University of Groningen

Functional carbohydrates from the red microalga Galdieria sulphurariaMartínez García, Marta

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:2017

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):Martínez García, M. (2017). Functional carbohydrates from the red microalga Galdieria sulphuraria.[Groningen]: University of Groningen.

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 19-06-2020

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.