4

REVIEWREVIEWREVIEWREVIEW OF LITERATUREOF LITERATUREOF LITERATUREOF LITERATURE

2.1 DESERT ECOSYSTEM AND THE EPS IN CYANOBACTERIA

2.1.1 Intracellular polysaccharide

2.1.2 Exocellular or exo-polymers

2.2 MAJOR CONSTITUENTS OF EXTRACELLULAR POLYMERS

IN CYANOBACTERIA

2.2.1 Neutral Polysaccharides

2.2.2 Acidic polysaccharides

2.2.3 Lipids

2.2.4 Protein

2.3 DETECTION OF POLYSACCHARIDE

2.4 STRUCTURAL CHARACTERIZATION OF EPS

2.4.1 Methodology

2.4.2 General characteristics of cyanobacterial extracellular

polymers

2.4.3 Conformation

2.4.4 Infra Red Spectroscopy

2.4.5 IR Spectroscopy in polymer charaterisation

2.4.6 UV-Visible spectroscopy

2.5 EXTRACELLULAR POLYSACCHARIDES IN

CYANOBACTERIA

2.5.1 Chemical composition of Cyanobacterial EPS

2.5.2 Filamentous heterocystous cyanobacteria

(a) Non descript

(b) Released EPS

5

(c) Capsule

2.5.3 Filamentous nonheterocystous cyanobacteria

(a) Released EPS

(b) Capsule

2.5.4 Unicellular cyanobacteria

(a) Non descript

(b) Released EPS

(c) Capsule

2.6 FACTORS AFFECTING EPS PRODUCTION

2.6.1 Age

2.6.2 Culture condition and Medium composition

2.6.3 UV radiation

2.7 OPTIMIZATION OF EPS PRODUCTION

2.8 BIOPOLYMER YIELD

2.9 PROPERTIES OF POLYSACCHARIDES

2.10 FUNCTIONS OF EPS

2.11 ROLES OF EPS IN ENVIRONMENT

2.11.1 Adhesion to and colonization of surfaces

2.11.2 Biochemical interactions

2.11.3 Cell protection

2.11.4 Hydrosorbing action

2.11.5 Cryoprotection

2.11.6 Ligand binding

2.12 INDUSTRIAL APPLICATIONS OF EPS

2.12.1 Rheology

2.12.1.1 Rheological Classifications

(a) Newtonian Fluids

(b) Non-Newtonian Fluids

6

2.12.2 Viscosity

2.12.3 Rheological behaviour of microbial polysaccharides

2.13 BIOTECHNOLOGICAL POTENTIAL OF EPS

2.13.1 Medical applications

2.13.2 Flocculation

2.13.3 Surfactant

2.14 COMMERCIALISATION PROSPECTS IN THE FOOD

INDUSTRY

7

2.1 DESERT ECOSYSTEM AND THE EPS IN CYANOBACTERIA

Microbial life in hot deserts is poised at the limits of survival. High

temperature and intense radiation coupled with extreme aridity impose so severe an

environmental stress that shrinks the diversity of eukaryotic organisms to almost

negligible. Due to this, hot desert microbial community is composed primarily of

prokaryotes (Friedmann, 1980). Cyanobacteria have a remarkable suite of attributes

and strategies, which enable them to colonize and survive where other organisms can

not. They are adapted to colonize two very different types of substrata, the unstable

sand dunes or soil that they actively stabilize and rocks to which they adhere or

penetrate. Many of the successful cyanobacterial colonizers in arid and semiarid areas

produce an abundance of sheath or capsular jelly.

Usually extracellular polymers do not contribute to the microbial structure, the

intracellular functions being not affected if they are absent (Sutherland, 1990).

However the characteristic globular shape of the Nostoc colony is due to the EPS. de

Philippis et al., (2000) tested 40 Nostoc strains for the presence of extracellular

polysaccharidic investments surrounding the trichomes of which twenty five strains

released water soluble polysaccharides. They classified EPS into three main types of

morphologies (i) capsules surrounded by an external pellicle, (ii) capsules with sharp

outlines but without an external pellicle, and (iii) slimy investments that surround the

trichomes loosely without following their shape or are organized in large globular

lumps. The terminology associated with mucilaginous layers in cyanobacteria is quite

dubious and confusing. As pointed out by Bertocchi et al. (1990), the structural and

functional differences between different sublayers of the mucilaginous layers, are

very arbitrary and thus are often not evident in the literature. These structures are

defined by Bertocchi et al. (1990), further confirmed by de Philippis and Vincenzini

(1998), and slightly modified by Adhikary (1998). In general, the external envelope

layers are divided into three structured or unstructured layers or zones (sheaths,

slimes, and capsules), and can be observed under the microscope using Nomarski

differential interference contrast microscope (schematic diagram is shown in Figure

2.1). The sheath is defined as a thin, electron dense layer that loosely surrounds cells

or cell groups. The layer is usually visible by light microscopy without staining. The

capsule generally consists of a thick and slimy layer intimately associated with the

cell surface, with sharp outlines, and is structurally coherent. Slime refers to the

8

mucilaginous material dispersed around the organism but not reflecting the shape of

the cells (de Philippis and Vincenzini, 1998).

Fig.2.1 Schematic diagram of the cell envelope. 1, cytoplasmic membrane; 2,

cell wall, 3, sheath; 4, capsule and slime

Painter (1993) classified polysaccharides in cyanobacteria into thrre groups:

storage, cell envelope and exocellular polysaccharides. Bertocchi et al. (1990) defined

them as follows:

2.1.1 Intracellular polysaccharide

Intracellular polysaccharide, known as storage form of carbon such as

glycogen that is located in the cytoplasm. Intracellular storage polysaccharides are

produced by plants (starch, inulin), green algae (starch), cyanobacteria and animals

(glycogen) and also by some other microorganisms.

Cell wall structural polysaccharides such as peptidoglycan or lipotechoic acids

of Gram positive bacteria and lipopolysaccharides of the Gram negative bacteria are

part of what is called as cell envelope. In some literature this is also discussed as

exocellular/exopolymer.

2.1.2 Exocellular- or exo-polymers

The exocellular polymers comprise of sheath (usually in filamentous forms)

and capsular polysaccharides in unicells (abbreviated as CPS) that impart a cohesive

9

layer, which is covalently linked to the cell surface. The sheath is a thin uniform layer

having homogeneous fribrillar structure that surrounds the cell wall and reflects the

shape of the organism and is visible even without staining. Sheath is usually extracted

using differential and sucrose gradient centrifugation of the homogenized cells

(Bertocchi et al., 1990).

The capsule is a non-uniform thick layer that may surround the sheath. It has a

more organized polymeric structure, densely packed, less diffusible and bound more

tightly to the cell than the slimes. Capsular exopolysaccharide is held to the cell wall

either by linkages between its carboxyl groups (hydroxyl groups in

lipopolysaccharids (LPS)) or by a covalent bonding through phospholipids and

glycoproteins. Solubilization of the polysaccharide contained in the capsule is

achieved by warm water treatment of the cellular mass.

The extracellular polymers include polysaccharide layers on the surface of

organisms together with some glycoproteins, termed ‘glycocalyx’. Extracellular

polymers in the microorganisms also termed released polymers (RPS) exist as slime

or as free dissolved matter (component of dissolved organic matter). Slime layer

loosely attached to the cell surface or secreted in the environment or growth medium

is included in this class of polymer (Sutherland, 1972; Madigan et al., 1997). The

slime is the most external layer lacking definite margins although it is physically

detectable as mucilage. Extracellular polymers occur widely among bacteria and

microalgae and less frequently among yeasts and fungi. Since it is already dissolved

in medium, the cellular mass can be separated by centrifugation and slime may be

obtained from the supernatant by alcoholic precipitation.

EPS

The abbreviation EPS has been used for “extracellular polymeric substances”,

“extracellular polysaccharides”, “exopolymers” “expolysaccharides”. It is used for

extracellular polymeric substances as a more general and comprehensive term from

different classes of organic molecules such as polysaccharides, proteins, nucleic acid

and lipids, which have been found to occur in the intracellular spaces of microbial

cells. Several species of cyanobacteria are characterized by the presence of thick

capsule/sheath surrounding the cells and by the ability to release polysaccharide

material in to the culture medium (de Philippisa and Vincenzinia, 2006).

10

Following is a schematic chart of extraction procedurs of different

polysaccharides modified after Bertochhi et al. (1990).

Cyanobacterial culture broth

Centrifugation

Cell pellet Supernatant

Hot water

Centrifugation

Homogenization: Alcohol

Pellet Supernatant differential sucrose precipitation

gradient centrifugation

Alcohol

Precipitation

Fine Chemical

Sheath polysaccharide

(exocellular polymer) Exocellular

polysaccharide

(slime)

Capsular

Polysaccharide

(CPS part of exocellular polymer)

Fig. 2.2 Schematic extraction procedures of the cyanobacterial

polysaccharides.

11

Desikachary (1959) in his monograph on cyanophyta described >63.5%

morphotypes of cyanobacteria as those showing thin or thick sheath or mucilaginous

film or slime.

In Bergey’s classification system, the polysaccharide producing cyanobacteria

belong to all 5 subsections (Castenholz and Phylum, 2001). In totality, 33 genera in 5

subsections have demonstrated the ability to release polysaccharides during their life

cycles (Table 2.1).

Table 2.1 Polysaccharide producing cyanobacteria in Bergey's classification system (Castenholz and Phylum, 2001)

Subsections No.of

total

genra

included

No.of

Polysaccaride

producing

Genera

Genus names

I 14 10 Chamaesiphon, Chroococcus, Cyanobium,

Cynotheca, Gloeobacter, Gloeocapsa,

Gloeotheca, Microcystis, Synechococcus

(including former Anacystis), Synechocystis

(including former Aphanocapsa)

II 7 6 Dermocarpella, Stanieria (inluding former

Dermocarpa), Xenococcus, Chrococcidiopsis,

Myxosarcina, Pleurocapsa

III 17 6 Geitlerinemia (including former phormidium),

Lyngbya, Microcoleus, Oscillatoria,

Pseudoanabaena,Spirulina,

IV 12 9 Anabaena, anabaenopsis, Cyanospira,

Cyclindrospermum, Nodularia, Nostoc,

Scytonema, Calothrix, Tolypothrix (Including

former Microchaete)

V 6

2 Chlorogloeopsis (including former

Mastigocladus), Fischerella

12

2.2 MAJOR CONSTITUENTS OF EXTRACELLULAR POLYMERS IN

CYANOBACTERIA

Extracellular polymers produced by the cyanobacteria are largely carbohydrates,

but proteins, lipids/or nucleic acids may also contribute to some extent in most of the

organisms (Wingender et al., 1999).

2.2.1 Neutral Polysaccharides

Generally the extra cellular polysaccharides are composed of

monosaccharides with hexoses (glucose, galactose, mannose, xylose, rhamnose and

arabinose) and/or pentoses forming the bulk of it. The monosaccharides are

significant factors in determining both the surface charge and settling properties.

They also determine rheological properties of suspensions.

2.2.2 Acidic polysaccharides

In acidic extra cellular polysaccharides the uronic acid is one of the

monomeric unit. It strongly influences the physical properties due to the presence of

charged anionic groups on these polysaccharides that can strongly interact with the

cations.

2.2.3 Lipids

Forster and Clarke (1983) and Goodwin and Forster (1985) reported presence

of lipids as one of the extracellular components in the ethanol extracts of activated

sludge. As much as up to 8% of cell mass has been reported being liberated by a

freshwater phytoflagellate Ochromonas danica by Aronson (1976). But reports of

such liberations by cyanobacteria were not found.

2.2.4 Protein

The amino acids and peptides although very common in algal filtrates

represent only a small fraction of the total cellular material (Gocke, 1970). On the

other hand cyanobacteria liberate very large portions of their assimilated nitrogenous

substances into the medium (Fogg, 1952, 1966; Jones and Stewart, 1969). The

13

dissociated carboxyl groups of these compounds give a negative charge to protein

thus enhancing their role in the floc structure as they are able to attach multivalent

cations (Ericksson and Alm, 1991).

Since cyanobacterial extracellular polymers are dominated by

polysaccharides, the review also has been targeted towards EPS only. The term EPS

therefore shall mean extracellular polysaccharides from here onwards.

2.3 DETECTION OF POLYSACCHARIDE

A number of observations have been reported concerning the visualization of a

mucopolysaccharide layer at the cell surface by staining with ruthenium red (Luft,

1965, 1966; Martinez-Palomo et al., 1969, Luft 1976). Waller et al. (2004)

demonstrated that ruthenium red is a polycationic stain used to visualize acid

polysaccharides on the outer surface of cells. Blanquet (1976 a, b) claimed that the

reaction visualizes acidic groups (such as carboxyl, sulphate and phosphate groups)

on the cell surface.

Strycek et al. (1992) studied the ability of freshwater algae and cyanobacteria

to form extracellular fibrils using ruthenium red (RR) staining on 28 species. Algae

with high proportions of RR-positive cells (M. radiata, Eremosphaera sp.,

Xanthidium sp., and M. aeruginosa) produced high levels of slime-like material and

distinct fibrils were often seen attached to the cell surface and only slowly released

into the medium. Ruthenium red staining revealed both the long and short classes of

cell surface fibril in thin sections of Streptococcus salivarius HB, indicating that the

fibrils contained polyanionic polymers, probably polysaccharides (Handley et al.,

1988). According to Ishii et al. (1984) an improved ruthenium red staining has been

applied to normal human skin and lichen myxoedematosus skin lesions for the

detection of the fine structure of proteoglycan aggregates. Overdahl and Zottola

(1991) evaluated seventeen strains of Lactobacillus acidophilus to determine the

relationship between bile tolerance and the presence of an outer polysaccharide layer

exterior to the cell wall.

14

2.4 STRUCTURAL CHARACTERIZATION OF EPS

2.4.1 Methodology

Polysaccharides are characterized by an extreme structural diversity. They are

seldom homopolymers. In most cases their backbone consists of several types of

sugar monomers linked in a chain. Moreover they are branched showing varying

structure and pattern of branching. Structural characterization of the polysaccharide

requires determination of composition (type and relative abundance of monomers),

structure (relative distribution of monomers and type of chemical bonds between

them), conformation (arrangement of monomeric chains and bonds between them),

relative molecular mass, type and arrangement of substitutes (Morin, 1998). This

information enables to correlate the functional properties of polysaccharides, like

solubility in water, relative viscosity and rheological behaviour (Stokke et al., 1998)

and ion(s) binding capacity (de Philippis and Vincenzini, 1998).

Table 2.2 shows different methods that are applied to determine main features

of the structure of the polysaccharide.

2.4.2 General characteristics of cyanobacterial extracellular polymers

Polysaccharides are composed of monosugars bound through glycosidic

bonds. They may possesss one (homopolysaccharides) or more structural units

(heteropolysaccharides) (Lindberg, 1990). The type of monomers is limited (neutral

hexoses, methylpentoses, oxisugars, aminosugars, uronic acids) yet the great variety

of EPS emanates from the number of combinations based on the possible bond

patterns (Sutherland, 1994) and the types of substitute radicals (if any) and their

arrangement. Most monosugar components of microbial polysaccharides are common

with plants. D-glucose, D-galactose and D-mannose in the pyranosic form are the

most frequent (Lindberg, 1998). Some polysaccharides also show the L-forms of

glucose, galactose and mannose, together with L-fucose and L-rhamnose (Thedieck

and Rausch, 2000). Some microorganisms produce EPS containing aminosugars

(glucosamine, galactosamine (Robijn et al., 1996a, b)) in the pyranosic form.

The monomeric sugar units may join to form linear chains (bacterial cellulose,

15

curdlan or pulullan) or ramified structures (xanthan, dextrans) (Ionescu, 2001). Helm

et al. (2000) reported that the N. commune DRH-1 EPS called nosturonic acid

contained a predominant repeat unit (1, 4-linked xylogalactoglucan backbone with D-

ribofuranose and 3-O-[(R)-1-carboxyethyl]-D glucuronic acid pendant groups).

However, the question of regularity of a unit structure (repeat unit or averaged

structure) in cyanobacteria is considered open for discussion as conflicting evidence

exists in literature.

Table 2.2: Principal characteristics of microbial polysaccharides and their methods of analysis. Characteristics Analysis methods References

Quantitative analysis of

polysaccharides

Gravimetric methods

Colorimetric methods

Ramus, 1977;

Bergmaier et al., 2001

Composition:Quantitative qualitat

-ive analysis of monosaccharide

components

High Performance Liquid

Chromatography (HPLC)

- Reverse-phase HPLC

- Ion-exchange HPLC

Unger and Weber, 1999;

Kaiser and Benner, 2000;

Meyer et al., 2001

Structure conformation NMR spectroscopy (NMR) Cowman et al., 2001;

Guetta et al., 2003

Differential Scanning

Calorimetry (DSC)

Spigno and de Faveri, 2004

X-ray diffraction spectroscopy Allen Busch et al., 1999

Rheological analysis Marques et al., 2002;

Picout and

Murphy,2002

Quantitative qualitative

analysis of substitutes

Ion-Exchange HPLC Thomas et al., 2003

NMR Pereira et al., 2002

Infrared Spectroscopy (IRS) Lijour et al., 1994

Unitary Sequence Linkage analyses of untreated

treated polysaccharide by

means of reduction of uronic

acids, mild acid hydrolysis with

oxalic acid or lithium degradation

Volk et al., 2007

EPS form dimension Dynamic Light Scattering or

Static Light Scattering

Ioan et al., 2001;

Santiago et al., 2002

16

Most of the polysaccharides are polyanionic. This character is another

important feature that is common to most cyanobacterial polysaccharides (de

Philippis and Vincenzini, 1998; Huang et al., 1998; Nicolaus et al., 1999; de Philippis

et al., 2000; Moreno et al., 2000; Shah et al., 2000). About 90% of these polymers are

characterized by the presence of uronic acids (glucuronic acid and galacturonic acid

being their usual ingredients (Lindberg, 1998)) that exceed 20% of the dry weight of

the released polysaccharide in half of the strains studied.

As substitutes, EPS show variety of compounds. The most usual being

pyruvate, sulphate and phosphate attached to a neutral hexose (Sutherland, 1990) that

contribute to the global charge of the macromolecules resulting in a rather high anion

density (de Philippis and Vincenzini, 1998). Sulphated polysaccharides are often

found in animals (heparins, condroitin sulphates, dermatan sulphates) (Medcalf, 1978)

and algae (agaragar or carrageenan in Gracilaria laminaria sp. Chapman, 1952),

however they are rare in eubacteria (Arad, 1988; Fattom and Shilo, 1985). Manca et

al. (1996) reported it in Bacillus species. They have been described in four

cyanobacteria viz. Phormidium sp. strain J-1, Anabaenopsis circularis PCC 6720,

Synechocystis PCC 6803 and 6714 and Gloeothece ATCC 27152 (Bar-Or and Shilo,

1987; Panoff et al., 1988; Tease and Walker, 1987). Other than cyanobacteria,

sulfated cell wall polymers also occur in certain archaebacteria viz. Halomonas sp.

(Bejar et al., 1998) and Haloferax mediterranei (Sutherland, 1994).

These substituents confer a specific charge on the macromolecule (Table 2.3).

Besides, they also determine properties of a biopolymer. Acyl groups affect the

rheological properties of EPS (Cadmus et al., 1976). Uronic acids but not sulphates

have been shown to participate in the metal-complexing capacities of the EPSs from

two Chlorella strains (Kaplan et al., 1987). The implication of sulfate residues in the

rheological characteristics of carbohydrate polymers is also established (Rees, 1969).

17

Table 2.3: List of substituents of extracellular polysaccharides in cyanobacteria along with linkages

Substituent Linkage Charge conferred on the polymer

Organic Acids

Acetate

Ester

None

Glycerate Ester Negative

Hydroxybutanoate Ester None

Propionate Ester None

Pyruvate Ketal Negative

Succinate Half ester Negative

Inorganic acids

Phosphate

---------

Negative

Sulphate --------- Negative

The presence of hydrophobic groups are reported for a rather large number of

cyanobacterial RPSs (Table 2.4). Ester-linked acetyl groups, ranging up to 12% of the

dry weight of RPS together with peptidic moieties and the deoxysugars (fucose and

rhamnose) may contribute to a significant hydrophobic behaviour of these otherwise

hydrophilic macromolecules (Flaibani et al., 1989; Shepherd et al., 1995).

2.4.3 Conformation

Conformation refers to the form of polysaccharide chains. It depends on the

monomers and their position and bond types in the polymeric chains (Belitz and

Grosch, 1999). The main conformations adopted by EPS (Belitz and Grosch, 1999)

are 1. ribbon-type chains (cellulose or alginate), 2. helix (lichenin) and 3. combined

conformation (majority of heteroglycans). Together with the structure, conformation

offers valuable information on the properties of polysaccharides and their practical

applications.

In solution, polysaccharides may form hydrogels. A gel is a soft material,

solid or solidified having two or more components (one of them being a solvent in

high quantity) (Picout and Ross-Murphy, 2002).

18

Table 2.4: Charged groups and hydrophobic constituents of the cyanobacterial RPSs described (de Philippis et al., 2001) (nd, not determined; p, present, but not quantified; in brackets, the number of strains investigated for presence of the specific constituent)

Genus Number

of

strains

Charged groups

(% of RPS dry wt)

Hydrophobic groups

(% of RPS dry wt)

Reference

Uro

nic

acid

s

Pyr

uvat

e

Sul

phat

e

Deo

xy*

suga

rs

Ace

tate

Pep

tidic

moi

ety

Subsection I

Aphanocapsa 1 0 nd 11.9 52.4 nd 10.3 Sudo et al., 1995

Anacystis 1 0 nd nd 0 nd nd Sangar and Dugan,1972

Chroococcus 1 5.9 nd nd 19.1 nd 3.2 Fischer et al., 1997

Cyanothece 16 9.8-80.1 0-3.9 P(13 strains) 3.4-73.0 0-4.2 1.4(1 Strain) de Philippis et al., 1993; 1998

Gloeothece 1 15.6 nd 13.8 8.8 nd p Tease et al .,1991

Microcystis 2 p-43.0 nd nd p-16.6 nd P (I strain) Plude et al., 1991; de Philippis 1998

Synechocystis 2 16.4-16.7 0 1.0-1.2 6.7-31.4 nd 20.0-40.0 Panoff et al ., 1988 Subsection III

Lyngbya 1 38.6 nd nd 0 nd nd Gloaguen et al., 1995

Microcoleus 2 0 nd nd p nd 6.0 Mazor et al ., 1996

Oscillatoria 5 6.7-24.2 nd nd 3.3-9.5 nd nd Bender et al., 1994; Gloaguen et al., 1995;

Phormidium 17 0.5-41.5 nd 0-1.6

(2 strains)

0-26.3 nd 4.4-13.0(2

strains)

Matulevicz et al ., 1984; Bar-Or and Shilo,

1987; Gloagnen et al., 1995

Contd.

19

Genus Number

of

strains

Charged groups

(% of RPS dry wt)

Hydrophobic groups

(% of RPS dry wt)

Reference

Uro

nic

acid

s

Pyr

uvat

e

Sul

phat

e

Deo

xy*

suga

rs

Ace

tate

Uro

nic

acid

s

Spirulina 2 20.0-40.0 nd 5.0 (1 strain) p-8.2 nd nd Filali Mohim et al., 1993; Tseng and Zhao,

1994 Subsection IV

Anabaena 8 0-51.5 nd P (1 strain)

0-19.1 nd 0-5.0

(2 strains)

de Philippis and Vincenzini, 1998; Nacolaus et

al., 1999; Moreno et al., 2000

Cyanospira 1 36.5 1.5 0 15.4 0 2.0 Vincenzini et al., 1990

Nostoc 37 0-69.4 0-6.2 P

(17 strains)

0-27.5 0-

12.9

0.6-69.5

(29 strains)

de Philippis and Vincenzini, 1998; Huang et

al., 1998;Nicolaus et al., 1999; de Philippis et

al., 2000

Scytonema 1 Nd nd nd 0 nd nd Nacolaus et al., 1999

Tolypothrix 1 Nd nd nd 14.3 nd nd Nacolaus et al., 1999 Subsection V

Chlorogloeop

sis

1 P nd nd 10.2 nd nd Nacolaus et al., 1999

Fischerella 1 Nd nd nd 17.6 nd nd Nacolaus et al., 1999

Mastigocladu

s

1 P nd nd p nd nd Morvan et al., 1997

*Amount expressed as percent of the RPS saccharide fraction.

20

When water is the liquid, a hydrogel is formed that can have four types of

structures (Picout and Ross-Murphy, 2002): well ordered lamellar structures;

completely disordered covalent polymeric networks (especially the synthetic

polymers); polymeric networks formed through physical aggregation, disordered with

ordered regions; and disordered particulate structures.

The structural characterization of extracellular polymeric substances (EPSs) is

performed qualitatively and quantitatively using various sophisticated techniques viz.

Gas chromatography, HPLC, GC-MS and NMR spectroscopy specifically to

determine unitary molecules and UV-visible, Fourier Transform Infrared

Spectrometry (FTIR) and NIR, NMR and X-ray spectroscopy for conformational

understanding. Following is the brief account of FTIR and UV-vis spectroscopy.

2.4.4 Infra Red Spectroscopy

Infrared (IR) radiation refers broadly to that part of the electromagnetic

spectrum which falls between the visible and microwave regions. Of greatest practical

use is the limited portion between 4000 and 400 cm-1. There has been some interest in

the near IR (14,290-4000 cm-1) and the far-IR regions 700-200 cm-1. Even a very

simple molecule can give an extremely complex IR absorbance spectrum. The organic

chemist takes advantage of this complexity to match the spectrum of an unknown

compound against that of an authentic sample.

Infrared radiations absorbed and converted by an organic molecule into

energy of molecular rotation. This absorption is quantized and thus a molecular

rotation spectrum consists of discrete lines. There are two types of molecular

vibrations; stretching and bending. A stretching vibration is rhythmical movement

along the bond axis such that the inter-atomic distance is increasing or decreasing. A

bending vibration may consist of a change in bond angle between bonds with a

common atom or the movement of a group of atoms with respect to the remainder of

the molecule without movement of the atoms in the group with respect to one another.

For example twisting, rocking and torsional vibrations involve a change in bond

angles with reference to a set of coordinates arbitrarily set up within the molecule.

Only those vibrations that results in a rhythmical change in the dipole moment of the

molecule are observed in the IR. The alternating electric field, produced by the

changing charge distribution accompanying a vibration, couples the molecule

vibration with the oscillating electric field of the electromagnetic radiation.

21

FTIR spectroscopy works by shining infrared radiation on a sample and seeing

which wavelength of radiation in the infrared region of the spectrum are absorbed by

the sample. Each compound has a characteristic set of absorption bands in its infrared

spectrum. Variation in stretching and bending modes of vibration with single

functional group is normally coupled with the vibration of adjacent group as well as

with the number of substitutions taking place on the molecule itself. This leads to the

shifting and overlapping of the peaks of two or more functional groups in the same

region of the IR spectrum.

2.4.5 IR Spectroscopy in polymer charaterisation

Infra red spectroscopy is probably the most extensively used investigative tool

for the structural analysis of polymer structure and the analysis of functional groups

(Zbinden, 1964; Koenig and Kendall, 1996; Nyquist, 1961). Since the intensity of

infra red absorption is a measure of the concentration of a group, the spectrum is also

used in quantitative analysis of polymers. In addition to structural and qualitative

analysis, infrared spectroscopy is also useful in the determination of number, average

molecular weight, degree of branching and in studying the course of chemical

reactions of polymer molecules (Sibilia and Paterson, 1965; Durbetaki and Miles,

1965; Arshady, 1984; Ayres and Mann, 1965). Another important application is the

determination of degree of regularity in the arrangement of the macro molecules as

degree of crystallinity and the degree of orientation of stretched films and fibers

(Ferruti et al., 1974; Stark weather and Moynihan, 1956; Zerbi et al., 1964). An

important factor which favours extension of infrared spectroscopy in polymer

chemistry is that both soluble and insoluble materials can be analysed (Crowley and

Rapoprt, 1976).

The FTIR spectrum of the polymer produced by the bacterium

Microbacterium sp. MC3B-10 displayed a broad O-H stretching band at 3421 cm-1

and at an intense band at 1070 cm-1, typical of carbohydrates. In addition, bands at

1650 and 1550 cm-1 were also detected. No doublet at 1250 and 1230 cm-1, indicative

of the presence of ester sulfate groups were seen (Ortega-Morales et al., 2007). The

IR spectrum of polymer MBFW31 of Vagococcus sp. W31 showed absorption peak at

3363 cm-1 (OH group) and 2980 cm-1 (CH stretching band). The spectrum also

displayed an asymmetrical stretching band at 1633 cm-1 and a symmetrical stretching

22

band at 1400 cm-1, which were consistent with the presence of carboxylate ion and

peak at 1150 cm-1 was caused by C-O stretching thus indicating the presence of a

methoxyl group. The absorption peak of OH (840 cm-1) showed that MBFW31 was

composed of sugar derivatives (Gao et al., 2006).

The infrared spectra of the biopolymer polysaccharide of Enterobacter

cloacae WD7 showed the presence of carbonyl (1716 cm-1), hydroxyl (3455 cm-1),

carboxyl (1608 and 1400 cm-1) and methoxyl (1136 and 1075 cm-1) groups. It was

concluded by Prasertsan et al. (2006) that the polysaccharide may be a partially

methylated polysaccharide containing uronic acid due to the presence of O-H

broadband at 3700-3000 cm-1 and the intensity of absorption due to O-CH3 at 1150-

1050 cm-1. Two absorption peaks at 1608 cm-1 and near 1400 cm-1 of carboxylate ions

were true to the characteristic pattern for uronate of the polysaccharides produced by

Butyvibrio fibrisolvens and Bacillus sp. (Prasertsan et al., 2006).

The biopolymer flocculant produced by a halothermophilic Bacillus sp. I-471

showed a broad stretching intense peak at 3420 cm-1, characteristics of OH group. A

symmetrical stretching peak at 1640 cm-1 and a weak symmetrical stretching peak

near 1450-1340 cm-1 suggested the presence of carboxyl group. Specifically, the

peaks at 1022-1100 cm-1 range and ~1600 cm-1 region indicated the presence of

guluronic acid or O-acetyl ester, which are the building blocks of alginic acid. The

bacterial alginate differs from algal alginate by having an additional peak at around

1160 cm-1 region due to the presence of O-acetyl ester (Kumar et al., 2004). The H12

polymer of Klebsiella pneumoniae showed distinct absorption wavelength at 3400 cm-

1, 1720 cm-1 and 1605 cm-1, indicating the presence of a hydroxyl, a carboxyl ester

moiety and a carboxylic acid respectively (Kobayashi et al., 2002).

Analysis principally based on IR spectrometric analysis for the

characterization of microbial polysaccharides are summarized in the Table 2.5.

23

Table 2.5 : Characteristics FTIR absorption spectrum of different EPS

isolated from bacteria and cyanobacteria.

Biopolymer from

Wave number/range

Characteristics peaks of IR

spectra

Reference

Vagococcus sp. W31

3363 cm-1 OH stretching Gao et al., 2006 2980 cm-1 CH stretching 1633 cm-1 asymmetrical

carboxylate group

1400 cm-1 symmetrical carboxylate group

Enterobacter cloacae WD7

1716 cm-1 carbonyl Prasertsan et al., 2006 3455 cm-1 hydroxyl

1608 and 1400 cm-1 carboxyl

1136 and 1075 cm-1 methoxyl

O-H broadband at 3700-3000 cm-1 and O-CH3 at 1150-1050 cm-1

partially methylated plysaccharide containing uronic acid

1608 cm-1 and near 1400 cm-1 of carboxylate ions

uronate

Cyanothece sp. and

Nostoc carneum

3400-3348 cm-1 OH stretching Parikh and Madamwar, 2006

2924 and 2854 cm-1 CH stretching 1040-1074 cm-1 C-O broad

stretching 1636 cm-1 carboxylate ions 840 cm-1 sugar derivatives

1259 and 1140 cm-1 sulfate group as S=0 and C-O-S (cyanothece sp.)

contd.

24

Biopolymer from

Wave number/range

Characteristics peaks of IR

spectra

Reference

Haloalkalophilic Bacillus sp.I-471

1022-1100 cm-1 and ~1600 cm-1

guluronic acid, mannuronic acid and O-acetyl ester

Kumar et al., 2004

Klebsiella pneumoniae H12

3400 cm-1 OH stretching Kobayashi et al., 2002 1720 cm-1 carboxyl ester

moiety 1605 cm-1 carboxylic acid

Penicillium vermiculatum

890 cm-1 β- linked polysaccharides

Kogan et al., 2002

805 cm-1 galactofuranosyl Pseudomonas sp. NCIMB 2021

1262-1251 cm-1 O-acetyl ester Beech et al., 1999 1661-1665 cm-1 protein-amide I

1547 cm-1 amide II

Microbacterium sp. MC3B-10

3421 and 1070 cm-1

1250 and 1230 cm-1

O-H stretching

ester sulfate groups

Ortega-Morales et al., 2007

Bacillus thermoantarticus

1240 cm-1

S=O

Manca et al., 1996

Bacillus sp. SPO18

3300 cm-1 OH stretching Pfiffner et al., 1986 2950 cm-1 CH stretching

1406 and 1619 cm-1 symmetrical and asymmetrical stretching of carboxylate ions respectively

Phormidium 94a 3430 cm-1 OH stretching Vicente-Garcıa et.al., 2003 2928 cm-1 C-H stretching

1600 cm-1 carboxylate group 1408 cm-1 symmetric CH3

Anabaena sp. ATCC 33047

3470 cm−1 O—H stretching Moreno et.al., 2000 1400 to 900 cm−1 C—O absorption

bands 1607 to 1663 and 1400 cm−1

carboxylate groups

Azotobacter vinelandii

3.0 µ ; 3.45µ ; 5.77 and 8.1 µ

OH ; C-H ; -CH2CO- O-R

Cohen and Johnstone, 1964

6.25 and 7.1 µ O=C-0 Cyanothece sp., Oscillatoria sp., Nostoc sp. and Nostoc carneum

3400–3448 cm-1 O–H stretching Parikh and Madamwar, 2006

2924 and 2854 cm-1 asymmetrical and symmetrical C–H

1040–1074 cm-1 C–O stretching

1636 cm-1 carboxylate group

25

Matsuhiro and Miller (2002) studied soluble polysaccharides of few species of

the family Rhodymenaceae (Rhodophyta). According to the literature members of this

family are agarophytes. Dried samples of tetrasporic, cystocarpic and gametophytic

Rhodymenia howeana and tetrasporic R. corallina when analysed by FT-IR

spectroscopy indicated that these seaweeds do not produce agar agar. Aqueous

extraction followed by chemical and FT-IR analysis showed that the extracts are very

similar in composition yet different from agar agar. Even carrageenotype structures in

the polysaccharides were not found.

The sudy conducted by Parikh and Madamwar (2006) showed a specific

absorbance of O–H stretching at 3448–3400 cm−1, asymmetrical and symmetrical C–

H stretching at 2924 and 2854 cm−1 respectively and a bending vibration of C–H at

1400–1380 cm−1 in the EPS of Cyanothece sp., Oscillatoria sp., Nostoc sp. and

Nostoc carneum. Absorbance at 1259 and 1140 cm−1 in EPS of Cyanothece sp.

indicated the presence of sulfur containing functional group.

2.4.6 UV-Visible spectroscopy

The measurement of absorption of ultraviolet and visible radiation provides a

convenient means for the analysis of numerous biomolecules. Yun and Park (2003)

reported that extracellular polysaccharides produced by Bacillus sp. CP912 showed

two peaks at 212 and 228 nm in UV absorption spectrum. The peak at 212 nm is

characteristic of carbohydrates especially ketoses. The exopolysaccharides of Bacillus

thermoantaricus EPS 1 and EPS 2 did not show any strong sbsorption peaks in the

range of 350 to 210 nm (Manca et al., 1996). The UV absorption spectrum of purified

polymer SPO18 obtained from Gram positive, motile, facultative spore-forming rod

similar to Bacillus sp. showed no absorbance at wavelength specific to nucleic acid

and proteins but strongly absorbed at 200 to 210 nm, which is characteristics of

carbohydrates (Pfiffner et al., 1986). The UV spectra of released and capsular

polysaccharide of Anabaena halophytica did not indicate any absorption peaks in the

range of 350 to 210 nm (Li et al., 2001). The ultraviolet spectrum of a

0.1% (w/v) aqueous solution of SP 3A revealed no maxima in the region 230 to 300

mµ (Cohen and Johnstone, 1964).

26

2.5 EXTRACELLULAR POLYSACCHARIDES IN CY ANOBACTERIA

Since early 1950s, more than one hundred cyanobacterial strains belonging to

twenty different genera have been investigated with regard to the production of

extracellular released polysaccharides into the culture medium.

Westiellopsis prolifica is reported to produce 3.5 mg biopolymer per mg dry

weight (Saxena and Kaushik, 1992) while Calothrix marchica produce 26.9mg EPS

(Ruangsomboon et al., 2007). Spirulina, Nostoc, Oscillatoria, Phormidium,

Anabaena, Scytonema, Tolypothrix, Fischerella and Chlorogleocopsis studied by

Nicolaus et al., (1999) produced 1-55.2 mg L-1 EPS with the maximum being

produces by Anabaena strain WSAF sp. A polymer producing strain of

Synechococcus sp. has been reported to produce 1 g polymer L-1 month-1 (Phlips et

al., 1989) while A. anomala produces 4.48g L-1 month-1 in batch conditions.

Extracellular polymers are generally heteropolymeric, unbranched or

branched polysaccharides that are rich in high molecular weight (10-30 KDa).

Molecular weight of the extracellular polymers in Microcoleus vaginatus, Scytonema

javanicum, Phormidium tenue and Nostoc sp. has been reported to be 4000 to 250,000

g mol-1 (Hokpusta et al., 2003).

2.5.1 Chemical composition of Cyanobacterial EPS

Almost all extracellular polymers have nonsaccharidic components, such as

peptidic moieties, acetyl, pyruvyl or sulphate groups. This quite unusual characteristic

is of great significance industrially (Sutherland, 1990). The chemical composition too

is very heterogeneous. In about 80% of the cases, they are composed of six to ten

different monosaccharides. The molecules may be neutral or acidic. Presence of

proteins, uronic acids,

pyruvic acid and O-methyl-, O-acetyl- and sulfate groups emphasizes the complex

nature of cyanobacterial EPS. Most of the cyanobacterial EPSs are composed of at

least one uronic acid and several neutral sugars (ranging from 2 to 10) in combination

with protein molecules (Otero and Vincenzini, 2003). The presence of pentoses,

which are usually absent in other polysaccharides of prokaryotic origin is found to be

unique among cyanobacteria (Otero and Vincenzini, 2003).

27

A large number of different monosaccharides in only one polymer can make

many structures and architectures possible, thus increasing the chance of having a

polymer with peculiar properties (Atkins, 1986). On the whole, there are ten common

sugars in cyanobacterial polysaccharides: the hexoses - glucose, galactose and

mannose; the pentoses - ribose, arabinose and xylose; the deoxyhexoses - fucose and

rhamnose and the acidic hexoses - glucuronic and galacturonic acid (de Philippis and

Vincenzini, 1998; Huang et al., 1998; Nicolaus et al., 1999; de Philippis et al., 2000;

Moreno et al., 2000; Shah et al., 2000). Additionally methyl and/or aminosugars are

found but only sporadically (Panoff et al., 1988; Tease et al., 1991; Filali Mouhim et

al., 1993; Bender et al., 1994; Gloaguen et al., 1995; Fischer et al., 1997; Nicolaus et

al., 1999). A large variation in the ratio among the monosaccharides has also been

found. In 60% of the cases glucose is the most abundant constituent.

The monosaccharide compositions of polysaccharides from different species

and strains of cyanobacteria are summarized in table 2.6. Given below is the

information on extracellular polysaccharide composition in major groups of

cyanobacteria, while maintaining the classification proposed by Bertocchi et al.

(1990).

28

Table 2.6 Monomeric Constituents of extracellular polysaccharides in some cyanobacteria

Organism Carbohydrate composition Reference Ribose Xylose Glucose Galactose Uronic

acid Arabinose Mannose Gal.

acid Glu.acid Rha. Fuc.

Nostoc commune DRH 1 + + + + + - + - + - - Helm et al., 2000 Nostoc commune + + + + Gunter et al., 1995 Phormidium 94 a + - - + - + + + - - - Vicente-Garcia et al., 2003 Nostoc sp. - + - + - - - - - + - Hough et al., 1952 N.muscorum - - + - - + - + + - - Biswas, 1957 N linkia f. muscorum + + + + - + - - - + - Kokyrsta and chekoi, 1972 Nostoc sp -221 - + + + - + - + + + - Mehta and vaidya, 1978 Nostoc calciola 79 WA 01

-- + + + -- + + + + + + Flaibani et al., 1989

N.commune UTEX584 -- + + + -- + + + + + + Nostoc sp. D + + + - - + - - - - - Cupac and Ganter, 1992 Nostoc commune - + + + - + + - + + + Huang et al., 1998 N.flagelliform - + + + - + + - + - - Huang et al., 1998 N.sphaeroids - + + + - - + - - + + Huang et al., 1998 Anabaena sp.ATCC33047

- + + + + - + - - - - Moreno et al., 2000

Nostoc strain of PCC - - - - + - - - - - - de Philippis et al., 2000 Azotobactor vinelandii - - + - + - - + - + - Cohen and Johnstone, 1964 Oscillatoria sp. + + + - - - - - - - - Parikh and Madamwar, 2006 Nostoc sp. - + - - - - + - - - - N.carneum - + - - - - + - - - - Cyanothece sp. + + + - - - - - - - Nostoc PCC strain 6302 - + - + - + + - + + + Dembitsky and Rezanka,

2005

Nostoc PCC strain 6310 + + - + - - + + + + +

contd.

29

Organism

Carbohydrate composition Reference

Ribose

Xylose

Glucose

Galactose Uronic acid

Arabinose

Mannose

Gal. acid

Glu.acid

Rha. Fuc.

Anacystis nidulans - - + + - - - - - + + Weise et al., 1970 Phormidium sp. - + + + - + + - - + - Mikheyskaya et al., 1977 Schizothrix calcicola - + + + - - + - - + - Keleti et al., 1979 Microcystis flos-aquae C30-40 + + + + + Plude et al., 1991 Nostoc insulare + + + Volk et al., 2007 Arthrospira + + + + + + + + Zhen-Yuan et al., 2002 G.gelatinosa + + + + + + + + + Ruangsomboon et al., 2006 Calothrix marchica + + + + + + + + + Ruangsomboon et al., 2007 Cyanothece strains + + + + + + + + + + + de Philippis et al., 1998 Spirulina platensis + + + + + + + Mouhim et al., 2004 Phormidium uncinatum + + + + + Hoiczyk, 1998 Cyanothece sp. PE13 + + + + + -- + -- -- + + de Philippis et al., 1998 Synechocystis sp. PCC 6714 -- + + + + + + -- -- + + Panoff et al., 1988 Synechocystis sp. PCC 6803 -- + + + + -- + -- -- + + Panoff et al., 1988 Oscillatoria amphibian PCC 7105 -- + + + + -- + -- -- + -- Gloaguen et al., 1995 Phormidium foveolarum C52 -- + + + + + + -- -- + + Gloaguen et al.,1995 Cyanospira capsulata ATCC 43193

-- -- + -- -- + + + -- -- + Vincenzini et al.,1990

Microcystis aeruginosa K-3A -- + + + -- + + + -- + + Nakagawa et al.,1987 Nostoc sp. - + + + - - + - + + - Hokputsa et al., 2003 M.vaginatus - + + + - + + - + + + Phormidium tenue - + + + - + + - - + + S.javanicum - + + + - + + - + + + Cyanothece sp.16Som2 -- + + + -- -- + + + -- + de Philippis et al., 1993 Aphanocapsa halophytica MN -- + + + -- -- + -- -- + + Sudo et al.,1995

30

2.5.2 Filamentous heterocystous cyanobacteria

(a) Non descript

Those polymers that cannot be distinguished on the basis of method described

that they are capsule or slime i.e., released polymer are categorized here as non

descript. Nostoc 221, IARI (Metha and Vaidya, 1978) produced both capsule and

slime. The hot water extract of the cell mass was very viscous and on concentration

formed a gel at <20oC. Capsule from Nostoc calcicola Gietler, strain 79WA01

(Flaibani et al., 1989) is composed of a glycuronoglycan moiety and an arginine rich

polypeptide moiety. The kitty of its sugars included glucose, galactose, xylose,

glucuronic acid, fucose, mannose, arabinose, galacturonic acid and rhamnose. They

found that their quantitative contribution varied in different fractions with glucose

being the major contributor, arabinose the most dominant pentose and Glucuronic

acid being twice the amount of galacturonic acid in all fractions.

Hokputsa et al. (2003) and Hu et al. (2003) described EPS from 5 desert

cyanobacteria, including Nostoc sp. They showed that the EPS contained up to 6

sugars viz. L-rhamnose (3.5%), D-xylose (20.9%), D-mannose (1.6%), D-galactose

(21.5%), D-glucose (44.0%) and 2-O-methyl-D-glucose (9.5%).

Nostoc insulare released an extracellular polymer with an average molecular weight of

2.8X106 Da (Volk et al., 2007 ). No sulfate and only traces of pyruvate and acetate

groups were detected. It consisted of glucuronic acid (GlcA), glucose (Glc), arabinose

(Ara) and 3-O-methyl-arabinose (3-O-Methyl-Ara). The following partial structure

was proposed for it: 1,3,4-Glcp with a side chain of 3-O-Methyl-Araf:→1)-Glcp-

(3→1)-Glcp-[(3→1)-3-O-Methyl-Araf](4→1)-GlcAp-(4→).

One of the earliest studies on exopolysaccharides include that of Bishop et al.

(1954) on Anabaena cylindrica which was shown to have glucose, galactose,

arabinose, xylose, rhamnose and uronic acids, but based on the extraction procedure

that they used it can not be distinguished whether this was only slime or both slime

and capsule, if any.

(b) Released EPS

Released EPS are the ones that are obtained from the supernatant after

removal of cells. Many cyanobacteria secrete polysaccharides soluble in the medium

and/or as a gelatinous layer around the cell. This imparts specific rheological behavior

to the medium. Perhaps this prompted Martin and Wyatt (1974) to classify Nostoc

31

species on the basis of the amount of slime that they produce, which may be extracted

by alcohol precipitation from cell free supernatants. Some of the strains in their study

could not produce any slime at all. The amount of slime was suggested to be

important for the motility of the cyanobacterium.

According to de Philippis et al. (2000) composition of the polysaccharides

released by the PCC Nostoc strains showed presence of uronic acids thus conferring

the acidic nature as evidenced by Alcian blue staining. The concentration of uronic

acids in the RPSs ranged from less than 1% to about 30% of the RPS dry weight.

Most RPSs also showed the presence of hexosamines at rather high concentrations,

exceeding 3.3% of RPS dry weight. The composition of the RPSs released by the

three symbiotic strains were characterized by low level of protein and a high level of

uronic acid content, while the strains previously described produced polysaccharides

with a low content of uronic acids and a high content of protein which was required

for good adhesion to plant roots (Gantar et al., 1995).

In Nostoc, slime was produced to the extent of 14-18% of its cell mass (Mehta

and Vaidya, 1978). It was observed that the amount of slime depended on cultutre

conditions as well as fermentation time. It contained glucuroninc acid, glucose and

xylose unlike the monosaccharides in capsule (as in the section on capsule) thus

implicating that excretion of exopolysaccharide involved some selective mechanism.

Similarly in Nostoc calcicola Geitler, strain 79WA01 Flaibani et al. (1989)

could co-precipitate a polypeptide in addition to the glycuronoglycan moiety on

addition of cetyltrimethylammonium bromide to the cell-free supernatant. They could

not separate the two components by physical methods. The sugar composition of

carbohydrate component was the same as of the capsule (listed in the section of

capsule) and here also the molar ratios of each of the sugar residue varied

considerably.

Parikh and Madamwar (2006) reported xylose in Nostoc sp. and Nostoc

commune. They also reported presence of ribose in N. commune and considered it a

novel feature. Ribose is a well known component of the lipo- and capsular

polysaccharides from many Gram-negative bacteria, where it is found exclusively as

a β-furanosyl residue (Gil-Serrano et al., 1998; Lindberg, 1990; Wolucka and

Hoffmann, 1995). It raises the question as to ‘why does a polysaccharide involved in

the protection of an organism inhabiting extreme environment have a carbohydrate as

labile as ribose ?’

32

Huang et al., 1998; Potts et al.,1983; Helm et al.,2000 suggested that perhaps

the RPS moiety protects neighbouring glycosidic bonds from the more common

glycan hydrolases. In which case the selective removal of the ribose group should

leave the polysaccharide more susceptible to enzymatic depolymerisation (Huang et

al., 1998; Potts et al., 1983; Helm et al., 2000). Another suggestion was that since N.

commune is restricted to neutral /or alkaline environments, the acid-labile nature of

ribose is never an important factor. Autoclaving the crude EPS results in a decrease in

solution viscosity and free ribose was detected in the resulting aqueous solution by

TLC. This supports the possibility that ribose is partially responsible for the

gelatinous consistency of the native material (viscosity modifier).

In a qualitative study on Nostoc sp. by Huang et al., 1998, released EPS of

tested strains contained xylose and glucose but the composition varied. EPS of N.

sphaeroides contained very little carbohydrate and thus could not be considered a

polysaccharide. The EPS of N. commune and N. flagelliforme were primarily

polysaccharides, but their monosaccharide compositions were quite different from

those of their cellular polysaccharides. In another study, sheath in Nostoc commune

was reported to be composed of cellulose-like homoglucan fibrils that were cross-

linked by minor monosaccharides (Frey-Wyssling and Stecher, 1954; Hoiczyk,1998).

Polymers containing nosturonic acid or uronic acids with lactyl moieties in N.

commune DRH-1 found in the so-called “extreme environments” are suggested

playing a pivotal role in the ability of organisms to survive under harsh conditions.

Such a functional group can act as a “spacer arm” or “linker” and could aid in

adherence of the EPS to inorganic or organic surfaces (biofilms)/or allow covalent

attachment of UV-absorbing pigments on adjacent polysaccharide chains (molecular

scaffold). Although lactyl-containing uronic acids were reported earlier in the

cyanobacteria Cyanospira capsulata (Garozzo et al., 1998), the 3-O-lactyl glucuronic

acid described by Helm et al. (2000) was reported in RPS only once before in the

exopolysaccharide produced by a strain of the bacterium Alteromonas (Dubreucq et

al., 1996).

Moore and Tischer (1965) and Wang and Tischer (1973) reported secretion of

two exopolysaccharides by Anabaena flos-aquae A37 to the yield of 250 mg L-1 of

the medium. Both polymers one neutral and the other acidic had glucose as the major

sugar.

33

The neutral polysaccharide was composed of glucose and xylose (ca. 8:1

molar ratio) and the acidic had glucose, xylose, ribose and an unidentified uronic acid

(6:1:1:10). The EPS from A. circularis had neutral and keto acid residues with no

sulfates. Its secretion occurred only under certain culture conditions (Bar-Or and

Shilo, 1987).

Qualitative composition of the RPS in Westiellopsis prolifica, Tolypothrix

ceylonica and Nostoc punctiforme was studied by Saxena and Kaushik (1992). They

showed that it comprised of galactose/fructose, arabinose, maltose, N-acctyl-N-

glucosamine and two unidentified sugars, including an unsaturated one. The presence

of galactose in Nostoc muscorum, fructose in Hapalosiphon fontinalis galactose and

arabinose in Tolypothrix ceylonica polysaccharides have been reported earlier as

sugar constituent of complex polysaccharides.

The crude polysaccharide preparation from Nostoc 2S9B (mucilaginous

sheaths) contained 2.8% (w/w) protein while in Anabaena C5 it was only 0.6%

(Ganter et al., 1995). The purified exopolysaccharide from Nostoc 2S9B contained

three neutral sugars and glucuronic acid, whereas fucose and uronic acid were the

main components of that from Anabaena C5. Removal of protein fraction by phenol

or pronase treatment in Nostoc 2S9B leads to the loss of the ability to adhere to the

root surface.

According to Plude et al., 1991 Anabaena flos-aquae synthesizes two

different RPS, a xyloglucan containing glucose and xylose in a molar ratio of 8:1 and

a more complex polysaccharide containing uronic acid, glucose, xylose and ribose in

the molar ratio of 10:6:1:1.

In Anabaena sp. ATCC 33047 the molecular weight of extracellular polymer

was estimated to be 1.35 MDa (Moreno et al., 2000). The RPS produced during the

stationary growth phase in batch culture was a heteropolysaccharide with xylose,

glucose, galactose and mannose (11:5:5:1) being the main neutral sugars (Moreno et

al., 2000). The uronic acid content of most cyanobacterial exopolysaccharides is

about 20% to 30%, similar to that found in Anabaena. Its carboxylate groups

conferred the EPS a polyelectrolyte nature that serve as binding sites for divalent

metal ions.

Two planktonic cyanobacteria, Anabaena sp. N1444 and Anabaena sp. PC-1,

and a green eukaryotic alga, Scenedesmus sp., produced extracellular flocculants. The

flocculant of Anabaena PC-1, when purified, was found to be a macromolecular

34

polysaccharide consisting of neutral sugars, uronic acids, and proteins, but not keto

acids, hexosamines nor fatty acids Choi et al. (1998).

Three of the five sections of cyanobacteria show genera exhibiting cellulose

biosynthesis (Nobles et al., 2001). It is found in slime tubes, sheaths and extracellular

slime, the three major classes of extracellular polysaccharides in the cyanobacteria.

The presence of cellulose in the sheath of Scytonema hofmanni has been reported by

Nobles et al. (2001). This is the first report of cellulose in slime tubes of motile

cyanobacterial trichomes. Based on the results of x-ray diffraction, electron

microscopy of microfibrils and cellobiohydrolase I-gold labeling, he reported the

occurrence of cellulose biosynthesis in nine species representing three of the five

sections of cyanobacteria viz. Oscillatoria sp. UTEX 2435, Nostoc sp. UTEX 2209,

Gloeocapsa sp. UTEX L795, S. hofmanni UTEX 2349, Anabaena sp. UTEX 2576, P.

autumnale UTEX 1580A, Synechocystis sp. and Oscillatoria princeps.

(c) Capsule

In 1985, Florenzano et al. observed that Cyanospira rippkae did not possess a

capsule, whereas C. capsulata (ex Anabaena spiroides) produced a thick layer of

capsule. This capsule was claimed to account for >90% of the total biomass

carbohydrates in C. capsulata.

Ruangsomboon et al. (2007) reported carbohydrates and protein (37.6%,

34.7% of total CPS, respectively) as the major components of capsular polysaccharide

(CPS) in Calothrix marchica. The neutral sugars in it were xylose, arabinose, ribose,

rhamnose, galactose, glucose, mannose and fructose. Acidic sugars including

galacturonic and glucuronic acids were also found.

The oligosaccharides released on subjecting to partial acid hydrolysis and N-

deacetylation-nitrous acid deamination, exocellular polysaccharide from Cyanospira

capsulata, the polysaccharide appeared to be an octasaccharide repeating unit

(Garozzo et al., 1998) with following structure:

35

2.5.3 Filamentous nonheterocystous cyanobacteria

(a) Released EPS

In Spirulina platensis, the soluble exocellular polysaccharide was reported to

be a primary metabolite. It was formed of ten different types of monomer units

including six neutral sugars (xylose, rhamnose, fucose, galactose, mannose and

glucose in the proportions 1.3/0.3/0.7/2.7/traces/2), two unidentified sugars, two

uronic acids and sulphate groups accounting for 40% and 5% respectively of the mass

of the molecule. This polysaccharide displayed a non Newtonian behaviour and a

strong pseudoplastic characteristic that could be attributed to its polyelectrolytic

property (Filali Mouhim et al., 1993).

Sodium spirulan (Na-SP)- a sulfated polysaccharide isolated from Spirulina

platensis consisted of two types of disaccharide repeating units, O-hexuronosyl-

rhamnose (aldobiuronic acid) and O-rhamnosyl-3-O-methylrhamnose (acofriose) with

sulfate groups, other minor saccharides and sodium ion (Toshiyuki et al., 2002).

Zhen-Yuan et al. (2002) characterized polysaccharides of Spirulina

(Arthrospira) maxima in a nitrogen-limited batch culture using GLC. Xylose,

rhamnose and glucose were the main sugars in the released polysaccharide. Two

uronic acids represented by glucuronic acid and galacturoic acid while six other

neutral monosaccharides including fucose, mannose, galactose and two possible

unidentified sugars were also found.

The EPS from Phormidium 94 a, had a molecular weight of 2,000 kDa while

that from Phormidium J-1 is 1,200 kDa (Bar-Or and Shilo, 1987) The highest

molecular mass values were in the range of 1,400–2,800 kDa (de Philippis and

Vincenzini, 1998). In Phormidium 94 a two other fractions (1,600 kDa and 12,000

36

Da) are also reported. Mac Cormick et al. (1996 suggested that the sample may be a

mixture of more than one polysaccharide.

The sheath of Phormidium uncinatum contains cellulose-like homoglucan

fibrils that are cross-linked by minor monosaccharides (Frey-Wyssling and Stecher

1954; Hoiczyk, 1998).The monosaccharide most frequently found in the

cyanobacterial exopolysaccharides is glucose (in more than 90% of the polymers),

although it is absent in Phormidium 94 a and in Phormidium J-1 (Bar-Or and Shilo,

1987) (Vicente-Garcia et al., 2003).

Phormidium sp. J-1, isolated form a drainage canal of Israel was found

producing a high molecular weight (1.2x106 D) exocellular polysaccharide called

emulcyan (Fattom and Shilo, 1984; Fattom and Shilo, 1985; Bar-Or and Shilo, 1987).

It had rhamnose, mannose and galactose (1:2:0.5 respectively) with a uronic acid that

was none of the following three- galacturonic, glucuornic or mannuronic acid.

Despite purification, it was found linked with proteins and fatty acids and contained

sulfate groups that were in fact responsible for its flocculating behavior as desulfation

lead to an 80% loss of flocculant activity.

In Oscillatoria sp. Parikh and Madamwar (2006) reported xylose and 6.5 to

7% ribose.

(b) Capsule

Zhen-Yuan et al. (2002) working on Spirulina (Arthrospira) maxima in

nitrogen-limited condition reported glucose to be the only monosaccharide in storage

granules, the cell wall and the most predominant one in the external layers of the cell.

Glucuronic acid, galacturoic acid, fucose, rhamnose, xylose, mannose, galactose,

glucose and two unidentified sugars were also found in the external layers of the cell.

2.5.4 Unicellular cyanobacteria

(a) Non descript

An exopolysaccharide from Microcystis flos-aquae contains levels of uronic

acid as high as 83% of the dry weight (Bender et al., 1994; Bertocchi et al., 1990;

Plude et al., 1991). As per Sutherland and Tait (1992) its composition is similar to that

of pectin, containing up to 83% galacturonic acid. Information about whether its

structure also resembles that of pectin is not yet available.

37

(b) Released EPS

The released polysaccharide from a halophilic Aphanothece halophytica

GR02 was separated into two main fractions by anion-exchange chromatography by

Li et al. (2001). The major fraction with an apparent molecular weight above 2.0 ×

106 Da consisted of glucose, fucose, mannose, arabinose and glucuronic acid. The

minor fraction consisted of rhamnose, mannose, fucose, glucose, galactose and

glucuronic acid with traces of arabinose. Methylation and GC-MS spectrometry

analyses of the major fraction revealed presence of 1-linked glucose, 1,3-linked

glucose, 1,3-linked fucose, 1,4-linked fucose, 1,3-linked arabinose, 1,2,4-linked

mannose, 1,3,6-linked mannose, 1-linked glucuronic acid and 1,3-linked glucuronic

acid residues. The major fraction was thought to originate from capsular

polysaccharide.

In a Cyanothece sp. Parikh and Madamwar (2006) reported xylose and 6.5 to

7% ribose.

Cyanobacterial strain Synechocystis aquatilis Sauvageau B90.79 was found to

release a fucose-rich sulfated polysaccharide, which was mainly composed of

arabinose (45%) fucose (47%) and had a degree of sulfation of 0.43. (Volk et al.,

2006).

In a study on Thorium (IV) binding exopolymers of marine microorganisms,

Nicolas (2004) found that the principal simple sugars in Synechococcus elongatus

were glucose, galactose, xylose galactoglucuronic acid.

(c) Capsule

According to Ruangsomboon et al. (2006) xylose, arabinose, ribose,

rhamnose, galactose, glucose, mannose and fructose were the neutral sugars present

in capsular polysaccharide (CPS) of Gloeocapsa gelatinosa. Acidic sugars including

galacturonic and glucuronic acids were also found in CPS. The amount and

composition of CPS varied according to its growth phase and culture conditions.

The slime material from Microcystis aeruginosa K-3A was found containing

66.9% (w/w) carbohydrate and 12.8% protein. Chemical analyses showed that the

carbohydrate was a heteropolysaccharide containing galacturonic acid, rhamnose and

xylose as the major constituents in addition to the sugars, fucose, arabinose, mannose,

galactose and glucose (Nakagawa et al., 1987).

Six cyanobacterial isolates recovered from Polynesian microbial mats by

38

Richert et al. (2005), called “kopara,” were cultured using laboratory-closed

photobioreactors. They were shown to produce exopolymers as released and capsular

exopolysaccharides. These polymers have been chemically characterized using

colorimetric elemental assays, infrared spectrometry and gas chromatography. Both

capsular and released EPS consisted of 7 to 10 different monosaccharides with neutral

sugars being predominant.

2.6 FACTORS AFFECTING EPS PRODUCTION

Researchers have suggested that each algal strain should be carefully tested in

order to envisage the right culture strategies aimed at optimizing polysaccharide

production. Because most cyanobacterial strains have only been tested in small

culture devices and in studies not oriented to maximizing its production, they attain

only moderate levels of polysaccharides except Cyanospira capsulata and some

Cyanothece strains (de Philippis et.al., 1991; de Philippis et.al., 1995).

2.6.1 Age

There is seemingly an absence of a common behaviour of polysaccharide

release among the polysaccharide-producing cyanobacteria described so far. Various

nutritional and environmental factors seem to control which type of exopolysaccharide

is formed by the Phormidium filaments. As in Gloeothece sp. (Tease and Walker,

1987) the sheath of Phormidium appears not to be a stable cell structure, and the

ability to form a sheath was frequently lost during repeated subculture, whereas the

ability to secrete slime was invariably found as long as the filaments displayed gliding

motility. The amount and composition of capsular polysaccharide (CPS) of

Gloeocapsa gelatinosa varied according to its growth phase and culture conditions

(Ruangsomboon et al., 2006). Gantar et al. (1995) reported that the mucilaginous

sheath of Nostoc 2S9B is mainly synthesized in the aseriate stage of the development

cycle and then released as empty shells, at the same instance when hormogonia are

liberated. Some other species viz. Cyanothece sp. BH68K, N.calcicola and

Phormidium sp. J-1, show a significant release of polysaccharides in the late

exponential growth phase, suggesting that cyanobacteria produce polysaccharides as a

typical secondary metabolite (de Philippis and Vincenzini, 1998). On the contrary, the

polysaccharides released by Spirulina platensis may be regarded as a primary

metabolite evident in the fact that polysaccharide production parallels biomass

39

production (Filali Mouhim et.al., 1993). This result is consistent with that of Mehta

and Vaidya (1978), in which the highest rates of polysaccharide synthesis and release

are achieved by young cultures in a Nostoc strain.

The kinetics of polysaccharide release has been considered as a complex

dynamic equilibrium among different processes, i.e. trichome elongation and akinete

germination (Vincenzini et.al., 1990). It has been concluded that the polysaccharide

synthesis is primarily directed toward the formation of the capsule, and when

trichome fragmentation and akinete differentiation takes place the polymer is released

into the culture medium. There are also reports that the sugar composition varies

quantitatively as well as qualitatively with the age of the culture (Panoff et.al., 1988),

however the released polysaccharide (RPS) from a halophilic cyanobacterium

Aphanothece halophytica GR 02 (Li et al., 2001) obtained at different ages of culture,

showed no striking variations in the monosaccharide composition and the relative

proportions of the monosaccharides.

In marine unicellular red alga Porphyridium sp., production of the cell wall

polysaccharide is enhanced during the stationary phase of growth, as has also been

found in other algae producing extracellular polysaccharides, as Chlamydomonas

mexicana (Kroen and Rayburn, 1984; Arad et al., 1988). The dependence of CPS

production on growth stage was also reported in red algae Porphyridium sp.

Exocellular polysaccharide production by Porphyridium cruentum was studied by

Thepenier and Gudin (1985) in fed-batch cultures. Growth of the culture was

characterized by a brief log phase, followed by a linear type phase. The duration and

the slope of the latter phase are largely dependent on culture conditions. The presence

of polysaccharide in the culture medium was observed as soon as the log phase was

over but the quantity did not become significant until the end of growth.

Polysaccharide production depended on the cell number and the specific productivity.

The cell number was affected mainly by the initial nitrogen and phosphorus

concentrations and the specific productivity by the aeration and agitation systems.

Under photoperiodic illumination the oscillations in cell volume were observed after

the log phase indicating at a synchronization of polysaccharide synthesis and/or

excretion with the light period and the solubilization with the dark period.

40

2.6.2 Culture condition and Medium composition

In cyanobacterial cultures, the production of polysaccharides appears to

depend on the culture conditions. A change in light intensity, temperature and the

concentrations of sulfur, iron, phosphate, and potassium affect polysaccharide

production (Sutherland, 1982; Myklestad et al., 1995; de Philippis and Vincenzini,

1998). Under nutrient starvation, especially under nitrate and sulfate limitation,

Synechococcus produces a large amount of exopolysaccharides (Adhikary, 1998).

However, the exudation of polysaccharide under special culture conditions is

demonstrated only for phycocyanin-rich cultures, suggesting different metabolic

pathways for phycocyanin and phycoerythrin rich Synechococcus species. More

important, the formation of substitutents of the polysaccharides, which may play a

crucial role in determining the physicochemical properties of the cyanobacterial

polysaccharides, can be manipulated by changing the growth conditions (Ehling-

Schulz and Scherer, 1999; Cesaro et.al., 1991).

The response to nitrogen starvation, is species specific in many cases and

depends on the nitrogen sources used (Lama et al., 1996; Tischer and Davis, 1971). In

some nitrogen fixing species, the production of polysaccharides is reported to be

associated with carbon metabolic flux or an impairment of balanced growth (Morvan

et al., 1997; de Philippis et al., 1991). Nicolaus et al. (1999) reports that the dramatic

decrease in polysaccharide yield occurs when the Phormidium sp. is grown with a

light dark cycle, in the absence of aeration and phosphorus. Likewise, the increase in

P and N in the medium, and the absence of combined nitrogen, have a similar effect

on the yield, giving rise to a one third decrease with respect to the amount obtained

under standard growth conditions. While phosphorus (P) is an abundant element in the

Earth’s crust, its availability can limit the growth of organisms present in both aquatic

and terrestrial environments. P is essential for many fundamental processes that

sustain life, including nucleic acid synthesis, membrane synthesis, energy metabolism,

signaling, redox reactions, and modification of protein activities. P limitation leads to

depletion of the pool of phosphorylated intermediates in the pentosephosphate cycle,

which results in a marked reduction in photosynthetic carbon fixation (Brooks, 1996;

Jacob and Lawlor, 1993). Studies with Chlamydomonas reinhardtii have

demonstrated that P and sulphur deprivation causes a loss of photosynthetic electron

transport activity, the consequence of a combination of reduced photosystem II (PS II)

41

abundance, accumulation of PS II QB nonreducing centers, an increase in

nonphotochemical quenching, and an increase in the tendency of the cells to be in

state II (Wykoff et al., 1998).

The carbon partitioning of the epipelic diatom Cylindrotheca closterium was

studied by Alcoverro et al. (2000) in the laboratory under varying scenarios of

phosphorous and nitrogen limitation. While dissolved carbohydrate remained the

same in all the treatments, EPS presented highest values under phosphorus limitation.

The production of EPS was closely linked to the periods of carbon assimilation

(transition phase) in the nutrient depleted treatments which pointed out the relevance

of the nutrient imbalance (nitrogen or phosphorus) in the production of EPS by the

benthic or resuspended diatoms. Under condition of phosphorus limitation, diatoms

inhibit or slow down the cellular divison while photoassimilation continues. This may

result in the release of a high proportion of assimilated carbon, mainly as

polysaccharides (Kuhl, 1968; Myklestad and Haug, 1972; Palmisano and Sullivan,

1985; Myklestad et al., 1989, Monti et al., 1992, Myklestad, 1995).

For Spirulina, on the other hand, lowering the nitrogen content and nitrogen

deprivation causes a strong increase in the total amount of polysaccharides even in the

absence of phosphate (Nicolaus et al., 1999). This finding has been confirmed in

polysaccharide analyses of three species of Nostoc (Huang et al., 1998), indicating

that combined nitrogen in the media has qualitative influence on the composition of

extracellular polysaccharide, but not on intracellular polysaccharides. It is noticeable

that the conclusions drawn from different researches working on different species or

strains are often confusing and sometimes even contradictory (Nicolaus et al., 1999).

The ambiguity in polysaccharide production is explained as the differences in

physiological roles played by the exocellular polysaccharides in different strains (de

Philippis and Vincenzini, 1998). For instance, shortage of metallic ions enhanced

secretion of polysaccharides, where it functions as a chelating agent for cations

essential for cell life. Likewise, when nitrogen limitation or starvation stimulates

polysaccharide release, the polymer may act as a product of overflow metabolism

which is exuded to allow cells to get rid of the carbon excesses (Dawes, 1986).

Another explanation is that nitrogen starvation or limitation causes a decrease in

photosynthetic pigments, leading to the decrease in photosynthetic efficiency, and

meanwhile, the response of microalgae to nitrogen deficiency is to degrade N-

containing compounds inside the cells, giving rise to the decrease in nitrogen content

42

and increase in carbohydrates and fatty acids (Morvan et al., 1997). Another approach

to increasing the amount of polysaccharide release is to supplement the algal culture

with intermediate metabolites such as glyoxylate in glycometabolism. Carbon flux

was altered by transferring the organism either to an argon atmosphere or to medium

containing the nitrogen assimilation inhibitors L-methionine-D, L-sulfoximine

(MSX), O-diazoacetyl-L-serine (AZAS) or D, L-7-azatryptophan (AZAT), or by

adding glyoxylate, known to stimulate carbon metabolism (de Philippis et al. 1991;

1996). When carbon flux is modified by interfering with nitrogen metabolism, the

concentration of total carbohydrates exceeded that of the control in the heterocystous

cyanobacterium Cyanospira capsulata. A 43% increase in EPS production was

observed on single addition of glyoxylate (30 mM) or by daily additions of 10 mM

glyoxylate without interfering with nitrogen metabolism. Thus a mere diversion of

carbon flux from the process of nitrogen assimilation, favours the synthesis of carbon

reserves.

Hoa et al. (2003) evaluated the effect of varying nutrient ratios of nitrogen and

phosphorus over EPS production and observed that EPS differed significantly with

variation in nitrogen concentration. However, this ratio was unaffected by different

phosphorus content.

Changes in pH and salinity over a wide range had little effect on the viscosity

and stability of EPS produced by marine bacteria in a study by Boyle and Reade

(1983). In Cyanospira capsulata, higher pH (10.7) did not show any significant effect

on both EPS productivity and cell viability (de Philippis et al., 1991). Saxena and

Kaushik (1992) reported enhancement of biopolymer production in Nostoc

punctiforme ARM 431, Tolypothrix ceylonica ARM 397 Westeillopsis prolifica ARM

365 with the increase in salt concentration. However growth was also adversely

affected by the salt concentration.

An exopolysaccharide, known as mauran, is produced by the halophilic

bacterium, Halomonas maura (Arias et al., 2003). When this strain was grown in

media containing salt (2.5%, w/v), it produced a high molecular weight (4.5 x 106

daltons) EPS that showed similarity in composition to that produced by Antarctic

marine isolates. It contained glucose, mannose and galactose as well as high amounts

of glucuronic acid (8%, w/w) and sulfates (6.5%, w/w) and was stable to salt, freezing

and thawing.

43

In Cyanospira capsulata, (de Philippis et al., 1991) the highest EPS yield was

obtained with the culture grown under Mg2+ deficiency. Under this condition, protein

synthesis was strongly hindered and the specific rate of EPS release was much higher

than in the other growth conditions. However, deficiencies of Ca2+ or PO3/4 – as well

as the addition of sodium chloride up to 0.35 M had no significant effects on EPS

production or protein synthesis. Polysaccharide released by a halophilic

cyanobacterium Aphanothece halophytica GR 02 (Li et al., 2000) exhibited gelling

properties and strong affinity for metal ions. The composition and ratio of galactose

and rhamnose was altered with salt concentration.

The highest amount of acidic sugars was produced in Gloeocapsa gelatinosa

when cultured under low light intensity (Ruangsomboon et al., 2006).

Sangar and Dugan, (1972) reported extensive accumulation of extracellular

polysaccharide as compared with capsular polysaccharide on agitation of culture of

Anacystis nidulans during growth and while the same has been reported with

Anabaena flos-aquae by Moore and Tischer (1964).

2.6.3 UV radiation

Long term exposure to UV-B is also known to induce synthesis of

extracellular polysaccharide in Nostoc commune DRH1 (Ehling - Schulz et al., 1997).

The UV absorbing mycosporines induced simultaneously are located in glycan sheath

(Bohm et al., 1995; Scherer et al., 1988) subsequently found non covalently attached

to the glycan thereby providing protection against radiation (Bohm et al., 1995; Hill

et al., 1994).

2.7 OPTIMIZATION OF EPS PRODUCTION

Optimization of culture conditions like growth medium composition, pH,

temperature, oxygen is a primary requirement in order to increase EPS production and

to determine the key factors involved in its synthesis. Exopolysaccharide producing

Pseudomonas and Arthrobacter strains isolated from soil showed maximal

polysaccharide yield when the concentration of the carbon source was lower than 2%.

Increasing the initial pH of the medium from 5.0 to 7.0 increased polysaccharide

production. Temperature 30º C showed maximum production, while higher and lower

temperatures had a negative effect on polysaccharides production (Bueno and Garcia-

cruz, 2006).

44

The carbon source added to the medium plays an important role in the

production of EPS in lactic acid bacteria (LAB), the total amount of polysaccharides

produced being strongly influenced by the added sugar. Production of EPS of

Lactobacillus casei CG11 in basal medium containing glucose was higher than in the

same medium containing lactose and galactose. In L. casei CRL87 EPS production

was 1.7 fold higher in galactose than glucose. Lactobacillus lactis sp. cremoris B40

produced higher amounts of EPS in glucose than in fructose. This was related to the

low activity of the enzyme fructose-6-phosphate, which catalyzes the conversion of

fructose-1,6-diphosphate into fructose-6-phosphate, an essential step in the

biosynthesis of sugar nucleotides from fructose but not from glucose (Raus-Madledo

and Reyes-Gavlian, 2005).

In bacterial strains RDL1 and RDL2, isolated from textile dyeing industry

named as Rainbow Denim Ltd. (Punjab, India) showed that carbon sources glucose

and fructose were effective for flocculant EPS production and as well for cell growth.

Organic nitrogen sources urea and peptone appeared favorable for both cell growth

and EPS production at an initial pH of 7.5 (Lachhawani, 2005).

Influence of different nutritional factors on the growth and EPS production by

Cyanothece sp. ATCC51142, Nostoc sp. and Oscillatoria sp. indicating the growth

linked EPS production, that higher the growth better the EPS production (Parikh and

Madamwar, 2006).

In Lactobacillus rhamnosus C83, an optimization of growth medium

composition led to an improvement in both growth and EPS production. The EPS was

stimulated three to four times respectively in the presence of 2% (w/v) glucose or

fructose or 4% (w/v) mannose when compared with the control. The inverse relation

between EPS yield and temperature were observed. The lower the temperature, the

higher the EPS yield. The EPS production was 37% higher when L. rhamnosus C83

was incubated at 25ºC instead of 37ºC (Gamar et al., 1997).

Berg et al. (1995) conducted a study evaluating the effects of temperature and

pH on EPS production by Lactobacillus sake 0-1. They found by using a one-

variable-at-a-time (OVAT) approach that optimal EPS production occurred at 20ºC

and pH 5.8. It was confirmed that unfavorable culture conditions might trigger slime

production by the cells as a form of self protection.

45

2.8 BIOPOLYMER YIELD

Various cyanobacteria and other bacteria grown either photoautotrophically or

in normally required growth conditions in liquid

cultures have been tested for the presence of exocellular polysaccharidic investments

surrounding the trichomes or vegetative cells. In Gloeothece magna, Mohamed (2001)

reported that the CPS production of 15.5 mg g-1 biomass.

Lewin (1956) studied green algae that produced extracellular polymer ranging

from 3 to 113 mg L-1, whereas mucilaginous species of green and bluegreen algae

analyzed by Moore and Tischer (1964) produced the polymers in concentrations from

174 to 557 mg L-1 of growth medium. In Anacystis nidulans Sangar and Dugan (1972)

determined the amount of extracellular polysaccharide gravimetrically after ethanol

precipitation and was 366 mg L-1 of growth medium after 21 days at 25oC while

capsular and water-soluble intracellular polysaccharide accounted for 10 and 15 mg

per liter, respectively.

Subsequent experiments with Anabaena flos-aquae after optimization gave an

average of 414 mg of extracellular polysaccharide L-1 and 12.5 mg of capsular

polysaccharide L-1.

Batch cultures of Cyanospira capsulata grown for 30-35 days in small open

ponds under continuous illumination and nitrogen fixing conditions on the standard

culture medium without pH control showed EPS productivities ranging from 5.6 to

6.0 g EPS m-2 day-1. (de Phillippis et al., 1991). These yields are well comparable

with those reported for other microalgae as Porphyridium sp. (Vonshak et al., 1985;

Arad et al., 1988; Thepenier et al., 1988), which is currently proposed for

polysaccharide production, indicating that C. capsulata can be regarded as a

promising polysaccharide producer.

Azotobacter vinelandii, grown in laboratory fermentor (Brunswick fermonters)

in a nitrogen-free, mineral broth medium with 2% sucrose , showed the yield of crude

slime polysaccharide, recovered after treatment with alcohol after 5 days growth was

740 mg per liter of supernatant culture medium (Cohen and Johnstone, 1964).

Various organisms with their respective biopolymer yield are tabulated in

table 2.7.

46

Table 2.7: Cyanobacteria, bacteria and their biopolymer yield

Organism Biopolymer yield Reference

Nostoc strain from PCC 30 to 50 mg L-1 D-1 de Philippis et al., 2000

Sphingomonas paucimobilis GS1 6.5 g L-1 Ashtaputre and Shah, 1995

Calothrix marchica 26.85 mg g-1 DW Ruangsomboon et al., 2007

Spirulina 9 µg mL-1 Nicolaus et al., 1999 Oscillatoria 1.5 µg mL-1

Phormidium 29 µg mL-1

Anabaena torulosa 3.7 µg mL-1

Anabaena sphaerica 1 µg mL-1

Anabaena strain WSAF sp. 55.2 µg mL-1

Anabaena variabilis 3.5 µg mL-1

Nostoc linckia 0.015 µg mL-1

Scytonema hofmanni 4 µg mL-1

Tolypothrix tenuis 7.4 µg mL-1

Fischerella muscicola 1.5 µg mL-1

Chlorogloeopsis sp. strain 6912 30 µg mL-1

Gloeocapsa gelatinosa 33.01 µg mL-1 Ruangsomboon et al., 2006

Lactic acid Bacillus (LAB) 60-400 mg L-1 Stingele et al., 1996

Westiellopsis prolifica 3.44 mg mg-1 DW

(0 mM NaCl)

14.93 mg mg-1DW

(400 mM NaCl)

Saxena and Kaushik, 1992

Synechocystis sp. 6803 and 6714 160-350 mg L-1 Panoff et al., 1988

Cyanospira capsulata ATCC

43193

3 g L-1. 9D-1 Vincenzini et al., 1990;

Florenzano et al., 1985

Anacystis nidulans 366 mg L-1. 21D-1 Sangar and Dugan, 1972

Cyanospira capsulata 32 mg L-1 D-1 de Philippis et al., 1993

Anabaena halophytica MN-11 47 mg L-1 D-1 Sudo et al., 1995

Nostoc insulare 54.79 36 mg L-1 D-1 Fischer et al., 1997

Anabaena floc aquae A 37 20 mg L-1 D-1 de Philippis and Vincenzini,

1998

Nostoc commune, N. sphaeroids 30 to 40 % DW Huang et al., 1998

Nostoc flagelliform 10 to 20 % DW

Microcystis aeruginosa K-3A 7.2% DW

Nakagawa et al., 1987

47

2.9 PROPERTIES OF POLYSACCHARIDES

The physical properties of the polysaccharides depend on their composition

and structure. Slight changes may cause considerable differences in physical

properties (Table 2.8). Acetyl groups play a major role in determining some of the

physical properties of bacterial exopolysaccharides (Sutherland, 1997, 1999b). Some

bacteria are capable of producing esterases which show wide specificity, removing

acyl groups from several bacterial polymers as well as from other esters (Cui et al.,

1999). Deacylation of the bacterial polysaccharide succinoglycan was shown to

improve pseudoplasticity in aqueous solution and an enhanced cooperativity of the

order-disorder transition (Ridout et al., 1997). On the other hand, deacylation of some

polysaccharides may lead to loss of any ordered conformation (Villain-Simonnet et

al., 2000).

Some polysaccharides preferentially bind ions from a mixture. Many undergo

transition from an ordered state at lower temperatures and in the presence of ions, to a

disordered state at elevated temperature in a low ionic environment (Nisbet et al.,

1984). In case of some polymers this represents change from a gel to a sol state.

EPS includes sulfate (Leppard et al., 1996), uronic acids (Decho, 1990) as

galacturonic acid, acetyl group, carboxyl group, succinyl group. These groups convey

an overall polyanionic quality to the EPS. The overall negative charge gives the

molecule a ‘sticky’ quality. The anionic charge is important in terms of the affinity of

these EPS for binding to cations such as dissolved metals (Brown and Lester, 1982).

Korstgens et al. (2001) reported, biofilms formed by Pseudomonas aeruginosa that

were dominated by polysaccharides with carboxyl groups. Calcium, copper and iron

provided stability to the network by acting as bridging ions. The presence of calcium

and magnesium salts resulted in increased viscosity in solutions of polysaccharide

from marine bacteria stored at low pH (Boyle and Reade, 1983). Divalent cations

provide stability to the polysaccharide gel matrix (Decho, 1990). Recent work with

the purified EPS from P. aeruginosa showed that there were strong electrostatic

interactions between divalent cation manganese and the carboxylate groups occurring

along the EPS chain.

48

Table 2.8: Effect of polysaccharide composition on physical properties

Polysaccharide

component

Effect Properties affected Example

Neutral sugars Uncharged Insoluble Cellulose, Biofilm EPS

Uronic acids Polyanionic Solubility, Ion binding Xanthan, Alginates

Pyruvate Polyanionic Ion binding, Transition Xanthan, Galactoglucans

Methyl pentoses Lipophilicity Soubility Biofilm EPS

Acetylation Solubility Gelation, Reduced ion

binding

Alginates, Gellan etc

Side-chains Various Solubility Scleroglucan, xanthan

1,3 or 1,4-Linkages Rigidity (In)solubility Curdlan, Cellulose

1,2 Linkages Flexibility Solubility, Stability Dextrans

This ‘stickiness’ is important in terms of the affinity of these EPS for binding

of cations such as dissolved metals (Brown and Lester, 1982). The EPS produced by

Antarctic bacterial isolates examined by Mancuso Nichols et al. (2005) appear to be

polyanionic and, therefore, ‘sticky’ with respect to cations such as trace metals.

The sulfated EPS-emulcyan from Phormidium sp. J-1 showed flocculating

activity (Fattom and Shilo, 1984; Fattom and Shilo, 1985; Bar-Or and Shilo, 1987).

Flocculating polysaccharides were also reported in the slime of Anabaenopsis

circularis and Calothrix desertica by Bar-Or and Shilo (1987). Cultures of two

different strains of Synechocystis sp. PCC 6803 and PCC 6714 (Panoff et al., 1988)

were anionic and showed gelling properties. Their composition showed a large

number of monosaccharides with sulphate residues. Cyanospira capsulata ATCC

43193 from an alkaline soda lake of Kenya, (Vincenzini et al., 1990; Florenzano et

al., 1985) produced a polysaccharide with viscoelastic properties (Navarini et al.,

1990) that were comparable to the microbial polymers already available in the

market.

49

2.10 FUNCTIONS OF EPS

Multiple roles have been suggested for EPS (Mazor et al., 1996). de Philippis

and Vincenzini (1998) have described the role of EPS in the attachment of cells to

sediment in benthic cyanobacteria, in facilitating the homogeneous dispersion of

trichomes, in the protection of nitrogenase from the harmful effects of oxygen.

Production of outer investments is a stable feature (de Philippis and Vincenzini, 1998;

de Philippis et al., 2000) while in some cases they may be synthesized as a

contrivance to stress. They serve as a protective boundary between the cell and its

immediate environment (de Philippis and Vincenzini, 1998). The cells embedded in

EPS in Nostoc colonies are highly desiccation tolerant (Tamaru et al., 2005). It plays

an important role in maintaining the structure and function of biological membranes

during desiccation (Dodds et al., 1995). EPS containing organisms exhibited

resistance to ultraviolet irradiation (Sinha et al., 1995). EPS aggregates soil particles

and is important for moisture retention (Flaibani et al., 1989). In N. commune it gives

resistance to heat in dry state (Helm et al., 2000). They play a major role in

determining the physical properties and structure of the microbial agglomerations

(Sutherland, 2001).

Salt tolerance of Microcoleus vaginatus Gom., a cyanobacterium isolated

from desert algal crust, was enhanced by exogenous carbohydrates (Chen et al.,

2003). Production of EPS responds to the growth regimen in some Nostoc species.

EPS may serve as a sink for excess fixed carbon under unbalanced C/N metabolism

conditions (Otero and Vincenzini, 2003; 2004). Some roles of exopolymers in

microorganisms are briefed in Table 2.9.

50

Table 2.9: Biological functions of EPS (modified after Wingender et al., 1999; Wolfraardt et al., 1999)

Function Relevance

Adhesion to surfaces Initial step in surfaces colonization, accumulation of bacteria on nutrient-rich surfaces (Tsuneda et al., 2003).

Protective barrier Resistance to pathogens (Bouarab et al., 1999; Potin et al., 1999) and grazers, non-specific and specific host defence, resistance to certain biocides including disinfectants and antibiotics.

Cell-to-cell recognition Symbiotic relationships with plants animals, initiation of pathogenic Processes.

Structural elements of biofilms

Mediation of biofilms mechanical stability, determination of the shape of EPS structure (capsule, slime, sheath).

Retention of water Prevention of desiccation under water-deficient conditions tolerance to desiccation (Hill et al., 1997; de Philippis and Vincenzini, 1998; Potts, 1999).

Sorption of exogenous organic inorganic compounds

Scavenging accumulation of nutrients from the environment, sorption of xenobiotics and toxic metal ions (detoxification), hypersalinity (Liu and Buskey, 2000), Promotion of polysaccharide gel formation.

Trace metal toxicity reduction

Under low water conditions, organisms have to face a high concentrations of dissolved molecules (Christensen, 1989; Pistocchi et al., 2000; Croot et al., 2000; de Philippis et al., 2001; Lombardi et al., 2002).

Enzyme activity Accumulation/retention and stabilization of secreted enzymes. Digestion of exogenous macromolecules for nutrient acquisition.

Protection from swelling and shrinkage

Inundation and desiccation exert very high pressure on cell envelop, ECP slows down the process and reduces its impact (Caoila et al., 1993).

UV protection Contain pigments that screen UV radiations (Franklin and Forster 1997; Ehling-Schulz and Scherer, 1999; Potts, 1999; Gröniger et al., 2000; Wolfe, 2000; Zimmer and Butman, 2000).

Prevention of soil erosion This act prevents unnecessary blowing away of the population along with sand (Falchini et al., 1996).

2.11 ROLES OF EPS IN ENVIRONMENT

Exopolymer production may require an energy expenditure of up to 70% and

this amounts to a significant carbon and energy investment for the bacterial cell

(Harder and Dijkhuizen, 1983; Wolfaardt et al., 1999). However benefits derived

from exopolymer production enhance the growth and survival of microbes and the

51

complex communities in which they are found (Wolfaardt et al., 1999). Extracellular

polymers augment the ability of microbes to compete and survive in changing

environmental conditions by altering the physical and biogeochemical micro-

environment around the cell (Costerton, 1974). In the marine environment, bacterial

exopolymers and EPS are essential in the production of aggregates (Biddanda, 1985;

Harris and Mitchell, 1973; Alldredge and Silver, 1988), adhesion to surfaces and other

organisms (Marshall, 1985; Fletcher and Floodgate, 1973; Paerl, 1975; Paerl 1976;

Vincent et al., 1994; Holmstrom and Kjelleberg, 1999), biofilm formation

(Sutherland, 2001; Sutherland, 1999), sequestering of nutrients (Decho and Herndl,

1995), as well as providing protection (Decho and Lopez, 1993; Bitton and Friehofer,

1978) and ecosystem stability (Uhlinger and White, 1983; Dade et al., 1990). The role

of microbial exopolymers in the ocean has been reviewed extensively (Decho, 1990;

Wolfaardt et al., 1999).

2.11.1 Adhesion to and colonization of surfaces

Surfaces exposed to seawater quickly adsorb and concentrate dissolved

organic compounds. Attachment to these surfaces by bacteria provides the opportunity

for growth in dilute solutions that would otherwise be unavailable (Zobell, 1943;

Paerl, 1975). Charged substrates including amino acids, sugars, fatty acids and

glycoproteins are often the first one to be concentrated on surfaces (Marshall, 1985).

Attachment can be reversible and is related to the electrostatic interactions and cell

wall hydrophobicity (van Loosdrecht et al., 1990; van Loosdrecht et al., 1987).

Irreversible binding may occur in some forms (Costerton, 1984) by cross-linking of

adjacent EPS chains (Marshall, 1980). This process is influenced by electrolyte

concentration (Fletcher, 1988). Some forms reversibly attach by secreting an

exopolymer allowing them to stick to a surface and use surface associated nutrients,

which is followed by the secretion of a second polymer, which releases the attached

bacterium (Hermannson and Marshall, 1985).

2.11.2 Biochemical interactions

Biochemical interactions between the bacteria and surrounding cells and

tissues may be made possible by exopolymeric material (Logan and Hunt, 1987;

52

Decho, 1990). Exopolymer slime and capsular material provide a biofilm matrix

around the cell. This is a hydrated layer, which can provide buffering against sudden

changes in the adjacent osmotic environment (Dudman, 1977). Such a stable

environment may aid in the localisation of secreted exoenzymes, which are essential

in the cycling of both organic and inorganic material in the marine environment

(Decho, 1990). The hydrated exopolymer matrix retains the exoenzymes activity in

close proximity to the cell thereby facilitating cellular uptake of small molecules for

metabolic conversion to energy and biomass (Decho and Herndl, 1995). Symbiotic

relationships may also occur between bacteria and other organisms. Bacteria adhere to

the site of nitrogen fixation on cyanobacterial heterocysts (Paerl, 1976) via the

heterocyst-produced EPS (Lupton and Marshall, 1984). These microzones around

cells facilitate the transfer of nutrients from one organism to another (Paerl, 1976).

The heavy metal binding properties of an EPS produced by a hydrothermal vent strain

was thought to be advantageous to the tubeworm host (Vincent et al., 1994). Members

of the genera Pseudomonas and Alteromonas produce polysaccharide-containing

exopolymers that potentially benefit the survival of other marine organisms by

facilitating attachment to surfaces (Szewzyk et al., 1991; Holmstrom and Kjelleberg,

1999).

2.11.3 Cell protection

Exopolymers may act as a physical barrier to grazers. In a study by Caron

(1987), microflagellates grazed more readily on freely suspended bacteria than those

on surfaces or enclosed in aggregates. The EPS may have provided protection to cells

within the aggregates, since the microflagellates were only able to graze the bacteria

on the surface of the aggregates. Slime exopolymer from one bacterial strain may be

preferred by consumers to the capsular exopolymer of the same strain (Decho and

Lopez, 1993). Such results suggest that these EPS may provide some buffering

against shifting environmental conditions in the natural environment. Bacteria isolated

from deep-sea hydrothermal vent communities displayed resistance to heavy metals

(Jeanthon and Prieur, 1990) and the purified EPS produced by these strains in

laboratory cultures showed very good metal binding properties (Loaec et al., 1997;

Loaec et al., 1998). Capsular polysaccharide may also provide the bacterial cells with

a protective barrier against toxic substances in the water column (Bitton and

Friehofer, 1978). In biofilm studies involving removal of organic and heavy metal

53

pollutants, exopolymeric substances removed the majority of organic pollutants while

heavy metals were taken up by the cellular fraction. These results indicated an

important role for cell wall components such as proteins in metal binding in complex

biofilm systems (Spaeth et al., 1998). These findings were confirmed in another study

that showed heavy metals were bound by cellular sorption as well as extracellularly

by polymeric substances such as polysaccharides in bacterial biofilms and microbial

flocs (Wuertz et al., 2000). Cells imbedded in the gel matrix of a biofilm are well

protected from biocidal treatments (Brown and Gilbert, 1993; McBain and Gilbert,

2001) thus current strategies to eliminate unwanted biofilms need antimicrobial agents

that can penetrate the gel matrix and target slow or dormant cells. Some success has

been achieved by incorporating transition metal catalysts into the substratum. These

generated biocidal species and killed the biofilm from the inside overcoming the

protection provided by the exopolymer matrix (Wood et al., 1998).

2.11.4 Hydrosorbing action

In natural aquatic environments, the nutrients required to support maximal

microbial growth rarely are present in sufficient quantities in the water column.

Microbial attachment to fixed surfaces, other cells and aggregates is a likely strategy

to increase the rate of substrate uptake (Logan and Hunt, 1987). Microbial cells

surrounded by a porous matrix of exopolymer sequester and concentrate dissolved

organic compounds (Decho and Lopez, 1993). The highly hydrated exopolymer

matrix act as a sponge to trap and concentrate nutrients in flowing liquids (Decho,

1990).

Nostoc commune has a marked capacity for desiccation tolerance. The cells

produce large amounts of an unusual excreted polysaccharide that contributes in many

ways to the marked stabilization of cells during prolonged storage in the air-dried

state, at low or high temperatures. First, the glycan inhibits fusion of membrane

vesicles during desiccation and freezedrying (Hill et al., 1997) and acts as an

immobilization matrix for a range of secreted enzymes which remain fully active after

long-term air-dried storage (Hill et al., 1994; Scherer and Potts, 1989; Shirkey et al.,

2000). Second, the glycan provides a structural and/or molecular scaffold with

rheological properties which can accommodate the rapid biophysical and

physiological changes in the community upon rehydration and during recovery from

desiccation. The glycan swells from brittle dried crusts to cartilaginous structures

54

within minutes of rehydration.

2.11.5 Cryoprotection

Freezing in sea ice results in brine channels where temperature is very low and

salinity is high due to brine. EPS may act as a protectant in these environments

(Krembs et al., 2002). Arctic studies (Krembs and Engel, 2001; Krembs et al., 2002)

have shown that large quantities of microbially produced exopolymeric substances

occur in sea ice and at the ice-water interface. This material was positively correlated

to bacterial abundances, although diatoms were thought to dominate the exopolymer

production in this system. These authors suggested high concentrations of exopolymer

with its high polyhydroxyl content would decrease the freezing point of water in the

low temperature, high salinity brine channels, especially near the cell, where

concentration of exopolymer are highest (Krembs et al., 2002). Exopolymer in the

brine channels might provide buffering against harsh winter conditions and high

salinity as well as cryoprotecting the microbes living there against ice crystal

formation by depressing the ice nucleation temperature of water (Krembs et al.,

2002). In sea ice isolates 5 – 50 times larger EPS with the average of 1 – 3 x105 D was

observed by Mancuso Nichols et al. (2005). The structure and properties of EPS are

influenced by the length of the polymer chain, that is the molecular weight

(Christensen, 1999). As the length of the polymer increases, there is a greater

opportunity for complex entanglement of the chains and intramolecular associations,

and these contribute to the tertiary structure and physical behavior of the polymer

(Sutherland, 1994).

In a study by Mancuso Nichols et al. (2004), a strain of Antarctic

Pseudoalteromonas isolated from sea ice, produced 30 times as much EPS at -2 and

10oC compared to 20oC in liquid culture. Bacterial EPS may provide a barrier against

the environmental extremes experienced by the bacterial cell in these environments by

modifying water properties near the cell. Exopolymer production is important in

temperate marine waters (Biddanda, 1986; Decho, 1990; Alldredge and Jackson,

1995; Passow, 2000; Kiorboe, 2001), which are centers of high microbiological

heterotrophic activity (Kiorboe, 2001).

Tamru et al. (2005) reported freezing tolerance in Nostoc commune by the

protection of oxygen evolution mechanism and photosynthesis by EPS.

55

2.11.6 Ligand binding

The ability of bacterial EPS to accumulate metals has been known for some

time (Bitton and Friehofer, 1978; Brown and Lester, 1979; Loaec et al., 1997) and at

the pH of ambient seawater (pH ~8), anionically charged EPS can remove >99% of

Zn and Ag (Harvey and Luoma, 1985). Exopolymer complexation with trace metals

may impact strongly on the availability of these micronutrients to marine organisms

and may be important in the downward transport of trace metals and micronutrients in

the ocean (Decho, 1990). Microbial EPS may also be a major component of the

colloidal matter which has been proposed to bind trace metals within an ‘onion’-like

matrix of metal oxides/hydroxides and organic compounds (Mackey and Zirino,

1994). Most (99%) dissolved iron in the ocean is bound to organic ligands in these

EPS aggregates that eventually aggregates and settles out of the photosynthetic zone

(Wu et al., 2001). On the other hand Geider (1999) argued that organic ligands

produced by microbes keep iron in solution and that microbes are important in the

conversion of iron from the particulate to the dissolved phase. As iron is essential for

nitrogen fixation, photosynthesis and respiration, the importance in these microbially

produced organic ligands to the biogeochemical iron cycle is important.

2.12 INDUSTRIAL APPLICATIONS OF EPS

Biopolymeric systems based on their microstructure are used in the food

industry to create products with specific characteristics. In such systems it is very

important to understand the relation between structure and functional properties

(Cesaro et al., 1992), to know the kinetics of structure formation and destruction and

how this can be influenced by the working conditions and composition (Aguilera and

Stanley, 1999). The functional properties are defined as the measurable properties that

correlate with the characteristics of the final product. The functional properties of a

compound can be identified as following

- Rheological behaviour (flow and viscosity functions, viscoelasticity);

- Behaviour towards water, depending on hydrogen and van der Waal’s interactions;

-Interactions of macromolecules with other macromolecules (properties of

polymerization through intermolecular ionic, hydrophobic or covalent associations);

-Interactions with small molecules, with molecules having little polarity (properties at

the interfaces, formation of polydispersed systems) (Linden and Lorient, 1999).

56

These functional properties can be modified to a certain extent. For example,

if the polysaccharide is a polyelectrolyte, viscosity can be controlled through

electrostatic repulsion, ionic strength or addition of di and polyvalent cations (making

possible the formations of gel through ionic bounds) (Mironescu, 2003). The molecule

size influences the rheological behaviour at various shear stress. Due to the length and

rigidity of hydrated alginate molecules, the aqueous solutions of this polysaccharide

are shear-thinning or pseudoplastic (Imeson, 1992). The alginate molecules are

disordered at small shear rates, whereas at high shear rates the parallel orientation of

polymeric chains occurs and the apparent viscosity decreases (Aguilera and Stanley,

1999). For the rheological analysis of EPS, two measuring systems are recommended:

parallel plate and cone-plate geometries (Steffe, 1996). Using these geometries,

steady-state and oscillatory (or dynamic) measurements can be performed; the first

one type of analyses gives informations on the flow behaviour of polysaccharides and

the second one characterises the viscoelastic behaviour (Hochstein, 2003).

The biopolymers produced by organisms are used to modify the flow

characteristics of liquids and serve as gelling agent in the industry. They are

renewable resource representing an important class of polymeric materials of

biotechnological interest offering a variety of potentially useful products to mankind.

Polysaccharidic biopolymers extracted from plants (Starch, cellulose, pectins,

glactomonas, gums), algae (carragenan, alginates, agar) and some bacteria (dextran,

levan) have already paved their way to the industry. Hogland et al. (1993) has

reviewed the structure, chemistry, and function of microbial extracellular

polysaccharide with reference to microbial ecology and industrial application.

2.12.1 Rheology

Rheology is still regarded by some as a “messy” science, partly by reason of

the nature of its materials but partly because of the lack of clear theory.

SCOTT BLAIR

The father of modern rheology is considered to be Eugene C. Bingham who

coined the word rheology from the Greek rheos meaning flow.

Rheology is the science of the deformation and flow of matter. It includes the

study of elastic deformation and other phenomena not necessarily associated with

flow. Matter is deformed, or starts to flow, only when it is acted by force. Rheology is

concerned with forces, deformation time, temperature and other secondary

57

parameters. For description of rheological behaviour of fluid, parameter of viscosity,

shear strength and tensile strength are generally considered.

Why is Rheology Important?

Controlling rheological properties is essential for polymeric processing and

application. Rheological behaviour of a polymer thus dictates its selection for a

different function in industry. Some of the basic applications affected include:

(a) Mixing - Rheology of a material has an impact on its ease of mixing

whether by hand, with a premeasured package, or with a static mix tube.

(b) Dispensing - Rheology determines how a material is applied whether by

spatula, spray,pump, syringe, cartridge, screen/stencil printing, or pouring. It is

influenced by how rapidly viscosity changes once mixed due to the mixture’s

chemistry.

(c) Settling - Rheology affects the degree and rate at which fillers settle. Also

important is filler type, particle size, shape, and size distribution. This is especially

important for coatings and heavily filled encapsulants.

(d) Flow Control - Rheology affects the way a material flows. High flow

allows easy entry into intricate spaces versus a low flow which prevents flow into

critical areas. Flow control is very important during initial application and during the

curing process as well.

It is well-known that, in concentrated polysaccharide dispersions, the increase

in viscosity with concentration is less pronounced as shear rate increases (Lapasin and

Pricl, 1995). This is necessary for their practical applications because marked

differences in viscosity must be expected at the different operative shear rates

associated with their performance as stabilizers, or with operations such as pouring,

mixing, and pumping.

Rheology in turn is affected by polymer type, its molecular weight,

formulation, size distribution, temperature and shear rate.

2.12.1.1 Rheological Classifications (Emerson and Cuming, 2000)

There are two types of fluids: Newtonian and Non-Newtonian.

58

(a) Newtonian Fluids

These are truly viscous “ideal liquids”, which means as the shear rate changes,

the viscosity remains constant (water, oils, solvents). Hence if you double the strain

rate you will double the stress required.

Newtonian flow can be described by considering two parallel plate A and B of

1 cm2 are (A), with the intervening 1 cm space being filled with the liquid under

consideration. Viscosity is equal to the force (F) that is required to induce a unit rate

of shear. The depth (d) of the substance in between is 1 cm. If a force (F) of 1 dyne is

required to move plate A with constant speed of 1 cm per second, then the viscosity of

this substance will be 1poise. The innumerable parallel layers of the substance must

move past each other once plate A is moved. If plate A is moved to the right, the layer

next to the stationery plate B remains without moving. The layers above it travel,

depending upon the distance from plate A with an increasing speed to the right.

Fig.2.3. Pictorial representation of Newtonian Flow (Ref. Brookfield Engineering,

Manual, 2006)

Each single layer of the substance, therefore, passes the one below it and

remains a little behind the one above it, Because the layer adhere to each other, a

force is encountered that opposes this side wise movement. This tenacity is called

viscosity (ƞ) or internal friction, of a system which per unit area is the same on each

layer. Viscosity then is a measurement of the combined effect of adhesion and

cohesion. The transmitted force (F) is therefore proportional to the coefficient of the

59

inner friction as follows:

F= ƞ A (dv / dr)

The (dv / dr) or D is the rate of shear and is directly proportional to the applied

force (F). Newtonian fluids exhibit direct proportionality between shear stress (F) and

shear rate (D). At any given temperature, these materials have a viscosity that is

independent of the rate of shear. Following figures show that the relationship between

shear force and rate of shear is a straight line and the viscosity in absolute units, is the

inverse slope of the line. The viscosity of Newtonian liquids remains constant as the

rate is changed.

Fig. 2. 4. Newtonian flow curve (Ref. Brookfield Engineering, Manual, 2006)

Newtonian behaviour has been found to be common to all gases and to all

liquids or solutions of low concentrations of some high molecular weight polymers.

(b) Non-Newtonian Fluids

These fluids are affected by shear and are divided into Power Law Fluids

(Pseudoplastic or Dilatant) and Time Dependent Fluids (Rheopectic or Thixotropic).

Most polymers are pseudoplastic and / or thixotropic.

Non Newtonian flow is characterized by the fact that the viscosity is not

directly proportional to the rate of shear but varies with the rate of shear and in certain

60

systems, it is also dependent upon the parameter of time. In non Newtonian systems,

the colloidal rather than the molecular properties are of major significance and the

relevant colloidal properties of the particles that determine their rheological behaviour

are

(1) particle shape

(2) particle size

(3) particle flexibility of ease of deformation

(4) solvation of particle

(5) presence and magnitude of electrical charges on the particles.

Non Newtonian behaviour is most often pronounced at intermediate shear rate.

Studies have shown that non-Newtonian flow behaviour occurs only when the

polymer chains are long enough to entangle (Brookfield Engineering, Manual, 2006).

. Pseudoplastic: As shear increases, viscosity decreases. No matter how long a

pseudoplastic material is sheared at a given shear rate, the viscosity will remain the

same. Many polymeric systems are pseudoplastic, perhaps the most classic example is

mayonnaise. Pseudoplastic properties are important in helping to provide good

sensory qualities, such as mouth feeling, flavour releasing, and suspending properties

of food products (Enriquez et al., 1989).

Dilatant: Opposite of pseudoplastic. These are shear thickening materials that is, as

shear increases the viscosity increases. Dilatancy is observed in fluids containing high

level of deflocculated solids such as clay slurries, candy compounds and corn starch

in water.

Rheopectic: Viscosity increases as a function of time. Examples would be a mixed

epoxy increasing in viscosity as curing takes place or a solvent based adhesive or

coating where the viscosity increases as the solvent evaporates.

61

Thixotropic: Viscosity decreases over time when using a constant shear rate. As shear

rate decreases the material will gradually recover the original internal structure before

shear, this can take seconds or days to fully recover. Many times, when a material sits,

it will “structure”, this will give a “false” high viscosity reading if the material is not

premixed.

2.12.2 Viscosity

Viscosity is a measure of the internal resistance of friction when a material

moves against itself. The internal resistance or viscosity is typically measured using a

rotating spindle instrument such as a Brookfield viscometer. The amount of force

needed to turn the spindle (torque) at a selected speed (RPM) is measured.

A simple calculation converts this “internal resistance” to viscosity.

Ƞ = viscosity = T /Y = shear stress / shear rate

There are different surface area spindles used to measure different viscosity

ranges. The higher the torque value, the higher the viscosity. The force is measured as

Pascal seconds, Poise or Newton seconds per meter2. One milli Pascal second (mpas.)

equals one centipoise (cp) or 1x10-4 Newton seconds per meter2. Rotational spindle

viscometers and cone and plate viscometers are used for most adhesives,

encapsulants, inks, and some coatings.

Low viscosity products like deploymerised galactomannas have attained

commercial importance in both food and non food applications (Zbinden, 1964).

These are required in dietic food products, dairy products cigarette paper and

pharmaceutical industry. Low viscosity depolymerised guaran finds application in

food industry, where the requirement for dietary fibre is high and a mild thickening

required for example, nonvegetarian diets, pet foods, soups, breads and noodles. Low

molecular weight guaran has also been used in the preparation of a sorbent in

chromatography, including chiral separation (Nyquist, 1961; Sibilia and Paterson,

1965). On the other hand high viscosity polymers are widely required in oil well

industries, where rapid hydration and maximum terminal viscosity is required with in

an hour of dispersion to achieve better work over completion. Chemically derivatized

biopolymers with altered viscoelastic properties like carboxymethyl, hydroxypropyl

and etherified derivatives are also in great demand in the industry.

High viscosity polymers though have widespread use in most industries

however their use in food and Pharmaceutical industries is limited due to these

62

polymers being a poor bulking agent.

In diary products such as pasteurized milk, yogurt and cheese, it produces a

thickening, which appears very unnatural. The consistency, body, stability and shelf

life achieved also are not satisfactory. The viscosity (thickening) produced in these

products is subject to variations, depending upon factors such as temperature, shear

rate and freeze thawing. This is because the high viscosity gum at concentration

higher than 0.5% has a non Newtonian (thixotropic) behaviour.

In case of frozen products, the shape, retention and melt down behaviour is not

much improved in case of low solid products.In Pharmaceutical industry, gum finds

application in improving the shelf life of the products, rate of their dissolution and

resorption, coating of granular and spray dried products. In these applications high

viscosity gum does not work well. In many non food uses, a viscosity builder with a

fast hydration rate is needed which should be able to maintain consistent viscosity

under variable shear rate and temperature. This is not possible with very high

viscosity gum products.

2.12.3 Rheological behaviour of microbial polysaccharides

Applicability of polysaccharide is largely dependent on its thermal and

rheological behavior (Marinho-Soriano and Bourret, 2005).

The rhelogical behaviour of polysaccharidic solutions and the influence of

physical or chemical factors on the rheological properties are important because they

offer information on the bioprocess, the biopolymer quality, texture (Moreno et al.,

2000), the relations between microstructure and physical properties (Pelletier et al.,

2001). Rheological characteristics depend on a large number of factors. The most

common industrial use of microbial polysaccharides is related to the capability of

these biopolymers to alter the rheological behaviour of water, food, cosmetic,

pharmaceutical and oil-well products, acting as thickening agents (Sutherland, 1996),

and to stabilise the flow properties of their aqueous solutions under drastic changes of

temperature, ionic strength and pH (Sutherland, 1998, de Vuyst and Degeest, 1999).

The viscosity of the product is affected by the amount and the type of EPSs

produced (Faber et al., 1998). The rheological properties of aqueous solutions of

levan from Bacillus sp. were studied at 20.0 °C over a wide range of concentrations.

At low concentrations the solutions were Newtonian, becoming shear thinning at

higher concentrations (Arvidson et al., 2006).

63

The rheological behaviour of the polysaccharides is also related to the three-

dimensional structure of polymer (Robijn et al., 1996 a, b). It was reported that in

food industries, skim milk fermented by ropy EPS producing strains exhibit similar

rheological properties and had greater viscosity than skim milk fermented by non ropy

strains.

In Pseudomonas mendocina P2d highly viscous pellet was obtained at low

temperature, the viscous nature was due to the gelling of the EPS produced by the

culture (Royan et al, 1999). Halomonas eurihalina strain F 2-7 produces large amount

of an extracellular polyanionic polysaccharide. This EPS was pseudoplastic with

viscosity of the solution decreasing as the shear rate increased. The authors proposed

potential applicability of the polymer in pharmaceuticals, food industry, and

biodegradation (Bejar et al., 1998; Calvo et al., 1995; Quesada et al., 1993).

The SPO18 polymer isolated from halotolerant, thermotolerant, facultative,

polymer producing Bacillus sp. had higher viscosities than commercially obtained

polymers. It also exhibited pseudoplastic behavior, however the viscosity of the

solution was restored when the shear rate was decreased. The polymer had lower

viscosities at higher temperature (Pfiffner et al., 1986).

Xanthan’s unique solution rheology, excellent compatibility with salts, and

thermal degradation have made it useful as an additive to drilling fluids.

The pseudoplasticity of its solution provides low viscosity at the drill bit,

where shear is high, and high viscosity in the annulus, where shear is low and is

responsible for effective removal of bit cuttings and other debris (Carico, 1976).

Therefore, xanthan serves a dual purpose in allowing faster penetration at the bit

because of the low solution viscosity in that area (Eckel, 1968) and, at the same time,

suspending cuttings in the annulus because of the high solution viscosity in the area.

Thus used to plug lost circulation zones, to repair damaged or corroded casing and, to

limit annular gas migration (Carico, 1976). Xanthan solutions are very stable to

shearing. Kenis (1971) reported 85 % regain in viscosity after 15 minute high speed

shearing. Shear resistance is a secondary benefit to friction reduction. Many polymers,

although initially more effective than xanthan in reducing hydrodynamic drag under

turbulent flow conditions, lose a larger proportion of their effectiveness after repeated

shearing (Kenis, 1971). Effecient and effective suspension results from the high yield

point, which provides a structure capable of supporting heavy particles such as barite

(Carico, 1976). Solutions of xanthan reduce friction pressure by 40-60 % in turbulent

64

flow areas, such as in drill string, and allow optimal use of equipment (Darley, 1976).

Xanthan in the aqueous fluids used for enhanced oil recovery, reduces the mobility of

water by increasing viscosity and decreasing permeability. Reviews of polymer

flooding and the application of xanthan in this technology have appeared in the past

(Chang, 1978; Sandvik, 1977; Sandford et al., 1984). The high solution viscosity,

unique rheology, broad compatibility with salts, and excellent stability to shear and

heat make xanthan solutions well suited to meet the requirements of polymer flooding

system.

In addition to xanthan, solutions of exopolysaccharides of Alcaligenes ATCC

31555 called welan and rhamsan (S-194) produce by submerged fermentation (Kang

and Veeder, 1982) are also known to show little change in viscosity even at

temperatures up to 100o C for extended period of time. Rhamsan, previously known as

S-194, possesses unusually good stability to shear, superb compatibility with high salt

concentrations, and excellent suspending ability in suspension fertilizer systems

(Veeder and Kang, 1982; Moorhouse, 1982). A new strain, Sphingomonas

paucimobilis GS1, studied by Ashtaputre and Shah (1995), produces an

exopolysaccharide at the rate of 4 g L-1 and its viscosity is 5.5 times (4200 cP) that of

xanthan gum and is stable over a wide pH and temperature range as well as in the

presence of NaCl. Deacetylated polymer produces a clear, agarlike, thermoreversible

gel in the presence of cations.

Viscosity values of Anabaena ATCC 33047 EPS (Moreno et al., 2000) and

Cyanothece EPS (de Philippis et al., 1998) dispersions are similar to those of a food-

grade commercial mixture of polysaccharides and not far from the values exhibited by

xanthan gum. While at high shear rates, the low viscosities of Anabaena EPS

dispersions enhances unit operations like pumping or mixing, and the large increase in

viscosity as shear rate decreases favors their application as a dispersion stabilizer,

thickening agent and formulation of oil drilling muds (Sutherland, 1996). These

promising properties are confirmed by their viscoelastic spectra. A comparison with

the viscoelastic spectra of Alkemir 110 and xanthan gum dispersions showed that

Anabaena EPS dispersions present a more “fluidlike” relaxation mechanism. The

rheology of the EPS dispersions seems to be consistent with the formation of an

intermediate structure between that of a random-coil polysaccharide and a weak gel.

Viscosimetric measurements demonstrated by de Philippis et al., 1991 in

Cyanospira capsulata shows a typical pseudoplastic behaviour, the viscosity

65

diminishing by increasing the rotation speed of the viscosimeter spindle, for both

whole cultures and cell-free supernatants. Pseudoplasticity increased with the age of

the cultures. The viscosities of the whole cultures were always significantly higher

than the corresponding values for the supernatant indicating that capsulated trichomes

participated in determining the flow properties of the cultures. In this respect, C.

capsulata is different from Porphyridium cruentum (Thepenier and Gudin, 1985) and

Synechococcous sp. (Phlips et al., 1989), whose cultures are reported to have flow

properties essentially depending on the dissolved polysaccharide. This different

behaviour possibly originates from the morphology of these microorganisms, the

capsulated tricomes of C. capsulata, having a greater size and being helicoidal,

occupy a bio volume much larger than the coccoid cells of P. cruentum and of

Synechococcous sp., so giving a significant contribution to the flow properties of

whole cultures. These findings suggest a utilization of whole C. capsulata cultures as

viscosifying agents in some technological processes such as enhanced oil recovery.

EPS from Phormidium 94 a shows a Newtonian behavior at low EPS

concentration changing to pseudoplastic above 0.1% solution (Vicente-Garcıa

et al., 2003). Aqueous dispersions (0.1% w/v) of polysaccharide produced by

Cyanothece strains were comparable to xanthan (de Philippis et al., 1998). Parikh and

Madamwar (2006) studied four cyanobacterial strains Cyanothece sp., Oscillatoria

sp., Nostoc sp. and Nostoc carneum. All the polysaccharides were low viscosity

products (6.9-18.4 cps) showed a decline in reduced viscosity with 0.1 M NaCl and in

the presence of 0.1 M CaCl2, they got precipitated. Calcium ions bond with

carboxylates, ring oxygen and bridging between oxygen of adjacent chains took place,

forming Ca2+-polysaccharide

aggregates (Yang et al., 1999; Parker et al., 1996).

Kinematic viscosity of Nostoc strains (Nostoc commune, Nostoc flagelliform

and Nostoc sphaeroides) grown in the field were found to be higher than the

corresponding suspension cultures grown under controlled condition (Huang et al.,

1998).

Table 2.10 demonstrates rheological behavior of various cyanobacteria and viscometeric methods.

66

Table 2.10 Rheological behavior of various cyanobacteria and viscometeric methods.

Organism Viscometeric method Rheological behavior Reference

Phormidium 94a • 20o C temp. Brookfield model DV-I,UL

adapter, 304 ss-1 Shear ramp 0.3-122 s-1 (6 min.

hold at each step)

Apparent viscosity shows shear thining with increase shear. 0.1 % soln shows Newtonian behavior. >0.1 % concn soln Non Newtonian, pseudoplastic behavior.

Vicente-Garcıa et al., 2003

Anabaena sp. ATCC 33047

Haake Rotovisco RV-20/CV-20N rheometer using an ME-31 Mooney–Ewart sensor system.

shear-rate ramp from 0.1 to 300 s−1 for 15 min.

maximum shear rate was maintained for 10 min to check for any shear-time effect.

Non-Newtonian, shear-thinning, pseudoplastic. The shear-thinning properties are less and less pronounced,

so that Newtonian behavior is achieved. No conformational changes occurred from 0.2% to 0.6% (w/w)

EPS. A marked shear-thinning response (n < 0.4). At higher EPS concentrations, the Anabaena dispersions turned

out to be more thixotropic. The rheological properties of Anabaena EPS dispersions reveal

they have an intermediate behavior between that typical of random coil polysaccharides and that of weak gels.

Moreno et al., 2000

Cyanospira capsulata

30oC temp. Brookfield L.V.T. viscometer,

spindle No.2.

A typical pseudoplastic behavior Pseudoplasticity was the more marked, the more aged the

cultures.

de Philippis et al., 1991

Cyanothece Brookfield LVT viscosimeter. 0.1% [wt/vol]

The viscosity dependence on shear rate of 0.1% aqueous solutions are comparable to xanthan.

de Philippis et al., 1998

contd.

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Organism Viscometeric method Rheological behavior Reference

Cyanothece sp., Oscillatoria sp., Nostoc sp. and Nostoc carneum

25° C temp. Ubbelohde capillary viscometer.

• EPSs solution 0.1% (w/v) EPS prepared in deionized water,

0.1M NaCl and 0.1M CaCl2 solution.

In all the aqueous polysaccharide solutions, reduced viscosity decreased with decreasing EPS concentration.

Significant decline in reduced viscosity is observed with 0.1M NaCl.

Intrinsic viscosity of the polysaccharides in different aqueous solutions was 18.4, 12.1 and 6.9 cp for Nostoc sp. Oscillatoria sp. and N. carneum decreasing to 11.7, 9.8 and 5.5 cp respectively in the presence of 0.1 M NaCl.

In the presence of 0.1M CaCl2, all the polysaccharides got precipitated.

Parikh and Madamwar, 2006

Nostoc commune vaucher, Nostoc flagelliforme and Nostoc sphaeroides

250C temp. Ostwald microviscometer in an

automated measuring unit AVS 310 (Schott-Gera¨te, Hofheim, Germany) by using capillary tube type I and 2 mL of sample.

The polymers (0.1%) were dispersed at room temperature for 15 h with continuous magnetic stirring prior to viscosity measurements.

N. spheroides exhibited the highest kinematic viscosity among all of the polymers tested.

The crude extracts of the field samples had higher viscosities than those of the corresponding suspension cultures and EPS.

Huang et al., 1998

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A study was made by Chen and Chen (2001) to explore the rheological

properties of the water-soluble mucilage of a green algae, Monostroma nitidium. The

apparent viscosity of the water-soluble mucilage decreased with increasing shear rate,

with increasing solution temperature (from 4 to 70 °C) and decreased slightly with a

shearing time of 60 min. The decreases in the apparent viscosity were more

pronounced for higher-concentration water-soluble mucilage.

The relationship of shear stress versus shear rate of myosin B, soya protein

components and their mixtures were investigated at varying temperatures. Myosin B

solution showed thixotropic flow behaviour at room temperature, while the solution of

soya protein components and the mixture of myosin B and soya protein components

were rheopectic. Upon heating, the rheopectic property of soya protein components

and the mixture of myosin B and soya protein components became thixotropic. The

results suggest that in these systems, thixotropic flow behaviour precedes the

formation of a protein gel on heating (Lin and Ito, 1986).

The rheological behavior of aqueous systems of scleroglucan, the neutral

polysaccharide secreted exocellularly by certain fungi of the genus Sclerotium showed

time-dependent properties, all scleroglucan aqueous systems exhibit a thixotropic

response. Stress transient experiments show that a delay time of approximately 4–5

min is necessary to remove shear history and to reconfigure the unperturbed state

(Grassi et al., 1996).

All known exopolysaccharides with industrial significance are produced in

aerobic submerged fermentation. Additionaly, in every case, the viscosity of the

fermentation liquor progressively increases to a high level as a result of product

formation. Viscosity makes microbial polysaccharide fermentation unique, as

compared with nonviscous fermentations, in that control of aeration and agitation

assumes special importance (Kang and Pettitt, 1993).

2.13 BIOTECHNOLOGICAL POTENTIAL OF EPS

In recent years, there has been a continuous search for new extracellular

polymeric substances (EPS), particularly those produced by microorganisms, offering

a wide variety of biotechnologically important products. These biopolymers have

69

been attracting interest due to their application potential in food, cosmetics,

pharmaceutical and other industries where they are used as thickening, stabilizing,

emulsifying and gelling agent and water binding agent. Several of the microbial EPS

have found use in industry due to their physiochemical properties that are similar to

plant (cellulose, pectin and starch) and seaweeds (alginate and carrageenan)

polysaccharides. However, microorganisms are better suited than higher plants, since

they exhibit high growth rate and are more amenable to manipulations of conditions

for enhancing growth and/or EPS production.

Various applications include improvement of water holding capacity of soil,

detoxification of heavy metals and radionucleoide contaminated waters and removal

of solid matter from water reservoirs. Physical and chemical characteristics of

microbial polysaccharides show little variability and are not vulnerable to variations

in climatic conditions of cultivation and production or pollution. Besides, gums of

microbial origin are susceptible to natural biodegradation, promoting little damage to

the environment and diminishing pollution.

Extracellular polysaccharides play a key role in texture of food products and

thus offer opportunities to develop a new generation of products, especially in

fermented milk manufacturing (Grobben et al., 1995; Robijn et al., 1995 a, b). Due to

their broad applicability, some bacterial polysaccharides are produced on an industrial

scale and used as raw material for processed foods, in medicine and in industrial

preparations.

Some examples of bacterial exopolymers that are commercially produced are

xanthan, acetan and gellan produced by the Xanthomonas campestris, Acetobacter

xylinum and Sphingomonas paucimobilis respectively and dextran produced by some

strains of lactic acid bacteria (LAB) like leuconostoc mesenteroides (Sutherland,

1998; van Kranenburg et al., 1997).

The species-specific structural heterogeneity and the many roles that EPS play

in the natural environment are reflected in the numerous existing and potential

applications for these bio-polymers (Weiner, 1997). Xanthan gum, the most well

known microbial polysaccharide, is produced by the plant pathogen Xanthomonas

campestris. Because of its physical properties it is commonly used as a thickener in

both food and non-food industries (Becker et al., 1998).

For food applications, guar gum (seed endosperm of Cyamopsis

tetragonolobus) is an effective water retention agent because it is intensely

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hydrophilic and extensively used as emulsifier, stabilizer and thickner. Unmodified

guar has been approved as a direct food additive by the WHO/FAO, and in the United

States by the FDA regulation 121.104- generally recognised as safe (Anon, 1952 a;

1952 b; 1958; 1966; Hutchins and Singiser, 1955 and Eatherton et al., 1955).

Table 2.11 summarizes some commercially available microbial polymers and

their potential applications.

2.13.1 Medical applications

Some polysaccharides have unique physiological activities as anti-tumor, anti-

viral and anti-inflammatory agents as well as an inducer of interferons, platelets

aggregation inhibition and colony stimulating factor synthesis. EPS isolated from

LAB may have anti-tumor activities and could be considered as a probiotic (Oda et

al., 1983). A probiotic is a mono-or mixed culture of living microorganisms which,

applied to animal or man, beneficially affects the host by improving the properties of

the indigenous population of gastrointestinal microorganisms (Salminen et al., 1998).

Probiotic effects are considered to include inhibition of pathogenic microorganisms,

antimutagenic and anticarcinogenic activity, increase of the immune response and

reduction of cholesterol levels (Nakajima et al., 1992).

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Table 2.11 : Commercialy used microbial polymers and their applications.

Polymer Composition Organism Application

Bacterial

cellulose

β-D-Glucan Acetobacter xylinum Improvement for latex binders, stabilizers of

emulsion in cosmetics and foods.

Xanthan β-1,4-linked D-Glucose backbone carrying

trisaccharide side chains composed of two

mannose and one glucuronic acid.

Xanthomonas campestris Special rheological properties, widely used in

food, cosmetics, pharmaceutical and paper

industry.

Pullulan ά- D-Glucan, 1,2-linked maltotriose and

maltotetrose units are coupled through 1,6- ά-

bonds.

Aureobasidium pullulans In food industry used as an edible film and a

starch replaced in low calorie food

formulations.

Dextran ά- 1,6 glycosidic linkages between glucose,

branches begin from ά- 1,3-linkages.

Leuconostoc

mesenteroides and

streptococcus mutans

Used as an antithrombotic (anti-platelet) and

to reduce blood viscosity. It is also used in

eye drops as a lubricant.

Gellan Linear tetrasacchride –4-L-rhamnopyranosyl-

(ά-1,3)D-glucopyranosyl- (β-1,4)-D-

glucuronopyranosyl- (β-1,4)D-glucopyranosyl

(β-1- with O(2) L-glyceryl and O(6) acetyl

substituents on the linked glucose.

Sphingomonas elodea Soluble in water, it is used primarily as an

alternative to agar as a gelling agent in

microbiological culture. Also used as a food

additive, thickener, emulsifier and stabilizer.

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Bacterial cellulose, produced by Acetobacter xylinum and other, mainly Gram-

negative bacterial species, has a high water binding capacity. This EPS is used to

make a type of wound dressing for patients with burns, chronic ulcers or extensive

tissue loss (Sutherland, 1998). Several Agrobacterium and Rhizobium species produce

curdlan and this improves the texture of tofu, bean jelly and fish pastes in Japan

(Sutherland, 1998). A promising new field of application for cyanobacterial RPSs that

recently attracted much attention is related to the presence in some polymers of

significant amounts of sulphate groups. Indeed, an increasing amount of data is

available on the antiviral activity of sulphated polysaccharides from cyanobacteria

(Hayashi and Hayashi, 1996; Hayashi et al., 1996), eukaryotic algae (Hasui et al.,

1995; Witvrouw and de Clercq, 1997) and red algae (Andrei et al., 1991).

In 1958, Gerber et al., reported that algal polysaccharides exhi-bited antiviral

activity towards mumps and influenza B viruses. Andrei et al. (1991) reported anti

human cytomegalovirus (HCMV) activity. According to Schaeffer and Krylov (2000)

anti-HIV active polysaccharides are also active against other retroviruses such as

Herpes Simplex Virus (HSV), but the amount of antiviral activity varies with the

compound and the virus. Sulfated homopolysaccharides are more potent than sulfated

heteropolysaccharides. The presence of the sulfate group is necessary for anti-HIV

activity, and potency increases with the degree of sulfation. Studies using nonsulfated

and sulfated homo and heteropolysaccharides isolated from algae or other natural

sources, or synthesized, have revealed the mechanisms of binding of drugs to the

virion, and the mechanisms of viral binding to host cells (Andrei et al., 1991).

Nagle and Jagtap (2004) studied antibacterial activity of cyanobacterial strains

viz. Phormidium tenue (Menegh.) Gomont, Synechococcus cedrorum Sauvg.,

Synechocystis pevalekii Ercegovic , Lyngbya sp., against five bacteria with maximum

activity (inhibition zone 22 mm) against Alcaligenes aquamarinus.

Blorr and England (1989) reported, a broad spectrum antimicrobial antibiotic

produced byNostoc muscorum during the post-exponential phase of growth. The

antibiotic inhibits the growth of bacteria, notably multiple-resistant Staphylococcus

aureus, and a biocide resistant Pseudomonas aeruginosa. Inoculation of a saline sodic

soil with an exopolysaccharide from Nostoc muscorum increased the soluble C by

100%, microbial activity by 366% and the amount of water soluble aggregates larger

than 250 mm by 12 times (de Caire et al., 1997).

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Heyduck-Soller and Fischer (2000) reported the use of spent medium in which

cyanobacteria (Anabaena sp. strain Hi 26, Oscillatoria subtilissima strain Bo 62,

Rivularia sp. strain Bo 85 and Oscillatoria limnetica strain Flo 1) were precultured,

inhibited the growth of gram-positive and gram-negative heterotrophic bacteria, as

well as of the yeast Saccharomyces cerevisiae.

2.13.2 Flocculation

Flocculation is achieved with the help of flocculants, which are the natural or

synthetic substances that facilitate the agglomeration or aggregation of the coagulated

particles to form floccules and thereby hasten the gravitational settling of suspended

solids in solution. Bioflocculation is a dynamic process resulting in the formation of

stable aggregates or flocs due to the synthesis of extracellular polymer by living cells.

Flocculation in microbial system was first reported by Louis Pasteur (Gao et al.,

2006). Conventionally, different flocculating agents such as organic flocculants

(polycrylamide, polyethylamine) inorganic flocculants (polyaluminium chloride,

aluminium sulphate) and natural flocculants (gelatin, chitosan, guar gum and

microbial flocculants) have been widely applied in different industrial process

including wastewater treatment, brewing, dredging, mineral industries, secondary oil

recovery, and chemical industries. It has also significant uses in food and fermentation

industries (Gutcho, 1977).

The organic flocculants widely applied in industrial fields have been shown to

be harmful to the environment and to be a dangerous source of pollution (Zhang et al.,

2002). Bioflocculants produced by micro-organisms have been expected to be

harmless to the environment because of their biodegradability. It has been reported

that algae, bacteria and fungi all produce flocculants with various properties (Kurane

et al., 1986; Kaplan et al., 1987; Kuniho and Ryuichiro, 1999). However,

bioflocculant is so expensive that its wide application has been restricted, mainly due

to the low productivity of the bioflocculant and the high cost of the medium (Kurane

et al., 1994).

Flocculants produced by Rhodococcus erythropolis S-1 (Kurane et al., 1986)

were predominantly proteinaceous in nature, whereas those produced by Alcaligenes

cupidus KT201 (Toeda and Kurane, 1991), Bacillus sp. 1471 (Kumar et al., 2004),

Bacillus subtilis 1103335 (Yokoi et al., 1996) and Bacillus sp. DP-152 (Suh et al.,

74

1997) was polysaccharidic in nature. On the other hand, the flocculants produced by

Arcuadendron sp. TS-4 (Lee et al., 1995) and Arthrobacter sp. (Wang et al., 1995)

were shown to be glycoproteins (Gao et al., 2006). Bioflocculation activity by

cyanobacterial release polysaccharides has been reported in owing to the presence of

both hydrophilic and hydrophobic groups by de Philippis et.al. (2001).

According to Gao et al. (2006) bioflocculant from Vagococcus sp. W31 was

heat-stable and had strong flocculating activity in a wide range of pH with relatively

low dosage requirement. MBFW31 was identified as a polysaccharide with molecular

weight over 2×106 D. It contained neutral sugar and uronic acid as its major and minor

components, respectively. They suggested that MBFW31 had potential application in

wastewater treatment.

Flocculation of yeast cells by flocculants obtained from Aspergillus sojaewas

has been explained in terms of a bridging phenomenon between discrete cells of yeast

and the linearly extended polymer chains forming a three dimensional matrix

(Nakamura et al., 1976).

The polygalactosamine chain of the flocculant PF-1 produced by

Paecilomyces, which absorbs anionic particles and neutralizes their charges, may

form intramolecular bridges that result in stable flocs and precipitation. Several

negatively charged polysaccharides isolated from bacteria, yeast cells and soil

fractions are known to be capable of flocculating suspended clay particles by

adsorbing onto the surfaces of neighboring negatively charged clay particles via

cation bridge. The effectiveness of such polymers depends on the length of the

molecule and the number of charged groups per unit length (charge density). These

factors determine the extent of inter-particle bridging by the flocculants. It was also

reported that the kaolin floc formation occurred by repeated adsorption of flocculants

produced by Alcaligenes cupidus KT201 on the kaolin particles by this bridging

mechanism (Zhang and Lin, 1999, cited in Sheng et al., 2006).

Cyanobacteria and algae have been reported to produce bioflocculants during

the stationary phase. Anabaena sp and Chlamydomonas maxicana have been reported

to produce bioflocculants (Kurane et al., 1994). Choi et al. (1998) reported 61, 83 and

46 % kaolin binding activity in 2,4 and 7 weeks old cultures of Anabaena N 1444,

Anabaena PC-1, Scenedesmus sp..

Bar-Or and Shilo (1987) screened several benthic, as well as planktonic,

cyanobacteria for production of extracellular flocculants. Significant amounts were

75

detected in the culture supernatants of Phormidium sp. strain J-1 and Anabaena

circularis PCC 6720 which, like Phormidium sp. strain J-1, is a benthic species

possessing hydrophobic cell surface properties (Fattom and Shilo, 1984). Another

benthic cyanobacterium, Calothrix desertica, produced small amounts of extracellular

flocculant at the late lag phase of growth (Bar-Or and Shilo, 1987).

2.13.3 Surfactant

Microbial compounds, which exhibit pronounced surface activity, are

classified as biosurfactant. Bioemulsifiers have received increasing attention in recent

years because of their role in the growth of microorganisms on water soluble

hydrophobic materials such as hydrocarbons. Bioemulsifiers are amphipathic

molecules that can be divided into (a) Low molecular weight compounds such as

glycolipids and phospholipids which lower the interfacial tension between

hydrophobic liquids and water and thus reduce the energy required to form emulsions

and (b) Polymers, which stabilize emulsions. Many amphipathic molecules produced

by living cells possess surface active characteristics observed in the form of lowering

of interfacial tension at the oil water interface, which leads the formation of

microemulsions. Emulsifying molecules are basically carbohydrate, lipid or protein in

nature and known to be released by microorganisms. Because of their high surface to

volume ratio, microorganisms are excellent source of potentially useful amphipathic

polymers. These molecules improve the availability of hydrocarbons to the microbial

cells by increasing the area at the aqueous hydrocarbon interfaces, which in turn

increases the rate of hydrocarbon dissolution and their utilization by microorganisms

(Tuleva et al., 2002; Maneerat, 2005).

Exopolymers may be of great value in enhanced oil recovery process because

of their surfactant activity and bioemulsifying properties. Altogether different group

of cyanobacteria that produces copious amount of exopolysaccharides with properties

of interest in biotechnology are the unicellular organisms designated as Aphanothece

halophytica, Aphanocapsa halophytica, Cyanothece etc. (de Philippis et al., 1998;

Morris et al., 2001; Sudo et al., 1995). RPSs produced by Aphanocapsa, Cyanothece,

Nostoc, Phormidium and Synechocystis strains, seems to be quite promising, owing to

large amounts of acetyl groups, deoxysugars and/or peptidic moieties the presence of

charged groups on the macromolecules may lead to other interesting industrial

76

applications: their capability to bind water molecules can be exploited by the cosmetic

industry for product formulations (Sutherland, 1994) or the ability of the charged

RPSs to trap metal ions can be exploited for the removal of heavy metals from

polluted waters (Urrutia, 1997).

Halomonas eurihalina strain F 2-7 produces large amount of an extracellular

polyanionic polysaccharide. This polymer is a potent emulsifying agent. Therefore the

polysaccharide may have a range of potential application in pharmaceuticals, the food

industry, and biodegradation (Bejar et al., 1998; Calvo et al., 1995; Quesada et al.,

1993). A Halomonas isolate from Morocco also produces interesting

exopolysaccharides, and may find applications as emulsifiers with potential in oil

industry (Bouchotroch et al., 2000).

The EPS from Pseudomonas mendocina P2d retained 61·9% and 57·14% of

the emulsion after 30 and 60 min, respectively (Rosenberg et al. 1979). Pseudomonas

putida 21BN produced surfactants ampholipids, the amphipathic surface active

glycolipids that substantially changes the surface tension of the culture medium when

grown in hexadecane (Tuleva et al., 2002). Acinetobacter junii SC14 from human

skin exhibited the emulsifier production (Patil and Chopade, 2001). Acinetobacter

radioresistens KA53 produce an extracellular emulsifying agent alasan, which

stabilized a variety of oil-in-water emulsions. Chemical characterization indicated that

it is a complex of an anionic, high molecular weight, alanine containing

heteropolysaccharides and protein (Navon-Venezia et al., 1995). The emulsifier

liposan isolated from yeast Candida lipolytica (Cirigliano and Carman, 1984) and

emulsans are amphipathic extracellular polyanionic polysaccharides and/or proteins

produced by Acinetobacter calcoaceticus RAG-1 that stabilize oil-in-water emulsions

(Rosenberg et al., 1979).

2.14 COMMERCIALISATION PROSPECTS IN THE FOOD INDUSTRY

Polysaccharides are derived primarily from the red and brown algae, and

higher plants, which have been widely employed in the food industry. These

polysaccharides serve as emulsifying agents, gelling agents, thickening agents, and

stabilizers (Sutherland, 1990). Microorganisms are a highly promising renewable

source for the production of polysaccharides. Microbial polysaccharides offer greater

77

advantages over the polysaccharide derived from other sources because they are

obtained from cultures of selected microbial strains with a high and stable yield in

controllable environment. Some cyanobacterial polysaccharides even possess a

superior property to Xanthomonas polysaccharides. Recent studies have indicated the

interesting properties of these polymers, which make them suitable for forming stable

gels, fibers, films and liquid crystals, stabilizing suspensions and emulsions,

enhancing viscosity of aqueous solutions, and serving as flocculants. All these

features make them particularly suitable for use in the food industry, although they

can be used equally well in the other industries. The assessment of their potential use

for various industrial purposes or specific applications is quite an arduous task.

One of the most important prerequisites of a polysaccharide, which determines

many of the properties generally considered useful for its industrial utilization (i.e.

high viscosity of its aqueous solutions, capability of forming gels with good tensile

strength, stabilizing emulsion), is that it possesses, together with an adequate

composition and structure, a high molecular weight (MW) (Shepherd et al., 1995).

Physical properties of certain polysaccharides must be seriously considered

before putting them into use in the food industry. The important properties of

cyanobacterial polysaccharides include high viscosity at a low concentration, high

pseudoplasticity (shear thinning), yield value and suspending power, and stable

viscosity towards salts, and a wide range of temperature and pH (Sandford et al.,

1984). In order to provide a scientific basis for industrial applications, future research

should focus on the alteration of rheological properties in different aqueous solutions,

the change in texture of the products, and the correlations between chemical

composition and physical properties.