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University of Groningen
Functional carbohydrates from the red microalga Galdieria sulphurariaMartínez García, Marta
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7
Chapter 1
General introduction
Chapter 1
8
1. Microalgal biotechnology
On a planet suffering the environmental consequences of unsustainable
economic growth, which has been relying on the exploitation of limited fossil
resources, the urge to shift to a bio-based economy has become a top priority. In
the search for new biomass resources, algae have emerged as a good alternative
to terrestrial crops due to the high biomass productivity achieved without
requiring arable land for growth, therefore eliminating the competition with
food production (Dismukes et al., 2008; Clarens et al., 2010, Posten & Chen,
2016).
Until recent years, algal biotechnology has been dominated by the cultivation of
certain species of marine macroalgae to be used directly as food source or for
the production of polysaccharides with gelling properties such as agar,
carrageenan and alginate, widely used as hydrocolloids in a range of food,
pharmaceutical and specialty products (Radmer, 1996). Microalgal
biotechnology is a branch of algal biotechnology that has taken advantage of the
faster growth rates and higher biomass productivity of unicellular algae
compared to multicellular species (Rosenberg et al., 2008, Posten & Chen,
2016). Although in the last years the use of microalgae for biodiesel production
has received much attention (Miao & Wu, 2006; Wijffels & Barbosa, 2010),
microalgae can actually produce a vast range of high value products, including
pigments, carotenoids, antioxidants, polysaccharides and polyunsaturated fatty
acids (Pulz & Gross, 2004; Borowitzka, 2013).
Most microalgae species are obligate phototrophs and their growth is
necessarily linked to the availability of light to perform photosynthesis. The
mass cultivation of these photosynthetic microalgae can be carried out in open-
ponds, where cells can grow utilizing sunlight, or in closed photobioreactors
with artificially supplied light (Lee et al., 2001). Usually, this type of
microalgae cultivation is associated with low biomass yields and the market
value of the product of interest needs to compensate for the high production
costs (Borowitzka, 1992). Heterotrophic cultivation of microalgae, by which
cells are grown in the dark using an organic compound as carbon and energy
source, results in much higher biomass productivities and is thus more cost-
effective (Perez-Garcia et al., 2011). However, the ability to grow
heterotrophically is only restricted to certain microalgae, such as for example
Hematococcus pluvialis, Aurantiocrytum limacinum and several Chlorella
Introduction
9
species (Miao & Wu, 2006; Bumbak et al., 2011; Sakarika & Kornaros, 2016;
Morales-Sánchez et al., 2016). Among the facultative heterotrophic microalgae,
we find Galdieria sulphuraria, a red microalga that grows optimally at pH 2
utilizing a wide range of organic substrates (Gross & Schnarrenberger, 1995).
Because of its acidophilic lifestyle - which minimizes considerably the risk of
contamination - and its ability to grow heterotrophically, G. sulphuria shows
desirable features for mass cultivation, which has until now only been explored
for the production of pigments (Graveholt & Erikssen, 2007). Research and
characterization of possible valuable compounds from G. sulphuraria will
contribute to further exploit the biotechnological potential of this microalga.
2. The extremophilic red microalga Galdieria sulphuraria
2.1. Red algae
Red algae (Rhodophyta) are a group of ancient photosynthetic eukaryotes which
some authors claim it diverged even before the appearance of the common
ancestor of plants and fungi (Stiller & Hall, 1997). However, this affirmation is
not widely supported by other phylogenetical analysis, and the classification of
red algae as a sister group sharing a common ancestor with plants, green algae
and fungi is still the most generally accepted today (Moreira et al, 2000).
Rhodophyta is a diverse group of organisms that contains both multicellular and
unicellular species that can colonize a wide range of habitats, including marine
and fresh waters, hot sulfur springs and volcanic environments. The taxonomic
classification of red algae has been revised and updated several times, with
more recent phylogenetic studies proposing a classification of red algae into two
subphyla: Cyanidiophytina and Rhodophytina (Yoon et al., 2006; Yang et al.,
2016). The subphylum Cyanidiophytina contains only one class,
Cyanidiophyceae, and this class contains only one order named Cyanidiales.
The subphylum Rhodophytina contains six classes: Floridiophyceae,
Bangiophyceae, Rhodellophyceae, Compsopogonophyceae,
Stylonematophyceae and Porphyridiophyceae. The class Floridiophyceae is the
most diverse and contains the majority of the currently described red algae
species (Guiry & Guiry, 2016), including economically relevant species such as
those used for the production of the polysaccharides agar and carrageenan, two
widely employed phycocolloids (Renn, 1997).
Chapter 1
10
A common characteristic of all red algae is the presence of chlorophyll a as the
only major photosynthetic pigment and phycobiliproteins as accessory pigments
to improve the efficiency of light harvesting. These phycobilliproteins are
arranged in big macromolecular complexes named phycobilisomes, similar to
those found in cyanobacteria, which are embedded in the membrane of the
choloroplasts (Gantt, 1981). Although most of red algae owe their color to the
orange-red pigment phycoerythrin, some species are blue-green because their
major photsynthetic pigment is the blue phycocyanin (Cole & Sheath, 1990).
2.2. The extremophilic order Cyanidiales
The order Cyanidiales groups the most ancient red algae that diverged from the
other species more than 1200 million years ago (Yoon et al. 2006, Yang et al.,
2016). This order consists of three genera: Cyanidium, Cyanidioschyzon and
Galdieria. The first two genera were the first to be described and contain only
one species each, Cyanidium caldarium (Hirose, 1958) and Cyanidioschyzon
merolae (De Luca et al., 1978), respectively. In 1981, it was revealed that more
than one species had been wrongly referred to as C. caldarium over the past
years, since another very similar microalga co-existed in the same habitat. A
new genus was created and the newly isolated species was given the name of
Galdieria sulphuraria (Merola et al., 1981). Because of that confusion, some
studies performed on G. sulphuraria prior to 1981 might have been wrongly
attributed to C. caldarium. Later on, three more species isolated from acid
thermal springs in Russia were included into the genus Galdieria on the basis of
morphological features, and were named Galdieria maxima, Galdieria partita
and Galdieria daedela (Sentsova, 1991). However, their status as new species
has been challenged by some authors who claim they are just strains of G.
sulphuraria (Cozzolino et al, 2000).
All Cyanidiales are unicellular and display a blue-green color due to the
presence of phycocyanin as their main accessory photosynthetic pigment. They
are extremophiles, thriving in environments with pH values between 0-4 and
temperatures up to 56 °C (Seckbach, 1999), a value close to the upper limit for
eukaryotic life (Rothschild & Mancinelli, 2001). These type of environments
are scattered around the world, and can be found in e.g. the hot sulfur springs of
Yellowstone National Park (USA) or volcanic areas in, Iceland, Italy, Indonesia,
New Zealand and Japan (Gross & Oesterhelt, 1999; Toplin et al., 2008), where
Introduction
11
the Cyanidiales represent the majority of the eukaryotic biomass and the only
photosynthetic organisms present. The Cyanidiales are also tolerant to high
concentrations of salts (Gross et al., 2002; Pade et al., 2015) and metals
(Yoshimura et al., 1999; Nagasaka et al., 2004), which are typical of these sites
(Gross, 2000).
Even though the three Cyanidiales genera diverged very early from each other -
for example, the evolutionary distance between C. merolae and G. sulphuraria
is the same as between the fruit fly and humans (Schönknecht et al., 2013) –
their physiological and morphological features are very similar and have been
conserved over the years, and no new species have been discovered and added
to the few already described. This is likely due to the extreme conditions
inhabited by the Cyanidiales, which puts the cells under a high selective
pressure that has minimized their diversity. However, some authors claim that
this diversity might be underestimated and that extensive sampling from
different natural habitats combined with cultivation-independent molecular
tools could result in the assignation of new species (Ciniglia et al., 2004).
Despite being very similar, the three Cyanidiales genera can be distinguished by
certain cytomorphological and ecophysiological features (Albertano et al.
2000). C. merolae cells are the smallest of all, with a size of 1.5 – 3.5 µm, and
have an oval, club-like shape. They lack a cell wall, contain one polymorph
chloroplast and one mitochondrion, and reproduce by binary fission. This
species is a strict autotroph and thus, it can only proliferate by performing
photosynthesis. C. caldarium cells are 2-5 µm in size, with a spherical form and
surrounded by a protein-rich cell wall (Bailey & Staehelin, 1968). They contain
a single spherical chloroplast and a concave mitochondrion and multiply
through the formation of 4 endospores. Like C. merolae, this species is also
restricted to phototrophic growth. The members of the genus Galdieria are also
spherical and have the biggest size of all Cyanidiales, reaching 11 µm. They
possess a cell wall, one multilobed chloroplast (Fig. 1), one branched
mitochondrion and one vacuole and proliferate through the formation of 4-32
daughter cells. Galdieria cells are facultative heterotrophs, being able to use
organic compounds as carbon source whenever environmental conditions limit
photosynthesis (Gross & Schnarrenberger, 1995). The differences in growth
regime between the Cyanidiales have influenced their distribution in natural
habitats. While C. merolae and C. caldarium - which rely on photosynthesis for
their survival – are predominantly found on the surface of rocks, G. sulphuraria
Chapter 1
12
has also been isolated from more interior layers where sunlight penetration is
insufficient, forcing the cells to switch to heterotrophy (Gross et al., 1998; Yoon
et al., 2006).
2.3. The metabolically flexible species Galdieria sulphuraria
Within the order Cyanidiales, the species belonging to the genus Galdieria
display a remarkable metabolic flexibility that has raised the interest of several
scientists and has been the subject of many investigations. The best established
species in this genus is Galdieria sulphuraria and, as such, it has been the most
studied over the years.
G. sulphuraria is the only member of the Cyanidiales reported to be able to
grow not only autotrophically, performing photosynthesis, but also
heterotrophically in complete darkness using many organic compounds as
carbon source, including a range of monosaccharides, sugar alcohols, organic
acids and amino acids (Rigano, 1976; Gross & Schnarrenberger, 1995), a
substrate spectrum matched by few organisms (Table 1). The possibility of
mixotrophic growth (simultaneous use of an inorganic and an organic carbon
Figure 1. Scanning electron microscopy pictures of G. sulphuraria cells growing
autotrophically (A) and heterotrophically (B). Note the clear absence of a lobed
chloroplast (CP) in the heterotrophic cell, where this organelle has
dedifferentiated into a proplastid, and the presence of what it seems a big vacuole
(V). For more information on changes in cell structure depending on growth
regime see Tischendorf et al., 2007.
Introduction
13
source) has been suggested by some authors (Stadnichuk et al., 1998), but it
seems that only heterotrophy and not true mixotrophy occurs in G. sulphuraria
(Oesterhelt et al, 2007). When cells are growing in the presence of an organic
carbon source, pigment synthesis is inhibited (Stadnichuck et al., 1998) and the
chloroplast of the cells dedifferentiates into a proplastid (Tischendorf et al.,
2007) (Fig. 1). As a result, the cells lose their characteristic blue-green color and
become yellow-green, although a strain that maintains its photosynthetic
apparatus under heterotrophic conditions has also been isolated (Gross &
Schnarrenberger, 1995). Since organic compounds are not abundant in the
natural habitat of G. sulphuraria, its heterotrophic capacity is regarded as a
survival strategy that enables the cells to use compounds released by
surrounding dying organisms when the amount of light is insufficient to sustain
photosynthesis (Gross et al., 1998). Genome analysis has revealed that the
ability of G. sulphuraria to grow heterotrophically is not conferred by the
presence of genes encoding essential enzymes for carbohydrate metabolism,
since these can also be found in the genome of the obligate autotroph C.
merolae, but by a multitude of genes encoding metabolite transporters, which
allow the uptake of compounds from the environment into the cells (Weber et
al., 2004; Barbier et al., 2005).
In 2013, the complete sequence of the highly compact genome of G.
sulphuraria (13.7 Mb of which 77.5% are coding genes) was published
(Schönknecht et al., 2013), providing tremendously valuable data to explain the
enormous metabolic versatility of this alga. In agreement with previous
observations, the authors found that the amount of genes encoding for
membrane transport proteins in G. sulphuraria was higher than in most other
eukaryotes, a feature that is essential for its ability to grow on so many different
compounds. Moreover, the study revealed that around 5% of the genes in G.
sulphuraria had been acquired by horizontal gene transfer (HGT) from archaea
and bacteria. These genes are directly involved in the adaptation of G.
sulphuraria to the harsh conditions present in its natural habitat, such as high
temperatures, low pH and high concentrations of salt and metals, being the first
reported case of HGT in eukaryotes involving genes linked to fitness-relevant
traits. Among the genes acquired by HGT there are several glycerol uptake
facilitators and glycerol dehydrogenases, which allow G. sulphuraria to grow
on glycerol as sole carbon source at rates comparable to those on glucose (Gross
& Schnarrenberger, 1995).
Chapter 1
14
Ta
ble
1.
Co
mp
ou
nd
s u
sed
as
carb
on s
ourc
e b
y G
. su
lph
ura
ria
gro
win
g h
eter
otr
op
hic
ally
in t
he
dar
k.
Typ
e o
f co
mpo
un
d
Ref
eren
ce
Mo
no
sacc
ah
rid
es
D-G
luco
se,
D-M
anno
se,
D-G
ala
cto
se,
D-F
ruct
ose
,
L-S
orb
ose
, D
-Fuco
se,
L-F
uco
se,
L-R
ham
no
se,
D-A
rab
ino
se,
L-A
rab
ino
se,
D-L
yxo
se,
D-R
ibo
se,
D-X
ylo
se
Gro
ss &
Sch
nar
renb
erger
, 1
995
;
Gro
ss e
t al
., 1
99
8;
Gro
ss &
Oes
terh
elt,
19
99
;
Oes
terh
elt
et a
l.,
19
99
; S
chm
idt
et a
l.,
20
05
;
Web
er
et
al.,
2
00
5;
Oes
terh
elt
et
al.,
2
00
7;
Gra
veh
olt
& E
rik
ssen,
20
07
;
Sch
ön
knec
ht
et al
., 2
01
3;
Pad
e et
al
., 2
01
5;
Sak
ura
i 2
01
6;
Sar
ian e
t al
., 2
01
6
Dis
acc
ha
rid
es
Sucr
ose
, L
acto
se
Gro
ss &
Sch
nar
renb
erger
, 1
995
;
Sch
mid
t et
al.
, 2
00
5;
Tis
chen
do
rf e
t al
., 2
007
Po
lyo
ls
Gly
cero
l, D
-Man
nit
ol,
D-S
orb
ito
l, D
ulc
ito
l, X
yli
tol,
L-A
rab
ito
l, D
-Ara
bit
ol,
Ad
onit
ol
Gro
ss &
Sch
nar
renb
erger
, 1
995
;
Gro
ss e
t al
., 1
99
8;
Gro
ss &
Oes
terh
elt,
19
99
;
Oes
terh
elt
et a
l.,
19
99
; T
his
thes
is
Am
ino
aci
ds
Glu
tam
ate,
Ala
nin
e, 2
-keto
-glu
tara
te
Rig
ano
et
al.,
19
76
By
-pro
du
cts,
wa
ste
stre
am
s
Mo
lass
es f
rom
su
gar
pro
duct
ion,
crud
e gly
cero
l fr
om
bio
die
sel
pro
du
ctio
n,
urb
an w
aste
wat
er e
fflu
ent
Sch
mid
t et
al.
, 2
00
5;
Hen
kan
atte
-Ged
era
et a
l.,
20
16
;
This
thes
is
Introduction
15
3. Funtional carbohydrates from G. sulphuraria
3.1. Short overview on biotechnological applications of
carbohydartes
Carbohydrates are the most abundant and naturally renewable group of
biomolecules. They are constituents of many biologically essential molecules
such as starch, cellulose, nucleic acids and glycoproteins. The physiological
functions of carbohydrates in organisms are very diverse, the major ones being
energy storage, energy transport between cells and structural support. Certain
types of carbohydrates such as starch, sucrose, cellulose and the hydrocolloids
agar, carrageenan and alginate are also biotechnologically relevant raw
materials and are widely used, together with their derivatives, in several
industries for a vast range of applications, some of which are summarized in
Table 2.
Agar, carrageenan and alginate are cell wall polysaccharides produced by
various species of red and brown macroalgae and are the only
biotechnologically relevant carbohydrates that do not originate from terrestrial
plants, with a global production of 100.000 tons (Kim & Chojnaka, 2015). They
are denominated phycocolloids and are widely employed in all kinds of food
products as gelling and thickening agents (Radmer, 1996; Renn, 1997). Three of
the main industrially relevant carbohydrates produced from terrestrial plants are
cellulose, sucrose and starch. Cellulose is a major component of the cell wall of
plants and most of its industrial production is directed to non-food applications,
mainly the production of paper and pulp (Klemm et al., 2005). Sucrose is the
major energy transport carbohydrate in plants. Annually, around 170×106 tons
of sucrose are extracted from sugar cane or beet in the world
(www.fas.usda.gov/commodities/sugar), and are employed by the food-industry
as sweetening agent. In the last years, sucrose has also gained attention as raw
material for the production of other chemicals thanks to the development of
enzyme technology, which takes advantage of the catalytic specificity of
enzymes for the conversion of sucrose into other valuable products (Röper,
2002; Lichtenthaler & Peters, 2004). One example is the use of sucrose as
glucosyl donor for the sucrose phosphorylase-mediated production of
glucosylglycerol, a moisturizing agent with application in the cosmetic industry
(Goedl et al., 2009).
Chapter 1
16
Starch is the energy storage carbohydrate accumulated by many plants,
including crops such as corn, wheat, rice and potato, and is thus one of the main
ingredients of the human diet. But starch is also the most industrially relevant
renewable raw material from which many derivatives can be generated via
physical, chemical or enzymatic methods (Ellis et al., 1998; Röper, 2002;
Buchholz & Seibel, 2008). In the European Union, around 10×106 tons of starch
is produced, of which 26% is used as native starches, 19% is modified and 55%
is converted to sweeteners (www.starch.eu). Starch and its derivatives are
widely used in numerous food and non-food applications (Table 2). Enzymatic
treatment of starch mainly consists of its hydrolysis to dextrins, oligosacharides
and glucose for the production of syrups and other sweeteners (Crabb &
Mitchinson, 1997; Buchholz & Seibel, 2008). Starch is also converted by
hydrolysis and re-arrangement of the glycosidic linkages to produce novel types
of molecules such as cyclodextrins (Biwer, 2002) or highly branched glucose
polymers (Backer & Saniez, 2005; van der Maarel & Leemhuis, 2013). As the
name indicates, highly branched glucose polymers are starch derivatives in
which the proportion of branching linkages is considerably increased with
respect to native starch. This characteristic confers them with properties that are
advantageous for certain applications, a topic further elaborated in section 3.2.4.
Introduction
17
Typ
e o
f ca
rbo
hyd
rate
A
pp
lica
tio
ns
Sta
rch
an
d d
eriv
ati
ves
Gra
nula
r st
arch
, p
re-g
ela
tiniz
ed
sta
rch,
oxid
ized
sta
rch,
cati
onic
sta
rch,
dex
trin
s, h
yd
roly
zed
sta
rch,
cycl
od
extr
ins,
bra
nch
ed g
luco
se p
oly
mer
s
Fo
od
ap
pli
cati
on
s
Gel
lin
g a
gents
, th
ickener
s, e
muls
ifie
rs,
swee
tener
s, f
lavo
ur
and
aro
ma
stab
iliz
ers
No
n-f
oo
d a
pp
lica
tio
ns
Co
atin
gs,
ad
hesi
ves
and
bin
ders
fo
r p
aper
and
co
rrugat
ed i
nd
ust
ry,
bio
pla
stic
s, s
urf
acta
nts
and
ble
ach a
ctiv
ato
rs f
or
det
ergen
t in
dust
ry,
feed
sto
ck f
or
pro
duct
ion o
f o
ther
chem
ical
s
Ph
arm
ace
uti
cal,
co
smet
ic a
nd
bio
med
ica
l a
pp
lica
tio
ns
Dru
g e
xci
pie
nts
and
car
rier
s, p
aren
tera
l nutr
itio
n s
olu
tio
ns,
per
ito
nea
l d
ialy
sis
solu
tio
ns
Su
cro
se,
der
iva
tiv
es a
nd
by
-pro
du
cts
Puri
fied
sucr
ose
, su
cro
se e
ster
s, m
ola
sses
,
bee
t p
ulp
Fo
od
ap
pli
cati
on
s
Sw
eete
ner
s, l
ow
cal
ori
e an
d n
on
-car
iogenic
sw
eete
ner
s, e
muls
ifie
rs
No
n-f
oo
d a
pp
lica
tio
ns
Anim
al f
eed
, fe
rmenta
tio
n f
eed
sto
ck
Cel
lulo
se a
nd
der
iva
tiv
es
cell
ulo
se,
cell
ulo
se e
ster
s, c
ellu
lose
eth
ers
Pap
er a
nd
pulp
pro
duct
ion,
fib
ers
for
texti
le i
nd
ust
ry,
coat
ings,
lam
inat
es
Ph
yco
coll
oid
s
Agar
, ag
aro
se,
carr
agee
nan,
algin
ate
Fo
od
ap
pli
cati
on
s
Gel
lin
g a
gents
, te
xtu
rize
rs,
em
uls
ion s
tab
iliz
ers
Bio
tech
no
log
ica
l a
nd
co
smet
ic a
pp
lica
tio
ns
Gel
mat
rice
s fo
r la
bo
rato
ry t
echniq
ues
, d
enta
l im
pre
ssio
n m
ed
ia,
too
thp
aste
bin
der
s
Ta
ble
2.
Bio
tech
no
logic
ally
rel
evan
t ca
rbo
hyd
rate
s an
d s
om
e ex
am
ple
s o
f ap
pli
cati
ons.
Chapter 1
18
3.2. The highly branched glycogen of G. sulphuraria
3.2.1. Basic structure of starch and glycogen
Many organisms use polysaccharides as a way of storing cellular energy. The
most widespread storage polysaccharides in nature are starch and glycogen.
Starch is mainly synthesized by photosynthetic eukaryotes, such as land plants
and green algae. Glycogen can be found in both prokaryotic microorganisms,
like bacteria and cyanobacteria, and eukaryotic cells, such as yeast and animal
muscle and liver cells. Both starch and glycogen have the same basic
composition, they are polymers of O-linked glucose units, but they differ in
their structure (Fig.2).
Starch is composed of two types of polymer fractions, amylose and
amylopectin, which are arranged in the complex quaternary structure that is the
semi-crystalline, insoluble starch granule. Amylose is a virtually linear polymer
of glucose units linked by α-(14) bonds. Amylopectin is a branched polymer
with a linear backbone of α-(14) linked glucoses and side chains attached
through α-(16) bonds. The proportion of branching linkages in amylopectin is
around 5% and the side chains can be classified into three types according to
their average degree of polymerization (DP): short chains with a DP of 14-18
glucose units, long chains with a DP of 45-55 and a few chains of DP> 60
(Buléon et al., 1998). The α-(16) linkages are distributed in a non-random
manner along the amylopectin molecule (Thompson, 2000), resulting in the
appearance of branched clusters in which the short chains are localized and
which are interconnected by the long chains, leaving an average chain length of
20-23 units between clusters. The side chains localized outside the branched
clusters interact with each other to form double helices. As a consequence, two
types of regions can be identified in the starch granule: crystalline regions,
corresponding to the double helices formed by the linear chains, and amorphous
regions, corresponding to the branched regions (Fig. 2). It is not clear how
amylose interacts with amylopectin in the starch granule but it is possible that it
participates in the formation of double helices with the amylopectin side chains
in the crystalline regions.
Like amylopectin, glycogen is also a branched polymer consisting of a linear
backbone of α-(14) linked glucoses with side chains attached through α-(16)
bonds. However, glycogen contains a higher number of branching linkages than
Introduction
19
amylopectin - in the order of 8-13%, depending on the glycogen source (Matsui
et al., 1993; Wang & Wise, 2011) – which are distributed irregularly along the
molecule. The most accepted structural model for glycogen describes it as
having a globular, tree-like conformation in which the chains are arranged in
concentric tiers (Manners, 1991), differing significantly from the structure of
starch amylopectin structure (Fig.2). The shorter length of the branches in
glycogen compared to amylopectin prevents chain interaction and the formation
of double helices and, as a result, glycogen is soluble in water. The maximum
number of tiers that a glycogen molecule (also referred as β-particle) can have is
determined by a “crowding” mechanism, by which the glycogen particle grows
during synthesis increasing its density exponentially with every new tier
formed, until this density is so high that the synthesis cannot proceed
(Meléndez, et al, 1993). Various glycogen molecules (β-particles) can group
together forming big complexes denominated α-particles or α-rosettes. The
mechanism by which this happens is not completely unraveled and it seems to
differ with the glycogen type, but some authors proposed a “crowding/budding”
mechanism involving glycosidic linkages, in which synthesis continues from a
branch that escapes the most outer tier of the glycogen molecule until a new β-
particle arises (Powell et al., 2015).
Although glycogen is composed mainly of glucose residues, some works have
reported the presence of other monosaccharides such as glucosamine or
galactose (Nordin & Hansen, 1963; Kirkman & Whelan, 1986), which could be
incorporated as a result of unspecific enzymatic activity during glycogen
synthesis. The incorporation of sulfate ester groups has been observed in the
glycogen of the marine sponge Aplysina fulva, where this modification could
render the molecule more resistant to degradative enzymes of competitor
organisms (Zierer et al., 1995).
Chapter 1
20
3.2.2. The storage polysaccharide of red algae: similarities and differences
with starch and glycogen
Floridean starch is the name given to the energy storage polysaccharide
synthesized by red algae and it derives from Florideophyceae, the class from
which this polysaccharide was first characterized (Barry et al., 1949). Despite
being commonly referred as a starch, this glucan shares metabolic and structural
features with both glycogen and plant starch.
A major difference between floridean starch and plant starch is the localization
within the cell. Even though red algae contain chloroplasts (sometimes referred
to as rhodoplasts), the synthesis and storage of floridean starch takes place in
the cytosol of the cells (Viola et al., 2001), thus differing from plant and green
algal starch, which is produced and accumulated in the plastids.
The metabolic pathway for floridean starch synthesis also shows some
differences to plant starch. The synthesis of both starch and glycogen requires
the same basic elements: a glucose donor in the form of sugar nucleotide, a
Figure 2. Structural models of amylopectin and glycogen. Discontinuous lines
represent the division between crystalline and amorphous regions in the
amylopectin molecule and the concentric tiers in the glycogen molecule.
Introduction
21
linear chain acceptor (primer) to which glucose molecules are added to form the
polymer, a synthesizing enzyme that catalyzes the formation of linear α-(14)
linkages and a branching enzyme responsible for the introduction of α-(16)
branching points.
The type of sugar nucleotide used as glucose donor depends on the specificity
of the enzyme responsible for the synthesis. In both glycogen-synthesizing
prokaryotes (bacteria and cyanobacteria) and starch-synthesizing eukaryotes
(green algae and plants) the enzyme uses adenosine-diphosphate-glucose (ADP-
glucose) as donor. It is widely accepted that this similarity derives from the fact
that plant chloroplasts evolved from a cyanobacteria-like ancestor that was
engulfed by a eukaryotic cell and, in consequence, certain metabolic traits have
been maintained through evolution (Battacharya & Medlin, 1995). Floridean
starch synthesis in red algae proceeds mainly via the use of uridine-
diphosphate-glucose (UDP-glucose) as donor, in analogy to glycogen synthesis
in other eukaryotes (fungi and animal cells). However, an ADP-glucose specific
synthase has also been reported in some species (Nagashima et al., 1971, Sheath
et al., 1981, Nyvall et al.1999), prompting some authors to hypothesize about
the coexistence of two independent pathways for floridean starch synthesis in
red algae: one in the cytosol and another one in the chloroplast (Viola et al.,
2001).
Regarding the primer needed to initiate polysaccharide synthesis, in glycogen-
synthesizing eukaryotes this role is played by a protein called glycogenin. This
protein displays autoglycosylation abilities and can catalyze the transfer of a
glucose molecule from the donor (the sugar nucleotide) to a tyrosine residue
localized in its own structure that acts as acceptor, forming a short α-(14)
glucan chain after several rounds of synthesis (Smythe & Cohen, 1991; Wilson
et al., 2010). Glycogen-synthesizing prokaryotes and starch-accumulating
eukaryotes do not possess a protein like glycogenin and it is unclear which
molecule acts as primer for polysaccharide synthesis. However, some authors
suggest that this role could be played by unbranched malto-oligosaccharides
that are constantly present in these cells as a result of glucan metabolism (Ball
& Morell, 2003). The existence of glycogenin in red algae is not well
documented, but it has been reported that the genome of G. sulphuraria
contains one gene encoding a putative glycogenin protein (Barbier et al, 2005).
Chapter 1
22
With respect to its structure, floridean starch was initially described as being a
type of starch containing only amylopectin (Fleming et al., 1956; Meeuse et al.,
1960; Greenwood & Thompson, 1961; Manners &Wright, 1962), but later
reports demonstrated the presence of amylose in some unicellular species
(McCracken & Cain, 1981; Shimonaga et al., 2007). The absence of amylose in
most of red algae species has been suggested to stem from the lack of synthase
activity embedded within the floridean starch granule, a requirement for
amylose synthesis in higher plants (Viola et al., 2001). After several studies
dealing with the structural characterization of floridean starch extracted from
different red algae species, both multicellular and unicellular, it seems now
inaccurate to speak of a unique and generalized structure for this storage glucan.
Instead, floridean starch shows a wide variation in structure depending on the
red algae species (Table 3). In some multicellular red algae, the structure of
floridean starch is similar to plant amylopectin in terms of average chain length,
chain length distribution, granule formation and crystallinity (Peat et al., 1959;
Greenwood & Thomson 1961; Yu et al., 2002). However, in some unicellular
red algae this polysaccharide is described as a semi-amylopectin or even as a
glycogen-type of molecule (Shimonaga et al., 2007; Shimonaga et al., 2008;
Hirabaru et al., 2010). Moreover, some early works even reported the presence
of α-(13) linkages in the polysaccharide (Barry et al., 1949; Peat et al., 1957),
a controversial observation that has not been confirmed in more recent studies
(Yu et al., 2002).
3.2.3. The highly branched glycogen of G. sulphuraria
The structural characterization of the storage polysaccharide accumulated by the
red microalgae of the order Cyanidiales has been reported in various works.
In the case of C. merolae, this storage glucan has been classified as a semi-
amylopectin type of molecule based on the comparison of the proportion of
long chains (DP≥37) versus short chains (DP≤8) with certain standards such as
plant amylopectin, cyanobacterial semi-amylopectin and cyanobacterial
glycogen (Hirabaru et al., 2010). The chain length distribution profile of the
storage polysaccharide of C. merolae showed a significant amount of chains
with DP around 40-50, but lower than what is characteristic for plant
amylopectin. These chains are considered essential in connecting the branched
clusters in amylopectin and therefore the authors concluded that C. merolae
accumulates a semi-amylopectin with a less organized cluster-like structure.
This semi-amylopectin glucan is stored in the form of small granules with low
Introduction
23
crystallinity that are free of amylose and display a higher gelatinization
temperature compared to those of other red algae, prompting the authors to
speculate that this could confer an advantage in granule stability in high
temperature environments like those inhabited by C. merolae. The storage
polysaccharides in C. caldarium and G. sulphuraria have been reported to be
similar to glycogen since they do not form an insoluble crystalline granule, and
possess high amounts of short chains and a low proportion of long chains with
DP≥20 (Shimonaga et al., 2007; Shimonaga et al., 2008). In particular, the chain
length distribution of the glycogen from G. sulphuraria showed a very
remarkable profile in which chains of DP 4-10 were the majority, with the most
abundant chain having a DP of 9, and chains with DP ≥ 15 were virtually absent
(Shimonaga et al., 2008, Martinez-Garcia et al. 2016). A similar chain length
distribution profiles for G. sulphuraria glycogen has been published in a later
work (Sakurai et al., 2016), hinting at a characteristic, very short-chained
structure for the storage polysaccharide of this microalga. In fact, the
polysaccharide accumulated by a similar species, Galdieria maxima, had been
previously characterized by methylation analysis and 1H-NMR and described as
a highly branched structure with an average chain length of 7 glucose residues,
shorter than reported for typical glycogens (Stadnichuk et al., 2007).
The difference in the structure of the storage polysaccharides between the
various Cyanidiales has helped to describe the evolution of glucan metabolism
in photosynthetic organisms. The fact that C. merolae - the most primitive of
the Cyanidiales and thus the most primitive of all photosynthetic eukaryotes
(Seckbach, 1987) - accumulates semi-amylopectin supports the hypothesis that
the single common ancestor of red algae, green algae and plants originally
accumulated starch as storage polysaccharide in the cytosol (Deschamps et al.,
2008). Later on, the storage polysacchride of C. caldarium and G. sulphuraria
might have reverted to glycogen due to environmental pressure (Shimonaga et
al., 2008).
Chapter 1
24
Red
alg
a s
pec
ies
Sto
rag
e po
lysa
cch
ari
de
stru
ctu
re
Ref
eren
ce
Dil
sea
ed
uli
s
Am
ylo
pec
tin
, no
am
ylo
se
Po
ssib
le p
rese
nce
of
α-(
1
3)
linkag
es
Gra
nu
le c
ryst
alli
nit
y c
har
acte
riza
tio
n n
ot
per
form
ed
Pea
t et
al.
, 19
59
Co
nst
an
tin
ea s
ubu
life
ra
Od
on
thali
a f
locc
osa
Am
ylo
pec
tin
, no
am
ylo
se
B-t
yp
e gra
nu
le c
ryst
alli
nit
y
Mee
use
et
al.,
19
59
Fli
nti
nie
lla
san
gu
ina
ria
Am
ylo
pec
tin
e +
am
ylo
se
Gra
nu
le c
ryst
alli
nit
y c
har
acte
riza
tio
n n
ot
per
form
ed
McC
rack
en &
Cai
n,
198
1
Gra
cila
riop
sis
lem
an
efo
rmis
A
mylo
pec
tin
, no
am
ylo
se
C-t
yp
e gra
nu
le c
ryst
alli
nit
y
Yu
et
al.,
200
2
Po
rph
yrid
ium
pu
rpu
reu
m
Sem
i-am
ylo
pec
tin
+ a
mylo
se
A-t
yp
e gra
nu
le c
ryst
alli
nit
y
Sh
imo
nag
a et
al.
, 2
00
7
Rh
od
oso
rus
ma
rin
us
Sem
i-am
ylo
pec
tin
+ a
mylo
se
Lo
w A
-typ
e gra
nu
le c
ryst
alli
nit
y
Sh
imo
nag
a et
al.
, 2
00
8
Rh
od
ella
vio
lace
a
Sem
i-am
ylo
pec
tin
+ a
mylo
se
B-t
yp
e gra
nu
le c
ryst
alli
nit
y
Po
rph
yrid
ium
so
rdid
um
M
ore
bra
nch
ed s
emi-
amylo
pec
tin
+am
ylo
se
Tw
o k
ind
s o
f gra
nu
le p
opu
lati
on
s w
ith
lo
w a
nd
hig
h A
-typ
e cr
yst
alli
nit
y
Cya
nid
iosc
hyz
on
mer
ola
e
Sem
i-am
ylo
pec
tin
, no
am
ylo
se
Lo
w A
-typ
e gra
nu
le c
ryst
alli
nit
y
Hir
abar
u e
t al
. 2
01
0
Cya
nid
ium
ca
lda
riu
m
Ga
ldie
ria s
ulp
hu
rari
a
Gly
cogen
No
gra
nu
le f
orm
atio
n
Sh
imo
nag
a et
al.
, 2
00
7
Sh
imo
nag
a et
al.
, 2
00
8
Ga
ldie
ria m
axi
ma
Hig
hly
bra
nch
ed g
lyco
gen
No
gra
nu
le f
orm
atio
n
Sta
dn
ich
uk e
t al
., 2
00
7
Tab
le 3
. V
aria
tion
in
th
e st
ruct
ure
of
the
sto
rage
po
lysa
cch
arid
e in
red
alg
ae.
Introduction
25
3.2.4. Properties and applications of highly branched glucose polymers
Among the wide variety of derivatives produced from starch (summarized in
section 3.1) we find the highly branched glucose polymers. These polymers can
be produced by treatment of the starch molecule with enzymes such as glycogen
branching enzyme, amylase, amyloglucosidase or glucanotransferase in order to
increase the branching density through hydrolysis of α-(14) linkages and/or
the creation of new α-(16) linkages. These features confer certain properties
to the highly branched glucose polymers that are advantageous compared to
those from starch. Because the semi-crystalline cluster structure of starch is
disrupted and the side chains are shortened, these polymers are readily soluble
in water and show slow or no retrogradation (Kim et al., 2008; Li et al., 2016).
The increase in the proportion of branching points makes these polymers more
resistant to digestive enzymes such as α-amylases and glucosidases, which
degrade the α-(16) linkages at a lower rate than the α-(14) linkages, and
turns them into slowly digestible carbohydrates (Lee et al., 2007; Ao et al.,
2007; Lee et al., 2013).
Two applications for which branched glucose polymers are already being
designed are in the formulation of peritoneal dialysis solutions and as ingredient
for sports drinks/foods (Backer et al., 2005; Fuertes et al., 2009). Peritoneal
dialysis is the introduction of a hypertonic solution in the peritoneal cavity of a
patient suffering from kidney failure in order to drain excess water and toxins
from the blood. Generally, glucose is employed as osmotic agent in the
preparation of these hypertonic solutions, since it is safe and can be completely
metabolized by the organism. However, the use of glucose as osmotic agent is
not effective when the treatment needs to last for long periods of time because
glucose can easily cross the peritoneal membrane and gets assimilated into the
bloodstream, therefore reducing the osmotic gradient inside the peritoneal
cavity. Branched glucose polymers represent a type of osmotic agent alternative
to glucose that is not easily absorbed into the bloodstream, since polymers
cannot cross the peritoneal membrane, therefore creating a long-lasting osmotic
gradient. Despite its polymeric nature, the branched glucose polymer is able to
create osmotic pressure and induce water filtration through the peritoneum by a
phenomenon known as colloid osmosis (Mistry et al., 1993). The company
Baxter Healthcare (USA) commercializes a peritoneal dialysis solution named
Extraneal® that contains the so-called icodextrin, a mixture of glucose polymers
produced from fractionation of hydrolyzed corn starch with a proportion of α-
Chapter 1
26
(16) linkages < 10% and weight-average molecular weight (Mw) of 1.3 -
1.9×104 Da (Moberly et al., 2002). A patent has been filed on the obtention of
glucose polymers for peritoneal dialysis with a much higher proportion of α-
(16) linkages (between 20-30%) via amylogluosidase treatment of starch
amylopectin. This higher degree of branching can improve their performance as
osmotic agents by making them more resistant to digestive enzymes, inducing a
lower glucose release into the bloodstream (Deremaux et al., 2013).
When carbohydrates are used as an ingredient in sports drinks, they should
delay the onset of fatigue by replenishing depleted body reserves and, at the
same time, allow fast fluid absorption from the stomach into the small intestine
to counteract dehydration (Maughan, 1998). Branched glucose polymers
represent a more optimal energy source than glucose or short linear
oligosaccharides because they are more slowly degraded by digestive enzymes
due to their high proportion of α-(16) linkages, leading to a more gradual
glucose appearance in the bloodstream and a lower insulin response (Takii, et
al., 1999). Because of their high molecular weight, branched glucose polymers
have a negligible contribution to the osmotic value of the solution even at high
carbohydrate concentrations, and can be combined with essential electrolytes to
produce hypotonic sports drinks with the optimum osmolality value to achieve
fast gastric emptying (Takii et al. 2005). The company Glico (Japan) produces a
highly branched cyclic dextrin with the tradename Cluster Dextrin® which is
generated by the cyclization reaction of a branching enzyme on corn
amylopectin. This cyclic dextrin is widely commercialized as ingredient for
sports meals as a quickly absorbed but slowly metabolized carbohydrate that
provides a constant supply of energy during exercise.
3.3. Floridoside, a compatible solute in G. sulphuraria
3.3.1. Compatible soultes
Compatible solutes are low molecular weight organic compounds accumulated
by most organisms (not necessarily extremophiles) in order to cope with
changes in water activity due to an increase in external osmotic pressure,
desiccation, freezing or high temperatures. The term ‘compatible’ derives from
the fact that these compounds can be accumulated to high intracellular
concentrations without disturbing cell metabolism (Brown, 1978) because they
are highly soluble in water and they stabilize the native conformation of
Introduction
27
proteins by preferential exclusion or direct interaction, depending on the solute
in question (Roberts, 2005). The different molecules that can act as compatible
solutes in nature can be divided in a few categories: betaines and ectoines,
amino acids and derivatives, carbohydrates and derivatives, and polyols (da
Costa et al., 1998). Certain compatible solutes are not restricted to specific
species but instead they are present in different taxonomic groups.
The category of carbohydrates and derivatives includes the non-reducing
disaccharides sucrose and trehalose and the glycosides glucosylglycerol,
galactosylglycerol, mannosylglyceramide and their negatively charged
derivatives, glucosylglycerate and mannosylglycerate. Trehalose and sucrose
are widely spread compounds accumulated by numerous organisms under
several stress conditions that not only act as compatible solutes but also fulfill
other major roles inside the cells (Elbein et al., 2003 ; Wind et al., 2010). The
glycosides that act as compatible solutes are compounds formed by a
monosaccharide unit (glucose, galactose or mannose) linked through the
anomeric carbon to a molecule of glycerol or a glycerol derivative (glycerate,
glyceramide). The use of glycosides as compatible solutes has some advantages
for the cells with respect to the use of either glycerol or a monosaccharide
alone. Glycerol is a powerful osmoregulator and it is effectively accumulated in
response to salt stress by yeast and some cyanobacteria and algae, but it can
easily diffuse through membranes, making its intracellular retention challenging
for the cells (Nevoigt & Stahl, 1997). Carbohydrates are able to stabilize
cellular membranes under low water activity due to the interaction of their
hydroxyl groups with the polar head of the lipids (Crowe et al., 1987), but the
reducing nature of monosaccharides limits its accumulation to high intracellular
concentrations. The structural conformation of the glycosides allows to combine
the advantages of both glycerol and carbohydrates as compatible solutes at the
same time that it circumvents the disadvantages (Hagemann & Pade, 2015).
Glucosylglycerol (2-O-α-D-glucopyranosylglycerol) (Fig. 3) is the most widely
spread glycoside used as compatible solute and it is accumulated by several
moderately halotolerant cyanobacteria and some species of bacteria (Roberts,
2005; Hagemann, 2011). Mannosylglyceramide, and the negatively charged
glycosides mannosylglycerate and glucosylglycerate are mainly found in
thermophilic and hyperthermophilic bacteria (Santos & da Costa, 2002;
Empadinhas & da Costa, 2006).
Chapter 1
28
3.3.2. Physiological roles of floridoside
Floridoside is the trivial name commonly used in literature for
galactosylglycerol (2-O-α-D-galactopyranosylglycerol) (Fig. 3), the main low
molecular weight compound in all red algae species except the members of the
order Ceramiales, which contain mannosylglycerate (also known by the name
digeneaside) (Kremer, 1978).
Floridoside was discovered in 1930 (Colin & Gueguen, 1930) and was first
described as being the primary photosynthetic product in red algae, following a
rapid labelling with 14
C during photosynthetic carbon assimilation (Bean &
Hassid, 1955). Later studies have supported this conclusion and have suggested
a role for floridoside as a dynamic carbon pool in red algae, in which
photosynthetically fixed carbon is transiently stored before being assimilated
into other cellular macromolecules such as the storage polysaccharide (floridean
starch) or the cell wall polysaccharide (Li et al., 2001; Li et al., 2002). An
isomeric form of floridoside, denominated isofloridoside (1-O-α-D-
galactopyranosylglycerol), has also been shown to originate as photosynthetic
product although in minor quantities, suggesting a non-equivalent metabolic
role for both glycosides despite their structural similarity (Craigie et al., 1968).
The role of floridoside as osmoregulator in red algae was first suggested by
Kauss upon observation that the 14
C labelling of this heteroside could be altered
under different salinity conditions (Kauss, 1968). This role was confirmed in
later works with several different red algae species, which showed a significant
relationship between the increase in floridoside content and the increase in
external osmotic pressure (Kirst & Bisson, 1979; Reed, 1985; Pade et al., 2015),
independently whether the solute causing that osmotic pressure was ionic or
non-ionic (Reed et al., 1980).
Because floridoside accumulation did not seem to produce complete recovery of
cell volume and turgor under hypersaline conditions, it was proposed that the
role of this heteroside was not only to regulate the osmotic potential inside the
cells but also to protect the enzymatic machinery, thus acting as a compatible
solute. Once more, the authors of these works also described the presence of
isofloridoside in osmotically stressed red algae cells, but both the low absolute
content and the slight changes in its concentration under hyperosmotic
conditions proved that this form of galactosylglycerol does not have a major
Introduction
29
role in osmotic adaptation like floridoside, although some exceptions exists
(Karsten et al., 1993). The increase in floridoside content in osmotically stressed
cells has been observed under conditions where photosynthetic activity is
significantly reduced, suggesting that this glycoside can be synthesized using
precursors derived from the degradation of the storage polysaccharide (floridean
starch) and not only from newly fixed carbon (Reed, 1985; Ekman et al., 1991;
Bondu et al., 2009).
The presence of floridoside in G. sulphuraria and the other Cyanidiales was
reported in 1983, and was used as an extra feature supporting the classification
of this particular microalgae as Rhodophytes (De Luca & Moretti, 1983).
Thanks to the availability of G. sulphuraria genome sequence, the enzymes
responsible for floridoside and isofloidoside synthesis in this microalga have
been identified (Pade et al., 2015). The authors confirmed that the two
glycosides are synthesized by independent enzymes and not by isomerization of
floridoside into isofloridoside, which had been previously suggested as one of
the options to explain the formation of this isomer (Meng & Srivastava, 1991).
The two enzymes use UDP-galactose and glycerol-3-phosphate as substrates to
synthesize a phosphorylated glycoside intermediate ((iso)floridoside-P) that is
later dephosphorylated to yield the final structure. In G. sulphuraria this
reaction can be performed in a single step since the two enzymes display both
synthase and phosphatase activity. In addition, the authors reported the presence
of the carbohydrate binding domain CBM20 in the protein sequence of the
enzyme responsible for isofloridoside synthesis. This domain would enable the
binding of the enzyme to the storage polysaccharide, which could facilitate the
availability of precursors for isofloridoside synthesis. However the enzyme
Figure 3. Structure of 2-O-α-D-galactopyranosylglycerol (floridoside)
and 2-O-α-D-glucopyranosylglycerol (glucosylglycerol).
Chapter 1
30
responsible for floridoside synthesis did not contain such domain, which
suggests that the presence of CBM20 is not essential for the production of
floridoside and isofloridoside from precursors derived from the degradation of
the storage polysaccharide in G. sulphuraria.
3.3.3. Properties and potential applications of floridoside
Floridoside has been the subject of several research works because, apart from
its in vivo role as transient carbon storage and compatible solute in red algae
cells, it possesses certain properties that make it attractive for application in
several fields.
In a work performed by Hellio and colleagues, floridoside was shown to possess
antifouling activity towards larvae of the marine barnacle Balanus amphitrite
(Hellio et al., 2004). Biofouling is the attachment and development of
organisms on the surface of underwater devices, such as the hull of ships and
oceanic measurement equipment, and it is a problem for the marine industry
which needs to invest large amounts of money in the prevention and cleaning of
fouled organisms (Callow & Callow, 2002). Current antifouling strategies
involve the use of biocide-based paints that can accumulate in coastal areas to
concentrations that are harmful for non-fouling marine organisms, so
considerable efforts are being made for the identification of environmentally
friendly antifouling agents by analyzing compounds extracted from the marine
organisms themselves (Qian et al., 2010). Even though floridoside showed
promising properties as antifouling compound, the authors admitted certain
limitations, such as lower levels of activity compared to other natural
antifouling agents. Nonetheless, these limitations could be overcome by
structure-activity analysis and the development of analogue molecules.
Different works have dealt with the characterization of floridoside as potential
therapeutic agent. Due to its ability to scavenge reactive oxygen species (Li et
al., 2009), floridoside was shown to inhibit the inflammatory response of
uncontrollably activated microglia cells, a phenomenon that is linked to the
development of various neurological diseases (Kim et al., 2013). The terminal
α-galactose in its structure allows floridoside to be recognized and bound by
antibodies involved in the classic complement pathway, suggesting its potential
use for immune system depletion during organ transplantation therapies
Introduction
31
(Courtois et al., 2008). Another study has suggested a possible application of
floridoside in bone formation treatments since this glycoside can enhance the
production of osteogenic differentiation markers in murine bone marrow cells
(Ryu et al., 2015).
Because floridoside shares structural similarity with glucosylglycerol (Fig. 3),
the most common compatible solute accumulated by moderately halotolerant
cyanobacteria, it is tempting to think that both glycosides might be functional in
the same type of applications. The main application developed so far for
glucosylglycerol is as ingredient in cosmetic and healthcare products, with
several patent applications related to this matter. Glucosylglycerol can enhance
the expression of cell protective enzymes, such as superoxide dismutase, and
therefore can be used in antiaging cosmetic products for protecting skin cells
from external aggressions (Klein et al., 2011). The moisturizing properties of
glucosylglycerol also contribute to its suitability as cosmetic ingredient (Thiem
et al., 1999), and a patent has been filed on the optimum composition of a
preparation containing glucosylglycerol which displays long-term stability
without appearance of microbial contamination (Schwarz & Klein, 2011).
Currently, glucosylglycerol is industrially produced by the German company
bitop AG via a patented enzymatic process catalyzed by sucrose phosphorylase
using sucrose and glycerol as substrates (Goedl et al., 2009). The product is
trademarked as Glycoin® and distributed as cosmetic ingredient by Jan Dekker
International under the name Glycoin Extremium®. Apart from these cosmetic-
related properties, glucosylglycerol has also attracted interest as potential
enzyme stabilizer (Sawangwan et al., 2010) and non-cariogenic, low calorie
sweetener (Takenaka & Uchiyama, 2000).
Compared to glucosylglycerol, the development of possible industrial
applications for floridoside is still in its infancy. In order to achieve progress in
this field, the obtention of sufficient amounts of floridoside for the analysis of
its properties would be a necessary first step. Enzymatic production of
floridoside has not been reported yet and chemical synthesis does not represent
the most desirable method, since yields are still insufficient and the process
requires a long sequence of steps to direct the reaction towards the
stereochemically pure product (Weïwer et al. 2008). Extraction of floridoside
from the natural producers (i.e. red algae) could represent a promising
alternative but requires screening of optimum species for production and
optimization of the cultivation and extraction techniques.
Chapter 1
32
4. Scope of the thesis
Galdieria sulphuraria is an ancient red microalga with the potential to become
an important name in the field of microalgal biotechnology. The ability of G.
sulphuraria to grow heterotrophically on a wide range of carbon sources under
acidic conditions (Gross & Scharrenberger, 1995) represents an advantageous
feature that can facilitate its mass cultivation. Being a microalga, G. sulphuraria
represents an interesting source of high-value products such as pigments
(Graveholt et al., 2007) and its extremophilic nature opens the door for its use in
many applications, such as e.g biosorption of metals in waste waters (Ju et
al.,2016). Furthermore, the complete genome sequence of G. sulphuraria is
already available (Schönknecht et al., 2013), providing a powerful tool that
could help in engineering this microalga to become an industrially relevant
species. Although no genetic transformation methods are developed yet for G.
sulphuraria, progress is being made with the related species C. merolae
(Minoda et al., 2004; Fujiwara et al., 2013). The identification of novel
interesting compounds from G. sulphuraria will also contribute to exploit its
biotechnological potential. This PhD thesis deals with the characterization of
two carbohydrate compounds with biotechnological prospects synthesized by G.
sulphuraria - its highly branched glycogen and floridoside - and the growth
conditions under which their accumulation is optimal.
Chapter 1 is an introductory chapter that provides some background
information on the physiological traits that contribute to the extremophilic
nature of G. sulphuraria and on the two types of carbohydrates that are the
subject of this thesis. A short overview of the importance of microalgae
biotechnology in the frame of a bio-based economy and of industrially relevant
carbohydrates is also included.
Chapter 2 reports the structural characterization of the glycogen accumulated
as storage polysaccharide by G. sulphuraria and the comparison with other
prokaryotic and eukaryotic glycogens. The results reveal that G. sulphuraria
glycogen differs from other glycogens because it possesses an unusually high
degree of branching - 18% of α-(16) linkages, the highest described to date -,
it is entirely composed of short chains (DP≤10) and it has a substantially
smaller molecular weight and particle size. The physiological role of this highly
branched glycogen in G. sulphuraria is also discussed.
Introduction
33
Chapter 3 analyzes the structure-properties relationship of the highly branched
glycogen of G. sulphuraria and a hyper-branched polymer produced from it via
enzymatic treatment, in terms of susceptibility to digestive enzymes, osmolality
and viscosity. Two branched polymers derived from potato starch are used for
comparison in order to evaluate the potential of G. sulphuraria glycogen as an
alternative substrate for the production of highly branched glucose polymers
with application in e.g. formulation of peritoneal dialysis solutions and sports
drinks. The results show that G. sulphuraria glycogen and the hyper-branched
polymer enzymatically produced from it display higher resistance to digestive
enzymes and a significantly decreased viscosity in solution compared to
polymers derived from starch, properties conferred by their shorter side chains
and higher branch density.
Chapter 4 describes the production of the compatible solute floridoside by G.
sulphuraria under different growth and osmotic stress conditions. This
glycoside has attracted attention for its potential antifouling and therapeutic
properties but research on industrial applications is hampered by limited
compound availability. Because a high-yielding production process for
floridoside has yet to be developed, the optimum conditions for the
accumulation of this glycoside in G. sulphuraria were explored to assess the
feasibility of using this microalga as a possible source for floridoside. The
results confirm the tolerance of G. sulphuraria to high salinity conditions and
reveal that an hyper-osmotic shock maintained for 24 h results in higher
floridoside accumulation than growing the cells in the presence of the osmotic
stress-causing agent. Among several parameters tested, the use of glycerol as
carbon source for cell growth has the most significant impact on floridoside
accumulation, which reached a maximum of 56.8 mg/g dry biomass.
Chapter 5 investigates the influence of media composition (level of nitrogen
supply and type of carbon source) and growth conditions on biomass, glycogen
and floridoside accumulation by G. sulphuraria grown under heterotrophic
conditions. Additionally, the effect of different extraction methods on
floridoside yields were also analyzed. Results demonstrate that G. sulphuraria
does not require growth-limiting conditions for the accumulation of high
amounts of glycogen (30-50% of the dry cell weight) and thus it is possible to
obtain high glycogen yields through the optimization of biomass yields. A first
attempt to cultivate G. sulphuraria in a 7L-scale bioreactor using a mineral
medium with high supply of carbon and nitrogen showed that high cell densities
Chapter 1
34
can be obtained by batch cultivation, even when using crude glycerol obtained
from the production of biodiesel as carbon source, and suggests that further
improvement of biomass productivity can be achieved if the media composition
is more finely adjusted. Floridoside yields obtained by ethanolic extraction or
by mechanical disruption of the cells are very similar and thus the advantages
and disadvantages of each method are discussed.
Chapter 6 discusses the results from the previous chapters in the light of the
available literature.
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