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Psychrophilic algae as candidates for outdoor bioreactors in cold countries

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Freshwater psychrophilic algae are promising candidates for usage in outdoor bioreactors of colder countries. Species live in extreme places like long lasting snowfields or glacier surfaces in alpine or polar regions. The photosynthetic performance and thus growth is optimised to temperatures below 20°C. A further aspect of extremophilic algae are specific metabolites. Due to the adaptation to their natural habitat, many species accumulate certain compounds in high concentrations. For example, protective pigments like secondary carotenoids (e.g. astaxanthin) or polyphenolic compounds (e.g. gallic acid derivatives) can occur. Intracellular anti-freezing agents like sugar-alcohols (" compatible solutes) are usually present. Not least, the level of antioxidatives like alpha-tocopherol can be significantly raised. Up to date, the applied use of psychrophilic algae is still limited, however culture collections have more and more according species in stock, as isolation of new strains from wilderness is still going on.
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PSYCHROPHILIC ALGAE AS CANDIDATES FOR OUTDOOR BIOREACTORS IN COLD COUNTRIES
Remias D., Kahr H., Jäger A.
University of Applied Sciences Upper Austria
Stelzhamerstr. 23, A-4600 Wels, Austria
Tel.: +43 (0)50804-44463
Fax.: +43 (0)50804-43166
E-mail: daniel.remias@fh-wels.at
ABSTRACT: Freshwater psychrophilic algae are promising candidates for usage in outdoor bioreactors of colder countries.
Species live in extreme places like long lasting snowfields or glacier surfaces in alpine or polar regions. The photosynthetic
performance and thus growth is optimised to temperatures below 20°C. A further aspect of extremophilic algae are specific
metabolites. Due to the adaptation to their natural habitat, many species accumulate certain compounds in high
concentrations. For example, protective pigments like secondary carotenoids (e.g. astaxanthin) or polyphenolic compounds
(e.g. gallic acid derivatives) can occur. Intracellular anti-freezing agents like sugar-alcohols (“compatible solutes) are usually
present. Not least, the level of antioxidatives like alpha-tocopherol can be significantly raised. Up to date, the applied use of
psychrophilic algae is still limited, however culture collections have more and more according species in stock, as isolation of
new strains from wilderness is still going on.
Keywords: algae, biomass, ecology, psychrophiles
1 INTRODUCTION
In regions with pronounced seasons like northern
Europe, the use of conventional microalgae in outdoor
bioreactors as energy source is limited during wintertime.
From October to March, the commonly used strains show
hardly any growth due to low temperatures. In this paper,
we propose freshwater psychrophilic algae as potential
alternative for biomass production in colder countries.
Compared to mesophiles (algae that preferably grow
around 20°C), temperature optima of psychrophilic
enzymes are significantly lowered. As a consequence,
primary production and thus biomass increase of cold-
adapted species behave the same way. Up to date, the
applied use of psychrophilic algae is still limited, and
only few culture collections have according species in
stock. Two main reasons for this situation have to be
considered. First, microalgae collected from natural snow
and ice habitats are usually found as spore stages. Such
dormant cells frequently do not divide when transferred
to culture media. Second, cultures have to be maintained
permanently at low temperatures (e.g. below 5°C),
because many true psychrophiles are subject to lethal
stress when exposed to mesophilic conditions for a longer
period of time.
The natural habitats of snow and freshwater ice algae
are harsh due to low temperatures, low level of nutrients
and/or high solar irradiation (VIS and UV region). The
cellular adaptation to cope with these factors includes the
accumulation of protecting compounds in high
concentrations, which may also be of biotechnological
interest [1].
2 MATERIAL AND METHODS
2.1 Source of microalgae
Field material of Chlamydomonas cf. nivalis
(Volvocales, Chlorophyta) was sampled in the European
Alps, province Tyrol, district Imst, Austria, where they
cause monospecific blooms during summer (Fig. 1). The
presence of algae (red spherical spores with a typical
diameter of approximately 15 µm) in snow fields with
red coloration at elevations of about 2400 m above sea
level was checked with a portable microscope (400 times
magnification). Red snow was harvested with a stainless
steel shovel into buckets and then covered with
aluminum foil. The snow was melted gently at 4°C and
the meltwater sieved with stainless steel sieves with 800,
400, 200 and 100 µm mesh aperture in a row to remove
any non-algal contaminants.
Figure 1: Natural red snow phenomenon caused by
psychrophilic microalgae during alpine summer.
Although they are green algae in a taxonomic point of
view, the cellular appearance is reddish, because the
chlorophylls are masked by secondary carotenoids.
2.2 Measurement of photosynthesis
Growth was measured as a function of short-term cell
respiration during darkness and oxygen evolution
(photosynthetic performance) at different light and
temperature levels. These values were normalized to the
amount of chlorophyll per sample. The freshly collected
microalgae were put into a thermostated 3 ml transparent
polyacry chamber, and the liquid was stirred permanently
during measurement. The oxygen content was logged
with a fluorescence-optic sensor (PreSens Fibox 3,
Regensburg, Germany). Further details about the setup
are described in [2].
23rd European Biomass Conference and Exhibition, 1-4 June 2015, Vienna, Austria
1911
3 RESULTS AND DISCUSSION
3.1 Photosynthetic optima for biomass generation
The photosynthetic performance of Chlamydomonas
cf. nivalis at different light- and temperature levels can be
seen in Fig. 2. Its shows that this organism can perform
well at quite different temperature levels from 2 to 10°C,
and may tolerate 20°C for a certain extent of time. This
goes along with a tolerance of a broad range of
irradiation up to 1800 µmol photons m
-2
s
-1
, which is
about full sunlight (no photoinhibition takes place). This
is in accordance to the ecology of this alga, which is
frequently exposed to high light conditions at the snow
surface.
Figure 2: Temperature and light dependent oxygen
turnover of Chlamydomonas cf. nivalis causing red snow
in the Alps. At darkness (y-axis: 0 µmol photons m
-2
s
-1
)
cell respiration was measured. At low light conditions
(below 50 µmol) best performance was at 2°C, followed
by 5°C. However under high light, best results were
achieved at 10°C. At 25°C, respiration was significantly
increased and photosynthetic performance was the
poorest.
3.2 Secondary metabolites of snow algae
Like many species found in snow, the green alga
Chloromonas cf. nivalis produces the protective
xanthophyll astaxanthin, a secondary keto-carotenoid (a
apoloar pigment causing the cells to become red) [2].
Cultures of algae initially isolated from snow, exposed to
elevated light and low nitrogen, show an increased
content of primary carotenoids, and some also elevated
levels of α-tocopherol [3]. In the lag-phase, several
strains of snow algae were demonstrated to accumulate
(poly-) unsaturated fatty acids in high ranges of 50 to 300
mg FA g C
−1
[4]. Streptophytic green algae, which are
taxonomically closer to the land plants (e.g.
Zygnematophyceae), produce (poly-) phenolic
compounds, such as glycolised derivatives of gallic acid
[5].
Moreover, cytosolic anti-freezing agents like glycerol
and sugar-alcohols have been detected. Recently, the
secretion of ice-binding proteins to prevent harmful
extracellular ice crystal formation has been described [6].
Two examples for abundant secondary pigments in
psychrophiles are given. First, the red snow alga
Chlamydomonas cf. nivalis has a modified secondary
pigment, which occurs as diglycoside ester of
astaxanthin, including and one fatty acid residue [7].
Second, a phenolic brownish compound (purpurogallin
carboxylic acid-6-O-b-D-glucopyranoside) in the glacial
ice alga Mesotaenium berggrenii [5], which probably
also occurs in Ancylonema nordenskioldii found at arctic
glaciers.
All these pigments have a broad spectral absorbance
in the UV A & B as well as VIS range, and are discussed
as natural source for skin protection agents.
3.3 Applied aspects
A main open task is to successfully culturing further
species, because many psychrophiles (especially spore
stages) refuse to grow when transferred to common media.
Maybe diluted media are needed, simulating low ion
concentrations that are common in snow meltwater, in
combination with adequate culture temperatures (>5°C).
Once spores germinate and sufficient biomass is achieved,
a depletion of the media’s nutrients can stimulate the
production of secondary metabolites before harvest, if
desired [3].
So far, one scientific institution in known to focus
exclusively on isolation and cultivation of eukaryotic
cryoflora (CCCryo at Fraunhofer IZI-BB, Potsdam,
Germany), and the strains can be ordered online [8].
4 REFERENCES
[1] P. Varshney, P. Mikulic, A. Vonshak, J. Beardall,
P.P. Wangikar, Extremophilic micro-algae and their
potential contribution in biotechnology. Bioresource
Technology (2015) 184: 363-372.
[2] D. Remias, U. Karsten, C. Lütz, T. Leya, T.,
Physiological and morphological processes in the
Alpine snow alga Chloromonas nivalis
(Chlorophyceae) during cyst formation. Protoplasma
(2010) 243: 73-86.
[3] T. Leya, A. Rahn, C. Lütz, D. Remias, Response of
arctic snow and permafrost algae to high light and
nitrogen stress by changes in pigment composition
and applied aspects for biotechnology. FEMS
Microbiology Ecology (2009) 67: 432-443.
[4] E. Spijkerman, A. Wacker, G. Weithoff, T. Leya,
Elemental and fatty acid composition of snow algae
in Arctic habitats. Frontiers in Microbiology (2012)
3: 380.
[5] D. Remias, S. Schwaiger, S. Aigner, T. Leya, H.
Stuppner, C. Lütz, Characterization of an UV- and
VIS-absorbing, purpurogallin-derived secondary
pigment new to algae and highly abundant in
Mesotaenium berggrenii (Zygnematophyceae,
Chlorophyta), an extremophyte living on glaciers.
FEMS Microbiology Ecology (2012) 79: 638-648.
[6] J.A. Raymond, The ice-binding proteins of a snow
alga, Chloromonas brevispina: probable acquisition
by horizontal gene transfer. Extremophiles (2014) 18:
987-994.
[7] T. Řezanka, L. Nedbalová, I. Kolouchová, K. Sigler,
LC–MS/APCI identification of glucoside esters and
diesters of astaxanthin from the snow alga
Chlamydomonas nivalis including their optical
stereoisomers. Phytochemistry (2013) 88: 34-42.
[8] T. Leya, A. Broedel, D. Connor, F. Jorde, D. Wenzel,
CCCryo - culture collection of cryophilic algae: a
bioresource for industrially relevant metabolites.
European journal of phycology (2011) 46: 83-84.
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