Arthrospira (Spirulina)

25
Claudio Sili, Giuseppe Torzillo and Avigad Vonshak

Contents Summary
Summary........................................................................................ 677
25.1 Introduction .................................................................. 677
The successful commercial exploitation of Arthrospira
because of its high nutritional value, chemical composition
25.2 Morphology................................................................... 678
and safety of the biomass has made it one of the most impor-
25.3 Taxonomy ...................................................................... 686 tant industrially cultivated microalgae. Knowledge of its
25.4 Occurrence and Distribution ...................................... 689 biology and physiology, which is essential for understand-
ing the growth requirements of this alkaliphilic organism,
25.5 Physiology ..................................................................... 691
25.5.1 Response to Environmental Factors ............................... 692 has been used in developing suitable technologies for mass
25.5.1.1 Effect of Light on Growth .............................................. 692 cultivation. The relationships between environmental and
25.5.1.2 Light Stress – Photoinhibition ....................................... 692 cultural factors, which govern productivity in outdoor cul-
25.5.1.3 Effect of Temperature on Photosynthesis tures, are discussed in connection with growth yield and
and Respiration .............................................................. 692
25.5.1.4 Effect of Temperature on Growth efficiency. The response of Arthrospira and its modification
and Cell Composition .................................................... 693 under stress is described, together with the strategy of
25.5.1.5 Interaction of Low Temperature osmotic adjustment and the mechanism of internal pH regu-
with Light and High Oxygen Concentration .................. 694 lation to alkalinity. The metabolic plasticity of the response
25.5.2 Effect of Salinity on Growth, Photosynthesis
and Respiration .............................................................. 695 to disparate environmental stimuli is demonstrated in the
25.5.3 Osmoregulation .............................................................. 695 natural environment, but is also well-expressed in the main-
25.5.4 Arthrospira as an Alkaliphile ......................................... 696 tenance of high productive monoculture in intensive outdoor
25.5.5 How Does Arthrospira Compete in Culture? ................. 696 cultivation systems.
25.6 Mass Cultivation of Arthrospira .................................. 697 While the confused taxonomy of Arthrospira and its
25.7 Market and Application .............................................. 699 relationship with Spirulina has been resolved by study of the
ultrastructural feature of trichomes and 16S rRNA sequence
References ...................................................................................... 701
analysis, the problem of species definition is still ongoing.
However, molecular methods such as total DNA restriction
profile analyses of a wide range of strains are helping to
resolve this.

25.1 Introduction

C. Sili (*) • G. Torzillo This account needs to start with a taxonomic comment.
Istituto per lo Studio degli Ecosistemi, CNR, Ever since Arthrospira was first reported in 1852 by
Via Madonna del Piano, 10, 50019, Sesto Fiorentino, Firenze, Italy Stizenberger, many species of this genus of helically coiled
e-mail: sili@ise.cnr.it; torzillo@ise.cnr.it
cyanobacteria have been described and isolated. However, its
A. Vonshak classification has long been a source of confusion. Geitler
Microalgal Biotechnology, The Jacob Blaustein Institute
(1925) invalidated the genus Arthrospira in his revision of
for Desert Research, Ben-Gurion University of the Negev,
Sede Boker Campus, Beersheba, 84990, Israel the Cyanophyceae and included all regularly helically coiled
e-mail: avigad@bgu.ac.il Oscillatoriales without firm sheaths in the previously described

B.A. Whitton (ed.), Ecology of Cyanobacteria II: Their Diversity in Space and Time, 677
DOI 10.1007/978-94-007-3855-3_25, © Springer Science+Business Media B.V. 2012
678
genus Spirulina Turpin 1829. Most authors followed Geitler
for a long while, but increasingly researchers realized that
genera are distinct and returned to using two names. The evi-
dence supporting this was summarized by Castenholz (2001)
and Komárek and Anagnostidis (2005). Many species cur-
rently listed as Spirulina should therefore be re-included in
Arthrospira and these include all those grown commercially
and sold as Spirulina. This material is now known so widely
under this name that it seems inevitable that the name will
persist; however, it should be written as Spirulina or spirulina
i.e. no italics.
Arthrospira has been reported to exist in environments
varying in their osmoticum, temperature and salt concentra-
tions, most being of high alkalinity (Iltis 1969a, b; Busson
1971). Filaments of (true) Spirulina also occur in many of
these environments, but apparently never forming the blooms
that often occur with Arthrospira.
There is great interest in past and present use of Arthrospira
as a food, though nowadays mostly as a “health” food.
Dangeard (1940), Brandily (1959) and Léonard and Compère
(1967) all described how African tribes living along Lake
Chad collect this alga. The biomass is harvested from water-
bodies near the lake and sun-dried on the shores to produce a
hardened dark cake called “dihé”, which is broken into small
pieces and used in different forms by the local populations as
part of their daily diet. At about the same time, Arthrospira
was also recorded in the water of Lake Texcoco, Mexico.
Here, it had been used as food by the natives living in
the area (Clément 1968). Travellers to Mexico during the
sixteenth century described how the Aztecs used a soft a
blue-green material, harvested with fine nets from the lake,
for making a kind of bread called “tecuitlatl” (Ciferri 1983).
It is striking that these two human populations, living far
apart, discovered the nutritional value of Arthrospira
independently.
Later, attention was refocused on “Spirulina” by the pio-
neering work of the Institute Française du Pétrole on
cyanobacterial blooms in the evaporation ponds of the indus-
trial soda production facility at Lake Texcoco near Mexico
City. This led to the first detailed study of the growth require-
ments and physiology of Arthrospira. The Ph.D. research of
Zarrouk (1966) was the basis for establishing the first large-
scale production plant. The work was followed up by several
groups in Italy, France and Israel and summarized in a review
by Ciferri (1983). The subsequent extensive research on cell
biology, biochemistry and biotechnology has been reviewed
in books edited by Vonshak (1997a), and Richmond (2004a).
This chapter provides a perspective on the biology and bio-
technology of the two most important species of Arthrospira
utilized for commercial mass cultivation, A. maxima and A.
fusiformis (usually reported as Spirulina maxima and
Spirulina platensis, respectively).
C. Sili et al.
25.2 Morphology
The main morphological feature of Arthrospira is the typical
arrangement of its multicellular cylindrical trichomes in an
open helix usually of relatively large diameter, sometimes
attenuated at the ends, and with evident cross-walls
(Fig. 25.1). In contrast, Spirulina presents a screw-like
trichome, generally with almost closed, uniform and narrow
diameter screws (0.5–3 mm), cells with cross-walls usually
invisible at light microscope, without gas vacuoles and with
prominent granules (Fig. 25.2). The trichomes of Arthrospira
are composed of cylindrical cells that undergo binary fission
in a single plane perpendicular to the main axis. Trichome
elongation occurs through multiple intercalary cell division
along the entire filament. Multiplication occurs only by frag-
mentation of a trichome, usually in correspondence of a
necridial cell (Fig. 25.3). The mechanism, has been described
in detail for both A. maxima and A. fusiformis by Tomaselli
et al. (1981). It consists in the destruction of an intercalary
sacrificial cell (necridium) that first becomes colourless and
finally biconcave due to the collapse of the lateral septa.
However, the presence of necridia become less evident when
cultures are subject to fast mixing as it occurs in mass cul-
tures. In contrast, the fragmentation of Spirulina trichomes
occurs always without the production of necridic cells
(Komárek and Anagnostidis 2005) (Table 25.2).
The trichome width of Arthrospira populations sampled
from nature ranges from about 2.5 to 16 mm, while the helix
pitch typically ranges from 0 to 80 mm and its diameter from
15 to 60 mm. The dimensions and other morphological fea-
tures of A. fusiformis (i.e. the well-known commercial
Spirulina platensis) not only vary markedly between popula-
tions, but also within one population (Fig. 25.4). Both under
laboratory and mass cultivation conditions, the helix architec-
ture (pitch and diameter) is highly dependent on growth and
environmental conditions. Observations on Arthrospira
fusiformis by the authors have shown that morphological
variability and trichome motility are especially evident during
the first weeks following trichome isolation, and that this is
usually maintained for some years in laboratory liquid culture
(Fig. 25.5). In contrast, morphological variation in A. maxima
following its isolation is much less marked (Fig. 25.6). Both
A. fusiformis (Fig. 25.7) and A. maxima typically have abun-
dant gas vacuoles, which help to position the organism in the
water column. They have an important role in the harvesting
of the bloom by African people who utilize A. fusiformis for
the preparation of dihè (Abdulqader et al. 2000).
The sheath of Arthrospira is usually absent in planktonic
populations. Where a sheath is present, it is colourless, tube-
like, adjacent to the trichome, open at the ends and contains
only one trichome (Komárek and Hauer 2011). In contrast, a
25 Arthrospira (Spirulina)
Fig. 25.1 Morphological aspects of the evident cross-walls in
Arthrospira fusiformis isolated from soda Lake Kailala (Chad). (a)
Fresh clonal trichome grown in laboratory; (b) wild trichome fixed in
Fig. 25.2 Clonal trichomes of +/− regularly screw-like coiled in (a) Spirulina subsalsa and (b) S. major. Note the absence of gas vacuoles and
visible cross-walls. Bar marker = 10 mm (Photos C. Sili)
considerable production of mucilage can be formed by a
strain of A. fusiformis, which was isolated from Lake Kailala,
on agar plates, particularly if the material starts to dry out
(authors, unpublished data). Under such conditions, cocoon-like
shapes, which are generally immersed in a polysaccharidic
matrix, with two or more entangled trichomes inside their
679
formaldehyde (2%); (c) fresh clonal trichome grown in mass culture;
(d) autoflorescence of trichome shown in (c). Bar marker = 10 mm
(Photos C. Sili)
capsular investment, start to increase (Fig. 25.8a). In some
cases, it is possible to observe a single wide mucilaginous
envelope surrounding each trichome (Fig. 25.8b). The rupture
of the capsule causes the release of 2–4 trichomes (Fig. 25.8c).
The polysaccharide slime produced by A. fusiformis on agar
plates can be dense (Fig. 25.8d). It seems likely that these
680
Fig. 25.3 Necridic cell formation in clonal: (a) Arthrospira fusiformis coiled trichome; (b) A. maxima straight trichome. Bar marker = 10 mm
(Photos C. Sili)
structures are an adaptation to cope with adverse environ-
mental conditions, such as those in the semi-dry zones at the
edge of the small alkaline lakes with A. fusiformis blooms,
which are often subject to rapid and extensive draining with
the onset of the dry season.
The effect of temperature on filament structure was stud-
ied by Van Eykelenburg (1979), who subsequently described
the rapid reversible change from the helix to the spiral shape
on solid media (Van Eykelenburg and Fuchs 1980). Although,
the simultaneous presence of straight and helicoidal forms of
A. fusiformis and A. maxima (Fig. 25.9) has been observed
frequently in the laboratory and in mass cultures, the trigger
factor and the mechanism for this morphological change still
remain obscure. A detailed study of the effect of physical
and chemical conditions on the helix geometry of Arthrospira
was done by Jeeji Bai and Seshadri (1980) and Jeeji Bai
(1985). They reported the occurrence of the straight or nearly
straight spontaneous culture variants also observed by Lewin
(1980). However, there are considerable differences in the
degree of coiling between different strains of the same spe-
cies and within the same strain, the helical shape of the
trichome is regarded as a peculiar property of the genus.
Once a strain has converted to the straight form, either natu-
rally or due to physical or chemical treatments, it does not
usually revert back to the helical form. Jeeji Bai (1985) sug-
gested that this may be due to a mutation affecting some
trichomes under certain growth conditions. The common
occurrence of straight trichomes in mass cultures of
Arthrospira also suggests that the helical character may be
carried on plasmids, but no one has yet demonstrated the
existence of plasmids in Arthrospira. It has been reported
that helical shape is related to protection of the organism
against photolysis (Fox 1996). Wang and Zhao (2005)
observed that the helical filaments originated from the linear
filaments 9 years old obtained with ionizing radiation.
C. Sili et al.
Further studies revealed that under growth conditions, the
linear trichomes could spontaneously revert to the helical
trichomes with the same morphology as the original ones.
These authors also observed, there were significant differ-
ences in morphology, ultrastructure, physiology, biochemis-
try, and genetic characteristics between the linear trichomes
and the original or reverted ones, but no differences were
found between the original (helical) and the reverted
trichome, indicating that both linearization and reversion the
helical shape of Arthrospira trichomes were due to genetical
variation. Occasional helical trichomes in cultures of
Arthrospira fusiformis strain D885/S which is a straight
form, have been also observed by Mühling et al. (2006).
However, in all cases the helical trichomes were outcom-
peted by straight ones and disappeared after subsequent sub-
culturing. In contrast, Noor et al. (2008) found that a stock
culture of Arthrospira (Spirulina) maintained it helical struc-
ture after 17 years of subculture, while filaments in pilot
ponds lost their helical structure. They concluded that the
prolonged period of acclimation to adverse climatic condi-
tions outdoors, may cause reversion of coiled filaments to
straight ones, and that this fact supports the idea that straight
filaments may have a higher survival capacity. However, bio-
chemical composition and nutritional properties were not
affected by the shape of the filaments (Noor et al. 2008;
G. Torzillo, unpublished).
Interestingly, Mussagy et al. (2006), starting from a culture
of hormogonia of the straight trichomes, obtained one with a
mix of loosely-coiled and straight ones. Finally, Hongsthong
et al. (2007), using a proteomic approach, concluded that the
linear morphology observed both in the laboratory and in
industrial production ponds was possibly induced firstly by
environmental stresses, such as oxygen level, carbon dioxide
level, nutrient availability and light, and secondly by the
change in a cell shape determination mechanism. This study
 25

Table 25.1 Morphology of Arthrospira species based on Komárek and Anagnostidis (2005) and Komárek and Hauer (2011)

Trichome Coils (mm) Gas Morphology

Arthrospira (Spirulina)

Species width (mm) Cell length (mm) Width Height Helix shape vacuoles of trichome end
Types without gas-vacuoles (forming mats)
A. balkrishnanii Kamat 1963 3.5–4 (5) 0.8–1.2 6–7 12–15 − Rounded. no calyptra
A. desikacharyensis Vasishta 1962 0.85–1.7 51–6.8 10.2–11.5 Regularly loosely spiral coils − Rounded
A gigantea (Schmidle) Anagnostidis 1998 (2.5) 3–4 (8) 11–16 7–12 (15) Regularly coiled − +/− conical
A gomontiana Setchell 1895 2.5–3 +/− isodiametric 6–6.5 15–19 −
A. jenneri Stizenberger ex Gomont 1892 (3.7) 4–6 (8) (7.4) 8–15 (17) (9.2) 12–25 (31) Regularly screw-like coils − Rounded, no calyptra
A. massartii Kufferath 1914 5–6 Loosely screw-like − Rounded conical
A. pellucidis Wang 1933
A. platensis (Nordstedt) Gomont 1892 (4) 6–7 (9?) Nearly (20?) 26–36 (24?) 30–57 +/− regularly loosely spiral − Rounded or flat calyptra
isodiametric coils
A. santannae Komárek et 2.8–3.8 +/− isodiametric 12.5–18.5 (loose) 20–40 (dense) +/− densely or loosely coil − Rounded, no calyptra
Komárková-Legnerová 2006 8.8–10.8 (dense) 5.5–5.8 (loose)
A. skujae Magrin, Senna et Komárek 1998 (2.7) 3.1–4.3 3.1–9.3 7.4–11.8 7–13 Regularly screw-like coil − Rounded
A. tenuis Brühl et Biswas 1922 About 2 2–3 20–35 −
Types with gas vacuoles (planktonic, solitary trichomes)
A. argentina (Frenguelli) 9–10.5 33–49 63–75 +
Guarrera et Kühnemann 1949
A. fusiformis (Voronichin) (3.4)5–9 (11?) 2–3 × shorter 15–50 0–80 Regularly screw-like coiled + Rounded
Komárek et Lund 1990 than wide intensely narrowed or
widened towards ends
A. indica Desikachary et Jeeji Bai 1992 8–9 1/3–1/2 as long 16–39 16–66 Spirally coiled and distinctly + Conical calyptra
as wide narrowed towards ends
A. ghannae Drouet et Stirickland 3–5.2 20–22 20–35 Regularly loosely screw-like + Slightly attenuated end
in Drouet 1942 coils and capitate
A. joshii Vasishta 1963 4.2–5.1 2.7–3.4 6.8–7.6 17–19.2 Regularly screw-like coils, + Rounded, no calyptra
not attenuated towards ends
A. maxima Setchell et Gardner (6.5) 7–9 (10) 5–7 40–60 70–80 Regularly loosely screw-like + Rounded with thickend
in Gardner 1917 coiled, gradually slightly outer cell wall
attenuated at ends
681
682
Fig. 25.4 Morphological variability in Arthrospira fusiformis in two
soda lakes at the irregular north-east fringe of Lake Chad. (a, b, c) Wild
fresh trichomes from Lake Kailala; (d, e, f) wild trichomes fixed in
was the first to show evidence at the protein level that could
explain this morphological transformation in Arthrospira.
The effect of solar UV radiation on morphology on
Arthrospira was studied by Wu et al. (2005) using an indoor
grown strain, which had not been exposed to sunlight for
decades, and an outdoor-grown strain grown under sunlight
for decades. The experiments confirmed what had already
observed by Jeeji Bai and Seshadri (1980) that the self-shad-
ing effect produced by compression of the spirals over adap-
tive time scales seem to play an important role in protecting
this species against deleterious UV radiation. Gao et al.
(2008) described interactions between temperature and UVR
on the morphology of A. fusiformis and found that UVR
caused a breakage of the spiral structure at 15°C and 22°C,
but not at 30°C. A. fusiformis was able to modify its spiral
structure by increasing helix tightness at the highest temper-
ature (30°C). PAR has been found to cause a decrease in the
helix pitch, while UVR increased the extent to which helices
are compressed (Ma and Gao 2009). In general it seems that
C. Sili et al.
formaldehyde (2%) from Lake Kossorom. (f) Shows that occasional ±
straight trichomes may also be present in a wild sample. Bar
marker = 40 mm (Photos C. Sili)
artificial laboratory conditions favour the development of
straight forms. After 8 years of repeated transplants in liquid
culture, two A. fusiformis strains (Kailala and Kossorom)
had about 30% had straight trichomes (Fig. 25.10a), 50%
with loosely-coiled trichomes (Fig. 25.10b), while only 20%
maintained their original or an elevated degree of helicity
(Fig. 25.10c) (C. Sili, unpublished data).
A survey of the morphological characters of 36 clonal
axenic strains of cultivated Arthrospira carried out by
Mühling et al. (2003) showed that of the 34 with helical
trichomes, five were right-handed and 29 left-handed. After
1 year of repeated subculture, the orientation of one helical
strain had changed from right to left-handed, suggesting a
probable genetic shift. In addition, a temperature shift from
30°C to 34°C for 7 days led to a change in orientation in three
strains, which was reversible if the temperature shift was
reversed. The fact that the orientation of trichome helix can be
reversed by genetic drift and by environmental factors such
as temperature upshift and mechanical stress demonstrates
Fig. 25.5 Morphological variability in fresh clonal trichomes of Arthrospira fusiformis isolated from soda Lake Kailala (Chad) and grown in the
laboratory. Bar marker = 20 mm (Photos C. Sili)

Fig. 25.6 (a) Morphological aspects of regular clonal trichomes of cells diameter at the ends. Bar markers correspond to 40 mm in a and
Arthrospira maxima isolated from the solar Lake Texoco evaporator. 20 mm in b (Photos C. Sili)
(b) Typical trichome regularly screw-like coiled with gradually attenuated
Fig. 25.7 (a) Clonal trichomes of Arthrospira fusiformis isolated from the soda Lake Kailala (Chad) with a typical spiral markedly narrowed
towards the ends. (b) Dark-field photo showing the abundant gas vacuoles. Bar marker = 20 mm (Photos C. Sili)

Fig. 25.8 Morphology of Arthrospira fusiformis grown on agar
Petri dishes. (a) Cocoon-like shape immersed in a polysaccharide
matrix, with two or more internal entangled motile trichomes; (b)
two single trichomes surrounded by mucilaginous envelopes that, in
turn, are surrounded by thick capsules; (c) clusters of trichomes of a
typical cocoon-like shaped after their release as single filaments;
(d) cocoon-like shaped and free trichomes immersed in a dense slime
of polysaccharides negatively stained with Indian ink. Bar markers
correspond to 40 m m in ( a ) and ( c ), 20 m m in ( b ) and 100 m m in
( d ) (Photos C. Sili)

Fig. 25.9 Straight and loosely-coiled clonal trichomes of Arthrospira maxima isolated from Lake Texoco (Mexico). Bar markers: A = 40 mm,
B = 20 mm (Photos C. Sili)
25 Arthrospira (Spirulina)
Fig. 25.10 Arthrospira fusiformis isolated from Lake Kailala (Chad) after 8 years of repeat transplants in liquid culture: (a) clonal straight
trichomes; (b) loosely coiled trichomes; (c) coiled trichomes. Bar marker = 20 mm (Photos C. Sili)
Fig. 25.11 Examples of different helix orientations observed in
Arthrospira fusiformis cultures. (A) left-handed helices, (a); right-handed
helices (b). (B) Example of reversal helix orientation due to mechanical
that care is needed in using this character for taxonomic pur-
poses (Mühling et al. 2003). Occasionally, reversed forms
from left-to- right handed orientation have been observed in
our Arthrospira fusiformis laboratory and mass cultures
(Fig. 25.11).
Sinking velocity is markedly influenced by the shape of
trichomes. If a planktonic species evolves towards a decreas-
ing sinking velocity, it has three options: (i) it may decrease
its body size (but this could increase the risk of being grazed
by zooplankton); (ii) it may decrease its specific gravity (e.g.
increasing gas vacuole density) or reducing the amount of
ballast substances (e.g. carbohydrate); (iii) or may increase
its form resistance. Padisák et al. (2003) investigated the
sinking velocities of different PVC models of phytoplankton
organisms. The sinking velocity was related to dimension-
less form resistance factor (F). It was observed that F
decreased in coiled shaped organisms, and increased with
the straight ones. As a result coiled forms tend to sink at a
faster speed than straight ones. These findings agree with
those made by Booker and Walsby (1979) on Anabaena flos-
aquae. They observed that helical filaments of all length
classes sank significantly faster than straight ones of corre-
sponding length. In the case of straight filaments there was a
685
stress; arrow indicates the position on the trichome where the helix
orientation reversed from left-hand (a) to right-hand (b). Bar marker = 20 mm
(Photos C. Sili)
slight progressive increase in mean sinking speed with
increasing length helical filaments though up to 10–20 cells.
The authors concluded the advantage of straight filaments
could not be in conferring a higher growth rate, but it might
be in resisting predation or affecting the rate of sinking and
floating. Similar conclusions were drawn by Mühling et al.
(2003). These authors reported that in one example of graz-
ing by large ciliate, the ciliate had to make a rotatory motion
almost following the coil of the Arthrospira, with up to 10
coils showing inside the ciliate. It was also suggested that,
perhaps the reversal of helix orientation at intervals within a
particular ecosystem in response to shifts between grazers or
changes within populations of one particular grazer.
The ultrastructural cell organization of Arthrospira is typ-
ical of prokaryotes, with a lack of membrane-bound organ-
elles (Van Eykelenburg 1979; Vonshak and Tomaselli 2000).
The thin cell wall has four layers, with an easily detectable
electron-dense layer corresponding to the peptidoglycan
layer (Van Eykelenburg 1977). The regularly spaced cross-
walls divide the trichome into cells connected by plasmodes-
mata. Cross-walls are formed by centripetal growth and
extensions of both the peptidoglycan and the more internal
layer of the cell wall toward the centre of the cell. The cell
686
has a number of inclusions mostly corresponding to the
typical ones for cyanobacteria described in previous books
(Fogg et al. 1973; Carr and Whitton 1973, 1982). Thylakoid
membranes with phycobilisomes are arranged in parallel
bundles. The other main subcellular inclusions are (in addi-
tion to carboxysomes, ribosomes and DNA fibrils) gas vacu-
oles, polyglucan granules, polyphosphate granules and large
cyanophycin granules. The extent of development of the last
three depends on the nutrient status of the trichome; for
instance N-rich conditions favour cyanophycin formation.
Structures like the cylindrical bodies reported for some
other members of the Oscillatoriaceae are often present.
These inclusions, whose explanation is still unknown, are
lacking in Spirulina (Tomaselli et al. 1996; Tomaselli 1997).
Studies carried out by Palinska and Krumbein (2000) on the
peptidoglycan cell wall of cyanobacteria confirmed the
observations made by Guglielmi and Cohen-Bazire (1982)
concerning different perforation patterns of Spirulina and
Arthrospira, which were used as a criterion in order to sepa-
rate them clearly into two different genera. Spiller et al.
(2000), using an x-ray microscope, showed that the cell end
walls were clearly visible in A. fusiformis, and that they
crossed the filament at intervals of about 2–3 mm. The images
also revealed two interesting structures characterised by a
spherical shape, as well as a looping strand that resembled
beads on a string.
25.3 Taxonomy
As discussed in the introduction section, the question of the
taxonomy and nomenclature of Arthrospira and Spirulina
was debated for almost the entire twentieth century. Several
questions have been solved only in recent decades with the
help of electron microscopy, biochemical analysis, and
recently, thanks to the support of molecular analysis, within
the context of what is commonly referred to as a “polyphasic
approach”. The genus Arthrospira has now become univer-
sally accepted, at least by taxonomists, as the name for the
organisms used in mass culture. With the aim of further clari-
fying the systematic position of the Arthrospira spp. com-
monly used for mass culture operations (basically speaking,
A. maxima and A. fusiformis), Komárek and Anagnostidis
(2005) and Komárek and Hauer (2011) have recently urged a
definitive abandoning of the commercial name Spirulina
(Arthrospira) platensis, recommending that it be replaced
with the taxonomically-correct name of A. fusiformis.
The long story concerning the name Spirulina started
about 130 years ago, when Wittrock and Nordstedt (1884)
reported the occurrence in a stagnant pond near Montevideo
of a blue-green alga with helical shaped filaments, which
they described as “Spirulina jenneri f. platensis”, even
though it possessed septa. At that time the genus Spirulina
C. Sili et al.
was considered to be unicellular. Some years later,
Stizenberger (1852), who had observed septa in some heli-
cally coiled oscillatoriacean forms, proposed that the forms,
with a multicellular structure, be included in a new genus,
Arthrospira. This distinction was confirmed later by Gomont
(1892) in his taxonomic study on the Oscillatoriaceae. He
left the apparently aseptate forms (trichomata unicellularia)
in Spirulina and placed the septate forms (trichomata pluri-
cellularia) in Arthrospira Stizenberger 1852.
However, Geitler’s revision (1925, 1932) of the Cyanop-
hyceae re-unified the members of these two genera in
Spirulina Turpin 1829. He based classification on the close
similarity in morphology and ignored the presence of septa
and thus made no distinction between the forms previously
recognized as separate genera. The genus was thus divided
by Geitler into two subgeneric taxa (Section I. Arthrospira
and Section II. Euspirulina) on the basis of the criterion orig-
inally used by Stizenberger (1852) to separate the genera
Spirulina and Arthrospira i.e. visible or non-visible cross-
walls. The forms with septa that were easily observable under
a light microscope were classified in Section Arthrospira and
those with unlikely or only artificially observable septa
(after trypsin treatment or staining with neutral red) in
Section Euspirulina.
In accordance with those authors who sustain that many
aseptate forms are only apparently so, including species that
reveal septa only after proper chemical treatment and stain-
ing, Desikachary (1959) returned to a separation of the two
genera: SpirulinaTurpin with invisible or unicellular cells of
trichome, and Arthrospira Stizenberger with cells of the
trichome clearly visible. Subsequently, Bourrelly (1970), by
following a radical taxonomy attribution, merged Spirulina
and Arthrospira in Oscillatoria, and remarked that new
understanding of cell colouration made it possible to show
also the otherwise invisible septa of Spirulina. Hindák (1985)
assigned all planktonic forms of Arthrospira (including those
previously described as “S. platensis”) in alkaline saline
lakes to “S. fusiformis” Voronichin 1934 (synonyms A. max-
ima Setchell et Gardner 1917, in Gardner 1917, and A. gei-
tleri De Toni 1935), since, in agreement with Fott and Karim
(1973), he considered “S. platensis” to be a benthic species.
In order to distinguish Spirulina and Arthrospira in
Bergey’s Manual of Systematic Bacteriology, Castenholz
(1989) suggested the use of three features of helically coiled
trichomes: (1) degree of inclination of the pitch of trichome
helix (from transverse axis); (2) aspect and visibility (LM) of
cross-walls between the cells in the filament; (3) distribution
(EM) of junctional pores in the cell wall. Thus Arthrospira
can be differentiated from Spirulina on the basis of the degree
of inclination of the pitch of the trichome helix, which forms
an angle >45° from the transverse axis, the presence of easy
visible septa and the distribution of junctional pores in one
circular row around the cross-walls. In 2001, with the Second
25 Arthrospira (Spirulina)
Fig. 25.12 Clonal trichomes of Arthrospira indica isolated from Lake Lonar. Arrows indicate the presence of a calyptra. Bar marker = 20 mm
(Photos C. Sili)
Edition of Bergey’s Manual of Systematic Bacteriology,
Castenholz updated the key to separate Spirulina from
Arthrospira. The two genera were distinguished according to
the trichome helix, cross-walls whether invisible or visible,
and cell diameter 2–4 mm in Spirulina versus typically
6–12 mm in Arthrospira.
An important revision of the taxonomy and nomenclature
for “Spirulina platensis”, “S. maxima” and “S. fusiformis”,
was suggested by Komárek and Lund (1990) who placed the
two taxa (“maxima” and “fusiformis”) within the planktonic
forms (i.e. with gas vacuoles). Furthermore, they suggested
that these two taxa, which may represent two species (A. fusi-
formis Komárek et Lund and A. maxima Setchell et Gardner),
have different geographical distributions. Hence, they consid-
ered A. maxima to be pantropical, while A. fusiformis was
limited to Africa and tropical and central Asia. Moreover, for
the first time they considered A. platensis (without gas vacu-
oles, similar to A. jenneri) to be a periphytic and benthic
species that is essentially restricted to South America.
Subsequently, Desikachary and Jeeji Bai (1992) and Jeeji
Bai (1999) proposed that all the calyptrate forms of A. fusi-
formis should be included in the new species A. indica
Desikachary. This implied that the thickening of the apical
cell wall (calyptra) should be considered a significant taxo-
nomic feature (Fig. 25.12). Komárek and Anagnostidis
(2005) concluded that Arthrospira has solitary and free-
floating trichomes in the planktonic forms of subtropical and
tropical saline lakes (A. maxima and A. fusiformis), while
freshwater benthic forms were united in a fine, mostly slimy,
thallus in A. platensis and A. jenneri. These authors placed
Arthrospira to the family Phormidiaceae (subfamily
Phormioideae) and recognized 17 species worldwide accord-
ing based on phenotypic markers. The main morphological
features of species without (i.e. benthic) gas vacuoles (ben-
thic) or with (i.e. planktonic) gas vacuoles, as distinguished
by Komárek and Anagnostidis (2005) and more recently by
Komárek and Hauer (2011), (http://www.cyanodb.cz). In the
light of this new taxonomic grouping, the Table 25.2
687
provides a summary of the synonyms of different Arthrospira
species recognised by Komárek and Anagnostidis (2005).
However, this classification which is based only on phenotypic
aspects needs to be supported, particularly for the cultivated
strains, by molecular analysis.
In the past 20 years, the great development of molecular
biology allowed to accompany this modern techniques to the
taxonomy based only on morphological features in accor-
dance with a combined methodology that Colwell (1970) for
the first time indicated as the “polyphasic approach”.
Viti et al. (1997), using total DNA restriction profile anal-
ysis, carried out on 10 strains belonging to Arthrospira max-
ima and Arthrospira platensis (fusiformis), found a great
consensus between these the genotypic clusters and those
grouped using morphological criteria. Subsequently,
Scheldeman et al. (1999), by means of phylogenetic study
using ARDRA (Amplified Ribosomal DNA Restriction
Analysis) of the ITS (Internal Transcribed Spacer) as a
molecular taxonomic marker, tested 51 Arthrospira cultures
that theoretically represented 37 unique genotypes obtained
from different culture collections coming from four conti-
nents. The studies confirmed a clear separation of Spirulina
from Arthrospira and the presence of two main genotype
clusters within Arthrospira which were unrelated as far as
the geographic origin of the strains, their denomination or
their phenotypic features.
In 2002 Manen and Falquet investigated the genetic diver-
sity of 23 natural, cultivated and commercial strains of
Arthrospira by comparing the genus with 20 other non-
Arthrospira cyanobacterial strains, utilizing the phycocyanin
locus (cpcB-cpcA). These studies confirmed once again, that
Arthrospira is not related to Spirulina and for the first time
showed that the former genus constitutes a strongly sustained
monophyletic group. Furthermore, the three genetically clus-
tered lineages according to which Arthrospira was divided
confirmed the general mismatch between their geographic
origin and morphology. Similar conclusions were reached by
Baurain et al. (2002), who found no clear relationships
688 C. Sili et al.

Table 25.2 Synonyms of main Arthrospira species, based on Komárek and Anagnostidis (2005)
Name Synonyms
Species of saline alkaline waterbodies, planktonic, with gas vacuoles (forming blooms)
A. fusiformis (Voronichin) Komárek et Lund (1990) Spirulina fusiformis Voronichin 1934;
Arthrospira platensis (Nordstedt) Gomont sensu Rich 1931, 1932, Thomasson 1960,
Léonard and Compère 1967, and later authors;
Arthrospira platensis (Nordstedt) Gomont f. minor Rich 1931;
Spirulina geitleri f. minor (Rich) Fott et Karim 1973;
Spirulina platensis (Gomont) Geitler sensu Thomasson 1960, Jeeji Bai and Seshadri 1980
Oscillatoria platensis (Nordstedt) Bourrelly 1970;
Oscillatoria platensis (Nordstedt) Bourrelly 1970 var. minor Rich in Iltis 1970;
Oscillatoria sp. sensu Iltis 1970;
Spirulina maxima (Setchell et Gardner) Geitler sensu Fott and Karim 1973
A. indica Desikachary et Jeeji Bai 1992 Spirulina fusiformis sensu Jeeji Bai and Seshadri 1980, non Voronichin 1934;
Arthrospira platensis f. granulata Desikachary 1959
A. maxima Setchell et Gardner in Gardner 1917 Spirulina maxima (Setchell et Gardner) Geitler 1932;
Spirulina geitleri De Toni 1935 and sensu Fott and Karim 1973;
Spirulina platensis (Gomont) Geitler sensu Welsh 1965, and later authors;
Oscillatoria pseudoplatensis Bourrelly 1970;
non Spirulina maxima Bernard 1909
Species of freshwaters, periphytic and benthic without gas vacuoles (forming mats)
A. jenneri Stizenberger ex Gomont 1892 Spirulina jenneri (Stizenberger) Geitler 1935;
Oscillatoria jenneri (Gomont) Compère 1974
A. platensis (Nordstedt) Gomont 1892 Spirulina jenneri var. platensis Nordstedt in Wittrock and Nordstedt 1884;
Spirulina platensis (Nordstedt) Geitler 1925 and later authors;
Oscillatoria platensis (Nordstedt) Bourrelly 1970;
Arthrospira platensis var. tenuis (Rao) Desikachary 1959

between ITS clusters and the denomination, morphology and
origin of the strains. A phenotypic analysis on 35 (33 helical
and 2 straight) axenic and clonal Arthrospira strains was
performed by Mühling et al. (2006). The strains included
28 features, 14 of which were morphological, 1 ultrastructural,
7 physiological, and 6 biochemical. This study reported two
phenotypic clusters that had a high correlation to the charac-
ters describing the helical trichome shape: cluster I had a
highly variable, always irregular, trichome helix that was
dumbbell-shaped, or a fusiform trichome helix with a fast-
diminishing helix attenuation toward the apices (A. maxima,
A. fusiformis, A. indica), and cluster II with a regular trichome
helix and fast-diminishing helix attenuation toward the
apices (A. platensis). These two clusters compared to the
molecular ones of the same strains reported by Scheldeman
et al. (1999) and Baurain et al. (2002) showed a high rate
correlation.
Thirty-three new strains of Arthrospira isolated from
plankton sampled in Mexico, East Africa and India were
investigated and compared with 53 strains or samples of ear-
lier studies (Dadheech et al. 2010). The study included mor-
phological features and molecular phylogenetic analyses on
the basis of nucleotide sequences of ITS between 16S rRNA
and 23S rRNA genes and partial sequences of beta and alpha
subunits including in their genes spaces (cpc BA-IGS) of
phycocyanin operon. It proved possible to divide the 53
strains into two main clusters: cluster I comprising sequences
of American strains, and cluster II containing the sequences
of the strains originating from Asia and Africa. Both genetic
regions of the strains in this study showed a significant
sequence divergence among Arthrospira strains from East
Africa, India and Mexico, indicating possible distinct evolu-
tionary lineages. It was concluded that there are deep geno-
typic divergences between Mexican and African/Indian
strains of Arthrospira, which represent two distinct geno-
types, which were also distinguishable on the basis of
trichome morphology, and referable to the two tropical main
planktonic species A. fusiformis and A. maxima.
In conclusion, it appears clear that Arthrospira is distinct
from Spirulina based on both morphological and molecular
criteria. However, because of the high morphological plastic-
ity observed particularly with wild and clonal forms (i.e.
laboratory and mass culture strains) of A. fusiformis, it is
sometimes difficult to distinguish the two edible species of
Arthrospira (A. fusiformis and A. maxima) by using only a
morphological approach. The continuous advancements
achieved by molecular analysis, provided that the origin of
strain is well known, can represent an important tool that
should contribute to better identification of the Arthrospira
species.
25 Arthrospira (Spirulina)
25.4 Occurrence and Distribution
Species of Arthrospira have been isolated mainly from alka-
line, brackish and saline waters in tropical and semitropical
regions (Castenholz 2001; Komárek and Anagnostidis 2005).
One of the striking facts about soda lakes is that, despite
apparently what might appear unfavourable conditions, these
environments are extremely productive because of high
ambient temperatures, high light irradiance and unlimited
supply of CO2. Primary production rates up to 10 g m−2 day−1
have been recorded, which is about one order of magnitude
more than most productive streams and lakes in the world,
making these the most productive aquatic environments any-
where. A characteristic feature of the majority of soda lakes
is the permanent or seasonal coloured blooms of phototrophic
microorganisms, as reported in a survey by Grant (2006).
Though most records of Arthrospira species have been for
mass growths in alkaline water, some species have been
occur occasionally in freshwaters. Species with apparently
more restricted distributions have been reported from pud-
dles (A. balkrishnaii), flowing and stagnant fresh water
(A. gigantea, A. jenneri), springs (A. massartii), stagnant
water (A. desikacharyensis, A. gomontiana), stagnant and
sulphur-containing water (A. platensis), ponds (A. khannae),
filter beds (A. tenuis), reservoirs (A. santannae) and tanks
(A. joshi). Less is known about these species, since they have
not been used for mass cultivation.
Among the various species, the most widely distributed,
A. fusiformis (A. platensis sensu Rich 1931), is mainly in Africa
(Chad, Kenya, Egypt, Ethiopia, Sudan, Libya, Algeria, Congo,
Zaire, Zambia, Mozambique), Asia (Russia, Pakistan, India,
Sri Lanka, Myanmar, Thailand) and South America (Uruguay,
Peru). A. maxima is in Central America (Mexico, California-
USA). Until a few years ago, this species was the main compo-
nent of the phytoplankton of the solar evaporation channels in
Lake Texcoco (Mexico), while A. fusiformis dominated the
alkaline saline lakes of the semi-desert Sudan-Sahel area
(Chad) and of the Rift Valleys (mainly Kenya). The frequent
occurrence of A. fusiformis and A. maxima in saline, alkaline,
tropical and subtropical waters has led to a widespread, but
possibly misleading, impression that these environments are
characteristic for the entire genus. The geographical distribu-
tion of the main populations of Arthrospira is given in
Table 25.3. Apparently none have been reported from marine
environments. However, when an adequate supply of bicarbon-
ate, N and P, together with a suitable pH and salinity, are pro-
vided, Arthrospira species can be grown in seawater (Tredici
et al. 1986), although the biomass productivity attained was
about 36% less than in Zarrouk’s medium. The biomass grown
in seawater had a reduced protein content (13%).
The most detailed studies on the ecology of Arthrospira
are those for A. fusiformis by Iltis (1968, 1969a, b, 1970,
689
1971) for tropical African lakes, and that by Busson (1971),
who described the ecology of A. maxima as a food source in
Mexico, together with its past and present use as a food
source. Other studies include those of Hindák (1985),
Komárek and Lund (1990) and Jeeji Bai (1999).
Most of the waterbodies in which A. fusiformis thrives are
permanent or temporary saline alkaline lakes (natron lakes).
These are scattered in the hollows of the fossil dune system of
the Sahara and the Sudan-Sahel areas fed by aquifers and the
scanty rain (about 300 mm per year). The salinity of these
lakes is highly variable: when evaporation exceeds inflow the
salinity rises and leads to an accumulation of salt deposits on
the shores (Iltis 1971). The mean water temperature is about
25°C, with oscillations of about ±10°C, although the temper-
ature in shallow lakes may reach 40°C during the warm sea-
son. The mean daylight length is about 12 h and solar radiation
reaches over 2,000 mmol photon m−2 s−1. The disposition and
slope of the impermeable clay and water-bearing sandy layers
favour subterranean seepage of water away from Lake Chad
(Maglione 1969). The relationships between the superficial
and the subterranean waters affect both the salinity and the
ionic composition. The latter is characterized by prevalence
of monovalent ions, with Na+ much higher than K+, and by
high concentrations of carbonate and bicarbonate, which lead
to pH values from 9.5 to 11.0. Ca2+ and Mg2+ concentrations
are low. SO42− concentration is usually high, while that of Cl−
is very low. The water of the lakes is often blue-green in
colour due to the mass development of phytoplankton.
A. fusiformis is present as nearly monospecific popula-
tions in the highly saline lakes (Iltis 1969a), only a few other
phototrophs being found. In contrast, in the mesohaline
lakes, Arthrospira co-exists with a number of other phototro-
phs, including other cyanobacteria, diatoms and
dinoflagellates. Among the cyanobacteria, Anabaenopsis
arnoldii and many Oscillatoria spp. are often important.
Several Spirulina species may be present, especially S. laxis-
sima, S. major and S. subsalsa. Studies by Margheri et al.
(1975) of two African natron lakes, Lake Mombolo and Lake
Rombou, in the region of Kanem north-east of Lake Chad,
found these Spirulina species as well as several Arthrospira
spp. Within the phototroph populations of both permanent
and temporary lakes, the rotifer density can be very high,
although varying seasonally, reaching several hundred indi-
viduals per litre (Iltis and Riou-Duwat 1971). In some lakes
Arthrospira constitutes an excellent feed for the cichlid fish
Tilapia (Coe 1966; Bergman et al. 2003). Another consumer
of Arthrospira and other large filamentous cyanobacteria
such as Anabaenopsis is the flamingo Phoenicopterus minor.
The pink colour of these birds comes from the carotenoid
pigments originating in cyanobacteria (Fox 1976).
The detailed studies by Iltis established a direct correlation
between the biomass density of Arthrospira and the salinity
of the water. The mass blooms occur at salinity values from
690 C. Sili et al.

Table 25.3 Geographical distribution of the main populations of Arthrospira

Country Location Taxon References
AFRICA
Chad Natron lakes (Bodou, Mombolo, A. platensis, A. platensis f. minor Léonard and Compère (1967),
Rombou, Yoan) and pools (Latir, Iltis (1972), and Rich (1931)
Iseirom, Latir, Liva), Kanem region
Lake Kailala, Lake Kossorom A. fusiformis Sili et al. (1999)
Kenya Natron lakes A. platensis Rich (1931) and Vareschi (1982)
(Bogoria, Crater, Elmenteita, Nakuru)
Rift Valley A. fusiformis Ballot et al. (2004)
Lake Bogoria A. platensis, A. platensis f. minor Tuite (1981)
A. fusiformis Hindák (1985)
Lake Simbi A. platensis Melack (1979)
A. platensis = A. fusiformis Kebede and Ahlgren (1996)
A. fusiformis Ballot et al. (2005)
Lake Sonachi A. fusiformis Ballot et al. (2005)
Lake Oloidien A. fusiformis Ballot et al. (2009)
Ethiopia Lake Aranguadi A. platensis Melack (1979)
Lake Chiltu, Green lake A. platensis = A. fusiformis Kebede and Ahlgren (1996)
Egypt Lake Maryut A. platensis El-Bestawy et al. (1996)
Sudan Lake Dariba, Jebel Marra A. geitleri Fott and Karim (1973)
Algeria Pond Tamanrasset A. platensis Fox (1996)
Congo Laka Mougounga Arthrospira sp. Fox (1996)
Lake Kivu Arthrospira sp. Fox (1996)
Zambia Lake Bangweolou Arthrospira sp. Fox (1996)
Tunisia Lake Korba Arthrospira sp. Fathi et al. (2001)
Mozanbique Wastewater ponds A. fusiformis Mussagy et al. (2006)
South Africa Lake Tswaing A. fusiformis Oberholster et al. (2009)
ASIA
India Ponds A. maxima Desikachary and Jeeji Bai (1996)
Lonar Lake; ponds; tank A. indica Desikachary and Jeeji Bai (1992)
(Madurai, MCRC isolate)
Myanmar Crater lake (Chapter 26) Arthrospira sp. Min Thein (1993)
Pakistan Fish pond, Lahore Arthrospira sp. Fox (1996)
Sri Lanka Lake Beria Arthrospira sp. Fox (1996)
China Fish ponds, Nanking A. platensis Tsen and Chang (1990)
Lake Bayannur A. platensis Zheng et al. (1992)
Thailand Tapioca factory effluent lakes, Bangkok Arthrospira sp. Fox (1996)
Russia Tunatan lake, Siberian steppe A. fusiformis Voronichin (1934)
Azerbaijan Water basin, Khumbasha Arthrospira sp. Fox (1996)
Pakistan Pond, Lahore Arthrospira sp. Fox (1996)
AMERICA
Mexico Lake Texcoco solar evaporator A. maxima Busson (1971)
Brazil Mangueira Lagoon Arthrospira sp. Greque de Morais et al. (2008)
California Pond, Oakland A. maxima Gardner (1917)
Coastal lagoon, Del Mar A. platensisa Lewin (1980)
Peru Lake Huachachina A. platensis Busson (1971)
A. maxima Desikachary and Jeeji Bai (1992, 1996)
Uruguay Montevideo A. platensisb Gomont (1892)
EUROPE
Spain Lake Santa Olalla Arthrospira sp. Rippka and Herdman (1992)
France Tiny lake, Camargue Arthrospira sp. Fox (1996)
Hungary Adasztevel-Oroshaz Arthrospira sp. Busson (1971)
Romania Alkaline pond near Cluj-Napoca A. fusiformis Aldea et al. (2002)
Serbia Salty puddles near river Tamiš A. fusiformis Fuzinato et al. (2010)
a
Reference strain PCC 7345
b
Holotype
25 Arthrospira (Spirulina)
22 to 60 g L−1, high carbonate-bicarbonate concentrations
(pH 8.5–11.0) and temperatures from 25°C to 40°C.
Generally, the waters populated by Arthrospira have a mean
salinity of 37 g L−1. However, Arthrospira has been found at
salinity levels ranging from 8.5 to 200 g L−1 and in at least
one case up to 270 g L−1 (Iltis 1968). Biomass density (as dry
weight) can exceed 1 g L−1. Sili et al. (1999) studied two soda
lakes (L. Kailala, L. Kossorom) on the north-east fringe of
Lake Chad, where a number of small bays and inlets between
the old sand dunes are located. Surveys showed that A. fusi-
formis was clearly the dominant (Fig. 25.4), with some
filaments of Anabaenopsis spp., Spirulina laxissima and
Synecochoccus sp., A. fusiformis is also found in some
lakes of the northern Algerian Sahara having saline waters
(1–8%) and pH levels from 7 to 9. These waters, which have
lower alkalinity (about 20 meq L−1 of HCO3−1 and CO3−2), but
Na+ levels similar to those of the highly alkaline saline
waters of the East African Rift Valley, may be considered to
be sulphate-chloride rich waters (with Cl− the major anion).
Over the past three decades the impact of excess water
abstraction and lack of care with pollution has had a marked
effect on some of the Rift Valley lakes, where Arthrospira
commonly is found, with a marked deterioration in their eco-
logical status and a progressive increase in nutrient concen-
tration. The two lakes Naivasha an Oloiden (Kenia) are
typical examples of this recent degradation and salinization
which has promoted the appearance of Arthrospira (Ballot
et al. 2009). The Lake Oloidien hypereutrophic alkaline
modification of the originally freshwater has caused a shift in
species dominance from coccoid Chlorophyceae to A. fusi-
formis and Anabaenopsis elenkinii. The main sources of
nutrient input are cattle and goat herds, watering at the lake
and local women washing clothes using detergents (Ballot
et al. 2009).
In Lake Nakuru, the dominance of A. fusiformis has been
documented since the 1960s (Talling and Talling 1965).
Okoth et al. (2009) demonstrated that A. fusiformis was the
most dominant species in terms of population density in
Lake Nakuru. However, the pollution effects due to the
urbanization process near the lake is expected to influence
the lake’s phytoplankton community strongly in the future.
As previously mentioned, a massive population of
A. maxima was found in some of the external sectors of the
gigantic spiral-shaped solar evaporator used to extract salts
from the saline carbonate-bicarbonate rich waters of Lake
Texcoco, Mexico. The evaporator lies 2,200 m above sea
level and is about 1 m deep. The alkaline (pH 10) saline
water is moved slowly from the outer part of the evaporator
towards the centre (Gallegos 1993). The water is high in
HCO3− and CO32−, Cl− and Na+ and the total salt concentra-
tion ranges from 11 to 39 g L−1 (Busson 1971). In the past
the company that operated the plant, Sosa Texcoco (and
later Spirulina Mexicana), had its “Spirulina” (A. maxima)
691
production facilities in the outside rim of the solar evapora-
tor, where it was present in almost monospecific populations,
being associated with only a few other cyanobacteria such as
Synechocystis aquatilis. In 1994 Sosa Texoco ceased cultiva-
tion operations of Arthrospira maxima in the external ponds
of the “El Caracol” solar evaporator (Alcocer and Williams
1996). At present Lake Texococo is reduced to several sepa-
rate waterbodies (Caracol, Lago recreativo and Nabor
Carillo) from where a number of A. maxima strains have
been isolated (Dadheech et al. 2010).
More recently, Arthrospira has been found occasionally
in domestic wastewater treatment plants. in Maputu,
Monzambique (Mussagy et al. 2006) and Turkey (G. Torzillo,
unpublished data), both characterised by pH ranging between
7.1 and 7.7 and a conductivity between 1,000 and 2,000 mS
cm−1. In contrast to what has sometimes been believed, this
suggests that Arthrospira can acclimate to new environments
not markedly or exclusively alkaline.
25.5 Physiology
The aim of this section is to point out relevant areas in which
Arthrospira has been used as a model organism, together
with other studies with data of significant importance in
understanding its growth, physiology and biochemistry,
especially when grown outdoors. One of the basic principles
of microalgal technology is to re-create the environmental
and cultivation conditions permitting high productivity in a
monoalgal culture. Without doubt light and temperature are
the most important. In nature, the organism uses its gas vacu-
oles to regulate its position within the underwater light gradi-
ent and follow daily and seasonal changes in light flux. In
shallow culture ponds, however, light availability has to be
optimized by modifying operational parameters such as opti-
mal cell density and the degree of mixing and culture depth
in order to obtain the highest productivity (Vonshak et al.
1982; Richmond 2004b; Belay 2008).
Adequate mixing coupled with maintenance of high cell
densities can to a certain extent reduce the effect of light satu-
ration of photosynthesis and damage to the photosynthetic
apparatus due to prolonged exposure to excess of light (pho-
toinhibition) (Torzillo and Vonshak 2003). Sufficient mixing
also ensures an even distribution of nutrients and oxygen
degassing of the culture, and at the same time prevents
filament aggregation and sinking (Torzillo et al. 2003a).The
fact that Arthrospira requires high alkalinity for growth
ensures selective conditions in the growth medium and
Arthrospira grown in outdoor open ponds remains a quasi-
monoculture (Vonshak et al. 1983). Moreover, when other
essential growth nutrients are present in sufficient concen-
trations, the elevated concentration of bicarbonate-carbonate
favours the high productivity observed in nature.
692
25.5.1 Response to Environmental Factors
25.5.1.1 Effect of Light on Growth
Zarrouk (1966) was the first to study the response of
Arthrospira maxima to light and concluded that growth rate
reached a maximum when cultures were grown under a light
irradiance between 25 and 30 klux (340–400 µmol photon
m–2 s–1). However, the lack of information about the strain
used, on how light was measured and its path in the culture
vessel, makes the results difficult to compare with later stud-
ies. Data gathered in our laboratory (Institute of Ecosystem
Study, Firenze, Italy) with A. fusiformis strain M2, which
was originally isolated from Mombolo Lake (Chad) (Balloni
et al. 1980), showed that growth becomes light-saturated in
the range of 150–200 mmol photon m−2 s−1 (Bocci et al. 1980);
this value for irradiance is about one order of magnitude less
than recorded outdoors in summer days (1,850–2,000 mmol
photon m−2 s−1). By using a turbidostatic culture, a maximum
growth rate of 0.063 h−1 was estimated for A. maxima strain
4 Mx, isolated from Texoco Lake, Mexico, and 0.059 h−1 for
A. fusiformis strain K4, isolated Rombou Lake, Chad (Balloni
et al. 1980), which correspond to doubling times of 11 and
11.7 h, and a light transformation efficiency of 7.2% and
7.8% of the photosynthetically active radiation (PAR, 400–
700 nm) for A. maxima and A. fusiformis, respectively (Bocci
et al. 1980). Based on a requirement of 20 quanta mol−1 CO2,
Ogawa and Aiba (1978) estimated the slightly higher value
for photosynthetic efficiency of 11.4% PAR.
25.5.1.2 Light Stress – Photoinhibition
Different strains of Arthrospira may differ in their sensitivity
to light stress (Vonshak et al. 1988b; Vonshak and Novoplansky
2008). Photoinhibition, which is the loss of photosynthetic
capacity due to damage to photosystem II (PSII) caused by
photon flux densities (PFD) in excess of those required to
saturate photosynthesis, can be manifested in various ways.
These include a decrease in the maximum quantum yield of
CO2 uptake or O2 evolution, a decrease in the convexity of the
photosynthesis light response curve (P/I), and, in the case of
prolonged exposure to excessive light, a decrease in the light-
saturated photosynthesis (Leverenz et al. 1990; Long et al.
1994). In at least one A. fusiformis, strain P4P, photoinhibi-
tion was probably due to a change in the turnover rate of a
specific protein, D1, which is part of PSII. Observed differ-
ences may be genotypic or environmental responses. Cultures
acclimated to high light intensities exhibit a higher resistance
to photoinhibition (Vonshak et al. 1996a). In a study of the
photosynthetic light response curve of A. fusiformis strain M2
cells exposed to strong light (Torzillo and Vonshak 1994),
photoinhibition caused a 48% reduction in the initial slope of
the P/I curve compared to the control (Fig. 25.13). However,
there was less effect on Pmax (maximum saturation rate), which
was reduced by about 25% (Table 25.4). These results empha-
size that photoinhibition not only affects Pmax, but actually has
C. Sili et al.
Fig. 25.13 Photosynthetic light response curves of A. fusiformis strain
M2 (○ photoinhibition; ● control) for 45 min at a PFD of 2,500 mmol
photon m−2 s−1
Table 25.4 Photosynthesis-irradiance (P/I) parameters estimated from
Arthrospira fusiformis strain M2 cultures before and after 45 min pho-
toinhibition at a PFD of 2,500 mmol photon m−2 s−1 at 35°C
Parameter Unit Control Photoinhibited
Pmax mmol O2 mg chl−1 h−1 800 600
IK mmol photon m−2 s−1 430 610
a mmol O2 (mg chl mmol 1.915 1.094
photon m−2 s−1)−1 h−1
R mmol O2 mg chl−1 h−1 −20.5 −26.5
Pmax light saturated rate, Ik saturation irradiance, a initial slope of the
P/I curve, R dark respiration rate
a stronger effect on the initial slope of the P/I curve i.e. on
light-limited photosynthetic activity. In contrast to higher
plants and microalgae, in the case of cyanobacteria it has
always assumed do not use an antenna-related quenching
mechanism to decrease the amount of energy funneled to
photosystem II (PSII) reaction centre (Campbell et al. 1998).
Recently, however, evidence has been presented for the exis-
tence of at least three distinct mechanisms for dissipating
excess energy in cyanobacteria. One of these photoprotective
mechanism is related to the phycobilisomes, the extramem-
branal antenna of cyanobacteria PSII. In this photoprotective
mechanism the orange carotenoid protein (OCP) in
Synechocystis sp PCC 6803 plays an important role
(Kirilowsky 2007; Wilson et al. 2006). The Arthrospira max-
ima OCP contains 3’-hydroxyechinenone (Kerfeld 2004).
However, mechanism of this novel photoprotective process in
cyanobacteria awaits elucidation.
25.5.1.3 Effect of Temperature on Photosynthesis
and Respiration
A detailed study of the response of an A. fusiformis strain
M2, to temperature was performed by Torzillo and Vonshak
(1994). They determined the optimal temperature for photo-
synthesis, and also measured the effect of temperature on the
25 Arthrospira (Spirulina)
Fig. 25.14 Effect of temperature on photosynthesis (a), and dark
respiration (b) rates of A. fusiformis strain M2. Cells were allowed to
equilibrate for 15 min at each temperature before measurement
dark respiration rate by following the O2 uptake rate in the
dark. The optimal temperature for photosynthesis was 35°C
(Fig. 25.14a), while dark respiration was highest at 45°C
(Fig. 25.14b). At temperature of 50°C the respiration rate
dropped to almost zero. At temperature extremes outside
those optimal for growth, both respiratory and photosynthetic
activity declined. However, the sensitivity of respiration to
such extremes was significantly greater than the sensitivity
of photosynthesis under the same conditions. Under conditions
where respiration was completely inhibited, photosynthetic
oxygen evolution was maintained at about 30% of the optimum
value. A temperature-dependent exponential relationship
between 15°C and 45°C was obtained, with the respiration
rate increasing as temperature increased. The temperature-
dependent dark respiration rate is given by:
R = 0.771.e (0.0636.T)
where R is the respiration rate (mmol O2 mg−1 chl h−1) and T
is the temperature (°C). An Arrhenius plot for respiration
showed an activation energy of 48.8 kJ mol−1 for Arthrospira.
The temperature coefficient (Q10) calculated for the 15–45°C
range was 1.85. The respiration-to-photosynthesis ratio in
Arthrospira was 1% at 20°C and 4.6% at 45°C (Torzillo and
Vonshak 1994). These low values confirmed the general
assumption that cyanobacteria have low respiration rates
(van Liere and Mur 1979). However, the respiration-to-
photosynthesis ratios measured in these experiments were
found to be much lower than those reported for outdoor cul-
tures of Arthrospira, where up to 34% of the biomass produced
693
Fig. 25.15 Temperature responses of three Arthrospira fusiformis iso-
lates measured as the increase in chlorophyll concentration after incu-
bating the cells for 4 days on a temperature block under constant light
(100 mmol photon m−2 s−1)
during the daylight period may be lost through respiration at
night (Guterman et al. 1989). Torzillo et al. (1991) concluded
that night biomass loss due to dark respiration depended on
the temperature and light irradiance to which the cultures
were exposed during the day, and that this occurred because
of the strong effect of temperature and light on cell composi-
tion. Analysis of the biomass harvested at sunset and sunrise
revealed that the protein content was ca. 57% and 71%, of
dry weight respectively, whereas the carbohydrate content
during the same periods was ca. 34% and 19%, of dry weight
respectively. An increase in carbohydrate in cells was observed
when these were grown at the suboptimal temperature of
25°C. Excess carbohydrate synthesized during the day was
then partially used for protein synthesis at night, while most
of them were lost by respiration (Torzillo et al. 1991).
25.5.1.4 Effect of Temperature on Growth
and Cell Composition
In nature Arthrospira is found in permanent or temporary
water bodies at relatively high temperatures. The optimal
temperature for laboratory cultivation of this organism is
about 35°C. However, many Arthrospira strains differ in
their optimal growth temperature as well as in their sensitiv-
ity to extreme values. For instance, in the comparison of
strains shown in Fig. 25.15, strain SA2 has a relatively low
optimal growth temperature (24–28°C), while the strain EY
grew well up to 38–40°C. The isolate marked K was charac-
terized by a relatively wide range of optimal growth tem-
peratures: 28–36°C. This is just one example of the variations
that exist among the different strains of Arthrospira, as
regards optimal growth temperatures. Significant differences
in the maximum temperature for growth have been also
found within the two species of A. maxima and A. fusiformis.
A. maxima strains were apparently unable to grow above
36°C, while some A. fusiformis strains were found to be
capable of growing at a temperatures higher than 40°C
(Tomaselli et al. 1987). Arthrospira fusiformis strain M2 was
694
able to grow up to 42°C and to withstand 46°C for several
hours. This strain has also been grown successfully in closed
photobioreactors (Torzillo et al. 1986). However, when
A. fusiformis strain M2 was grown under light-limited tur-
bidostatic conditions at its maximum tolerable growth tem-
perature (42°C), there was a marked decrease in photosynthetic
pigment and protein levels. Under these conditions, an increase
in carbohydrate cell content associated with changes in the
degree of fatty acid saturation (i.e. reduction in g-linolenic
acid synthesis in favour of linoleic acid accumulation) was
also observed (Tomaselli et al. 1988). It has been shown that
Arthrospira is able to accumulate glycogen, under high light
and nitrogen limitation, therefore it has been proposed as a
potential feedstock for fermentative production of biofuels
(Aikawa et al. 2012).
25.5.1.5 Interaction of Low Temperature with Light
and High Oxygen Concentration
It is suggested that Arthrospira cultures grown at suboptimal
temperatures are more sensitive to photoinhibition than those
grown at the optimal value. The latter will be able to better
handle excessive light energy, since they have a higher rate
of electron transport, an active repair mechanism and more
efficient means of energy dissipation (Vonshak 1997b). The
saturation pulse method was applied in order to study the
synergistic effect of high oxygen concentration and low tem-
perature in outdoor cultures of A. fusiformis strain M2
(Torzillo et al. 1998). It was observed that the combination of
low temperature (25°C) and high oxygen concentration (70–
80 mg L−1) had considerable impact of PSII photoinhibition
evaluated as changes in the Fv/Fm ratio (variable to maximum
fluorescence). The Fv/Fm ratio decreased down to 64% of the
morning value in the middle of day in the high oxygen culture
(Fig. 25.16). When high oxygen concentration was combined
with low temperature, the Fv/Fm ratio decreased to up to 45%
of the morning value. Complete recovery of the Fv/Fm ratio
occurred in the afternoon only in the low oxygen cultures, while
it reached about 80% of the morning value in the high oxy-
gen culture, and only 63% in the culture exposed to combina-
tion of high oxygen and low temperature (Fig. 25.15).
Photoinhibition studies in the cyanobacterium Synechocystis
sp. PCC 6803 have indicated that photodamage is initiated
by the direct effect of light on the oxygen evolving complex
(OEC) and that reactive oxygen species (ROS) inhibit the
repair of photodamaged photosystem II primarily suppress-
ing the synthesis of proteins de novo (Murata et al. 2007).
The photosynthetic electron transport system represents
the major source of ROS, that is, singlet oxygen, hydrogen
peroxide and the superoxide radical (Asada 1994; Krause
1994). When scavenging of potentially damaging oxygen spe-
cies is insufficient, photoinhibition can occur. High oxygen
concentration itself can influence the growth and the photo-
synthetic activity of the cultures even when the light inten-
C. Sili et al.
Fig. 25.16 Effect of oxygen concentration and temperature on maximum
quantum yield of PSII (Fv/Fm) of Arthrospira fusiformis strain M2
cultures grown outdoors in photobioreactors. (■) Low oxygen- optimal
temperature; (▲) high oxygen – optimal temperature; (●) high oxygen-low
temperature; (Δ) PFD
sity is relatively low, as demonstrated by several experiments
carried out both in the laboratory and outdoors (Torzillo et al.
1984; Marquez et al. 1995; Vonshak et al. 1996a). The com-
bination of high oxygen concentration and high light inten-
sity can be a very common event in outdoor cultures,
particularly in closed systems. For example, in well growing
A. fusiformis strain M2 cultures in tubular photobioreactors
(i.d. 5 cm), the oxygen concentration can increase at a rate of
2–3 mg L−1 min−1. This results in an oxygen concentration of
up to 70–80 mg L−1 (Torzillo et al. 1998).
Sensitivity to stress is strongly influenced by the light
acclimation conditions of the cells. Cells acclimated at low
light usually saturate photosynthesis at lower light intensities
(Ramus 1981). Measurements of chlorophyll fluorescence
quenching in outdoor cultures of Arthrospira showed that
sensitivity to photoinhibition strongly increased in A. fusi-
formis strain M2 cultures acclimated to low light. Yet, despite
a 40% reduction in the Fv/Fm ratio observed in cells exposed
to high stress conditions i.e. a combination of suboptimal
temperature (25°C) and high dissolved oxygen concentra-
tions (up to 60 mg L−1), no significant loss in the D1 content
was found. By contrast, under the same stress conditions, the
D1 content was reduced by 50% in low-light acclimated cul-
tures (Torzillo et al. 2003a). Productivity of low-light accli-
mated cultures was lower than that obtained in high-light
acclimated cultures. Light acclimation also strongly
influenced the effect of stress on the pigment composition of
the A. fusiformis strain M2 cells. Pigment analysis showed
that ß-carotene decreased during the day in high stress cul-
tures, while the mixoxanthophyll and zeaxanthin content
increased. The considerable presence of mixoxanthophyll
and zeaxanthin may provide a source of defense for cells
against photooxidation (Torzillo et al. 2003a).
25 Arthrospira (Spirulina)
25.5.2 Effect of Salinity on Growth,
Photosynthesis and Respiration
The occurrence of Arthrospira spp. in marine habitats has
not been documented, at least not as dominant blooms like
those found in the natron lakes. This is most likely due more
to the very low content of bicarbonate rather than to the high
content of NaCl. Arthrospira, unlike marine organisms, does
not require high NaCl concentration for growth; rather, it
only tolerates the salt. The exposure of Arthrospira cultures
to high NaCl concentrations at first results in an immediate
cessation of growth; after a lag period, a new steady state
of growth is established (Vonshak et al. 1988a). The length
of the time lag is exponentially correlated to the degree of
salinity stress imposed on the cells. In many cases, this lag
is associated with a decline in chlorophyll and biomass
concentrations in the culture (Vonshak et al. 1988a).
When the biomass composition of two strains grown
under different salt stress conditions was compared,
significant changes, mainly an increase in carbohydrate and
a decrease in the protein levels, were observed. These
changes correlated with the degree of stress imposed (i.e.
higher level of carbohydrate at a higher salt concentration).
The difference in the level of carbohydrate accumulated by
the two strains may also reflect a difference in their ability to
acclimate to salt stress. Changes in lipid synthesis have also
been demonstrated: in salt-stressed cells, with an increase in
lipid content and in the degree of fatty acid saturation is
observed with specific modifications in the long-chain fatty
acids (C18). In A. fusiformis strain M2 the oleic acid content
doubled when the organism was grown in presence of 0.5 M
NaCl (Tomaselli et al. 1993).
It has been suggested that exposure to high salinity is
accompanied by a higher demand for maintenance energy by
the stressed cells (Blumwald and Tel-Or 1982). Changes in
the photosynthetic and respiratory activities of an Arthrospira
strain were measured over a period of 48 h beginning 30 min
after exposure to 0.5 and 1.0 M NaCl. A marked decrease in
the photosynthetic oxygen evolution rate was observed
30 min after exposure to the salt. This decline was followed
by a recovery period, characterized by a lower steady state
rate of photosynthesis (Vonshak et al. 1988a). The immedi-
ate inhibition of the photosynthetic and respiratory systems
after exposure to salt stress has been explained by Ehrenfeld
and Cousin (1984) and Reed et al. (1985), in, respectively,
Dunaliella and in Synechocystis. A short-term increase in
cellular sodium concentration was due to a transient increase
in the permeability of the plasma membrane during the first
seconds of exposure to high salt concentration. It has been
suggested that the inhibition of photosynthesis due to the
rapid entry of sodium, might be the result of the detachment
of phycobilisomes from the thylakoid membranes (Blumwald
et al. 1984).
695
High rates of dark respiration in cyanobacteria due to
salinity stress had been reported previously (Vonshak and
Richmond 1981; Fry et al. 1986; Molitor et al. 1986). This
high activity may be associated with the increased level of
maintenance energy required for pumping out the excess of
sodium ions. Variable chlorophyll fluorescence was applied
by Lu et al. (1999) in order to study the kinetics of the response
of A. fusiformis strain M2 cells to salinity stress. The study
revealed that the response of PSII photochemistry during the
first 12 h of their exposure to high salinity consisted of two
phases. The first phase, which took place during the first 4 h,
was characterized by an immediate drop in Fv/Fm during the
first 15 min (about 26%) after exposure. This was followed by
a recovery to around 92% of the original level after 2 h incu-
bation. After 4 h from the beginning of the salt treatment,
another decline in Fv/Fm was observed, which reached about
70% of the initial value after 12 h (Lu et al. 1999). According
to Lu and Vonshak (2002), salt stress in A. fusiformis strain
M2 inhibited the electron transport at both the donor and
acceptor sides of PSII, and resulted in damage to the phyco-
bilisomes and a partial disconnection of these from PSII
which shifted the distribution of excitation energy in favour
of the PSI (state 2). An alteration in the structure of the phy-
cobilisomes of A. fusiformis in response to an enhanced Na+
level was also reported by Verma and Mohanty (2000). The
effect of salt stress on A. fusiformis was further investigated
by Gong et al. (2008). The results confirmed previous obser-
vations that salt stress leads to damage on the reducing side of
PSII and, in particular, to a modification of the QB niche.
Moreover, the authors confirmed that also the donor side of
PSII was affected by salt stress, and that this damage was
associated with a dissociation of the PsbO protein from the
thylakoid membranes (Gong et al. 2008).
Another modification in salt-stressed cells is an increase in
the respiration rate (Vonshak et al. 1988a). In A. fusiformis
strain M2, this increase was two- and three-fold when the con-
centration of salt in the medium was increased from 0.5 to
0.75 mM, respectively (Zeng and Vonshak 1998). Sensitivity to
photoinhibition was found to be enhanced when A. fusiformis
strain M2 cells were exposed to sizable combinations of high
light and salt stress (Zeng and Vonshak 1998). It seems that
many cyanobacteria are capable of compensating the reduction
in the energy supply coming from the photosynthetic pathway
by significantly increasing their respiratory activity, which in
addition can provide carbon skeletons for the synthesis of
organic osmolytes and for the extrusion of Na+ in cells in order
to maintain the osmotic balance (Peschek et al. 1994).
25.5.3 Osmoregulation
During the course of acclimation to salinity, an osmotic
adjustment is required (Borowitzka 1986). In Arthrospira
696
this involves the accumulation of low molecular weight car-
bohydrates. These have been identified as a nine carbon het-
eroside called glucosyl-glycerol, plus a trehalose (Martel
et al. 1992). Changes in the amount of cellular trehalose and
in the activity of the enzyme malthooligosyl trehalose hydro-
lase (Mth), the enzyme that catalyses the formation of treha-
lose from maltooligosyl trehalose, were recently investigated
in A. fusiformis by Ohmori et al. (2009). These authors
observed that the amount of trehalose in the cells increased
rapidly when a high concentration of NaCl was added to the
culture medium. The inhibition of sodium ion transport by
means of amiloride, a sodium channel blocker, and mon-
ensin, a cation ionophore, significantly decreased the amount
of cellular trehalose, suggesting that the influx of Na+ into
the cells was connected with an accumulation of trehalose.
The synthesis of trehalose was comparable when KCl was
used to replace NaCl, while the effect on cellular trehalose
synthesis was only half as much by the same amount of LiCl.
It was suggested that the cells accumulated trehalose mainly
via ionic stress, rather than by osmotic stress. It was also sug-
gested that the influx of Na+, and not the mere presence of
Na+ in the medium, is connected with the physiological effect
of NaCl. It was concluded that the addition of NaCl activated
Mth and, at the same time, triggered a transcription of the
mth gene. Disruption of the mth gene, if such is possible,
may provide further evidence on the role of Mth in the stress-
response accumulation of trehalose in Arthrospira.
25.5.4 Arthrospira as an Alkaliphile
Most Arthrospira species currently grown in mass culture
were isolated from alkaline and saline or brackish waters
characterized by high levels of carbonate-bicarbonate and
high pH levels (Sect. 25.4). The physiological characteristics
which enable a microorganism to thrive in such an extreme
environment are still not fully understood. Arthrospira not
only survives at high pH values, but actually thrives in such
conditions. Belkin and Boussiba (1991), who compared
the growth pH optima for the two cyanobacteria Anabaena
and Arthrospira, demonstrated that while the optimal pH
for Anabaena was in the range of 6.8–7.2, the maximal
growth rate for Arthrospira was obtained in the 9.5–9.8
range. When incubated at pH 7.0, the growth rate of
Arthrospira was severely inhibited and was only 20% of that
under the optimal conditions. This high pH (>8) requirement
clearly defines Arthrospira as an obligatory alkaliphile
(Grant et al. 1990).
One of the major problems faced by cells in a highly alka-
line environment is that of regulating their internal pH.
Belkin and Boussiba (1991) were the first to measure the
ability of Arthrospira to maintain a pH gradient across its
cytoplasmic membrane. It was shown that, at external pH
C. Sili et al.
values of 10.0 and 11.5, the intracellular pH values were only
8.0 and 8.5, respectively.
Many alkaliphiles require sodium in order to survive in
extreme alkaline environments (Horikoshi and Akiba 1982).
Padan et al. (1981) demonstrated that an active sodium – pro-
ton antiporter is required in order to maintain a low internal
pH. Most cyanobacteria require sodium in an mM or sub-
millimolar concentration range in order to maintain viability,
especially at an alkaline pH (Miller et al. 1984; Espie et al.
1988). Some reports have indicated, in cyanobacteria incubated
in a sodium-deficient media, the loss of the photosynthetic
oxygen evolution (Zhao and Briand 1988) and a degradation
of the thylakoid membranes (Averdano et al. 1989). The effect
of sodium deprivation is very dramatic in A. fusiformis.
In this obligate alkaliphilic cyanobacterium, a strict Na
dependence has been found for both the growth and photo-
synthetic activity of the cells (Schlesinger et al. 1996). It has
been demonstrated that concentrations of Na+ no less than
50 mM in the medium are required in order to maintain
growth at a high pH. Depletion of Na+ causes a light-depen-
dent inhibition of PSII and a loss in the phycocyanin content
of A. fusiformis cells (Schlesinger et al. 1996). It has been
suggested that pigment bleaching and a loss of PSII activity
are due to the inability on the part of the cells to maintain an
appropriate intracellular pH. Pogoryelov et al. (2003) theo-
rized that the specific site of inactivation of the photosyn-
thetic electron transport at alkaline pH lies in the
water-splitting enzyme (oxygen evolving complex) and, in
contrast with earlier reports, that the inactivation occurs in
the dark and for short periods, without any detectable dam-
age to the photosynthetic chain. A very intriguing question is
how Arthrospira is capable of coping simultaneously with
two extreme environmental situations: a high pH and a high
concentration of Na+. In fact, not only does the adaptation of
cells in this organism permit growth at high pH and high Na+
concentrations, as in the case of many other alkali-tolerant
cyanobacteria, but also the cell functions are optimized to
these extreme conditions (pH 9.5–11.5 and 150–250 mM
NaCl). Further studies on the energy requirement and
involvement of light in the regulatory process are needed.
25.5.5 How Does Arthrospira Compete
in Culture?
Studies performed on different Arthrospira isolates from
various sources demonstrate highly variable responses to dif-
ferent ecological factors (Tomaselli et al. 1987; Vonshak
et al. 1996a, b). It is on the basis of this variability that strains
suitable for the biotechnological applications are selected:
i.e. photo-, thermo- or osmotolerant strains (Tomaselli et al.
1993; Vonshak and Guy 1992; Vonshak et al. 1988b).
Detailed studies aimed at measuring the ecological specificity
25 Arthrospira (Spirulina)
of the different species, particularly of those utilized for
commercial purposes, are scarce (Kebede and Ahlgren 1996).
Nevertheless, full knowledge of the ecological demands of
the chosen species and the use of more productive selected
strains is important if high productivity is to be achieved in
intensive outdoor cultivation systems. The use by commer-
cial producers of multistrain inocula which respond posi-
tively to changes in abiotic factors (due to their heterogeneity)
represents another way of compensating for the present lack
of well-characterized strains.
Like other components of the phytoplankton, Arthrospira
plays a role in cycling nutritional elements and in their
passage into different food chains; moreover, it provides an
important direct food source for zooplankton, fishes, birds
and humans. The same factors that in the original habitat
regulate the occurrence of Arthrospira in monospecific pop-
ulations are those required for both successfully maintaining
a monoculture in intensive production ponds outdoors and
for achieving high productivity. Factors such as high alkalin-
ity and salinity are the key features that selectively limit the
growth of other organisms and predators. Moreover, once the
optimal conditions for the growth of Arthrospira are main-
tained in ponds, this organism successfully outcompetes
other occasionally occurring phototrophs for nutrient and
light. This may be attributable to its fast growth rate and pho-
toacclimation capability, and its high tolerance to environ-
mental stress (irradiance, salinity, temperature).
25.6 Mass Cultivation of Arthrospira
At present Arthrospira (commercially indicated as Spirulina)
represents the second most important commercial microalga
in term of US$ (after Chlorella), while in terms of total bio-
mass produced, the Arthrospira market is twice or more of
that occupied by Chlorella (Torzillo and Vonshak 2003).
Commercial production takes two broad approaches: that of
industrialised countries who interested in producing the bio-
mass for health food market, as well as for the extraction of
fine chemicals, and that of developing countries looking for
a rich source of protein which can be produced locally, using
marginal land and saline water unsuitable agriculture, and
the opportunity for treating animal and human waste
(Laliberté et al. 1997; Habib et al. 2008). A major producer
of Arthrospira is the DIC group of companies, Earthrise in
California, USA, Hainan DIC Marketing in Hainan Island,
China. These facilities produce about 1,000 t Arthrospira
annually (Belay 2008). Another important Arthrospira pro-
ducer is Cyanotech Corporation of Hawaii with an annual
production of 300 t (dw). Other producers are located mainly
in the Asia-Pacific region, particularly China and India (Lee
1997) and the highest production capacity of Arthrospira
biomass takes place in China. Recent estimates are that the
697
total potential of the different sites in China now exceeds
2,000 t (dw), but the situation is changing rapidly due to the
development of production on the Ordos Plateau of Inner
Mongolia (Lu et al. 2011). Although the average annual tem-
perature in the region is only 6.4°C (Qiao et al. 2001), which
poses a challenge (Li et al. 2003), commercial production
under greenhouses permits lower costs than elsewhere in
China and it is aimed to reach an annual output of 3,000 t
within a few years. Lu et al. (2011) state that a 1,000,000-t
plan for production has been sketched out.
Arthrospira production is mostly carried out in raceway
ponds of 2,000–5,000 m2 and may contain between 400 and
1,000 m3 of culture according to the depth adopted, which
can vary between 15 and 40 cm depending on season, desired
algal density and, to a certain extent, the desired biochemical
composition of the final product (Shimamatsu 2004; Belay
2008). Further information on commercial production of
Arthrospira (Spirulina) the reader can be found in more spe-
cialized books (Vonshak 1997b; Richmond 2004a; Gershwin
and Belay 2008).
The major share of the market for this organism is for
health food involving crude biomass production. This has the
advantages of simple processing (harvest and rudimentary
handling) keeping production costs reasonably low, and to
elude the competition with chemical industry which cannot
match the wealth in nutritional bioactive components and
attractiveness of natural products (Boussiba and Affalo 2005).
The experience gathered over many years of industrial
production of Arthrospira has allowed to individuate the
main problems related with commercial scale production
(Shimamatsu 2004). They are: (1) how to maintain an unial-
gal production, that is, without sizeable presence of contami-
nants all over the year, and (2) how to maintain a consistent
quality of the product. These two aspects are strongly depen-
dent on an appropriate strain selection and adequate formula-
tion of the culture medium. Major criteria for the selection of
strains are growth rate, biochemical composition and resis-
tance to environmental stress, usually high light and low and
high temperatures at each production site. The chemical
composition of culture medium is similar to than used for
laboratory culture (Zarrouk’s medium) with a high content
of NaHCO3 (16.8 g L−1), K2HPO4 (0.5 g L−1) and KNO3
(2.5 g L−1), pH is maintained at 9.5–10.3 by artificial CO2
supply. The high alkalinity and high pH represent an efficient
barrier against contamination by other microalgae, permit-
ting the maintenance of a unialgal culture in open ponds. The
cost of nutrients usually dictates the choice of nutrient to be
used. For example, the use of ammonia, which is cheaper
than nitrate, is restricted by the fact that it can be toxic to the
cultures. The predominance of NH3 over NH4+ at pH values
greater than 9.25 (the pKa of the ammonia system at 25°C),
together to the high permeability of the NH3, causes uncou-
pling of photosynthesis in Arthrospira cells (Boussiba 1989;
698
Boussiba and Gibson 1991). At pH values of 9.5 which is
considered optimal for the growth of Arthrospira, concentra-
tion of free ammonia above 2.5 mM is reported to be toxic to
many algae (Abeliovich and Azov 1976; G. Torzillo, unpub-
lished data). For this reason, the fed-batch addition of
ammonia-based nitrogen sources is mandatory to effectively
prevent any inhibiting effect (Rodrigues et al. 2011).
Moreover, the fed-back addition of ammonia-nitrogen can
also reduce the risk of nitrogen deficiency which may occur
when ammonium salts are used as the only source of nitro-
gen and ammonium is lost by out-gassing. For this reason the
simultaneous use of nitrates and ammonium salts are often
used Arthrospira farms to avoid the risk of nitrogen
depletion.
Another important nutrient component of the culture
medium is the carbon source. The carbon content of the
Arthrospira biomass is about 50% (Torzillo et al. 1991),
meaning that a minimum of 1.8 g of CO2 to synthesize 1 g
(dry weight) of biomass is required. Therefore, the utiliza-
tion of CO2 in the flue gas may help to reduce the cost of the
biomass production (Ferreira et al. 2012). Strategies for sup-
plying CO2 in large-scale cultivation ponds to increase the
CO2 absorptivity have been proposed (Bao et al. 2012).
Cultures are operated according to a semi-continuous
regime where a constant cell concentration is maintained in
the ponds, usually in the range of 400–600 mg L−1 dry weight,
by daily harvesting of biomass to the extent that it has grown
over the last 24 h. This concentration range is mainly dictated
by harvesting efficiency. However, a concentration below
100 mg L−1 dry weight can make cells susceptible to photo-
inhibition (Vonshak and Guy 1992; Torzillo et al. 1998).
This phenomenon can occur at the start of the pond inocu-
lation, when the culture is too dilute and not sufficiently
acclimated to high light conditions, or when cultures are
subject to a combination of high light and low temperature
(Sect. 25.5.1).
Maintaining a culture in large open ponds without any
contamination by other organisms is one of the major chal-
lenge. Vonshak and Richmond (1988) estimated the overall
annual loss of productivity due to contamination and subse-
quent discarding of culture to be in the order of 15–20%.
Dilution of the cultures caused by rainfall and low light con-
ditions often facilitates the onset of microalgae (Chlorella in
particular), protozoa and insects. Continuous recycling of
medium can easily result both in the concentration of con-
taminants like Chlorella or Arthrospira strains characterized
by having short filaments, that are not arrested by the filters,
and in the accumulation of organic matter because of decom-
position of death algae. This situation usually stimulates the
growth of graze particularly protozoa. In order to prevent this
situation it is mandatory to maintain the cultures at the opti-
mum growth conditions, by continuous monitoring of the
physiological state of the cultures using fast and reliable
C. Sili et al.
measurements such as chlorophyll fluorescence technique
(Sect. 25.5.1), and to reduce as much as possible the stress
damage mechanically created by excessive stirring or by use
of centrifuge pumps for culture transfer (e.g. during the har-
vesting). In most cases, the steps which proved effective in
preventing contamination by Chlorella were: maintaining a
high bicarbonate concentration (e.g. 0.2 M); and taking pre-
cautions to maintain the dissolved organic load in the culture
medium as low as possible. Amoeba grazing on Chlorella,
and Arthrospira have been also observed in some improperly
operated commercial ponds. Addition of ammonia (2 mM)
arrested the development of these grazers (Vonshak et al.
1983). Similar observations have been reported by Lincoln
et al. (1983), who showed that the population of grazers was
significantly reduced when ammonia was used as the main N
source. However, ecological control by predators, although
this has occasionally been observed in natural lakes domi-
nated by Arthrospira (e.g. Lake Ankorongo, Madagascar),
has been never applied in industrial cultures (Shimamatsu
2004).
Safety and quality control of biomass represent another
important issue for the industrial production of Arthrospira
(Belay 2008). Toxic species of other cyanobacteria may be
present (Chap. 24) and some Arthrospira biomass companies
have developed methods for their determination and actually
certify their products to be free of toxins, such as microcys-
tins. Arthrospira biomass does not contain microcystin (An
and Carmichael 1996), but contamination by other cyanobac-
teria cannot be ruled out. A potential problem of open ponds
of Arthrospira production is that the water may be contami-
nated with pathogenic organisms. Handling of the product
during processing can also result in microbial contamination
(Belay 2008). Production cost is an area where little infor-
mation is available as companies are usually not available to
provide this information. However, cost of production is esti-
mated to range from US$ 6–12 kg−1 dry matter. The lower
figure is based on a “feed” grade product, not for direct
human consumption. The product is sun-dried and contains a
high ash and low chlorophyll content. The demand for such
a product is continually growing, as its relatively low price is
making it more attractive to the feed industry as an additive
to fish and chicken feed. This demand is reflected in the
reports of an increase in the production of feed grade prod-
ucts up to 60 t year−1 by Earthrise Farms, compared to only a
few tonnes of feed grade produced some years ago. A starch-
producing factory in Thailand has started to use its water
refuse from an anaerobic digester to grow Arthrospira bio-
mass and then sun-dry the product. Cost estimates indicate
that the product can be sold for less than US$ 6 kg−1. This is
a very important step towards the production of Arthrospira
biomass, not only as a health food product, but for a much
larger market such as for feed additives. Further reduction of
production cost depends upon several factors: (1) increase in
25 Arthrospira (Spirulina)
Fig. 25.17 (a) An overview of the soda Lake Kossorom (Chad); (b) a
bloom of Arthrospira fusiformis (Lake Kossorom); (c) accumulation of
A. fusiformis biomass at the edge of the lake; (d) harvesting A. fusi-
productivity of the organism; (2) control of contamination
and hence the reduction on the culture renewable time; (3)
increasing of harvesting efficiency; (4) reduction of the over-
all operational efficiency of the farm (Shimamatsu 2004).
25.7 Market and Application
The biomass produced is mainly sold to the health food addi-
tives market in the form of powder or pills. It has also been
selected by the NASA (National Aeronautics and Space
Administration) and by the European Space Agency as one
of the primary foods for long-term space missions. In many
cases the term “Spirulina” has become a practical synonym
for cultivated microalgae. Attempts have been made by
Proteous (a marketing company mainly associated with
Earthrise Farms in the USA) to incorporate Arthrospira bio-
699
formis blooms from the surface of Lake Kossorom; (e) young women
collecting pieces of sun-dried Arthrospira biomass (dihé) after drying
them on sand (Photos M.R. Tredici)
mass into a variety of food products such as granola bars and
various kinds of pasta. In Mexico and China, subsidized by
the government, Arthrospira powder is added to child food
such as biscuits and chocolate (Fox 1985). Another available
product is a protein extracted from Arthrospira biomass con-
taining mainly the blue pigment phycocyanin and marketed
under the “Lima Blue” brand name (Dainippon Ink 1980,
1981). The product is mainly used as a colourant for the food
market to provide an edible dye for ice cream and as a natural
dye in the cosmetics industry. The main problem is that the
pigment is light sensitive and special care has to be taken in
handling the dye to protect it from bleaching. Full accounts
of the potential application of Arthrospira as a nutritional
and therapeutic supplement in health management are given
by Belay (2002) and Gershwin and Belay (2008).
Beside its commercial production, Arthrospira still has an
important role as food in the economy of some African
700
Fig. 25.18 (a) Harvesting Arthrospira fusiformis from the surface of Twin Taung Lake, Myanmar. (b) Noodle-like filaments of Arthrospira paste
dried in the sun on transparent plastic sheets. (c, d) Sun-dried trichomes of Arthrospira fusiformis (Photos a, b: A. Vonshak; c, d: C. Sili)
countries. In 2000 a delegation funded by the African
Development Bank visited a number of African countries in
an attempt to study to what extent the local tribes use
Arthrospira biomass as a part of their local food in order to
supplement their protein requirements (Sodelac 2000). The
internal report issued indicated that the harvesting of natu-
rally-occurring blooms of Arthrospira from lakes around the
Rift Valley is a very common tradition. A well-defined social
and practical structure has been reported. Only Kanembu
women carry out the harvesting, while men are banned from
entering the water, since it is a deep-rooted belief that they
would make the lake barren. Arthrospira biomass is har-
vested from Lake Kossorom throughout the year, with mini-
mum yield in December and January, and a maximum from
June to September during the rainy season. The sun-dried
product is used by the people who harvest it, as well as being
sold on many local markets. It has been estimated that up to
4,000 t dried Arthrospira biomass are harvested every year
by local tribes in different countries from 14 wadis flooded
by Lake Chad during the rainy season. According to Sodelac
(2000) in 2000, over 90% of the production was sold by
female producers. The price of traditional dihé at production
sites in 2009 ranged between US$ 0.678 and 1.1 kg−1.
A survey carried out among the Kanembu who harvest
Arthrospira from Lake Kossorom in the Prefecture of Lac
(Chad) has added considerable understanding of local eco-
nomics (Abdulqader et al. 2000). The trading value of the
C. Sili et al.
dihé annually harvested from Lake Kossorom (about 40 t)
amounted to more than US $ 100,000 which represents an
important income for the economy of the area (Fig. 25.17).
Natural production of Arthrospira has been also documented
in Myanmar. Four volcanic lakes with natural Arthrospira
bloom were studied beginning in 1984. Production began at
Twin Taung Lake in 1988, and in 1999 increased to
100 t year−1. About 60% is harvested from boats on the sur-
face of the lake, while the other 40% is cultivated in outdoor
ponds alongside the lake. During the bloom season in sum-
mer, when Arthrospira forms thick mats on the lake, people
in boats collect a dense concentration of Arthrospira in buck-
ets (Fig. 25.18a). This biomass is harvested on inclined
filters, washed with fresh water, dewatered and pressed again.
The paste is then extruded into noodle-like filaments which
are dried in the sun on transparent plastic sheets (Fig. 25.18b).
Dried chips are taken to a pharmaceutical factory in Yangoon,
pasteurized, and pressed into tablets (Habib et al. 2008). A
more detailed study on the consumption patterns in these
various countries, as well as on the strains used, could A
more detailed study on the consumption patterns, as well as
on the strains used, could provide valuable information for
the further development of the Arthrospira production, pro-
cessing and consumption industry.
The accumulated knowledge of the ecology, physiology
and biochemistry of Arthrospira has made its commercial
production feasible. At present the photosynthetic efficiency
25 Arthrospira (Spirulina)
of Arthrospira is still well below (2–3%) of the theoretical
maximum, which can be set at about 10% total solar light.
Light saturation it has indicated to be one of the most important
factors which limit the full exploitation of the photosynthetic
capacity. More work is required to provide strain selection
and better understanding of the factors governing growth,
especially light utilization in dense cultures, to support this
growing agro-industry.
Acknowledgements The authors (TG and SC) thank the Italian Space
Agency for financial support through the MoMa project (contract number
I/014/06/0). Partial funding support was provided for joint projects in
the framework of Bilateral Scientific Agreement (2010–2012) between the
National Research Council of Italy and the Czech Academy of Science.
We thank Prof. Brian Whitton for his critical and helpful comments.
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