Overview of Microalgal Extracellular Polymeric Substances (EPS) and Their

Applications

Rui Xiao, Yi Zheng

PII: S0734-9750(16)30105-7
DOI: doi: 10.1016/j.biotechadv.2016.08.004
Reference: JBA 7069

To appear in: Biotechnology Advances

Received date: 24 February 2016

Revised date: 1 August 2016
Accepted date: 24 August 2016

Please cite this article as: Xiao Rui, Zheng Yi, Overview of Microalgal Extracellular
Polymeric Substances (EPS) and Their Applications, Biotechnology Advances (2016), doi:
10.1016/j.biotechadv.2016.08.004

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 ACCEPTED MANUSCRIPT

Overview of Microalgal Extracellular Polymeric Substances (EPS) and Their Applications

Rui Xiao, Yi Zheng*

Department of Environmental Engineering and Earth Sciences, Clemson University,

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342 Computer Court, Anderson, SC 29625, USA
*
Corresponding author. Tel.: +1 864 656 7468; fax: +1 864 656 1041.

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E-mail address: zheng9@clemson.edu (Y. Zheng).

Abstract

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Microalgae have been studied as natural resources for a number of applications, most particularly
food, animal feed, biofuels, pharmaceuticals, and nutraceuticals. In addition to the intracellular

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compounds of interest, microalgae can also excrete various extracellular polymeric substances
(EPS) into their immediate living environment during their life cycle to form a hydrated biofilm
matrix. These microalgal EPS mainly consist of polysaccharides, proteins, nucleic acids and
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lipids. Most notably, EPS retain their stable matrix structure and form a 3-D polymer network for
cells to interact with each other, and mediate their adhesion to surfaces. EPS also play a role as
extracellular energy and carbon sinks. They are also abundant source of structurally and
compositionally diverse biopolymers which possess unique bioactivities for special high-value
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applications, specifically as antivirals, antitumor agents, antioxidants, anticoagulants and anti-

inflammatories. Their superior rheological properties also make microalgal EPS particularly
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useful in mechanical engineering (e.g., biolubricants and drag reducers) and food
science/engineering (e.g., thickener and preservatives) applications. The chemical composition
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and structure of EPS appear to correlate with their applications, but the fundamentals of such
relationship are not well understood. This article summarizes previous research on microalgal
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EPS derived from green algae, diatoms and red algae, including compositions/functions/structure,
production, and potential applications. The importance of exopolysaccharides and EPS proteins,
with their particular metabolic characteristics, are also described because of their potential high-
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value applications. This review concludes with potential future research areas of microalgal EPS.

Keywords: Microalgae; Extracellular polymeric substances; Exopolysaccharide; Protein;

Bioactivity

1. Introduction

Instead of living as pure cultures of dispersed single cells, microalgae always assume the form of
polymicrobial aggregates (e.g., flocs, sludge, mats, granules, or as biofilms) at the interface. The
materials that hold these cells together to form a heterogeneous matrix are excreted by algae
themselves into their immediate environment. These excretions, known as extracellular
polymeric substances (EPS) are composed of a complex high-molecular-weight (HMW) mixture
of biopolymers (e.g., EPS from microalgae as shown in Figure 1). These EPS in turn
significantly affect the physicochemical properties of algae aggregates, particularly the surface
charge, viscosity, flocculation, structure, and settling properties. EPS are versatile, natural
biopolymers that may function as anticoagulants that can protect cells against dewatering and
toxic substances, and that can serve as energy and carbon sinks responding to stress. They also
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promote the formation of algae aggregates, immobilize cells in close proximity in a matrix,
initiate cell adhesion to a substrate, promote gliding motility along the substrate, and create
ensheathment within the biofilm matrix (Parker, 2013). The microalgae aggregates formed by the
EPS create a completely different lifestyle from the planktonic state.

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A B C
) ) )

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Figure 1. Images of microalgal EPS: A) Atomic force microscopy of a released polymer image (500 nm) of the
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whole cell of a diatom, Cylindrotheca closterium (Paniagua-Michel et al., 2013) ; B) The fibrillar EPS from Cyl.
closterium mainly extends from the apices of the rostra and appears bound to the cell (Pletikapić et al., 2011) ; and C)
the living cells of a red microalga, Rhodella maculata in a weak suspension of Indian ink showing the extent of the
encapsulating mucilage (×900) (Evans et al., 1974).
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Microalgal biomass and intracellular compounds have been studied and used as renewable
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natural resources for a wide spectrum of applications, including food (e.g., emulsion stabilizer
and anti-crystal formation), animal feed, biofuels, pharmaceuticals/cosmetics (e.g., hydrating
agents), nutraceuticals (e.g., eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA), and
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so on (Cheng et al., 2011, 2013; Zheng et al., 2015, 2016). Some of such applications have been
commercialized, e.g., algal EPA and DHA. Although microalgal EPS have received extensive
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research on their compositions, properties, functions, production, and applications, the

knowledge about the interactions between EPS component polymers, the interactions between
EPS and cells, and the contributions of polymers to overall EPS properties and functions are
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poorly understood at molecular level. EPS have been a topic of current research interest and
more research is still needed to fully understand the precise roles of EPS in the biological
processes and potential applications. For example, it was found that microalgal EPS have great
potential for use in the creation of novel anti-adhesives, antitumor agents, anti-inflammatories,
antivirals, antibacterials, immunomodulatory agents, biosurfactants, and bioemulsifiers. These
possibilities make microalgal EPS another promising natural bioactive compound resource in
addition to their biomass and intracellular compounds. Therefore, making the in-depth study of
EPS is also important in terms of the development of value-added EPS applications. Compared
to bacteria and cyanobacterial EPS, much less is known about microalgal EPS (cyanobacteria are
not considered as microalgae in this review). The emphasis is placed on reviewing EPS from
different microalgae taxa (green algae, diatoms and red algae), including the compositions,
functions, structure, production, and applications.

2. Microalgal EPS Composition and Functions

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EPS are an abundant source of structurally and compositionally diverse biopolymers.

Investigations on the compositions and characteristics of EPS is urgently important to understand
production mechanism and their properties for potential applications. The major components of
EPS include polysaccharides, proteins (i.e., enzymes and structural proteins), nucleic acid (DNA)
and lipids. Humic substances, uronic acid and inorganic components are also found within EPS.

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Although nonsugar components are less than polysaccharides in quantity, they are generally
attached to the sugar residues and are important in conveying unique characteristics to the EPS
(Decho, 1990; Sutherland, 1979). The range of polymers in EPS and the fractions of specific

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components are influenced by various factors, including the species, strain, substrate type,
nutrient availability, operation conditions (e.g., temperature, pH, shear force and salinity),
physiology, and age of the culture. EPS perform a variety of functions, and are involved in

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diverse biological processes, e.g., transport and transformation of organic matter, complexation
of dissolved metals and biogeochemical cycling of elements. In addition, EPS are an important
carbon source for different organisms in the food chain because they are rich in organic carbon.

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EPS are also involved in flocculation and the nature of EPS controls the floc formation rate, e.g.,
sulfates are able to generate flocs in the presence of deoxy sugars (Zhou et al., 1998). Sulfates in
EPS also impart EPS hydrophilicity so that EPS or in part can have gel-like consistency by
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holding water molecules (Wingender et al., 1999). Apart from other components, cations such as
Ca2+ and Mg2+ were also found in EPS which are involved in multiple cross-linkages between
sugar molecules of different polysaccharide chains of EPS, resulting in high consistency and
stability of EPS (Smidsrod, 1974). EPS were also found to possess unique properties for special
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high-value applications such as antivirus and antitumor and wastewater treatment. Indeed much
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effort has entailed the exploitation of valuable EPS derived from bacteria and cyanobacteria for
industrial applications, over the last few years, particularly with the urgency of developing
renewable and sustainable non-petroleum bio-based chemicals and polymers. Therefore, new
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research to create natural polymers for various industrial applications, now requires deriving EPS
from such novel sources as microalgae. This review on composition is focused on only
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exopolysaccharides and protein because little information is available for other component such
as lipid and nucleic acid of microalgal EPS.
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2.1. Isolation and analysis of EPS

In the study of EPS, the isolation and analysis of EPS is a most challenging endeavor. The
separation method for microalgal EPS can in turn influence the compositions, contents, structure,
properties, and functions of EPS (Takahashi et al., 2009). Specifically, current extraction
methods (e.g., alkaline, high temperature and organic solvent) can change the properties of EPS,
making any precise analysis of the individual components of EPS difficult (Takahashi et al.,
2009). The difficulty of purifying polymers also greatly complicates the process, which means
that EPS may exhibit multiple properties and functions that are actually reflected by the
collective EPS characteristics of the compositional polymers (Table 1). This shifting landscape
consequently makes it difficult if not impossible to identify a component for a specific function.
A comprehensive evaluation of different methods by Sheng et al. (2010) and Flemming and
Wingender (2010) determined the cation exchange resin method to be the most effective EPS
extraction method in that it avoids chemical contamination and property change of the product
EPS. Gas chromatography (GC), high pressure liquid chromatography (HPLC), GC-mass
spectrometry (GC-MS), X-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance

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(NMR), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) are
all common methods used in EPS analysis. A new method however, developed by Badel et al.
(2011) and known as BioFilm Ring Test (BFRT) can detect the EPS of red microalgae such as
Rhodella violacea and Porphyridium purpureum, but the effectiveness of BFRT for other
microalgal EPS is unknown. Stewart et al. (2013) recently developed a new protocol called

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liquid chromatography-organic carbon detection-organic nitrogen detection (LC-OCD-OND) to
extract, fractionate, and analyze EPS. This method can simultaneously identify and quantify
organic carbon fraction and nitrogen with respect to protein and polysaccharide content as well

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as molecular weight distribution. It can be also used to study biofilm community structure and
metal-EPS binding. Even though many technologies have been developed for EPS isolation and
analysis, there is no universal method, i.e., the extraction and analysis procedures have to be

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adapted to the specific type of EPS under investigation.

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Table 1. Example EPS functions and related components (Adapted from Flemming and Wingender, 2010)
Functions EPS components
Adhesion, cohesion of biofilm and cell aggregation Polysaccharides, protein and DNA
Water retention Polysaccharides, protein
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Sorption of organic compounds and inorganic ions Charged Polysaccharides and protein
Nutrient source All EPS
Energy source Polysaccharides (degraded by enzymes)
Enzyme activity Protein
Enzyme generation Polysaccharides and enzymes
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Antivirus, antioxidant, antitumor, and anti- Polysaccharides

inflammatory
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Surfactant, emulsifier Substituents (polysaccharide-link methyl and acetyl

groups) and lipid
Export of cell components Nucleic acid, enzyme, lipopolysaccharides and
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phospholipids
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2.2. Exopolysaccharides

Of all the known EPS components, exopolysaccharides (very often called EPS because they are
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the highest content in EPS. In its broad sense, EPS include all extracellular polymeric
substances), particularly the sulfated polysaccharides (sPS) have been the subject of recent and
intense research because of their versatile roles in EPS functions and possible use in
pharmaceuticals, therapeutics, regenerative medicine, mechanical and food industry applications.
Polysaccharides are linear or branched long molecules with molecular weight of (0.5-2)×106 Da
(Flemming and Wingender, 2010). It seems that only exopolysaccharides from Gyrodinium
impudicum and cell wall polysaccharides from Chlorella vulgaris contain homopolymer of
galactose and β-(1,3)-glucose, respectively (Nomoto et al., 1983; Yim et al., 2007), while the
exopolysaccharides from other microalga species are all heteropolysaccharides with different
substituents in various carbons of their backbone and side-sugar components (Raposo et al.,
2014a). Organic or inorganic substituents often exist in exopolysaccharides affecting the physical
and biological properties of EPS. Glucuronic acid and sulfate residues, often present in the
backbone of polysaccharides, appears crucial for biomedical and anticoagulation activities
(Cumashi et al., 2007; Moore and Tischer, 1964; Raposo et al., 2015). Many exopolysaccharides
also contain polyanionic and polycationic compounds such as xanthan and N-acetylglucosamine
which contribute to adhesion and cell aggregations.

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The component sugars of exopolysaccharides mainly include glucose, galactose, fucose, xylose,
arabinose, rhamnose, mannose, fructose, and/or sugar derivatives (Table 2). However,
microalgal exopolysaccharides are highly diverse so that their monomers and proportions can
vary significantly as compared to various species of microalgae, even between strains of a single

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species (Hanlon et al., 2006). The variation in physiological conditions greatly influences their
composition, suggesting that microalgae modulate their polysaccharide biosynthesis machinery
to adapt to environmental conditions. Indeed, the structural and compositional features of

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microalgal exopolysaccharides can significantly affect the physicochemical properties and
biological activities of EPS, including the molecular weight, sugar residue composition, sulfation
level, distribution of sulfate groups in the polysaccharide backbone, and stereochemistry,

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antimicrobial activity, and so on (Damonte et al., 2004; Ghosh et al., 2009). For example, the
presence of arabinose in EPS helps in cell aggregation (Efrat et al., 2004), whereas deoxy sugars
such as fucose and rhamnose in diatom EPS affect foaming, flocculation and cell adhesion (Zhou

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et al., 1998; Willis et al., 2013; Wustman et al., 1998). In addition, the abundance of galactose in
the EPS was hypothesized to have a role to maintain the stationary phase of Botryococcus
braunii (Tanoi et al., 2011). It was reported that Penium margaritaceum produces a large amount
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of EPS rich in xylose, fucose and glucuronic acid and such EPS functioned by linking cells and
substrate together, with the EPS secretion occurring in different modes and contributing to initial
adhesion, capsule formation and gliding (Domozych et al., 2005; Domozych, 2014).
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Table 2. Summary of monosaccharide and glucuronic acid contents of EPS from different microalgae *
Microalgae Species Monosaccharide content References
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Dunaliella salina Glu, Gal, Fru, and Xyl=105, 130, 160, and 110 µg/La, respectively Mishra and Jha (2009)
C. vulgaris Glu, Xyl and GlcUA
Chlorella ellipsoidea Glu, Ara, GlcUA, and Fuc
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Moore and Tischer

Chlamydomonas sp. Glu, Xyl and Fuc
(1964)
Oocystis sp. Gal, Ara, GlcUA, and Fuc
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Chlorella sp. Glu, Ara, GlcUA, and Rha

Chlorella sp. Glu (193 g/kg), Fuc (45 g/kg), Ara (325 g/kg), and GlcUA (385 g/kg) Yalcin et al. (1994)
Green algae B. braunii Glu, Gal, Fuc, Ara, Rha, and GlcUA Allard and Casadevall
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(1990); Weiss et al.

Scenedesmus acuminatus
Spondylosium panduriforme
Hyalotheca dissiliens
Chaetoceros affinis var. willei Mainly: (Rha + Fuc=63%)b, Ara and Gal
(Gran) Hhustedt
C. closterium
Navicula salinarum
Achnanthes longipes
Diatom Amphora coffeaeformis
Cymbella cistula
Cymbella mexicana
Coscinodiscus nobilis
Cylindrotheca fusiformis
Pinnularia viridis
(2012)
Man (76), Xyl (3), GlcUA (9), Gal (2.5), Ara (0.5), Fuc (3.5), Rha
Lombardi et al. (2005)
(2.5), and GalUA (3) b
Glu, Ara, Fuc, Xyl, Gal, Rha, and GlcUA Paulsen and Vieira
(1994)
Glu, Ara, Fuc, Xyl, Gal, Rha, and GlcUA Vieira and Paulsen
(1994)
Myklestad and Haug
Others: Glu, Man and Xyl (1972)
Glu/Gal/Man/Rha/Ara/Xyl=22.9/12.2/4.1/14.7/0/46.1 c
Glu/Gal/Man/Rha/Ara/Xyl=41.6/19.1/13.8/5.6/0/20.2 c Staats et al. (1999)
Glu/GlcUA/Gal/Man/Gul/Xyl/Ara/Fuc/Rha=11/10/25/16/0/10/3/23/3 b
Glu/GlcUA/Gal/Man/Gul/Xyl/Ara/Fuc/Rha=40/7/13/13/0/9/0/9/9 b
Wustman et al. (1997)
Glu/GlcUA/Gal/Man/Gul/Xyl/Ara/Fuc/Rha=4/0/69/3/0/18/1/0/2 b
b
Glu/GlcUA/Gal/Man/Gul/Xyl/Ara/Fuc/Rha=5/1/57/12/0/16/1/2/6
Fuc, Rha, Man, Glu, Xyl, Gal, Ara, and GlcUA Percival et al. (1980)
d
Gal/Glu/Man/Xyl/Rha/Fuc=37.7/26.5/5.4/13/12.9/4.4 Magaletti et al. (2004)
Rha/Fuc/Xyl/Ara/Glu/Man/Gal=79/32/62/5/80.5/51.5/52c Chiovitti et al. (2003)

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C. closterium Rha/Fuc/Xyl/Man/Gal/Glu=7/5/9/8/14/29b Underwood et al.

(2004)
Stauroneis amphioxys Glu/GlcUA/Gal/Xyl/Rha/Man/Ara/Fuc=13/25/21/11/5/9/2/7b McConville et al.
Gregory (1999)
Porphyridium sp. Xyl=40~44%, Gal=30~32% and Glu=26~29%b Singh et al. (2000)
Porphyridium sp. Xyl/Glu/Gal=2.5/1/1.8e and GlcUA=8~10 %b
Geresh and Arad

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Porphyridium aerugineum Xyl/Glu/Gal=1.7/1/1.1e and GlcUA=9~10 %b
(1991)
Rhodella reticulata Xyl/Glu/Gal=1.2/1/1.3eand GlcUA=7.8 %b
Porphyridium cruentum Glu, Gal and Xyl Jones (1962)
P. cruentum Xyl:Glu:Gal:GlcUA=3:1:2.5:0.8e

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Percival and Foyle
Red algae P. aerugineum Xyl:Glu:Gal:GlcUA=1.7:1:1.1:0.5e (1979)
Porphyridium sp. Xyl:Glu:Gal:GlcUA=2.1:1.1:1.0:0.2e Geresh et al. (2009)

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P. aerugineum Glu, Gal and Xyl Ramus (1972)
R. maculata Xyl (major), Gal and Glu (trace) Evans et al. (1974)
P. cruentum Gal:Xyl:Glu:GlcUA=2.12:2.42:1.00:1.22e Heaney-Kieras and
Chapman (1976)

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*
Glu=glucose, Gal=galactose, Gul=gulose, Fru=fructose, Xyl=xylose, Ara=arabinose, Rha=rhamnose,
Man=mannose, Fuc=fucose, GlcUA=glucuronic acid, and GalUA=Galacturonic acid
a
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unit is micrograms of monosaccharide per liter of supernatant
b
weight % in total sugar
c
mole %
d
weight % in total EPS
e
mole ratio
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2.2.1. Green microalgae

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Many green microalgae species (e.g., Chlorella sp., D. salina, Ankistrodesmus angustus, and B.
braunii) can produce EPS containing a large amount of exopolysaccharides. The monosaccharide
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components of the exopolysaccharides are hexose, pentose, uronic acid, methyl pentose, methyl
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hexose and/or sulfate. The most common monosugars were glucose, galactose, arabinose, fucose,
xylose, mannose, glucuronic acid, and galacturonic acid (Table 2). Among all studied five green
algae, no exopolysaccharides contain more than four major monosaccharide components (Moore
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and Tischer, 1964). Except for glucose, S. acuminatus has all the common sugars. Mannose has
been found a typical and important monosaccharide of exopolysaccharides from Chlorophyceae
including S. acuminatus, Ankistrodesmus densus and Kirchneriella aperta (Lombardi and Vieira,
1999; Lombardi et al., 2005; Paulsen et al., 1998). Mannose seems to be one of key sugars
(among mannose, rhamnose and uronic acid) responsible for the complex with heavy metals, e.g.,
copper and lead. Allard and Casadevall (1990) found that five strains of B. braunii can produce
exopolysaccharide with concentration in the range of 0.25 to 1 g/L. The results of their GC and
GC-MS analysis showed galactose as the main component of the polysaccharides accompanied
by fucose, rhamnose, and glucose (Allard et al., 1987; Allard and Casadevall, 1990). In addition,
the 3-O-methyl fucose and 3-O-methyl rhamnose were also detected in B. braunii
polysaccharides, which may be of significance in that very few studies have reported the
presence of methyl sugars in other green microalgae EPS. It was found that C. vulgaris did not
have arabinose or fucose, but C. ellipsoidea did not have xylose in its exopolysaccharides
(Moore and Tischer, 1964). In addition, Chlamydomonas sp. lacked the uronic acid moiety in its
exopolysaccharide which was present in other green algae. Studying the same species, however,
Lewin (1956) revealed that the exopolysaccharides from eighteen green algae strains of
Chlamydomonas species consisted of similar monosugar compositions, including galactose,

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arabinose, glucose, and xylose with other sugar moieties, particularly fucose, rhamnose, mannose,
and uronic acids. Sulfate is very common in exopolysaccharides, but it was not found in EPS
from A. densus (Paulsen et al., 1998) or B. braunii (Bayona and Garces, 2014). Different sugar
compositions of exopolysaccharides of the same species from different research groups was due
to the different experiment conditions and analysis methods, which makes comparisons difficult.

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To date, only limited studies have been done on the fine structure of exopolysaccharides of green
algae on linkages. For example, the main chain of exopolysaccharides of S. acuminatus consists
of 1-2/1-4 linked fucose and 1-2/1-4 linked mannose (Lombardi et al., 2005); however, C.

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vulgaris exopolysaccharides mainly consists of 1-6 galactose (67% of total polysaccharides) (i.e.,
beta-1, 6-D-galactopyranose backbone) (Noda et al., 1996). Exopolysaccharides from
Dictyospheaerium chlorelloides is a novel biopolymer which could potentially have industrial

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applications due to its unusual structure of α-glucan with large amount of galactose and
rhamnose residues. It could provide a perfect basis for its application for the selective
immobilization of particles because of their high porosity of its matrix with regular elongated

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pores (Cybulska et al., 2016).

2.2.2. Diatoms
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Diatoms are found in almost every aquatic and moist habitat, particularly in fresh and marine
waters and soils. Both benthic and planktic forms exist and both are ecologically important in
their living environment in terms of carbon flux. The annual primary production of diatoms is
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estimated at 1 G tons of carbon which is equivalent to 50% of marine and 25% of the global
primary production (Ansell et al., 1999; Pletikapić et al., 2011; Underwood and Kromkamp,
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1999; Urbani et al., 2012). Although unicellular, diatom could form colonies via EPS. Their
motility is limited however, in that they can only move to the substrate along a slit-like groove or
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channel formed by EPS secreted within (Underwood et al., 2004). This means that diatoms are
restricted to the photic zone. Diatoms are a group of the most abundant EPS producers that
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release a large amount of organic carbons in the form of EPS into their growth environment.

EPS from diatoms have been extensively studied because of their multiple functions in aquatic
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systems, including aggregation of particles, influence of organic carbon flux, part of gliding
locomotion and habitat aggregation, protection against desiccation, aid in colony formation,
energy storage, mechanism of motility, and loose symbiosis with bacteria (Magaletti et al., 2004;
Staats et al., 1999; Underwood et al., 2004). Both pelagic and benthic diatoms can exude EPS
during attachment, colonization and population growth to form biofilms and mucilage on the
sediment surface to stabilize sediment.

Relatively, diatoms received more research than green algae on the chemical structure of
exopolysaccharides. The chemical characteristics of EPS from different diatom species revealed
that the EPS consisted predominantly of polysaccharides which mainly contain neutral sugars,
uronic acid and sulfate residues and small quantities of proteins; lipids however have not been
detected in the diatom EPS (Hoagland et al., 1993; Urbani et al., 2012). Diatom
exopolysaccharides have three general compositional features, including (1) they are all
heteropolysaccharides which are sulfated; (2) they contain fucose, rhamnose, mannose, glucose,
xylose, arabinose, galactose, and/or glucuronic acid; (3) substituted mannan that is closely
associated with the cell wall appear to be universal features of diatom frustules (Chiovitti et al.

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2005). Diatom exopolysaccharides have high proportions of methylpentose (Gugi et al., 2015).
As shown in Table 2, the constituent sugars of diatoms are more than those of green microalgae
in general. They are highly diverse and consist of 6-9 different monosaccharides excluding sugar
derivatives, but different species have many monosaccharides in common with varied
proportions. In their study of the EPS of P. viridis, Chiovitti et al. (2003) found the EPS were

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composed of carbohydrate, protein, and sulfate. Of carbohydrate contents, nineteen sugars were
identified, including pentoses, hexoses, 6-deoxyhesoses, O-methylated sugars, aminohexoses,
and traces of uronic acids. The exopolysaccharides from C. nobilis consisted of fucose, rhamnose,

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mannose, glucose, xylose, glucuronic acid, galactose (trace) and half ester sulfate (Percival et al.,
1980). These researchers also observed that the different fractions of EPS derived from different
extraction methods contained similar constituent monosaccharides, with a significant variance in

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proportion, however. Also, EPS production rate and monosaccharide compositions differ
according to the growth phase and physiological status of the cells. McConville et al. (1999)
reported that the proportion of monosaccharides varied during the growth of S. amphioxys while

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the total amount of EPS stayed constant. Gugi et al. (2015) summarized monosugar composition,
sulfate substitution and linkage of exopolysaccharides of 23 diatoms and found that a large
diversity of linkages were present in exopolysaccharides of 6 diatoms reported, with many
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glycosyl residues being typical of branched structures.

2.2.3. Red microalgae

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Most studies on red microalgae EPS have been conducted on the Porphyridium genus with a
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particular emphasis on the purpureum, cruentum and aerugineum species, and with lesser
research on the Rhodella and Rhodosorus species. Most research entailed the determination of
the compositions, chemistry and properties of exopolysaccharides of EPS which were found to
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have a broad range of pharmaceutical/biomedical (e.g., antivirus, antibacterial, antitumor,

antioxidant, anti-inflammatory) and industrial (e.g., drag reducer and biolubricant) applications.
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Although the chemical compositions of red alga exopolysaccharides are similar among different
species, the relative percentages vary significantly (Table 2). Major monosaccharides, including
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glucose, xylose, galactose, and glucuronic acid were identified in exopolysaccharides from red
algae. The exopolysaccharide of Porphyridium sp., which was a heteropolymer with molecular
weight as high as 7×106 Da, consists of 10 different monosugars with xylose (40%), galactose
(18%) and glucose (15%) mostly present (Geresh et al., 1992). Geresh and Arad (1991) also
found the predominance of monomers in the exopolysaccharides of P. cruentum included xylose,
galactose and glucose as well as 10% of glucuronic acid and 7.6% of sulfate, plus protein moiety
attaching to the polysaccharide molecules. Most reported red algae have less than four major
component monosaccharides in their exopolysaccharides. Compared to green algae and diatoms,
red alga exopolysaccharide composition is much simpler and consistent among different species.
Under certain conditions, the exopolysaccharides from some species or strains lack of glucuronic
acid (Geresh et al., 2009; Ramus, 1972; Singh et al., 2000). In their use of NMR technology for
chemical characterization of EPS from Porphyridium sp., Gloaguen et al. (2004) found that the
EPS primarily included proteins and polysaccharides (composed of the three neutral
monosaccharides xylose, glucose, galactose, and glucuronic acid. Neutral monosugars and
glucuronic acid also exist as methylated and sulfated residues such as 2-, 3-, and 4-O-

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methylsugars, 2-O-methlylglucuronic acid and sulfated galactose/glucose in the EPS from P.

cruentum and P. aerugineum (Percival and Foyle, 1979).

In contrast to green algae and diatoms, red algae received more research on the structural
characteristics of their exopolysaccharides because their EPS has been recognized to have

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variable high-value biomedical and pharmaceutical applications. Percival and McDowell (1967)
revealed that the characteristic exopolysaccharides of red algae consisted of alternating 1, 3- and
1, 4-linked galactose residues carrying variable proportions of half ester sulfate and methyl ether

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residues. Turvey and Williams (1970) found that xylans which comprised 1, 3- and 1, 4-linked
xylose units had been found in a number of species red algae. Medcalf et al. (1975) and Percival
and Foyle (1979) made a conclusion that the presence of both L- and D-forms of galactose

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appears to be a characteristic common to the exopolysaccharides of red algae. Arad and
coworkers had done a lot of research to elucidate the fine structure of exopolysaccharides from
red algae including Porphyridium sp., P. cruentum, P. aerugineum, R. reticulata, and R.

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maculata. Their results indicated that the presence of aldobiuronic acid 3-O-(α-D-
glucopyranosyluronic acid)-L-galactopyranose seems to be a characteristic common to all of the
polysaccharides of the studied red unicells (Arad et al., 1993; Dubinsky et al., 1992; Evan et al.,
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1974; Geresh et al., 1990, 1992; Geresh and Arad, 1991; Jaseja et al., 1989; Lupescu et al., 1991,
1992; Simon et al., 1992, 1993). Gloaguen et al. (2004) reported that Porphyridium sp. contained
xylose, glucose, galactose, and uronic acid in its exopolysaccharides and the structure of the
polysaccharide was established by NMR (Figure 2):
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A). B).
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Figure 2. Structure of exopolysaccharide of Porphyridium sp. (Gloaguen et al., 2004)

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Similar to Gloaguen et al. (2004), Geresh et al. (2009) studied the chemical structure of acidic
crude extract of exopolysaccharides from Porphyridium sp. (UTEX 637) and revealed that the
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almost linear backbone was composed of (1-2)- or (1-4)-linked D-xylopyranosyl, (1-3)-linked L-

galactopyranosyl, (1-3)-linked D-glucopyranosyl and (1-3)-linked D-glucopyranosyluronic acid
and comprised a possible acidic building unit:[(2 or 4)-β-D-Xylp-(l-3)]m-α-D-Glcp-(1-3)-α-D-
GlcpA-(1-3)-L-Galp. Attached to the backbone are sulfate groups and nonreducing terminal D-
xylopyranosyl and galactopyranosyl residues, which occurred at the O-6 positions of Glc-derived
moieties in the main chain.

2.3. Extracellular protein

Except for polysaccharides, microalgal EPS also consist of protein moiety that has
physicochemical interactions with polysaccharides and other components (e.g., extracellular
DNA) to form a stable EPS matrix. Extensive studies on extracellular proteins in bacterial and
fungal biofilms found that extracellular enzymes are involved in the degradation of biopolymers,
including polysaccharides, proteins, and nucleic acids. They also degrade EPS components so
that EPS matrix can serve as carbon and energy sources for host cells (Flemming and Wingender,
2010). Excretion of low-molecular-weight (LMW) polypeptide materials by algal cells grown
under normal conditions commonly occurs (Newell et al., 1972). The authors has done research

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on extracellular protein of microalgae and found a wide spectrum of enzymes were present in
EPS and possibly responsible for degrading EPS to provide energy and carbon sources for host
cells to grow (unpublished). Therefore, polymer degradation enzymes in EPS are important in
the life cycle of host algal cells. Other enzymes of importance can promote cell attachment and
detachment. Interactions between enzymes and exopolysaccharides can protect enzymes from

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thermal deactivation and proteolysis. The non-enzymatic structural proteins, which are integral
to the formation and stabilization of an EPS matrix network, form a link between the host cell
surface and EPS. The proteins bound to carbohydrates are called lectins. Other extracellular

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protein could include cell-associated surface proteins and amyloids (Flemming and Wingender,
2010). Little is known however, either of the composition structure, chemistry, and function of
proteins in microalgal EPS or of the extracellular enzymes of microalgae. Consequently, the

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interactions between protein and polysaccharides/host cell surfaces are also nearly unknown.
Limited available results on protein cover its content in EPS and its amino acid component are
summarized in Table 3. It can be seen that both protein content and compositional amino acids

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vary from species to species.

Table 3. Extracellular proteins and amino acid contents of protein from different microalgae
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Microalgae Species Total protein Amino acid content References
Dunaliella tertiolecta 13.4% a Goo et al. (2013)
C. vulgaris 1.45 mg/L b Chiou et al. (2010)
Chodatella sp. 0.5 mg/L b Chiou et al. (2010)
Green algae Chlorella sp. 10 mg/L b ND Wang et al. (2014)
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Micractinium sp. 11 mg/L b Wang et al. (2014)

2.4 mg/L b
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Chlamydomonas Baba et al. (2011)

reinhardtii
C. fusiformis ND proline (22), serine (11%), cysteine (11%), and aspartate Kröger et al. (1997)
(9%) c
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a
Amphora sp. 12.6% ND Zhang et al. (2008)
A. longipes 5% a ala (17%), gly (13%), val (13%), ser (13%), and asp/asn Wustman et al. (1997)
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(13%) d
Diatoms a
A. coffeaeformis 13% ND Wustman et al. (1997)
C. cistula 1.5% a gly (23%), arg (9%), ala (8%), glu/gln (18%), asp/asn Wustman et al. (1997)
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(8%), and ser (7%) d

a
C. closterium 16.9% ND Staats et al. (1999)
N. salinarum 5.4% a ND Staats et al. (1999)
R. maculata 16% a ND Evans et al. (1974)
P. cruentum 1~2% a asp (14.2), gly (17.0), glu (14.1), ala (11.5), and ser (11.5) Heaney-Kieras et al. (1977)
Red algae e
a
Porphyridium sp. 5.5 % ND Gloaguen et al. (2004)
a
weight % of total EPS
b
mg/L of supernatant
c
% in each domains
d
mole %
e
nmol/mg of biomass
ND=Not determined

2.3.1. Green microalgae

Glycoproteins are quite common in the EPS of green algae (Paulsen et al., 1998) and their
proportion in EPS from green algae is much higher than that from other microalgae. Allard and
Casadevall (1990), studying the composition of the exopolysaccharides released by three

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different chemical races of B. braunii, verified that the polysaccharides contained proteins as a
part of the molecules, which indicated that proteins connected to polysaccharides in EPS
somehow. Noda et al. (1996) showed C. vulgaris EPS contained 35% of glycoprotein which was
found to have antitumor activities. The extracellular protein from Chodatella sp. and C. vulgaris
were approximately 30% and 50% of EPS, respectively, which caused membrane fouling

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during the treatment of algae-laden eutrophic source waters using membrane technology (Chiou
et al., 2010). In the EPS from D. tertiolecta, the protein content reached 13% even though it was
deemed an impurity because the purpose of this research was to convert polysaccharides in EPS

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to bioethanol (Goo et al., 2013). In their use of Chlorella sp. and Micractinium sp. to treat N-rich
wastewater, Wang et al. (2014) found that the protein contents in EPS were significantly affected
by the N content (i.e., a high N led to a high protein content) and the algae cultivation stage (i.e.,

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the total protein contents in the EPS reached the highest level of 60% and 63%, respectively at
day 15 over 31-day period). It was reported that C. reinhardtii can produce a variety of
extracellular proteins, specifically enzymes and structural proteins, and the environmental CO2

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level during photosynthesis induced the change in protein compositions (Baba et al., 2011;
Hanawa et al., 2004; 2007). These researchers pointed out extracellular proteins were involved in
the determination of CO2 metabolism pathways.
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2.3.2. Diatoms

Diatoms can produce extracellular proteins in their EPS, especially A. coffeaeformis (Wustman et
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al., 1997), Amphora sp. (Zhang et al., 2008) and C. closterium (Staats et al., 1999) with protein
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contents that exceed 10% of the total EPS (Table 3). It was determined from the adhesive
modular and silica-associated extracellular proteins derived from diatoms (including
Phaeodactylum tricornutum, C. fusiformis, Amphora sp., A. longipes, A. coffeaeformis, C. cistula,
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C. closterium, and N. salinarum) that the protein percentage varied from species to species, and
the protein compositional amino acids also differed (Staats et al., 1999). The extracellular
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proteins of C. cistula were composed of various amino acids such as gly, arg, ala, glu/gln,
asp/asn, and ser while A. longipes contained amino acids including ala, gly, val, ser, asp and asn
in its extracellular proteins (Wustman et al., 1997). Both species shared some amino acids such
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as gly and ala. The protein content has been reported in microalgal EPS, but rarely been
characterized, except for frustulins, a family of proteins that coat the silicified cell wall, and
silaffins which catalyze silica deposition (Willis et al., 2013). These proteins were initially found
in C. fusiformis and subsequently identified in the EPS from a number of other diatom species
(Kroger et al., 1994, 1996, 1997, 1999; Poulsen et al., 2003; Sumper and Kroger 2004).

2.3.3. Red microalgae

Red algae also produce extracellular proteins in their EPS, the most studied of which include R.
maculata; P. cruentum and Porphyridium sp. The extracellular proteins from R. maculata
reached 16% of the total EPS (Evans et al., 1974). However, other genus of red algae produced
much less protein, less than 10%. Studying the synthesis and composition of EPS from Rhodella,
Evans et al. (1974) found ducts from the subplasmalemmal endoplasmic reticulum (ER) were
frequently seen fused to the plasmalemma, thus providing an open channel from the interior of
the ER to the outside. They also found that electron-dense material was occasionally detected at
the exit of such ducts, and this was probably the origin of the protein detected in the solubilized

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EPS. The major compositional amino acids of extracellular proteins from P. cruentum included
asp, gly, glu, ala, and ser (Heaney-Kieras et al., 1977). These researchers also found that the
extracellular protein-polysaccharide from P. cruentum had a covalent linkage of protein to
polysaccharide similar to that of the chondroitin sulfate portion of cartilage proteoglycan.
Shrestha et al. (2004) studied the protein in EPS from Porphyridium sp. (UTEX 637), P.

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cruentum Naegeli, P. aerugineum Geitler and R. reticulata Deason (UTEX LB 2320) and found
a number of noncovalently associated proteins using SDS-PAGE, but no covalently bound
proteins were detected, which is in opposition to the results obtained by Heaney-Kieras et al.

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(1977). The possible reasons may be different strains, experiment conditions and/or analytical
methods. All studied red algae were found to have a protein in EPS which was 66-kDa
glycoprotein consisting of a polypeptide of approximately 58 kDa and a glycan moiety of

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approximately 8 kDa containing N-linked terminal mannose (Shrestha et al., 2004). It was also
revealed that the glycoprotein was specific to Porphyridium sp. and P. cruentum, because it was
not detected in the other species of red microalgae examined.

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3. Microalgal EPS Production MA
EPS from microalgae are one of groups of compounds with wide valuable industrial applications
in food, pharmaceutical, nutraceutical, cosmetic, wastewater treatment industries, agriculture,
and so on. The quantity, composition, structure, and properties of EPS produced are influenced
by many factors such as species, strain, nutrient, cultivation condition, age, bioreactor used and
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even toxicity in environment.

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3.1. Green microalgae

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In their study of EPS production from five green algae, C. vulgaris, C. ellipsoidea,
Chlamydomonas sp., Oocystis sp., and Chlorella sp., Moore and Tischer (1964) achieved total
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exopolysaccharide yields of 235, 234, 224, 197 and 174 mg/L, respectively. Lewin (1956)
screened a number of Chlamydomonas spp. for EPS production and found Chlamydomonas
mexicana was the most useful species which yielded up to 25% of its total organic production as
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polysaccharides. L-race strain of B. braunii can produce EPS up to 1 kg/m3 whereas race-A and
race-B strains only produced up to 250 g/m3 (Banerjee et al., 2002).

During the study on the effect of salinity on EPS production from D. chlorelloides (CCALA 330),
Koyun et al. (2015) found that salinity stress (below growth inhibition level) did not have
significant stimulatory effect on EPS production. However, Mishra and Jha (2009) found the
total EPS from D. salina increased from 56 to 944 mg/L concomitantly with the increase of salt
concentration from 0.5 to 5 M NaCl, but four monosaccharides including glucose, galactose,
fructose, and xylose in the EPS reached their maximum values of 105 µg/L, 130 µg/L, 160 µg/L,
and 110 µg/L, respectively at the salt concentration at 3 M NaCl. B. braunii also favors high
salinity to produce high concentration of EPS (Bayona and Garces, 2014; Rao et al., 2007).

Nitrogen source was found to have significant effect on the growth and EPS production from D.
chlorelloides that was able to produce EPS in the late stationary phase of growth (Koyun et al.,
2015). Ammonium nitrate achieved the fastest initial growth rate because it can induce earlier
start of stationary phase resulting in the highest EPS concentration of 9.3 g/L after 16-day

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cultivation. Similar to Koyun et al. (2015) on nitrogen effect, Domozych (2007) reported that
substitution of NaNO3 with ammonium salts NH4Cl and NH4NO3 results in noticeable decreases
in cell number but increases in EPS production from P. margaritaceum. Corresponding
significant increases in the EPS yield were noted in high-nitrate medium, while decreases in EPS
yield were seen under low-phosphate conditions. However, EPS content from B. braunii

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decreased when either urea or ammonia was used as nitrogen source in replace of nitrate because
the consumption of urea and ammonia caused pH drop leading to low EPS yield (Lupi et al.,
1994). Also, both Chlorella sp. and Micractinium sp. grown in the wastewater with high nitrogen

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concentration and P-limited condition produced a large amount of protein-rich EPS due to
nitrogen over-uptake (Wang et al., 2014). Parker (2013) also found nitrogen depletion did not
increase EPS production from C. vulgaris. In contrast, Bayona and Garces (2014) found that low

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nitrogen caused high EPS yield and no amino- or sulfated polysaccharides were present in EPS
during the experiments for comparing different growth media on EPS production by B. braunii.
Studying EPS from B. braunii, Fernandes et al. (1989) also observed that nitrogen deficiency

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conditions resulted in enhanced EPS and the EPS yield was higher in the decline phase of alga
growth compared to exponential and stationary phases. The contradict results on the effect of
nitrogen deficiency on EPS production could be due to the different species used and/or non-
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Nitrogen nutrient depletion (e.g., P-limit used in Wang et al., 2014).

Cordoba-Castro et al. (2012) found cultivation conditions including light intensity, agitation,
carbon concentration and their interaction had significant effects on the specific growth rate and
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EPS production of Scenedesmus obliquus (UTEX 393). The maximum specific growth rate and
EPS concentration were 0.64 d-1 and 24.7 mg/L, respectively achieved at 180 µE/m2/s, 4% air-
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CO2 and 1200 rpm. Dayananda et al. (2007) reported that continuous light plus shaking achieved
much higher EPS yield from B. braunii than either continuous or intermittent light without
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agitation. The possible reason could be shaking promotes photon distribution in the medium.
Lupi et al. (1991) also found EPS synthesis under continuous illumination was higher than that
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under cyclic illumination. However, Parker (2013) found both short and long light exposures can
significantly increase EPS secretion by C. vulgaris and no stress occurred during growth.
Increased turbulence decreased growth while increasing superoxide production, thus induced
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EPS production with high yield at both early and late growth stage (Parker, 2013). In addition,
algal response to environmental stress varies according to the applied stress and EPS production
does not correlate with a particular stress indicator such as growth inhibition, cell size changes,
and production of reactive oxygen species (ROS).

High temperature (i.e., 30 °C) and high level Ca2+ led to high growth rate, whereas EPS yield per
cell decreased significantly (Domozych, 2007), however, both cell numbers and EPS production
decreased when temperature reduced to 4 °C. The optimal temperatures for growth and EPS
synthesis of B. braunii UC 58 concurred in the ranges of 25-30 °C at which 4.5-5 g/L of EPS
was produced, leading to high broth viscosities (Lupi et al., 1991). However, EPS synthesis was
negligible when temperature was reduced below 23 °C, although the specific growth rate
maintained high values; at supraoptimal temperatures EPS synthesis and alga growth rate
decreased simultaneously. Another important finding was that the molecular weight of EPS
reached maximum at temperature within the optimum range for EPS production, however, the
molecular weight of EPS synthesized at suboptimal and supraoptimal temperatures became
significantly lower than the values within the optimal range (Lupi et al., 1991).

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Green algae including Anabaena PCC 7120, Oscillatoria angustissima, S. obliquus, and C.
vulgaris produced as high as 3 mg/mL EPS at high concentration of cyanobacterial toxin (200
µg/mL), which indicated particular algal species exuded EPS as a defense response to
environmental stress (El-Sheekh et al., 2012). The presence of toxic ionic and nanoparticle silver
can change the EPS composition from C. reinhardtii, i.e., greater levels of LMW materials were

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produced in EPS (Taylor et al., 2016). It was reported that glyoxylate, a stimulator of carbon
metabolism is able to inhibit photorespiration and increase photosynthesis in higher plants
(Oliver and Zelitch, 1977) and some cyanobacteria (Bergman, 1981). Supplementing S. obliquus

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with glyoxylate, Liu et al. (2010) achieved increased EPS production probably because
glyoxylate promoted photosynthesis of this green alga.

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Cultivation reactor system can impact EPS production. When scale up to pilot scale
photobioreactor (PBR), the algae growth and EPS production decreased significantly probably
due to the mixing limitation and/or insufficient light penetration into PBR (Koyun et al., 2015).

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3.2. Diatom MA
The production of EPS by diatom is a function of their mobility system and a response to its
growth environment. Maximum EPS production generally occurs at the end of the growth phase,
coinciding with depletion of nutrients such as N and P, i.e., diatoms tend to produce more EPS
under N- and/or P-limiting conditions (Bhaskar and Bhols, 2005), however, green algal EPS
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usually peaks at stationary phase. The reason for this difference is unknown. Other factors such
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as salinity, temperature and irradiance also influence the EPS production. The EPS excretion
pattern and the effects of nutrient limitation are species-specific. Thus, it is difficult to
distinguish the influence of nutrient limitation and the age of the cultures on the EPS production
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rate, especially in batch processes (Liu and Buskey, 2003). The sensitivity of EPS production
from diatoms could be used as an indicator for environment change (e.g., water pollution), which
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has not received much studies.

Nitrogen abundance and source had significant effect on EPS production from Thalassiosira
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pseudonana, i.e., nitrate depletion was more effective than phosphate depletion to stimulate EPS
production (Ai et al., 2015). It was also found that ammonia rather than nitrate/urea and sodium-
glycerophosphate rather than phosphate induced higher EPS yield, the degree of polymerization
of EPS varied and the proportion of low and high molecular weight polymer increased in EPS
under the nitrate and phosphate depletion. Abdullahi et al. (2006) and Urbani et al. (2005) found
P-limitation resulted 1.2 to 2.5-fold increase of EPS production from P. tricornutum, C.
closterium, T. pseudonana, and Skeletonema costatum depending on the growth phase and the
EPS abundance and maximum production rate decreased significantly from logarithmic to
stationary phase. In addition, P-limitation also reduced glucose content in the EPS of C.
closterium. Magaletti et al. (2004) studied the effect of P- and N-depletion on the production and
the molecular-level compositions of EPS released by C. fusiformis. They found that P-depletion
stimulated a higher release of EPS than both nutrient-replete and nitrogen deficiency, and P-
limitation increased the galactose proportion but decreased glucose while the remaining
monosaccharides were almost unaffected in EPS. It appears glucose in EPS from the genus of
Cylindrotheca always decreased under P-limitation condition (Magaletti et al., 2004; Urbani et
al., 2005). Similarly, the diatom C. affinis released significantly more EPS under severe P-

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limitation than under severe N-limitation (Myklestad, 1977; Myklestad and Haug, 1972). Three
epipelic diatoms such as C. closterium, Navicula perminta, and Amphora exigua produced their
EPS following the same pattern in response to varied nutrient conditions (Underwood et al.,
2004). Specifically, cellular rates of colloidal carbohydrates, EPS and glucan production were
significantly higher during nutrient-replete compared to nutrient-limited, but the proportion of

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polysaccharides in EPS increased significantly when the nutrient was limited. These researchers
also found that C. closterium produced two types of EPS with different sugar composition and
production patterns, i.e., nutrient-replete resulted in EPS containing rhamnose, fucose, xylose,

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mannose, galactose, glucose, and uronic acids. However, EPS under nutrient-limit conditions
lacked rhamnose and fucose. Underwood et al. (2004) used inhibitors and 14C-labelling methods
to elucidate two pathways for EPS synthesis: 1) photosynthesis produces intracellular glucan

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which is a precursor for EPS; and 2) glucan from photosynthesis serves as a carbon store to
provide energy for EPS synthesis.

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In their study of the influence of nutrient ratios (N/P/Si) on EPS production, Penna et al. (1999)
observed differences among diatoms such as Nitzschia closterium, Chaetoceros sp. and S.
costatum. At a high N/P ratio, N. closterium had higher polysaccharide releasing rate than the
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other two species in both exponential and stationary phases. In contrast, Chaetoceros sp. and S.
costatum exuded more polysaccharides than N. closterium at a low N/P ratio during the
stationary phase. In addition, Si limitation had little effect on the polysaccharide production.
Although the EPS from N. salinarum and C. closterium differed in their composition, both EPS
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consisted predominantly of polysaccharide (80-86%) with glucose and xylose being the main
constituents while small quantities of protein, uronic acids and SO4-2 moieties were also present
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(Staats et al., 1999). These researchers also found the transition from exponential to stationary
growth phase resulted in the highest EPS production rate.
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Salinity can effectivity simulate EPS production by 2 to 4-fold by P. tricornutum when it was
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increased from 5 to 65 psu even though the number of algal cells slightly decreased (Abdullahi et
al., 2006). The effect of temperature and irradiance on EPS production from C. closterium
depended on the growth stage, i.e., the highest EPS was produced at 15 °C and 25 °C (Wolfstein
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and Stal, 2002). In addition, temperature and irradiance had strong interaction on EPS production,
i.e., temperature had slight effect at low irradiance; however high temperature (e.g., 35 °C) had
inhibitory effect on EPS production at high irradiance. At low temperature (e.g., 15 and 25 °C),
EPS yield reached the maximum value at 571 µmol photons/m2/s and decreased at higher
irradiance; however EPS yield kept increasing in the tested range of irradiance at temperature
35 °C.

3.3. Red microalgae

The unicellular red microalgae belong to the order Porphyridiales that includes about eight
genera, the best known of which are Porphyridium, Rhodella and Rhodosorus. Porphyridium has
been the most studied red algae especially the strains of P. cruemtum and P. aerugineum for they
produce a large quantities of EPS with commercial value although some work has also been done
on R. maculate and R. reticulata (Ramus, 1972). The factors being studied to induce EPS
production from red algae include nitrate, sulfate, light irradiation, cell age, and so on.

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A two-stage cultivation process was patented to enhance EPS production by P. aerugineum. In

this process, fresh nutrient medium is supplied to the culture in the first stage and a portion of the
culture medium is transferred from the first stage to the second stage in which a nitrogen
deficiency is created to shift the culture to a senescent phase to enhance biopolymer production
(Ramus, 1980). Arad et al. (1992) reported that the highest exopolysaccharide contents per cell

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from R. reticulata (LB 2320) were obtained in the nitrogen-deficient and sulfate-deficient
cultures and both nitrate and sulfate starvation enhanced 14C and 35S incorporation into the
exopolysaccharides. Working with immobilized cells of Porphyridium, Thepenier et al. (1985)

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found that nitrate-depletion caused an increase in polysaccharide production, resulting in a sharp
increase of viscosity of the medium. Within a certain range of nitrate concentrations (5-20 mM),
depletion of nitrate from the medium also caused an increase in the production of

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exopolysaccharides from Porphyridium sp. (Adda et al., 1986). Different nitrogen sources,
nitrate and ammonium did not show difference on the pattern of cell growth and polysaccharide
production by Porphyridium sp. (UTEX 637) (Arad et al., 1988). The batch cultivation achieved

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the highest cell growth and polysaccharide production (either per volume or per cell), and the
nitrogen deficient-mode had the lowest biomass growth (per volume). Per cell, the continue-
mode had similar polysaccharide yield to the deficient-mode. Nitrogen affected the
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polysaccharide distribution between soluble and bound polysaccharides and most
polysaccharides were dissolved in the deficient-mode (Arad et al., 1988). In addition to N/S,
magnesium shortage and high ion concentration was found to be able to induce EPS production
by P. cruentum and MgSO4 addition in medium increased the sulfate and protein contents
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(carbohydrate and uronic acid contnents did not change) in EPS resulting in higher antivirus
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activity of EPS (Raposo et al., 2014b).

Evans et al. (1974) reported that light can accelerate the uptake of 35SO42- by R. maculate to
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produce more EPS containing with 10% sulfate. The major sugars are xylose and uronic acid
accompanied by small amount of galactose and glucose. Arad et al. (1985) showed that the
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amount of exopolysaccharides produced by Porphyridium sp. cells increased with the increase in
light irradiance. When light intensity increased from 75 to 300 µmol quanta/m 2/s, the
polysaccharide of Porphyridium sp. (UTEX 637) and P. aerugineum were increased by 1.5 and 3
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folds (Friedman et al., 1991). The production rate per cell of exopolysaccharides from P.
aerugineum were the greatest in stationary phase light-grown cultures and the Golgi complex
was found to play a crucial role in the production of exopolysaccharides by red algae (Ramus,
1972).

Given the important functions of EPS from red algae for potential high-value applications, the
bulk production of EPS and proper preparation methods will be needed. To the best of our
knowledge, however, only one publication studied details about the preparation of EPS from red
algae. Ginzberg and Korin (2008) reported that the bioactivities of EPS from red algae,
Porphyridium sp. was sensitive to temperature, i.e., high temperature (over 90 °C) drying
significantly destroyed biological activities of the EPS from Porphyridium sp. Therefore, they
invented a low temperature two-step drying process including convection and lyphophilization to
prepare dry EPS for storage and end-applications.

4. Applications of Microalgal EPS

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Microalgal EPS already proved to have important physicochemical and biological properties
(e.g., high viscosity and bioactivities) and promising applications which derive from their
chemical composition and structure. For example, microalgal EPS possess a wide spectrum of
bioactivities as shown in Table 4, which have received rapidly increased research interests.
Often, it is difficult to identify their chemical structure, thus their properties for applications were

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not being thoroughly understood. Few research has been done to establish a correlation between
the composition/structure of EPS and their applications. The properties of EPS normally result
from a complex interaction of many structural features, e.g., molecular weight, sugar residue

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composition, sulfation level, distribution of sulphate groups along the polysaccharide backbone,
protein moiety, and stereochemistry (Raposo et al., 2015). For instance, the anticoagulation
activity of fucan resulted from the sugar composition, molecular weight, sulfation level and

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sulfur group position; galactans, fucans and galactofucans are typical polysaccharides of EPS
and were reported to inhibit herpes simplex virus (HSV) types 1 and 2 infection even though
they are different in structure, sulfation level and molecular weight (Raposo et al., 2015). It was

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found that branched fucoidan oligosaccharides had higher anti-inflammatory activity than the
linear counterpart (Clement et al., 2010). Although research on microalgal EPS is still at the
early stage, they have already exhibited to be a large group of potential natural resources for
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producing a bulk amount of biopolymers for various applications in the medical, biomedical,
food, mechanical engineering, wastewater treatment areas and so on. In addition, the exploration
of EPS as valuable by-products of microalgae would enhance the cost-competitive alga-based
biorefinery for producing multiple products when microalgae are used as the third generation
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biomass feedstock.
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Table 4. Bioactivities of EPS from different microalgae

Microalgae Species Functions References
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• Antivirus: retroviruses, HSV, and varicella zoster Huleihel et al. (2001);

viruses (VZV) Talyshinsky et al. (2002)
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Porphyridium sp. • Antioxidant property Tannin-Spitz et al. (2005)

• Anti-inflammatory property: anti human skin Matsui et al. (2003)
inflammation symptom
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• Increase viscosity Yaron et al. (1992)

Huleihel et al. (2001);
• Antivirus: retroviruses, HSV and VZV
Talyshinsky et al. (2002)
P. aerugineum • Anti-inflammatory property: anti human skin
Matsui et al. (2003)
inflammation symptom
• Increase viscosity Yaron et al. (1992)
Huleihel et al. (2001);
Red algae • Antivirus: retroviruses, HSV and VZV
Talyshinsky et al. (2002)
R. reticulata • Antioxidant property Chen et al. (2010)
• Anti-inflammatory property: anti human skin Matsui et al. (2003)
inflammation symptom
• Increase viscosity Yaron et al. (1992)
Ertesvåg and Valla (1998);
• Antivirus: encephalomyocarditis virus and
Gehrke et al. (2001); Yim
G. impudicum KG03 influenza virus
et al. (2004)
• Anti-tumor property Yim et al. (2005)
• Antivirus: haemorrhagic septicaemia virus
P. cruentum (VHSV) for salmonid fish and mammalian viral Fábregas et al. (1999)
diseases

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• Antivirus: VHSV, African swine fever virus Fábregas et al. (1999);

Cochlodinium (ASFV) and human immunodeficiency virus
Hasui et al. (1995)
Polykrikoides (HIV)
• Anticoagulant property Hasui et al. (1995)
• Antivirus: VHSV and ASFV Fábregas et al. (1999)
Guzman-Murillo and
• Anti-adhesive

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Ascencio (2000)
P. tricornutum Guzman et al. (2001,
• Anti-inflammatory and analgesic
2003)
Ford and Percival (1965a,

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Diatoms • Free radical scavenging
b); Guzman et al. (2001)
Isochrysis galbana

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Isochrysis galbana var • Antivirus: VHSV and ASFV Fábregas et al. (1999)
Tiso
• Antivirus: HSV 1&2, influenza A virus (IFV-A)
Navicula directa Lee et al. (2006)
and HIV

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Tetraselmis tetrathele
Tetraselmis suecica
Chlorella autotrophica
• Antivirus: VHSV and ASFV Fábregas et al. (1999)
Dunaliella bardawil
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D. tertiolecta
Ellipsoidon sp.
Green algae • Anti-allergic Borowitzka (1995)
D. salina
• Anti-inflammatory Fujitani et al. (2001)
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• Anti-inflammatory and analgesic

Chlorella stigmatophora Guzman et al. (2001)
• Free radical scavenging
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S. obliquus
Oocystis sp. Abedin and Taha (2008);
• Antifungi
Chlorella pyrenoidosa Ghasemi et al. (2007)
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Chroococcus dispersus
C. reinhardtii •
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Antifungi
Ghasemi et al. (2007)
C. vulgaris • Antibacteria

According to literatures, the type of applications varies significantly among different taxa
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probably due to different properties of their EPS or no research being done on some applications
for specific alga taxa or species. For example, wastewater treatment has been one of the major
applications of the EPS from green algae and diatoms, while the major application of the EPS
from red algae is focused on the medical and biomedical areas. Another example, no drag
reduction is reviewed in diatom EPS application, but green and red algae have. Therefore, this
section tries to comprehensively cover all applications available in literatures for individual
microalga taxa.

4.1. Green microalgae

4.1.1. Medical and biomedical applications

EPS of many species (e.g., C. stigmatophora, C. autotrophica, C. vulgaris, T. tetrathele, D.

tertiolecta, and D. salina) of green algae had a high sulfation level, which were of use in anti-
inflammatory/immunomodulatory, antimicrobial (e.g., antibacterial, antifungal and antivirus,)
and infection prevention (Amaro et al., 2011; Fábregas et al., 1999; Guzman et al., 2003;

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Guzman-Murillo and Ascencio, 2000; Ogawa et al., 1997, 1999; Raposo et al., 2013). EPS from
C. stigmatophora showed both anti-inflammatory activity against the carrageenan-induced paw
edema and the immunosuppressant effects on phagocytic activity assayed in vivo and in vitro
(Guzman et al., 2003). The IC50 for paw edema was only 2.25 mg/kg, much lower than IC50=8.50
mg/kg of anti-inflammatory indomethacin, a commercial non-steroidal anti-inflammatory drug.

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During the production and expression of interleukins, dectin-1 and toll-like receptors-2 on
macrophages and dendritic cells, respectively, (1, 3)-β-glucans from C. vulgaris induced
antifungal and antibacterial responses in rats (Rice et al., 2005), and towards infections by E. coli

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on mammal organisms (Verma and Gu, 2012). Ghasemi et al. (2007) screened 60 green alga
strains of microalgae and found 21 antibacterial strains and 17 antifungal strains. Among all
these species, Chroococcus disperses, C. reinhardtii and C. vulgaris appeared to be the most

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promising strains with a broad spectrum of antibacterial and antifungal activities. Fábregas et al.
(1999) reported EPS from C. autotrophica and Ellipsoidon sp. inhibited virus replication of
VHSV and ASFV up to 44-67%. The EPS from Tetraselmis spp. and D. tertiolecta were found

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to effectively inhibit Helicobacter pylori adhesion to HeLa S3 cells and fish pathogens adhesion
(Vibrio campbellii, Vibrio ordalii, Streptococcus saprophyticus, and Aeromonas veronii) to
tissue culture cells, respectively (Guzman-Murillo and Ascencio, 2000). However, EPS from
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different alga species act differently even from the same genus, e.g., T. suecica EPS promoted
adhesion of H. pylori to HeLa S3 cells, in contrast to the inhibition effect of EPS from
Tetraselmis spp. (Guzman-Murillo and Ascencio, 2000). The differences in EPS chemical
composition and/or structure could be the reasons. As a result, although algal EPS promise to be
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good antimicrobial agents, they should be accessed specifically on invasive microbes. In addition
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to pharmaceutical applications, EPS can be also used as biomaterials for additional biomedical
applications. Lewin (1956) revealed that the polysaccharides of EPS from Gloeocystis gigas
showed phase transition from soluble (when heated) to gel states (when cooled), which could
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make the EPS from G. gigas good thermoresponsive macromolecules for applications in drug
delivery, gene transfection, bioseparation, and sensor development, and so on (Cohen Surat,
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2010).

4.1.2. Industrial applications

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Except for the production of intracellular compounds (e.g., β-carotene, protein and glycerol), D.
salina was also thought to be a promising EPS producer for industrial applications, e.g.,
biosurfactants and bioemulsifiers. The emulsifying activity of EPS from D. salina was 85.76%
retention (Mishra and Jha, 2009), which was comparatively high than that of EPS produced by
Vibrio harveyi (40% retention) (Bramhachari and Dubey, 2006), Halomonas (25-60% retention)
and commercially available surfactants including Tween 20 (65%), Tween 80 (60%) and Triton
X-100 (65%) in tetradecane (Mata et al., 2006). An energy-dispersive X-ray (EDX) analysis
showed that D. salina EPS contained sulfate, and a 1H NMR analysis indicated the presence of
uronic acid, primary amine, aromatic-compounds, halides, aliphatic alkyl and sulfides (Mishra et
al., 2011). The stable emulsifying activity may make D. salina EPS a renewable alternative of
the commercial chemical surfactants and emulsifiers. The EPS from D. salina were also found
thermostable up to 270 °C and they had high viscosity of their solution at acidic pH, which
enables them for additional characteristic for further applications (Mishra et al., 2011). In their
study of EPS from Cosmarium turpinii (UTEX LB 1042) and Palmella texensis (UTEX 1708),
Ha et al. (1988) reported that the EPS which consisted of fucose, xylose, galactose, glucose and

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glucuronic acid had pseudoplastic properties due to their high viscosity, indicating an ability to
function as a thickening agent. P. texensis produced low-viscosity homogenous galactose-based
polysaccharides which showed Newtonian-like flow properties at pH (3.5-9.5) and temperature
(20-80 °C), thus EPS from P. texensis could be used as a stabilizer in food industry.
4.1.3. Drag reducer

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Hoyt (1984) showed that EPS from C. stigmatophora and P. cruentum had prominence as drag
reducers. EPS from Schizochlamydella capsulate and C. stigmatophora were studied for their

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functions of drag reducing and the inherent drag-reducing ability was measured by drag
reduction as a function of polysaccharide concentration in the water solution (Gasljevic et al.,

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2008). EPS from S. capsulate performed better than that from C. stigmatophora, i.e., to achieve a
level of drag reduction of 15% at the given Reynolds number, the necessary concentration of
EPS from S. capsulata was 23 ppm, while EPS from C. stigmatophora was 175 ppm (Gasljevic

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et al., 2008). Comparatively, EPS from P. cruentum and R. maculata were better drag reducers
than those from S. capsulate and C. stigmatophora (Gasljevic et al., 2008); however, Ramus et al.
(1989) and Ramus and Kenney (1989) found S. capsulata EPS performed better than R.
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reticulata and P. cruentum on drag reduction. The disparity may be attributed to the difference in
cultivation condition and analytical method. Due to the drag-reducing property, algal EPS could
be applied to the hull of the vessels to reduce friction losses by reducing flow turbulence (due to
the elasticity of EPS); as such fuel consumption and propelling power for a ship to achieve a
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certain velocity can be lowered (Gasljevic et al., 2008).

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4.1.4. Wastewater treatment

The large quantity of nitrogen and phosphorus in wastewater is one of the main factors resulting
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in eutrophication (Singh and Thomas, 2012). Since algae have excellent performance in
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removing nitrogen and phosphorus without additional carbon sources or nutrients (Xin et al.,
2010), algae-based technologies have been adopted as a crucial process in wastewater treatment
(de-Bashan and Bashan, 2010; Wang et al., 2010). The toxicants, such as heavy metals (Cd2+,
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Pb2+, Cu2+, etc.), usually coexist with nitrogen and phosphorus in wastewater, leading to toxic
effects on algal growth and EPS production (Cooper et al., 2010; Sheng et al., 2013). Green
algae are the most commonly used species and are able to adsorb metals, thus they have
considerable potential to treat metal-contaminated wastewater (Mehta and Gaur, 2005). They can
effectively remove metals from multi-metal solutions. EPS are believed to play an important role
in metal biosorption by algae and major influential factors include EPS composition, metal
species, solution chemistry and operating conditions. Various pretreatments such as CaCl2
pretreatment can enhance metal sorption capacity of algae probably because pretreatment can
induce EPS production or interact with EPS leading to high affinity of EPS to heavy metals
(Mehta and Gaur, 2005). EPS can chemically and/or physically bind with many exogenous
organic and inorganic compounds because of their abundant functional groups (e.g., -COOH, -
NH, -OH, -CO-, etc.) (Chen et al., 2015). For instance, EPS can stimulate the uptake of NH4+-N
and PO43--P by algae, and react with heavy metals (e.g., Cu2+ and Cd2+) as ligands via ion
exchange, complexation, surface precipitation, electrostatic, etc. (Yin et al., 2011; d’Abzac et al.,
2013; Li and Yu, 2014). The interactions between EPS and heavy metals could reduce the
toxicity of heavy metals for algae (Chen et al., 2015). Under certain concentrations (<0.5 mg/L),
heavy metal such as Cd2+ can promote the production of EPS by C. vulgaris leading to increased

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exopolysaccharides and protein (e.g., tryptophan-like and tyrosine-like components) contents,

which accelerated the interactions of Cd2+ with exopolysaccharides and protein in EPS fractions
and such Cd2+-EPS interactions enhanced the removal of NH4+-N and PO43-P by C. vulgaris
(Chen et al., 2015). Algae-based techniques could be a stable treatment of wastewater with the
presence of some toxic heavy metals.

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Recent increased interest in algal biofilms has been driven by the need for wastewater
remediation strategies for nutrient control, alternative biofuel feedstock, and effective low cost

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biomass harvesting techniques (Kesaano and Sims, 2014). Chemical- and physical-based
technologies are available to remove excess nitrogen and phosphorus from wastewaters; however,
they often consume a large amount of energy and chemicals, leading to high cost. Algae are

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capable of metabolizing these pollutants, and if harvested, can be utilized as a feedstock for
biofuel production. When algae are grown as surface-attached biofilms glued by EPS, the
biomass could be naturally concentrated and more easily harvested, leading to less expensive

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removal from treated water, and less expensive downstream processing for biofuel production.
Compared to suspended algae systems, the attached algal biofilm led to increased biomass
production and improved water quality and an algae feedstock suitable for further processing in
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the production of biofuels (Christenson, 2011). In alga-based wastewater treatment systems, EPS
from Scenedesmus sp. (UTEX # 1589) and C. vulgaris played an important role in the settling of
algal biomass via bioflocculation for alga harvesting for downstream applications (Manheim and
Nelson, 2013).
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However, algal EPS could cause problems when algal biomass need to be separated from treated
water. When membrane filtration is used to remove algal biomass from wastewater treatment
plant (e.g., Pseudo-coccomyxa ellipsoidea) for its utilizations (e.g., for biofuel production), EPS
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were found to be a major negative factor causing membrane fouling, i.e., soluble EPS for
irreversible fouling and bound EPS for reversible fouling (Matsumoto et al., 2014). Chiou et al.
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(2010) used ultrafiltration (UF) to treat algae-laden eutrophic source waters and found membrane
fouling caused by EPS from C. vulgaris and Chodatella sp. was a major obstacle to the smooth
operation of membrane processes and the constituents of EPS may also play a role. Lee et al.
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(2006) studied the fouling of low-pressure membrane (MF/UF) with algal EPS, and reported that
protein and/or polysaccharide in EPS were responsible for significant fouling, especially when
they existed as organic colloids. It was also found that filtration resistance caused by Chlorella
algae under nutrient-deficient condition was increased likely due to an increase of EPS
production and/or a change in EPS characteristics such as sugar content (Babel et al., 2002).

4.2. Diatom

4.2.1. Adhesive activity

Diatom EPS can function as adhesives directly involved in initial active attachment and cell
motility. The responsible adhesives have extraordinarily high cohesive strength and binding
strength to the solid surfaces, enabling the organisms to remain attached under tensional
conditions. Understanding how diatoms adhere to surfaces will facilitate the development of
adhesives and of anti-biofouling surfaces for use in aquatic environments, and will provide a
model to investigate the nutrient flux between biofilm and surrounding media. Wustman et al.

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(1997) characterized the extracellular adhesives from the diatoms A. longipes, A. coffeaeformis,
C. cistula, and Cy. Mexicana, and indicated that polysaccharides of EPS were the major
component of adhesives formed during cell motility, synthesis of a basal pad, and production of
a highly organized shaft. Rincé et al. (1999) developed a method using artificial cell-
immobilization to successfully simulate the immobilization of a diatom Haslea ostrearia in

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natural environment. Staats et al. (1999) studied EPS from two benthic marine epipelic diatoms
such as N. salinarum and C. closterium, and revealed that negatively charged groups such as
uronic acids and sulfated sugars were determinant factors for adhesion capacity of EPS and

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probably played an important role in intertidal sediment stabilization. Due to its high adhesive
quality, diatom EPS could be more effective tissue adhesives for medical use, for example,
ophthalmic therapies, bone gluing and soft tissue closure after surgery (Laurienzo, 2010).

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In addition to the benefits of adhesive properties, diatom EPS also play an import role in
biofouling, especially on man-made surfaces in the marine environment. Biofouling diatoms can

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tenaciously adhere to and colonize on even the most resistant artificial surfaces, causing serious
problems (increased drag, fuel consumption and exhaust gas emission associated with high cost)
for the navies and for marine industries around the world (Hunsucker and Swain, 2016). For
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example, a 1-mm thick slime film can lead to 80% increase in skin friction coefficient, 15% loss
in ship speed, and the powering penalty can be up to 86% at cruising speed (Lewthwaite et al.,
1985; Schultz, 2007); and it has been estimated that the overall cost associated with hull fouling
for the Navy's present coating, cleaning, and fouling level to be $56 M per year for the entire
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DDG-51 class or $1 B over 15 years (Schultz et al., 2011). Although various antifouling paints
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and fouling-release coatings (either toxic or environmentally friendly) are effective against most
fouling organisms, fail badly to diatoms (Molino and Wetherbee, 2008). To develop effective
and nontoxic/less toxic antifoulants, it is important to find out diatom species which have high
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biofilm adhesion strength, the basic biology of fouling diatoms, the nature of their adhesives, and
their mechanisms of adhesion. Besides chemical antifoulants, it is of interest to find natural
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antifoulants, e.g., marine microbes are promising potential sources of non-toxic or less-toxic
antifouling compounds as they can produce substances that inhibit not only the attachment
and/or growth of microorganisms but also the settlement of invertebrate larvae and algal spores
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(Dobretsov et al., 2006).

4.2.2. Anti-adhesive activity

EPS from diatoms are capable of blocking the adhesion of pathogenic microorganisms, thus they
can be used in anti-adhesive therapies against pathogen infections (Guzman-Murillo and
Ascencio, 2000). It was reported that sPS of EPS from P. tricornutum can effectively inhibited
the cytoadhesion of the human pathogen H. pylori to the HeLa S3 cell line, and adhesion of the
fish pathogens (V. campbellii, V. ordalii, S. saprophyticus, and A. veronii) to spotted sand bass
primary tissue (gills, gut and skin) cultured cells (Guzman-Murillo and Ascencio, 2000).
Microbial infection occurs usually after binding to the host cell membrane. Carbohydrates (e.g.,
heparin sulfate glycosaminoglycan) have been recognized as receptors in host cells (Bellamy et
al., 1993; Bergey and Stinson, 1988) and their net charge and molecular stereochemistry seem
important to the interaction between host and pathogen (Ascencio et al., 1993). No direct
correlation has been established between concentration of the sulfated residues in the EPS and
the inhibition efficacy of bacterial adhesion. EPS from diatom may compete with carbohydrates

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on the host cell surfaces as recognition sites to which pathogens can attach to (Ofek et al., 1978).
Thus, it is likely to use diatom-derived carbohydrate-based drugs for blocking the early stages of
an infection process. Diatom EPS could be alternatives to antibiotics to treat infectious diseases,
thus eliminating the use of antibiotics with the subsequent problem of resistant strains and
stimulating the nonspecific immune system of the treated organisms (Guzman-Murillo and

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Ascencio, 2000). However, further studies are needed to determine the cytotoxic and
anticoagulant risk and bioavailability as to block pathogen adhesion and colonization of the host
as well as distinct biochemical mechanisms of anti-adhesive action of EPS.

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4.2.3. Medical application

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EPS from diatoms especially sulfated EPS were found to have multiple bioactivities including
anti-inflammatory, antivirus, anti-adhesive, and free radical scavenging (Table 4). For example,
P. tricornutum, the most studied diatom for its EPS bioactivities has already demonstrated to

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have anti-inflammatory activity against carrageenan-induced paw edema with IC50 value of 2.92
mg/kg, compared with the anti-inflammatory indomethacin with an IC50 of 8.50 mg/kg (Guzman
et al., 2003). The anti-inflammatory efficacy was determined in vivo (by intraperitoneally
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injecting the crude EPS in female rats and mice) and in vitro (the phagocytic activity being
evaluated in macrophages from mice). The immunostimulatory effects of EPS from P.
tricornutum on immune cells was indicated by the positive phagocytic activity tested in vivo and
in vitro. Another diatom species N. directa was found a potent antivirus agent against broad
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spectrum of enveloped viruses including HSV 1, HSV 2, IFV-A, and HIV with high selectivity
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(cytotoxicity/IC50 ratio) (e.g., HSV 2=1400 and HIV=540) (Lee et al., 2006). It was detected that
N. directa EPS inhibited hyaluronidase and the virus-cell interaction, i.e., virus binding onto host
cell and penetration into host cell were blocked by EPS (Lee et al., 2006).
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4.2.4. Erosion control

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The adhesive properties render diatom EPS important applications in sediment erosion control.
Among benthic habitats, diatoms are the most frequent early algal colonizers of natural and
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artificial substrata, where they adhere and produce a great number of EPS during the formation
of biofilms (Wetherbee et al., 1998). Adhesion properties of diatom EPS seem to play an
important role in either the locomotion or in the aggregation of soil and sand particles (Paterson,
1989; Sutherland et al., 1998), influencing stability and cohesiveness of sediments (de Boer,
1981; Frostick and McCave, 1979; Holland et al., 1974; Vos et al., 1998). The presence of the
diatom, Nitzschia curvilineata affected the erosion characteristics of the sediment by increasing
erosion threshold and decreasing erosion rate. During the diatom growth period, variations in
erosion rate were significantly greater than those in erosion threshold. It was found that there was
a strong negative correlation between erosion rate and EPS concentration, and sediment stability
increased with successive stages of diatom growth, especially in the stationary phase of growth
(EPS reached maximum during stationary phase) (Sutherland et al., 1998). It was also reported
that diatom-inhabited sediments increased significantly in surface stability in tidal exposure as
compared with control sediments (Paterson, 1989). The EPS matrix produced by diatoms was
considered as part of their locomotive mechanism. Diatom binding can increase the resistance of
the seabed to erosion and maintain the protection during tidal surge.

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4.2.5. Wastewater treatment

The growing diatoms can efficiently adsorb CO2 (given the fact that diatoms contribute up to 20%
of global CO2 fixation) and consume nutrients (e.g., N and P) in water bodies while release O2
which can promote aerobic bacteria breakdown organics into its constituents thus cleaning up the

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water (Gugi et al., 2015; Kumar, 2008). The organics constituents serve as nutrients further
absorbed by diatoms to grow. During growth, diatom cells can quickly form biofilms by
excreting EPS which can immobilize and accumulate noxious compounds like a natural

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molecular sieve or ion exchanger, thus diatoms are useful for bioremediation purposes such as
wastewater treatment (Craggs et al., 1996; Wingender et al., 1999). Given these characteristics of
diatoms, a patented product called Nualgi which is a plant growth nutrient was invented and has

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been used to specifically induce diatom domination in wastewater treatment process (Kumar,
2008). Nualgi technology has been successfully used to treat brewery wastewater and sewage. In
phototrophic biofilms from wastewater treatment plants, diatoms were always found a dominant

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taxa existing together with cyanobacteria and/or green algae, and the total amount of EPS present
and the proportion of uronic acids in EPS were positively correlated with diatom biomass
(Congestri et al., 2005; 2006). The negatively charged groups and conformational behavior of
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diatom biofilm matrix are important for the removal of nutrients and noxious cations in
wastewaters, indicating the potential use of autochthonous, diatom biofilms in tertiary
wastewater treatment (removal of nutrients and pollutants) alternatives to conventional physico-
chemical technologies with respect to the protection of receiving water bodies and the
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development of sustainable waste treatment (Congestri et al., 2005; 2006; Pippo et al., 2009).
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Similar to green algae, diatoms can be used as biosorbent materials to remove heavy metal ions
with the contribution of the interaction between EPS and heavy metals. For example, some
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diatoms such as Achnanthes brevipes, Asterionellopsis glacialis, Cyclotella cryptica, C.

closterium, C. fusiformis, P. tricornutum, S. costatum, Thalassiosira sp., T. pseudonana have
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been used to adsorb high amount of heavy metals such as Cu, Zn and Cd (Bræk et al., 1976;
Morelli and Pratesi, 1997; Pistocchi et al., 2000; Sbihi et al., 2012). Planothidium lanceolatum
which is widely available diatom can effectively remove heavy metals, especially Cu and Zn
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from contaminated water. In comparison with other studied algae (e.g., P. tricornutum and C.
vulgaris), P. lanceolatum is more resistant to Cu, Cd and Zn and can accumulate high amounts
of these elements (Sbihi et al., 2012). The increased production and release of EPS in the
presence of these metals was common to all the species investigated. Some diatoms like C.
fusiformis and P. tricornutum have capability of phytochelatin synthesis on exposure to Cu and
Cd and their EPS represent specific ligands produced in response to the presence of a toxic
metals, i.e., EPS chelates the metals in the extracellular environmental (Morelli and Pratesi, 1997;
Pistocchi et al., 2000).

In addition to the role in pollutant removal, diatom EPS is also important for flocculation and
sedimentation of algal biomass in wastewater treatment. EPS aggregated diatom cells and other
material into larger flocs, usually leading to an increased particle size and settling velocity of a
diatom bloom, e.g., Chaetoceros spp (Leung, 2003; Passow and Alldredge, 1995). Shipin et al.
(1999) found EPS produced by algal biofilms in a trickling filter enhanced solid flocculation in a
later clarifier operation. However, it was reported that the increased amount of EPS resulted in

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deflocculation of S. costatum (Leung, 2003). Therefore, the effect of EPS on flocculation

characteristics of diatoms seems to vary between species.

4.3. Red microalgae

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4.3.1. Antivirus

The antiviral activity of EPS from red microalgae was well reviewed by Amaro et al. (2011),

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Carlucci et al. (2012), Wijesekara et al. (2011), and Raposo et al. (2014a). The EPS from red
algae have attracted more and more research interests in their antiviral activity against a variety
of viruses to inhibit cytopathic effect, either mammalian or otherwise, especially after EPS from

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C. polykrikoides was found to be able to inhibit HIV (Hasui et al., 1995). Red alga species
including P. cruentum, P. purpureum, R. reticulata, G. impudicum, and C. polykrikoides were
reported to produce EPS with antiviral activity (Table 4). Of them, EPS from Porphyridium

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received the most studies showing their antiviral activity against a wide range of viruses,
including influenza virus (Flu), vaccinia virus (VACV), HSV, human cytomegalovirus (HCMV),
HIV, hepatitis B virus (HBV), murine sarcoma virus (MuSV), measles, mumps, a variola-related
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virus, retrovirus, and so on. Huleihel et al. (2001) found that EPS from Porphyridium sp., P.
aerugineum and R. reticulata exhibited antiviral activity against HSV and VZV, but
Porphyridium sp. derived EPS were the most effective with no cytotoxic effects. EPS from P.
purpureum proved to be active against VACA and Ectromelia orthopoxvirus infection; the IC50
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for VACA inhibition was only 0.65 µg/mL, significantly lower than the 1.24 µg/mL of dextran
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sulfate (Radonić et al., 2010).

The antiviral activity of EPS from Porphyridium depends on culture medium, strains, dosage,
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cell lines used for testing, but also on EPS extraction methodology, the size of the molecules, the
degree of sulfation and uronic acid content (Ghosh et al., 2009; Huleihel et al., 2001, 2002;
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Raposo et al., 2014b). The monosaccharide compositions and the linkage types also influence the
functional properties of EPS. Raposo et al. (2013) pointed out that acidic property along with
half-ester sulfate and carboxyl groups of the EPS contribute to their anionic characteristics,
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making them good antiviral agents. However, there was a contradictory conclusion on the effect
of the degree of sulfation. When testing on VHSV and ASFV, Fábregas et al. (1999) found the
inhibition of viral replication did not correlate with the percentage of sulfation of EPS from P.
cruentum. The reason may be different viruses were used for tests by Ghosh et al. (2009) and
Raposo et al. (2014b). Therefore, the tests for antiviral activity of the EPS should be conducted
with a wide range of viruses specifically. Talyshinsky et al. (2002) revealed that EPS of
Porphyridium sp. should be added 2-h before or at the time of virus infection in order to
effectively inhibit viruses, which deduced that part of the inhibitory effect could be ascribed to
the functions of EPS to block viral receptors and impede the penetration of the virus into the
cells.

The antivirus may be the most studied medical application of EPS from red algae, especially
Porphyridium. Several possible mechanisms have been proposed to elucidate these antiviral
activities (Hasui et al., 1995; Hayashi et al., 1996a, b; Kim et al., 2012; Martinez et al., 2005): (1)
anionic nature of EPS against viruses; (2) inhibition of attachment/adsorption; (3) inhibition of
the penetration of viral particles into host cells; (4) inhibition of virus replication during the early

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phases of the virus infection; and (5) prevention of the formation of various retroviral reverse
transcriptase without cytotoxic effects against the host cells. However, it has not been clearly
identified which mechanism(s) is the most useful against specific viruses because it was believed
that multiple mechanisms could be involved in antivirus activities.

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4.3.2. Anti-inflammatory

EPS from G. impudicum KG03 can activate the production of nitric oxide (NO) and

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immunostimulates the production of cytokines in macrophages (Bae et al., 2006). In their
analysis of the anti-inflammatory property of the EPS on human skin inflammation, Matsui et al.
(2003) found the anti-inflammatory property of Porphyridium EPS was due to the inhibition of

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the migration/adhesion of polymorphonuclear leukocytes (PMNs) and the development of
erythema by the EPS. The inhibition of chemotaxis increased from 38% to 100 % when the
concentration of EPS increased from 0% to 0.5 % (w/v), and there was a positive correlation of

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the sulfate content with the immunomodulatory activity (Sun, 2010). This correlation indicates a
direct stimulation of the immune system by EPS via triggering cells and stimulating humors
(Namikoshi and Rinehart, 1996). In addition to the inhibition of tissue oxidative damages, EPS
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from P. cruentum can inhibit biomembrane peroxidation to enhance in vitro immunomodulatory
activity via the stimulation of macrophages (Sun et al. 2009; 2012). These researchers found low
molecular weight EPS can stimulate the proliferation of macrophages and the production of NO
which is involved in human immune system response, i.e., the activity strength increased with
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the decrease of EPS molecular weight. Both proteoglycans of Porphyridium (sulfated

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glycosaminoglycans covalently linked to proteins) and non-covalently linked protein moieties

showed anti-inflammatory properties (Raposo et al., 2014a).
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4.3.3. Antibacterial
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The antibacterial activity of the EPS from P. cruentum depends on the solvent used to extract the
EPS. Ethanolic extracts did not show significant antibacterial activity on the growth of E. coli
and Staphylococcus aureus, but some activity against Salmonella enteritidis (Raposo et al.,
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2014b). Similar results were found by Amaro et al. (2011) and Ghasemi et al. (2007) who
summarized antimicrobial activities of EPS from microalgae and reported antifungal and
antimicroalgal activities were affected by the solvents used. The possible reason can be the
bioactive portions of the EPS have different affinities to the extraction solvents, being highly
affected by mutual interactions (Basedow et al., 1980), and ethanol might not be able to extract
sufficient bioactive ingredients from EPS and maintain their antibacterial activity. It should be
also aware that different bacteria react differently to the same EPS (Raposo et al., 2014b), which
is probably related to the composition of the bacterial cell wall, the specific structure in the
bacteria, and/or the ability of the bacteria to change the chemical structure of the EPS (Michael
et al., 2002). The antibacterial mechanisms of the EPS could be: antibiofilm formation, modify
the physical properties of the bacterial cell surfaces, and block adhesion of pathogen (e.g., lectin-
dependent adhesion) to host cells (Raposo et al., 2013; 2014a).

4.3.4. Antioxidant

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The EPS from Porphyridium showed the capacity to prevent the accumulation and the activity of
free radicals and reactive chemical species, therefore having antioxidant activity. Tannin-Spitz et
al. (2005) evaluated the antioxidant properties of EPS from Porphyridium sp. (UTEX 637) to
inhibit the auto-oxidation of linoleic acid/ferrous oxidation and oxidative damage to 3T3 cells.
They determined that the antioxidant activity of the EPS can protect the algae against ROS,

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possibly by scavenging the free radicals and transporting them from the cell to the medium. The
EPS of P. cruentum inhibited oxidation in a dose-dependent manner and the antioxidant activity
correlated positively with the sulfate content of the EPS, but negatively with the molecular

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weight; glycoprotein may also contribute to antioxidant properties (Tannin-Spitz et al., 2005).
The LMW EPS from P. cruentum had stronger antioxidant activity than the HMW ones on
scavenging ability of free radicals and inhibitory effects on lipid peroxidation in liver

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homogenates and hemolysis of mouse erythrocytes, thus giving better protection to mouse cells
and tissues against oxidative damage (Sun et al., 2009). The reason could be that antioxidant
activity involves crossing the cellular membrane of tested cells, thus HMW of EPS can be

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shortcoming to pursue their properties. In addition, uronic acid content, the structure and
conformation of EPS seem to influence the radical scavenging properties and antioxidant
capacity (Sun et al., 2009; Tao et al., 2007). Both crude and deprotenized (via autoclave and
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sonication) EPS from R. reticulata demonstrated the free radical scavenging and antioxidant
activity in a dose-dependent manner; they were twice as strong as standard antioxidant α-
tocopherol against superoxide anion radical scavenging (Chen et al., 2010). However, the crude
EPS exhibited higher free radical scavenging capacity and better antioxidant activity than the
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deprotenized samples, which indicated that protein content of EPS could also contribute to the
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antioxidant activity. Therefore, the EPS from R. reticulata can be used as a potent natural
antioxidant.
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4.3.5. Antitumor
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EPS also exhibited inhibitory actions against tumor growth (in vivo) and antiproliferative activity
in cancer cell lines (in vitro). The EPS from G. impudicum KG03 are also good
immunostimulators for strengthening the tumoricidal activity of macrophages and NK cells to
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suppress tumor cell growth through the activation of NO production, which stimulated the
innate immune system and increased the production of cytokines interleukin-1 (IL-1), IL-6 and
THF-α in macrophages (Bae et al., 2006; Namikoshi and Rinehart, 1996; Yim et al., 2005)

The molecular weight and sulfation level are critical in effecting the antitumor activity of the
EPS in that the HMW EPS showed little antitumor activity (Geresh et al., 2002; Sun, 2010).
Only the EPS with molecular weight of 6.53-1,002 kDa from P. cruentum was found inhibit
tumor cell proliferation on S180 tumors in mice models (Sun et al., 2012). However, increasing
the degree of sulfation can greatly improve the cancer inhibition property of the HMW EPS
(Geresh et al., 2002). It has been demonstrated that oversulfated (using agents such as
pyridine•SO3, DMFP•SO3 and ClSO3H) HMW EPS from Porphyridium sp. and R. reticulata
showed inhibitory effects on neoplastic mammalian cell growth and colon cancer proliferation in
rat (Shopen-Katz et al., 2000; Geresh et al., 2002). It is thought that the main mechanism of
antitumor activity of the EPS is a reinforcement of immune system induced by the EPS (Sun et
al., 2012; Zhou et al., 2004). By changing the biochemical characteristics of the cell membrane,
the EPS could also induce tumor cell differentiation and apoptosis, regulate the cell signaling

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pathways, and block the interaction or adhesion between cancer cells and the host cells (Raposo
et al., 2014a; Zhou et al., 2004).

4.3.6. Antihyperlipidaemic activity

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EPS from red algae have the strong potential to be hypolipidemic, hypoglycemic and
hypocholesterolemic agents. They could serve as functional additives to food and feed in
nutraceutical and food industry. They can inhibit human pancreatic cholesterol esterase, an

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enzyme responsible for promoting the intestinal absorption of cholesterol and fatty acids
(Laurienzo, 2010). Increased molecular weight and sulfation at a specific positon (e.g., 3-sulfate
on the sugar ring) can enhance such inhibitory effects (Laurienzo, 2010). The natural features of

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HMW and sulfation render the EPS non-absorbable anticholesterolemic activities. In addition,
Dvir et al. (2000, 2009) found the EPS from Porphyridium sp. can reduce coronary heart disease
because of their hypocholesterolemic properties. Ginzberg et al. (2000) used Porphyridium sp.

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biomass containing EPS to feed chickens and observed a decreased cholesterol level in the blood
specimens and egg yolk as well as modified fatty acid profile and increased carotenoid content in
egg yolk. Using rat models, Dvir et al. (1995, 2000, 2009) found that the whole algal biomass of
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Porphyridium sp. (UTEX 637) and R. reticulata reduced cholesterol, triglyceride and VLDL
levels in serum, but improved the hepatic cholesterol levels with no toxic effects noticed. More
interestingly, the EPS and biomass from P. cruentum and Rhodella respectively can lower the
insulin and/or glucose levels in diabetic rodents without modifying the pancreatic island cells,
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fibrosis or hemorrhagic necrosis in cells (Dvir et al., 1995; Huang et al., 2006). The mechanisms
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of antihyperlipidaemic activity could include (Dvir et al., 2009; Glore et al., 1994; Marlett, 2001;
Oakenfull, 2011): (1) EPS increase the viscosity of intestinal contents, affecting nutrient
absorption and micelle formation and decreasing lipid absorption; (2) EPS disrupt the
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enteropathic circulation of bile acids, leading to the decrease of cholesterol and the increase of
bile excretion; (3) EPS affect the lipid metabolism, resulting in intestinal metabolic and
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morphological changes, thus reduce serum cholesterol.

4.3.7. Biolubricant
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In their study of the chemistry and rheology of EPS from Porphyridium sp., P. aerugineum and
R. reticulata, Geresh and Arad (1991) found rheological characteristics of these EPS were
similar to surfactant properties and non-Newtonian rheological behavior in aqueous solutions,
even though the differently sourced EPS differed in composition. These researchers also found
the rheological properties of EPS aqueous solutions were resistant to pH and temperature change
in a certain range and were compatible with monovalent cations. Due to its rheological properties
and thermal stability, EPS from Porphyridium has already shown good lubrication capacity
(Arad and Weinstein, 2003). Arad et al. (2006) have proved the superior lubricant power of EPS
from Porphyridium by comparing the lubricating properties of EPS with the most used hydrogel
lubricant, hyaluronic acid. The viscosity of EPS from Porphyridium were more stable over a
wide range of temperatures, pH, and salinities; moreover the resistance of EPS to hyaluronidase
degradation makes them capable of reducing friction and wear (Arad et al., 2006). For example,
simulating efforts of joints (both when walking and running), Arad et al. (2006) found the EPS
from Porphyridium had a better quality as its rheological characteristics were stable at higher
temperatures than most lubricants used, whose lubricity viscosity decreased simultaneously. It

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was also found that a 1% solution of EPS presented the best friction properties under high loads,
and its viscosity did not have any significant change even with the presence of hyaluronidase
(Arad et al., 2006). These results reflect the potential of EPS from Porphyridium to be an
excellent biolubricant to substitute hyaluronic acid. Such EPS may also be a promising joint-
lubricating substance for the development of visco-supplements to mitigate degenerative joint

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disorders caused by arthritis (anti-arthritic medications) (Arad and Atar, 2015). The effects of the
solutions containing EPS from Porphyridium were tested by injecting them into the joints of
rabbits’ knees (Arad and Atar, 2015).

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4.3.8. Drag reducer

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This is one of lesser known applications for red algae EPS, and only a few studies were
conducted to determine whether EPS could be used as drag-reducing agents to broaden their
applications in engineering areas, e.g., ship and machine/mechanical engineering. Gasljevic et al.

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(2008) studied ten strains of microalgae (3 reds, 2 greens and 5 diatoms) for the potential of
drag-reducing ability. Only red algae such as P. cruentum and R. maculate and green algae such
as C. stigmatophora and S. capsulate had superior EPS productivity and their EPS showed
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prominence to be drag-reducers. Overall, EPS from P. cruentum and R. maculata had higher
drag-reducing power at lower concentrations, followed by S. capsulate and C. stigmatophora in
that order (Gasljevic et al., 2008). To achieve the same level of drag-reduction effectiveness, 25%
more EPS of R. maculata is required than of P. cruentum, and almost three and twelve times as
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much as the EPS of S. capsulate and C. stigmatophora, respectively. Eteshola et al. (1998)
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reported the unique rheological properties of the Porphyridium sp. (UTEX 637) EPS including a
relatively high viscosity and shear thinning and reversible thermal gelation upon heating in the
presence of a weak gel-like structured network. The rheological properties of Porphyridium sp.
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aqueous preparations ranked favorably with characteristics of microbial gums such as xanthan
gum which has been the most used natural drag-reducing additive (Kim et al., 1998). Polymers
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with HMW are usually more efficient as drag-reducers, thus the HMW is an important
requirement for microalgal EPS to be good drag-reducing agents (Gasljevic et al., 2008). To this
point, EPS from red algae are promising candidates, as their molecular weight is similar to or
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even higher than that of industrial counterparts (e.g., xanthan gum).

4.3.9. Other applications

The algal EPS, especially the sPS is also known to exhibit anticoagulant/antithrombotic activities.
Of the little research undertaken to elucidate the anticoagulant properties of the sPS from red
algae, it was determined that the anticoagulant activity from seaweed is associated with a high
sulfate content of the polysaccharide (Wijesekara et al., 2011). However the sPS from the
microalga, C. Polykrikoides, did not exhibit anticoagulant activity regardless of the high sulfate
percentage (Hasui et al., 1995). Consequently, it was hypothesized that the anticoagulant activity
could be affected by both the sulfate residue content, and the sulfate groups distribution/positions
and/or by the polymer chain configurations (Ginzberg and Korin, 2008; Pereira et al., 2002). On
the other hand, the anticoagulant feature and antiviral activity could conflict because antiviral
activity may not need high degree of sulfation (Fábregas et al., 1999). Also, some care should be
taken on hemorrhagic strokes when EPS is used as an antithrombotic agent to break down the
clot.

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EPS from red algae are quite useful in cosmetics. For example, the addition of EPS from
Porphyridium sp., P. aerugineum and R. reticulata to aloe vera gel resulted in increased viscosity
of aloe vera gel, which revealed that the EPS could stabilize the network structure of aloe vera
gel and extend its shelf life (Yaron et al., 1992). Geresh and Dawadi (2000) quaternized the EPS

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from Porphyridium sp. and achieved a greater elasticity of the quaternized EPS than the native
material. These modified EPS can be used as a naturally-derived ingredient in cosmetic
formulations. The EPS of red algae have also been used in wastewater treatment, but to a less

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extent than green algae. The negative charge (due to the presence of glucuronic acid and half
ester sulfate groups) of EPS from both Porphyridium sp. and R. reticulata was used to prepare
ion exchangers which successfully removed toxic ions from industrial wastewater (Geresh et al.,

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1990, 1997; Lupescu et al., 1991). In addition, red algae EPS have other various promising
applications in drugs, crop growth promoters and health food markets (Pignolet et al., 2013).

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5. Conclusions

EPS are an essential group of metabolites of microalgae (e.g., green algae, diatoms and red algae)
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that form an extracellular biofilm matrix in the immediate environment of host cells. In terms of
chemical composition, microalgal EPS are composed of polysaccharides, protein, DNA, lipid,
and other trace moieties. However, almost all previous research focused on determining the
chemistry, structure, properties, and applications of polysaccharides of EPS. Although the
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polysaccharides from different microalgae may have some similarities in composition, they can
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be very heterogeneous and structurally different. While this diversity makes research very
challenging, successful studies have shown the efficacy of polysaccharides in the production of
bio-based materials for pharmaceutical, therapeutic, regenerative medicine, nutraceutical,
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cosmetic, and wastewater treatment applications. Many beneficial bioactivities of microalgal

exopolysaccharides (e.g., antiviral, antitumor, antibiotics, anti-inflammatories, and
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anticoagulants) have been demonstrated, either in vitro or in vivo. Despite this compelling set of
characteristics for use in applications for human health, further studies of the microalgal
exopolysaccharides are needed to determine their toxicity and bioavailability on humans.
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Furthermore, the rheological and biochemical characteristics of polysaccharides could be useful

in creating applications in the machine/mechanical concepts and in ship engineering (e.g., as
drag-reducers), and in food science/engineering (e.g., as thickeners to increase viscosity). Unlike
polysaccharides, microalgal extracellular proteins (enzymatic and structural proteins) have also
been the subject of little research. Many compelling questions remain unclear, however, all of
which are the subject of very compelling research (e.g., the protein type, the role of proteins in
the EPS generation and degradation during the cell life cycle, the enzyme activity and the
quantitative contribution to the carbon cycle and energy flux, and enzyme applications). These
extracellular proteins may be another promising biopolymers, thus enhancing the value of EPS
from microalgae. Consequently, elucidating the extracellular proteins in these algae is another
realm of as yet undiscovered knowledge. In addition, the overall amount and the dynamic of EPS
formation in response to various physical and biological conditions are largely unknown. The
greatest deficit in current EPS research is the inability to elucidate the underlying fundamental
regulatory mechanism, making the prediction and manipulation of EPS production impossible.
Overall, microalgae are promising biological resources to produce high-value EPS. They can
grow under controlled conditions at industry scale making chemical composition, structure and

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properties of algal EPS more stable along the several harvesting periods without concerning the
climate condition. Connecting with algae-based biorefinery, the EPS is a most promising high-
value coproduct enabling the commercialization of alga biofuels.

Acknowledgments

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The authors wish to thank Mr. Godfrey Kimball (Grant Editor in the College of Engineering and
Science at Clemson University) for critical review.

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