Chapter I Introduction
2
1.1 Introduction
Carbohydrates are naturally occurring and well distributed, and are the most
important building blocks of the biosphere. These evolutionary and biologically
important organic compounds are present on Earth in different forms. Traditionally,
on the basis of the number of sugar units, carbohydrates are classified into three
groups: monosaccharides, oligosaccharides, and polysaccharides. The natural
macromolecules composed of several monosaccharide units (more than ten) are
known as polysaccharides and are synthesized at different stages of life cycle of
every living organisms for different purposes. The monosaccharide units of
polysaccharides are joined to each other by an acetal linkage. These acetal linkages
are formed by the reaction of a hemiacetal hydroxyl group of one unit with an
alcohol group of another unit which liberates water to give a glycosidic bond.
Polysaccharides not only have different sequences of monomeric units but also have
different sequences of glycosidic linkages and different types of branching. They
may be amorphous or even insoluble in water. All these factors together give
polysaccharides a great diversity of structure, property, and functions.
Polysaccharides can be classified into two separate groups based upon their
monomeric composition. Homopolysaccharides contain only one type of sugar
moiety, while heteropolysaccharides contain two or more (Sutherland 1982; Purama
et al., 2009; Sutherland 2007).
In recent years, the increased demand for natural polymers or biopolymers for
various industrial and biotechnological applications has led to a renewed interest in
exopolysaccharides or extracellular polymeric substances (EPS) production by
microorganisms as soluble or insoluble polymers. Different types of polysaccharides
produced by plants (cellulose, pectin and starch), algae (agar, alginate and
carrageenan) and bacteria (alginate, dextran, gellan, pullulan and xanthan gum) are
commonly used as food additives for their gelling, stabilizing or thickening
properties (Sutherland 1998). These EPS produced by both prokaryotes (eubacteria
and archaebacteria) and eukaryotes (phytoplankton, fungi, and algae), have a great
deal of research interest (Kumar et al., 2007). The growing environmental concerns
and increasing demands from end-use sectors are expected to increase the global
market for microbial products to about 250 billion US dollars by 2016 (McVilliams
Chapter I Introduction
3
2011). Exopolysaccharides are the metabolic product that accumulate on the cell
surface and play roles in various biological mechanisms such as immune response,
adhesion, infection, and signal transduction (Sutherland 1998; Kumar et al., 2007).
Table 1.1 shows the biological functions of microbial exopolysaccharides.
Table 1.1 Biological functions of EPS (Wingender et al., 1999; Wolfraardt
et al., 1999)
Microbial polysaccharides are high molecular weight carbohydrate polymers
present either at the outer membrane as lipopolysaccharides (LPS) that mainly
determine the immunogenic properties or secreted as capsular polysaccharides
(CPS) forming a discrete surface layer (capsule) associated with the cell surface or
excreted as EPS that are only loosely connected with the cell surface (Cuthbertson et
al., 2009). Whereas CPSs are assigned with functions directly related with
pathogenicity like resistance to specific and nonspecific host immunity, and
adherence (Taylor and Roberts 2005).
An important distinction of polysaccharide is based on their charge properties
they may be as naturally anionic and neutral. Microbial EPS like xanthan,
Function Relevance
Adhesion to surfaces Initial step in surfaces colonization, accumulation of
bacteria on nutrient-rich surfaces
Protective barrier Resistance to non-specific and specific host defense,
resistance to certain biocides including disinfectants
and antibiotics
Cell-to-cell recognition Symbiotic relationships with plants and 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
Sorption of exogenous
organic and inorganic
compounds
Scavenging and accumulation of nutrients from the
environment, sorption of xenobiotics and toxic metal
ions (detoxification).
Promotion of polysaccharide gel formation
Interaction with enzyme
and enzymatic activities
Accumulation/retention and stabilization of secreted
enzymes.
Digestion of exogenous macromolecules for nutrient
acquisition
Chapter I Introduction
4
phosphomanan an alginate belongs to anionic group while EPS like levan,
scleroglucan pullulan and dextran are belongs to neutral group. Some
polysaccharides have anionic properties and they contain acidic groups, such as
carboxyl, phosphate or sulfate.
1.2 Exopolysaccharide producing microorganisms
The diversity of various EPS produced by microorganisms is often stressed. At
present a considerable number of bacteria Lactic Acid Bacteria (LAB), higher
basidiomycetes, lower filamentous fungi and yeasts from different ecological niches
are known for their ability to synthesize EPS in nature as well as in laboratory
culture system. However, many still remain uninvestigated or unexplored. For a
better observation at a glance a list of EPS producers is represented in Table 1.2.
1.2.1 Basidiomycetes fungi
Fungi from the division basidiomycota have been producer of one of the
most powerful secondary metabolites, and these metabolites have been researched
and developed into therapeutic agents. (Gan et al., 1998a; Eo et al., 1999b; Hatvani
2001).
1.2.1.1 Fungi derived β-glucans
Glucans are polysaccharides that contain glucose as a sole monomer unit
(Murray et al., 2002). This group of polysaccharides involves glycogen, cellulose,
and dextran. Their general formula is (C6H12O5)n (Duchon 1985). Polysaccharides
called β-glucans as well as β-1,3-D glucans or β-1,4-D-glucans (earlier, also
lichenins) are present in the cell walls of higher plants and also in the seeds of some
cereals (e.g., barley and oats). Related polymers, which are also called β-glucans
and/or β-1,3-D glucans and β-1,6-D-glucans, are synthesized by fungi, molds, and
yeasts.
Chapter I Introduction
5
Table 1.2 List of principal bacterial and fungal exopolysaccharide producing
microorganisms
Exopolysaccharides Organisms
Bacteria
Alginate Pseudomonas aeruginosa (Hay et al., 2009), Azotobacter
vinelandii (Gaona et al., 2004)
Cellulose Gluconabacter xylinus (Nguyen et al., 2008)
Curdlan Alcaligenes faecalis (Matsushita 1990)
Cellulomonas fauigena (Kenyon and Buller 2002)
Dextran Leuconostoc mesenteroides (Sarwat et al., 2008)
Gellan Sphingomonas paucimobilis (Bajaj et al., 2007)
Hyaluronan Pseudomonas aeruginosa (Bonde 1957)
Pasteurella multocida (DeAnglis et al., 1998)
Levan Bacillus subtilis (Kang et al., 2009)
Zymomonas mobilis (Poli et al., 2009)
Xanthan Xanthomonas campestris (Palaniraj and Jayaraman 2011)
Fungi
Pullulan Aureobasidium pullulans (Singh et al., 2008)
Scleroglucan Sclerotium glutanicum (Schmid et al., 2011)
Schizopyllan Schizophyllum commune (Kumar and Singhal 2011)
Lentinan Lentinula edodes (Zhang et al., 2007)
Grifolan Grifola frondosa (Tada et al., 2009)
Pleuran Pleurotus ostreatus (Hozova et al., 2004)
Krestin Coriolus versicolar (Ooi and Liu 2000)
Ganoderan Ganoderma lucidum (Volman et al., 2008)
Natural products containing β-glucan have been consumed for thousands of
years. Anecdotally specially in china and Japan for their healing powers as well as to
increase human longevity (Borchere 1999). Today, fungi are appreciated and
consumed primarily for their sensory and nutritional properties and are also used
increasingly in medicine and pharmaceutical industry. Fungi show favorable dietetic
properties with respect to their low fat and caloric value, and high levels of proteins,
minerals, and certain polysaccharides.
Chapter I Introduction
6
Higher Basidiomycetes represent a taxonomically, ecologically, and
physiologically extremely diverse group of eukaryotic organisms. Medicinal
mushrooms belonging to Basidiomycetes are abundant yet largely untapped source
of useful natural products with various biological activities (Wasser and Weis 1999;
Reshetnikov et al., 2001; Kirk et al., 2001; Xu et al., 2011). It is estimated that
about 650 mushrooms possess medicinal properties, but only several edible
(Flammulina velutipes, Grifola frondosa, Hericium erinaceus, Lentinus edodes,
Pleurotus spp., and Tremella spp.) and non-edible mushroom species (Ganoderma
lucidum, Schizophyllum commune and Trametes versicolor) have been investigated.
Presently, increasing attention is being paid to polysaccharides, which are an
integral component of fungi (Wasser and Weis 1999). The cell walls of fungi
contain two polymers, chitin and β-glucan. Glucans β (1→ 3), β (1→ 4), and β (1→
6) are a key reason that fungi are used as food additives and in pharmacology and
they have also shown beneficial effects when used in the treatment of various
diseases (Pelley and Strickland 2000). The medicinal properties of theses fungi are
due to the secondary metabolites which have been isolated and identified from the
fruiting bodies, mycelia and culture broth of mushrooms. These biologically active
molecules (BAM) belong to various functional polysaccharides, proteins and their
complexes and low molecular weight metabolites such as phenolic compounds,
polyketides, triterpenoids, steroids, alkaloids, nucleotides, lactones, and fatty acids
(Lindequist et al., 2005; Quang et al., 2006; Wasser 2010). Some of these
compounds have cholesterol-lowering, anti-diabetic, antioxidant, antitumor,
immunomodulating, antimicrobial, and antiviral activities ready for industrial trials
and further commercialization while others are in various stages of development.
The best example is Ganoderma lucidum, which contains more than 400 different
biologically active molecules (Kim and Kim 1999; Zhou and Gao 2002). Ohno et
al., (2005) reported the promising results for oncological treatments using β-glucan
isolated from Agaricus brasiliensis. β-glucan found in fungi differ in structure, water
solubility, molecule size and molecular mass. These features lead that not all β-
glucan in fungi show equally strong healing property (Zhang et al., 2007). Wasser
(2002) reported high molecular weight β-glucan as more effective than low
molecular mass. Ohno (2005) also reported high healing activity of high molecular
mass scleroglucan from genus Sclerotium. However, by Zhang et al., (2007)
Chapter I Introduction
7
reported higher antitumor activity by lentinan (Lentinula edodes) with a low
molecular weight. Furthemore, Wasser (2011) reported that efficiency of β-glucans
is influenced by length of side-chain, number of backbone branches and ratio of
(1,4) bonds to (1,6) and (1,3). Soluble β-glucans are strong immunomodulators than
the insoluble ones, was reported by Ishibashi et al., (2001) and Wasser (2011). The
antitumor activities of β-glucans refered by (1-3)/(1-6)-β form are and further related
to their ability to neutralize free radicals are considered to be vital reasons of cancer
occurrences (Chen and Seviour 2007). Jeurink et al., (2008) revealed in clinical
trials that stronger antitumor activitiy was shown by β-glucans linked with protein
than free β-glucans. Antibacterial, antiviral and antiallergic activities in fungi
derived β-glucans have also reported by Kumar et al., (2004).
The solubility of β-g1ucans is associated with the degree of polymerization
(DP). β-glucans are completely insoluble in water when DP>100. Solubility
increases as DP decreases. β-glucans can be classified according to their solubility
properties: (a) alkali-insoluble, acetic acid insoluble (1-3)-β-g1ucan; (b) alkali-
soluble (1-3)-β-g1ucan; and (c) highly branched (1-6)-β-g1ucan (Zekovic et al.,
2005). Among the natural β-g1ucans of clinical interest are lentinan, schizophyllan
and krestin (PSK).
1.2.1.2 Fungi derived β-glucans characteristic
Lentinan, produced from Shiitake mushroom, Lentinus edodes, is a β (1-3), β
(1-6) glucan (Fig. 1.1). Chihara et al., (1970) isolated lentinan and demonstrated its
antitumor effects were greater than other mushroom polysaccharides. It has a
molecular weight ranging from 400-1000 kDa (Kidd 2000) and has proved
successful in prolonging the overall survival of cancer patients, especially those with
gastric and colorectal carcinomas (Furue et al., 1981; Taguchi et al., 1985 a,b).
Adotey et al., (2011) lentinan showed also immunostimulative effect in case of
AIDS patients.
Chapter I Introduction
8
Fig. 1.1 Structure of Lentinan
Another important glucan is schizophyllan, produced and derived from
Schizophyllum commune, which has a β-g1ucopyranosyl group linked (1-6) to every
third or fourth residue of the main chain (Fig. 1.2). It has a triple-helix structure and
molecular weight of approximately 450 kDa. Schizophyllan also has shown to
increase overall survival of patients with head and neck cancers (Kimura et al.,
1994).
Fig. 1.2 Structure of Schizophyllan
Pleuran, is isolated from oyster mushroom Pleurotus ostreatus. This
compound is built of molecules of glucose linked by (1-3)-β bounds (Fig. 1.3). Such
backbone with line structure is linked with side-chains built of glucopyranose
molecules (Hozova et al., 2004). It has molecular mass between 600-700 kDa.
Pleuran demonstrates antitumor properties, lowers concentration of lipids in blood,
and antifungal properties (Chu et al., 2005).
Chapter I Introduction
9
Fig. 1.3 Structure of Pleuran
Krestin (PSK), is a β-glucan/protein compound consisting of 25-38% protein
residues and (1-4)-β-g1ucan with (1-6)-β-glucopyranosidic lateral chains (Fig. 1.4).
It is produced from Coriolus versicolus and has a molecular weight of 94 kDa (Ooi
and Liu 2000).
Fig. 1.4 Structure of Krestin
Grifolan, is derived from Grifola frondosa and commonly named GRN (Fig.
1.5). This glucan includes (1-3)-β linkages and has a molecular weight
approximately 450 kDa (Tada et al., 2009). It is present in both mycelium and
fruiting bodies of G. frondosa (Minato 2010). Grifolan is a polysaccharide that
demonstrates high biological activity like immunomodulator and antitumor agent
(Nie et al., 2006). Deng et al., (2009) reported that polysaccharides content from G.
frondosa confirmed immunomodulative properties in clinical trials of patients with
breast cancer. Grifolan is also used in treatment of HIV, hyperlipidaemia,
hypertension and virus hepatitis (Mayell 2001).
Chapter I Introduction
10
Fig. 1.5 Structure of Grifolan
1.3 Biosynthesis of polysaccharide in mushrooms
EPS production can be influenced by growth phase, medium composition
(carbon source nitrogen source), pH and temperature (De Vuyst and Degeest 1999;
Petry et al., 2000). The biosynthesis of EPS is related to the primary carbohydrate
metabolism of the producing cells (De Vuyst and Degeest 1999; Levander et al.,
2002). In general EPS production is expected to take place during active sugar
consumption, as it requires large numbers of activated nucleotide sugars, energy for
building the repeating units, for polymerization and transmembrane translocation. It
has been reported that EPS is a growth-associated product.
Although structure and functions of polysaccharides have been well known,
biosynthesis pathway of polysaccharide has not been fully understood, especially in
mushrooms. Sutherland (1977) and Sutherland (1994) suggested a general pathway
for the biosynthesis of extracellular polysaccharides in three major steps: (i)
substrate uptake, (ii) intracellular formation of polysaccharide, and (iii) extrusion
from the cell. A variety of enzymes are involved in the overall synthesis, each with
specialized functions in the process are shown in Figure 1.6.
According to Sutherland (1982), the enzymes involved in exopolysaccharide
biosynthesis may be classified into four types:
GROUP 1- those enzymes which are involved in initial metabolism of the substrate,
such as hexokinase.
Chapter I Introduction
11
GROUP 2- enzymes responsible for the synthesis and inter-conversion of sugar
nucleotides, (examples include the enzymes UDP-glucose pyrophosphorylase and
UDP-glucose dehydrogenase.
GROUP 3- transferases, which are enzymes responsible for formation of repeating
monosaccharide unit attached to the carrier lipid.
GROUP 4- translocases or polymerases which form the exopolysaccharide
biopolymer molecule.
Similarly, Fig. 1.7 depicts, the intracellular biosynthesis of bacterial EPS
regulated by enzymes located in various regions of the cell (Kumar et al., 2007)
Fig. 1.6 Biosynthesis of an exopolysaccharide (1) Hexokinase,
(2) Phosphoglucomutase, (3) Phosphoglucoisomerase (Wang and McNeil 1996)
Chapter I Introduction
12
Fig. 1.7 Catabolic mechanism representing the sugar nucleotide synthesis and
the interconversion of various monosaccharides through epimerization,
dehydrogenation and decarboxylation, occurred in the cell cytoplasm (Kumar
et al., 2007)
1.4 Fermentative production of exopolysaccharides
Fermentation for EPS production are batch, fed batch or continuous
processes depending on the microbial system used. In view of the importance of
EPS attempts have been made to produce them under submerged cultivation. Many
investigators have tried to cultivate mushrooms on solid artificial media for fruiting
body formation in order to obtain polysaccharides (Bae et al., 2000; Bhargava et al.,
2002). Furthermore, the cultivation of mushrooms for fruiting bodies production is a
long-term process needing from one to several months for the first fruiting bodies to
appear, depending on species and substrate (Pfefferle et al., 2000).
1.5 Factors influencing exopolysaccharide production
Fermentation is a very versatile process technology for producing value
added products such as microbial biopolymers. The fermentation parameters have a
high impact upon the viability and economics of the bioprocess, their optimization
holds great importance for process development. The production of microbial
polysaccharide production is greatly influenced by fermentation conditions such as
Chapter I Introduction
13
pH, temperature, oxygen concentration and agitation as well as by the composition
of the culture medium (Sutherland 2007; Nicolaus et al., 2010; Kazak et al., 2010).
The production of EPS is tightly regulated and is a carbon and energy-intensive
process resulting in a wide range of nutritional and environmental requirements.
Generally, media containing high carbon to limiting nutrient ratio (often
nitrogen) is favored for polysaccharide production. Conversion of 60–80 % of the
utilized carbon source into crude polymer is commonly found in high yielding
polysaccharide fermentations.
The optimal design of the medium is very important in the growth of
microorganisms, stimulating the formation of products and providing the necessary
energy for metabolic purposes. The nutrients required by a fungus include
macronutrients such as carbon, oxygen, nitrogen, phosphorus, sulphur, potassium
and magnesium, which comprise an average 98 % of dry cell mass of fungi
(Sutherland 1982).
1.5.1 Carbon source
Exopolysaccharides can be formed from a variety of carbon substrates. It is
widely accepted that EPS yields vary based upon carbon substrate utilized (De and
Basu 1996; Datta and Basu 1999; Degeest and De Vyust 2000). However, the
impact of carbon substrate on the chemical composition of exopolysaccharides
formed is a point of contention. Many studies have been conducted, utilizing a wide
variety of bacterial species, to evaluate the role of carbon source in
exopolysaccharide chemical composition. Several studies have found
exopolysaccharide composition to be unchanging with different carbon substrate
utilization (Breedveld et al., 1993; Degeest and De Vyust 2000), while others have
found exopolysaccharide composition to be variable upon utilization of different
carbon substrates (Petit et al., 1991; Pirog et al., 2003).
In general, glucose, sucrose, maltose, lactose, fructose, galactose, xylose,
cellobiose, sorbitol, xylitol, mannitol, and different types of agricultural byproducts
are used as a carbon source in the culture medium. In most of the cases glucose,
sucrose, and maltose have been selected as the most influential carbon sources for
Chapter I Introduction
14
the production of fungal EPSs. However, reasons are not clearly understood as in
most cases fungal growth and EPS production are not directly proportional.
Elisashvili et al., (2009) studied eight different basidiomycetes for EPS production,
and reported that G. lucidum, Inonotus levis, and Phellinus robustus produced
maximum EPSs in media containing glucose as a carbon source. Elisashvili et al.,
(2004) reported an interesting observation that L. edodes and Pleurotus spp. strains
produced maximum EPSs in culture media containing sodium gluconate as a carbon
source but the reasons were not clearly understood. The concentration of selected
carbon source in the culture media is another critical factor for EPS production. In
most of the findings, carbon at a concentration of 30-60 g/l was suggested to support
EPS production from fungi, although a few exceptions were also reported by Farina
et al., (1998), Sudhakaran and Shewale (1988), Xu et al., (2003), and Tavares et al.,
(2005).
1.5.2 Nitrogen source
Nitrogen comprises 8–14 % of the dry cell mass of bacteria and fungi. It is a
component of proteins and enzymes, and is necessary in cellular metabolism. EPS
producing microorganisms can utilize a variety of organic and inorganic nitrogen
sources. Among the various inorganic nitrogen sources ammonium chloride,
ammonium sulfate, sodium nitrate, potassium nitrate, urea, and di-ammonium
oxalate monohydrate are commonly used. However, high amount of EPS is
produced when or organic sources peptone, yeast extract poly peptone, martone A-1,
Soybean meal, and corn steep powder are supplemented. Many observations
suggested that in the presence of inorganic nitrogen sources, fungi produce less
EPSs in comparison to organic nitrogen supplements. Among the inorganic nitrogen
sources, ammonium salts are found to be efficient than other inorganic salts.
Sutherland (1996) reported that EPS production generally occurred in
nitrogen limiting conditions. Generally, the addition of extra nitrogen favors the
biomass concentration, but diminishes glucan formation. In the case of a glucan such
as pullulan, the production of polysaccharide is stimulated by depletion of nitrogen
source. Similarly, high concentrations of nitrogen in the form of ammonium
sulphate have been reported to reduce the scleroglucan production (Farina et al.,
1998).
Chapter I Introduction
15
1.5.3 Carbon/Nitrogen ratio
Carbon and nitrogen are both required for normal cellular metabolism, the
ratio in which they occur influences polysaccharide production. The maximum EPS
production is favored under conditions of nitrogen limitation (high C/N ratios)
(D’Haeze et al., 2004; Quelas et al., 2006). Under such nitrogen-limited conditions,
any excess sugars remaining can be used specifically for polysaccharide synthesis.
Generally exopolysaccharide yields can be maximized when the ratio of carbon to
nitrogen set as 10:1.
1.5.4 Medium pH
The pH of culture broth influences the physiology of a microorganism
significantly by affecting nutrient solubility and uptake, enzyme activity, cell
membrane morphology, byproduct formation and oxidative reductive reactions.
Culture pH can have profound effects on both the rate of production and the
synthesis of polysaccharides. However, the effect of pH on the biosynthesis of
exopolysaccharides and cell growth varies with different microorganisms (Lee et al.,
1999), operational conditions (Eugenia et al., 1995) and medium compositions
(Lacroix et al., 1985). In case of xanthan production, Moraine and Rogovin (1971)
observed that culture pH influenced polysachharide production more than cell
growth. Shu and Lung (2004) developed a two-stage processes, with the first stage
designed for optimal culture growth and the second stage for maximum
polysaccharide production.
1.5.5 Temperature
The internal temperature of the microorganism must be equal to that of its
environment. Like many chemical reactions, the microbial activity is sensitive to
environmental temperature. Temperature is crucial parameter that affects both
culture growth and polysaccharide production. The optimal temperature for cell
growth and EPS formation are predominantly different, especially in non growth
associated product, secondary metabolites (Wang and McNeil 1996). Temperature
can affect the availabilities of oxygen and substrate due to solubility changes in the
medium.
Chapter I Introduction
16
1.5.6 Aeration and agitation
Functions of aeration and agitation in fermentation can be described as
follows: (1) to provide adequate mixing of the contents, sufficient mass and heat
transfer rate, (2) to increase the transfer rate of O2 from gas bubbles to the cells, (3)
to increase the nutrient uptake rate and release rate of metabolic products from cells
to medium, (4) to promote CO2 removal from medium. Therefore, the rates of
aeration and agitation greatly influence the cell growth and EPS formation (Wang
and McNeil, 1996).
Availability of oxygen is limited by the rate of diffusion from the atmosphere
and is dependent upon the solution’s viscosity. During the polysaccharide
production the fermentation medium becomes more viscous and shows non-
Newtonian (pseudoplastic) behavior. However, mycelial pellet formation may occur
at low agitation rates and result in restriction of mass transfer regarding nutrient,
oxygen, product penetration and toxic metabolic products. Consequently, substrate
diffusion may be the rate-limiting process in filamentous fungi fermentation with the
form of pellets. The optimum agitation rate represents a balance between oxygen
transfer into the medium and shear stress, both of which increase with increasing
agitation rate. Higher shaking speeds favor EPS production because they decrease
the adsorption of the secreted extracellular polysaccharides on the cell wall,
providing stimulus for further EPS synthesis.
1.5.7 Metal ions
Phosphorus is an important element for secondary metabolism in fungi and
also regulates lipid and carbohydrate uptake by the cells. Phosphate salts, such as
K2HPO4 or KH2PO4, notably serve as a pH buffer in the fermentation medium and
they are also reported to be most efficient PO4 supplement (Dube 1983). Farina et
al., (1998) indicated an increase of total phosphorus from 0.12 to 0.28 g/l improved
the scleroglucan production (from 4 to 5 g/l). Potassium is the principal inorganic
cation in the cell; it is usually added as an inorganic salt (e.g. K2SO4, K2HPO4, or
KH2PO4). Potassium is a co-factor for some enzymes, required in the carbohydrate
metabolism and in many transport processes. Magnesium is also required by fungi, it
functions as an enzyme cofactor, and is present in cell walls and membranes. It is
Chapter I Introduction
17
usually supplied as MgSO4·7H2O. Pilz et al., (1991) noticed that thiamine and zinc
addition in a defined mineral medium could replace yeast extract. Their results
indicate the importance of meeting the zinc requirement of the microorganisms.
1.6 Recovery of exopolysaccharide
The extraction procedure and the method used for the purification of the
obtained fractions of active polysaccharides depend in many aspects on the type of a
fungus and physical properties of polysaccharides, such as solubility, molecule
conformation, branching level, molecular weight. The selection of extraction
methods includes concentration, isolation and purifications. The extraction of
polysaccharides is commonly performed in aqueous solutions. Water or basic
solutions may not be strong enough to separate water insoluble polysaccharides, for
this purpose acidic solutions may be employed. The extraction may also include
successive extraction with water at room temperature followed by centrifugation.
The main objectives of the recovery process are:
Concentration of fermentation broth or extract to a form, usually solid which
is microbiologically stable, easy to handle, transport and store and can be
readily redissolved or diluted for use in particular application.
Purification to reduce the level of non-polymer solids, such as cells or salts,
and to improve the functional performance, color, odor, or taste of the
product.
Deactivation of undesirable contaminating enzymes, such as cellulases,
pectinases, etc.
Modification of the chemical properties of the polymer to alter either the
functional performance, the solids handling properties of the dried product or
the dispersion and solution rate characteristics.
Isolation of polysaccharide is achieved by lowering the solubility of the polymer.
It results in either a precipitate or a phase consisting of a concentrated solution, by
either addition of a water miscible solvent, (e.g. methanol, ethanol, isopropanol,
acetone) or salt or acid. The method of choice for isolation of polysaccharide is
determined by economics, practicability and the final specification of the product,
with alcohol precipitation being widely favored.
Chapter I Introduction
18
Polysaccharides extracted from the source material are generally dissolved in
water or aqueous solutions. Nevertheless, other macromolecules, such as proteins,
may be also present in the medium. Therefore, several purification steps must be
carried out to remove other substances. The samples may be subjected to a previous
step consisting of a methanolic extraction in order to remove phenolic compounds,
monosaccharides, amino acids and other related molecules. The elimination of
phenolic derivatives is successful (Palacios et al., 2011), and increases the
effectiveness of extraction (Park et al., 2009). Proteins can be removed by
precipitation with trifluoroacetic acid (20%, w/v) or by treatment with the enzyme
protease at 40 °C for 1 h (pH 7.5). Then, proteins are separated by centrifugation.
After protein removal, polysaccharides are precipitated from the supernatants by the
addition of ethanol in 2:1 ratio (v/v). Concentrated sodium chloride solutions can be
added to favor the precipitation, and the solid can be washed with organic solvents,
such as acetone or ethanol.
Size exclusion chromatography (SEC) allows the separation of polysaccharides
according to their size, and the subsequent determination of the molecular weight.
Columns composed of hydroxylated polymers with residual carboxyl functionality
are usually employed for the separation of polysaccharides. Elution is performed
with diluted NaCl, NaNO3 or NaH2PO4 solutions at low flow rates. Polysaccharide
detection is achieved by refractive index detectors or by post column derivatization
with calcofluor (Bao et al., 2001; Ye et al., 2009). Table 1.3 depicts the common
methods used for isolation and purification of EPS.
Chapter I Introduction
19
Table 1.3 Common methods for isolation and purification of EPS
1.7 Characterization of exopolysaccharides
Exopolysaccharides are defined in terms of composition (type and relative
abundance of monomers), structure (relative distribution of monomers and type of
chemical bounds between them), conformation (arrangement of monomers chains
and bounds between them), relative molecular mass and type and arrangement of
substitutes (Morin 1998). Table 1.4 depicts the various characteristics and methods
of analysis of exopolysaccharides. These informations are used to analyze the
functional properties of polysaccharides, like solubility in water, relative viscosity
Methods Applied Contaminants Removed
Dialysis or dialfiltration
against distilled water
Any contaminants with molecular weight less
than membrane cut off
Trichloroacetic acid
precipitation (final
concentration ranging from 4
to 14%)
Amino acids, peptides, proteins. Some
polysaccharides might also be co-precipitated.
Enzyme digestion Proteins and nucleic acids by protease and
nuclease, respectively
Alcohol (e.g., ethanol,
methanol, acetone, etc.)
precipitation
Small molecules like ions could be eliminated,
but proteins and other macromolecules could also
be co-precipitated.
Anion exchange
chromatography
Any macromolecules with surface net charges
different from target compounds
Single dimension gel
electrophoresis (i.e.,
isoelectric focusing)
Any macromolecules with isoelectric point
different from target compounds
Two-dimension SDS-PAGE Any macromolecules with surface net charges
and molecular masses different from target
compounds
Size exclusion
chromatography
Any contaminants with molecular weights
different from target compounds
Chapter I Introduction
20
Table 1.4 Principal characteristics of microbial polysaccharides and analysis
methods
Characteristics Analysis method
Quantitative analysis of polysaccharides Gravimetric methods
Colorimetric methods
Quantitative and qualitative analysis of
monosaccharides components of EPS
High-Performance
Liquid Chromatography(HPLC)
-Reverse- phase HPLC
-Ion exchange HPLC
EPS structure and conformation
Nuclear magnetic resonance (NMR)
Differential Scanning Calorimetry
(DSC)
X- ray diffraction
Rheological analysis
Quantitative and qualitative analysis of
substitutes
Ion exchange HPLC
NMR
Infrared spectroscopy IR
EPS form and dimension
Dynamic light Scattering or Static
Light Scattering
and rheological behaviour (Stokke et al., 1998), ions binding capacity (De Philippis
and Vincenzini 1998). Relationship exists between the glycoside linkage, geometry
of polysaccharide and their conformation. Polysaccharides undergo transition from
an ordered state at lower temperature in the presence of ions, to a disordered state at
elevated temperature under low ionic environments (Nisbet et al., 1984). In case of
some polymers, this represents conversion from a gel to a sol state. The side-chains
attached to many linear polysaccharides promote conformational disorder and
inhibit any ordered assembly resulting in solubility in aqueous solutions. Thus,
xanthan, which possesses a cellulose backbone with trisaccharide side-chains on
alternate glucose residues, has been described as a natural water-soluble cellulose
derivative. Charged residues, found on the exterior of the extended molecules, may
readily promote interaction with ions and macromolecules. This may account for the
Chapter I Introduction
21
extensive binding of heavy metal cations ascribed to Zoogloea flocs and
polysaccharide (Friedman et al., 1968). Conformation refers at the form of
polysaccharide chains; it depends on the monomers and their position and bound
types in the polymeric chains (Belitz and Grosch 1999).
The main conformations adopted by EPS are:
1) Ribbon-type chains, like cellulose or alginate;
2) Helix conformation, founded to lichenin;
3) Combined conformation i.e. heteroglycans.
1.8 Functional properties of exopolysaccharide
The functional properties are defined as the properties measurable using
instruments and correlated with the final product characteristics through these
measurements. Linden and Lorient (1999) identified functional properties of a
compound on the basis of the following characteristics:
1) Rheological behavior (flow and viscosity functions, viscoelasticity)
2) Behavior towards water, depending on hydrogen and van der Waals interactions
3) Interactions of macromolecules with other macromolecules (properties of
polymerization through intermolecular ionic, hydrophobic or covalent associations)
4) Interactions with small molecules, with molecules having little polarity
(properties at the interfaces, formation of polydispersed systems).
1.9 Rheological properties of polysaccharides
The rheological behavior of polysaccharides solutions and the influence of
physical or chemical factors on the rheological properties are important because they
offer informations on the bioprocess, the biopolymer quality, the relations between
microstructure and physical properties (Pelletier et al., 2001), textural analysis
(Moreno et al., 2000). Rheological characteristics depend on a large number of
factors, as observed in Figure 1.8.
Chapter I Introduction
22
Fig. 1.8 Factors influencing the rheological characteristics of biopolymeric
aqueous solutions (Steffe 1996)
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 bonds). The
molecules size influences the rheological behavior at various shear stress. For
example, due to the length and rigidity of hydrated alginate molecules, the aqueous
solutions of this polysaccharide are shear-thinning or pseudoplastic (Imeson 2002).
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 behavior of polysaccharides and the second one characterizes the
viscoelastic behavior (Hochstein 2005).
1.10 Association properties of polysaccharides
The tendency of molecules to associate when the solutions are destabilised is
due to the destruction of the equilibrium between the attraction and the rejection
forces given by changes in medium (pH, ionic strength, temperature) and to the
Chapter I Introduction
23
formation of new bonds. For example, polysaccharides form gels in the presence of
water and under temperature influence; the gelling process implies interactions
between polymer chains with formation of complex structures (double helix) where
the solvent molecules are entrapped (Fig. 1.9). Some EPS form gels in the presence
of ions, like alginates at the interaction with calcium ions (Bracccini and Perez
1999) (Fig. 1.10)
Salts ions added in solutions act as flocculants by neutralizing the repulsive
charge at the surface. The flocculation efficiency is given by the critical coagulation
concentration of counter-ions, but is also dependent from the atomic number (Belitz
and Grosch 1999).
Fig. 1.9 Schematic representation of gel formation process
(Belitz and Gorsch 1999)
Fig. 1.10 Representation of coordinative bounds between Ca+2
and
alginate (Bracccini and Perez 1999)
Chapter I Introduction
24
1.11 Applications
Microbial exopolysaccharides including fungal EPS have gained an
importance due to their wide range of potential applications in various industries and
notably in therapeutics, food, petroleum, agronomy and pharmaceuticals.
1.11.1 EPS in food applications
EPS may function as viscosifying agents, stabilizers, emulsifiers, gelling
agents, or water-binding agents in food (Van den Berg et al., 1995). Majority of
polysaccharides used in foods are of plant origin. Most of them are chemically or
enzymatically modified in order to improve their rheological properties, e.g.
cellulose, starch, pectin, alginate and carrageenan. EPS produced by microorganism
have unique rheological properties. Dextran is the first industrial polysaccharide
produced by lactic acid bacteria. Dextran may be used in confectionary to improve
moisture retention, viscosity and inhibit sugar crystallization. In gum and jelly
candies it acts as gelling agents. In ice-cream it inhibits crystal formation, and in
pudding mixes it provides the desirable body and mouth feel.
1.11.2 Cosmetics
Glucan, mainly scleroglucan may be used in hair control compositions and in
various skin care preparations, creams and protective lotions. Polysaccharide
extracts of fungi with antioxidative properties have commercial application in
creams that provide protection from ultraviolet rays and from other detrimental
effects caused by activities of free radicals that lead to damages and aging of the
skin. Hyaluronan from G. frondosa used as ingredients for enhancing collagen
biosynthesis in skin cells (Kougias et al., 2001).
1.11.3 Emulsifiers
Surfactants and emulsifiers from the microbial sources have received
attention due to their biodegradability and possible production from renewable
resources. The polysaccharide, Emulsan produced by Acinetobacter calcoaceticus
stabilized emulsion more effectively than other commercial gums such as arabic,
tragacanth, karaya and xanthan (Ashtaputre 1995).
Chapter I Introduction
25
1.11.4 Biomedical applications
Antitumor, antiviral and immunomodulatory activities of polysaccharides
have been reported from fungi and bacteria.
1.11.4.1 Antitumor activity
Polysaccharides from fungi have been widely employed in tumor therapies
due to their properties as immunological enhancers. for instance, Agaricus,
Calocybe, Ganoderma, Grifola, Inonotus, Lentinus, Phellinus, Pholiota, Pleurotus,
etc. are different mushrooms genera capable of providing antitumor activity (Hsieh
and Wu 2011; Lee et al., 2011; Selvi et al., 2011; Zhang et al., 2011). The mitogenic
activity of fungal polysaccharides involves several mechanisms, such as the
antiproliferation of cancer cells and induction of apoptosis, regulation of the immune
system and the antimetastatic effects. Polysaccharide can depress the E-selectin
protein and gene expression which in turn inhibit tumor cell to cell adhesion (Yue et
al., 2012). Fungal polysaccharide widely employed as co-adjuvant in tumoral
therapies. Lentinan, a β-(1→3),(1→6)-linked from Lentinus edodes, has been used
in a combined treatment of patients with advanced or recurrent gastric or colorectal
cancer.
1.11.4.2 Antioxidant activity
Oxidative damage caused by free radicals may be related to prevalence of
common diseases such as aging and chronic degenerative diseases, such as diabetes,
cancer, arthritis, cardiovascular disease, Alzheimer, Parkinson, Down syndrome and
multiple sclerosis. An antioxidant has been defined as any substance that when
present at low concentrations compared to those of an oxidizable substrate,
significantly delays or prevents oxidation of the substrate. Polysaccharides from
different mushrooms showed free radical scavenging activity (Asantiani et al.,
2007), superoxide radical scavenging activity, reducing properties, lipid
peroxidation inhibition, suppression of proliferation and oxidative stress, etc.
(Asatiani et al., 2007). These activities are not only dependent on fungal species but
also on the chemical structure and arrangement of active polysaccharides (Heleno et
al., 2012; Wang et al., 2012). Similarly, mixed carbohydrates, such as
polysaccharide–peptide complexes, have also shown a potent antioxidant activity
Chapter I Introduction
26
(Li et al., 2012). The most widely used synthetic antioxidants are butylated
hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and tert-butylated
hydroxyquinone (TBHQ), which are applied in fat and oily foods to prevent
oxidative deterioration (Grice 1988; Kahl and Kappus 1993).
1.11.5 Other pharmaceutical applications
Fungal exopolysaccharides are effective in promoting the treatment of
diseases like microbial infections, diabetes and hyper-cholesterolemia. Curdlan is
used to lower the cholesterol concentration in liver. Gellan gum is used in oral drug
deliver, and in nasal spray pumps. Pullulan is also being investigated for its drug
delivery applications, hydrophobized pullulan as drug delivery carriers. Dextran is
also explored as potential plasma expanders. Scleroglucan include the use in tablet
coating, ophthalmic solutions, injectable antibiotic suspensions and calamine lotion.
1.11.6 Microbial Enhanced oil recovery
In oil recovery, scleroglucan increases the viscosity, hence the hydraulic
pressure of sea water or brine used to extract oil. Scleroglucan lubricates the drill
and controls the backpressures created during drilling. In addition, scleroglucan
exhibited high thermostability in drilling fluids when it was associated with
polyglycols through intermolecular interactions (Mahapatra and Banerjee 2013).
1.11.7 Bioremediation and waste water treatment
An expanding area of biotechnology is the application of EPS producing
microorganisms in the remediation of environmental effluents (Iyer et al., 2006;
Valenzuella et al., 2006). Biofilm-mediated bioremediation has been found to be a
more effective and safer alternative to bioremediation with planktonic bacteria
(Donlan 2002). The pressure of biofilm helps maintain optimal chemical and
physiological conditions, localized solute concentrations and redox potential,
allowing cells to improve mineralization processes (Singh et al., 2006).
Fungi have been proved to be efficient and economical or the removal of
toxic metals from dilute aqueous solutions by biosorption because fungal biomass
offers the advantage of having a high percentage of cell wall material, which shows
excellent metal binding properties (Das et al., 2008). Moreover, large quantity of
Chapter I Introduction
27
fungal biomass is available from antibiotics and food industries. Ultimately, the
biosorption results not only in metal removal but also provides an eco-friendly
environment. A. niger has been found capable of removing heavy metals like Pb,
Cd, and Cu (Kapoor and Viraraghvan 1995). Agaricus macrospores efficiently
extract Cd, Hg and Cu from contaminated wastes. Huang et al., (1988) also studied
removal of Cd using nine different species of fungi both in batch and continuous
reactors. Table 1.5 presents the exhaustive list of microorganisms used for the
removal for heavy metals.
Table 1.5 Biosorbent uptake of metals by microbial biomass
Metal Biomass Type Biomass class Reference
Ag
Freshwater alga Biosorbent Brierley and Vance 1988
Streptomyces noursei Filamentous fungus
bacteria Mattuschka et al., (1993)
Sacchromyces
cerevisiae Yeast Brady and Duncan (1993)
Au
Sargassum natans Brown alga Volesky and Kuyucak (1988)
Aspergillus niger Fungus Kuyuack and Volesky (1988)
Bacillus subtilis Bacteria Cell wall Beveridge (1986)
Cd
Penicillium
spinulosum Fungus Townesley et al., (1986)
Spirulina sp. Blue green algae Chojnacka et al., (2005)
Co
Ascophyllum
nodosum
Brown marine
algae Kuyucak and Volesky (1989)
Ulva reticulata Marine green
algae Vijayaraghavan et al., (2005)
Enterobacter
cloaceae Marine bacterium Iyer and Jha (2005)
Cr
Bacillus biomass Bacterium Brierley and Brierley (1993)
Pantoea sp. TEM 18 Bacteria Ozdemir et al., (2004)
Spirulina sp. Cyanobacteria Chojnacka et al., (2005)
Spirogyra sp. Filamentous algae Gupta et al., (2001)
Cu
Pencillium
chrysogenum Fungus Niu et al., (1993)
Ulva reticulata Marine green alga Vijayaraghavan et al., (2005)
Spirulina sp. Blue green algae Chojnacka et al., (2005)
Fe Bacillus subtillis Bacterial cell wall Beveridge (1986)
Sargassum fluitans Brown alga Figueira et al., (1995)
Hg
Pencillium
chrysogenum Fungus Nemec et al., (1977)
Cystoseira baccata Marine alga Herrero iet al., (2005)
Chlamydomonas
reinhardtii Algae Tuzun et al., (2005)
Ni Ulva reticulata Marine green algae Vijayaraghavan et al., (2005)
Polyporous versicolor White rot fungus Dilek et al., (2002)
Chapter I Introduction
28
Aim and scope of the study
During the past three decades many biological active compounds like
polysaccharides and polysaccharide-protein complexes have been isolated from
fungi, algae, lichens and plants. Furthermore most of them which have various
biological activities were originated from fungi, especially mushrooms. Mushrooms
in submerged culture are more preferably used to produce EPS which is gradual
substitute for solid culture in recent years. It takes several months for the solid-
culture mushrooms to grow into the fruiting bodies on solid substrates. Moreover,
submerged culture gave rise to many potential advantages of higher mycelial
biomass or EPS production in a compact space and shorter time with less chances of
contamination. Exopolysaccharides from mushrooms have found a wide range of
applications as thickeners, bioadhesives, stabilizers, probiotic, and gelling agents in
food and cosmetic industries. The use of β-(1,3)-D-glucans produced from
mushrooms have attracted attention in the last few years with the exploitation of
their biological activities i.e. antitumor, immunomodulating, antibacterial, antiviral
and antioxidative properties. The use of white rot fungi for the removal of heavy
metals also deserves great attention. Hence, the techniques such as submerged
cultivation and biosorption have been used by various researchers for the treatment
of heavy metals contaminated water.
Many microbial EPS provide properties that are almost identical to the gums
currently in use. With innovative approaches, efforts are underway to supersede the
traditionally used plants and algal gums by their microbial counterparts. β-glucans
have been shown to possess various industrial applications. This finding calls for
future perspectives into wide production of EPS using white rot fungi for their
application in many quite distinctive areas, such as food industry, biomedicine,
cosmetology, agriculture, environmental protection and waste water management.
Chapter I Introduction
29
Objectives of the present study
The objectives of the present study were following:
Isolation and screening of potential EPS producing white rot fungi.
Optimization of various physico-chemical parameters for EPS production
using one-factor-at-a-time method and statistical methodology under
submerged cultivation of S. commune AGMJ-1.
Purification and characterization of EPS.
Removal of hexavalent chromium from fortified solution by S. commune
AGMJ-1 and phytotoxicity study.
Biosorption of Cr(VI) using dried EPS and mycelial biomass of S. commune
AGMJ-1
Biosorption of Cr(VI) using pretreated wet mycelial biomass of S. commune
AGMJ-1
Assessment of antioxidant properties, antiproliferative activity and
genotoxicity study of EPS.