanothece ET 2 7.3 2.28 3 0.1Cyanothece ET 5 12.6 0.39 3 6 0.1Cyanothece IR 20 4.6 2.11 + 1.3Cyanothece PE 14 9.4 0.17 tr 1.6Cyanothece TI 4 24.7 1.37 tr 1.5Cyanothece VI 22 17.5 0.23 + 2.2Cyanothece 16Som2 11.2 3 + 1.8C. capsulata ATCC 43193 13.6 1.5 3 1.2

Abbreviations: tr = traces; + = present; 3= absent.Notes: a: mg of Alcian blue linked per mg of crude polysaccharide.


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containing high levels of rhamnose did not show anyparticular emulsifying activity [119]. It has also beenreported that the presence of acetyl groups on themacromolecules may hinder cation binding, as oc-curs in bacterial alginates [120], or gel formation,as was observed in gellan [121], but may facilitategel swelling, as in the case of alginate gel beads[102]. Moreover, acetyl groups may contribute tothe stabilisation of the ordered form of the polymers,as has been reported for the xanthan structure [69].

Polysaccharides characterised by high concentra-tions of charged components (like uronic acids, sul-fate or phosphate groups, pyruvate ketals) usuallyform stable gels in the presence of metallic ions[69] and are the most promising for the removal oftoxic metals from polluted waters [40,68,122]. How-ever, the mere determination of the quantity ofcharged groups is not enough for anticipating theactual metal binding capability of a polymer, be-cause, depending on the conformational structureof the macromolecules, some of the charged groupscould be hardly accessible for the ions. Indeed, theion uptake depends on both charge density andcharge distribution on the polymers [69]. Useful in-formation about the actual metal binding capabilityof a polysaccharide can be obtained by the determi-nation of the anion density through simple methods,

like that based on the ability of the negativelycharged polymers to link the Alcian blue pigment[123]. Using this method as modi¢ed by Bar-Orand Shilo [30], for instance, no evident correlationswere found between the quantity of charged groupsin some cyanobacterial RPSs and their anionic den-sity (Table 7). In any case, the number of chargedgroups in the macromolecules, and particularly ofsulfate substituents [18], has been reported to playa key role in £occulating suspended clay particles[30]. Charged groups also signi¢cantly contribute tothe solubility in water of the polysaccharides, whichotherwise would be insoluble [69], and improve theability of the macromolecules to bind water mole-cules. Consequently, the viscosity of their aqueoussolutions, tightly related to the e¡ective volume oc-cupied by the macromolecules, increases [124]. Onthe other hand, the protective role against desicca-tion, suggested for microbial exopolysaccharides[37,85,98], is particularly e¡ective as the presence ofacidic components in the macromolecules increasestheir water-retaining capability [125].

If the above reported considerations are useful togive an idea of the expected behaviour of a polysac-charide, on the other hand they underline the lack ofexperimental evidence on the properties of cyanobac-terial RPSs. Indeed, a very small number of thesepolysaccharides has been tested with regard to theirphysico-chemical and rheological properties.

Two cyanobacterial strains, studied with regard tothe viscosity of their cultures, underlined the impor-tance of cell morphology in determining culture vis-cosity. In the case of the coccoid strain Synechococ-cus BG0011, the increase of culture viscosity,observed during its photoautotrophic growth, wasmainly due to the released polysaccharide, the di¡er-ence in viscosity between whole culture and cell-freemedium being less than 5% [88]. In contrast, in C.capsulata the capsulated trichomes contributed sig-ni¢cantly to the overall viscosity of the culture, par-ticularly in the ¢rst period of growth [39]. In a fur-ther study [126], it was shown that whole cultures ofC. capsulata are characterised by a pseudoplastic be-haviour, the viscosity decreasing with the increase inshear rate, the more marked the older the culture.

A larger, but still limited, number of studies isavailable on the rheological properties of aqueoussolutions of cyanobacterial RPSs, most of them


Fig. 4. Viscosity dependence of aqueous solutions of polysacchar-ides (0.1% w/v) on shear rate: xanthan gum (F), RPS producedby Cyanospira capsulata (E) and RPSs produced by Cyanothecestrains CA 3 (R) ; ET 2 (b) ; IR 20 (a) and PE 14 (P).


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only considering the viscosity dependence on shearrate. All the RPSs tested showed the pseudoplasticbehaviour typical of this kind of biopolymer, but, iftheir viscosity dependence on shear rate is comparedwith that of solutions of xanthan gum at the sameconcentrations, many di¡erences become evident.The RPS produced by Synechococcus BG0011showed a more marked shear thinning behaviourthan solutions of xanthan gum but, at the same con-centrations, lower values of viscosity [88]. The poly-mer produced by S. platensis PCC8005 showed apseudoplastic behaviour at very low concentrationsand was also characterised by a signi¢cant decreaseof viscosity with increasing ionic strength of the sol-ution [55]. On the other hand, a rather wide range ofrheological behaviours has been observed in aqueoussolutions of the RPSs produced by several Cyano-thece strains or by C. capsulata (Fig. 4). Indeed,the £ow properties highly di¡erentiated these poly-mers: some of them showed a behaviour quite com-parable to or even better than xanthan gum, whilesome of the others showed a more marked shearthinning behaviour, a property that could be of par-ticular interest for some applications, e.g. for theformulation of oil drilling muds [102]. Finally, theremaining RPSs showed very low viscosities.

Deeper investigations on the £ow properties of theaqueous solutions of RPS are available only for thepolymer produced by C. capsulata. In this case, itwas demonstrated that the pseudoplastic behaviouris the more marked the higher the polymer concen-tration and that the performances, at least with re-gard to the viscosity dependence on shear rate, are

comparable with systems possessing a high suspend-ing capability [127]. Moreover, it was suggested thatthe polymer has a random-coil behaviour, but withrelevant interactions among the macromolecules[127] ; the chain £exibility was considered compara-ble to that of alginate [78]. Other comparative databetween xanthan gum and the RPS from C. capsu-lata, concerning the dependence of viscosity on pH,NaCl concentration and temperature, demonstrateda very promising behaviour of the cyanobacterialpolymer (Table 8).

As a concluding remark, it has to be kept in mindthat all the studies available on cyanobacterial RPSsinvolve laboratory experiments and there is an abso-lute lack of information on technological, economicand applicative aspects of these polymers. Hence, thevery small number of patents issued during the last10 years on cyanobacterial exopolysaccharides (Ta-ble 9) should not surprise.

6. Future prospects

The present knowledge of cyanobacterial RPSs,described in the previous sections, suggests that thesepolymers may cover a broad range of complex chem-ical structures and consequently di¡erent properties.Moreover, it is reasonable to anticipate that furtherstudies on RPS-producing cyanobacteria will lead tonew polymers possessing properties di¡erent fromthose of the polymers already available. This greatdiversity per se does not imply that cyanobacterialRPSs will ¢nd applications as integrative or alterna-


Table 9Patents on cyanobacterial RPSs issued in the last 10 years (information drawn from J. Appl. Phycol., section `Patents' compiled bi-monthly by M.A. Borowitzka)

Year Patent no. Country Title RPS producer strain

1989 4,826,624 USA Bioemulsifyer for dispersing liquid hydrocarbon(s) in a secondliquid ^ is a polymeric product produced by cyanobacteria.

Phormidium spp.

1990 4,894,161 USA Clari¢cation of particulate containing liquid with bio£occulantproduced by Phormidium genus cyanobacterium.

Phormidium spp.

1992 4,370,098 Japan Continuous production of polysaccharide used as agar-agarsubstitutes etc.

Spirulina platensis

1993 5,049,491 Japan High yield polysaccharide production. Aphanocapsa halophytica1993 5,250,201 USA Secondary recovery of petroleum from subterranean

formations.Phormidium spp.

1994 6,040,880 Japan Cosmetic material with su¤cient skin-whitening e¡ect andhigh stability and safety.

Aphanocapsa spp.


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tive options to biopolymers currently in use, so hav-ing actual industrial perspectives for uses with alarge market or for pro¢table market niches. Thepolymers already on the market (of microbial orplant origin) appear quite adequate for most appli-cations and requirements of the industries; further-more, they can rely on a mature industrial technol-ogy for their production and, some of them, on thesanitary authorisations necessary for their use infoods. Notwithstanding, it has been envisaged thatthe use of `greener' technologies will most probablyincrease in the immediate future [102], so that thepossibility of utilising photoautotrophic microorgan-isms to produce natural biopolymers from renewableresources will very likely attract more and more at-tention. However, as emphasised at the internationalsymposium recently held in Israel (`PolysaccharideBiotechnology: an interdisciplinary approach'; BeerSheva, 18^19 May 1998), one should bear in mindthat the development of any microbial polysacchar-ide into biotechnological products requires co-ordi-nated e¡orts of both basic and applied research, in-volving all the necessary expertise to move from thelaboratory culture of a polysaccharide-producingstrain to the biopolymer market. Unfortunately, inthe case of cyanobacterial polysaccharides, a widegap exists between the current knowledge and whatis needed to carry out a complete biotechnology pro-gramme. In this context, any research activity aimedat assessing and/or promoting the e¡ectiveness of acyanobacterial RPS will be greatly appreciated aswell as any practical suggestion on the propertiesthat a new polymer should possess in view of itsspeci¢c applications.

Cyanobacterial RPSs destined to special applica-tions, such as those in the biomedical sector, deserveseparate consideration, owing to their particularlyhigh added value. Indeed, cyanobacterial RPSs couldhave a great biomedical signi¢cance, as demon-strated in the case of the sulfated polysaccharide iso-lated from S. platensis that showed anti-herpes sim-plex virus and anti-human immunode¢ciency virusactivities [128,129]. In this perspective, cyanobacteri-al strains of saline or hypersaline origin, amongwhich the ability to synthesise and to release sulfatedexocellular polysaccharides seems to be frequent [47],may provide a great resource for the production ofnew bioactive polymers.

Acknowledgments

The authors would like to acknowledge the fruitfulcollaboration over the years with their colleagues,Drs. Maria Cristina Margheri and Claudio Sili.Some of the recent results described here, from theauthors' laboratory, have been obtained in theframework of the EC Project `Biodiversity: Appliedand Systematic Investigations of Cyanobacteria'(BASIC), Contract BIO4-CT96-0256, and in theframework of the `Progetto Coordinato POLISA(Nuovi polisaccaridi microbici di potenziale interesseper l'industria alimentare)' funded by the NationalResearch Council of Italy (CNR).

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