arthrospira (spirulina) 25 · 2018-04-30 · b.a. whitton (ed.), ecology of cyanobacteria ii: their...
TRANSCRIPT
677B.A. Whitton (ed.), Ecology of Cyanobacteria II: Their Diversity in Space and Time,DOI 10.1007/978-94-007-3855-3_25, © Springer Science+Business Media B.V. 2012
Contents
Summary ........................................................................................ 677
25.1 Introduction .................................................................. 677
25.2 Morphology ................................................................... 678
25.3 Taxonomy ...................................................................... 686
25.4 Occurrence and Distribution ...................................... 689
25.5 Physiology ..................................................................... 691 25.5.1 Response to Environmental Factors ............................... 692 25.5.1.1 Effect of Light on Growth .............................................. 692 25.5.1.2 Light Stress – Photoinhibition ....................................... 692 25.5.1.3 Effect of Temperature on Photosynthesis
and Respiration .............................................................. 692 25.5.1.4 Effect of Temperature on Growth
and Cell Composition .................................................... 693 25.5.1.5 Interaction of Low Temperature
with Light and High Oxygen Concentration .................. 694 25.5.2 Effect of Salinity on Growth, Photosynthesis
and Respiration .............................................................. 695 25.5.3 Osmoregulation .............................................................. 695 25.5.4 A rthrospira as an Alkaliphile ......................................... 696 25.5.5 How Does Arthrospira Compete in Culture? ................. 696
25.6 Mass Cultivation of Arthrospira .................................. 697
25.7 Market and Application .............................................. 699
References ...................................................................................... 701
Summary
The successful commercial exploitation of Arthrospira because of its high nutritional value, chemical composition and safety of the biomass has made it one of the most impor-tant industrially cultivated microalgae. Knowledge of its biology and physiology, which is essential for understand-ing the growth requirements of this alkaliphilic organism, has been used in developing suitable technologies for mass cultivation. The relationships between environmental and cultural factors, which govern productivity in outdoor cul-tures, are discussed in connection with growth yield and ef fi ciency. The response of Arthrospira and its modi fi cation under stress is described, together with the strategy of osmotic adjustment and the mechanism of internal pH regu-lation to alkalinity. The metabolic plasticity of the response to disparate environmental stimuli is demonstrated in the natural environment, but is also well-expressed in the main-tenance of high productive monoculture in intensive outdoor cultivation systems.
While the confused taxonomy of Arthrospira and its relationship with Spirulina has been resolved by study of the ultrastructural feature of trichomes and 16S rRNA sequence analysis, the problem of species de fi nition is still ongoing. However, molecular methods such as total DNA restriction pro fi le analyses of a wide range of strains are helping to resolve this.
25.1 Introduction
This account needs to start with a taxonomic comment. Ever since Arthrospira was fi rst reported in 1852 by Stizenberger, many species of this genus of helically coiled cyanobacteria have been described and isolated. However, its classi fi cation has long been a source of confusion. Geitler ( 1925 ) invalidated the genus Arthrospira in his revision of the Cyanophyceae and included all regularly helically coiled Oscillatoriales without fi rm sheaths in the previously described
Arthrospira (Spirulina)
Claudio Sili , Giuseppe Torzillo and Avigad Vonshak
25
C. Sili (*) • G. Torzillo Istituto per lo Studio degli Ecosistemi, CNR , Via Madonna del Piano, 10, 50019 , Sesto Fiorentino, Firenze , Italy e-mail: [email protected] ; [email protected]
A. Vonshak Microalgal Biotechnology, The Jacob Blaustein Institute for Desert Research , Ben-Gurion University of the Negev , Sede Boker Campus, Beersheba , 84990 , Israel e-mail: [email protected]
678 C. Sili et al.
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 fi ne 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 fi rst detailed study of the growth require-ments and physiology of Arthrospira . The Ph.D. research of Zarrouk ( 1966 ) was the basis for establishing the fi rst 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).
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 m m), 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 fi ssion in a single plane perpendicular to the main axis. Trichome elongation occurs through multiple intercalary cell division along the entire fi lament. 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 sacri fi cial cell (necridium) that fi rst becomes colourless and fi nally 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 m m, while the helix pitch typically ranges from 0 to 80 m m and its diameter from 15 to 60 m m. 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 fi rst 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
67925 Arthrospira (Spirulina)
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
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
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 fi xed in
formaldehyde (2%); ( c ) fresh clonal trichome grown in mass culture; ( d ) auto fl orescence of trichome shown in ( c ). Bar marker = 10 m m (Photos C. Sili)
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 m m (Photos C. Sili)
680 C. Sili et al.
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 fi lament 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 fi laments originated from the linear fi laments 9 years old obtained with ionizing radiation.
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 signi fi cant 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 fi laments 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 fi laments to straight ones, and that this fact supports the idea that straight fi laments may have a higher survival capacity. However, bio-chemical composition and nutritional properties were not affected by the shape of the fi laments (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 fi rstly 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
Fig. 25.3 Necridic cell formation in clonal: ( a ) Arthrospira fusiformis coiled trichome; ( b ) A. maxima straight trichome. Bar marker = 10 m m (Photos C. Sili)
68125 Arthrospira (Spirulina)
Tab
le 2
5.1
M
orph
olog
y of
Art
hros
pira
spe
cies
bas
ed o
n K
omár
ek a
nd A
nagn
ostid
is (
2005
) an
d K
omár
ek a
nd H
auer
( 20
11 )
Spec
ies
Tri
chom
e w
idth
( m m
) C
ell l
engt
h ( m
m)
Coi
ls (
m m)
Hel
ix s
hape
G
as
vacu
oles
M
orph
olog
y of
tric
hom
e en
d W
idth
H
eigh
t T
ypes
wit
hout
gas
-vac
uole
s (f
orm
ing
mat
s)
A. b
alkr
ishn
anii
Kam
at 1
963
3.5–
4 (5
) 0.
8–1.
2 6–
7 12
–15
−
Rou
nded
. no
caly
ptra
A
. des
ikac
hary
ensi
s V
asis
hta
1962
0.
85–1
.7
51–6
.8
10.2
–11.
5 R
egul
arly
loos
ely
spir
al c
oils
−
R
ound
ed
A g
igan
tea
(Sch
mid
le)
Ana
gnos
tidis
199
8 (2
.5)
3–4
(8)
11–1
6 7–
12 (
15)
Reg
ular
ly c
oile
d −
+
/− c
onic
al
A g
omon
tian
a Se
tche
ll 18
95
2.5–
3 +
/− is
odia
met
ric
6–6.
5 15
–19
−
A. j
enne
ri S
tizen
berg
er e
x G
omon
t 189
2 (3
.7)
4–6
(8)
(7.4
) 8–
15 (
17)
(9.2
) 12
–25
(31)
R
egul
arly
scr
ew-l
ike
coils
−
R
ound
ed, n
o ca
lypt
ra
A. m
assa
rtii
Kuf
fera
th 1
914
5–6
Loo
sely
scr
ew-l
ike
−
Rou
nded
con
ical
A
. pel
luci
dis
Wan
g 19
33
A. p
late
nsis
(N
ords
tedt
) G
omon
t 189
2 (4
) 6–
7 (9
?)
Nea
rly
isod
iam
etri
c (2
0?)
26–3
6 (2
4?)
30–5
7 +
/− r
egul
arly
loos
ely
spir
al
coils
−
R
ound
ed o
r fl a
t cal
yptr
a
A. s
anta
nnae
Kom
árek
et
Kom
árko
vá-L
egne
rová
200
6 2.
8–3.
8 +
/− is
odia
met
ric
12.5
–18.
5 (l
oose
) 8.
8–10
.8 (
dens
e)
20–4
0 (d
ense
) 5.
5–5.
8 (l
oose
) +
/− d
ense
ly o
r lo
osel
y co
il −
R
ound
ed, n
o ca
lypt
ra
A. s
kuja
e M
agri
n, S
enna
et K
omár
ek 1
998
(2.7
) 3.
1–4.
3 3.
1–9.
3 7.
4–11
.8
7–13
R
egul
arly
scr
ew-l
ike
coil
−
Rou
nded
A
. ten
uis
Brü
hl e
t Bis
was
192
2 A
bout
2
2–3
20–3
5 −
T
ypes
wit
h ga
s va
cuol
es (
plan
kton
ic, s
olit
ary
tric
hom
es)
A. a
rgen
tina
(Fr
engu
elli)
G
uarr
era
et K
ühne
man
n 19
49
9–10
.5
33–4
9 63
–75
+
A. f
usif
orm
is (
Vor
onic
hin)
K
omár
ek e
t Lun
d 19
90
(3.4
)5–9
(11
?)
2–3
× s
hort
er
than
wid
e 15
–50
0–80
R
egul
arly
scr
ew-l
ike
coile
d in
tens
ely
narr
owed
or
wid
ened
tow
ards
end
s
+
Rou
nded
A. i
ndic
a D
esik
acha
ry e
t Jee
ji B
ai 1
992
8–9
1/3–
1/2
as lo
ng
as w
ide
16–3
9 16
–66
Spir
ally
coi
led
and
dist
inct
ly
narr
owed
tow
ards
end
s +
C
onic
al c
alyp
tra
A. g
hann
ae D
roue
t et S
tiric
klan
d in
Dro
uet 1
942
3–5.
2 20
–22
20–3
5 R
egul
arly
loos
ely
scre
w-l
ike
coils
+
Sl
ight
ly a
ttenu
ated
end
an
d ca
pita
te
A. j
oshi
i Vas
isht
a 19
63
4.2–
5.1
2.7–
3.4
6.8–
7.6
17–1
9.2
Reg
ular
ly s
crew
-lik
e co
ils,
not a
ttenu
ated
tow
ards
end
s +
R
ound
ed, n
o ca
lypt
ra
A. m
axim
a Se
tche
ll et
Gar
dner
in
Gar
dner
191
7 (6
.5)
7–9
(10)
5–
7 40
–60
70–8
0 R
egul
arly
loos
ely
scre
w-l
ike
coile
d, g
radu
ally
slig
htly
at
tenu
ated
at e
nds
+
Rou
nded
with
thic
kend
ou
ter
cell
wal
l
682 C. Sili et al.
was the fi rst 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 con fi rmed 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
arti fi cial 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, fi ve 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. 25.4 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 fi xed in
formaldehyde (2%) from Lake Kossorom. ( f ) Shows that occasional ± straight trichomes may also be present in a wild sample. Bar marker = 40 m m (Photos C. Sili)
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 m m (Photos C. Sili)
Fig. 25.6 ( a ) Morphological aspects of regular clonal trichomes of Arthrospira maxima isolated from the solar Lake Texoco evaporator. ( b ) Typical trichome regularly screw-like coiled with gradually attenuated
cells diameter at the ends. Bar markers correspond to 40 m m in a and 20 m m in b (Photos C. Sili)
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- fi eld photo showing the abundant gas vacuoles. Bar marker = 20 m m (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 fi laments; ( 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 m m, B = 20 m m (Photos C. Sili)
68525 Arthrospira (Spirulina)
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 in fl uenced 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 speci fi c 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 fi ndings agree with those made by Booker and Walsby ( 1979 ) on Anabaena fl os-aquae . They observed that helical fi laments of all length classes sank signi fi cantly faster than straight ones of corre-sponding length. In the case of straight fi laments there was a
slight progressive increase in mean sinking speed with increasing length helical fi laments though up to 10–20 cells. The authors concluded the advantage of straight fi laments could not be in conferring a higher growth rate, but it might be in resisting predation or affecting the rate of sinking and fl oating. 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
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 m m (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
stress; arrow indicates the position on the trichome where the helix orientation reversed from left-hand ( a ) to right-hand ( b ). Bar marker = 20 m m (Photos C. Sili)
686 C. Sili et al.
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 fi brils) 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 con fi rmed 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 fi lament at intervals of about 2–3 m m. 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 de fi nitive 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 fi laments, which they described as “ Spirulina jenneri f. platensis ”, even though it possessed septa. At that time the genus Spirulina
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 con fi rmed 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-uni fi ed the members of these two genera in Spirulina Turpin 1829 . He based classi fi cation 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 classi fi ed in Section Arthrospira and those with unlikely or only arti fi cially 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: Spirulina Turpin 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 fi lament; (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
68725 Arthrospira (Spirulina)
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 m m in Spirulina versus typically 6–12 m m 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 fi rst 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 signi fi cant taxo-nomic feature (Fig. 25.12 ). Komárek and Anagnostidis ( 2005 ) concluded that Arthrospira has solitary and free- fl oating trichomes in the planktonic forms of subtropical and tropical saline lakes ( A. maxima and A. fusiformis ), while freshwater benthic forms were united in a fi ne, 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
provides a summary of the synonyms of different Arthrospira species recognised by Komárek and Anagnostidis ( 2005 ) . However, this classi fi cation 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 fi rst time indicated as the “polyphasic approach”.
Viti et al. ( 1997 ) , using total DNA restriction pro fi le 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 (Ampli fi ed 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 con fi rmed 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 con fi rmed once again, that Arthrospira is not related to Spirulina and for the fi rst 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 con fi rmed the general mismatch between their geographic origin and morphology. Similar conclusions were reached by Baurain et al. ( 2002 ) , who found no clear relationships
Fig. 25.12 Clonal trichomes of Arthrospira indica isolated from Lake Lonar. Arrows indicate the presence of a calyptra. Bar marker = 20 m m (Photos C. Sili)
688 C. Sili et al.
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 signi fi cant 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 Arthrospir a 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 dif fi cult 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 identi fi cation of the Arthrospira species.
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
68925 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 CO
2 . 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 ), fl owing 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 ), fi lter 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,
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 in fl ow 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 m mol 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 super fi cial 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. Ca 2+ and Mg 2+ concentrations are low. SO
4 2− 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 monospeci fi c 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 dino fl agellates. 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 fi sh Tilapia (Coe 1966 ; Bergman et al. 2003 ) . Another consumer of Arthrospira and other large fi lamentous cyanobacteria such as Anabaenopsis is the fl amingo 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,
Rombou, Yoan) and pools (Latir, Iseirom, Latir, Liva), Kanem region
A. platensis, A. platensis f. minor Léonard and Compère ( 1967 ) , Iltis ( 1972 ) , and Rich ( 1931 )
Lake Kailala, Lake Kossorom A. fusiformis Sili et al. (1999) Kenya Natron lakes
(Bogoria, Crater, Elmenteita, Nakuru) A. platensis Rich ( 1931 ) and Vareschi ( 1982 )
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 (Madurai, MCRC isolate)
A. indica Desikachary and Jeeji Bai ( 1992 )
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 ef fl uent 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. platensis a Lewin ( 1980 ) Peru Lake Huachachina A. platensis Busson ( 1971 )
A. maxima Desikachary and Jeeji Bai ( 1992, 1996 ) Uruguay Montevideo A. platensis b 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
69125 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 fi laments 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 HCO
3 −1 and CO
3 −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 modi fi cation 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 in fl uence 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 HCO
3 − and CO
3 2− , 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 )
production facilities in the outside rim of the solar evapora-tor, where it was present in almost monospeci fi c 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 m S 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 signi fi cant 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 fl ux. 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 2004 b ; 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 ) . Suf fi cient mixing also ensures an even distribution of nutrients and oxygen degassing of the culture, and at the same time prevents fi lament 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 suf fi cient concen-trations, the elevated concentration of bicarbonate-carbonate favours the high productivity observed in nature.
692 C. Sili et al.
25.5.1 Response to Environmental Factors
25.5.1.1 Effect of Light on Growth Zarrouk ( 1966 ) was the fi rst 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 dif fi cult 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 m mol 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 m mol 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 ef fi ciency 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 CO
2 ,
Ogawa and Aiba ( 1978 ) estimated the slightly higher value for photosynthetic ef fi ciency 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 fl ux 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 CO
2 uptake or O
2 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 speci fi c 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 P
max (maximum saturation rate), which
was reduced by about 25% (Table 25.4 ). These results empha-size that photoinhibition not only affects P
max, but actually has
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
Fig. 25.13 Photosynthetic light response curves of A. fusiformis strain M2 (○ photoinhibition; ● control) for 45 min at a PFD of 2,500 m mol 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 m mol photon m −2 s −1 at 35°C
Parameter Unit Control Photoinhibited
P max
m mol O 2 mg chl −1 h −1 800 600
I K m mol photon m −2 s −1 430 610
a m mol O 2 (mg chl m mol
photon m −2 s −1 ) −1 h −1 1.915 1.094
R m mol O 2 mg chl −1 h −1 −20.5 −26.5
P max
light saturated rate, I k saturation irradiance, a initial slope of the
P/I curve, R dark respiration rate
69325 Arthrospira (Spirulina)
dark respiration rate by following the O 2 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 signi fi cantly 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:
where R is the respiration rate ( m mol O 2 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 coef fi cient (Q
10 ) 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 con fi rmed 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
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. Signi fi cant 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
= (0.0636.T)R 0.771.e
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
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 m mol photon m −2 s −1 )
694 C. Sili et al.
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 ef fi cient 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 F
v /F
m ratio (variable to maximum
fl uorescence). The F v /F
m 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 F
v /F
m ratio decreased to up to 45%
of the morning value. Complete recovery of the F v /F
m 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 insuf fi cient, photoinhibition can occur. High oxygen concentration itself can in fl uence the growth and the photo-synthetic activity of the cultures even when the light inten-
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 in fl uenced 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 fl uorescence 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 F
v /F
m 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 signi fi cant 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 in fl uenced 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 ) .
Fig. 25.16 Effect of oxygen concentration and temperature on maximum quantum yield of PSII (F
v /F
m ) of Arthrospira fusiformis strain M2
cultures grown outdoors in photobioreactors. (■) Low oxygen- optimal temperature; (▲) high oxygen – optimal temperature; (●) high oxygen-low temperature; (Δ) PFD
69525 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 fi rst 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, signi fi cant 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 re fl ect 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 speci fi c modi fi cations 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 fi rst 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 ) .
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 fl uorescence 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 fi rst 12 h of their exposure to high salinity consisted of two phases. The fi rst phase, which took place during the fi rst 4 h, was characterized by an immediate drop in F
v /F
m during the
fi rst 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 F
v /F
m 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 con fi rmed previous obser-vations that salt stress leads to damage on the reducing side of PSII and, in particular, to a modi fi cation of the Q
B niche.
Moreover, the authors con fi rmed 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 modi fi cation 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 signi fi cantly 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 C. Sili et al.
this involves the accumulation of low molecular weight car-bohydrates. These have been identi fi ed 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, signi fi cantly decreased the amount of cellular trehalose, suggesting that the in fl ux 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 in fl ux 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 de fi nes 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 fi rst to measure the ability of Arthrospira to maintain a pH gradient across its cytoplasmic membrane. It was shown that, at external pH
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-de fi cient 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 speci fi c 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 speci fi city
69725 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, fi shes, birds and humans. The same factors that in the original habitat regulate the occurrence of Arthrospira in monospeci fi c 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 fi ne 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-Paci fi c region, particularly China and India (Lee 1997 ) and the highest production capacity of Arthrospira biomass takes place in China. Recent estimates are that the
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 m 2 and may contain between 400 and 1,000 m 3 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 fi nal 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 NaHCO
3 (16.8 g L −1 ), K
2 HPO
4 (0.5 g L −1 ) and KNO
3
(2.5 g L −1 ), pH is maintained at 9.5–10.3 by arti fi cial CO 2
supply. The high alkalinity and high pH represent an ef fi cient 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 NH
3 over NH
4 + at pH values
greater than 9.25 (the pKa of the ammonia system at 25°C), together to the high permeability of the NH
3 , causes uncou-
pling of photosynthesis in Arthrospira cells (Boussiba 1989 ;
698 C. Sili et al.
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 de fi ciency 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 CO
2 to synthesize 1 g
(dry weight) of biomass is required. Therefore, the utiliza-tion of CO
2 in the fl ue gas may help to reduce the cost of the
biomass production (Ferreira et al. 2012 ). Strategies for sup-plying CO
2 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 ef fi ciency. 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 suf fi ciently 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 fi laments, that are not arrested by the fi lters, 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
measurements such as chlorophyll fl uorescence 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 signi fi cantly 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 fi gure 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 fi sh and chicken feed. This demand is re fl ected 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
69925 Arthrospira (Spirulina)
productivity of the organism; (2) control of contamination and hence the reduction on the culture renewable time; (3) increasing of harvesting ef fi ciency; (4) reduction of the over-all operational ef fi ciency 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-
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
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-
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)
700 C. Sili et al.
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-de fi ned 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 fl ooded 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
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 fi lters, washed with fresh water, dewatered and pressed again. The paste is then extruded into noodle-like fi laments 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 ef fi ciency
Fig. 25.18 ( a ) Harvesting Arthrospira fusiformis from the surface of Twin Taung Lake, Myanmar. ( b ) Noodle-like fi laments 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)
70125 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 fi nancial support through the MoMa project (contract number I/014/06/0). Partial funding support was provided for joint projects in the framework of Bilateral Scienti fi c 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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