International Conference on Geothermal Energy 1

Applications in Agriculture, May 2004, Athens, Greece

Mass culture of the microalga Spirulina using geothermal fluids in Greece -

Antioxidant activities of Spirulina powder extracts

A. Arvanitis
Institute of Geological and Mineral Exploration, Reg. Dept. of C. Macedonia, Thessaloniki, Greece

T. Sotiroudis
Insitute of Biological Research & Biotechnology, The National Hellenic Research Foundation, Ath-
ens, Greece

E. Nerantzis
Technological Educational Institution of Athens, Faculty of Food Science and Nutrition, Laboratory
of Biotechnology and Industrial Fermentations, Athens, Greece

S. Fournadzhieva
Institute of Plant Physiology, Bulgarian Academy of Sciences, Sofia, Bulgaria

E. Koultsiakis
ALGAE A.C., Therma - Nigrita, Greece

ABSTRACT tivities. Thus this microalgae food product of-

fers valuable nutritional antioxidant components
Spirulina sp. has a long history of use as food
with potential benefits in human health.
because of its rich content of protein, vitamins,
essential amino acids and fatty acids and a host
of other phytochemicals that have potential 1. INTRODUCTION
health benefits. It can also be used as protein
Spirulina, now named Arthrospira, is a micro-
supplement in feeds for poultry, cattle, pig and
scopic and filamentous cyanobacterium (blue-
aquaculture, as food colourant and in cosmetics.
green alga) found in the highly alkaline lakes of
A cost reduction application of geothermal flu-
Africa and Mexico. Its name derives from the
ids for commercial production of the microalga
spiral or helical nature of its filaments. The na-
cyanobacterium Spirulina sp. (Arthrospira) in
tives of these places have been using Spirulina
Nigrita-Serres, is presented. The microalgal cul-
as part of their diet for centuries. Today,
tivation is based on a photosynthetic process us-
Spirulina cultivation is becoming a worldwide
ing sunlight, CO2 and nutrition elements con-
phenomenon owing to its extraordinary nutri-
tained in a fresh water culture medium heated
by geothermal waters. The geothermal energy tional qualities. Interest in Spirulina focuses
mainly on its rich content of protein, vitamins,
used in this way as well as setting up the culti-
essential amino acids, minerals and essential
vation ponds in a greenhouse contribute to in-
fatty acids. Spirulina is 60-70% protein by
creasing the daily yield by 20-30% (spring and
weight and contains a rich source of vitamins,
autumn), and the cultivation season is prolonged
especially vitamin B12 and provitamin A (-
(from March until November). The use of freely
carotene), and minerals, especially iron. It is one
released geothermal CO2 reduces the production
of the few sources of dietary -linolenic acid
costs of Spirulina biomass by over 25%. Ex-
and it also contains a host of other phytochemi-
tracts of Spirulina powder produced by ALGAE
cals that have potential health benefits. The
A.C., Therma, Nigrita, contain sulphated poly-
biliprotein phycocyanin, represents a major pro-
saccharides and polyphenols, which express
tein of Spirulina, which is not only an effective
strong antioxidant/free radical scavenging ac-
natural blue colorant, but also a compound with
2 International Conference on Geothermal Energy
Applications in Agriculture, May 2004, Athens, Greece
strong antioxidant and a free radical scavenging
capacities, beneficial to the health of the con-
sumer (Belay, 2002). Recently, a sulfated poly-
saccharide named calcium spirulan, was isolated
from Spirulina platensis and was proved to ex-
press antiviral activity (Hayashi and Hayashi,
1996). Moreover, despite the few human studies
done so far on the health benefits of spirulina,
the evidence for its potential theurapeutic appli-
cation is overwhelming in the areas of immu-
nomodulation, anticancer, antiviral, antidiabetic,
antihypertensive and cholesterol-reduction ef-
fects as well as kidney and liver detoxification,
while its safety as food has been established
through centuries of human use and through
numerous toxicological studies (Belay, 2002).
World market evolution of Spirulina involves
among others: (a) protein supplements in feeds
for poultry, cattle, pig and aquaculture, (b)
products for the improvement of health of cats
and dogs (c) products for the improvement of
health, beauty and color of ornamental birds, (d)
tonic for horses, cows and breeding bools, (e)
natural colors for foods and cosmetics, (f) fluo-
rescent markers for medical tests and (g) en-
zymes for genetic research. Recently, several
pharmaceutical drug companies have become
interested in growing spirulina and other micro-
algae for extracts to be used in new pharmaceu-
tical drugs. Cultivation biotechnology at exist-
ing commercial farms and new photobioreactor
systems will produce these products (Henrikson,
2000).
The role of dietary antioxidants and their po-
tential benefits in health and disease have at-
tracted great attention (Kehler and Smith, 1994).
Although the occurrence of phenolic antioxidant
compounds in plants and plant foods is well
known (Sotiroudis et al., 2003) the antioxi-
dant/free radical scavenging characteristics of
Spirulina sp. are poorly known, except those
concerning the protein phycocyanin constituent
of the microalga (Romay et al., 1998). In this
respect, the evaluation of free radical scaveng-
ing capacities of Spirulina extracts produced
from a Spirulina commercial food product de-
veloped in Greece would be important.
Another reason accounting for the increasing
interest of the private sector for investments on
Spirulina production is that commercial
Spirulina can be normally produced in large
outdoors ponds under controlled conditions es-
tablished in desert environments normally not
used for agricultural purposes. Nevertheless, the
price per kilogram of Spirulina biomass is cur-
rently, high on account of the relatively large
investments, costliness of pure CO2, expensive
mineral nutrients, low conversion efficiency in
comparison with bacteria, seasonal character of
the production, etc. This necessitates the search
for technology optimization.
The geothermal field of Therma - Nigrita lies
at the central-western part of the Strymon basin,
at a distance of 25 km south of the Serres city in
Northern Greece (Macedonia), extending to an
area of 20 km2. It is the most exploited low en-
thalpy field in Greece. The geothermal waters
have temperatures up to 64oC and they come
from the basal conglomerates at depths of 100-
400 m. The geothermal anomaly in the area oc-
curs mainly to fault systems trending NE-SW
and NW-SE. The geochemical study proved that
the geothermal waters with TDS between 1.9
and 2.5 g/l are classified as Na-HCO3 type of
waters and they are rich in Na+, K+, Mg2+,
HCO3-, CI- and SiO2 contents. They contain
large amounts of dissolved gases (Arvanitis et
al., 1998). The dominant gas is CO2 (>97%
mol/mol). The presence of CO2 in combination
with the temperature and hydrostatic pressure
create a constantly artesian flow of geothermal
aquifers. Therefore, most wells exhibit artesian
flow between 80-150 tn/h, their wellhead pres-
sures range from 3 to 7 bar and two-phase flow
(H2O+CO2) during pumping. Thus, this geo-
thermal source of CO2 can be used for a drastic
reduction of the costs of Spirulina production in
Greece.
This paper focuses on the large-scale cultiva-
tion of Spirulina in Nigrita Serres and the re-
sults obtained in terms of technology optimiza-
tion and cost reduction through the use of geo-
thermal fluids. Moreover experimental findings
concerning important antioxidant properties of
the Spirulina product are presented.
2. EXPERIMENTAL
Spirulina powder was a product of ALGAE
A.C. Spirulina sulfated polysaccharide was ex-
tracted with boiling water and the extract was
treated with 10% trichloroacetic acid and dia-
lyzed against distilled water (Hayashi and Ha-
yashi, 1996). Algal sulfated polysaccharide con-
International Conference on Geothermal Energy 3
Applications in Agriculture, May 2004, Athens, Greece

centrations were determined with methylene

blue (Jiao and Lieu, 1999). Total sugars of the
polysaccharide were estimated by the phenol-
sulfuric acid assay with glucose standard (Du-
bois et al., 1956). The antioxidant activity of
polysaccharide and polyphenol extracts were
determined using N,N-dimethyl-p-phenylene-
diamine (DMPD) radical (Fogliano et al., 1999),
DPPH (2,2-diphenyl-1-picrylhydrazyl) radical
(Nenadis and Tsimidou, 2002) and ABTS (2,2-
azino-bis-(3-ethylbenzthiazoline-6sulfonic acid)
radical (Schlesier et al., 2002) assays. Determi-
nation of total phenolics was performed by the Figure 1: Location map of the Nigrita area with the sites
use of the Folin-Ciocalteau method. of the geothermal production well (Th-13b) and the plant
for cultivation of Spirulina.

3. RESULTS AND DISCUSSION Th-13 is presented in Table 1. The geothermal

waters contain small quantities of As (~0.5
3.1 Production of Spirulina
ppm) and Sr and they are not used directly for
Microalgal cultivation is based upon the logic of Spirulina cultivation. So, fresh water is used
the photosynthetic process: solar energy is used which is heated by the geothermal fluids using
for the synthesis of organic compounds out of simple heat exchangers, locally constructed. As
non-organic substances. The amount of micro- mentioned above, CO2 is the most abundant gas
algae produced depends mainly on the ge- in the geothermal field of Therma-Nigrita. The
nus/species, photoperiod and total amount of composition of the gases from the well Th-13 is
light, temperature, pH, rate of removal of cells presented in Table 2.
from the medium, turbulence and nutrient com- The installations are oval and made of con-
position of the medium, CO2-supply and others. crete. Their area totals 1,950 m2, split in instal-
These considerations have been taken into ac-
count in the creation and exploitation of the in- Table 1: Results of chemical analysis of geothermal water
from the well Th-13.
stallations in Nigrita. To optimize the process
Temperature (oC) 47.0
and reduce costs, the Nigrita base was set up
Conductivity (S/cm) 3490
near geothermal wells containing freely emitted
pH 6.6
CO2 (3-4 kg/tn of geoth. fluid). The cultivation a+ (mg/l) 620.7
parameters have been improved in accordance K+ ( -//- ) 78.2
with the qualities of the geothermal source, such Ca2+ ( -//- ) 148.3
as capacity, temperature, water composition, Mg2+ ( -//- ) 116.7
CO2 amount, etc. Fe ( -//- ) 0.03
The plant for cultivation of the microalga 2+
Mn ( -//- ) 0.042
Spirulina is located at a distance of 1.5 km north Li+ ( -//- ) 0.88
of the Therma village. For its energy require- Sr2+ ( -//- ) 0.79
ments, geothermal water with temperature of NH4+ ( -//- ) 0.28
51.2 C is used from a neighboring production CI- ( -//- ) 177.2
well (Th-13b), 300 m deep, at a distance of CO3- ( -//- ) 2252.6
about 500 m (Fig. 1). Until 2 years ago, the geo- CO32- ( -//- ) 0.00
thermal well Th-13, 372 deep, producing waters SO42- ( -//- ) 129.7
at 47oC was being used for the energy require- F- ( -//- ) 0.70
ments of the installations. Today the well Th-13 NO3- ( -//-) 1.5
is plugged with cement and sealed and certainly NO2- ( -//- ) 0.03
out of operation. The distance between the wells SiO2 ( -//- ) 85.0
Th-13 and Th-13b is about 50-60 m. The B ( -//- ) 3.42
chemical composition of the water from the well As ( -//- ) 0.50
4 International Conference on Geothermal Energy
Applications in Agriculture, May 2004, Athens, Greece
Table 2: Chemical composition (% by volume) of the
gases from the geothermal well Th-13.
CO2 98.70 %
N2 1.30 %
O2 ---
CH4 ---
H2S ~ 3 ppm
lations of various sizes in view of their gradual
inoculation (2 x 1.8 m2, 2 x 4.5 m2, 1 x 50 m2, 1
x 100 m2 and 8 x 225 m2) (Fig. 2).
The installations are located in a greenhouse
covered with French foil allowing for 80%
transparency. There is no doubt that covering
the cultivation installations is of crucial impor-
tance for the protection of the algal culture from
harmful weather conditions (rain, dust or others)
and, in particular, contributes for optimum heat-
ing effectiveness.
Optimization of photosynthesis was achieved
by supplying the inorganic carbon substrate
necessary for the photosynthesis (geothermal
CO2) and optimizing of the temperature-
radiation regime by heating with thermal en-
ergy. The mass algal cultivation is accompanied
by daily and seasonal changes in the tempera-
ture and radiation. The nutrition medium for the
cultivation of Spirulina in Nigrita by ALGAE
A.C. is based on the classical recipe (Aiba and
Ogawa, 1977) but has been modified, depending
on mineral salts prices and results on the growth
rate of Spirulina. High NaHCO3 content is of
vital importance for Spirulina to maintain a pH
between 9 and 10.5.
The overall cultivation process includes the
following stages: (a) museum algal culture of
Spirulina (extensive conditions), (b) inoculation
(intensification of the cultivation process), (c)
cultivation in production ponds (Fig. 2), (d)
Figure 2: ALGAE A.C. production pond.
Figure 3: Microscopic view of cultured Spirulina sp.
separation and (d) drying and packing. The
laboratory collection of different kinds of
Spirulina strains is maintained as liquid cultures
under suitable extensive conditions (illumina-
tion of approximately 2,000 lx, temperature 20-
22C). The algal culture is transferred from the
collection to an intensive cultivation installation
(bubbling with air containing 2% of CO2, t 34-
36C, illumination of 8,000 lx). As a result of its
growth and dilution with nutrition medium, sus-
pension volume continually increases up to
reaching the amount necessary for inoculation at
semi production installations (50 and 100 m2). It
is then transferred to the production ponds (225
m2) (Fig. 2). Pedal wheels do stirring. Velocity
of suspension circulation is 15 m/min. Suspen-
sion layer thickness is 8 10 cm (80 100
l/m2). Figure 3 shows a microscopic view of
Spirulina sp. cultured in the installations of
ALGAE A.C. in Nigrita.
The heating of the suspension is extremely
important for the growth of Spirulina and the
entire cultivation process, especially in spring
and autumn. Thus, the cultivation season is pro-
longed (from March until November) and the
daily yield increases by 20 to 30%. The heating
of the suspension is important for maintaining
an optimum night temperature and an effective
start of the cultivation day. Using geothermal
energy as well setting up installations for
Spirulina cultivation in greenhouse (covering
with plastic foil) contribute to optimizing the
temperature and radiation regime because the
effect of temperature and light on algal growth
are in correlation. According to data of Becker
and Venkataraman (1980), the CO2 supply ac-
counts for 27.4% of the algal cultivation ex-
International Conference on Geothermal Energy 5
Applications in Agriculture, May 2004, Athens, Greece

Table 3: Typical analysis of Spirulina biomass from AL- centrifugation has a high density. The biomass
GAE A.C., Nigrita, Greece. is then milled. A spray dryer is about to be im-
Minerals plemented. The biomass produced by Algae
General Analysis (%)
(per 10g) A.C. in Therma - Nigrita has an ISO9002 stan-
Proteins 54.5 Ca 58 mg dard, which is a guarantee for the quality of this
Carbohydrates 20.5 Fe 5 mg product. Typical analysis of Spirulina biomass
Lipids 9.4 Mg 51 mg is presented in Table 3.
Moisture 9.0 Zn 1.5 mg The average output of Spirulina biomass is
Minerals (Ash) 8.3 Se 8 mcg estimated to be 30-40 kg/day or 10-12 tn/year.
Vitamins Cu 26 mcg Annual output is trending upwards (4, 7 and 10
Vitamin A, (mg %) 0.1 Mn 0.4 mg tn/year for the last 3 years correspondingly) al-
Vitamin E, mg % 1.2 Cr 46 mcg though according to the first estimations an an-
Vitamin D3, mg % 0.6 K 146 mg
nual output of approximately 2000 kg/decare (1
-Carotene, mg % 80.75 Na 340 mg
decare = 0.2471 acres, 1 acre = 4046.86 m2) or 2
Vitamin B2, mg % 0.63 Ni Trace
kg/m2 was possible. The price of the produced
Pigments Hg Absent
<0.06
biomass as raw powder is about 40 /kg. This
Chlorophyll (%) 1.5 Pb
mg/kg price increases according to the process and
<0.02 packing.
Phycobiliproteins (%) 12.8 Cd The total cost of the investment was about
mg/kg
Carotenoids (%) 0.89 As Absent 700,000 in the year 1999 and it was partially
Pheophorbides (mg %) 0.7 financed (60% by the amount of 235,000 ) by
E.U. (LEADER II). The maximum time for am-
penses. Comparing this number with the low ortization is estimated to be 5 years.
cost when using geothermal CO2 supply in con- 3.2 Antioxidant properties of Spirulina powder
trolled amounts clearly emphasizes the eco-
nomic advantage of using the geothermal car- The antioxidant properties of Spirulina and its
bon dioxide source. The carbon dioxide from extracts have attracted the attention of research-
the geothermal well Th-13b in Nigrita does not ers recently, especially those concerning phyco-
need any compression because its amount is cyanin (Belay, 2002), however little is known
very high. A separator water-CO2 is used. The about the antioxidant properties of commercial
CO2 required for the photosynthesis is provided Spirulina formulations. In this respect, we have
through bubbling tubes immersed in the suspen- examined the free radical (DMPD, DPPH,
sion next to the turbine in the production ponds. ABTS) scavenging activity of water and solvent
Control over CO2 supply is implemented extracts of the Spirulina powder produced by
through pH (maintained between 9.0 and 10.5). ALGAE A.C. in order to evaluate if the drying
A high quality control over the cultivation and packaging processes destroy the antioxidant
process requires measurements of the following properties of the microalgae.
parameters: algal suspension density, pH of the We found that the hot water extract of this
algal suspension, algal suspension temperatures product contained high molecular weight sul-
(maintained between 20 and 38oC), biological fated polysaccharide component(s), as revealed
state of the culture (physiological conditions of by the formation of specific methylene blue
the culture contaminants), algal purity, O2 and dye-polysaccharide complexes. These Spirulina
CO2 concentrations, moisture in the final prod- polyanions are able to drastically reduce the
uct. DMPD and ABTS free radicals. Accordingly,
Every endeavour was made to find an opti- they represent major antioxidant components of
mum solution for the daily separation of the al- Spirulina powder. We have further examined
gal yield. A centrifuge of the Alpha Laval the antioxidant activities of Spirulina powder
type proved to be the best solution. Drying is constituents, extracted with various organic sol-
done in natural conditions. Geothermal energy vents, that is, acetone, acetone/water (80%) and
can be successfully used for algal biomass dry- methanol, expressed as polyphenol (4-
ing especially when algal slurry obtained after methylcatechol) antioxidant equivalents and
found that all extracts contained polyphenols as
6 International Conference on Geothermal Energy
Applications in Agriculture, May 2004, Athens, Greece

determined by Folin-Ciocalteu reagent. For the
determination of the antioxidant activity of the
extracts, we have evaluated the amount of the
dry mass of the extracts needed to reduce by
50% the initial concentration of DPPH. The or-
der of the efficiency of the extracts for free radi-
cal reducing capacity was acetone extract> ace-
tone/water extract> methanol extract. On the
other hand, the order of the efficiency of the
solvents for the extraction of polyphenols, as
determined by Folin-Ciocalteu reagent, was ace-
tone/water > methanol > acetone.
ACKNOWLEDGMENTS
TGS and ETN were supported by the Greek
GSRT (01PRAXE11).
Kehler, J.P. and C.V. Smith, 1994. Free radical in biol-
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Nenadis N. and M. Tsimidou, 2002. Observations on the
estimation of scavenging activity of phenolic com-
pounds using rapid 1,1-diphenyl-2-picrylhydrazyl
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Romay, C., J. Armesto, D. Ramirez, R. Gonzalez, L. Le-
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inflammatory properties of c-phycocyanin from blue-
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Schlesier, K., M. Harwat, V. Bohm and R. Bitsch, 2002.
Assessment of antioxidant activity by using different
in vitro methods, Free Rad.Res., 36, pp. 177-187.
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Sotiroudis, 2003. Chemopreventive potential of minor
components of olive oil against cancer, Ital. J. Food
Sci., 15, pp. 169-185.

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